Objectives. During open orthopaedic surgery, joints may be exposed to air, potentially leading to cartilage drying and chondrocyte death, however, the long-term effects of joint drying in vivo are poorly understood. We used an animal model to investigate the subsequent effects of joint drying on cartilage and chondrocytes. Methods. The patellar groove of anaesthetised rats was exposed (sham-operated), or exposed and then subjected to laminar airflow (0.25m/s; 60 minutes) before wounds were sutured and animals recovered. Animals were monitored for up to eight weeks and then sacrificed. Cartilage and chondrocyte properties were studied by histology and confocal microscopy, respectively. Results. Joint drying caused extensive chondrocyte death within the superficial regions of cartilage. Histology of dried cartilage demonstrated a loss of surface integrity at four weeks, fibrillations at eight weeks, and an increased modified Mankin score (p < 0.001). Cartilage thickness increased (p < 0.001), whereas chondrocyte density decreased at four weeks (p < 0.001), but then increased towards sham-operated levels (p < 0.01) at eight weeks. By week eight, chondrocyte pairing/clustering and cell volume increased (p < 0.05; p < 0.001, respectively). Conclusions. These in vivo results demonstrated for the first time that as a result of laminar airflow, cartilage degeneration occurred which has characteristics similar to those seen in early osteoarthritis. Maintenance of adequate cartilage hydration during open orthopaedic surgery is therefore of paramount importance. Cite this article: Dr A. Hall. Drying of open animal joints in vivo subsequently causes cartilage degeneration. Bone Joint Res 2016;5:137–144. DOI: 10.1302/2046-3758.54.2000594

Design of bone tissue engineering scaffolds imposes a number of requirements for their physical properties, in particular porosity and mechanical behaviour. Alginates are known as a potential material for such purposes, usually deploying calcium as a cross-linker. Calcium over-expression was reported having proinflammatory effect, which is not always desirable. Contrary to this, barium has better immunomodulatory outcome but data for barium as a cross-linker are scarce. In this work the objective was to produce Ba-linked alginates and compare their viscoelastic properties with Ca-linked controls in vitro. Sodium alginate aqueous solution (1 wt%) with 0.03 wt.% CaCl. 2. is gelled in dialysis tubing immersed in 27 mM CaCl. 2. (controls) or BaCl. 2. , for 48 h, followed by freeze-drying and rehydration (with 0.3 wt.% CaCl. 2. and 0.8 wt.% NaCl). Hydrogel discs (diameter 8-10 mm, thickness 4-6 mm) were assessed in dry and wet (DMEM immersed) states by dynamic mechanical analysis (DMA) under compressive creep conditions with increased loads, frequency scans and strain-controlled sweeps in physiological range (0.1-20 Hz) at 25°C and 37°C. Resulting data were analysed by conventional methods and by a model-free BEST (Biomaterials Enhanced Simulation Testing) to extract invariant values and material functions. Significant differences were observed in properties of Ba-linked hydrogel scaffolds vs. Ca-linked controls. Specifically, for the similar porosity Ba-samples exhibited lower creep compliance, higher dynamical stiffness and lower loss factor in the whole studied range. Invariant modulus exhibited a non-linear decay vs. applied stress. These differences were observed in both dry and wet states and temperatures. Use of barium as a cross-linker for alginates allows further modification of biomechanical properties of the scaffolds for better compliancy to the tissues in the application. Barium release might have an immunomodulating effect but also promote ion exchange for osteogenesis due to additional Ca/Ba concentration gradient

Introduction. Histology is still considered the gold standard method for the evaluation of soft tissues in the musculoskeletal field, thanks to the possibility of studying structures using different staining and high magnification microscopy. To overcome the intrinsic limits of this method, contrast enhanced microtomographic (CE- microCT) protocols are constantly evolving to allow 3D study of soft tissues. However, no standardized approaches are available, and many concerns exist about the alterations induced to the samples. Method. microCT/histology protocols were explored on human tendons and menisci. To enhance contrast tissues for microCT scanning 1) examethyldisilazane drying 2) 2% phosphotungstic acid (PTA) in alcoholic solution exposition and 3) 2% PTA in aqueous solution exposition were performed; to observe PTA contrast progression, three exposition and scanning times were selected. microCT images were compared to histological slices obtained from the same samples, after rehydration protocols, or from adjacent tissues portion, stained with Picrosirius red to highlight the peculiar collagenic structures. Result. Exposition times influence PTA diffusion and tissue contrast; its specificity for collagenic structure allow a clearer contrast of the tissues. Histological processing on the same samples is possible: PTA removal requires careful washing in basic solution to reduce the hardening of the sample, while drying can be reverted applying inverse protocol. Comparison with microCT images is really accurate if histology is performed on the same sample, although all protocols induce tissue shrinkage with relative packing of collagen fibers. Conclusion. The contrast approaches tested proved effective in highlighting the structures of both tendons and menisci, but the structural effects induced by tissue shrinkage do not allow a completely real microCT visualization of native tissue. Histology can be the reference method to monitor the efficacy of the contrast methods and the alterations induced to define the possibility of improvement of the technique. Acknowledgement. PR23-PAS-P4 “ADJOINT 2”- INAIL

Abstract. Objective. In this systematic review we aim to compare wound complication rates from Negative Pressure Wound Therapy (NPWT) to dry sterile surgical dressings in primary and revision total knee arthroplasty (TKA). Methods. A search was performed using PubMed, Embase, Science Direct, and Cochrane Library. Eligible studies included those investigating the use of NPWT in primary and revision TKA. Exclusion criteria included studies investigating NPWT not related to primary or revision TKA; studies in which data relating to NPWT was not accessible; missing data; without an available full text, or not well reported. We also excluded studies with poor scientific methodology. All publications were limited to the English language. Abstracts, case reports, conference presentations, and reviews were excluded. Welch independent sample t-test was used for the statistical analysis. Results. Our review identified 11 studies evaluating 1,414 patients. Of the 1,181 primary TKA patients analysed (NPWT = 416, surgical dressing = 765), the overall wound complication rates in patients receiving NPWT ranged from 0% – 63% (Median 7.30%, SD ± 21.44) This is in comparison to complication rates of 2.8% – 19% (Median 6.50%, SD ± 6.59) in the dry dressing group. The difference in complication rates between the two groups was not statistically significant (p =0.337). In the revision TKA cohort of 279 patients (NPWT group = 128, dry dressing group = 151), the overall wound complication rates in the NPWT group ranged between 6.7% – 12% (Median 9.80%, SD ± 2.32) vs 23.8% – 30% (Median 26.95%, SD ± 2.53) in the dry dressing group. This difference was statistically significant (p<0.001). Conclusion. NPWT dressing demonstrated statistically significant reduction in wound complication rates when used in revision TKA but not primary TKA when compared to dry sterile dressings. This is probably due to higher wound related risks encountered with revision TKA surgery compared to primary TKA surgery. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications

The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models

Introduction. In tissue engineering, the establishment of sufficient vascularization is essential for tissue viability and functionality. Inadequate vascularization disrupts nutrients and oxygen supply. Nonetheless, regenerating intricate vascular networks represents a significant challenge. Consequently, research efforts devoted to preserving and regenerating functional vascular networks in engineered tissues are of paramount importance. The present work aims to validate a decellularisation process with preservation of the vascular network and extracellular matrix (ECM) components in fasciocutaneous flaps. Method. Five vascularized fasciocutaneous flaps from cadaveric donors were carefully harvested from the anterolateral thigh (ALT), preserving the main perforator of the fascia lata. The entire ALT flap underwent decellularization by perfusion using a clinically validated chemical protocol. Fluoroscopy and computed tomography (CT) were used to analyze the persistence of the vascular network within the flap, pre- and post-decellularization. Histological analysis, including hematoxylin and eosin staining, and quantitative DNA assessment evaluated decellularization efficacy. Further qualitative (immunohistochemistry, IHC) and quantitative analyses were conducted to assess the preservation of ECM components, such as collagen, glycosaminoglycans, and elastin. Result. On average, the ALT flap maintains 82% of the perfusion area (p = 0.094) post-treatment. Histological analysis confirmed decellularization efficacy and revealed structural rearrangement. Paired analysis revealed a significant decrease in DNA levels (<14.8 ng/mg of dry weight, p****< 0.0001) and well-maintained ECM. IHC indicated the persistence of elastine, collagen IV and laminin. Quantitative analysis confirmed elastin (p = 0.44) and collagen persistence (+74%, p*** = 0.001, albeit with a decrease in matrix glycosaminoglycans (-41%, p*** = 0.01). Conclusion. Decellularization effectively removed cells, while preserving the ECM overall and maintaining some vascular network integrity. Yet, further study is needed to validate these findings, involving microCT examination of the vascular network and its ability to support cell colonization and viability

Osteosarcoma and other types of bone cancers often require bone resection, and backfill with cement. A novel silorane-based cement without PMMA's drawbacks, previously developed for dental applications, has been reformulated for orthopedic use. The aim of this study is to assess each cement's ability to elute doxorubicin, maintain its potency, and maintain suitable weight-bearing strength. The silorane-based epoxy cement was synthesized using a platinum-based Lamoreaux's catalyst. Four groups of cement were prepared. Two PMMA groups, one without any additives, one with 200 mg of doxorubicin. Two silorane groups: one without any additive, one with doxorubicin, added so that the w% of drug into both cements were equal. Pellets 6 × 12 mm were used for testing (ASTM F451). n=10. Ten pellets from each group were kept dry. All others were placed into tubes containing 2.5 mL of PBS and stored at 37 °C. Elution from doxorubicin-containing groups were collected every day for 7 days, with daily PBS changeout. Antibiotic concentrations were determined via HPLC. Compressive strength and compressive modulus of all groups were determined for unsoaked specimens, and those soaked for 7 and 14 days. MTT assays were done using an MG63 osteosarcoma cell line. Both cements were able to elute doxorubicin over 7 days in clinically-favorable quantities. For PMMA samples, the incorporation of doxorubicin was shown to significantly affect the compressive strength and modulus of the samples (p<0.01). Incorporation of doxorubicin into silorane had no significant effect on either (p>.05). MTT assays indicated that doxorubicin incorporated into the silorane cement maintained its effectiveness whereas that into PMMA did not. At the dosing used, both cements remained above the 70 MPa. Both PMMA and silorane-based cements can deliver doxorubicin. Doxorubicin, however, interacts chemically with PMMA, inhibiting polymerization and lowering the chemotherapeutic's effectiveness

According to the latest report from the German Arthroplasty Registry, aseptic loosening is the primary cause of implant failure following primary hip arthroplasty. Osteolysis of the proximal femur due to the stress-shielding of the bone by the implant causes loss of fixation of the proximal femoral stem, while the distal stem remains fixed. Removing a fixed stem is a challenging process. Current removal methods rely on manual tools such as chisels, burrs, osteotomes, drills and mills, which pose the risk of bone fracture and cortical perforation. Others such as ultrasound and laser, generate temperatures that could cause thermal injury to the surrounding tissues and bone. It is crucial to develop techniques that preserve the host bone, as its quality after implant removal affects the outcome of a revision surgery. A gentler removal method based on the transcutaneous heating of the implant by induction is proposed. By reaching the glass transition temperature (T. G. ) of the periprosthetic cement, the cement is expected to soften, enabling the implant to be gently pulled out. The in-vivo environment comprises body fluids and elevated temperatures, which deteriorate the inherent mechanical properties of bone cement, including its T. G. We aimed to investigate the effect of fluid absorption on the T. G. (ASTM E2716-09) and Vicat softening temperature (VST) (ISO 306) of Palacos R cement (Heraeus Medical GmbH) when dry and after storage in Ringer's solution for up to 8 weeks. Samples stored in Ringer's solution exhibited lower T. G. and VST than those stored in air. After 8 weeks, the T. G. decreased from 95.2°C to 81.5°C in the Ringer's group, while the VST decreased from 104.4°C to 91.9°C. These findings will be useful in the ultimate goal of this project which is to design an induction-based system for implant removal. Acknowledgements: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB/TRR-298-SIIRI – Project-ID 426335750

Bone is a dynamic tissue that undergoes continuous mechanical forces. Mechanical stimuli applied on scaffolds resembling a part of the human bone tissue affects the osteogenesis [1]. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a piezoelectric material that responds to mechanical stimulation producing an electrical signal, which in turn promotes the osteogenic differentiation of bone-forming cells by opening voltage-gated calcium channels [2]. In this study we examined the biological behavior of pre-osteoblastic cells seeded onto lyophilized piezoelectric PEDOT-containing scaffolds applying uniaxial compression. Two different concentrations of PEDOT (0.10 and 0.15% w/v) were combined with a 5% w/v poly(vinyl alcohol) (PVA) and 5% w/v gelatin, casted into wells, freeze dried and crosslinked with 2% v/v (3-glycidyloxypropyl)trimethoxysilane and 0.025% w/v glutaraldehyde. The scaffolds were physicochemically characterized by FTIR, measurement of the elastic modulus, swelling ratio and degradation rate. The cell-loaded scaffolds were subjected to uniaxial compression with a frequency of 1 Hz and a strain of 10% for 1 h every second day for 21 days. The loading parameters were selected to resemble the in vivo loading situation [3]. Cell viability and morphology on the PEDOT/PVA/gelatin scaffolds was determined. The alkaline phosphatase (ALP) activity, the collagen and calcium production were determined. The elastic modulus of PEDOT/PVA/gelatin scaffolds ranged between 1 and 5 MPa. The degradation rate indicates a mass loss of 15% after 21 days. The cell viability assessment displays excellent biocompatibility, while SEM images display well-spread cells. The ALP activity at days 3, 7 and 18 as well as the calcium production are higher in the dynamic culture compared to the static one. Moreover, energy dispersive spectroscopy analysis revealed the presence of calcium phosphate in the extracellular matrix after 14 days. The results demonstrate that PEDOT/PVA/gelatin scaffolds promote the adhesion, proliferation, and osteogenic differentiation of pre-osteoblastic cells under mechanical stimulation, thus favoring bone regeneration

Introduction. The incidences of fragility fractures, often because of osteoporosis, are increasing. Research has moved towards bioresorbable scaffolds that provide temporary mechanical stability and promote osteogenesis. This research aims to fabricate a 3D printed composite Poly (l-lactic-co-glycolic acid)-strontium doped tricalcium phosphate (PLGA-SrTCP) scaffold and evaluate in an in vitro co culture study containing osteoporotic donor cells. Method. PLGA, PLGA TCP, and PLGA SrTCP scaffolds were produced using Fused Filament Fabrication (FFF). A four-group 35-day cell culture study was carried out using human bone marrow derived mesenchymal stem cells (hMSCs) from osteoporotic and control donors (monoculture) and hMSCs & human monocytes (hMCs) (Co culture). Outcome measures were biochemical assays, PCR, and cell imaging. Cells were cultured on scaffolds that had been pre-degraded for six weeks at 47°C prior to drying and gamma sterilisation. Result. 3D printed scaffolds were successfully produced by FFF. All groups in the study supported cell attachment onto the scaffolds, producing extracellular matrices as well as evidence of osteoclast cell structures. Osteoporotic cells increased CTSK activity and CAII activity and decreased ALP activity compared to controls. In control cultures, the addition of bTCP and bTCP/Sr to the PLGA reduced TRAP5b, CAII and ALP activity compared to PLGA alone. The addition of Sr did not show any differences between donors. Conclusion. This study details suitability of 3D printed polymer scaffolds for use in bone tissue applications. Both composite and pure polymer scaffolds promote osteogenesis in vitro. The introduction of ceramic filler and ion doping does not beneficially effect osteogenic potential and can reduce its ability compared to pure polymer. This study suggests the behaviour of control and osteoporotic cells are different and that osteoporotic cells are more prone to bone resorption. Therefore, it is important to design bone scaffolds that are specific to the patient as well as to the region of fracture

Current strategy for orthopedic tissue engineering mainly focusses on the regeneration of the damaged tissue using cell-seeded three-dimensional scaffolds. Biocompatible scaffolds with controllable degradation and suitable mechanical property are required to support new tissue in-growth and regeneration . [1]. Porous composite scaffolds made from organic and inorganic materials are highly preferred, which can mimic the natural bone in their composition as well can enhance tissue repair . [2]. Scaffolds with optimum mechanical strength in both dry and wet state are more suitable for in vivo orthopedic application. Biphasic calcium phosphate (BCP), a biocompatible ceramic and carboxymethyl cellulose (CMC), a semi-natural polymer are used in the study to prepare composite scaffolds. Citric acid is used as a crosslinking agent for the polymer to improve its stability . [3]. Stability, mechanical property in dry and wet conditions and cytocompatibility of the scaffolds were investigated. Cellulose-BCP (BC25) and crosslinked cellulose-BCP (BC25CA) scaffolds are fabricated by freeze-drying method. The stability of the scaffolds was assessed in phosphate buffered saline (PBS) and compressive modulus was measured in dry and wet condition. Cytocompatibility was assessed by culturing pre-osteoblast cells at a density of 2.5×10. 4. on crosslinked scaffold and cell proliferation was measured by performing MTT assay on day 4 and 7. Crosslinked scaffold was more stable than non-crosslinked scaffold in aqueous environment as the latter disintegrated within few hours in the solution. Non-crosslinked scaffold showed higher compressive modulus of 116.3±14.8 kPa in dry condition but is reduced to 1.2±0.7 kPa in hydrated state. Though the crosslinked scaffold shows low compressive modulus of 37.67±6.7 kPa in dry state, it exhibited appreciable compressive moduli of 17.15±1.3 kPa in hydrated state. Thus, the crosslinking of the scaffolds improved the stability as well as the mechanical strength in wet condition. Cytocompatibility was assessed by culturing pre-osteoblast cells and from the MTT assay, it is shown that the cells are proliferating on the crosslinked scaffolds with time which indicates that the scaffolds are non-toxic and cytocompatible. Stability and optimum mechanical property for scaffold in aqueous environment are highly crucial for in vivo hard tissue regeneration. This study demonstrated the preparation of crosslinked scaffolds which exhibited good stability and mechanical strength in wet condition along with a porous architecture, controlled degradability and cytocompatibility, hence, crosslinked cellulose-BCP scaffold can be used for orthopedic application

Saline (0.9%) is typically used to rinse joints during osteo-articular surgery. It is not unusual for cartilage to then be exposed to the air of the operating theatre for 1-2hrs, which can lead to chondrocyte death. We have compared the survival of in situ chondrocytes within bovine cartilage which has been rinsed in various solutions or simply drained of synovial fluid (SF) and then allowed to dry, to identify approaches that could reduce chondrocyte death arising from cartilage drying. Metacarpophalangeal joints from 3yr-old cows were opened under aseptic conditions. The joints were then (a) rinsed with saline (Baxter's Healthcare, Newbury), (b) rinsed with saline+glucose (20mM; both 300mOsm) or (c) drained of SF, and allowed to dry at room temperature. Full depth cartilage explants were taken after 2hrs, placed into Dulbecco's modified Eagle's medium and incubated with CMFDA (5-chloromethyl-fluorescein diacetate; 10microM) and propidium iodide (10microM) for the identification/quantification of living and dead cells respectively by confocal scanning laser microscopy and image analysis. After 2hrs, the appearance and properties of the cartilage of the drying joints were clearly different. Saline-rinsed cartilage was dark purple and appeared dull with the cartilage difficult to sample. However when the rinsing solution was saline+glucose, or when joints were drained of SF, the cartilage was almost identical to the freshly-opened joint with a pearly-blue, shiny appearance, and cartilage sampling was easy. Chondrocyte death was markedly increased in saline rinsed/dried joints after 2hrs (21±9% cell death). In contrast, there was no significant (P>0.05) death in saline+glucose rinsed/dried (2±1%) or SF-drained joints (3±2%;means±s.e.m.;n=5). The loss of cartilage wet weight over 2hrs (time=0 taken as 100%) was almost identical between cartilage rinsed in saline (73.6±1.6%), saline + glucose (78.6±1.1%) or SF (75.0±0.2%; data means±s.d.;n=2). These results suggest that it was not the loss of water per se during cartilage drying that was the key determinant of chondrocyte viability. As chondrocytes are normally anaerobic, the rise in cartilage pO2 which occurs during exposure to air could have a deleterious effect on cell viability however the presence of glucose or SF protects through an anti-oxidant effect

Summary. Our study shows that a tendon rupture can be successfully augmented with Demineralised Cortical Bone (DCB) giving initial appropriate mechanical strength suitable for in vivo use providing the biological reactions to the graft are favourable. Introduction. Treatment of tendon and ligament injuries remains challenging; the aim is to find a biocompatible substance with mechanical and structural properties that replicate those of normal tendon and ligament. Because of its structural and mechanical properties, we proposed that DCB can be used in repair of tendon and ligament as well as regeneration of the enthesis. DCB is porous, biocompatible and has the potential to be remodelled by the host tissues. 2 studies were designed; in the first we examined the mechanical properties of DCB after gamma irradiation (GI) and freeze drying (FD). In the second we used different techniques for repairing bone-tendon-bone with DCB in order to measure the mechanical performance of the construct. Methods. In the first study we allocated the DCB specimens into 4 groups; group-A non-freeze dried non-gamma irradiated, group-B freeze dried non-gamma irradiated, group-C non-freeze dried gamma irradiated and group-D freeze dried and gamma irradiated. The 4 groups were tested for maximum tensile strength. In the 2nd study, patella - patellar tendon - tibia construct of mature ewes were harvested and the distal 1cm of the patellar tendon was excised, 4 models of repair were tested;. • Model-1, DCB was used to bridge the gap between the tendon and the tibial tuberosity. The DCB strip was stitched to the tendon using one bone anchor. • Model-2, similar to model-1 with the use of 2 bone anchors. • Model-3, similar to model-2, construct was offloaded by Fiberwire continuous thread looped twice through bony tunnels sited in the patella and in the tibial tuberosity. • Model-4, similar to model-3 with 3 hand braided fiberwire threads as offloading loop. All 4 models were tested until failure and force displacement curves used to investigate the structural properties of the reconstruction. Results. The Median of maximum tensile force for group-A was 218N [95%C.I.=147.9–284.7N], group-B was 306N [95%C.I.=154.1–488.6N], group-C was 263N [95%C.I.=227.8–315.6N], group-D was 676N [95%C.I.=127-1094.9N]. Group-D results were statistically higher (p=<0.05) compared to group-A and group-C, while there was no statistical significance compared to group-B. The median failure force for model-1 was 250N, (95%C.I.=235-287), model-2 was 290N (95%C.I.=197-396), model-3 was 767N (95%C.I.=730-812) and for model-4 was 934N (95%C.I.=867-975). There was no statistical significance between model-1 and model-2 (p=0.249), however statistical significance was found between other models (p=<0.006). Discussion. Demineralised Bone is widely used as a bone graft substitute and may be used to augment bone formation in load bearing applications. In this study we focus on the potential use of demineralised bone in ligament and tendon repair. A previous animal study by our group found that the use of demineralised bone can enhance healing of the enthesis. Other published studies suggested the possibility of using DCB as ligament substitute. We examined the effect of gamma radiation as the most common sterilisation technique in medical field and the freeze drying as a possible technique for long term storage on the tensile strength of the DCB

Abstract. Introduction. Bone grafts are utilised in a range of surgical procedures, from joint replacements to treatment of bone loss resulting from cancer. Decellularised allograft bone is a regenerative, biocompatible and immunologically safe potential source of transplant bone. Objectives. To compare the structural and biomechanical parameters of decellularised and unprocessed (cellular) trabecular bone from the human femoral head (FH) and tibial plateau (TP). Methods. Bone pins were harvested from 10 FHs and 11 TPs (27, 34 respectively). Pins were decellularised (0.1% w/v sodium dodecyl sulphate) or retained as cellular controls. QA testing was carried out to assess protocol efficacy (total DNA and histological analysis). Cellular and decellularised FH (n=7) and TP (n=10) were uCT scanned. Material density (MD); apparent density (BV/TV); trabecular connectivity; trabecular number; trabecular thickness (Tb-t) and trabecular spacing were measured. Pins were then compression tested to determine ultimate compressive stress (UCS), Young's modulus and 0.2% proof stress. Results. Total DNA levels of decellularised bone were below 50 ng.mg. −1. dry weight. Cell nuclei and marrow were largely removed. No significant differences in properties were found between decellularised and cellular bone from either anatomical region (p>0.05, Mann-Whitney). No significant differences in biomechanical properties were found between cellular FH and cellular TP (p>0.05) though significant differences in structural properties were found (MD: TP>FH, p=0.001; BV/TV: FH>TP, p=0.001; and Tb-t: FH>TP, p=0.005). Significant differences were found between decellularised FH and decellularised TP (UCS: FH>TP, p=0.001; Young's modulus: FH>TP, p=0.002; proof stress; FH>TP, p=0.001; MD: TP>FH, p<0.001; BV/TV: FH>TP, p<0.001 and Tb-t: FHT>P p<0.001. Conclusion. Decellularisation did not affect the properties of human trabecular bone. Differences were found between the mechanical and structural properties of decellularised FH and TP which could facilitate stratified bone grafts for different applications. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction and Objective. Knee osteoarthritis (KOA) is a frequent disease for which therapeutic possibilities are limited. In current recommendations, the first-line analgesic is acetaminophen. However, low efficacy of acetaminophen, frequently leads to the use of weak opioids (WO) despite their poor tolerance, especially in elderly patients. The primary objective was to compare the analgesic efficacy and safety of a new wearable transcutaneous electrical nerve stimulation (W-TENS) to weak opioids (WO) in the treatment of moderate to severe, nociceptive, chronic pain in knee osteoarthritis patients. Materials and Methods. ArthroTENS study is a phase 3, non-inferiority, multicentric, prospective, randomized, single-blinded for primary efficacy outcome, controlled, in 2-parallel groups, clinical study comparing W-TENS versus WO over a 3-month controlled period with an additional, optional, non-controlled, 3-month follow-up for patients in W-TENS group. The co-primary outcome was KOA pain intensity (PI) at month 3 and the number of adverse events (AEs) over 3 months. Results. The non-inferiority of W-TENS was demonstrated in both the PP and ITT populations. At M3, PI in PP population was 3.87 (2.12) compared to 4.66 (2.37) (delta: −0.79 (0.44); 95% CI (−1.65; 0.08)) in W-TENS and WO groups, respectively. Since the absolute value of the 95% CI of the between-treatments mean PI difference [−1.71, – 0.12] was above 0 in ITT set, the planned superiority analysis was performed, demonstrating that W-TENS was significantly superior to WO at M3 (P=0.0124). At M1 and M3, the W-TENS group reached the absolute minimal clinically important difference (MCID) for an analgesic (1.8 (2.1) and 2.1 (2.3), respectively), corresponding to a 20 mm reduction in PI (interquartile range: 15–30) on a 0–100 mm visual analogic scale – i.e. 2 points on a numerical rating scale – which equates to “much better”. Conversely, in the WO group, a 0.5 (1.8) and a 1.1 (2.1) reduction in PI were observed at M1 and M3, respectively, while a 1-point reduction in PI is required to be considered as a “slightly better” improvement. In WO group, AEs were the common systemic AEs reported with WO (nausea, constipation, drowsiness, dizziness, pruritus, vomiting, dry mouth). AEs in W-TENS group were local, such as local cutaneous reaction (erythema). Thirty-nine (70.9%) patients wished to extend W-TENS treatment for 3 additional months. Only one patient discontinued this additional period and results were maintained at M6. Conclusions. W-TENS was more effective and better tolerated than WO in the treatment of nociceptive KOA chronic pain and could represent an interesting non-pharmacological alternative to WO

Introduction and Objective. Guided Bone Regeneration (GBR) uses biodegradable collagen membranes of animal origin tissues (dermis and pericardium). Their barrier effect prevents soft tissues to interfere with the regeneration of alveolar bone. However, their xenogeneic origin involves heavy chemical treatments which impact their bioactivity. Wharton's Jelly (WJ) from the umbilical cord is a recoverable surgery waste. WJ is mostly made from collagen fibers, proteoglycans, hyaluronic acid, and growth factors. WJ with immunologically privileged status and bioactive properties lends credence to its use as an allograft. Nevertheless, low mechanical properties limit its use in bone regenerative strategies. Herein, our objective is to develop a crosslinked WJ-based membrane to improve its strength and thus its potential use as a GBR membrane. Materials and Methods. The umbilical cords are collected after delivery and then stored at −20°C until use. The WJ membranes (1 × 5 × 12 mm) were obtained after the removal of blood vessels and amniotic tissue, washed, lyophilized, and stored at −20°C. WJ membranes were incubated in genipin solutions in decreasing concentrations (0.3 g / 100 mL − 0.03 g / 100 mL) for 24 hours at 37°C. The crosslinking degree was estimated by ninhydrin and confirmed by FTIR (Fourier-transform infrared spectroscopy) assays. The swelling rate was obtained after the rehydration of dry crosslinked WJ-membrane for 10 min in D-PBS. The mechanical properties were assessed in hydrated conditions on a tensile bench. The resistance to the degradation was evaluated by collagenase digestion (1 mg/mL for 60 hours) assay. The cytotoxicity of crosslinked WJ-membrane was evaluated in accordance with the standard ISO.10993-5 (i.e. Mitochondrial activity and Lactate Dehydrogenase release) against Mesenchymal Stem Cells (MSCs). Finally, the MSCs colonization and proliferation were followed after 21 days of culture on crosslinked WJ-membranes. Results. The increase of crosslinking rates from 30% to 90% of the WJ membrane was demonstrated by the ninhydrin assay. FTIR analysis showed a prominent peak at 1732 cm. -1. , confirming the incorporation of genipin in the WJ. The swelling rate of crosslinked WJ-membrane decreased with an increase of the crosslinking rate. An increase in elastic modulus and an increase in the resistance to the collagenase degradation were observed along with an increase in the crosslinking degree. Cytotoxicity investigations did not elicit a harmful effect of the genipin, however, a poor MSCs adhesion on the crosslinked membrane was observed. Conclusions. Our results show that a membrane can be developed from Wharton's jelly. The mechanical and degradation properties can be improved by crosslinking with genipin without inducing any cytotoxicity effect. However, the percentage of crosslinking has an influence on the adhesion of the cells to the membranes. The crosslinked WJ-membrane bioactivity and the osteo-regenerative potential in vitro/in vivo will be evaluate

Abstract. Objectives. Assess and characterise the suitability of a novel silk reinforced biphasic 3D printed scaffold for osteochondral tissue regeneration. Methods. Biphasic hybrid scaffolds consisted of 3D printed poly(ethylene glycol)-terephthalate-poly(butylene terephthalate)(PEGT/PBT) scaffold frame work (pore size 0.75mm), which has been infilled with a cast and freeze dried porous silk scaffold (5×5×2mm. 3. ), in addition to a seamless silk top layer (1mm). Silk scaffolds alone were used as controls. Both the biphasic and control scaffolds were characterised via uniaxial compression testing (strain rate 0.1mm/min), and the potential biocompatibility of the scaffolds was tested via in vitro culture of seeded bone marrow stromal cells post fabrication. Results. Uniaxial compression testing showed that the biphasic scaffolds (N=4) initially demonstrated similar behaviour on a stress-strain curve to a silk scaffold alone control group (N=6), until a strain of 30% was reached. After 30% strain, load was transitioned from the silk only chondral layer to the 3D printed PEGT/PBT scaffold which resisted further compression and exhibited a significantly greater compressive modulus of 12.6±0.9MPa compared to 0.113±0.01MPa (p<0.001) in the silk scaffold control group. Following 24hours of seeding, no difference was noticed in cell adhesion behaviour under fluorescent microscopy between silk scaffolds and biphasic scaffolds (n=5). Discussion. The use of 3D printing within this novel scaffold provides a solid framework and increases its versatility. The reinforced silk not only provides the secondary Porous structure to the 3D printed scaffold for the bone phase, but also a superficial layer for the cartilage phase. This unique structure has the potential to fill a niche within osteochondral tissue regeneration, especially with the possibility for its use within personalised medicine. Conclusions. These results demonstrate that the novel silk reinforced biphasic 3D printed scaffold is biocompatible and has suitable mechanical properties for osteochondral tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Background:. The Lateral Intercondylar Ridge (LIR) gained notoriety with arthroscopic trans-tibial Anterior Cruciate Ligament (ACL) reconstruction where it was mistakenly used to position the ‘over the top’ guide resulting in graft malposition. With anatomic ACL reconstruction some surgeons use the same ridge to define the anterior margin of the ACL femoral insertion in order to guide graft placement. However there is debate about whether this ridge is a consistent and reliable anatomical structure. The aim of our study was to identify whether the LIR is a consistent anatomical structure and to define its relationship with the femoral ACL insertion. Methods:. In the first part, we studied 23 dry bone specimens. Using a digital microscribe, we created a 3D model of the medial surface of the lateral femoral condyle to evaluate whether there was an identifiable bony ridge. In the second part, we studied 7 cadaveric specimens with soft tissues intact. The soft tissues were dissected to identify the femoral ACL insertion. A 3D reconstruction of the femoral insertion and the surface allowed us to define the relationship between the LIR and the ACL insertion. Results:. All specimens (23 dry bones; 7 intact soft tissues) had a defined ridge on the medial surface of the lateral femoral condyle. The ridge extends from the apex point of the lateral intercondylar notch, where the posterior condyle meets the femoral shaft, and extends obliquely to the articular margin. The mean distance from the midpoint of the posterior condylar articular margin was 10.1 mm. The ridge was consistently located just anterior to the femoral ACL insertion. Conclusion:. This study shows that the LIR is a consistent anatomical structure that defines the anterior margin of the femoral ACL insertion. This supports its use as a landmark for femoral tunnel placement in ACL reconstruction surgery. Abstract 28

Collagen is a key component of the extracellular matrix in a variety of tissues and hence is widely used in tissue engineering research, yet collagen has had limited uptake in the field of 3D printing. In this study we successfully adapted an existing electronic printing method, aerosol jet printing (AJP), to print high resolution 3D constructs of recombinant collagen type III (RHCIII). Circular samples with a diameter of 4.5mm and 288 layers thick, or a diameter of 6.5mm and 400 layers thick were printed on glass cover slips with print lines of 60µm. Attenuated Total Reflectance Fourier-Transorm Infa-red (ATR-FTIR) spectroscopy performed on the 4 of the printed samples and dried non-printed RHCIII samples showed that no denaturation had occurred due to the printing process. Printed samples were crosslinked using EDC [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, Sigma Aldrich] to improve their stability and mechanical strength. Differential scanning calorimetry (DSC) performed showed a marked difference in the denaturation temperature between crosslinked printed samples and fibrillar non-printed samples and nano-indentation showed that the construct was relatively stiff. Previous results with similar samples have shown that mesenchymal stem cells (MSCs) align with and travel parallel to print direction. Results obtained from these samples show signs that they might be applied in other areas such as bone tissue engineering