In Displaced Intracapsular Hip Fractures (ICHF) in young active patients, preservation of the femoral head and its blood supply are of high importance and urgent surgical treatment with anatomic reduction and internal fixation is the preferred intervention. Due to the strong varus displacement shear forces exerted across the hip, there are relatively high complication rates after fixation. There is no consensus regarding the optimal fixation device or technique. This retrospective study compared closed reduction internal fixation method using cannulated cancellous screw (CCS) with the Targon Femoral Neck (TFN) hip fixed angle screw. Data regarding, gender, operational data, duration of surgery, complications, NAS (Numerical Analogue Scale) pain score, Modified Harris Hip Score (MHHS) and SF-12 scores were retrieved for patients younger than 65 with displaced ICHF. Eighty-two patients were included in the study, 30 patients treated with CCS were compared to 52 patients treated with TFN. Fracture configuration (Garden and Pauwel classifications), mean time to surgery and complication rate did not differ significantly. Operative time did differ significantly between groups (CCS 56 minutes, TFN 92 minutes, p<0.001). At final follow-up the CCS group reported less pain (NAS 2.3 vs 3.5, p< 0.049) and better Mental Health Composite score of SF-12 (p=0.017) compared to the TFN group. Complication rates for the treatment of displaced ICHF with TFN and CCS showed no significant differences; however, the functional outcomes, as presented by the NAS and Mental Health Composite score of SF-12, showed superiority for CCS treatment. As this fixation method is related to reduce costs, we suggest CCS for the treatment of displaced ICHF in the young population.

With increasing numbers of total joint arthroplasties being performed, peri-prosthetic fracture incidence is rising, and operative management remains the gold standard. Short-term survivorship up to 12 months has been well-documented but medium to long-term is almost unknown. We present survivorship review from a district general hospital, undertaking 800 primary hip and knee arthroplasties per year. Patients with peri-prosthetic fractures and background total knee replacements were identified using our computer database between 2006–2011. All patients were operated on our site; methods used include open reduction, internal fixation (ORIF) using Axsos (Stryker Newbury) locking plates (28), intra-medullary nailing (1) or complex revision (6) depending on fracture and patient factors and surgeon's preference. Mortality was assessed at 30 days, 12 months and 5 years. Thirty-four patients were identified with a 7:1 female to male ratio and mean age of 76. 75% of patients had their primary arthrodesis at our hospital. There was only 1 plate failure noted requiring revision plating. Mortality at 30 days, 12 months and 5 years were 3.2, 12.5% and 50% respectively. When compared to the literature our time interval from index surgery to fracture is considerably longer (115 vs 42 months). Further multi-centre reviews are required to further asses this unexpected finding. Overall mortality is better than our hip fracture cohort, suggesting that good results can be achieved in District Hospital. The longer-term results are encouraging and can act as a guide for patients with this injury. We recommend that patients are managed in consultant-led, multi-disciplinary teams.

Inflammation has been associated with immunological dysfunctions and chronic inflammatory diseases but is important for normal repair processes like bone healing. Macrophages (mØ) are important for bone growth, maintenance, and regeneration. MØ are distinct from other bone cells and play an important role in the inflammatory stage of bone healing. Previous data has shown that ablation of mØs during the inflammatory stage can severely impair bone healing and exacerbate bone loss in osteoporotic models. However, little research has focused on characterizing the mØ subtypes found during the inflammatory stage. We hypothesized that different mØ subtypes are activated during inflammation and release factors to regulate bone repair. Therefore, bone marrow was collected from mice femurs at days 0, 1, 2, 4, and 7 after fracture and mØ were isolated using established methods. MØ subtypes were identified using anti-F4/80, anti-CD80, and anti-CD86 antibodies via flow cytometry and cytokine expression was quantified using Luminex. When compared to unfractured controls, a 40–50% increase in MHC class II+/CD80+ double positive mØs and MHC class II+/CD86+ double positive mØs were found on day 2 post-fracture, which remained elevated through day 4 or 7, respectively. No differences were found in mØ populations between femurs in naïve (unfractured) mice. mØs of the fractured limbs expressed higher levels of cytokines overtime. Our results suggest that different subtypes of mØs are present during the inflammatory stage and may support diverse functions such as effertocytosis, chemotaxis, and tissue anabolism or catabolism, which provides insight into their contribution in normal or uncontrolled inflammatory related processes and conditions.

A significant number of fractures develop non-union. Stem cell therapy may be beneficial in their treatment, however this requires acquisition, culture and delivery of stem cells. Stem cell homing and migration is regulated through SDF-1 and its receptor CXCR4. Studies have demonstrated endogenous mobilisation of different populations of stem and progenitor cells by administering growth factors with a pharmacological antagonist of CXCR4, AMD3100. This may therefore be a means to improve compromised fracture healing. A 1.5mm femoral osteotomy in adult female Wistar rats was stabilised with an external skeletal fixator. After osteotomy, saline/PBS (P) VEGF (V), IGF-1 (I) or GCSF (G) (100ug/kg, 0.5ml/100g i.p.), were administered daily for 4 days. On day 5, a single 5mg/kg i.p. dose of AMD3100 was given. Control group (C) did not receive growth factors or AMD 3100. At 5 weeks, the femur was retrieved and microCT scanned. Compared to group C (n=7), group P (n=5) had a significant increase in bone volume (P=0.01) 8.9±2.2um∧3 (control 4.3±3.1um∧3) and trabecular thickness (P=0.03). Group I (n=6) also had a significant increase in bone volume (P=0.035) 5.1±4.2um∧3 and trabecular thickness 0.062±0.008um (control 0.042±0.01um) (P=0.01). Group V (n=8), showed a non-significant increase in bone volume; 5.22±1.7um∧3 and trabecular thickness 0.048±0.007um. Group G (n=5) showed a significant decrease in bone volume (2.5±2.6um∧3) (P=0.048). AMD3100 alone and IgF1-AMD3100, showed the greatest increase in bone formation, presumably through mobilisation of beneficial combinations of stem and progenitor cells. GCSF-AMD3100, which is expected to mobilise hematopoietic progenitors inhibited bone healing.

Total ankle replacement (TAR) is contraindicated in patients with significant talar collapse due to AVN and in these patients total talus body prosthesis has been proposed to restore ankle joint. To date, five studies have reported implantation of a custom-made talar body in patients with severely damaged talus, showing the limit of short-term damage of tibial and calcaneal thalamic joint surfaces. Four of this kind of implants have been performed. The first two realized with “traditional” technology CAD-CAM has been performed in active patients affected by “missing talus” and now presents a survival follow-up of 15 and 17 years. For the third patient affected by massive talus AVN we designed a 3D printed porous titanium custom talar body prosthesis fixed on the calcaneum and coupled with a TAR, first acquiring high-resolution 3D CT images of the contralateral healthy talus that was “mirroring” obtaining the volume of fractured talus in order to provide the optimal fit. Then the 3D printed implant was manufactured. The fourth concern a TAR septic mobilization with high bone loss of the talus. The “two-stage” reconstruction conducted with the implant of total tibio-talo-calcaneal prosthesis “custom made” built with the same technology 3D, entirely in titanium and using the “trabecular metal” technology for the calcaneous interface. Weightbearing has progressively allowed after 6 weeks. No complications were observed. All the implants are still in place with an overall joint mobility ranging from 40° to 60°. This treatment requires high demanding technical skills and experience with TAR and foot and ankle trauma. The 15 years survival of 2 total talar prosthesis coupled to a TAR manufactured by a CAD-CAM procedure encourages consider this 3D printed custom implant as a new alternative solution for massive AVN and traumatic missing talus in active patients.

Physiotherapy is generally accepted as an important component in the care pathway surrounding total knee replacement. Therapy interventions can be delivered prior to surgery, as part of the inpatient stay, and post-operation through outpatient appointments. Though ‘physiotherapy’ is generally promoted there is considerable national and international variation in actual therapy provision. Specific rehabilitation protocols are strongly entrenched at individual physiotherapy departments however the wider efficacy of varying physiotherapeutic interventions is poorly established. This uncertainty as to effectiveness of physiotherapy makes it difficult for commissioning organisations, healthcare providers, and patients to make decisions as to what therapy is ‘needed’ and therefore the correct level and mechanism of funding for such services. This talk will explore the variation in physiotherapy service provision and evidence for different interventions surrounding total knee replacement.

ONFH with large or lateral-located lesion is challenging due to difficulty of regeneration. We introduce novel tissue engineering technique using ex vivo expanded bone marrow stromal cell seeded on calcium metaphosphate (CMP) scaffold to regenerate dead bone for these challenging cases. Ten millilitres of bone marrow was aspirated from iliac crest and mononuclear cells were collected. These cells were expanded and differentiated to osteoblast-lineage cells using osteogenic media and autologous serum for 2–4 weeks ex vivo. Porous bead-form scaffolds were made of CMP and cells were seeded in a density of million/ml³ into 20 to 30 beads for 1 hour. The necrotic area was curetted and the beads were implanted through core tract in 9 hips (Steinberg IIc in 5 hips and IVc in 4 hips which involved greater than 30% of whole head; JIC classification C1 in 4 hips, and C2 in 5 hips which involved weight bearing area). The tract was blocked with a CMP rod. The age of patients ranged from 16 to 37. Associated factors were; steroid in 4, idiopathic in 3, alcoholic in 1 and traumatic in 1 hip, respectively. Kerboul combined necrotic angle was more than 200° in all hips. We compared preoperative and annual radiographs and MRI images to check dome depression of femoral head and signal change of osteonecrotic area. Follow-up period ranged from 8 to 14 years. Two IIc lesions progressed and were converted to THA at two and six years postoperatively. We could get clinical and radiographic success in 7 hips (78%). Follow-up radiographs and MRI showed partial or nearly complete regeneration of necrotic bone, prevention of collapse, and reduction in necrotic lesion. This can be a good strategy for bone regeneration of unmet need as in a human model.

The aim of this study was to determine the outcomes and survivorship of the Triathlon knee replacement at 7 years after surgery. A cohort of 266 patients receiving a Triathlon knee replacement were assessed before surgery and at 3 months, 1 year, 2 years, 3 years, 5 years and 7 years post-operation. Patient-reported outcomes were assessed using the WOMAC, KOOS Knee-Related Quality of Life scale, Satisfaction Scale and questions on kneeling ability and whether they regretted having the operation. Data on survivorship was collected from self-report and medical records. At 7 years after surgery, 32 patients were deceased, and 17 patients were withdrawn. Of the 217 patients remaining in the study, 164 (76%) returned a completed study questionnaire. At 7 years after surgery, 92% of patients reported an improvement in their WOMAC Pain score greater than the minimally clinically important improvement (defined as improvement of ≥9 points from before surgery) and 82% reported this in their WOMAC Function score (defined as improvement of ≥12 points). Knee-related quality of life was good, with a mean score of 66.8 (SD 26.0) (0–100 scale, worst to best). A high percentage of patients (89%) were somewhat or very satisfied with their outcome at 7 years. Survivorship with revision as the endpoint was 96.4% (95% CI 93.2–98.1%) at 7 years post-operation. Five percent of patients regretted having their operation and 68% reported much difficulty or an inability to kneel. In conclusion, this study observed good long-term patient outcomes and survivorship of the Triathlon knee replacement.

Though there are many techniques utilised in the correction of hallux valgus (HV), no single approach has been reported to be ideal for all patients to date. A great deal of controversy remains concerning the type of osteotomy, method of fixation, and inclusion of soft tissue procedures. Herein, we compared the outcomes of two different operative techniques, the minimally-invasive modified percutaneous technique and the distal chevron osteotomy, used to treat mild to moderate hallux valgus. This study was conducted in line with the CONSORT 2010 guidelines. 41 patients (58 feet) with mild to moderate hallux valgus were randomly assigned by computer to two different groups. The first group containing 24 patients (33 feet) was treated by the modified percutaneous technique, whereas the second group included 17 patients (25 feet) treated by distal chevron osteotomy. In the modified percutaneous group, after a mean follow up of 43 months, the mean correction of hallux valgus angle (HVA) was 26.69° (P=0.00001), the mean correction of intermetatarsal angle (IMA) was 9.45° (P=0.00001), and the mean improvement of AOFAS score was 47.94 points (P=0.00001). In the chevron osteotomy group, after a mean follow up of 44 months, the mean correction of hallux valgus angle was 26.72° (P=0.00001), the mean correction of intermetatarsal angle was 9° (P=0.00001), and the mean improvement of AOFAS score was 44.76 points (P=0.00001). In our study, the modified percutaneous technique proved to be equally effective as the distal chevron osteotomy, but with fewer complications and a higher rate of patient satisfaction.

Restoration of anatomy is paramount in total hip arthroplasty (THA) to optimise function and stability. Leg-length discrepancy of ≥10mm is poorly tolerated and can be the subject of litigation. We routinely use a multimodal protocol to optimise soft tissue balancing which involves pre-operative templating, leg-length measurement supine and in the lateral position after positioning, and the use of an intra-operative leg-length measurement device to ensure optimisation of leg-length. We have analysed the results of our protocol in restoring leg-length in primary THA. Radiological leg-length was measured in a consecutive series of 50 patients who had THA for unilateral arthritis by an independent observer pre- and post-operatively using validated methods utilising radiological software. The measurements pre- and post-operative were compared. Patients with bilateral hip arthritis and poor imaging were excluded. Leg-length was successfully restored to within 5.0mm of the target leg-length in 84.0% of patients (mean +0.7mm (95% CI +0.2 to +1.1)). The other 14.0% of patients were restored to within 5.1–8.0mm (mean +2.2mm (95% CI −2.7 to +7.1)) and 2.0% of patients were restored to within 8.1–10.0mm. Leg length was accurately restored across the subset of patients within a narrow range of either side of the mean target leg length. Intra-operative measurement of leg length can be difficult but is vital in ensuring appropriate restoration of leg-length. We recommend a similar multimodal protocol to ensure restoration of leg-length within narrow limits to maximise function and patient satisfaction.

Neck modularity has been proposed to improve THA accuracy, thanks to the close restoration of anatomy, however it has been associated with issues like early breakages or corrosion. Our Hospital has been using neck modularity since the 90s, so we analyzed retrospectively implants performed between January 2000 and December 2014. The minimum follow up was 1Y. The cohort was composed of 1,033 THAs or 951 patients (82 bilateral), of which 643 females and 390 males. Average patient age was 67.7Y. THA indications were primary Osteoarthritis (80.9%), Fracture (9.0%), Congenital Dysplasia or Congenital Luxation (4.2%), Osteonecrosis (3.2%), other causes (2,7%). The stems used were all cementless, 381 anatomically shaped (36.9%), 635 straight (61.5%), 17 short MIS (1.6%). All necks used were made of Titanium alloy. 419 implants (40.5%) were manufactured by Wright Medical, while 614 (59.5%) were produced by Adler Ortho. A total of 37 revisions has been reported, mainly due to periprosthetic fractures (32.4%), luxation (24.3%), implant mobilization (18.9%) and implant breakage (16.2%). We have recorded 3 modular neck breakages. 4 patients required re-revisions, because of luxations (3) and neck breakage (1). The overall survival rate was 96.4%. We did not observe any component corrosion, probably thanks to the exclusive use of Titanium necks. We had a neck breakages rate of 0.29% and a luxation rate of 0.87%, lower than normally reported in the literature. In conclusion, our experience suggests as neck modularity could be a safe and effective way to reconstruct the proximal femur in THA patients.

Orthopaedic surgical site infections (SSI's) prolong total hospital stays by a median of 2 weeks per patient, approximately double re-hospitalization rates, and increase healthcare costs by more than 300%. Patients with orthopaedic SSI's have significant reductions in their health-related quality of life. We performed a systematic review and meta-analysis to compare differences in outcomes between use of sutures and non-absorbable staples for closure of orthopaedic surgical wounds in adults. The primary outcomes were rates of superficial and deep SSI. Secondary outcomes included wound dehiscence, length of hospital stay, patient satisfaction and pain during removal of closure material. Data sources including PubMed, EMBASE, Scopus, Web of Science, Cochrane Library, clinicaltrials.gov, National Institute for Health and Research, UK clinical trials gateway were searched for randomised controlled trials (RCT's) meeting inclusion criteria. Sixteen RCT's published between 1987 and 2017 were included. Overall, wound infection outcomes (superficial and deep infections combined) showed no statistically significant difference between closure with staples compared with sutures with arelative risk of 1.17 (95% CI 0.59–2.30, p=0.66). A subgroup was performed specific to hip wound infection outcomes. Interestingly, a sensitivity analysis demonstrated sutures to be statistically favourable (p=0.04) in terms of hip wound infection outcomes. There was no statistically significant difference among secondary outcomes between sutures and staple groups. Overall it appears the choice of sutures or staples in closure of orthopaedic wounds has no effect on wound complications. However, caution is needed in applying the findings to different population groups due to heterogeneity across studies.

The Ottawa Rules were developed in 1992 by Stiell et al. to “assist clinicians in being more selective in their use of radiography” in suspected ankle fractures. They have a sensitivity of almost 100% and should reduce the number of unnecessary radiographs by 30–40%. We aimed to determine the application of the Ottawa Rules in ankle plain radiograph requests in Accident and Emergency (A&E) and determine the number of unnecessary plain radiographs requests. We carried out a retrospective analysis of 366 ankle plain radiographs, request forms and reports, in A&E over 3 months. We implemented a reminder on the electronic requesting system to prompt clinicians to apply the Ottawa rules and analysed a further 226 scans over the next month. Unnecessary scans were calculated by determining the false negatives. i.e. requests did not fulfil Ottawa Rules and scan showed no fracture. Of the 336 original requests, 45% fulfilled the Ottawa Rules and 43% of all scans were unnecessary. Following our intervention, only 42% of requested scans fulfilled the Ottawa rules and 52% were unnecessary. We concluded that Ottawa rules are knowingly not applied in A&E. Reasons may include low cost and ease of scan, patient and clinician reassurance and perceived low risk of scanning. There are a huge number of unnecessary ankle plain radiographs and soft interventions do not impact on this. We have implemented a simplified version of the text reminder and are re-auditing the data.

The treatment of extremity ballistic injury is challenging in that the zone of injury can be extensive and determining the surgical exposure can be difficult. We describe a method of pre-operative evaluation of the zone of injury in conjunction with the regional anesthesiologist utilizing ultrasound to determine the presence of nerve disruption. This non-invasive method of examination may elucidate whether significant nerve exists and may also serve to pinpoint the location of injury. Such information allows the surgeon to more effectively and efficiently surgically expose the zone of injury and understand the boundaries of the nerve outside the zone of injury. Moreover, such preoperative evaluation may at times obviate the need for exploratory surgery at all. It is important for the anesthesiologist and surgeon to work together with respect to the ability to both interpret the ultrasound images and to clinically correlate the findings. The zone of tissue disruption in ballistic injuries is extremely variable. It is beneficial to both the surgeon and patient to engage in a collaborative effort with an experienced regional anesthesiologist who is well-versed in interpretation of ultrasound images and tissue plane disruption in an effort to minimize surgical time and the potential unintended consequences of unnecessary exploration. We present a series of cases representing instances wherein the zone of injury was small, extensive, and a unique situation in which there was in fact no injury present despite clinical symptoms and MRI consistent with radial nerve disruption.

Tissue engineering and regenerative medicine are increasingly taking advantage of active materials, allowing to provide specific clues to the cells. In particular, the use of electroactive polymers that deliver electrical signals to the cells upon mechanical solicitation, open new scientific and technological opportunities, as they in fact mimic signals and effects present in living tissues, allowing the development of suitable microenvironments for tissue regeneration. In fact, electrical and electromechanical clues are among the most relevant ones in determining tissue functionality in tissues such as muscle and bone, among others, indicating their requirement for proper tissue regeneration. Piezoelectric polymers have already shown strong potential for novel tissue engineering strategies, once they can account for the existence of piezoelectricity within some specific tissues and also can modulate the electrical signals existing in tissue development and function. In this context, this talk reports on piezoelectric and magnetoelectric materials used for tissue engineering applications. The most used materials and morphologies for tissue engineering strategies are reported, together with the need of novel bioreactor designs allowing to take full advantage of those materials. Further, the main achievements, challenges and future needs for research and actual therapies will be presented and discussed.

By definition, a smart biomaterial is a material, such as a ceramic, alloy, gel or polymer, that can convert energy from one form into another by responding to a change in a stimulus in its environment. These stimuli may involve temperature, pH, moisture, or electric and magnetic fields. In particular, thermoresponsive biomaterials have been successfully employed to host mammalian cells with a view to musculoskeletal tissue engineering. The presentation provides an overview of the use of thermosensitive polymers for the non-enzymatic stem cell harvesting, cell sheet engineering, three-dimensional scaffolds fabrications and organ-printing materials.

One of the latest trends in the field of tissue engineering is the development of in vitro 3D systems mimicking the target tissue or organ and thus recapitulating the tridimensional structure and microenvironment experienced by cells in vivo. Interestingly, certain tissues are known to be regulated by endogenous bioelectrical cues, in addition to chemical and mechanical cues. One such tissue is the bone. It has, indeed, been demonstrated to exhibit piezoelectric properties in vivo, with electrical signaling playing a role in its formation during the early embryo developmental stages. Electrical stimulation has been proven to sustain cell proliferation and to boost the expression of relevant genes and induce higher levels of enzymatic activities related to bone matrix deposition. Herein, we describe the development of a 3D model of bone tissue based on the conductive polymer PEDOT:PSS and human adipose derived stem cells. 3D electroactive porous scaffolds have been produced using the ice-templating technique, and different compositions (different ratios of conductive polymer to Collagen Type 1) have been explored. The developed scaffolds as well as cells interaction and response have been characterized. Overall, the results obtained so far highlight the usefulness of the porous conductive scaffolds as an in vitro platform for the development of 3D models for bone tissue engineering.

There is a growing interest in the development of tissue engineering (TE) therapies to repair damaged bone. Among the scaffolds for TE applications, injectable hydrogels have demonstrated great potential as three-dimensional cell cultures in bone TE, owing to their high water content, porous structure that allows cell transplantation and proliferation, similarity to the natural extracellular matrix and ability to match irregular defects. We investigated whether fibrin-based hydrogels capable of transducing near infrared (NIR) energy into heat can be employed to lead bone repair. Hollow gold nanoparticles with a plasmon surface band absorption at ∼750 nm, a NIR wavelength within the so called “tissue optical window”, were used as fillers in injectable fibrin-based hydrogels. These composites were loaded with genetically-modified cells harbouring a heat-activated and rapamycin-dependent gene circuit to regulate transgenic expression of the reporter gene firefly luciferase (fLuc). NIR-responsive cell constructs were injected to fill a 4 mm diameter critical-sized defect (CSD) in the parietal bone of mouse calvaria. NIR-irradiation in the presence of rapamycin triggered a pattern of fLuc activity that faithfully matched the illuminated area of the implanted hydrogel. Having shown that this platform can control the expression of a transgene product, we tested its effectiveness on regulating the secretion of transgenic bone morphogenetic protein 2 (BMP-2) from NIR-responsive hydrogels implanted in CSD. The spatiotemporal pattern of transgenic BMP-2 secretion induced by NIR-irradiation in the presence of rapamycin significantly stimulated bone regeneration from the edge of osteotomy in the CSD practiced, validating the therapeutic approach.

The field of nanoparticle related research for the diagnosis and therapy of diseases evolves rapidly. Magnetic nanoparticles in combination with magnetizable implant materials for the treatment of implant related infections present a possible implementation in orthopedics. Magnetic nanoporous silica nanoparticles (MNPSNPs) were developed and equipped with fluorescent dyes. In vitro/in vivo biocompatibility and in vivo biodistribution were examined to appraise their potential applicability. Cell culture tests with NIH-3T3 and HepG2 cell lines indicated a good in vitro biocompatibility. Ferritic and titanium alloy (control) plates were implanted subcutaneously at the hind legs of Balb/c mice. Immediately after i.v. or s.c. injection of MNPSNPs, the caudal half of the mice was placed between the poles of an electro magnet. Exposure to the electromagnetic field of approx. 1.7 T was maintained for 10 minutes. 10 animals each were euthanized at days 0, 1, 7, 21 or 42, respectively. Quantity of MNPSNPs in liver, spleen, kidney, lung and skin/muscle samples was assessed by fluorescent microscopic methods. MNPSNP existence on the implant surface was also appraised after several steps of detachment. MNPSNPs showed a time-dependent accumulation in the organs after i.v. injection with initial accumulation in the lungs followed by redistribution to liver and spleen. After s.c. injection no systemic distribution but local appearance of MNPSNPs could be found. First histological evaluation showed no pathological changes after i.v. injection. With good in vivo biocompatibility, future focus will be laid on increasing circle life time of MNPSNPs and evaluation in an infection model.

Tissue engineering by self-assembly offers the possibility to fabricate contiguous cell sheets that are stabilised by intact cell-cell contacts and endogenously produced extracellular matrix (ECM) However, these systems lack the possibility to introduce topographical cues, that are fundamental for the organisation of many types of tissues. Herein we venture to fabricate aligned electrospun thermoresponsive nanofibres to sustain growth and detachment of ECM-rich living substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted to estimate matrix deposition and composition. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes. The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability revealed that hADSCs were able to grow on the scaffold. The cells aligned on the fibres after 3 days and they were able to detach as intact cell sheets in presence of MMC. Moreover, it was demonstrated that MMC, by a volume extrusion effect, enhances collagen type I deposition, one of the main components of the ECM. Collectively the pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich cell sheets.

Compressive fracture of osteoporotic vertebrae has been one of the most important health problems in aged societies because severely injured spin might be a reason of bedridden for elderly people. Osteoporosis has been widely assessed by averaged bone mineral density of vertebrae measured using DEXA, however, BMD sometimes does not reflect the strength of vertebrae. CT imaged based finite element method (CT-FEM) has been applied to evaluate the strength of vertebrae based on the biomechanics theory and approved by a part of the highly advanced medical treatment in Japan. In the present study, compressive strength of more than 100 vertebrae were evaluated using CT-FEM, and the correlation between BMD and the strength was thoroughly investigated. It was found that some vertebrae with high BMD could have low strength which may cause fracture easily. Thus, a controversial point of the BMD based diagnosis of osteoporosis was clearly indicated. In this invited talk, some basic theories of CT-FEM and fracture assessment and some key results from the recent study will be presented.

Biomechanical analysis is important to evaluate the effect of orthopaedic surgeries. CT-image based finite element method (CT-FEM) is one of the most important techniques in the computational biomechanics field. We have been applied CT-FEM to evaluate resorptive bone remodeling, secondary to stress shielding, after total hip arthroplasty (THA). We compared the equivalent stress and strain energy density to postoperative BMD (bone mineral density) change in the femur after THA, and a significant correlation was observed between the rate of changes in BMD after THA and equivalent stress. For periacetabular osteotomy cases, we investigated mechanical stress in the hip joint before and after surgery. Mechanical stress in the hip joint decreased significantly after osteotomy and correlated with the degree of the acetabular coverage. For arthroscopic osteochondroplasty cases, we examined mechanical strength of the proximal femur after cam resection using CT-FEM. The results suggested that both the depth and area of the resection at the distal part of femoral head-neck junction correlated strongly with fracture risk after osteochondroplasty. This talk consists of our results of clinical application studies using CT-FEM, and importance of application of CT-FEM to biomechanical studies to assess the effect of orthopaedic surgeries.

Latarjet procedure (transfer of coracoid process to the anterior glenoid rim) has been widely used for severe anterior shoulder instability. The purpose of the present study was to investigate the intraarticular stress distribution after this procedure to clarify the pathomechanism of its postoperative complications. CT-DICOM data of the contralateral healthy shoulder in 10 patients with unilateral anterior shoulder instability (9 males and 1 female, age: 17–49) was used for the present study. Three-dimensional finite element models of the glenohumeral joint was developed using software, Mechanical Finder (RCCM, Japan). In each shoulder, a 25% bony defect was created in the anterior glenoid cavity, where coracoid process was transferred using two half-threaded screws. The arm position was determined as 0-degree and 90-degree abduction. While medial margin of the scapula was completely constrained, a standard compressive load (50 N) toward the centre of the glenoid was applied to the lateral wall of the greater tuberosity. A tensile load (20N) was also applied to the tip of coracoid process along the direction of conjoint tendon. Then, elastic analysis was performed, and the distribution pattern of Drucker-Prager equivalent stress was investigated in each model. The proximal half of the coracoid represented significantly lower equivalent stress than the distal half (p < 0.05). In particular, the lowest mean equivalent stress was seen in its proximal-medial-superficial part. On the other hand, a high stress concentration newly appeared in the antero-inferior aspect of the humeral head exactly on the site of coracoid bone graft. We assumed that the reduction of mean equivalent stress in the proximal half of the coracoid was caused by the stress shielding, which may constitute one of the pathogenetic factors of its osteolysis. A high stress concentration in the humeral head may eventually lead shoulder joint to osteoarthritis.

Smith's fractures generally occur when falling on a flexed wrist; however, orthopedic trauma surgeons often encounter distal radius fractures with volar displacement in patients who have allegedly fallen on the palm of their hands. This study aimed to reveal both the basic and clinical pathogenesis of Smith's fracture through a step-by-step investigation. We enrolled 17 patients with Smith's fractures, of which 71% fell on the palm and only 6% on the dorsum of the hand. First, we interviewed the outpatients to determine the mechanics of the injury and the position of their arm during injury. Second, we created a three-dimensional (3D) finite element model to predict the arm's position when the Smith's fracture occurred, which finite element analysis revealed as a 30° angle between the long axis of the forearm and the ground in the sagittal plane. Third, using this predicted position, we conducted experiments on 10 fresh frozen cadavers to prove the possibility of causing a Smith's fracture by falling on the palm of the hand. The results showed Smith-type fractures in seven of 10 wrists, whereas Colles-type fractures did not occur. Finally, we analyzed stress distribution in the distal radius when a Smith's fracture occurs using the 3D finite element model. In conclusion, this study demonstrates that Smith's fractures can also occur by falling on the palm of the hand.

The reduction for unstable femoral intertrochanteric fracture should be extramedullary, which means that the proximal fragment protrudes for the distal fragment. However, only few articles have compared extramedullary and intramedullary reductions in a biomechanical study. Thus, we created unstable femoral intertrochanteric fracture models using imitational bone (extramedullary and intramedullary groups, each with 12 cases) and evaluated their biomechanical stabilities. The fracture type was 31-A2 according to the AO-OTA Classification of Fractures and Dislocations and greatly lacked bone on the posterior side. We performed compression examination and evaluated stiffness. The implant used for fixation was TFNA (DePuy Synthes). We applied axial compression with 20 adduction in the standing position. Statistical analysis was performed using the Mann-Whitney U test. No significant difference in initial loading force was found between the two groups. However, the axial stiffness of the extramedullary bone showed a significant increase (p < 0.05) in high loading force (800–1000 N). This means that the stability of the extramedullary reduction was superior to that of the intramedullary reduction in terms of high loading force in the standing position. We suggest that antero-medial bony buttress is important for unstable femoral intertrochanteric fractures. These data indicate that extramedullary reduction and fixation for unstable femoral intertrochanteric fractures increase stability.

The pathology of the posterior acetabular legion in femoroacetabular impingement (FAI) syndrome, so called “contre-coup region”, is still unclear. ¹⁸F-fluoride positron emission tomography (PET) is a functional imaging modality, which reflects the osteoblast activity. Recent technological advances in PET combined with computed tomography (CT) imaging allowed us to obtain detailed 3-dimensional (3D) morphological information. We evaluated the abnormal uptake of ¹⁸F-fluoride PET/CT on posterior acetabular lesion in FAI syndrome cases. We enrolled forty-one hips from 41 patients who were diagnosed as FAI syndrome and were performed ¹⁸F-fluoride PET/CT between October 2014 and October 2016. In each hip, the maximum standardized uptake value (SUV_max) on the posterior acetabular was measured. The cases were divided into 4 groups; cam-type (11 cases), pincer-type (7), combined-type (11), dysplastic developmental hip (DDH) with cam morphology (12). The average SUV_max of the pincer-type was significantly smaller than that of the other 3 groups (p < .05). The percentage of the cases with SUV_max ≥ 6 was 81.8% in cam-type, 28.6% in pincer-type, 90.9% in combined-type, 91.7% in DDH with cam morphology. Furthermore, the average degree of α angle of the cases of SUV_max ≥ 6 was significantly higher than that of the cases of SUV_max < 6 (p = .005). Although actual biomechanical mechanism in contre-coup region is still controversial, this result indicated that the cam morphology related to the posterior acetabular lesion with accelerated bone metabolism.

Osteoarthritis is the most prevalent joint disease, causing severe pain, deformity and a loss of mobility. Low back pain (LBP), frequently associated with degeneration of the intervertebral disc (IVD), is the No.1 cause of Years Lived with Disability. Age is a major risk factor for both conditions. However, the reasons why susceptibility to these conditions increases with age are poorly understood. The circadian (24 hourly) clocks in the brain and periphery direct key aspects of physiology through rhythmic control of tissue-specific sets of downstream genes. Work from our group focuses on the roles of circadian clocks in the articular cartilage and IVD. We show that the daily rhythm in these tissues becomes dampened and out-of-phase during ageing. Further, our data identify circadian clock disruption in cartilage and IVD as a new target of inflammation. Moreover, we show that mice with targeted knockout of an essential clock gene (BMAL1) in chondrocytes and disc cells have profound, yet tissue-specific degeneration in the articular cartilage and IVD. These findings implicate the local skeletal clock as a key regulatory mechanism for tissue homeostasis. This new avenue of research holds potential to better understand, and eventually treat these debilitating conditions.

Degeneration of intervertebral disc (IVD) Nucleus Pulposus (NP) is a major cause of low back pain (LBP). Healthy NP contains two cell types: notochordal cells (NTC) and nucleopulpocytes (NPCytes). While NTC are embryonic notochord derived cells that are regarded as the resident stem cells of NP, NPCytes are considered the mature NP cells responsible for extracellular matrix (ECM) synthesis. During IVD aging, some still unknown cues drive NTC disappearance. This loss of NTC alters their dialog with NPCytes thereby jeopardizing cell viability and ECM homeostasis, which in turn drives NP degeneration. In this context, NP regeneration by re-establishing this NTC/NPCytes dialog has been contemplated with clinical interest. We will first share our view of the mesenchymal stem cells (MSC)-based therapies that have been preclinically and clinically assessed in LBP. We will then comment on the biomaterial-assisted MSC therapies that recently enter the scene of IVD regeneration. Finally, we will present our REMEDIV project that aims at developing a NP substitute containing stem cells-derived NPCytes and NTC within an injectable hydrogel. We will share our results regarding the generation of NPCytes from adipose-derived MSC and our recent unpublished evidences that human induced-pluripotent stem cells can be differentiated into NTC. Finally, we will consider our ability to transplant these regenerative cells using hydrogels in various animal models. Whether this concept could open new therapeutic windows in the management of discogenic low back pain will finally be discussed.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However, to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration. Immunohistochemistry for the senescence marker: p16INK4A was firstly utilised to screen human intervertebral discs for discs displaying at least 30% immunopostivity. These discs were then subsequently analysed for immunopostivity for DNA damage markers γH2AX and cGAS and the presence of cytoplasmic DNA. The number of immunopositive cells for p16 INK4A positively correlated with the expression of γH2AX and cGAS. Senescent cells were also associated with the presence of cytoplasmic DNA. These new findings elucidated a role of cGAS and γH2AX as a link from genotoxic stress to cytokine expression which is associated with senescent cells. The findings indicate that cellular senescence in vivo is associated with DNA damage and presence of cytoplasmic DNA. Whether this DNA damage is a result of replicative senescence or stress induced is currently being investigated in vitro.

Clinical trials are underway to elucidate a successful MSC-based therapy for the repair and regeneration of intervertebral disc (IVD) tissue. Currently, there is a lack of knowledge surrounding the relationship between naïve MSCs and the inflammatory microenvironment of the degenerate disc. To inform a phase II clinical trial, this study tests the hypothesis that cytokines, IL-1ß and TNFα regulate the expression of neuropeptides and neurotrophic factors from MSCs, thus exacerbating pain in those patients that have the presence of sensory nerve fibres within the IVD. Patient-matched MSCs derived from bone marrow (BM) or adipose (AD) tissue were stimulated with IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours in either 21% or 5% O2. qRT-PCR was performed to assess expression of trophic factors involved in the survival or nerves (NGF & BDNF), blood vessels (VEGF) as well as pain related peptides (SP & CGRP) and inflammatory factors. Conditioned culture medium was analysed using ELISAs to identify secretion of soluble factors. IL-1β did not regulate neurotrophic factor expression from BM-MSCs under normoxic or hypoxic conditions. However, TNFα increased NGF, BDNF, SP and CGRP under normoxic conditions. In ADMSCs, VEGF was increased following IL-1β and TNFα stimulation; with TNFα also increasing NGF and CGRP under normoxic conditions. When exposed to hypoxia, the trophic effect of TNFα on human BM-MSCs was reduced. Overall this data suggests a role for priming or pre-stimulation of naïve MSCs prior to implantation to prevent exacerbation of pain from sensory nerve fibres.

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6.

The most challenging complications in orthopaedic trauma surgery are fracture-related infections (FRI). The incidence ranges from approximately 1% after closed fractures or joint replacement, to more than 30% in complex open limb fractures. Despite tremendous efforts with prolonged antibiotic therapy and multiple revision surgeries, these complications are associated with considerable rates of recurrent infections as well as permanent functional impairment. The primary aim for the clinician is to prevent infection, because once established, an infection is difficult to eradicate. The main reason for this is biofilm formation on the implanted device, which allows pathogens to protect themselves from host immune response and antimicrobial therapy. In open fractures with a considerable wound contamination and soft- tissue damage, systemically-delivered antibiotics may not reach sufficient local concentrations to eradicate the bacteria. Locally delivered antibiotics can overcome this problem by providing high local concentrations. Currently, several antibiotic loaded biomaterials for local infection prophylaxis and/or treatment are available. In this talk, next to the diagnostic challenges of FRIs, the currently available antimicrobial-loaded biomaterials will be described. Against a backdrop of increasing infection and antimicrobial resistance, the prudent use and availability of such materials will become even more important.

Device-associated infection remains a serious clinical problem in orthopaedic and trauma surgery. The emergence of resistant organisms such as methicillin resistant Staphylococcus aureus (MRSA) has further exacerbated this problem by limiting the range of treatment options. Currently, systemic antibiotic therapy is the cornerstone of treatment, alongside surgical resection of infected tissues and implant removal. The potential for antibiotic loaded biomaterials to support the prevention and treatment of infection is significant, although the currently available options are limited in number and often re-purposed from other applications e.g. antibiotic loading of bone cement. The first part of the talk will cover the basic concepts involved in antibiotic treatment, with an emphasis on the ideal antibiotic release kinetics from biomaterials, and how bacterial biofilms and antibiotic resistance influence antimicrobial efficacy. The next generation of biomaterials for antibiotic delivery should be specifically designed with this knowledge in mind. Regulatory approval of antimicrobial combination devices, however, is an evolving process as regulatory bodies seek more robust and clinically relevant efficacy data. Approval will require preclinical efficacy using standardized animal models that recapitulate the key features of the clinical disease. The second part of this talk will cover best practice in this important stage of development.

Thermostability is a key property in determining the suitability of local delivery of antibiotics in the treatment of orthopaedic infections. Herein, we aimed to assess the thermal stability and antibacterial activity of ciprofloxacin, ceftriaxone, gentamycine and vancomycine in high temperature conditions. Using a standardized E-test method, minimally inhibited concentration of each antibiotic substance against Staphylococcus aureus cultures were determined. The solutions of antimicrobial drugs ciprofloxacin 2 mg/ml, ceftriaxone 200 mg/ml, gentamycine 40 mg/ml and vancomycine 200 mg/ml were diluted twofold in deionised water. Acquired solutions were divided into three aliquots. The first aliquot was held at 40°C for 30 min in a waterbath, the second and the third aliquots were exposed to 80 and 100°C for 30 min in hot-air sterilizer, respectively. The treated solutions were tested for residual activity against S. aureus using a standardized disk diffusion method. Mediums with untreated antibiotic solutions and S. aureus were used as control. Plates were incubated at 37°C, at which time zones of inhibition (ZoI) were measured to the nearest whole millimeter for 14 days. The investigation indicated that the temperature elevation impacted considerably on antimicrobial activity and antibiotic stability overall. The in vitro temperature-response curves showed that ZoI diameter decreases logarithmically with elevated temperatures. Gentamicin was the only drug that was found to be affected to some extent. Results from the study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics and methods of antibiotic impregnation.

Implant-related infections pose a severe economical and societal burden, hence solutions capable of exerting suitable efficacy while not causing toxicity and/or development of resistant bacterial strains are needed. Thus, inorganic antibacterial coatings, and in particular silver coatings, have been extensively studied and used in the clinical practice. However, some drawbacks such as scarce adhesion to the substrate, delamination, or scarce control over silver release have been evidenced. Here, antibacterial nanostructured silver thin films have been developed by a novel plasma-assisted technique. The technique allows deposition on several substrates, including heat sensitive materials and objects of complex shape. Thanks to nanostructured surface, a tuned release can be achieved, preventing citoxicity. Composition (grazing incidence XRD, XPS) and morphology (SEM, AFM, ASTM) of the obtained coatings were characterized, then, their efficacy was validated in vitro against relevant bacterial strains (gram+ S. Aureus and gram– E. Coli). Live/dead kit and confocal microscopy were used to evaluate antibacterial efficacy. Super resolution imaging in the Structured Illumination Microscopy (SIM) setup was used to investigate damage to the bacterial wall. Results indicate that the coatings are composed of nanosized aggregates of metallic silver, indicating a perfect transfer of composition from the deposition target to the coating. Because of the sub-micrometric thickness, they do not alter the micro- and macro- morphology and surface finishing of the implants, developed by the manufacturers to ensure optimal integration in the host bone. Finally, remarkable efficacy was found against both gram+ and gram- bacteria, indicating that the developed coatings are promising for antibacterial applications.

Orthopedic metallic medical devices are essential in the treatment of a wide range of skeletal diseases and disabilities. However, they are often related with surgery complications due to acute prosthetic joint infections (PJI) causing devastating complications. Gallium (Ga) antibacterial activity has been recently demonstrated: in aqueous solutions, Ga ionize in a trivalent form Ga³⁺ that can replace Fe³⁺ in bacterial metabolism thus leading to bacteria death. However, it is not yet clear whether such effect is typical to Ga³⁺ release, and how this would affect longer term performance. Here we investigated Ga addition into titanium alloys using metallurgical methods. The study has confirmed that metallurgical addition of gallium even in small amounts (1–2% wt.) to titanium alloys have highly efficient antibacterial function without any visible cytostatic or cytotoxic effects. The presence of gallium within the metal matrix might ensure that antibacterial effect will persist for a long time towards multi-drug resistant S. aureus, which might not be possible if gallium or other metal are only in thin degradable coatings or similar formulations. A 5-logs decrease in CFU number was detected for alloys with 2% Ga and more after 72 h (alamar blue and CFU count assays). The alloys also show to be in vitro cytocompatible with both mature U2OS osteoblasts and progenitor pre-osteoblasts hFOB. Since gallium is metallurgically analogous to aluminium in titanium alloys, it might be used without affecting other alloy properties.

Aseptic loosening is a major cause of revision surgeries and occurs when osteolysis is stimulated around the implant by pro-inflammatory cytokines including IL-1β. Production of active IL-1β in response to orthopedic wear particles depends on processing by the NLRP3 inflammasome which requires priming followed by activation. We found that pathogen associated molecular patterns (PAMPs) adherent to wear particles are necessary to prime the NLRP3 inflammasome. In contrast, in pre-primed macrophages, particles themselves are sufficient to activate the NLRP3 inflammasome and induce secretion of active IL-1β. Particles themselves also induce cell death, kinetically preceding the release of active IL-1β. Phagocytosis of particles is required to initiate both responses as the phagocytosis inhibitor cytochalasin blocks cell death and IL-1β release. Lysosome membrane destabilization is also critical as inhibition of lysosomal function with bafilomycin or chloroquine significantly abrogated the release of active IL-1β and cell death in response to wear particles. The pan-cathepsin inhibitors Ca-074-Me or K777 also inhibit cell death and IL-1β release indicating that cathepsin release from lysosomes is also a necessary step in the particle-induced response. Our results open the possibility of clinical intervention with lysosomal or cathepsin inhibitors to treat aseptic loosening as these drugs have better specificity and less in vivo toxicity than the phagocytosis inhibitors. Testing of these inhibitors in vivo in models of particle induced osteolysis is a key future direction.

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Existing regenerative approaches rely on high doses of growth factors, such as BMP-2 in bone regeneration, which can cause serious side effects. We describe an ultra-low-dose growth factor technology yielding high bioactivity based on a simple polymer, poly (ethyl acrylate) (PEA), and report its translation to a clinical veterinary setting. This polymer-based technology triggers spontaneous fibronectin organization and stimulates growth factor signaling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, we use plasma-polymerized PEA on 2D and 3D substrates to enhance cell signaling in vitro, showing the complete healing of a critical-size bone injury in mice in vivo. We demonstrate its safety and efficacy in a Münsterländer dog with a non-healing humerus fracture, establishing the clinical translation of advanced ultra-low-dose growth factor treatment.

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with ranging Young's modulus values for bulk rigidity, at the sub-cellular level, and particularly at the micro- and nanoscales, tissues are comprised of heterogeneous distributions of rigidity.

Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, we show for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric PDMS substrates and develop a new class of two-dimensional elastomeric substrates with controlled patterned rigidity ranging from the micron to the nanoscale. The mechano-response of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces.

Promising work on bioactive glasses (BAGs) in bone defect regeneration has led to their clinical implementation. However, the effects of the ionic dissolution products of different types and the physical interaction modalities of BAGs on the behavior and function of mesenchymal stromal cells (MSCs) of human patients have not received sufficient attention. Recently, we showed that the in vitro response of hMSC to micron-sized, monodispersed BAGs is dependent on dosage, composition, and mode of interaction¹. Two commercially available and widely used types of BAGs, namely the silicate BAGs 45S5 and 1393, were used to study hMSC cell behavior. Interestingly, exposure to 1393 BAG resulted in superior metabolic activity, proliferation, and cell spreading compared to 45S5 BAG in similar dosage, suggesting that additional cellular functions could also be differentially modulated by both glasses¹. In the context of bone regeneration, the hMSCs’ potential to secrete angiogenic factors as well as deposit mineralized matrix upon exposure to BAG dissolution products was investigated in the present study. Aside from dose-dependent effects of both glasses, 45S5 BAG induced a significant pro-angiogenic response, demonstrated by robust tube formation in HUVECs in the presence of MSC conditioned media. 1393 BAG, on the other hand, stimulated osteogenesis by upregulating osteogenic gene expression and mineralized matrix deposition. Based on these results, combining the pro-angiogenic 45S5 BAG and the pro-osteogenic 1393 BAG might be an attractive strategy to target the multiple processes underlying bone regeneration. These results highlight how different BAGs can be utilized to promote MSC-mediated bone regeneration.

Among the innovative therapeutic techniques in orthopedics, a considerable interest arose around Mesenchymal Stem Cells (MSCs) - based therapies for one-step clinical applications. In order to achieve a better cell targeting at the injury site, these applications would need a specific cell delivery system. Hence, in this study a protocol for an efficient cell delivery based on the rapid cell adhesion on the surface of lyophilized fibroin-coated alginate microcarriers (L-FAMs) was optimized by the Design of Experiment (DoE) method in accordance with the minimum requirements for one-step clinical application. Specific parameters (seeding time, intermittent or not dynamic culture, stirring speed and volume of cell suspension) were combined in 13 different protocols, tested on human Adipose derived stem cells - ASCs (n=3). Cell adhesion rate in term of DNA quantification and metabolic activity of cells adhered on L-FAMs, and their qualitative observations by Calcein Staining were evaluated. The data showed that a suspension of 3.75 × 10⁵ cells/ml and 10 mg/ml of FAMs, 12.3 rpm of stirring speed and 85.6 minutes of seeding time are the most performing combination of parameters. The final protocol was then tested and validated on both hASCs (n=3) and human bone marrow derived stem cells - BMSCs (n=3). The results confirmed a high adhesion rate of cells, homogenously arranged on the surface of L-FAMs without cell cluster formation. Even though further optimizations are still needed, the present protocol may represent the proof of concept for the introduction of L-FAMs as carriers in one-step intraoperative applications.

Bone regenerative medicine aims at designing biomimetic biomaterials able to guide stem cells fate towards osteoblast lineage and prevent orthopaedic common pathogen adhesion. Owing to bone inorganic/organic composition, we herein report, using a versatile process based on simultaneous spray coating of interacting species, a calcium phosphate (CaP) / chitosan (CHI) / hyaluronic acid (HA) functionalized collagen membrane as a new strategy for bone regenerative medicine. Physicochemical characterizations of CaP-CHI-HA coating were performed by scanning electron microscopy, X-ray photoelectron and infrared spectroscopies and high-resolution transmission electron microscopy, revealing the formation of a thin coating mainly composed of non-stoichiometric crystalline hydroxyapatite dispersed into polymorphic organic film. Biocompatibility of CaP-CHI-HA coated membrane, evaluated after 7 days in contact with human mesenchymal stem cells (MSCs), showed spread, elongated and aligned cells. Metabolic activity and DNA quantification studies showed an increase in MSCs proliferation on coated membrane compared to uncoated membrane over the study time. Similarly, cytokines (IL-6, IL-8, osteoprotegerin) and growth factors (VEGF, bFGF) release in supernatant, as well as endothelial cells recruitment, were significantly increased in presence of CaP-CHI-HA coated membrane. Thus, CaP-CHI-HA coated membrane provides a suitable environment for MSCs to induce bone healing. Moreover, pro-inflammatory cytokines (IL-1β and TNF-α) secretion by human monocytes was significantly reduced on CaP-CHI-HA coating compared to LPS stimulation. CaP-CHI-HA coating also reduced significantly Staphylococcus aureus and Pseudomonas aeruginosa adhesion on the membrane, conferring a bacterial anti-adhesive surface. Based on our results, CaP-CHI-HA functionalized collagen membrane provides an interesting material for bone regeneration.

Extensive annulus fibrosus (AF) radial tears lead to intervertebral disc (IVD) herniation. While unrepaired defects in the AF are associated with postoperative reherniation and high IVD degeneration prevalence, current surgical strategies are limited to symptomatic treatment of pain and disregard the structural integrity of the AF. For all these reasons, this study is focused on i) designing polycaprolactone (PCL) electrospun implants that mimic the multi-lamellar fibrous structure of the native tissue and ii) assessing their ability to properly close and repair an AF defect in a sheep in vivo model. Oriented PCL mats were produced by electrospinning with average fiber diameters of 1.3µm and a tensile modulus (55±1MPa) matching the one of a native human AF lamella (∼47MPa). In vitro experiments demonstrated a spontaneous colonization of PCL mats by human and ovine AF cells. In vivo study was carried out on 6 sheep in which 5 lumbar discs were exposed using a left retroperitoneal approach. Defects (2×5mm, 2mm depth) were created in the outer annulus, with randomized distribution of conditions including 10-layer oriented or non-oriented mats, untreated and healthy groups. X-ray and MRI examinations were performed every month until explantations at 1, 3 and 6 months, followed by immuno-histological analysis. Data showed no dislocation of the implants, cell infiltration between the PCL mats and within the mats, and a continuous type I collagen tissue formation between the implants and the surrounding AF tissue. These results highlight that multi-layer PCL electrospun mat is a promising biomaterial for AF repair.

The initiation and progression of malignant tumors are supported by their microenvironment: cancer cells per se cannot explain growth and formation of the primary or metastasis, and a combination of proliferating tumor cells, cancer stem cells, immune cells, mesenchymal stromal cells and/or cancer-associated fibroblasts all contribute to the tumor bulk. The interaction between these multiple players, under different microenvironmental conditions of biochemical and physical stimuli (i.e. oxygen tension, pH, matrix mechanics), regulates the production and biological activity of several soluble factors, extracellular matrix components, and extracellular vesicles that are needed for growth, maintenance, chemoresistance and metastatization of cancer. Both in osteosarcoma and bone metastases from carcinomas this aspect has been only recently explored. In this lecture, I will discuss the role of tumor microenvironment, with a particular focus on the mesenchymal stroma, contributing to bone tumor progression through inherent. The most recent advances in the molecular cues triggered by cytokines, soluble factors, and metabolites that are partially beginning to unravel the axis between stromal elements of mesenchymal origin and bone cancer cells, under different microenvironmental conditions, will be reviewed providing insights likely to be used for novel therapeutic approaches.

Osteomyelitis is an infection of bone or bone marrow with a concomitant inflammation involving the bone marrow and the surrounding tissues. Chronic osteomyelitis is historically treated in a two-stage fashion with antibiotic-loaded polymethylmethacrylate as local antibacterial therapy. Two-stage surgeries are associated with high morbidity, long hospitalization and high treatment costs. Next to antibiotic releasing biomaterials, S53P4 bioactive glass is a biomaterial that enables one-stage surgery in local treatment of chronic osteomyelitis. S53P4 bioactive glass is gaining interests in recent years in clinical treatment of chronic osteomyelitis in a one-stage fashion due to its antibacterial and bone regenerating capacities. By changing local pH and osmotic pressure S53P4 bioactive glass attack bacteria in a different way as compared to antibiotics. In this presentation, we will present current clinical treatment options for osteomyelitis, clinical results and level of evidence of various biomaterials used in osteomyelitis treatment. In addition, the clinical results and health-economic results of S53P4 bioactive glass will be detailed. Thereafter a summary of the current standing across the board in osteomyelitis treatment will be provided.

Our goal is to repurpose drugs to block the growth of lung metastases, the lethal process in osteosarcoma. We therefore screened the NCI-panel of 114 FDA-approved oncology drugs to identify agents that potently reduce growth of osteosarcoma spheroids (sarcospheres). We first developed a system to routinely generate large numbers of highly-uniform spherical sarcospheres (1/well) with a 400um diameter, to most closely simulate micrometatases. Our primary drug screen (Z’-factor=0.70+0.10) utilized sarcospheres from three highly-metastatic human osteosarcoma cell lines (LM7, 143B, and MG63.3) in the presence and absence of MAP chemotherapeutics. Dose-response experiments with 13 of the most effective drugs confirmed initial results and allowed comparison with each drug's toxicity on normal human osteoblasts and normal small airway epithelial cells. Romidepsin, a HDAC inhibitor (HDACi), had the most favorable toxicity/efficacy ratios (TD₅₀/IC₅₀=57–580, depending on cell line). The only other HDACi in the panel of FDA-approved drugs (vorinostat) also ranked highly in the screen. Since newer HDACi's may have improved toxicity/efficacy ratios, we compared romidepsin and vorinostat with the three other HDACi's that are FDA-approved (belinostat, panobinostat, and valproic acid) plus one that is in clinical trials (entinostat). Romidepsin (C_max/IC₅₀=36–360) and belinostat (C_max/IC₅₀=14–20) reduced sarcosphere growth at clinically-achievable levels, in the presence or absence of MAP. Importantly, both romidepsin and belinostat were synergistic with MAP (BLISS scores=5–15). Propidium iodide staining showed that both romidepsin and MAP substantially induced cell death throughout the sarcospheres. Our results strongly support future studies to determine effects of romidepsin and belinostat on growth of lung metastases in vivo.

Total knee arthroplasty (TKA) is a common orthopaedic procedure with over 1,500 done in 2016 in Ireland alone. 96% of all TKAs are due to pain in the knee associated with osteoarthritis. According to the UK National Joint Registry (NJR), there is a 0.47%, 1.81%, 2.63% and 4.34% probability risk of undergoing a revision TKA within one, three, five and ten years respectively post-index surgery. A variety of reasons for failure of TKA have been described in the literature including infection, aseptic loosening, pain, instability, implant wear, mal-alignment, osteolysis, dislocation, peri-prosthetic fracture and implant fracture. The NexGen Posterior Stabilised Fixed has NJR revision rates of 0.44%, 1.61% and 2.54% at years one, three and five respectively. A retrospective review was carried out of 350 NexGen TKAs that were performed directly by, or under the supervision of, a fellowship trained arthroplasty surgeon in a dedicated orthopaedic hospital between April 2013 and December 2015. 26 (7.4%) of these were revised as of 31 December 2017. Three were for septic arthritis with the remaining 23 (6.6%) for aseptic loosening. Patients typically started to experience symptoms of medial tibial pain with supra-patellar swelling from a combination of effusion and synovial thickening at 12–24 months. Inflammatory markers were normal in all cases. Radiographs of symptomatic knee replacements showed bone loss on the medial tibia with a tilt of the tibial component into a varus alignment. The high number of revisions of this particular prosthetic has led to its use being discontinued at this centre.

Although osteoarthritis (OA) is characterized by articular cartilage damage, synovial inflammation is a prominent feature contributing to disease progression. In addition to synovial tissue resident macrophages, infiltrating macrophages and monocytes, their lineage precursors, may also contribute to pathological processes. In mice, peripheral blood monocytes may be categorized according to pro-inflammatory/classical and patrolling/non-classical subsets. The aim of this study was to identify profiles of peripheral blood monocyte subsets as well as different synovial macrophage phenotypes during disease development. OA was induced in knees of C57BL/6 mice by destabilization of the medial meniscus (DMM). Blood was harvested from the facial vein 7 days prior to and 1, 7, 14, 28, and 56 days post induction of OA. Separate mice were sham-operated as a control. Monocyte subsets and synovial macrophage populations were identified by flow cytometry. Levels of classical monocytes were significantly higher at day 14 (p<0.001) and day 28 (p=0.031) in peripheral blood of DMM-operated mice compared to control. Furthermore, the percentage of non-classical monocytes was significantly lower in DMM-mice at day 14 (p=0.026). At day 56 post OA-induction, an increase in total synovial macrophages (CD11b+F4/80+ cells) was observed between DMM and sham operated knees (p=0.021). The ratio between pro-inflammatory (CD11b+F4/80+CD86+) and tissue repair (CD11b+F4/80+CD206+) synovial macrophage subsets tended to be higher in DMM knees, however this finding was not statistically significant (p>0.05). In light of the present findings, further investigation is required to elucidate the relationship of peripheral blood monocyte subsets to synovial inflammation and features of OA pathogenesis.

Several previous pathoanatomical and biomechanical studies focused primarily on the cam morphology as the primary contributor to symptoms of femoroacetabular impingement (FAI) and limited range of motion. However, there is a growing population of individuals with asymptomatic cam morphologies who show no clinical signs; thus, the cam deformity, alone, may not fully delineate an individual's symptomatology or limited motion. These studies expanded beyond the cam morphology, to determine how additional anatomical characteristics could contribute to symptoms and influence functional mobility, using: 1) in vivo analyses, where we asked how specific anatomical parameters (in addition to the cam morphology) can predict individuals at risk of symptoms; 2) In silico simulations, where we examined how pathoanatomical features contributed to adverse loading conditions, resulting in higher risks of hip joint degeneration; 3) In vitro cadaveric experiments, where we examined the contributions of the cam morphology and encapsulating ligaments to joint mechanics and microinstability. This research further highlights that more emphasis should be placed on proper patient selection. There are implications of how structural anatomy can affect musculature, joint loading and stability, which should all be closely examined to improve the effectiveness of hip preservation surgery as well as the understanding of non-surgical management.

Osteoarthritis is a multifactorial disease in which altered mechanical loading is one of the agreed contributing factors. Whereas in the past, altered mechanical loading was merely deferred from static, image-based evaluations of malalignment, the recent use of 3D motion capture allowed dynamic evaluation of joint loading in terms of dynamic alignment (e.g. varus trust) and even joint loading strategy (merely using proxy measures like knee adduction moment.) Combining these measurements with musculoskeletal models, the overall loading distribution in the joint due to muscle action underlying the patient's motion pattern can be quantified. Using this approach, our group showed the potential of this technique to differentiate between control subjects and subjects with early medial knee OA before the presence of radiographic evidence of structural joint degradation. Nevertheless, no changes in loading distribution could be detected in a cohort of subjects suffering of local cartilage defects in an otherwise healthy knee joint, indicating that patients did not present active unloading strategies despite the presence of clinical symptoms. Furthermore, subject-specific strategies aiming contributing to modified loading of the hip joint have been evaluated.

Bi-cruciate stabilized (BCS) TKA is the prosthesis that aims to substitute bi-cruciate ligament with post-cam engagement. We estimated to describe the in vivo kinematics during deep knee bending in BCS and Cruciate retaining (CR) TKA with the same articular geometry. We analyzed 26 knees who agreed to the current investigation under institutional review board approval. 17 knees were implanted with BCS (Journey ∥BCS, Smith & Nephew. Memphis, US) and 9 knees with CR (Journey∥CR). Each patient was asked to perform deep knee bending under weight-bearing condition. To estimate the spatial position and orientation of the TKA, 2D/3D registration technique with single fluoroscopy was used. We evaluated anteroposterior (AP) translation of the nearest point from femoral component to tibial axial plane for medial and lateral sides, femoral external rotation relative to tibial component and post-cam engagement in BCS. Measurement results were analyzed using Wilcoxon test. Values of P<0.05 were considered statistically significant. Medial AP translation indicated 11.7±5.1% posterior movement in BCS and 4.0±6.6% anterior movement in CR from minimum flexion to 130°. Lateral AP translation indicated 28.9±11.4% posterior movement in BCS and 18.3±6.2% posterior movement in CR from minimum flexion to 130°. Femoral external rotation were observed in both group and the amount of rotation were 5.2°±4.5° in BCS and 8.2°±4.0° in CR. Anterior post-cam engagement was not observed in all cases (76.5%). But medial AP translation in BCS was anteriorly in shallow flexion angles compared to CR. It suggested that anterior post-cam engagement couldn't work in valid.

There are few studies that have compared between continuous flexion activities and extension activities of normal knees. The purpose of this study is to compare in vivo kinematic comparison of normal knees between flexion activities and extension activities. Total of 8 normal male knees were investigated. We evaluated in vivo three-dimensional kinematics using 2D/3D registration technique. We compared femoral rotation angle relative to tibia, anterior/posterior (AP) translation of medial femoral sulcus (medial side) and lateral femoral epicondyle (lateral side) onto tibial plane perpendicular to tibial functional axis between flexion activities (F groups) and extension activities (E groups). Femoral external rotation was observed with the knee bending during both groups. The external rotation angle of F group was larger than that of E group significantly from 20 to 30 degrees with flexion (p < 0.05). Regarding medial side, anterior translation was observed up to 40 degrees in F group. From 40 to 140 degrees, posterior translation was observed. In E group, anterior translation was observed from 140 to 40 degrees with extension. From 40 degrees, posterior translation was observed. From 30 to 40 degrees, F group located anterior than E group (p < 0.05). Regarding lateral side, posterior translation was observed with flexion in F group. On the other hand, anterior translation was observed with extension in E group. Regarding AP location with flexion angle, there was no significant difference between two groups. In conclusion, there were different kinematics between flexion activities and extension activities.

Tibial bone density may affect implant stability and functional outcomes following total knee replacement (TKR). Our aim was to characterise the bone density profile at the implant-tibia interface following TKR in mechanical versus kinematic alignment. Pre-operative computed tomography scans for 10 patients were obtained. Using surgical planning software, tibial cuts were made for TKR either neutral (mechanical) or 3 degrees varus (kinematic) alignment. Signal intensity, in Hounsfield Units (HU), was measured at 25,600 points throughout an axial slice at the implant-tibia interface and density profiles compared along defined radial axes from the centre of the tibia towards the cortices. From the tibial centre towards the lateral cortex, trabecular bone density for kinematic and mechanical TKR are similar in the inner 50% but differ significantly beyond this (p= 0.012). There were two distinct density peaks, with peak trabecular bone density being higher in kinematic TKR (p<0.001) and peak cortical bone density being higher in mechanical TKR (p<0.01). The difference in peak cortical to peak trabecular signal was 43 HU and 185 HU respectively (p<0.001). On the medial side there was no significant difference in density profile and a linear increase from centre to cortex. In the lateral proximal tibia, peak cortical and peak trabecular bone densities differ between kinematic TKR and mechanical TKR. Laterally, mechanical TKR may be more dependent upon cortical bone for support compared to kinematic TKR, where trabecular bone density is higher. This may have implications for surgical planning and implant design.

A defining characteristic of the Nucleus Pulposus (NP) and the inner AF is the very limited vascular supply and low pH that imposes metabolic constraints on the disc cells. Interference with the normal physiology of the NP niche, by activities linked to changes in oxygen diffusion across the endplate leads to dysregulated niche function. Hypoxia Inducible Factor-1 (HIF-1) and HIF-2 are robustly and constitutively expressed by cells of the NP. Our recent work has shown that expression of HIF-1 is indispensable for NP cell survival in vivo and suggests an important role of HIF-1 in NP cell metabolic program. This talk will discuss central role of HIF-1 as metabolic and pH homeostatic regulator of NP cells and possible implication for a therapeutic strategy to treat disc degeneration

Current medical treatments for IVD degeneration rely on conservative therapies or surgery. Surgical treatments (e.g. spinal fusion,) have shown satisfactory results in alleviating pain, but long-term clinical outcomes remain poor. Thus, there is an urgent need for alternative cell based regenerative therapies focussed on correcting the underlying pathogenesis of IVD degeneration. However, for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. We have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 results in improved differentiation to a nucleus pulposus (NP)-like phenotype and synthesis of proteoglycan rich matrix with micromechanical properties akin to the healthy IVD. Significantly, these studies have highlighted that AD-MSCs are the more appropriate cell source. Furthermore, our studies have shown hat GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate atrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based therapy may be beneficial both in directing lineage-specific MSC differentiation, but also in restoring a more anabolic phenotype in native NP cells, thereby having a dual regenerative effect.

Early clinical studies investigating the effects of delivery of mesenchymal stromal cells (MSCs) to degenerated intervertebral discs have shown promising results, but with an incomplete understanding of the therapeutic mechanism(s) of action. To address this, we have developed a 3D co-culture system to unravel the biological interaction between MSCs and nucleus pulposus (NP) cells. Alginate constructs were created using a biphasic configuration consisting of a cylindrical shell with an encapsulated bead. Human NP cells were seeded in monolayer or encapsulated within alginate and cultured in hypoxia with variations of pH, osmolarity and growth factors (n = 6) to replicate healthy or degenerative conditions. Wells and gels were fixed and stained for ECM content, and retrieved cells and media were analysed for ECM and inflammatory factor expression. Encapsulated hNPCs showed no migration from either alginate structure and full bead separation was achieved over 14 days, reinforcing the construct as a separable 3D co-culture method. Addition of the degenerative growth factors TNFα and IL-1β as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. TGF-β3 addition showed higher incidence of aggrecan deposition over addition of IL-1β. Addition of FGF2 altered cell morphology and ECM deposition including formation of pseudo lamellae, indicating a phenotype shift toward annulus fibrosis cells as shown in late-stage degenerative disc disease. The data from this study will be used in future MSC:NPC co-cultures to determine immunoregulatory interactions in a degenerative environment.

The objective of this study was to investigate the effects of different doses rhBMP-2 on bone healing in an ovine lumbar interbody fusion model. In this study 22 sheep underwent two level lumbar interbody fusion using a ventrolateral approach with secondary dorsal fixation at L1/2 and L3/4. After randomization in one level a PEEK-cage was implanted filled with one of three doses rhBMP-2 (0,5mg; 1mg; 2mg) delivered on an ACS. The other level received an empty PEEK-cage or ACS filled cage. Animals were sacrificed after 3 and 6 months and decalcified histology was performed. This included histomorphological analysis well as histomorphometry of the tissues within the cage.

At 3 months after surgery the groups treated with rhBMP-2 showed higher amounts of bone tissue within the cage. At 6 months the amounts of bone tissue increased in all groups, were still lower in the groups without growth factor. At 3 months there was only one active osteolysis in the cage/ACS. 7 of 8 segments of the rhBMP-2 groups had a compromised bone structure around the implant. These areas were filled with fibrous tissue and fibrocartilage. This finding was not detected in the groups without rhBMP-2 at 3 months. At 6 months most of the segments with an empty cage or cage/ACS showed a chronic inflammation. Predominant cells were macrophages and giant cells. The groups treated with rhBMP-2 showed only a few mild chronic inflammatory reactions. The well-known dose dependent effect of rhBMP-2 on bone healing could also be recognized in our study. Attention has to be payed to the proinflammatory properties of the growth factor. Consistent with other studies we found 2 strong inflammatory reactions, each one in the lowest and highest dose group. Also, the potential for causing transient bone resorptions, according to the results of others, was demonstrated. At 3 months 7 of 8 segments treated with rhBMP-2 showed compromised peri-implant bone. Osteoblasts, but not osteoclasts, were seen in the periphery of these areas. It can be concluded that there where bone resorptions which already merged into an increased osteoblastic activity. Usually resorptions occur between 2 and 12 weeks and are followed by a period of increased osteoblastic activity. This finding wasn't recognized at 6 months anymore. Striking is that at 6 months most of the segments without rhBMP-2 showed a compromised bone structure around the implant with a mild to mainly moderate chronic inflammatory reaction. This cannot be attributed to the growth factor. Also, the ACS is degraded at 6 months and is unlikely a possible explanation. Therefore, the cage as a reason must be considered and it has to be questioned whether PEEK is the optimal material for interbody cages.

Aging has been associated with decreases in muscle strength and bone quality. In elderly patients, paravertebral muscle atrophy is accompanied by vertebral osteoporosis. The purpose of this study was to use paravertebral injection of botulinum toxin-A (BTX) to investigate the effects of paravertebral muscle atrophy on lumbar vertebral bone quality. Forty 16-week-old female SD rats were randomly divided into four groups: (1) a control group (CNT); (2) a resection of erector spinae muscles group (RESM); (3) a botulinum toxin-A group (BTX) that was treated with local injection of 5U BTX into the paravertebral muscles bilaterally; and (4) a positive control group (OVX) that underwent bilateral ovariectomy. At 3 months post-surgery the lumbar vertebrae (L3 – L6) were collected. The BMDs of the RESM and BTX groups were significantly lower than that of the CNT group (P < 0.01). Micro-CT scans showed that rats in the three experimental groups had fewer trabeculae and trabecular connections than rats in the CNT group. The bone loss trend of the trabecular networks was most obvious in the OVX rats. Vertebral compression testing revealed that the three experimental groups had significantly lower maximum load, energy absorption, maximum stress, and elastic modulus values than the CNT group (P < 0.01), and these parameters were lowest in the OVX group (P < 0.05). Our results demonstrate that the new paravertebral muscle atrophy model using local BTX injection causes sufficient muscle atrophy and dysfunction to result in local lumbar vertebral bone loss and quality deterioration.

By modifying only the nanofeatures on material surfaces without changing surface chemistry, it is possible to increase tissue growth of any human tissue by controlling the endogenous adsorption of adhesive proteins onto the material surface. In addition, our group has shown that these same nanofeatures and nano-modifications can reduce bacterial growth without using antibiotics, which may further accelerate the growth of antibiotic resistant microbes. Inflammation can also be decreased through the use of nanomaterials. Finally, nanomedicine has been shown to stimulate the growth and differentiation of stem cells, which may someday be used to treat incurable disorders, such as neural damage. This strategy also accelerates FDA approval and commercialization efforts since new chemistries are not proposed, rather chemistries already approved by the FDA with altered nanoscale features. This invited talk will highlight some of the advancements and emphasize current ceramic nanomaterials approved by the FDA for human implantation. It will also emphasize the future of nanomaterials in medicine, such as their use in personalized medicine as internal sensors to detect and fight alterations in health.

When joints sustain injury, the release of inflammation cytokines can cleavage matrix proteins and result in cartilage degradation and the subsequent osteoarthritis. RNA therapeutics emerging recently is a very promising approach to efficiently and specifically inhibit disease gene expression. However, the major challenge is how to deliver therapeutic RNA into joint and cartilage. Janus base nanotubes are self-assembled from synthetic Janus bases inspired from DNA base pairs. Based on the charge interaction, we are able to “sandwich” small RNAs among Janus base nanotubes to form tiny, nano-rod shaped delivery vehicles. Such vehicles can be engineered into different sizes and shapes. We have found that short and slim morphologies can greatly increase their penetration to extracellular matrix and delivery into “difficult-to-reach” tissues, such as cartilage and brain. Moreover, by delivering therapeutic siRNA, we have demonstrated its high-efficacy in inhibiting expression of an inflammatory regulator, Interleukin-1 receptor (IL-1R) in articular cartilage. Moreover, the inhibition effect is long-lasting so that joint inflammation and cartilage degradation caused by meniscus injury are greatly inhibited in a mouse model. Therefore, the Janus base nanotubes present a great potential in engineering into nano-structures for RNA delivery. Such approach may become an effective therapeutic against joint inflammation and arthritis.

Implant infection is an increasing problem in orthopedic surgery, especially due to progressive antibiotic resistance and an aging population with rising numbers of implantations. As a consequence, new strategies for infection prevention are necessary. In the previous study it was hypothesized that laser-structured implant surfaces favor cellular adhesion while hindering bacterial ongrowth and therewith contribute to reduce implant infections. Cuboid titanium implants (0.8 × 0.8 × 12 mm³, n=34) were used. Seventeen were laser-structured by ultra-short pulsed laser ablation to create a spike structure; the others were polished and served as controls. In general anesthesia, implants were inserted in rat tibiae and infected with a S. aureus suspension. During a 21 day postoperative follow-up, daily clinical control was performed. Radiographs were taken at day 14 and day 21. After euthanasia, bacterial load and biofilm formation on the implant surface was evaluated semi quantitatively by confocal laser scanning microscopy and computational acquisition of bacteria and cells by Imaris®-software. Additionally, histology of the surrounding bone was performed. Clinically, no differences were observed between the groups. However, contrary to our hypothesis, bacterial load was increased in the laser-structured implant group although cellular adhesion was even more pronounced. Radiographical and histological evaluations showed increased bone alterations in the group with laser-structured implants compared to the control group. These findings did not confirm prior in vitro studies, where a reduction of bacterial load was found for similar surfaces and demonstrate the necessity of in vivo trials prior to the clinical use of new materials.

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm², pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 10⁹ CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.

Long-term survival and favourable outcome of implant use are determined by bone-implant osseointegration and absence of infection near the implants. As with most diseases, prevention is the preferred approach. Silver ion doped calcium phosphate based ceramic coating (Silveron®) for implant coating has been shown previously to be a potent antimicrobial agent as indicated by in vitro testing. The present study reports on clinical experience using silver ion doped calcium phosphate based ceramic coated external fixator pins as surgical treatment in the management of chronic osteomyelitis and open fractures. Ten patients had external fixators: six for open fractures of ankle, three for chronic osteomyelitis of the femur, one for tibia pseudoarthrosis. The electrospray method was used for coating the external fixator pins with silver ion doped calcium phosphate-based ceramics. A radiofrequency energy source was used to sinter the coated pins. Microbiological, roentgenographic, toxic and biochemical analyzes of patients were carried out. Wound debridement, and subsequent wound care resulted in control of the infection in three chronic osteomyelitis and in healing of seven fractures after follow-up ranging from three to six months. In total 67 pins were used in 10 patients but only one pin was positive microbiologically in one patient. Collectively, these data clearly illustrate that the toxic effects of silver were not observed at the doses used. Silver ion doped calcium phosphate based ceramic coating (Silveron®) can be used to prevent infection associated with the implant.

Transverse patella fractures are commonly encountered in trauma surgery, open reduction and internal fixation are considered the gold standard treatment modality that could permit early knee motion and immediate rehabilitation. Many fixation methods had been defined and compared to each other's in many clinical and biomechanical studies. The aim of this study was to assess the safety and stability of our novel anatomical patella plate and to compare its stability with tension band-wire technique. A total of 12 cadaveric preserved knees (six right and six left patellae) with close patellar size were chosen to form two groups of six samples. Each group received either plate or tension band-wiring fixation for an experimentally created patella fracture. Cyclic load of an average of 350 N was applied for all specimens and after accomplishing 50 cycles the displacements of all fracture edges were recorded. After completing 50 cycles in each group, the average fracture edges displacement measured in the plate group was 1.98 ± 0.299 mm, whereas the average fracture edges displacement measured in the tension band-wire group was 2.85 ± 0.768 mm (p = 0.016). In the operative treatment of displaced transverse patellar fractures, the strength of fixation obtained by titanium curved plates is highly stronger when compared to the fixation with a tension band-wire technique. Fixation with titanium curved plates provides satisfactory stability at the fracture site which allow withstanding the cyclic loads during the postoperative rehabilitation.

Scaffold-based bone tissue engineering holds great promise for the future of osseous defects therapies. Prepare the suitable scaffold properties are physiochemical modifications in terms of porosity, mechanical strength, cell adhesion, biocompatibility, cell proliferation, mineralization and osteogenic differentiation are required. We produce various bone tissue scaffolds with different techniques such as lyophilization, 3D printing and electrospinning. We wish to overview all the different novel scaffold methods and materials. To improve scaffolds poor mechanical properties, while preserving the porous structure, it is possible to coat the scaffold with synthetic or natural polymers. An increasing interest in developing materials in bone tissue engineering is directed to the organic/inorganic composites that mimic natural bone. Specifically, bone tissue is a composite of an organic and inorganic matrix. Using PLLA, loofah, chitin and cellulose biomaterials we produced bone tissue scaffold with lyophilization technique. Also, using fish scale powder and wet electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) a sponge structure had created. Using MRI image data and 3D printer method, a bone tissue scaffold is created by PLA filament. Their mechanical properties had analysed with compression tests and their biocompatibilities had investigated. In order to provide novel strategies for future treatment of bone tumours, the properties of the scaffold, including its in vitro extended-release properties, the inhibition effects of chemotherapeutic agent on the bone tumours and its bone repair capacities were investigated in vitro by using MG63 cells. To develop chemotherapeutic agent-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles in a porous nano-hydroxyapatite scaffold we aimed to use double emulsion method.

Meniscus is mainly composed of three different cell types; chondrocytes(Ch) situate in the superficial zone, whereas fibroblast-like cells locate in the peripheral region having long cell extensions in contact with different parts of the matrix, fibrochondrocytes(FC), is from the inner part of the meniscus and show a clear cell associated matrix. The aim of this study is to develop meniscus cell population using with mesenchymal stem cells (MSCs). For this purpose, MSCs were isolated from rabbit bone marrow and verified by flow cytometry analyses using cell surface markers (CD73APC, CD90FITC, CD34PE, CD45PE/Cy5.5). The results indicate that CD73 and CD90-positive cells were 92.8%, and CD 45 and CD 34-negative cells were 52.4%. Differentiation potential of MSCs were also evaluated by differentiating into Ch, osteoblasts (Ob), adipocytes (Ad), fibroblasts (Fb). Histology stainings showed that differentiated Ch can produce proteoglycans, Ob have mineralization property, Fb have spindle shape and Ad have oil drops morphology. Afterwards Fb, Ch and undifferentiated MSCs (for formation of the FC) were seeded in same plate in cocktail medium and Fb, Ch, seeded individually, were used as control group. Proliferation activity of the cells was analyzed by XTT assay at 3^th,7^thand14^thdays. In addition, cells were analyzed by flow cytometry with identical surface markers at 3^th,7^thand14^thdays. Results show that cell cocktail have the greatest proliferation ability with a greater speed than the individual Ch or Fb cultures. In addition, FC formation was identified by histological staining. In conclusion, meniscus specific cell population has been successfully generated from the cell cocktail containing rabbit MSCs.

After total hip replacement, force generating capacity of gluteal muscles is an impotant parameter on joint contact forces and primary fixation of total hip replacement. Femoral offset is an option to optimize muscle moment arms, especially main abductor Gluteus Medius and Minimus. To investigate relationship with weak gluteal muscles (Gluteus Medius and Minimus) and increased femoral offset, we build a musculoskeletal model. Creating of three-dimensional femur geometry and scaling of the musculoskeletal model according to the subject were performed with computed tomography data. Obtained gait kinematic and kinetic data were applied and to mimic gluteal muscle weakness, the force generating capacities of Gluteus Medius and Minimus reduced (%20-%80). Analysis were done for both anatomical and +10mm offset. Then, muscle and joint reaction forces obtained from musculoskeletal analysis transfered to CT based finite element model to evaluate changes in maximum principle stresses on femur. According to the results of the musculoskeletal analysis, the weakness of the gluteal muscles caused an increase in the activation of Gluteus Maximus, Rectus Femoris and Tensor Fasciae Latae. Effects of +10 mm femoral offset on total abductor muscle activity increased with reduced muscle strength. As a result of the finite element analysis, no significant difference was observed for maximum principle stresses on femur with varying muscle activites. The results of these analyses are important to understand weakness of gluteal muscles and for planning hip surgery.

Cell micro-environment and biochemical, physical and mechanical signals coming from their micro-environment orientate specific functions of cells. In this study, we prepared novel hydrophilic and hydrophobic amino acids conjugated self-assembled molecules (AA-SAMs) modified Polydimethylsiloxane (PDMS) in order to observe the effect of hydropathy on osteoblasts behaviour. PDMS cell substrates were prepared with a prepolymer cross linker ratio of 10:1. Hydrophobic leucine amino acid (Leu-SAM) and hydrophilic histidine amino acid (His-SAM) conjugated SAMs were produced and characterized by using ¹H Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FTIR) Spectrophotometers. AA-SAMs have ethoxy surface active head group to form SAMs on plasma oxygenated PDMS and functional head group to interact with cells. Hydrophilic 3-Aminopropyltriethoxysilane (APTS) modification was also done as a control group. Modifications of PDMS substrates were confirmed by using water contact angle measurements and X-ray Photoelectron Spectroscopy (XPS) analysis. In order to investigate cellular behaviour, as a preliminary experiment, human osteoblasts were cultured on PDMS substrates at 15.000 cells/cm² in 48 well plates with DMEM-F12 (Sigma Aldrich, D6421) medium supplemented with 10% FBS. Cell viability and proliferation were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay after 1, 4 and 7 days. MTT assay showed a significant increase in cell proliferation in both AA-SAMs modified PDMS, in comparison to plain PDMS (p < 0,01). Among AA-SAMs and hydrophilic APTES, hydrophilic His-SAM modification was observed to provide a better cellular metabolic activity (p < 0,01). Hence, these novel AA-SAMs modified PDMS substrates are promising cell substrates to enhance osteoblast behaviour in vitro.

We define regenerative engineering as a convergence of advanced materials science, stem cell science, physics, developmental biology, and clinical translation. Stem cells play an important role. Work in the area of musculoskeletal tissue regeneration has focused on a number of paradigms. Polymer and polymer-ceramic systems can be utilized for the regeneration of bone. Direct induction can be controlled through material characteristics. Through the use of inducerons, small molecules fostering induction, the design of regeneration-inducing materials can be realized. We believe the medicinal use of stem cells will be of critical importance in the design of next generation systems answering grand challenges to musculoskeletal regeneration.

Recent advances in tissue engineering have made progress towards the development of biomaterials with the capability for delivery of growth factors to promote enhanced tissue repair. However, controlling the release of these growth factors is a major challenge and the associated high costs and side effects of uncontrolled delivery of has proved increasingly problematic in clinical orthopaedics. Gene therapy might be a valuable tool to avoid these limitations. While non-viral vectors are typically inefficient at transfecting cells, our group have had significant success in this area using a scaffold-mediated gene therapy approach for regenerative applications. These gene activated scaffold platforms not only act as a template for cell infiltration and tissue formation, but also as a ‘factory’ to provoke autologous host cells to take up specific genes and then engineer therapeutic proteins in a sustained but eventually transient fashion. Alternatively, scaffold-mediated delivery of siRNAs and miRNAs can be used to silence specific genes associated with pathological states in orthopaedics. This presentation will provide an overview of some of this research with a particular focus on gene-activated biomaterials for promoting stable cartilage formation in joint repair and on scaffold-based delivery of therapeutics for enhancing vascularization & bone repair.

Tissue regeneration using growth factors has disadvantages while needing to use supraphysiological growth factor concentrations. Gene therapy has been proposed as alternative. Unfortunately, drawbacks such as the use of viruses and the inefficiency of non-viral systems jeopardize clinical translation. mRNA-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. mRNA molecules can be chemically modified in order to improve stability and immunogenicity. Chemically modified mRNA (cmRNA) is much more efficient than pDNA in delivering genes into the cell. The combination of biomaterials with cmRNA is interesting for the tissue engineering and regenerative medicine field. The resulting construct, known as Transcript-Activated Matrix, may act as a cmRNA delivery platform while supporting cell proliferation, extracellular matrix deposition and ultimately de novo tissue formation. Our work and the work of others demonstrated that the use of Transcript-Activated Matrix prolonged transgene expression and enhanced protein translation. This presentation will provide an overview of ongoing research from our group on cmRNA for improving bone repair with a particular focus on Transcript-Activated Matrix for enhancing osteogenesis. Results of our investigation in vitro with stem cells, ex vivo using tissue culture and in vivo using rat models will be presented.

Healthy bone metabolism is a tightly coupled dynamic process that relies on a balance between bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Traditionally, tissue-engineering approaches for non-union fracture repair employ local anabolic therapeutic delivery strategies that target mesenchymal stem cells (MSCs) and osteoblasts to induce bone formation, however, the challenge of healing non-union defects depends on the cause of defect e.g. trauma or disease, and targeting bone formation alone is often not sufficient. Our research focuses on utilising both anabolic therapeutics, including recombinant human bone morphogenic protein (rhBMP) −2 and parathyroid hormone (PTH)_(1–34), and anti-catabolic bisphosphonates (BPs) to target bone metabolism. A major challenge with harnessing a combined dosing regimen is controlling the release of the individual therapeutics to target cells. We have developed a number of polymer-ceramic based biomaterial delivery systems, including injectable and implantable scaffolds, for the controlled release of rhBMP-2 and the BP zoledronic acid (ZA) and demonstrated their efficacy in vivo. A dual therapeutic load provided a synergistic enhancement of bone regeneration, demonstrating significantly increased bone formation and remodelling compared to anabolic therapies alone. Utilising hydroxyapatite as the ceramic phase in our scaffolds further increased bone formation, demonstrating the polymer-ceramic scaffolds to be osteoconductive in the absence of therapeutics. In addition, we have demonstrated the manipulation of bone metabolism through a specific dosing regimen of PTH_(1–34), a therapeutic traditionally used as an anabolic, to induce bone remodelling and drive healing in BP loaded fractures. Our research to date has shown that optimising the delivery and regimen of anabolic and anti-catabolic therapeutics to control bone metabolism, augments the bone regenerative potential of these therapeutics in orthopaedic applications.

The potential of piezoelectric biomaterials for bone tissue engineering is demonstrated. This work proves that the use of piezoelectric poly(vinylidene fluoride) (PVDF), able to provide electrical stimuli upon mechanical solicitation to the growing bone cells, enhances the bone regeneration in vivo. Poled and non-poled PVDF films, with and without macroscopic piezoelectric response, respectively and randomly oriented piezoelectric electrospun fiber mats have been used as substitutes for bone to test their osteogenic properties in Wistar rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. After 4 weeks, the qualification of the regenerated bone was performed according the H&E staining. Defect implanted with poled PVDF films demonstrated significantly more defect closure and bone remodeling, showing the large potential of piezoelectric biomaterials for bone repair, as well as for other electromechanical responsive tissues such as muscle and tendon.

Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects the whole joint. Mesenchymal stem cells ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of OA. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, we developed a micromolding protocol of encapsulation that allows to obtain particles that (i) could be injected with a 26G needle into a mouse joint and (ii) could provide a 3D microenvironment supporting cell biological activity. Polydimethylsiloxane (PDMS) chips containing circular micromolds were manufactured and a solution of alginate (2% w/v) containing human adipose stem cells (3 millions/mL) was deposited on the chips. Cell loading into the micromolds was performed either by sedimentation or by centrifugation. Following Ca2+ crosslinking, alginate particles (diameter 150±0.7μm) were obtained. The number of cells per particle was 5 times higher when the micromolds were loaded by centrifugation. Cell number and metabolic activity remained stable for 7 days after encapsulation and injection through a 26G needle had no impact on cell viability. When cells were stimulated with TNF-alpha and INF-gamma, prostaglandin E2 (PGE2) concentration in the supernatant was multiplied by 13 and 7 and indoleamine2,3-dioxygenase (IDO) activity was 2 and 4 times higher when cell loading was performed by sedimentation or centrifugation, respectively. We have demonstrated that encapsulated cells were able to sense and respond to an inflammatory stimulus and their therapeutic potential will be evaluated in a murine model of osteoarthritis.

Osteoporosis affects millions globally and current anti-catabolic treatments are limited by significant side-effects. Osteoporosis arises when skeletal stem cells (SSC) no longer sufficiently replenish osteoblasts, leading to net bone loss. A key regulator of SSC behaviour is physical loading, yet the mechanisms by which SSCs sense and respond to changes in their mechanical environment are virtually unknown. Primary cilia are nearly ubiquitous ‘antennae-like’ cellular organelles that have very recently emerged as extracellular chemo/mechano-sensors and thus, are strong candidates to play an important role in regulating SSC responses in bone. This paper will demonstrate that the SSC primary cilium plays an important role in loading-induced bone formation via initial chemosensation and transduction of the potent chemokine TGFβ1 regulating SSC recruitment to the bone surface and secondly it will be shown that the primary cilium is a cAMP responsive mechanosensor directly regulating SSC mechanotransduction via localisation of adenylyl cyclase 6 to the ciliary microdomain. Finally, it will be shown that targeting the cilium therapeutically can be an effective approach to enhance both biochemical and biophysically induced SSC osteogenesis contributing to bone formation, demonstrating a novel anabolic therapy for bone loss diseases such as osteoporosis.

Clinical translation of MSC therapies in orthopaedics has been hampered by heterogeneity and a lack of standardised and validated testing protocols for quality assurance. Although minimal criteria have been proposed¹, it is apparent that these do not predict performance in vivo. We used a combinatorial antibody profiling tool to probe the surface immunophenotype of human bone marrow derived MSCs and used this to define new marker panels. Cells were cultured from three marrow donors using specified expansion conditions and probed by high throughput flow cytometry using a panel of 230 antibodies. Analysis of expression of the surface proteins revealed significant variation in response to culture conditions and considerably less variation between donors. Of the panel of 230 markers 107 were negative, 24 had high expression in all samples, 1 had low expression and 98 displayed significant differences between cell preparations. Cluster analyses revealed that marker expression in one culture condition varied considerably from the other two. Phenotypic characterization of the cell preparations, assessed by analysis of differentiation propensity, showed similar patterns of variability between these samples. This suggests that the selected panel may be used as phenotypic MSC markers. Ongoing work involves the generation of novel antibody arrays which will be used as quality tests in a manufacturing environment. These tests will be used for in-process and product release applications for enhanced cell manufacturing and improved clinical outcomes.

Mesenchymal stem cells (MSCs), characterised by their self-renewal and multidifferentiation potential, are a favoured cell population for future tissue engineering applications. Differentiation of MSCs towards a specific lineage has been extensively studied, mainly through the use of growth factors or conditioned media. However, growth factor supplementation is a mono-domain approach and considering the number of permutations, it is unlikely to find the optimal cocktail. Although PRPs are used extensively, its use is controversial, and standardization is impossible. Conditions media have various limitations, including how much, when and how effective it is at the time that it would be aspirated. Thus, co-culture systems are at forefront of scientific research and technological innovation. Co-culture system gives access to the complete cell secretome and offers the advantages of autologous therapy. However, several weeks of co-culture are necessary to observe stem cell differentiation. We hypothesize that, by using macromolecular crowding, which has been shown to recapitulate the dense in vivo microenvironment of the extracellular area and enhance matrix deposition in vitro with its excluded volume effect, it will accelerate stem cell differentiation towards tenogenic lineage. Further, we will assess if tendon specific extracellular matrix deposited by tenocytes is sufficient for stem cell differentiation without the necessity of cell contact between tenocytes and stem cells.

Mesenchymal stromal cells (MSCs) are promising candidate for cell therapy in osteoarthritis (OA) patients since that they exert anti-inflammatory, immunomodulatory, anti-fibrotic and anti-hypertrophic effects in the joint tissues. However, little is known about the OA milieu factors that could enhance the migration and tissue specific engraftment of exogenously injected MSC for successful regenerative cell therapy. GMP-clinical grade adipose stromal cells (ASC) were evaluated both in normoxic and hypoxic (2%O₂) conditions, with or without OA synovium milieu. We found that both OA synovial fluids and OA synoviocytes derived conditioned medium (CM) contain approximately the same amounts of different cytokines/chemokines (i.e. IL6, CXCL8, CXCL10, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11). ASC migration was significantly increased by both OA synovium milieu and not affected by normoxic or hypoxic condition. We identify that ASC migration was mainly influenced by different macrophage chemokines (i.e. CCL2, CCL3, CCL4). In hypoxic condition basal GMP-ASC showed an increase of CXCR3 and CCR3, a decrease of CCR1 and CCR5 receptors, while CXCR1, CXCR4, CXCR7, CCR2 and IL6R were not modulated. The addition of OA synovium milieu induced CCR3, CXCR3 and IL6R and decreased CCR1 and not affected CCR2, CCR5, CXCR1, CXCR4, CXCR7 in hypoxic condition. Our data demonstrated that GMP-ASC chemotaxis was mainly induced by macrophage chemokines. Moreover, we evidenced that hypoxia, as better condition to mimic the OA milieu, affected some GMP-ASC cytokine/chemokine receptors, suggesting the involvement of specific chemokine-receptor axis.

Human in vitro models of the neuromuscular junction (NMJ) are currently moving from embryonic stem cells to induced Pluripotent Stem Cells (iPSCs). With this, a robust model could be optimised for physiology and pathophysiology studies, as well as representing a drug screening platform. For this reason, the work presented here represents the optimisation of a human co-culture model of skeletal muscle (hSkM)/ iPSC-derived motor neurons (MNs) both in monolayer and in 3D tissue engineering collagen constructs. Firstly, human iPSC-derived motor neurons (MNs) were characterised over a period of 35 days to test their cholinergic potential. Then, primary human skeletal muscle (hSkM) and MNs were co-cultured on different substrates (gelatin and SureBond+ReadySet (Axol Bioscience)) and differentiated in various combinations of media to allow both myotube formation and neurite extension. Morphological (β-III Tubulin and Rhodamine Phalloidin) and interaction (α-Bungarotoxin and Synaptic Vesicle 2) immunofluorescent stainings were used to evaluate cell differentiation and co-localisation of pre and post-synaptic markers. Results from this study showed that the MNs presented a cholinergic phenotype up to 21 days; hSkM and MNs co-existed in culture and differentiated in neuronal Maintenance Medium (MM, Axol Bioscience); the 3D constructs allowed alignment and maturation of the muscle tissue, while providing a matrix for neurite extension and NMJ formation. This model has the potential to become a valid tool for in vitro drug screening while reducing the use of animals in research and providing the scientific community with a platform for personalised medicine.

Distraction histogenesis (DH) techniques have been widely accepted and practiced in orthopaedics, traumatology, and craniofacial surgery over the last two decades. Using DH methods, many previously untreatable conditions have been successfully managed with outstanding clinical outcomes. The biological mechanisms underlying DH have been studied and the tension-stress principles of tissue regeneration are attributed to upregulated gene expression, enhanced cell proliferation, angiogenesis and tissue remodelling and endogenous stem cell mobilization. The new methods of enhancing bone consolidation in DH are proposed and need further clinical studies. The novel applications of DH have now been extended for the treatment of vascular diseases, cranial defect (with neuronal disorders), hip and spinal deformity corrections and soft-tissue defects in addition to various bone defects and deformities. There are more surprises and novel mechanisms yet to be discovered for these novel applications of DH.

Locking plates have led to important changes in bone fracture management, allowing flexible biological fracture fixation based on the principle of an internal fixator. The technique of locking plate fixation differs fundamentally from conventional plating and has its indications and limitations. Most of the typical locking plate failure patterns are related to basic technical errors, such as under-sizing of the implant, too short working length, and imperfect application of locking screws. After analysis of the fracture morphology and intrinsic stability following fracture reduction, a meticulous preoperative planning is mandatory under consideration of the principles of the internal fixator technique to avoid technical errors and inaccuracies leading to early implant failure.

Musculoskeletal diseases are leading causes of disability, morbidity and economic loss across the globe today. Yet for much of the world's population access to cheap, safe and effective intervention is lacking, while others choose not to accept best practice and best evidence, or significantly more expensive treatment. Great advances have been made in some diseases like rheumatoid arthritis, but the cost of many new treatments is unaffordable, and individuals, insurance and governments struggle to, or cannot fund it. Anchor bias and politics determines national policies and research funding, often favouring other illnesses while musculoskeletal disorders lack the support proportional to their frequency and impact. This is not appreciated by policy makers and governments, and the consequences of lack of care or poor-quality care. The need has never been greater for a treatment for osteoarthritis, the most common disease in the world; but the search for a cure needs funding, and if discovered, who will pay for it?

The biological reaction in metallosis and pseudotumor generation after metal on metal total hip arthroplasty or corroding metal implants remains unsettled. Clinically, still lethal cases appear with massive bone loss and metal ions are suspected to be responsible for this inflammatory reaction, solid metal wear particles instead are usually not observed in the common literature. The aim of this study was to compare the biological reactions of metal ions and metal wear particles in a murine in vivo model. Metal ions (CoCr), metal particles (CoCr), polyethylene particles (UHMWPE) and phosphate buffered saline (PBS) were injected into the left knee joint of female BALB/c mice. 7 days after injection, the microcirculation was observed using intravital fluorescence microscopy, followed by euthanasia of the animals. After the assessment of the knee diameter, the knees underwent histological evaluations of the synovial layer. Throughout all recorded data, CoCr particles caused higher inflammatory reactions compared to metal ions and UHMWPE particles. The mice treated with the solid particles showed enlarged knee diameters, more intensive leukocyte–endothelial cell interactions and an elevated functional capillary density. Pseudotumor-like tissue formations in the synovial layer of the mice were only seen after the exposition to solid CoCr particles. Even if the focus of several national guidelines concerning metallosis and pseudotumor generation is on metal ions, the present data reveal that solid CoCr particles have the strongest inflammatory activity compared with metal ions and UHMWPE particles in vivo.

Mesenchymal stem cells (MSCs) have been long studied for their role in skeletal development. MSCs are unique in adult physiology in that they exhibit pluripotency and differentiate into cells that can evolve into various skeletal tissue as a result have been extensively employed as a viable alternative to terminally differentiated cells in engineering of cartilage and bone tissue ex vivo and in vivo. In spite of decades of effort in this direction, our understanding of what drives MSC fate choices is rather narrow in that it places heavy emphasis on a role for morphogens and cytokines (TGF-beta super family, FGF-2). In recent years it has become evident that MSCs also play an important role in wound healing, immunomodulation (immune suppression) and in tumour progression. However, what becomes of an MSC when it arrives at or exits an environment is less understood. We hypothesize that activation of differentiation programs in MSCs have an autocrine and paracrine component involving interplay between MSC-MSC (cell-cell contact) and MSC-(environment), and in this signalling paradigm the biophysical aspects of their microenvironment play a dominant role. We have tested this premise in several aspects of MSC behaviour (proliferation, migration, differentiation, chondrogenesis) and have gathered compelling evidence for biophysics and mechanobiology in MSC fate decisions. This talk will present some of our latest findings in this broad arena.

As stem cells and primary cells hold potential for improving disease outcomes and patient lives, methods for steering cell fate are of considerable importance. In this context, an emerging method is directing cell function through controlling cellular shape. The talk will discuss how cell functions are based on mechano-transduction events related to the balance of intra- and extracellular forces. The talk will explore the multiple biophysical cues that affect cell shape and present methods for directly generating cell shape, e. g. micro-contact printing used for directing the differentiation lineage of stem cells. Based on our own work, the talk will introduce the novel concept that specific biomaterial types and stiffnesses can be chosen for generating specific cellular “baseline shapes” and associated function. As our cells are exposed to continuously changing biomechanical forces, the talk will also report how specific forces can be used for engineering shape. The talk will explore how biomaterial stiffness and biomechanical forces act together on cellular shape, and whether one of the two stimuli is able to override the other. The novel insights reported here are fundamental for designing cell shape-instructive 3D biomaterials in the context of steering cell function in situ for regenerative medicine.

Cartilage-bone interactions play a critical role in joint diseases and the osteochondral junction has been identified as a locus of osteoarthritis development. However, it is challenging to study osteochondral (OC) interaction in vitro, since cartilage and bone require very different environments. We developed a new medium-to-high throughput osteochondral microphysiological system bioreactor to culture biphasic native or engineered constructs and that can be used to study any musculoskeletal tissue interfaces. We developed engineered constructs from hMSCs on a porous polymeric matrix with a gradient in pore size to assess the supportive effect of the local topology on cartilaginous and osseous differentiation. Furthermore, we developed a triphasic, vascualized osteochondral constructs based on porous polycaprolactone and methacrylated gelatin scaffolds to study the specific effects of vasculature on cartilage and bone. We also cultured native OC tissues from postmenopausal women, exposing either cartilage or bone to sex hormones studying their protective effects. Finally, our bioreactor is being implemented for use on the International Space Station to study countermeasures against microgravity bone loss. Overall, our bioreactor maintains media separation for in vitro culture and engineering of OC tissues and constructs of progressively greater complexity, and it preserves the possibility of direct cartilage-bone crosstalk opening new opportunities to study interactions across the osteochondral junction.

The successful application of smart implantable devices requires materials used to easily adapt and respond to their microenvironment via physical and chemical cues. Nanotopography, a known important factor in cellular processes (i.e. cellular adhesion, proliferation, and, differentiation), has become a central approach to imparting clinically relevant materials with bioactive and biomimetic properties. This work focuses on the use of Directed irradiation synthesis (DIS), to create nanostructures on dissimilar materials including surfaces of metals, semiconductors, and polymers. DIS is a novel method that allows for the tuning of both surface nanoscale topography and surface chemistry through the tailoring of ion beam parameters, including energy and fluence. The application of DIS to direct cellular interactions on Ti6Al4V, MgAZ31, and PEEK is presented. Topography and chemistry changes at the nanoscale were characterized by SEM, XPS, AFM, and Contact Angle. In vitro tests were performed using macrophages (JJ741A) and human aortic and bone marrow mesenchymal stem cell (MSCs). DIS promotes an advanced cell adhesion state where cells are orientated following the designed nanofeatures in all irradiated specimens. A delay on immune response due to low levels of TNFa and higher levels of IL10 on irradiated Ti6Al4V were observed. Modified PEEK showed 3-fold higher ALP content at 7 days compared to pristine samples, and porous MgAZ31 treated with DIS revealed lower corrosion state and increased cell proliferation of HBMMSCs. Controlling the nanopatterning in biomaterials using DIS enables the design of bioactive surfaces to highly promote implant integration and tissue regeneration.

Bioactive glasses were first discovered in the late 1960s by Larry Hench. In the 1980s and 1990s bioactive glasses experienced a surge of research interest, an interest which has since declined. This talk will examine the current status of bioactive glasses and discuss future roles and applications for bioactive glasses in regenerative medicine, specifically those related to orthopaedic tissue engineering. Bioactive materials are often considered as those that have the ability to bond to mineralised bone tissue in the physiological environment, however, this talk, as well as examining this aspect, will consider the broader sense of bioactive as ‘having or eliciting a biological effect’. It will examine the role of bioactive glasses as active drug carriers and the influence which enhanced nanotechnology will have on the application of bioactive glasses in vivo.

20 years ago, we designed injectable bioactive suspensions in water of calcium phosphate ceramics for bone and periapical regenerations. Because of leakage of these suspensions, we focused on injectable hydrogels before to set in situ by chemical crosslinking to form 3D scaffolds. We set up a platform to develop a series of innovative hydrogels for bone, cartilages and periodontal tissue regeneration. We based our strategy on polysaccharides macromolecules because they are renewable materials, that originate from biological sources and generally are biocompatible, non-toxic and biodegradable. We developed a family of silated macromolecules able to react forming biocompatible hydrogels. The silated polymers are self-setting hydrogels able to covalently crosslink under pH variation, without addition of toxic crosslinking agent. All these macromolecules could be combined in multicomponent hydrogels, representing a strategy for improving mechanical properties of biomaterials or to tailor particular properties to meet specific needs. For mineral scaffolding, we realized composites of calcium phosphates particles or cements with hydrogel, increasing the ductility and creating macroporous scaffold to propose foam bone cements well adapted to bone biomaterials and Bone tissue engineering. Perspectives are 3D printing and bio printing techniques. We will use our hydrogels platform to prepare tunable (bio)inks in skeletal medicine.

The AMPA/kainate glutamate receptor (GluR) antagonist NBQX reduced bone destruction when injected intra-articularly, in rat antigen induced arthritis (AIA) and is similarly protective in rodent models of osteoarthritis. NBQX reduced bone turnover in vivo and reduced mineralization in human primary osteoblasts (HOBs) in vitro. We are developing sustained release GluR antagonist delivery methods, to improve therapeutic effect. DNQX loaded Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were synthesized via double emulsion. DNQX loaded thermosetting hydrogels were synthesised by dissolving Pluronic-F127 (22% w/v) and Carbopol 934 (0.5% w/v) in dH₂O, homogenising with DNQX/NBQX and set in dialysis cassettes at 37˚C. Supernatants from nanoparticles and hydrogels suspended in PBS (37˚C) were analysed using high performance liquid chromatography to determine drug release. Y201 MSCs were differentiated to osteoblasts (DMEM+10% FBS, Dexamethasone, β-Glycerophosphate and Ascorbic acid-2-phosphate) in sustained presence/absence of NBQX (200µM) or DNQX (200 and 400µM). Alizarin red staining quantified mineralisation at 14 days. Nanoparticles encapsulated 2.5mM DNQX (encapsulation efficiency=22%) and released encapsulated drug over 4 weeks. Hydrogels released 2.5mM DNQX load over 24 hours in 37˚C PBS. Y201 alizarin red staining was significantly reduced by both DNQX (p<0.01) and NBQX (p<0.05), compared to untreated controls. PLGA nanoparticles and hydrogels revealed different sustained release profiles. Sustained treatment with GluR antagonists reduced mineralisation in Y201 derived osteoblasts, consistent with effects of NBQX in HOBs. Sustained release of NBQX and DNQX in nanoparticles and hydrogels may improve efficacy of AMPA/kainate GluR antagonists in reducing bone remodelling and enhancing their bone protective potential in the treatment of joint disease.

Skeletal sequels of traumatisms, diseases or surgery often lead to bone defects that fail to self-repair. Although the gold standard for bone reconstruction remains the autologous bone graft (ABG), it however exhibits some drawbacks and bone substitutes developed to replace ABG are still far for having its bone regeneration capacity. Herein, we aim to assess a new injectable allogeneic bone substitute (AlloBS) for bone reconstruction. Decellularized and viro-inactivated human femoral heads were crushed then sifted to obtain cortico-spongious powders (CSP). CSP were then partly demineralized and heated, resulting in AlloBS composed of particles consisting in a mineralized core surrounded by demineralized bone matrix, engulfed in a collagen I gelatin. Calvarial defects (5mm in diameter, n=6/condition) in syngeneic Lewis1A rats were filled with CSP, AlloBS±TBM (total bone marrow), BCP (biphasic calcium phosphate)±TBM or left unfilled (control). After 7 weeks, the mineral volume/total volume (MV/TV) ratios were measured by µCT and Movat's pentachrome staining were performed on undemineralized frontal sections. The MV/TV ratios in defects filled with CSP, AlloBS or BCP were equivalent, whereas the MV/TV ratio was higher in AlloBS+TBM compared to CSP, AlloBS or BCP (p<0.01; Mann-Whitney). Histological analyses exhibited a collagen-rich matrix in all the defects, and osteoid at the surface of all implanted biomaterials. Our data indicates that AlloBS is a promising candidate for bone reconstruction, with ease of manipulation, injectability and substantial osteogenic capacity. Further experiments in larger animal models are under consideration to assess whether AlloBS may be a relevant clinical alternative to ABG.

The hydrolysed collagen has a molecular weight of 3–6 KDa, is soluble in water, colourless and odourless. Hydrolysed collagen was obtained by proteolysis of the native ovine collagen. The enzymatic treatment was carried out with Heliozym under alkaline treatment (pH 8) for different periods of time (0 min, 10 min, 20 min, 30 min, 1 h, 2h, 3h y 4h) at 60°C. The hydroxyproline concentration increased significantly from time 0 min (11.44±2.81 mg/L) to the 4 h (24.47±1.60 mg/L); this change in concentration was observed in the FTIR spectra at a length of 1,037 cm-1 for OH group as well a change in the Amide I (1641 cm-1). The viscosity showed significant differences (P≤ 0.05) between the different hydrolysis times. This parameter was correlated with the molecular weight; when the viscosity was 0 cP the molecular weight showed the lowest value at 5.62 KDa. The antioxidant activity for ABTS radical scavenging showed significant differences (P≤ 0.05) between the times of hydrolysis, the greater the time, the higher the inhibition resulting with 67.61% at the end of the treatment. The DPPH radical scavenging resulted with 27.89 % at the beginning of the hydrolysis. However, the end of the hydrolysis (4h) showed inhibition at 52.75%. The antioxidant activity increased when molecular weight decreased, and this is related with the amino acids present in the peptides obtained for the hydrolysis of the collagen.

As near-infrared (NIR) photothermal agents, copper sulfide nanoparticles (CuSNP) offer several advantages over plasmonic gold nanoparticles (GNP), the most widely used photothermal nanotransducers in biomedical applications. CuSNP exhibit strong optical absorption at NIR wavelengths (650–1100 nm) and convert it into heat due excitation of electronic transitions or plasmonic photoexcitation. In contrast with GNP, CuSNP are degradable, readily prepared, inexpensive to produce, efficiently cleared from the body and their photothermal efficiency is less sensitive to the dielectric constant of the surrounding medium. We explored the feasibility of CuSNP to function as degradable NIR nanotransducers within fibrin-based cellular scaffolds, paying great attention to the stability and photothermal efficiency of the composite. We tested in vitro and in vivo whether NIR-responsive fibrin hydrogels comprising CuSNP (CuSNP hydrogels) are reliable platforms for triggering transgene expression in cells harboring a gene circuit activatable by heat and dependent of rapamycin. NIR laser irradiation of the CuSNP hydrogels increased local temperature and, in the presence of rapamycin, triggers the gene switch based on the promoter of the highly heat-inducible HSP70B gene (HSPA7). After implantation of such a cell-containing CuSNP hydrogel, transgenic expression can be remotely triggered by NIR-irradiation. Interestingly, we found that CuSNP hydrogels induce remodeling activity in stem cells and stimulate an angiogenic response. In short, CuSNP hydrogels offer compelling features for tissue engineering applications, as fully degradable implants with enhanced integration capacity in host tissues that can provide for remote control in the deployment of therapeutic gene products.

While the phenomena of bone adaption to mechanical loading has been long observed, the mechanisms governing bone mechanotransduction during health and disease are not well understood. Our multidisciplinary experimental and computational research strives to enhance understanding of bone mechanobiology, and in particular how this process is affected at the onset of osteoporosis. We have provided an enhanced understanding of bone cell mechanosensation. We have characterised the local mechanical environment of MSCs, osteoblasts and osteocytes in vivo. Most importantly, we have discovered that the matrix composition, expression of mechanosensors and the mechanical environment of osteocytes is altered during osteoporosis. Interestingly, a mechanobiological response restores the homeostatic mechanical environment of the cells in the longer term. Our recent in vitro studies have revealed that estrogen withdrawal from bone cells alters calcium signalling, mineralisation, biochemical responses and osteogenic gene expression when these cells are exposed to an applied fluid shear stress. Our ongoing research is investigating mechanobiology-based therapeutic approaches for treatment of bone pathologies, by (1) targeting mechanoregulatory signalling pathways and (2) developing in vitro tissue regeneration strategies that seek to optimise the mechanical environment (through matrix stiffness, bioreactors) to stimulate osteogenesis.

By combining cells, biological factors, and biomaterials the field of tissue engineering has generated technologies capable of supporting regeneration. However, the regulatory hurdles associated with the use of cell-based therapies often hinder translation. Consequently, to meet the growing demand for regenerative technologies new approaches are needed. Emerging evidence suggests that cell-derived extracellular vesicles (EVs) are critical in cell-cell communication and regulation of bone formation. This talk will explore the role of osteoblast EVs in directing stem-cell differentiation in-vitro. EVs were isolated from cell culture media by ultracentrifugation and profiled for size and composition using a range of techniques. Notably, proteomic analysis revealed the presence of calcium channelling annexins and bridging collagens that may be key to their role in mineralisation. To minimise the concentration of EVs required to induce a pro-osteogenic effect we propose that they may be locally delivered. Opportunities to incorporate these pro-osteogenic EVs into injectable biomaterials will be discussed, in particular the formulation of microcapsules and fluid-gels. In summary, incorporation of EVs in tissue-engineered scaffolds has the potential to deliver all the advantages of a cell-based therapy but without using viable cells. The advantages of this approach may represent a new phase of tissue engineering.

Nitrogen-containing bisphosphonates such as Zoledronic Acid (ZA) are used clinically for the treatment of skeletal diseases related with increased bone resorption. The gold standard is to administrate the drug through a systemic pathway, however this is often associated with high dosages, risk of side-effects, reduced site-specific drug delivery and hence, limited drug-effectiveness. A controlled local drug delivery, via a biomimetic bone graft, could be beneficial by direct and time-regulated application of significantly lower drug dosage at the site of interest. Thus, higher efficacy and reduced side-effects could be expected. In this experimental in vivo study, we examined the effect of ZA when used together with a Calcium Sulphate/Hydroxyapatite biomaterial in a femoral condyle bone defect in rats and compared local to systemic administration. The following groups were used: group1: empty defect (no biomaterial & no treatment), group2: biomaterial alone, group3: biomaterial + systemic ZA (0.1mg ZA/kg – single subcutaneous injection), group4–6: biomaterial conjugated with ZA at different concentrations, (0.07 to 0.70 mg ZA/mL of paste, corresponding to 0.0024 to 0.024 mg ZA/kg). The animals were sacrificed at 6 weeks and toxicological examination was performed. Bone regeneration was evaluated using qualitative and quantitative micro-CT analysis and Histomorphometry. The results showed a significant difference between the groups, suggesting that ZA has an overall effect on bone healing. The most pronounced effect was seen with the local application of approximately 10 times less ZA-dosage when compared to systemic use (p<0.001). This study demonstrates the importance of local ZA administration in bone regeneration.

Osteoporosis has long been associated with weak bones but recent studies have shown that bone tissue mineral becomes more heterogeneous and the expression of mechanosensors are altered during estrogen deficiency in an animal model of osteoporosis. However, whether these changes occur as a primary response to estrogen deficiency is unknown. In this study we investigate whether matrix production and mineralisation by mechanically-stimulated osteoblasts are impaired as a direct consequence of estrogen depletion. Osteoblast-like MC3T3-E1 cells were cultured for 14 days with 10⁻⁸M of 17β-estradiol and subsequently cultured with osteogenic media only, or supplemented with estrogen or an estrogen antagonist (Fulvestrant, 10⁻⁷M). Physiological shear stress (1Pa) was applied using an orbital shaker (290rpm, 40min/day), which allows long-term culture and induces oscillatory flow on cells. Osteoblasts phenotype, extracellular matrix (ECM), mineralisation and mechanosensors were tracked by qRT-PCR (Runx2, Col1a1, Col1a2, Cox2, Bglap2, FN1), by biochemical assays (ALP activity, DNA and calcium content), by immunostaining (integrin α_v, BSP2, fibronectin) and by labelling with calcein the calcium. The results of this study demonstrate that after 7 days, estrogen depleted cells had less integrin α_v mechanosensors compared to those that received continuous estrogen treatment. By 14 days the ECM formation (calcium, fibronectin) by osteoblasts was altered under estrogen depletion, when compared to cells that were cultured continuously with estrogen. This study provides evidence of changes in osteoblast behaviour under estrogen depletion, which might explain the alteration in tissue mineral content and the decrease of integrins observed previously in ovariectomized rats in vivo.

Osteocytes are terminally differentiated long-lived cells and account for greater than 95% of the bone cell population. It has been established that osteocytes are connected through their highly developed dendritic network, which is necessary for the maintenance of optimal bone homeostasis. However, little is known on how osteocytes use the network to coordinate their cellular function and communication that requires energy and protein turnover. Here using super-resolution confocal imaging on both live and fixed osteocytes, we demonstrated conclusively that mitochondria are widely distributed and dynamically shared between osteocytes. Using confocal live cell imaging analysis we showed that inhibiting the contact between mitochondria and endoplasmic reticulum (ER) by the knockdown of MFN2 in osteocytes impedes the transfer of mitochondria suggesting the involvement of ER contact with mitochondria in the transfer process. Moreover, we showed that transport of mitochondria between osteocytes within the network enables rescue of osteocytes with dysfunction of mitochondria. Using the 3D tetraculture system with confocal imaging, we identify the transfer of mitochondria from healthy osteocytes enables recovery of mitochondria activities in osteocytes that devoid of mitochondrial DNA by ethidium bromide. The results indicated that when osteocytes are depleted of functional mitochondria, normal parental osteocytes can transfer mitochondria to these stressed osteocytes to provide them with energy. Collectively we show for the first time that the utilisation of mitochondrial transfer enables osteocytes to function with a network and coordinate their cellular activities in response to different energy demands.

To prevent bone loss, OPG/RANK/RANKL signalling pathway is a key in keeping the balance between the action of osteoblasts and osteoclasts. Aim of this study is to assess the influence of long-term nicotine exposure on bone mineral density (BMD) scores, RANKL and OPG levels of plasma and RANKL and OPG immunoreactivities of tissue in rats. In this study, totally 36 Swiss Albino rats (70±10 g) were used in three groups. Whereas normal drinking water was given for the control group (n:12), 0.4 mg/kg/day and 6.0 mg/kg/day nicotine was added to drinking water for low-dose nicotine (LDN) group (n:12) and high-dose nicotine (HDN) group (n:12), respectively for 12 months. At the end of 12^th month, BMD scores were measured via X-ray absorptiometry and then bone turnover was assessed via measuring both RANKL, OPG levels in plasma and RANKL, OPG immunoreactivities in tail vertebrae of all rats. Lumbar spine and femoral regions BMD scores of the control group and the nicotine groups were not significantly difference. In HDN group, OPG levels of plasma were found significantly higher when compared with the control and LDN groups (p=0.001) unlike RANKL levels of plasma. RANKL and OPG immunoreactivities of tissue were found significantly lower in both LDN and HDN groups (p<0.001, p=0.004, respectively) in comparison to control group. No correlation was found between plasma levels and tissue immunoreactivities of RANKL and OPG. As a result, this study indicates that nicotine is not primarily responsible for the decline of BMD frequently seen in smokers.

Osteonecrosis is a potentially devastating condition with poorly defined pathogenesis that can affect several anatomical areas with or without a previous traumatic insult. Post traumatic osteonecrosis (PON) in the foot and ankle has been commonly described in the talus and navicular but rarely in the distal tibia. PON of the distal tibia is a rarely reported and infrequent complication of fracture dislocations of the ankle. Its scarcity can lead to misdiagnosis and inappropriate management due to a lack of clinical knowledge or suspicion with resultant severe functional compromise. We aim to highlight the clinical and radiological features of PON of the distal tibia and report the findings in a series of four patients following a fracture dislocation of the ankle. Three patients sustained a SER4 fracture dislocation and one patient sustained a PER4 fracture dislocation in keeping with standard patterns of injury seen in most trauma units. In each case, PON of the distal tibia presented with progressive anterolateral tibial plafond collapse and valgus deformity of the ankle. The radiological features previously reported in the literature are based on plain film x-ray, CT and MRI but no description of SPECT-CT findings. One of the patients in the series underwent SPECT-CT following clinical suspicion of PON and thus we describe the findings not previously reported. Our objective is to highlight this rare condition as a potential cause for ongoing pain following fracture dislocation of the ankle as well as advocating the use of SPECT/CT as a useful imaging modality to aid in the diagnosis.

It is well documented that implant loosening rate in sickle cell disease patients is higher than that seen in patients with hip arthroplasty from other indications. The Hypoxic inducible factor(HIF) - is activated in the microcellular hypoxic environment and this through a cascade of other enzymatic reactions promotes the activity of other factors and further help enhance angiogenesis and osteogenesis. The aim of this study was to investigate and propose a potential model for investigating osseointegration in a hypoxic microcellular environment using osteoblasts(MG63).

Human MG63 osteoblastic cells were cultured under normoxia and hypoxic conditions (20%; and 1% oxygen saturation) for 72 hours under two different condition- with and without cobalt chloride. The samples cultured under normoxic condtions without cobalt chloride acted as control. Using qualitative polymerase chain reaction-(qPCR) - HIF expression was assessed under the above conditions in relation to the control.

The results showed there was significant expression of the HIF 1 alpha protein under hypoxic condition with cobalt chloride in comparison with the control samples- all at 72hours incubation. Mann-Whitney U test was used to deduce level of significance of fold change.(p=0.002; <0.05). This was deemed as being a significant difference in the level of expression of HIF compared to the control.

The results show that the hypoxic inducible factor can be expressed using the above tested

experimental invitro-model with significant results which can be a foundation for further research into improving hip implant prosthesis design to help enhance osseo-integration in sickle cell disease patient with AVN.

Side-effects associated to the use of bone morphogenetic proteins into scaffold-based devices for bone repair highlight the necessity for identifying new therapeutic targets that potentially improve bone healing in adults. In this sense, we recently demonstrated the age-associated decrease in the mechanosensitivity of bone marrow mesenchymal stromal/stem cells (MSCs) and identified c-Jun N-terminal kinase 3 (JNK3) as a mechanically-activated modulator of the superior osteogenic potential of MSCs derived from children (C-MSCs) in comparison to adults (A-MSCs). Building on this work, the aim of this study was to design a JNK3-activated collagen-nanohydroxyapatite (coll-nHA) scaffold that restore the child bone healing capacity in adults. Results revealed that JNK3 activator (JNK3*) enhanced A-MSC’ alkaline phosphatase (ALP) activity to the same extent of C-MSCs by facilitating the activation of JNK3. Moreover, A-MSCs cultured on the coll-JNK3* scaffold (collagen-scaffold containing JNK3*) showed positive uptake of the JNK3*, upregulation of early osteogenic markers as well as increased ALP activity and mineralization. More importantly, rat critical calvarial defects treated with coll-JNK3* for 28 days showed a significantly higher 18.07 % bone volume fraction in comparison to rats treated with Coll-nHA −6.04%- and empty defects −2.58%. Which correlated with the presence of a larger amount of blood vessels and mineralized tissue in samples treated with coll-JNK3* when compared with coll-nHA and empty defects. In conclusion, the coll-JNK3* capacity to enhance osteogenesis and bone healing by activating JNK3 highlights how by understanding the stem cell mechanobiology we can improve the development of next generation therapeutics for tissue repair.

Adipose derived mesenchymal stromal cells (ASC) are adult stem cells exhibiting functional properties that have open the way for cell-based clinical therapies. Primarily, their capacity of multilineage differentiation has been explored in a number of strategies for skeletal tissue regeneration. More recently, MSCs have been reported to exhibit immunosuppressive as well as healing capacities, to improve angiogenesis and prevent apoptosis or fibrosis through the secretion of paracrine mediators. Among the degenerative diseases associated with aging, osteoarthritis is the most common pathology and affects 16% of the female population over 65 years. Up to now, no therapeutic option exists to obtain a sustainable improvement of joint function beside knee arthroplasty. This prompted us to propose adipose derived stem cells as a possible cell therapy. We performed pre-clinical models of osteoarthritis and showed that a local injection of ASC showed a reduction of synovitis, reduction of osteophytes, joint stabilization, reducing the score of cartilage lesions. This work was completed by toxicology data showing the excellent tolerance of the local injection of ADSC and biodistribution showing the persistence of cells after 6 months in murine models. The aim of the ADIPOA trial is to demonstrate the efficacy of adipose derived stem cells therapy in knee osteoarthritis (OA) in a phase 2/3 controlled multicenter study controlled against standard of care. Safety and feasibility as well as dose response was previously assessed in the ADIPOA FP7 project. The bi-centric phase I clinical trial in Montpellier (France) and Würzburg (Germany) included 18 patients with moderate to severe knee OA, each patient received a single injection of autologous ADSC, in a open scale up dose trial, starting form 2 10 6 cells to 50 106 cells. The 107 dose appears to be well tolerated and showed preliminary response in terms of decreasing local inflammation. This first study confirmed the feasibility and safety of local injection of ADSC in knee OA and suggested the most effective dose (107 autologous ADSC). This work constituted a significant step forward treating this disease with ADSC to demonstrate safety of the procedure.

we conduct a prospective multicenter randomized Phase 2/3 study with 86 patients with moderate to severe knee OA to demonstrate superiority of stem cell-based therapy compared to standard of care (SOC) in terms in reduction in clinical symptoms (WOMAC score) and structural benefit (assessed by T1rhoMRI that allow quantification of cartilage proteoglycan content). This project will offer EU a unique leadership in OA with strong positions in EU and US due to patents and quality of the methodology to demonstrate efficiency of ADSC. ADIPOA brings together a unique combination of expertises and leaders in clinical rheumatology, MRI specialists, Stem cell Institutes, national GMP grade adipose derived stem cell production platform (ECELLFRANCE) and SME specialized in cell therapy trials in the EU. The production of the cells will be granted to EFS through ECELLFRANCE national platform, which have the GMP facility and will work as a contracting manufacturing organization. The expertise, leadership and critical mass achieved by this Consortium should enable breakthroughs in ASC engineering directly amenable for clinical applications in OA.

Among all stem cell based clinical trials in the world, most of them are related to Mesenchymal Stem Cells whatever the tissue origin. Over time, the uses of cultured cells have increased greatly, particularly since 2009. Cells derived from adipose tissue are also increasingly used in trials compared with bone marrow cells. No real specificity emerged as to the therapeutic uses of the different types of stem cells and the more than half the MSC studies concerned allogeneic MSCs. With the maturation of this field, the requirements of relevant safety and potency cell control assays are now absolutely required for the future phase III and IV but quite different according to the autologous or allogenic setting. If for autologous setting, such assays have to be defined to identify MSC batches not to inject (for safety or lack of efficacy), in allogenic setting, potency assays are required to select the best donor with the maximum of safety. Up to now, most of assays are based on pre-clinical animal studies but need to be largely improved for a better relevance and accuracy. Their development stumbles on two difficulties: MSC themselves and our limited knowledge of their pleiotropic action mechanisms in conjunction with MTI regulatory rules. This indicates that we have to move from simple tests to multi-modal and combinatory approaches. We propose to discuss and illustrate these different points in view of the different clinical trials and how they inform us.

Synovitis has been shown to play a role in pathophysiology of OA promoting cartilage destruction and pain. Synovium is mainly composed of synovial fibroblast (SF) and macrophage (SM) that guide synovial inflammation. Adipose stromal cells (ASC) promising candidate for cell therapy in OA are able to counteract inflammation. Two different subsets of macrophages have been described showing a pro-inflammatory (M1) and an anti-inflammatory (M2) phenotype. Macrophage markers: CD68, CD80 (M1-like) and CD206 (M2-like) were evaluated in osteoarthritic synovial tissue. GMP-clinical grade ASC were isolated from subcutaneous adipose tissue and M1-macrophages were differentiated from CD14+ obtained from peripheral blood of healthy donors. ASC were co-cultured in direct and indirect contact with activated (GM-CSF+IFNγ)-M1 macrophages for 48h. At the end of this co-culture we analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassay. PGE2 blocking experiments were performed. In moderate grade OA synovium we found similar percentages of CD80 and CD206. M1-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both co-culture conditions. Moreover, ASC induced the typical M2 macrophage markers IL10, CD163 and CD206. Blocking experiments showed that TNFα, IL6, IL10, CD163 and CD206 were significantly modulated by PGE2. We confirmed the involvement of PGE2/COX2 also in CD14+ OA synovial macrophages. In conclusion we demonstrated that ASC are responsible for the switching of activated-M1-like to a M2-like anti-inflammatory phenotype, mainly through PGE2. This suggested a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

Mesenchymal stem cells (MSC) are multipotent cells that possess regenerative functions that are of interest for in osteoarticular diseases such as osteoarthritis (OA). These functions are thought to be primarily mediated by mediators released within extracellular vesicles (EV). The aim of this study was to compare the immunomodulatory effects of two major types of EV, exosomes and microparticles, secreted by MSCs. EV subsets were isolated from murine primary MSCs by ultracentrifugation. Size and structure were evaluated by Dynamic Light Scattering and electron microscopy. Expression of membrane and endosomal markers was tested by flow cytometry. Proliferation of murine splenocytes was quantified after 72h of incubation with EVs after CFSE-labelling. Phenotypic analysis of T lymphocyte subpopulations was also performed by flow cytometry. In vivo, EVs were injected in the knee joint in the collagenase-induced osteoarthritis (CIOA) model and histological score was performed. In vitro functional analysis indicated that addition of microparticles or exosomes in proliferative assays inhibited the proliferation of total splenocytes in a dose-dependent manner. Analysis of T cell subpopulations revealed a decrease in CD8⁺IFNγ⁺ lymphocytes and an increase in both CD4⁺IL10⁺ Tr1 and CD4⁺CD25⁺FOXP3⁺ Treg cells. This immunomodulatory function of EVs was also observed in vivo in the CIOA model. In summary, our data indicated that the immunosuppressive effect of MSCs is in part mediated by exosomes and microparticles that play in vivo a major role in MSC-mediated therapeutic effect by reducing osteoarthritic symptoms.

Bioactive functional scaffolds are essential for support of cell-based strategies to improve bone regeneration. Adipose-tissue-derived-stromal-cells (ASC) are more accessible multipotent cells with faster proliferation than bone-marrow-derived-stromal-cells (BMSC) having potential to replace BMSC for therapeutic stimulation of bone-defect healing. Their osteogenic potential is, however lower compared to BMSC, a deficit that may be overcome in growth factor-rich orthotopic bone defects with enhanced bone-conductive scaffolds. Objective of this study was to compare the therapeutic potency of human ASC and BMSC for bone regeneration on a novel nanoparticulate β-TCP/collagen-carrier (β-TNC). Cytotoxicity of β-TCP nanoparticles and multilineage differentiation of cells were characterized in vitro. Cell-seeded β-TNC versus cell-free controls were implanted into 4 mm calvarial bone-defects in immunodeficient mice and bone healing was quantified by µCT at 4 and 8 weeks. Tissue-quality and cell-origin were assessed by histology. β-TNC was non-toxic, radiolucent and biocompatible, lent excellent support for human cell persistence and allowed formation of human bone tissue by BMSC but not ASC. Opposite to BMSC, ASC-grafting significantly inhibited calvarial bone healing compared to controls. Bone formation progressed significantly from 4 to 8 weeks only in BMSC and controls yielding 5.6-fold more mineralized tissue in BMSC versus ASC-treated defects. Conclusively, β-TNC was simple to generate, biocompatible, osteoconductive, and stimulated osteogenicity of BMSC to enhance calvarial defect healing while ASC had negative effects. Thus, an orthotopic environment and β-TNC could not compensate for cell-autonomous deficits of ASC which should systematically be considered when choosing the right cell source for tissue engineering-based stimulation of bone regeneration.

Mesenchymal stem/stromal cells (MSC) have the ability to home and migrate towards injured and inflamed tissues which can be useful as a minimally invasive systemic approach to deliver MSC to the site of damaged articular surface in arthritis in human and veterinary patients. From a molecular point of view, the CXCR4/SDF-1 plays an important role in this phenomenon and can be used as a target to enhance the therapeutic efficacy of culture expanded MSC. It has been demonstrated that extensive in vitro expansion down-regulates CXCR4 expression in human, murine and canine MSCs hindering their therapeutic efficacy. Therefore, the aim of the present study was to assess the effect of hypoxia and basic fibroblast growth factor (bFGF) pre-conditioning on CXCR4 and SDF-1 expression in canine adipose derived MSC (cAT-MSC). MSC were isolated from subcutaneous adipose tissue of two adult Beagle dogs (n=2; 3–5 years old, 9–12kg) and cultured under standard conditions (5%CO₂, 37°C). Cells at passage 3 were then cultured in hypoxia (2%O₂) and normoxia, with supplementation of 1 and 5 ng/ml bFGF for 24h. MTT assay, flow cytometry, immunohistochemistry and qRT-PCR analysis were conducted to assess respectively the modulation effect on cell proliferation, CXCR4 protein expression and CXCR4 and SDF-1 gene expression. Cell proliferation increased proportionally with the increasing bFGF concentrations, with a statistically significant higher proliferative rate in normoxic conditions (p<0.05). The gene expression of CXCR4 and SDF-1 increased in hypoxic conditions with bFGF supplementation (p<0.05). bFGF supplementation increased cytoplasmatic expression of CXCR4 in hypoxic conditions (p<0.05), however the surface expression remained low in all culture conditions. The described pre-conditioning method can be used for the enhancement of the therapeutic potential of systemically administered canine AT-MSC and can have a relevant translational character for the optimization of culturing protocols of human adipose derived MSC.

Bone formation proceeds through two distinct processes. One involves the deposition of bone by osteoblasts (intramembranous ossification) and another through the remodeling of an intermediate cartilaginous matrix formed by chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) aggregates – a process called endochondral ossification (EO). EO is responsible for formation of long bones during development and most prevalent during facture repair upon callus formation. In adult bone injuries MSCs from periosteum form bone via EO whereas MSCs from bone marrow are primarily differentiate to osteoblast in vivo. We hypothesized that the unique biophysical and biochemical properties of bone mineral phase has a role in programming MSCs. Using a biomimetic bone like apatite (BBHAp) as surrogate for bone mineral phase, we studied the chondrogenic differentiation of human marrow derived MSCs and observed that the BBHAp dictates MSCs fate and strictly dictates the pathway of bone formation in vivo. Through exhaustive dissection of the signaling pathways at play, a prominent role of PTH1R in modulating the effects imposed by the BBHAp has been unraveled. These fundamental insights gained in how bone microenvironment might alter fate of MSCs has important implications for bone repair and regeneration therapies.

Cell-based therapies have taken the emerging field in many clinical directions. Among them, orthopaedic surgery is one of the most promising directions – due to the clinical needs, and because of the availability of the advanced cell-based constructs dedicated to bone and cartilage regeneration. The current practical clinical input is, however, below expectations – because of numerous difficulties which have their source in scientific, practical, finance and legal issues. Regarding legal issues, Advanced Therapy Investigational Medicinal Products (ATIMP) are regulated by three different legal orders. As medicines (according to the EU law, ATIMP is a pharmaceutical) – they are subject to pharmaceutical law; as cell-containing specimens – to cell and tissue banking regulations; as tested by registered clinical trials - they are subject to Good Clinical Practice rules and regulations. Formal requirements coming from these three areas are completely different, sometimes contradictory and incompatible with the specific nature of cell-based products. At the same time they involves the need for huge financial expenditures. We discuss these issues from the perspective of the university laboratory, which currently conducts clinical trials of the ATIMPs for three different clinical indications and, at the same time, has experience in the basic and applied scientific work at the laboratory level – towards improvement of osteogenic capacity of stem cells. With the undoubtful need of well documented scientific results, which is accompanied by complicated and imperfect regulations, we think that the scientific community focused around cellular therapies is now facing challenges that may determine the future of this field.

Osteoarthritis (OA) is an inflammatory degenerative disease that affects every fourth person with irreversible damage to the articular. Mesenchymal stem cells (MSCs) have been shown to affect host cells by paracrine stimulation in regenerative environments. Here we apply hyaluronic acid (HA), an essential part of the extracellular matrix in cartilage, for MSC immobilization. The aim was to investigate long-term MSC survival and paracrine effect on chondrocytes in an inflammatory co-culture environment. We hypothesized that MSCs immobilized in a HA hydrogel could provide a long-term immunomodulatory effect on chondrocytes in vitro. Human MSCs were seeded in a HA hydrogel and co-cultured with non-osteoarthritic human chondrocytes in biphasic wells inhibiting cellular contact. An inflammatory environment was induced by IL1-beta and compared with standard culture medium. Relative gene expressions of collagen types I, II and X, aggrecan, SOX9, MMP-13 and ADAMTS-5) were examined at day 3,7,14 and 28. Significant up-regulation of SOX9 at day 7, 14 and 28 and a significant down-regulation of ADAMTS-5 (day 14 and 28) was observed with co-culture of HA-immobilized MSCs and MSCs compared with controls with or without HA (without MSCs)No changes in expression was observed for aggrecan and collagen type 1. We showed that MSC affect the expression of SOX9 and ADAMTS-5 in a paracrine manner when co-cultured with chondrocytes in an inflammatory environment. MSCs immobilized in HA hydrogels survived and were contained in the hydrogel for up to 28 days. This suggests that HA-immobilized MSCs could potentially be used as adjuvant treatment of OA.

Cartilage lesions occur as a result of joint trauma, and progressively degenerate over time leading to osteoarthritis (OA). Early intervention therapies to repair the initial tissue damage have the potential to delay or prevent the onset of OA. We have developed two acellular treatments; 1) an injectable proteoglycan-like self-assembling hydrogel for the repair of ICRS grade 1 lesions, and 2) a decellularised xenogeneic osteochondral scaffold for surgical grafting in grade 2–4 lesions. We produced an in vitro glycosaminoglycan depleted grade 1 lesion model using porcine cartilage. Peptide-chondroitin sulphate mixture was injected and spontaneously gelled in situ. Cartilage resistance to deformation was increased by 50 %. Decellularised porcine osteochondral scaffolds which maintain the native tissue composition and architecture whilst being immunocompatible were successfully developed and are currently undergoing in vivo assessment in an ovine critical size condylar defect model. Incorporation of the subchondral bone in osteochondral scaffolds is intended to improve osseointegration; implanted decellularised bone-only scaffolds in sheep exhibited superb osteoinductive and osteoconductive properties in a proof-of-concept study. We envisage that our early intervention therapies will be employed clinically to maintain or restore functional hyaline-like cartilage across the whole range of early chondral pathologies and prevent the onset of OA.

In recent years, novel therapies for intervertebral disc (IVD) regeneration have been developed that are based on the delivery of cells, biomaterials or bioactive molecules. The efficacy of these biological therapies depends on the type and degree of IVD degeneration. Whole organ culture bioreactors provide an attractive platform for pre-clinical testing of IVD therapeutics, since the cells are maintained within their native extracellular matrix, and the endplate remains intact to fulfil its function. Moreover, defined regimes of mechanical stress are applied to the IVD, representing either physiological or degenerative, detrimental loading. Different degrees of degeneration can be induced by high load, low nutrition, enzyme injection, and/or mechanical damage; while recent organ culture models also implement an inflammatory component. Using whole organ culture models, we found that mesenchymal stem cell injection into nucleotomized IVDs had an anabolic effect on the IVD cells. Furthermore, hyaluronan hydrogels were beneficial for cell delivery and mechanical support. We also found that anti-inflammatory treatment could partially prevent the induction of cytokines in an inflammatory model. However, chemokine delivery did not induce a significant repair response in an annulus fibrosus defect. In line with 3R principles, relevant ex-vivo models are essential to reliably test biological IVD treatments.

Articular cartilage repair is assumed to improve by covering the cartilage lesion with a biomaterial scaffold tailored to the specific requirements of the weight-bearing joint surface. We have tested the feasibility of a novel composite collagen-polylactide scaffold rhCo-PLA in cartilage repair. To confirm these results and further challenge the scaffold, we tested it in a large porcine cartilage defect. A critical-sized full-thickness chondral defect was made in the medial femoral condyle of 18 domestic pigs. This technically widest possible defect size of 11×17 mm was determined in a pilot test. Five weeks later, the defect was either treated with the novel rhCo-PLA scaffold or left untreated to heal spontaneously. After four months, the medial condyles were evaluated macroscopically using Goebel's score, in which the worst possible result receives a total of 20 points and imaged with µCT to evaluate subchondral bone. Macroscopic score and subchondral bone microstructure were similar in both study groups. The total Goebel score was higher in spontaneous group (9.75±3.9 for spontaneous and 9.1±3.7 for rhCo-PLA, respectively) but differences between individual animals were large. Subchondral bone volume fraction was 48.2±3.6% for rhCo-PLA and 44.2±3.4% for spontaneous. Trabecular thickness was greater in operated joints (207.9±18.8 µm for spontaneous and 242.9±32.9 µm for rhCo-PLA) than in contralateral non-operated joints (193.3±15.1 µm and 213.4±33.2 µm, respectively). These preliminary data demonstrate that individual differences in the macroscopic appearance were large but there were no significant differences between the two study groups in the score or subchondral bone structure.

Intervertebral disc (IVD) degeneration presents a harsh microenvironment characterised by low glucose, low oxygen and matrix acidity posing a significant challenge for cell-based therapies. The objective of this work was to assess the effect of primed bone marrow derived stem cells (BMSC) and articular chondrocytes (AC) in different pH (7.1, 6.8 and 6.5) conditions and assess metabolic activity in terms of oxygen (O₂) and glucose consumption as well as lactate production. Secondly, we investigated pH effects on cell viability and matrix accumulation capacity. Primary cells were encapsulated in alginate beads and cultured in disc-like conditions (5% O₂, 5mM glucose, pH 7.1, 6.8 and 6.5). For growth factor priming, cells were cultured with 10ng/ml TGF-β3 at a pH of 7.4 for 14 days prior to being subjected to acidic pH conditions. AC exhibited superior cell viability and sGAG deposition compared to BMSC at all pH levels which was further enhanced after priming. Priming also reduced O₂ consumption of AC for all pH conditions while lactate production profiles of both cell types were altered with decreasing extracellular pH. This work demonstrates the importance of cell type selection to sustain disc-like microenvironmental conditions. Results show that BMSCs that have not been primed may need additional factors to sustain the harsh acidic microenvironment. In contrast, AC were capable of sustaining the low pH conditions better than BMSC and accumulated more similar disc-like matrix in all conditions. Overall this study highlights that AC may be advantageous for disc regeneration and warrant further investigation for disc repair.

Osteophyte deposition and subchondral bone damage are notable features of osteoarthritis (OA). Deregulated mineralization contributes to osteophyte and subchondral irregularity. The microRNA-29 (miR-29) family is associated with arthritic disorders. This study is aimed to investigate miR-29a function to OA osteophyte formation and subchondral integrity. Intact and damaged articular cartilage in patients with end-stage knee OA who required total knee arthroplasty were harvested to probe miR-29a, cartilage, and mineralized matrix expression using RT-PCR and in situ hybridization. Osteophyte volume and subchondral morphometry of collagenase-induced OA knees in mice were quantified using μCT and histomorphometry. Increased bone matrix expression (collagen I and bone alkaline phosphatase) and reduced cartilage matrix (collagen II and aggrecan) along with low miR-29a expression existed in human OA specimens. Aged miR-29a knockout mice showed spontaneous osteophyte formation and articular cartilage erosion. In primary articular chondrocytes, miR-29a deficiency significantly reduced cartilage matrix synthesis, whereas von Kossa staining-positive mineralized matrix production was increased. Of interest, the severity of collagenase-induced osteophyte accumulation and subchondral damage along with serum cartilage breakdown products CTX-II and COMP levels were significantly compromised in mice overexpressing miR-29a. Intra-articularly injecting miR-29a significantly reduced osteophyte volume and subchondral integrity and retained cartilage morphology in collagenase-injured knees. Reduced miR-29a signalling worsens osteophyte and subchondral destruction in OA through increasing mineralized matrix formation of chondrocytes. Restoring miR-29a shields joints from cartilage degradation, osteophyte and subchondral destruction. This study conveys new mechanistic underlying OA osteophyte pathogenesis and shines light on the remedial potential of miR-29a to OA.

Discogenic low back pain affects 42% of patients suffering low back pain. Degenerative disc disease is described as failure in cellular response to external stresses leading to physiologic dysfunction. Glycosylation patterns of tissues give insights into the spatially and temporally regulated inflammatory and degenerative processes. These glycoconjugates participate in many key biological processes including molecular trafficking and clearance, receptor activation, signal transduction, and immunomodulation. We hypothesise that glycoprofile of the the intervertebral disc(IVD) is temporally and spatially distinct in health and degeneration. The glycoprofile of the IVD has been studied in murine, bovine and ovine models for injury and aging. In this study, healthy(n=2) and degenerated(n=2) human IVD samples received from Utrecht(UU, ND) with ethical approval(NUIG), were compared using lectin histochemistry. The N-glycan profile of human degenerated IVD samples was characterised by high resolution quantitative UPLC-MS. Healthy and degenerated human discs present distinct glycosylation trends intracellularly/extracellularly in annulus fibrosus(AF) and nucleus pulposus(NP) tissue. There are quantitative and spatial differences in glycosylation in healthy and degenerated tissue. These findings are consistent with previous studies of IVD in murine, bovine and ovine models. The human N-glycan profile of degenerated surgical tissues is distinct from other cited tissue profiles such as human plasma⁵. These studies offer validation of previous animal models of IVD injury and degeneration, demonstrating similar changes in the glycoprofile in both animals and humans. Glycoprofiling may offer insight into disease progression, offering new realms of disease classification in patient specific manner while also elucidating potentials therapeutic targets, inhibiting disease progression.

The IVD is a highly hydrated, hyperosmolar tissue that allows the correct biomechanical function of the spine. When degenerated, water and ions are lost from the disc, especially within the central nucleus pulposus (NP), producing a hypoosmotic environment in which the resident cells can no longer function correctly, exacerbating the degenerative cascade. One potential way that IVD cells may adapt to their environment is through the expression and regulation of aquaporin (AQP) channels that control the movement of water in and out of cells. During human IVD degeneration AQP1 and 5 expression is decreased, highlighting AQPs may be of importance for the correct function of NP cells. The regulation of AQPs in NP cells by healthy and degenerate conditions, and the potential underlying molecular mechanisms, were investigated in both human and rat IVD cells. The gene and protein expression of AQP1 and AQP5 was upregulated by hyperosmotic conditions (425mOsm/kg H₂O) in rat and human NP cells. Lentiviral knockdown of tonicity enhancer binding protein (TonEBP), a transcription factor responsible for maintaining the function of NP cells, resulted in the loss of AQP1 and 5 gene expression under hyperosmotic conditions. The maintenance of the IVD environment and adaptation of cells is vital for the function of the IVD. The regulation of AQPs by physiological conditions and TonEBP suggests a role for these water channels related to the adaptation of disc cells to their environment, which is dysregulated during degeneration.

The degeneration of the intervertebral disc (IVD) is the primary cause for low back pain, which is treated with surgical interventions such as spinal fusion. A strategy to develop a regenerative and non-invasive treatment requires an injectable cell carrier system. Our efforts have focussed on developing a hyaluronan (HA)-based hydrogel system that can be used as a carrier for therapeutic agents in annulus fibrosus (AF) repair. High molecular weight HA at 20mM is chemically crosslinked with varying concentrations of 4-arm PEG. Hydrogels were optimised for degree of crosslinking, stability and rheological properties. Subsequently, the morphology and viability of the human AF cells encapsulated in the hydrogels were studied. The highest crosslinking was seen with 4-arm PEG at 1:1 HA:PEG molar ratio. This was the most stable against enzymatic and hydrolytic degradation, and had greater swelling property, which is desired as the degeneration decreases the water retention capability of the IVD. The gelation time, important for in situ injectability, was under five minutes for all formulations. Storage modulus was between 0.4–1.1 kPa. Compared to 2D cultures, cells were rounder after encapsulation, mimicking the native microenvironment, and had the similar metabolic activity for seven days. AF cells encapsulated in HA/4-arm PEG hydrogel were stiffer compared to the nucleus pulposus (NP) cells encapsulated similarly as measured with Brillouin microscopy. The 4-arm PEG crosslinked HA-based hydrogel system promises to be a candidate for an injectable carrier for cells for AF repair and regeneration.

Phenotypic drift of stem cells and insufficient production of extracellular matrix (ECM) are frequently observed in tissue-engineered cartilage substitutes, posing major weaknesses of clinically relevant therapies targeting cartilage repair. Microenvironment plays an important role for stem cell maintenance and differentiation and therefore an optimal chondrogenic differentiation protocol is highly desirable. Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates biological processes by several orders of magnitude. MMC was recently shown to significantly increase ECM deposition and to promote chondrogenic differentiation of stem cells. We hypothesise that the addition of sulphated high-molecular weight polysaccharides (carrageenan) to the media positively affects stem cell maintenance and chondrogenic differentiation. Herein, we venture to assess the impact of MMC on the maintenance of stem cell phenotype and multipotency, and ECM deposition in xeno-free human bone marrow mesenchymal stem cell (BMSCs) cultures. We investigate different xeno- and serum-free stem cell media with MMC for expansion of BMSCs, assessing multipotency maintenance (FACS analysis), cell viability, metabolic activity, proliferative capacity and matrix deposition (SDS-PAGE, ICC) at day 4 and day 10. Experiments will be conducted at 2 different passages (p3, p7). Medium without MMC will be used as control. Based on these results, cells expanded with the best protocol will be subsequently investigated for chondrogenic differentiation comparing different xeno-/serum-free and serum containing differentiation media. Chondrogenic differentiation will be assessed via Alcian blue and Safranin O stainings, gene expression for chondrogenic marker genes and quantification of GAG content. Finally, these findings will pave the way for developing more effective strategies for cartilage tissue engineering.

All cells exist within a 3D microenvironment where they are exposed to a multitude of mechanobiological cues, from nano-level cell/matrix interactions, to tissue-level mechanical strain. These cues are fundamental to maintaining tissue homeostasis, but when disrupted during disease, can promote pathological outcomes and impair healing. This is particularly true in tendons; 3D load bearing connective tissue structures composed of a complex arrangement of matrix proteins, organised in a highly aligned manner and maintained by tendon cells (tenocytes). When diseased or injured (termed tendinopathy), the tendon begins a journey of poor healing, characterised by mechanically inferior disorganised scar tissue which ultimately results in compromised or total loss of function. In both health and disease, the mechanobiological stimuli experienced by tenocytes will directly affect their behaviour, yet this is a poorly studied area of research. We have used decellularised tendon slices to mimic the structure of healthy tendon, and induced degradation to mimic tendinopathic tendon. We have re-seeded these slices with tenocytes or immune cells and are building a greater picture of the role that the structure and stiffness of the matrix has on cell behaviour in health and disease.

Ligaments and tendons are vital musculoskeletal soft tissues, which are commonly injured due to overuse and trauma. Their distinct functions are well known however their unique structure and biochemical composition and how they change with disease is poorly described. The most commonly injured ligament in the dog and man is the cranial cruciate (CCL) and anterior cruciate ligament (ACL) respectively. Therefore, the structure, function and pathophysiology of disease of this ligament has been most commonly studied in both species. Canine cranial cruciate ligament rupture (CCLR) most commonly occurs following gradual ligament degeneration or disease (CCLD) followed by a non-contact injury or a minor trauma. Several studies have described marked degenerative histological changes in ligament structure prior to and following rupture which consist of loss of the collagen fascicular structure, areas of poor collagen fibril staining, a marked increase in “chondroid” type cells and mineralisation. The ECM protein profile is also altered with increased sulphated glycosaminoglycans content, increased immature collagen cross-links as well as enzymes involved in collagen remodelling. In man, similar findings have been described in the ACL with age and in osteoarthritis (OA). Previously it had been thought that ligament degeneration occurred following OA but these more recent studies suggest that ligament degeneration can lead to joint destabilisation and OA. Being able to determine early degenerative ligament changes in spontaneous clinical cohorts and the mechanisms which cause them are ideal starting points to determine targets for future therapies in the prevention of ligament degradation and rupture. Further identification of ligament cell types in terms of degenerative, responsive and regenerative (stem) types is essential to try and alter ligament cellular and extracellular matrices harnessing their therapeutic potential.

Electromechanical coupling (piezoelectricity) is present in all living beings and provides basis for sense, thoughts and mechanisms of tissue regeneration. Herein, we ventured to assess the influence of MMC in mesenchymal stem cell culture. In this study, we fabricated piezoelectric regenerative scaffolds to assess the role of electromechamical stimulation on tendon regeneration. Tendon cells were selectively stimulated in vitro by mechanical or electromechanical cues using non-piezoelectric or piezoelectric scaffolds and optimal mechanical loading (4% deformation at 0.5 Hz). This was followed up with an in vivo study to assess tendon regeneration in a rat Achilles tendon injury model. P(VDF-TrFE), scaffolds were observed to mimic the fibrous structure of tendon tissue (figure 1) and were capable of producing electrical charges up to 17 pC/N when mechanically loaded (figure 1. Genes associated with tendon specific markers (Col.I/Col III, Scx and Mkx) and mechanosensitive ion channels such as PIEZO1, TRAAK and TRPV1 were significantly upregulated (figure 2). The upregulated genes were validated with individual real time Q-PCR and bioinformatics revealed a possible regulated function. Those results were further validated in vivo. Protein expression of repaired tendons showed a correlation between increase in expression of tendon related proteins SCX, TNMD, Decorin and expression of ion channels KCNK2, TRAAK and TRPV1. Collectively, these data clearly illustrate that scaffolds made of PVDF-TrFE can produce electrical charges when mechanically loaded. Moreover, gene and protein analyses showed a positive regulation of tendon specific markers through activation mechanosensitive voltage-gated genes.

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Tendon injuries constitute a major healthcare burden owing to the limited healing ability of these tissues and the poor clinical outcomes of surgical repair treatments. Recent advances in tendon tissue engineering (TTE) strategies, particularly through the use of biotextile technologies, hold great promise toward the generation of artificial living tendon constructs. We have previously developed a braided construct based on suture threads coated with gelMA:alginate hydrogel encapsulating human tendon cells. These cell-laden composite fibers enabled the replication of cell and tissue-level properties simultaneously. Based on this concept, in this study we explored the use of platelet lysate (PL), a pool of supra-physiological concentrations of growth factors (GFs), to generate a hydrogel layer, which is envisioned to act as a depot of therapeutic factors to induce tenogenic differentiation of encapsulated human adipose stem cells (hASCs). For this purpose, commercially available suture threads were first embedded in a thrombin solution and then incubated in PL containing hASCs. Herein, thrombin induces the gelation of PL and consequent hydrogel formation. After coating suture threads with the mixture of PL-ASCs, cells were found to be viable and homogeneously distributed along the fibers. Strikingly, hASCs encapsulated within the PL hydrogel layer around the suture thread were able to sense chemotactic factors present in PL and to establish connections between adjacent independent fibers, suggesting a tremendous potential of PL cell-laden hydrogel fibers as building blocks in the development of living constructs aimed at tendon repair applications.

Extensor tendon attachment to the dorsum of the proximal phalanx may fully extend the finger metacarpal phalangeal joint (MPJ). 15 fresh-frozen cadaveric hands were axially loaded in the line of pull to the extensor digitorum comunis of the index, middle, ring and small finger at the level just proximal to the MPJ. We measured force of extension at the MP joint in 3 groups: 1) native specimen, 2) extensor tendon release at the proximal interphalangeal (PIP) joint with release of lumbricals/lateral bands, 3) extensor tendon release at the PIP joint and dorsal proximal phalanx and lumbrical/lateral band release. Degree change of extension was calculated using arctan function with height change of the distal aspect of the proximal phalanx, and the length of the proximal phalanx. We used Student T-test to determine significant decrease in the extension of the phalanges. Extension of all fingers decreased slightly when the extensor tendon were severed at the PIP joint with release of the lateral bands/lumbricals (8deg+/−2deg). After this release, the finger no longer extended. Slight loss of extension was not statistically significant (p >.05) between group 1 and group 2. Groups 1 and 2 were significantly different compared to group 3. In summary, distal extensor tendon transection and release of lateral bands/lumbricals resulted in little change in force and degree of finger extension. The distal insertion of the extensor, released when exposing the PIP joint dorsally, may not need to be repaired to the base of the middle phalanx.

Achilles tendinopathy is classically defined as a tendinosis devoid of an inflammatory cell population. However, recent literature suggests inflammation as a mediator in the pathogenesis. These finding were mainly based on semi-quantative immunohistochemistry. We therefore used flow cytometry to obatain a more accurate identification and quantification of the different cell types involved. Thirty-two samples were obtained from twelve patients with chronic tendinopathic lesions undergoing Achilles tendon surgery. Samples obtained from three patients with hemiplegia requiring surgical release due to spastic Achilles tendons served as control. We used two panels to identify the myeloid and lymphoid population targeting the following markers: CD45, CD3, CD8, CD4, CD19, CD11b, CD56, CD14, CD16, Vα7.2, 6b11, CD161, TCRγδ. To assess the presence of fibroblasts CD90 was targeted. The mean count of CD45+ hematopoietic cells in the tendinopathic samples was significantly higher than in the control samples, respectively 13.27% and 3.24% of the total cell count (P<0.001). The mean fraction of CD3+ cells present in the complete cell population was significantly higher in pathological samples than in control samples, respectively 1.70% and 0.37% (P<0.05). Presence of CD19+ B cells was not reported. The mean fraction of γδ T cells was significantly higher in tendinopathic samples compared to blood samples of the same patient and consisted of 12.9% and 5.8% γδ T cells respectively (P<0.05). These findings support an inflammatory cell infiltration in midportion Achilles tendinopathy that show similarities to enthesitis in SpA. This implies a potential target to investigate in novel treatment modalities.

Cultured primary cells have a limited life span and undergo dedifferentiation. Tissue engineering (TE) approaches require high cell numbers, but availability of human derived cells is limited and animal cells show inter-species differences. The advantages of immortalized cells are delayed senescence and phenotypic stability. The present study was undertaken to validate key properties of immortalized human anterior cruciate ligament (ACL) fibroblasts in direct comparison with non-immortalized cells from the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40 years old female donor) were either immortalized using repeated transient transfection with a simian virus SV40 plasmid or remained untreated. Both cell populations were analyzed for cell survival, DNA content, tendon marker, extracellular matrix (ECM) and cytoskeletal protein expression. Cell spheroids of both populations were seeded on scaffolds embroidered either from polylactic acid (PLA) threads alone or combined PLA- and PLA-co-caprolacton-(P(LA-CL)) threads, functionalized with fluor treatment and collagen foams. Cell survival on the scaffolds was monitored for up to 5 weeks. In contrast to non-immortalized ligamentocytes, immortalized cells reflected some chaotic and incomplete cell divisions, higher DNA content, numbers of dying cells and nucleoli, reduced vimentin and vinculin-associated focal adhesions. Analysed markers, other cytoskeletal and ECM components were similarly expressed. Compared to the non-immortalized ligamentocytes immortalized formed instable spheroids and died on the scaffolds after 21 d. Both cell populations reflected superior growth on the PLA-P(LA-CL) compared with PLA scaffolds. Immortalized cells share crucial properties with their non-immortalized counterparts, but TE is only possible for limited culturing periods.

Acute lateral ankle sprain accounts for 85% of sprains. The lateral sprain is associated with other ligament injuries e.g. medial and syndesmosis sprain. Long-term, approximately 20% of acute lateral sprains develop into chronic lateral ankle instability (CLAI) which includes persistent pain, and recurring ankle sprains. This study evaluated the grade of an ankle ligament injury by ultrasonography (US) and compared the findings to the outcome of patient-reported questionnaires. 48 subjects (18–40 years) diagnosed with an ankle sprain attended a clinical and US examination of ankle ligaments within two weeks after the sprain. Evaluation was done by US of acute lateral ligament injuries (ATFL, CFL), syndesmosis injury (AiTFL), and medial injury (dPT, TCt) only in participants with the positive clinical signs of medial injury. Participants were then mailed a questionnaire (PROMQ) every third month for a year. 29 women and 19 men participated with a mean age at 26.50 years. One-year follow-ups need to be analyzed further for final results. Temporary results include data based on the initial 26 patients: Two clinical signs statistically correlated. Multiple logistic regression analysis confirmed the results. Positive palpated tenderness AiTFL predicted with partial ruptured ATFL and reported pain during active plantar flexion of ankle predicted with normal CFL confirmed by the US. Patients with partial rupture of ATFL presented with tenderness at AiTFL point. Patients presenting with intact CFL reported pain during active plantar flexion. Compared to the US findings, the overall examinations were inconclusive in predicting ATFL, CFL, AiTFL, and medial ligament injuries.

Collagen scaffolds are generally characterized by their random fibre distribution and weak mechanical properties, which makes them unsuitable as substitutes for highly anisotropic tissues such as cornea or tendon. Recently, we developed a technique to create collagen type I scaffolds with well-defined anisotropic micro-patterns. Porcine collagen was mixed with PBS10X, NaOH and one of the following cross-linkers: glutaraldehyde (GTA), genipin and 4-arm polyethylene glycol (4SP). The resulting mixture was casted on micro-grooved (2×2×2 μm) polydimethylsiloxane (PDMS) moulds and allowed to dry in a laminar flow hood to obtain 5mg/ml collagen films. Different pH, temperatures (Tº), and cross-linker concentrations were tested in the process. Collagen gelation kinetics was analysed with rheometry and surface topography was assessed with scanning electron microscopy (SEM). Human bone marrow stem cells (HBMSCs) were seeded on the films and cell alignment was analysed by rhodamine/phalloidin staining and imaged with fluorescence microscopy. From all three cross-linkers tested, only 4SP cross-linked scaffolds showed a well-defined micro-grooved pattern. Increasing pH and Tº on 4SP-treated collagen decreased gelation time, which resulted in complete inhibition of the pattern, suggesting that an initial low viscous solution is required for a correct PDMS pattern infiltration. A wide range of 4SP concentrations (0.5, 1, 1.5 mM) maintained the well-defined topography on the films, opening the door to future fine-tuning of the stiffness sensed by cells. hBMSCs seeded on top of the scaffolds aligned along the pattern for 14 days in culture. Collectively, this data highlights the potential of these collagen scaffolds as tendon substitutes.

Patient reported outcomes have become validated objective measures of success in research studies. They take time and effort to develop and administer. However, to remain relevant and universal PROMS should be gathered routinely and used to manage evidence-based change in healthcare systems. To ensure that they are adopted individual clinician involvement is key however a framework for comparison and relevance promotes engagement. Several examples will be presented of system change using PROMS and PREMS as well as using routine data to defend patient selection. How and what we present depends on whom we are expecting to influence.

Patient-reported outcomes (PROs) are widely used in the orthopaedic field to assess the impact of conservative and surgical interventions from a patient's perspective. Available instruments cover a range of outcome parameters, such as pain, function, stiffness, quality of life or joint awareness. Choice of instrument for a specific study for clinical practice should include the appraisal of the psychometric characteristics of the measure. The presentation will focus on the assessment of the psychometric characteristics of PRO instruments and provide criteria for evaluating those. The concepts of objectivity, reliability and validity will be explained in the context of PRO instrument and the interpretation of score points derived from PRO instruments will be discussed detailing concepts such as minimal important change/difference, norm data, and thresholds based on external criteria. Finally, international guidelines that define standards for the various procedures on development, validation and translation of PRO instruments will be summarised.

Patient-reported outcome measures are a cornerstone of outcome assessment in orthopaedics. However, completing the pencil and paper questionnaires in clinic is something of a burden to the individual patient and the health care institution. We do not provide much in the way of incentives to collect PRO data. Lengthy questionnaires and hidden data analysis offer no direct benefit to the individual patient nor the clinician. Employing ePRO, utilising tablet PCs for questionnaire completion, can improve this situation considerably. Swift and cost-effective data management and instant availability of results using intuitive graphical display make questionnaire completion more rewarding. Direct feedback of PRO data during the consultation can inform the individual's care. Completing electronic questionnaires also makes computer-adaptive testing (CAT) possible. CAT creates dynamic questionnaires, adapting to the individual symptom burden of the individual patient. CAT both increases measurement precision and reduces the number of questions required. As such, ePRO assessment may help to maximise the efficiency and the utilisation of PRO data.

Over the past two decades much has been written regarding pain and disability following whiplash injury. Several authors have reported on the relationship between insurance claims and whiplash-associated disorders. Our own experience of over 10-years suggests that fracture may be protective of whiplash injury following road traffic accident (RTA). We exported all ‘medical legal’ cases due to RTA from our EMR system and combined this with patient-reported outcome measures. 1,482 (57%) of all medicolegal cases are due to RTA: 26% ‘head-on’, 34% ‘side-impact’ and 40% ‘rear-ended’. Over half of the vehicles involved are subsequently written-off. While the mean BMI is 27.1, ¼ of this cohort has a BMI over 30 (obese). 163 (11%) patients report a fracture occurring as a result of RTA. Type of impact is significant for fracture (p < 0.05). 47% of RTA which result in fracture are due to ‘head-on’ collision; conversely only 21% are due to ‘rear-ended’ impacts. In 1,324 (89%) of RTA without fracture, patients are twice as likely to report whiplash injury as one of their top-3 sources of pain (p < 0.01). Gender is statistically significant for age (M 44.4, F 38.6, p < 0.05). While the BMI of this cohort is alarming, it is consistent with Irish obesity statistics. Type of impact, in particular ‘head-on’ collision (high kinetic energy event), is significant for fracture. Finally, we report that fracture is significantly protective (p < 0.01) of whiplash injury following RTA.

The use of a tourniquet during total knee arthroplasty (TKA) is controversial. Return to function and pain are believed to be affected by the use of a tourniquet. The hypothesis of this study was that use of a tourniquet (T) would delay postoperative functional recovery and increase pain as compared to no tourniquet use (NT). 200 patients were recruited for this prospective, double-blinded, randomized controlled trial. All surgeries were performed by one of two fellowship trained arthroplasty surgeons at our institution. Patients were randomized to either undergo TKA with T or NT and blinded to group allocation. An otherwise standardized perioperative protocol was followed. The primary outcome measures were functional assessment testing using the timed up-and-go (TUG) and stair-climb (SC) tests and visual analog scale pain (VAS-P) scores. Secondary outcome measures included blood loss and range-of-motion (ROM). Patients completed outcomes measures preoperatively, in hospital, and postoperatively at 4–6 weeks and 6–8 months. Minimal detectable change (MDC) and Student's T-test, alpha of p < 0.05, were used to determine significance. No significant differences were seen in postoperative TUG, SC, VAS-P, or ROM at any time point. NT patients were seen to have significantly more calculated blood loss (means: T 1,370.04mL, NT 1,743.85mL; p < 0.001), without a significant increase in transfusion events. Tourniquet use during TKA significantly decreases blood loss and does not adversely affect early postoperative outcomes. Tourniquet use during routine TKA is safe and effective and concerns over deleterious effects on function and pain may not be justified.

Various arthroscopic techniques using differing graft materials have been described and present a potential alternative to arthroplasty for rotator cuff arthropathy. We describe the short-term outcomes of allograft reconstruction, having evolved of our surgical technique from graft interposition to superior capsule reconstruction (SCR). All patients with an irreparable tear, in the absence of clinical and radiograph evidence of osteoarthritis, who underwent an allograft (Graft Jacket^TM) reconstruction with either an arthroscopic interposition or SCR technique within our institution were included. A retrospective case note analysis was performed to ascertain perioperative details including total operating and consumable implant costs. 15 patients were in the interposition group, mean age 66 years (48–77). Mean postoperative follow-up time was 17 months (1.9 −27.8). The mean OSS improved from 30.6 to 35.7 (p<0.05). Additionally, mean pain scores out of 10 improved from 7.7 to 1.5 (p<0.01). Mean satisfaction for the surgery was 7.8 out of 10. Complications included 2 re-ruptures (13.3%), 1 infection (6.7%) and 1 case of no improvement (6.7%). In the SCR group, there were 10 patients, mean age 64.5 (56– 68 years). Half of these patients had previous rotator cuff surgery. Mean postoperative follow-up time was 8.7 months (1.9 – 16.3). The mean OSS improved from 24 to 32.9 (p<0.01). Similarly, pain scores decreased from 7.9 to 3.5 (p<0.01). Mean satisfaction was 7.2. Complications included 1 case of no improvement (10%) resulting in a reverse TSR and 1 re-rupture (10%). A formal, prospective comparison trial is advocated to determine if SCR is superior.

Late presentation of DDH continues to remain a major problem particularly in the developing countries. Femoro-Acetabular Zones (FAZ) system is created to find a relation between acetabular maturity and severity of dislocation, in one hand, and the success of closed reduction, on the other hand. We hypnosis that the lower the acetabular index and the closer the femoral head to the acetabulum, the more likely the success of treatment. Thus, a retrospective study was performed on late diagnosed DDH hips that underwent closed treatment at a particular hospital in the Middle East. FAZ are drawn on the AP view of the pelvic x-ray and is based on a perpendicular from the acetabular index at the lateral margin of the superior acetabular rim then another perpendicular to Perkin's line is drawn. This gives three zones, graded I-III. The center of femoral metaphysis is identified denoting the position of the femoral head in relation to the zone classification. FAZ system was applied on 65 pelvic radiographs; mean patient age was 24 months (range: 12 to 36 months) with a minimum follow up of 3 years. Overall, 37 of 65 hips (57%) achieved a satisfactory outcome (Severin I&II), while 22 hips (33%) were found to be unsatisfactory (Severin III). 6 hips (10%) needed an open reduction (p-value 0.001). FAZ could perfectly predict the successful cases. FAZ system is a simple and novel classification and if employed, could reasonably predict the outcome of non-surgical treatment of DDH after walking age.