Vertebral metastases are the most common type of malignant lesions of the spine. Although this tumour is still considered incurable and standard treatments are mainly palliative, the standard approach consists in surgical resection, which results in the formation of bone gaps. Hence, scaffolds, cements and/or implants are needed to fill the bone lacunae.

Here, we propose a novel approach to address spinal metastases recurrence, based on the use of anti-tumour metallic-based nanostructured coatings. Moreover, for the first time, a gradient microfluidic approach is proposed for the screening of nanostructured coatings having anti-tumoral effect, to determine the optimal concentration of the metallic compound that permits selective toxicity towards tumoral cells.

Coatings are based on Zinc as anti-tumour agent, which had been never explored before for treatment of bone metastases.

The customized gradient generating microfluidic chip was designed by Autodesk Inventor and fabricated from a microstructured mould by using replica moulding technique. Microstructured mould were obtained by micro-milling technique. The chip is composed of a system of microfluidic channels generating a gradient of 6 concentrations of drug and a compartment with multiple arrays of cell culture chambers, one for each drug concentration. The device is suitable for dynamic cultures and in-chip biological assays. The formation of a gradient was validated using a methylene blue solution and the cell loading was successful.

Preliminary biological data on 3D dynamic cultures of stromal cells (bone-marrow mesenchymal stem cells) and breast carcinoma cells (MDA-MB-231) were performed in a commercial microfluidic device.

Results showed that Zn eluates had a selective cytotoxic effect for tumoral cells. Indeed, cell migration and cell replication of treated tumoral cells was inhibited. Moreover, the three-dimensionality of the model strongly affected the efficacy of Zn eluates, as 2D preliminary experiments showed a high cytotoxic effect of Zn also for stromal cells, thus confirming that traditional screening tests on 2D cultured cells usually lead to an overestimation of drug efficacy and toxicity.

Based on preliminary data, the customized platform could be considered a major advancement in cancer drug screenings as it also allows the rapid and efficient screening of biomaterials having antitumor effect.

Our knowledge of primary bone marrow edema (BME) of the knee is still limited. A major contributing factor is that it shares several radiological findings with a number of vascular, traumatic, and inflammatory conditions having different histopathological features and etiologies.

BME can be primary or secondary. The most commonly associated conditions are osteonecrosis, osteochondritis dissecans, complex regional pain syndrome, mechanical strain such as bone contusion/bruising, micro-fracture, stress fracture, osteoarthritis, and tumor. The etiology and pathogenesis of primary BME are unclear. Conservative treatment includes analgesics, non-steroidal anti-inflammatory drugs, weight-bearing limitations, physiotherapy, pulsed electromagnetic fields, prostacyclin, and bisphosphonates. Surgical treatment, with simple perforation, fragment stabilization, combined scraping and perforation, and eventually osteochondral or chondrocyte transplant, is reserved for the late stages. This retrospective study of a cohort of patients with primary BME of the knee was undertaken to describe their clinical and demographic characteristics, identify possible risk factors, and assess treatment outcomes.

We reviewed the records of 48 patients with primary BME of the knee diagnosed on MRI by two radiologists and two orthopedists. History, medications, pain type, leisure activities, smoking habits, allergies, and environmental factors were examined. Analysis of patients’ characteristics highlighted that slightly overweight middle-aged female smokers with a sedentary lifestyle are the typical patients with primary BME of the knee. In all patients, the chief symptom was intractable day and night pain (mean value, 8.5/10 on the numerical rating scale) with active as well as passive movement, regardless of BME extent. Half of the patients suffered from thyroid disorders; indeed, the probability of having a thyroid disorder was higher in our patients than in two unselected groups of patients, one referred to our orthopedic center (odds ratio, 18.5) and another suffering from no knee conditions (odds ratio, 9.8). Before pain onset, 56.3% of our cohort had experienced a stressful event (mourning, dismissal from work, concern related to the COVID-19 pandemic). After conservative treatment, despite the clinical improvement and edema resolution on MRI, 93.8% of patients described two new symptoms: a burning sensation in the region of the former edema and a reduced ipsilateral patellar reflex. These data suggest that even though the primary BME did resolve on MRI, the knee did not achieve full healing.

Rotator cuff repair has excellent clinical outcomes but continues to be a challenge when it comes to large and massive tears as well as revision procedures. Reported symptomatic retear rates are still too high to be acceptable. The purpose of the present study was to evaluate the effectiveness of a combination of augmentation techniques consisting of microfractures of the greater tuberosity, extracellular matrix (ECM) patch graft and subsequent platelet concentrate (PC) subacromial injections in revision rotator cuff repair.

The study was designed as a retrospective comparative study on prospectively collected data from a consecutive cohort of patients. All patients who underwent arthroscopic revision rotator cuff repair for symptomatic failure of previous posterosuperior rotator cuff repair were considered eligible for the study. Symptomatic failure had been diagnosed according to clinical examination and confirmed by magnetic resonance imaging (MRI). Structural integrity had been assessed on MRI and classified according to Sugaya classification. Only patients affected by stage IV-V were considered eligible. Tear reparability was confirmed during arthroscopy. Only patients with a minimum 2 years follow-up were included. Patients were divided in two groups. In group 1 (control group) a standard arthroscopic revision and microfractures of the greater tuberosity were performed; in group 2 (experimental group), microfractures of the greater tuberosity and a ECM patch graft were used to enhance tendon repair, followed by postoperative PC injections. Minimum follow-up was 12 months. Primary outcome was the Constant-Murley score (CMS) normalized for age and gender. Subjective outcome was assessed with the Disabilities of the Arm, Shoulder and Hand (DASH) score in its short version (Quick-DASH). Tendon integrity was assessed with MRI at 6 months after surgery. Comparison between groups for all discrete variables at baseline and at follow-up was carried out with the Student's t-test for normally distributed data, otherwise Mann-Whitney U-test was used. Within-group differences (baseline vs follow-up) for discrete variables were analyzed by paired t-test, or by Wilcoxon signed-rank test in case of data with non-normal distribution. Differences for categorical variables were assessed by chi-squared test. Significance was considered for p values < 0.05.

Forty patients were included in the study (20 patients for each group). The mean follow-up was 13 ± 1.6 months. No patients were lost at the follow up. Comparison between groups did not show significant differences for baseline characteristics. At follow-up, mean CMS was 80.7 ± 16.6 points in group 1 and 91.5 ± 11.5 points in group 2 (p= 0.022). Mean DASH score was 28.6 ± 21.6 points in group 1 and 20.1 ± 17.4 points in group 2 (p= 0.178). Post-operative MRI showed 6 healed shoulders in Group 1 and 16 healed shoulders in Group 2 (p<0.004). No postoperative complications were reported in both groups.

The combination of microfractures of the greater tuberosity, ECM patch graft, and subsequent PC subacromial injections is an effective strategy in improving tendon healing rate.

Prosthetic joint infections represent complications connected to the implantation of biomedical devices. Bacterial biofilm is one of the main issues causing infections from contaminated orthopaedic prostheses. Biofilm is a structured community of microbial cells that are firmly attached to a surface and have unique metabolic and physiological attributes that induce improved resistance to environmental stresses including toxic compounds like antimicrobial molecules (e.g. antibiotics). Therefore, there is increasing need to develop methods/treatments exerting antibacterial activities not only against planktonic (suspended) cells but also against adherent cells of pathogenic microorganisms forming biofilms. In this context, metal-based coatings with antibacterial activities have been widely investigated and used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing the biofilm formation prevention efficacy. Additionally, standardized and systematic approaches to test antibacterial activity of newly developed coatings are still missing, while standard microbiological tests (e.g. soft-agar assays) are typically used that are limited in terms of simultaneous conditions that can be tested, potentially leading to scarce reproducibility and reliability of the results.

In this work, we combined the Calgary Biofilm Device (CBD) as a device for high-throughput screening, together with a novel plasma-assisted technique named Ionized Jet Deposition (IJD), to generate and test new generation of nanostructured silver- and zinc-based films as coatings for biomedical devices with antibacterial and antibiofilm properties. During the experiments we tested both planktonic and biofilm growth of four bacterial strains, two gram-positive and two gram-negative bacterial strains, i.e. Staphylococcus aureus ATCC 6538P, Enterococcus faecalis DP1122 and Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, respectively. The use of CBD that had the only wells covered with the metal coatings while the biofilm supports (pegs) were not sheltered allowed to selectively define the toxic effect of the metal release (from the coating) against biofilm development in addition to the toxic activity exerted by contact killing mechanism (on biofilms formed on the coating). The results indicated that the antibacterial and antibiofilm effects of the metal coatings was at least partly gram staining dependent. Indeed, Gram negative bacterial strains showed high sensitivity toward silver in both planktonic growth and biofilm formation, whereas zinc coatings provided a significant inhibitory activity against Gram positive bacterial strains. Furthermore, the coatings showed the maximal activity against biofilms directly forming on them, although, Zn coating showed a strong effect against biofilms of gram-positive bacteria also formed on uncoated pegs.

We conclude that the metal-based coatings newly developed and screened in this work are efficient against bacterial growth and adherence opening possible future applications for orthopedic protheses manufacturing.

Adverse events (AEs) are still a major problem in spinal surgery, despite advances in surgical techniques, innovative technologies available and the introduction of checklist and predictive score systems aimed at reducing surgical complications.

We previously analysed the results of the introduction of the WHO Safety Surgical Checklist (SSC) in our Institution, comparing the incidence of complications between two periods: from January to December 2010 (without checklist) and from January 2011 and December 2012 (with checklist), in order to assess the checklist effectiveness.

The sample size was 917 patients with an average of 30 months of follow-up. Complications were observed in 107 patients (11.6%) among 917 spinal surgery procedures performed, with 159 (17.3%) complications in total. The overall incidence of complications for trauma, infectious pathology, oncology, and degenerative disease was 22.2%, 19.2%, 18.4%, and 15.3%, respectively. We observed a reduction of the overall incidence of complications following the introduction of the WHO Surgical Checklist: in 2010 without checklist, the incidence of complications was 24.2%, while in 2011 and 2012, following the checklist introduction, the incidence of complications was 16.7% and 11.7%, respectively (mean 14.2%) (p<0.0005).

Thus, the SSC appeared to be an effective tool to reduce complications in spinal surgery and we proposed to extend the use of checklist system also to the pre-operative and post-operative phases in order to further reduce the incidence of complications.

We also believe that a correct capture and classification of complications is fundamental to generate a clinical decision support system aimed at improving patients’ safety in spinal surgery.

In the period between January 2017 and January 2018 we prospectively recorded the adverse events and complications of patients undergoing spinal surgery in our department, without using any collection system. Then we retrospectively recorded the intraoperative and postoperative adverse events of surgically treated patients during the same one-year period, using the SAVES v2 system introduced by Rampersaud and collaborators (Rampersaud YR et al. J Neurosurg Spine 2016 Aug; 25 (2): 256-63) to classify them.

In the one-year period from January 2017 to January 2018 a total of 336 patients underwent spinal surgery: 223 for degenerative conditions and 113 for spinal tumors. Comorbidities were collected (Charlson Comorbidity Index [CCI]).

Overall, a higher number of adverse events (AEs) was recorded using SAVES compared to the prospective recording without the use of any capture system and the increased number was statistically significant for early postoperative AEs (138/336 vs 44/336, p<0.001).

210 adverse events were retrospectively recorded using the SAVES system (30 intraoperative adverse events, 138 early postoperative and 42 late postoperative adverse events). 99 patients (29.5%) on the cohort had at least one complication.

Furthermore, the correlation between some risk factors and the onset of complications or the prolonged length of stay was statistically analyzed. The risk factors taken into account were: age, presence of comorbidities (CCI), ASA score, previous surgery at the same level, type of intervention, location of the disease, duration of the surgery.

In particular, the duration of the surgery (more than 3 hours) and the presence of previous surgeries resulted to be risk factors for complications in multivariate analyses

Open limb fractures are typically due to a high energy trauma. Several recent studied have showed treatment's superiority when a multidisciplinary approach is applied. World Health Organization reports that isolate limb traumas have an incidence rate of 11.5/100.000, causing high costs in terms of hospitalization and patient disability.

A lack of experience in soft tissue management in orthopaedics and traumatology seems to be the determining factor in the clinical worsening of complex cases. The therapeutic possibilities offered by microsurgery currently permit simultaneous reconstruction of multiple tissues including vessels and nerves, reducing the rate of amputations, recovery time and preventing postoperative complications.

Several scoring systems to assess complex limb traumas exist, among them: NISSSA, MESS, AO and Gustilo Anderson. In 2010, a further scoring system was introduced to focus open fractures of all locations: OTA-OFC. Rather than using a single composite score, the OTA-OFC comprises five components grades (skin, arterial, muscle, bone loss and contamination), each rated from mild to severe. The International Consensus Meeting of 2018 on musculoskeletal infections in orthopaedic surgery identified the OTA-OFC score as an efficient catalogue system with interobserver agreement that is comparable or superior to the Gustilo-Anderson classification. OTA-OFC predicts outcomes such as the need for adjuvant treatments or the likelihood of early amputation. An orthoplastic approach reconstruction must pay adequate attention to bone and soft tissue infections management. Concerning bone management: there is little to no difference in terms of infection rates for Gustilo-Anderson types I–II treated by reamed intramedullary nail, circular external fixator, or unreamed intramedullary nail. In Gustilo-Anderson IIIA-B fractures, circular external fixation appears to provide the lowest infection rates when compared to all other fixation methods.

Different technique can be used for the reconstruction of bone and soft tissue defects based on each clinical scenario. Open fracture management with fasciocutaneous or muscle flaps shows comparable outcomes in terms of bone healing, soft tissue coverage, acute infection and chronic osteomyelitis prevention. The type of flap should be tailored based on the type of the defect, bone or soft tissue, location, extension and depth of the defect, size of the osseous gap, fracture type, and orthopaedic implantation. Local flaps should be considered in low energy trauma, when skin and soft tissue is not traumatized. In high energy fractures with bone exposure, muscle flaps may offer a more reliable reconstruction with fewer flap failures and lower reoperation rates. On exposed fractures several studies report precise timing for a proper reconstruction. Hence, timing of soft tissue coverage is a critical for length of in-hospital stay and most of the early postoperative complications and outcomes. Early coverage has been associated with higher union rates and lower complications and infection rates compared to those reconstructed after 5-7 days. Furthermore, early reconstruction improves flap survival and reduces surgical complexity, as microsurgical free flap procedures become more challenging with a delay due to an increased pro-thrombotic environment, tissue edema and the increasingly friable vessels.

Only those patients presenting to facilities with an actual dedicated orthoplastic trauma service are likely to receive definitive treatment of a severe open fracture with tissue loss within the established parameters of good practice. We conclude that the surgeon's experience appears to be the decisive element in the orthoplastic approach, although reconstructive algorithms may assist in decisional and planification of surgery.

With the coronavirus disease 2019 (COVID-19) pandemic, remote working has been ubiquitously implemented to reduce disease transmission via minimization of in-person interactions. Low back pain (LBP) is the first cause of disability worldwide and is frequently reported by workers with sedentary occupations. This cross-sectional study aimed to assess the role of remote working in a population of adults affected by LBP through an online questionnaire.

We enrolled 136 teleworkers affected by LBP. A total of 101 responses were received and 93 suitable questionnaires were included in the final analysis. Demographic data, remote working features and tasks, and LBP burden were analysed. The psychological burden of remote working was evaluated with the World Health Organization Five Well-Being Index (WHO-5) and the Patient Health Questionnaire-2 (PHQ-2). LBP severity was evaluated using a visual analog scale (VAS). LBP-related disability was assessed using the Oswestry Disability Index (ODI). The effect of LBP on working capacity was examined with the Occupational Role Questionnaire (ORQ). Independent risk factors related to LBP worsening were identified using a multivariate logistic regression model.

LBP severity was significantly higher compared to previous in-person working (p<0.0001) as well as average weekly work hours (p<0.001). Furthermore, the risk of LBP deterioration was associated with being divorced (OR: 4.28, 95% CI: 1.27-14.47; p=0.019) or living with others (OR: 0.24, 95% CI: 0.07-0.81; p=0.021), higher ill-being (OR: 0.91, 95% CI: 0.83-0.99; p=0.035) and depression scores (OR: 1.38, 95% CI: 1.00-1.91; p=0.048), as well as having reported unchanged (OR: 0.22, 95% CI: 0.08-0.65; p=0.006) or decreased job satisfaction (OR: 0.16, 95% CI: 0.05-0.54; p=0.003) and increased stress levels (OR: 3.00, 95% CI: 1.04-8.65; p=0.042).

These findings highlight key factors to consider for improving remote workers’ physical and mental wellbeing and decrease their LBP burden.

Bone metastases radiographically appear as regions with high (i.e. blastic metastases) or low (i.e. lytic metastases) bone mineral density. The clinical assessment of metastatic features is based on computed tomography (CT) but it is still unclear if the actual size of the metastases can be accurately detected from the CT images and if the microstructure in regions surrounding the metastases is altered (Nägele et al., 2004, Calc Tiss Int). This study aims to evaluate (i) the capability of the CT in evaluating the metastases size and (ii) if metastases affect the bone microstructure around them.

Ten spine segments consisted of a vertebra with lytic or mixed metastases and an adjacent control (radiologically healthy) were obtained through an ethically approved donation program. The specimens were scanned with a clinical CT (AquilionOne, Toshiba: slice thickness:1mm, in-plane resolution:0.45mm) to assess clinical metastatic features and a micro-CT (VivaCT80, Scanco, isotropic voxel size:0.039mm) to evaluate the detailed microstructure. The volume of the metastases was measured from both CT and micro-CT images (Palanca et al., 2021, Bone) and compared with a linear regression. The microstructural alteration around the metastases was evaluated in the volume of interest (VOI) defined in the micro-CT images as the volume of the vertebral body excluding the metastases. Three 3D microstructural parameters were calculated in the VOI (CTAn, Bruker SkyScan): Bone Volume Fraction (BV/TV), Trabecular Thickness (Tb.Th.), Trabecular Spacing (Tb.Sp.). Medians of each parameter were compared (Kruskal-Wallis, p=0.05).

One specimen was excluded as it was not possible to define the size of the metastases in the CT scans. A strong correlation between the volume obtained from the CT and micro-CT images was found (R2=0.91, Slope=0.97, Intercept=2.55, RMSE=5.7%, MaxError=13.12%). The differences in BV/TV, Tb.Th. and Tb.Sp. among vertebrae with lytic and mixed metastases and control vertebrae were not statistically significant (p-value>0.6). Similar median values of BV/TV were found in vertebrae with lytic (13.2±2.4%) and mixed (12.8±9.8%) metastases, and in controls (13.0±10.1%). The median Tb.Th. was 176±18 ∓m, 179±43 ∓m and 167±91 ∓m in vertebrae with lytic and mixed metastases and control vertebrae, respectively. The median Tb. Sp. was 846±26 ∓m, 849±286 ∓m and 880±116 ∓m in vertebrae with lytic and mixed metastases and control vertebrae, respectively.

In conclusion, the size of vertebral metastases can be accurately assess using CT images. The 3D microstructural parameters measured were comparable with those reported in the literature for healthy vertebrae (Nägele et al., 2004, Calc Tiss Int, Sone et al., 2004, Bone) and showed how the microstructure of the bone tissue surrounding the lesion is not altered by the metastases.

Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test, gene expression profile, histology and immunohistochemistry. Results showed the maintenance and preservation, at time 0 and after 40 days of culture, of the tissue viability, biological activity, as well as molecular markers, i.e. several key genes involved in the complex interactions between the tumour cells and bone able to drive cancer progression, cancer aggressiveness and metastasis to bone. A good tis sue morphological and microarchitectural preservation with the presence of lacunar osteolysis, fragmented trabeculae locally surrounded by osteoclast cells and malignant cells and an intense infiltration by tumour cells in bone marrow compartment in all examined samples. Histomorphometrical data on the levels of bone resorption and bone apposition parameters remained constant between T0 and T40 for all analysed patients. Additionally, immunohistochemistry showed homogeneous expression and location of CDH1, CDH2, KRT8, KRT18, Ki67, CASP3, ESR1, CD8 and CD68 between T0 and T40, thus further confirming the invasive behaviour of breast cancer cells and indicating the maintaining of the metastatic microenvironment. The novel tissue culture, set-up in this study, has significant advantages in comparison to the pre-existent 3D models: the tumour environment is the same of the clinical scenario, including all cell types as well as the native extracellular matrix; it can be quickly set-up employing only small samples of breast cancer bone metastasis tissue in a simple, ethically correct and cost-effective manner; it bypasses and/or decreases the necessity to use more complex preclinical model, thus reducing the ethical burden following the guiding principles aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes; it can allow the study of the interactions within the breast cancer bone metastasis tissue over a relatively long period of up to 40 days, preserving the tumour morphology and architecture and allowing also the evaluation of different biological factors, parameters and activities. Therefore, the study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumour subtypes so predicting patient specific responses.

The in vitro mimicking of bone microenvironment for the study of pathologies is a challenging field that requires the design of scaffolds with suitable morphological, structural and cytocompatible properties. During last years, 3D in vitro tumour models have been developed to reproduce mechanical, biochemical and structural bone microenvironment elements, allowing cells to behave as in vivo.

In this work, gas foamed polyether urethane foams (PUF) and 3D printed thermoplastic polyether urethane (3DP-PU) designed with different patterns are proposed as scaffolds for in vitro model of bone tissue. Surface coatings for a biomimetic behaviour of the 3D scaffold models were also investigated. Morphological, chemico-physical, mechanical properties, and biological in vitro behaviour were investigated.

PUFs for metastases investigation. The suitability of PUF as 3D in vitro model to study the interactions between bone tumour initiating cells and the bone microenvironment was investigated. PUF open porosity (>70%) appeared suitable to mimic trabecular bone structure. Human adipose derived stem cells (ADSC) were cultured and differentiated into osteoblast lineage on the PU foam, as confirmed by Alizarin Red staining and RT-PCR, thus offering a bone biomimetic microenvironment to the further co-culture with bone derived tumour-initiating cells (MCFS). Tumour aggregates were observed after three weeks of co-culture by e-cadherin staining and SEM; modification in CaP distribution was identified by SEM-EDX and associated to the presence of tumour cells.

3DP-PU as tumour bone model. 3D printed scaffolds have pores with a precise and regular geometry (0°-90°, 0°-45°-90°-135°, 0°-60°-120°). PU scaffold porosity evidenced values from 55 to 67%, values that belong to the porosity range of the trabecular bone tissue (30-90%). The compressive modulus varied between 2 and 4 MPa, depending on the printed pattern. Biomimetic nanostructured coating was performed on 0-90° 3DP-PU by Ionized Jet Deposition. Coatings had a submicrometric thickness, variable tuning deposition time, nanostructured surface morphology and biomimetic composition. Coating on 3DP-PU promoted cells colonization of the whole porous scaffolds, compared to the controls, where cells concentrated mostly on the outer layers.

In conclusion, based on the obtained results, scaffolds with different geometries have been successfully produced. Morphological and structural properties of the scaffolds here presented are suitable for mimicking the bone tissue, in order to produce a 3D in vitro model useful for bone pathologies research.

Recent studies on animal models focused on the effect of preserving tendon remnant of rotator cuff on tendon healing. A positive effect by combining tendon remnant preservation and small bone vents on the greater tuberosity in comparison with standard tendon-to-bone repair has been shown. The purpose of the present clinical study was to evaluate the efficacy of biologic augmentation of arthroscopic rotator cuff repair by maintaining tendon remnant on rotator cuff footprint combined with small bone vents of the greater tuberosity.

A retrospective study was conducted. All patients who underwent arthroscopic rotator cuff repair associated with small bone vents (nanofractures) and tendon footprint preservation were considered eligible for the study. Inclusion criteria were: diagnosis of full-thickness rotator cuff tear as diagnosed at preoperative magnetic resonance imaging (MRI) and confirmed at the time of surgery; minimum 24-month of follow-up and availability of post-operative MRI performed not earlier than 6 months after surgery. Exclusion criteria were: partial thickness tears, irreparable tears, capsulo-labral pathologies, calcific tendonitis, gleno-humeral osteoarthritis and/or previous surgery.

Primary outcome was the ASES score. Secondary outcomes were: Quick-DASH and WORC scores, and structural integrity of repaired tendons by magnetic resonance imaging (MRI) performed six months after surgery. A paired t-test was used to compare pre- and postoperative clinical outcomes. Subgroup analysis was performed according to tear size. Significance was set at p < 0.05.

The study included 29 patients (M:F = 15:14). Mean age (+ SD) of patients was 61.7 + 8.9 years. Mean follow-up was 27.4 ± 2.3 months. Comparison between pre- and postoperative functional scores showed significant clinical improvement (p < 0.001). Subgroup analysis for tear size showed significant differences in the QuickDASH score (0.04). Particularly, a significant difference in the QuickDASH score could be detected between medium and large tears (p=0.008) as well as medium and massive lesions (p=0.04). No differences could be detected between large and massive tears (p= 0.35). Postoperative imaging showed healed tendons in 21 out of 29 (72%) cases.

Preservation of tendon remnant combined with small bone vents in the repair of medium-to-massive full-thickness rotator cuff tears provided significant improvement in clinical outcome compared to baseline conditions with complete structural integrity in 72% of the cases.

Paediatric musculoskeletal (MSK) disorders often produce severe limb deformities, that may require surgical correction. This may be challenging, especially in case of multiplanar, multifocal and/or multilevel deformities. The increasing implementation of novel technologies, such as virtual surgical planning (VSP), computer aided surgical simulation (CASS) and 3D-printing is rapidly gaining traction for a range of surgical applications in paediatric orthopaedics, allowing for extreme personalization and accuracy of the correction, by also reducing operative times and complications. However, prompt availability and accessible costs of this technology remain a concern. Here, we report our experience using an in-hospital low-cost desk workstation for VSP and rapid prototyping in the field of paediatric orthopaedic surgery.

From April 2018 to September 2022 20 children presenting with congenital or post-traumatic deformities of the limbs requiring corrective osteotomies were included in the study. A conversion procedure was applied to transform the CT scan into a 3D model. The surgery was planned using the 3D generated model. The simulation consisted of a virtual process of correction of the alignment, rotation, lengthening of the bones and choosing the level, shape and direction of the osteotomies. We also simulated and calculated the size and position of hardware and customized massive allografts that were shaped in clean room at the hospital bone bank. Sterilizable 3D models and PSI were printed in high-temperature poly-lactic acid (HTPLA), using a low-cost 3D-printer.

Twenty-three operations in twenty patients were performed by using VSP and CASS. The sites of correction were: leg (9 cases) hip (5 cases) elbow/forearm (5 cases) foot (5 cases) The 3D printed sterilizable models were used in 21 cases while HTPLA-PSI were used in five cases. customized massive bone allografts were implanted in 4 cases. No complications related to the use of 3D printed models or cutting guides within the surgical field were observed. Post-operative good or excellent radiographic correction was achieved in 21 cases.

In conclusion, the application of VSP, CASS and 3D-printing technology can improve the surgical correction of complex limb deformities in children, helping the surgeon to identify the correct landmarks for the osteotomy, to achieve the desired degree of correction, accurately modelling and positioning hardware and bone grafts when required. The implementation of in-hospital low-cost desk workstations for VSP, CASS and 3D-Printing is an effective and cost-advantageous solution for facilitating the use of these technologies in daily clinical and surgical practice.

C. Difficile infections in elderly patients with hip fractures is associated with high morbidity and mortality. Antibiotic regimens with penicillin and its derivatives is a leading cause. Antibiotic prophylactic preferences vary across different hospitals within NHS. We compared two antibiotic prophylactic regimens - Cefuroxime only prophylaxis and Teicoplanin with Gentamicin prophylaxis in fracture neck of femur surgery, and evaluated the incidence of C. Difficile diarrhea and Surgical Site Infection (SSI).

To assess the Surgical Site Infection and C. Difficile infection rate associated with different regimens of antibiotics prophylaxis in fracture neck of femur surgery.

Data was analyzed retrospectively. Neck of femur fracture patients treated surgically from 2009 in our unit were included. Age, gender, co morbidities, type of fracture, operation, ASA grade was collected. 1242 patients received Cefuroxime only prophylaxis between January 2009 and December 2012 (Group 1) and 486 patients received Teicoplanin with Gentamicin between October 2015 and March 2017 (Group 2). There were 353 males and 889 female patients in Group 1 and 138 males and 348 female patients in Group 2. The co morbidities in both groups were comparable. Incidence of C. Difficile diarrhea and Surgical Site Infection (SSI) was noted. Statistical analysis with chi square test was performed to determine the ‘p’ value.

C. Diff diarrhea rate in Group 2 was 0.41 % as compared to 1.29 % in Group 1. The Surgical Site Infection (SSI) rate in Group 2 was 0.41 % as compared to 3.06 % in Group 1. The comparative results were statistically significant (p = 0.0009).

Prophylactic antibiotic regimen of Teicoplanin with Gentamicin showed significant reduction in C. Difficile diarrhea & Surgical Site Infection in fracture neck of femur patients undergoing surgery.

Cannulated hip screws are frequently used in the management of hip fractures. There have been concerns over the failure rate of the technique and the outcomes of those that subsequently require conversion to total hip replacement (THR).

This study utilised a database of over 600 cannulated hip screw (CHS) fixations performed over a 14-year period and followed up for a minimum of one year (1-14). We identified 57 cases where a conversion to THR took place (40 females, 17 males, mean age: 71.2 years). Patient demographics, original mechanism of injury, fracture classification, reason for fixation failure, time until arthroplasty, implant type and post-arthroplasty complications were recorded. Clinical outcomes were measured using the Oxford Hip Score.

The failure rate of cannulated screw treatment was 9.4% and the mean time from initial fixation to arthroplasty was 15.4 (16.5) months. Thirty six fractures were initially undisplaced and 21 were displaced. As one might expect the displaced cases tended to be younger but this didn't reach statistical significance [66.5(14.3) vs 72.7(13.1), p=0.1]. The commonest causes of failure were non-union (25 cases, 44%) and avascular necrosis (17 cases, 30%). Complications after THR consisted of one leg length discrepancy and one peri-prosthetic fracture. The mean Oxford score pre-arthroplasty was 12.2 (8.4), improving to 38.4 (11.1) at one-year. Although the pre op Oxford scores tended to be lower in patients with undisplaced fractures and higher ASA scores, the improvement was the same whatever the pre-op situation. The one-year Oxford score and the improvement in score are comparable to those seen in the literature for THR in general.

In conclusion, CHS has a high success rate and where salvage arthroplasty is required it can provide good clinical outcomes with low complication rates.

Abstract

Plantar fasciitis (PF) is one of the widespread conditions causing hindfoot pain. The most common presenting symptoms are functional limitation and pain (first step and activity) on plantar surface of the foot. The non-operative treatments provide complete resolution of pain in 90% of patients, but functional limitation still remains as a risk factor for recurrency of PF. Although the number of non-operative treatment options showing efficacy on pain and functional limitation are excessive, the evidences are limited for functional limitation. Additionally, Mulligan mobilization with movement (MMWM) in Chronic Plantar Fasciitis has been poorly studied in the literature. According to these findings, the study was aimed to determine effectiveness of Mulligan mobilization with movement on Chronic Plantar Fasciitis.

A total of 25 patients (40 feet) with chronic PF were included in the study. The patients were randomly divided into Mulligan concept rehabilitation group (PF-M, n=20 feet) and Home Rehabilitation group (PF-H, n=20 feet). (MMWM), Foot and ankle exercises program were applied to PF-M, twice a week totally 8 week (16 sessions) and foot- ankle exercises as a home program were given for PF-H, 8 weeks. The range of motion (ROM) for dorsiflexion and plantar flexion was measured by using a manual goniometer. Pain, disability and activity restriction were assessed by Foot Function Index (FFI) . The first step morning pain was evaluated by Visual Analogue Scale (VAS) and Kinesiophobia was also reported by using Tampa Scale (TSK). Patients were evaluated at baseline and 8 weeks.

FFI, VAS, TSK, ROM values improved in all groups (intragroup variability) at 8th week (P < .05). The other result indicated that ROM values for DF and PF and TSK scores in PF-M had more significant improvement than PF-H (p<.05).

To the best of our knowledge this is the first randomised controlled trial for investigating Mulligan Concept efficiancy on chronic PF. Both Mulligan mobilization with movement (MMWM) and exercise protocols are effective for chronic PF. Furthermore, The Mulligan concept seems more effective treatment option in reducing kinesiophobia and improving functional capacity.

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro.

Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6).

Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001).

CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris.

Meniscal tears are the most common injury in the knee, affecting 66/100,000 people/year. Surgical treatment includes arthroscopic meniscectomy or meniscal repair. Little is known regarding medium-term outcomes following these procedures in isolated meniscal tears. This study aims to quantitatively evaluate patients with meniscal tears, and those who have undergone meniscectomy and meniscal repair using validated patient reported outcome measures (PROMs), further exploring factors which affect surgical outcomes.

This observational study screened 334 patients who underwent arthroscopic surgery at South Tyneside Hospital since August 2013. 134 patients with isolated meniscal tears were invited to complete postal PROMs. A combination of patient notes and radiological imaging was used to collect information of interest including age, gender, knee-laterality, injured meniscus, tear pattern, procedure performed, complications, and associated injuries.

A total of 115 patients (pre-operative patients with current meniscal tear (n=36), meniscectomy (n=63), meniscal repair (n=16)) were included in the analysis with 96% successful PROM completion. Both meniscectomy and meniscal repairs (mean 55-months follow-up) showed better outcomes than pre-operative patients with meniscal tears. Meniscal repairs demonstrated superior outcomes across all PROMs when compared to meniscectomy, with a greater mean overall KOOS score of 17.2 (p=0.009). Factors including higher pre-operative Kellgren-Lawrence Grade, pre-operative articular cartilage lesions and bilateral meniscectomies were shown to negatively influence outcomes.

Both meniscectomy and meniscal repair maintain clinical benefit at mean 55-months follow-up, affirming their use for treatment of meniscal tears. When feasible, meniscal repair should be performed preferentially over meniscectomy in isolated meniscal tears. Identified predictive factors allow adequate treatment stratification in specific patient groups.

Abstract

Critical-sized bone defects can result from trauma, inflammation, and tumor resection. Such bone defects, often have irregular shapes, resulting in the need for new technologies to produce suitable implants. Bioprinting is an additive manufacturing method to create complex and individualised bone constructs, which can already include vital cells.

In this study, we established an extrusion-based printing technology to produce osteoinductive scaffolds based on polycaprolactone (PCL) combined with calcium phosphate, which is known to induce osteogenic differentiation of stem cells.

The model was created in python based on the signed distance functions. The shape of the 3D model is a ring with a diameter of 20 mm and a height of 10 mm with a spongiosa-like structure. The interconnected irregular pores have a diameter of 2 mm +/− 0.2 mm standard deviation.

Extrusion-based printing was performed using the BIO X6. To produce the bioink, PCL (80 kDa) was combined with calcium phosphate nanopowder (> 150 nm particle size) under heating. After printing, 5 × 10⁶ hMSC were seeded on the construct using a rotating incubator.

We were able to print a highly accurate ring construct with an interconnected pore structure. The PCL combined with calcium phosphate particles resulted in a precise printed construct, which corresponded to the 3D model. The bioink containing calcium phosphate nanoparticles had a higher printing accuracy compared to PCL alone. We found that hMSC cultured on the construct settled in close proximity to the calcium phosphate particles. The hMSC were vital for 22 days on the construct as demonstrated by life/dead staining.

The extrusion printing technology enables to print a mechanically stable construct with a spongiosa-like structure. The porous PCL ring could serve as an outer matrix for implants, providing the construct the stability of natural bone. To extend this technology and to improve the implant properties, a biologised inner structure will be integrated into the scaffold in the future.

Abstract

Abstract

A hot swollen joint is a commonly encountered condition in clinical practice. With a broad range of differentials, septic arthritis (SA) is perhaps one of the most concerning. Treated by culture-specific antibiotics and arthroscopic lavage, some patients require multiple washouts. We aimed to determine:

(1) What are the risk factors for development of SA?

(2) What are the risk factors for repeat washout in SA patients?

(3) What are the important clinical differences between a periprosthetic joint infection (PJI) and SA cohort?

All patients presenting to the emergency department, orthopaedic, and rheumatology clinics between January 2020 to January 2021 with a hot, swollen joint were retrospectively evaluated. Patients with previous trauma on the ipsilateral joint, with data missing from their medical records in any of the variables required for analysis, <24 months follow-up were excluded.

Variables of interest in the three-month period preceding the diagnosis of SA were compared between SA and non-SA patients. Factors with a p-value of p<0.100 in univariate analysis were included in a stepwise multivariate logistic regression model. Similar analyses were performed to compare SA patients with multiple washouts with those needing one washout. Demographical and clinical data for PJI patients were collected to delineate important differences with SA cohort.

211 patients were included (SA:28;PJI:24;pseudogout:32;gout:26;others:101). Multivariate analysis showed rheumatoid arthritis (RA), skin infection, and liver disease were risk factors for SA. Amongst patients with septic arthritis, multivariate analysis showed that WBC levels above normal limits (3.6-10.5×109 cells/L) and RA were risk factors for multiple washouts.

Between the SA and PJI cohorts, BMI (p=0.002) was significantly lower in the former, whilst WBC level (p=0.023) and CRP (p<0.0001) was significantly higher in the former.

Early diagnosis of septic arthritis requires understanding the risk factors, namely RA, skin infection, and liver disease. Considering PJI and septic arthritis as the same entity can lead to wrong clinical judgement, and clinicians should be aware of important differences. We believe that the models in this study are of prognostic value to clinicians who are presented with the common presenting compliant of a hot swollen joint.

In this study, we aimed to investigate tibiofemoral and allograft loading parameters after OCA transplantation using tibial plateau shell grafts to characterize the clinically relevant biomechanics that may influence joint kinematics and OCA osseointegration after transplantation. The study was designed to test the hypothesis that there are significant changes in joint loading after tibial plateau OCA transplantation that may require unique post-operative rehabilitation regimens in patients to restore balance in the knee joint.

Fresh-frozen cadaveric knees (n=6) were thawed and mounted onto a 6 DOF KUKA robot. Specimens were size matched to +2 mm for the medial-to-lateral width of the medial tibial hemiplateaus. Three specimens served as allograft recipient knees and three served as donor knees. Recipient knees were first tested in their native state and then tested with size-matched medial tibial hemiplateau shell grafts (n=3) prepared from the donor knees using custom-cut tab-in-slot and subchondral drilling techniques. Tekscan sensors were placed in the joint spaces to evaluate the loading conditions under 90N biaxial loading at full extension of the knee before and after graft placement. The I-Scan system used in conjunction analyzed the total force, pressure distribution, peak pressure, and center of force within the joint space.

Data demonstrated significant difference (p<0.05) in joint space loading after graft implantation compared to controls in both lateral and medial tibial plateaus. The I-Scan pressure mapping system displayed changes in femoral condylar contact points as well.

The results demonstrated that joint space loading was significantly different (p<0.05) between all preoperative and postoperative cadaveric specimens. Despite the best efforts to size match grafts, slight differences in the host's joint geometry resulted in shifts of contact areas between the tibial plateau and femoral condyle therefore causing either an increase or decrease in pressure measured by the sensor. This concludes that accuracy in graft size matching is extremely important to restoring close to normal loading across the joint and this can be further ensured through postoperative care customized to the patient after OCA surgery.

To analyze the effect of tooth extraction site preservation on bone mineral density 6 months after surgery.

From 2020 to 2021, two adjacent teeth (37, 38) of the same patient were extracted at the same time, and then 37 were selected for site preservation, implanted with Bio-oss bone powder, covered with double Bio-gide membrane, reduce tension and sutured. After 6 months of self-healing, 38 was taken CBCT, and the gray value measurement tool in the software was used to measure the bone mineral density of 37 bone graft areas and 38 extraction sockets.

Bone density was high in the center of the bone graft area after the extraction site, and the density decreased in the adjacent alveolar socket, but the gray value was still higher than 38 for natural healing.

Extraction site surgery can improve bone mass and quality at the extraction site. It is good for implanting.

For clinical movement analysis, optical marker-based motion capture is the gold standard.

With the advancement of AI-driven computer vision, markerless motion capture (MMC) has emerged. Validity against the marker-based standard has only been examined for lightly-dressed subjects as required for marker placement. This pilot study investigates how different clothing affects the measurement of typical gait metrics.

Gait tests at self-selected speed (4 km/h) were performed on a treadmill (Motek Grail), captured by 9 cameras (Qualisys Miqus, 720p, f=100Hz) and analyzed by a leading MMC application (Theia, Canada). A healthy subject (female, h=164cm, m=54kg) donned clothes between trials starting from lightly dressed (LD: bicycle tight, short-sleeved shirt), adding a short skirt (SS: hip occlusion) or a midi-skirt (MS: partial knee occlusion) or street wear (SW: jeans covering ankle, long-sleeved blouse), the lattern combined with a short jacket (SWJ) or a long coat (SWC). Gait parameters (mean±SD, t=10s) calculated (left leg, mid-stance) were ankle pronation (AP-M), knee flexion (KF-M), pelvic obliquity (PO-M) and trunk lateral lean (TL-M) representing clinically common metrics, different joints and anatomic planes. Four repetitions of the base style (LD) were compared to states of increased garment coverage using the t-test (Bonferroni correction).

For most gait metrics, differences between the light dress (LD) and various clothing styles were absent (p>0.0175), small (< 2SD) or below the minimal clinically important differences (MCID). For instance, KF-M was for LD=10.5°±1.7 versus MD=12.0°±0.5 (p=0.07) despite partial knee cover. AP-M measured for LD=5.2°±0.6 versus SW=4.1°±0.7 (p<0.01) despite ankle cover-up. The difference for KF-M between LD=10.5°±1.7 versus SWL=6.0°±0.9, SW and SWJ (7.6°±1.5, p<0.01) indicates more intra-subject gait variability than clothing effect.

This study suggests that typical clothings styles only have a small clinically possibly negligible effect on common gait parameters measured with MMC. Thus, patients may not need to change clothes or be instructed to wear specific garments. In addition to avoiding marker placement, this further increases speed, ease and economy of clinical gait analysis with MMC facilitating high volume or routine application.

X-Linked Hypophosphataemia (XLH) is a rare, progressive, hereditary phosphate-wasting disorder characterised by excessive activity of fibroblast growth factor 23. The International XLH Registry was established to provide information on the natural history of XLH and impact of treatment on patient outcomes. The cross-sectional orthopaedic data presented are from the first interim analysis.

The XLH Registry (NCT03193476) was initiated in August 2017, aims to recruit 1,200 children and adults with XLH, and will run for 10 years. At the time of analysis (Last Patient In: 30/11/2020; Database Lock: 29/03/2021) 579 subjects diagnosed with XLH were enrolled from 81 hospital sites in 16 countries (360 (62.2%) children, 217 (37.5%) adults, and 2 subjects of unknown age).

Of subjects with retrospective clinical data available, skeletal deficits were the most frequently self-reported clinical problems for children (223/239, 93.3%) and adults (79/110, 71.8%). Retrospective fracture data were available for 183 subjects (72 children, 111 adults); 50 had a fracture (9 children, 41 adults). In children, fractures tended to occur in tibia/fibula and/or wrist; only adults reported large bone fractures. Joint conditions were noted for 46 subjects (6 children, 40 adults). For adults reporting osteoarthritis, knees (60%), hips (42.5%), and shoulders (22.5%) were the most frequently affected joints. Retrospective orthopaedic surgery data were collected for 151 subjects (52 children, 99 adults). Osteotomy was the most frequent surgery reported (n=108); joint replacements were recorded for adults only.

This is the largest set of orthopaedic data from XLH subjects collected to date. Longitudinal information collected during the 10-year Registry duration will generate real-world evidence which will help to inform clinical practice.

Authors acknowledge the contribution of all International XLH Registry Steering Committee members.

Common tendon injuries impair healing, leading to debilitation and an increased re-rupture risk. The impact of oxygen-sensing pathways on repair mechanisms, vital in regulating inflammation and fibrosis, remains unclear despite their relevance in tendon pathologies. Recent studies show that pulsed electromagnetic field (PEMF) reduce inflammation in human tendon cells (hTDCs) and in hypoxia-induced inflammation. We investigated the hypoxia's impact (1% and 2% oxygen tension) using magnetic cell sheet constructs (IL-1β-magCSs) primed with IL-1β. IL-1β-magCSs were exposed to low OT (1h, 4h,6h) in a hypoxic chamber. To confirm the role of PEMF (5Hz, 4mT, 50% duty cycle) on hypoxia modulation, IL-1β-magCSs, previously exposed to OT, were 1h-stimulated with PEMF. Our results show a significant increase in HIF- 1a and HIF-2a expression on IL-1β-magCSs after exposure to 2%-OT at all time points, compared to 1%- OT and normoxia. TNFa, IL-6, and IL-8 expression increased after 6 hours of 1%-OT exposure. PEMF stimulation of hypoxic IL-1β-magCSs led to decreased pro-inflammatory genes and increased anti-inflammatory (IL-4,IL-10) expression compared to unstimulated magCSs. IFN-g, TNF-α, and IL-6 release increased after 6 hours, regardless of %-OT, while IL-10 levels tended to rise after PEMF stimulation at 2%-OT. Also, NFkB expression was increased on IL-1β-magCSs exposed to 4 h and 6 h of 2%-OT, suggesting a link between NFkB and the production of pro-inflammatory factors. Moreover, PEMF stimulation showed a significantly decreased NFkB level in IL-1β-magCSs.

Overall, low OT enhances expression of hypoxia-associated genes and inflammatory markers in IL-1β-magCSs with the involvement of NFkB. PEMF modulates the response of magCSs, previously conditioned to hypoxia and to inflammatory triggers, favouring expression of anti-inflammatory genes and proteins, supporting PEMF impact in pro-regenerative tendon strategies.

Acknowledgements: ERC CoG MagTendon(No.772817), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND. Thanks to Hospital da Prelada for providing tendon tissue samples (Portugal), and TERM

RES Hub (Norte-01-0145-FEDER-022190).

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM.

A spine compression fracture is a very common form of fracture in elderly with osteoporosis. Injection of polymethyl methacrylate (PMMA) to fracture sites is a minimally invasive surgical treatment, but PMMA has considerable clinical risks. We develop a novel type thermoplastic injectable bone substitute contains the proprietary composites of synthetic ceramic bone substitute and absorbable thermoplastic polymer.

We used thermoplastic biocompatible polymers Polycaproactone (PCL) to encapsulate calcium-based bone substitutes hydroxyapatite (Ca10(PO4)6(OH)2, HA) and tricalcium phosphate (TCP) to form a biodegradable injectable bone composite material. The space occupation ration PCL:HA/TCP is 1:9. After heating process, it can be injected to fracture site by specific instrument and then self-setting to immediate reinforce the vertebral body.

The thermoplastic injection bone substitute can obtain good injection properties after being heated by a heater at 90˚C for three minutes, and has good anti-washout property when injected into normal saline at 37˚C. After three minutes, solidification is achieved. Mechanical properties were assessed using the material compression test system and the mechanical support close to the vertebral spongy bone.

In vitro cytotoxicity MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed and no cell cytotoxicity was observed.

In vivo study with three New Zealand rabbits was performed, well bone growth into bone substitute was observed and can maintain good mechanical support after three months implantation.

The novel type thermoplastic injection bone substitute can achieve (a) adequate injectability and viscosity without the risk of cement leakage; (b) adequate mechanical strength for immediate reinforcement and prevent adjacent fracture; (c) adequate porosity for new bone ingrowth; (e) biodegradability. It could be developed as a new option for treating vertebral compression fractures.

Articular cartilage (AC) and subchondral bone (SB) are intimately intertwined, forming a complex unit called the AC-SB interface. Our recent studies have shown that cartilage and bone marrow are connected by a three-dimensional network of microchannels (i.e. cartilage-bone marrow microchannel connector; CMMC), which differ microarchitecturally in number, size and morphology depending on the maturation stage of the bone and the region of the joint. However, the pathological significance of CMMC is largely unknown. Here, we quantitatively assessed how CMMC microarchitecture relates to cartilage condition and regional differences in early idiopathic osteoarthritis (OA).

Two groups of cadaveric female human femoral heads (intact cartilage vs early cartilage lesions) were identified and biopsy-based high-resolution micro-CT imaging was used. Subchondral bone (SB) thickness, CMMC number, maximum and minimum CMMC size, and CMMC morphology were quantified and compared between the two groups. The effect of joint region and cartilage condition on each dependent variable was examined.

The number and morphology of CMMCs were influenced by the region of the joint, but not by the cartilage condition. On the other hand, the minimum and maximum CMMC size was modified by both joint location and cartilage condition. The smallest CMMCs were consistently found in the load bearing region (LBR) of the joint. Compared to healthy subjects, the size of the microchannels was increased in early OA, most notably in the non-load bearing region (NLBR) and the peripheral rim (PR) of the femoral head. In addition, subchondral bone thinning was observed in early OA as a localized event associated with areas of partial chondral defect.

Our data suggest an enlargement of the SB microchannel network and a collective structural deterioration of the SB in early idiopathic OA.

A painful “dreaded black line” (DBL) has been associated with progression to complete fractures in atypical femur fractures (AFF). Adjacent sclerosis, an unrecognized radiological finding, has been observed in relation to the DBL. We document its incidence, associated features, demographics and clinical progression.

We reviewed plain radiographs of 109 incomplete AFFs between November 2006 and June 2021 for the presence of sclerosis adjacent to a DBL. Radiographs were reviewed for location of lesions, and presence of focal endosteal or periosteal thickening. We collected demographical data, type and duration of bisphosphonate therapy, and progression to fracture or need for prophylactic stabilization, with a 100% follow up of 72 months (8 – 184 months).

109 femurs in 86 patients were reviewed. Seventeen sclerotic DBLs were observed in 14 patients (3 bilateral), involving 15.6% of all femora and 29.8% of femora with DBLs. Location was mainly subtrochanteric (41.2%), proximal diaphyseal (35.3%) and mid-diaphyseal (23.5%), and were associated with endosteal or periosteal thickening. All patients were female, mostly Chinese (92.9%), with a mean age of 69 years. 12 patients (85.7%) had a history of alendronate therapy, and the remaining 2 patients had zoledronate and denosumab therapy respectively. Mean duration of bisphosphonate therapy was 62 months. 4 femora (23.5%) progressed to complete fractures that were surgically managed, whilst 6 femora (35.3%) required prophylactic fixation.

Peri-lesional sclerosis in DBL is a new radiological finding in AFFs, predominantly found in the proximal half of the femur, at times bilateral, and are always associated with endosteal or periosteal thickening. As a high proportion of patients required surgical intervention, these lesions could suggest non-union of AFFs, similar to the sclerotic margins commonly seen in fractures with non-union. The recognition of and further research into this new feature could shed more light on the pathophysiological progression of AFFs.

µCT images are commonly analysed to assess changes in bone density and architecture in preclinical murine models. Several platforms provide automated analysis of bone architecture parameters from volumetric regions of interest (ROI). However, segmentation of the regions of subchondral bone to create the volumetric ROIs remains a manual and time-consuming task. This study aimed to develop and evaluate automated pipelines for trabecular bone architecture analysis of mouse proximal tibia subchondral bone.

A segmented dataset involving 62 knees (healthy and arthritic) from 10-week male C57BL/6 mice were used to train a U-Net type architecture, with µCT scans (downsampled) input that output segmentation and bone volume density (BV/TV) of the subchondral trabecular bone. Segmentations were upsampled and used in tandem with the original scans (10µ) as input for architecture analysis along with the thresholded trabecular bone. The analysis considered the manually and U-Net segmented ROIs using two available pipelines: the ITKBoneMorphometry library and CTan (SKYSCAN). The analyses included: bone volume (BV), total volume (TV), BV/TV, trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp), and bone surface density (BSBV).

There was good agreement for bone measures between the manual and U-Net pipelines utilizing ITK (R=0.88-0.98) and CTan (R=0.91-0.98). ITK and CTan showed good agreement for BV, TV, BV/TV, TbTh and BSBV (R=0.9-0.98). However, a limited agreement was seen between TbN (R=0.73) and TbSb (R=0.59) due to methodological differences in how spacing is evaluated.

This U-Net/ITK pipeline seamlessly automated both segmentation and quantification of the proximal tibia subchondral bone. This automated pipeline allows the analysis of large volumes of data, and its open-source nature may enable the standardization of stereologic analysis of trabecular bone across different research groups.

There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture.

To assess the optimal concentration of MSCs for promoting fracture healing in a rat model.

Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time polymerase chain reaction.

Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post fracture, SDF-1, TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-β1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture.

The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures.

The objective of this study was to investigate how a new customizable light-curable osteosynthesis method (AdFix) compared to traditional metal hardware when loaded in torsion in an ovine phalanx model.

Twenty-one ovine proximal phalanges were given a 3mm transverse osteotomy and four 1.5mm cortex screws were inserted bicortically on either side of the gap. The light-curable polymer composite was then applied using the method developed by Hutchinson [1] to create osteosyntheses in two groups, having either a narrow (6mm, N=9) or a wide (10mm, N=9) fixation patch. A final group (N=3) was fixated with conventional metal plates. The constructs were loaded in torsion at a rate of 6°/second until failure or 45° of rotation was reached. Torque and angular displacement were measured, torsional stiffness was calculated as the slope of the Torque-Displacement curve, and maximum torque was queried for each specimen.

The torsional stiffnesses of the narrow, wide, and metal plate constructs were 39.1 ± 6.2, 54.4 ± 6.3, and 16.2 ± 3.0 Nmm/° respectively. All groups were statistically different from each other (p<0.001). The maximum torques of the narrow, wide, and metal plate constructs were 424 ± 72, 600 ± 120, and 579 ± 20 Nmm respectively. The narrow constructs were statistically different from the other two (p<0.05), while the wide and metal constructs were not statistically different from each other (p=0.76).

This work demonstrated that the torsional performance of the novel solution is comparable to metal fixators. As a measure of the functional range, the torsional stiffness in the AdhFix exceeded that of the metal plate. Furthermore, the wide patches were able to sustain a similar maximum toque as the metal plates. These results suggest AdhFix to be a viable, customizable alternative to metal implants for fracture fixation in the hand.

Due to their immunomodulatory and regenerative capacity, human bone marrow-derived mesenchymal stromal cells (hBMSCs) are promising in the treatment of polytrauma patients. However, few studies evaluated the effects of sera from polytraumatized patients on hBMSCs. The aim of this study was to explore changes in hBMSCs exposed to serum from polytrauma patients from different time points after trauma.

Sera from 84 patients on day 1 (D1), 5 (D5) and 10 (D10) after polytrauma (ISS ≥ 16) were pooled respectively to test the differential influence on hBMSC. As a control, sera from three healthy age- and gender-matched donors (HS) were collected. The pooled sera were analyzed by Multicytokine Array for pro-/anti-inflammatory cytokines. For the cell culture experiments, hBMSCs from four healthy donors were used. The influence of the different sera on hBMSC regarding cell proliferation, colony forming unit-fibroblast (CFU-F) assay, cell viability and toxicity, cell migration, as well as osteogenic and chondrogenic differentiation was analyzed. One-Way-ANOVA and LSD-test were used for the parametric, Kruskal-Wallis-test for non-parametric data. p≤0.05 was considered as statistically significant.

The results showed that D5 serum reduced hBMSCs cell proliferation capacity by 41.26% (p=0.000) compared with HS and increased the proportion of dead cells by 3.19% (p=0.008) and 2.25% (p=0.020) compared with D1 and D10. The frequency of CFU-F was reduced by 49.08% (p=0.041) in D5 and 53.99% (p=0.027) in D10 compared with HS, whereas the other parameters were not influenced.

The serological effect of polytrauma on hBMSCs was related to the time after trauma. It is disadvantageous to use BMSCs in polytraumatized patients five days after the incidence as obvious cytological changes could be found at that time point. However, it is promising to use hBMSCs to treat polytrauma after 10 days, combined with the concept of “Damage Control Orthopaedics” (DCO).

Following anterior cruciate ligament reconstruction (ACLR) using a semitendinosus (ST) autograft measures such as length, cross-sectional area, and volume may not fully describe the effects of tendon harvest on muscle morphology as these discrete measures cannot characterize three-dimensional muscle shape. This study aimed to determine between-limb ST shape similarity and regional morphology in individuals with a unilateral history of ACLR using a ST graft, and healthy controls.

A secondary analysis of magnetic resonance imaging was undertaken from 18 individuals with unilateral history of ST ACLR and 18 healthy controls. ST muscles were manually segmented, and shape similarity were assessed between limbs and groups using Jaccard index (0-1) and Hausdorff distance (mm). ST length (cm), peak cross-sectional area (CSA) (cm²), and volume (cm³) was compared between surgically reconstructed and uninjured contralateral limbs, and between the left and right limbs of control participants with no history of injury. Cohen's d was reported as a measure of effect size.

Compared to healthy controls, the ACLR group had significantly (p<0.001, d= −2.33) lower bilateral ST shape similarity. Furthermore, the deviation in muscle shape was significantly (p<0.001, d= 2.12) greater in the ACLR group. Within the ACLR group, maximum Hausdorff distance indicated ST from the ACLR limb deviated (23.1±8.68 mm) from the shape of the healthy contralateral ST, this was observed particularly within the distal region of the muscle. Compared to the uninjured contralateral limb and healthy controls, deficits in peak cross-sectional area and volume in ACLR group were largest in proximal (p<0.001, d= −2.52 to −1.28) and middle (p<0.001, d= −1.81 to −1.04) regions.

Findings highlight morphological features in distal ST not identified by traditional discrete morphology measures. ST shape was most different in the distal region of the muscle, despite deficits in CSA and volume being most pronounced in proximal and middle regions. ST shape following ACLR may affect force transmission and distribution within the hamstrings and contribute to persistent deficits in knee flexor and internal rotator strength.

Knee swelling is common after injury or surgery, resulting in pain, restricted range of movement and limited mobility. Accurately measuring knee swelling is critical to assess recovery. However, current measurement methods are either unreliable or expensive [1,2]. Therefore, a new measurement method is developed. This wearable (the ‘smart brace’) has shown the ability to distinguish a swollen knee from a not swollen knee using multi-frequency-bio impedance analysis (MF-BIA) [3].

This study aimed to determine the accuracy of this smart brace. The study involved 25 usable measurements on patients treated for unilateral knee osteoartritis with a 5mL injection of Lidocaïne + DepoMedrol (1:4). MF-BIA measurements were taken before and after the injection, both on the treated and untreated knee. The smart brace accurately measured the effect of the injection by a decrease in resistance of up to 2.6% at 100kHz (p<0.01), where commonly used gel electrodes were unable to measure the relative difference. Remarkably, both the smart brace and gel electrodes showed a time component in the MF-BIA measurements.

To further investigate this time component, 10 participants were asked to lie down for 30 minutes, with measurements taken every 3 minutes using both gel electrodes and the smart brace on both legs. The relative change between each time step was calculated to determine changes over time. The results showed presence of a physiological aspect (settling of knee fluids), and for the brace also a mechanical aspect (skin-electrode interface) [4]. The mechanical aspect mainly interfered with reactance values.

Overall, the smart brace is a feasible method for quantitatively measuring knee swelling as a relative change over time. However, the skin-electrode interface should be improved for reliable measurements at different moments in time. The findings suggest that the smart brace could be a promising tool for monitoring knee swelling during rehabilitation.

Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats.

Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT.

Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling.

The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects.

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells.

For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed.

We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored.

A Morel-Lavallee lesion (MLL) is a benign cystic lesion that occurs due to injury to the soft-tissue envelope's perforating vascular and lymphatic systems, resulting in a distinctive hemolymphatic fluid accumulation between the tissue layers. The MLL has the potential to make a significant impact on the treatment of orthopaedic injuries.

A 79-year-old male patient community ambulatory with assisting aid (cane) known case of Diabetes mellitus, hypertension, bronchial asthma and ischemic heart disease. He was brought to the Emergency, complaining of right hip discomfort and burning sensation for the last 5 days with no history of recent trauma at all. Patient had history of right trochanteric femur fracture 3 years ago, treated with DHS in a privet service. Clinical and Radiological assessment showed that the patient mostly has acute MLL due to lag screw cut out. We offered the patient the surgical intervention, but he refused despite explaining the risks of complications if not treated and preferred to receive the conservative treatment. Compression therapy management explained to him including biker's shorts (instructed to be worn full-time a day) and regular follow up in clinic. Symptom's improvement was reported by the patient in the subsequent visits.

In the polytrauma patient, a delayed diagnosis of these lesions is conceivable due to the presence of more visible injuries. It's located over the greater trochanter more commonly, but sometimes in other areas such as the lower lumbar region, the thigh, or the calf. Incorrect or delayed diagnosis and care can have unfavorable outcomes such as infection, pseudocyst development, and cosmetologically deformity. Magnetic resonance imaging (MRI) and ultrasound will aid in MLL diagnosis. However, the effectiveness of MLL therapy remains debatable.

We strongly believe that the MLL caused due to tangential shear forces applied to the soft tissue leads to accumulation of the blood and/or lymph between the subcutaneous and overlying fascia and it often misdiagnosed due to other distracting injuries. Nontheless, in our case we reported MLL occur due to internal pressure on the fascia caused by cut out of DHS lag screw.

To quantify bone-nail fit in response to varying nail placements by entry point translation in straight antegrade humeral nailing using three-dimensional (3D) computational analysis

CT scans of ten cadaveric humeri were processed in 3D Slicer to obtain 3D models of the cortical and cancellous bone. The bone was divided into individual slices each consisting of 2% humeral length (L) with the centroid of each slice determined. To represent straight antegrade humeral nail, a rod consisting of two cylinders with diameters of 9.5mm and 8.5mm and length of 0.22L mm and 0.44L mm respectively joined at one end was modelled. The humeral head apex (surgical entry point) was translated by 1mm in both anterior-posterior and medio-lateral directions to generate eight entry points. Total nail protrusion surface area, maximum nail protrusion distance into cortical shell and top, middle, bottom deviation between nail and intramedullary cavity centre were investigated. Statistical analysis between the apex and translated entry points was conducted using paired t-test.

A posterior-lateral translation was considered as the optimal entry point with minimum protrusion in comparison to the anterior-medial translation experiencing twice the level of protrusion. Statistically significant differences in cortical protrusion were found in anterior-medial and posterior-lateral directions producing increased and decreased level of protrusion respectively compared to the apex. The bottom anterior-posterior deviation distance appeared to be a key predictor of cortical breach with the distal nail being more susceptible. Furthermore, nails with anterior translation generated higher anterior-posterior deviation (>4mm) compared to posterior translation (<3mm).

Aside from slight posterolateral translation of the entry point from the apex, inclusion of a distal posterior-lateral bend into current straight nail design could improve nail fitting within the curved humeral bone, potentially improving distal working length within the flat and narrow medullary canal of the distal humeral shaft.

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD.

Mechanical loading is important to maintain the homeostasis of the intervertebral disc (IVD) under physiological conditions but can also accelerate cell death and tissue breakdown in a degenerative state. Bioreactor loaded whole organ cultures are instrumental for investigating the effects of the mechanical environment on the IVD integrity and for preclinical testing of new therapies under simulated physiological conditions. Thereby the loading parameters that determine the beneficial or detrimental reactions largely depend on the IVD model and its preparation. Within this symposium we are discussing the use of bovine caudal IVD culture models to reproduce tissue inflammation or matrix degradation with or without bioreactor controlled mechanical loading. Furthermore, the outcome parameters that define the degenerative state of the whole IVD model will be outlined. Besides the disc height, matrix integrity, cell viability and phenotype expression, the tissue secretome can provide indications about potential interactions of the IVD with other cell types such as neurons. Finally, a novel multiaxial bioreactor setup capable of mimicking the six degrees-of-freedom loading environment of IVDs will be introduced that further advances the relevance of preclinical ex-vivo testing.

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration.

Recent researches indicate that both M1 and M2 macrophages play vital roles in tissue repair and foreign body reaction processes. In this study, we investigated the dynamics of M1 macrophages in the induced membrane using a mouse femur critical-sized bone defect model.

The Masquelet method (M) and control (C) groups were established using C57BL/6J male mice (n=24). A 3mm-bone defect was created in the right femoral diaphysis followed by a Kirschner wire fixation, and a cement spacer was inserted into the defect in group M. In group C, the bone defect was left uninserted. Tissues around the defect were harvested at 1, 2, 4, and 6 weeks after surgery (n=3 in each group at each time point). Following Hematoxylin and eosin (HE) staining, immunohistochemical staining (IHC) was used to evaluate the CD68 expression as a marker of M1 macrophage. Iron staining was performed additionally to distinguish them from hemosiderin-phagocytosed macrophages.

In group M, HE staining revealed a hematoma-like structure, and CD68-positive cells were observed between the spacer and fibroblast layer at 1 week. The number of CD68-positive cells decreased at 2 weeks, while they were observed around the new bone at 4 and 6 weeks. In group C, fibroblast infiltration and fewer CD68-positive cells were observed in the bone defect without hematoma-like structure until 2 weeks, and no CD68-positive cells were observed at 4 and 6 weeks. Iron staining showed hemosiderin deposition in the surrounding area of the new bone in both groups at 4 and 6 weeks. The location of hemosiderin deposition was different from that of macrophage aggregation.

This study suggests that M1 macrophage aggregation is involved in the formation of induced membranes and osteogenesis and may be facilitated by the presence of spacers.

The aim of the ongoing projects was to demonstrate the efficacy of autologous bone marrow derived stem cells (MSC) combined with biomaterial to induced new bone formation in a randomized multicenter controlled clinical trial.

Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8–12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the projects.

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel.

ASCs at 2*10⁶ cells/mL were embedded in a 3D VitroGel RGD^® hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated.

Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis.

These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE.

Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis).

Management of ankle arthritis in young patients is challenging. Although ankle arthrodesis gives consistent pain relief, it leads to loss of function and adjacent joint arthritis. Ankle joint distraction (AJD) has been shown to give good outcomes in adults with osteoarthritis or post-traumatic arthritis. The efficacy in children or young adults and those with juvenile idiopathic arthritis is less well evidenced.

Clinical notes and radiographs of all patients (n=6) managed with AJD in one tertiary referral centre were retrospectively reviewed. Radiographs were taken pre-surgery, intra-operatively, 1 month following frame removal and at the last follow up, tibiotalar joint space was assessed using ImageJ software. Measurements were taken at the medial, middle and lateral talar dome using frame components as reference. Radiographic data for patients with a good clinical outcome was compared with those who did not.

At time of surgery mean age was 16.1 years (12 – 25 years). Mean follow up was 3.4 years (1.5 – 5.9 years). Indications were juvenile idiopathic arthritis (4) post-traumatic (1) and post-infective arthritis (1). Three patients at last follow up had a good clinical outcome. Two patients required revision to arthrodesis (1.3 and 2.4 years following distraction). One patient had spontaneous fusion. One patient required oral antibiotics for pin site infection.

Inter-observer reliability was 95%. Mean joint space was 1.17mm (SD = 0.87mm) pre-operatively which increased to 6.72mm (SD = 2.23mm) at the time of distraction and 2.09mm (SD = 1.14mm) at the time of removal. At one-year follow up, mean joint space was 1.96mm (SD = 1.97mm).

Outcomes following AJD in this population are variable although significant benefits were demonstrated for 50% of the patients in this series. Radiographic joint space preoperatively did not appear to be associated with need for arthrodesis. Further research in larger groups of young patients is required.

Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages.

Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by flow cytometry (FC) after staining with cell-mask-green and anti-CD81 antibody. EV cargo analysis was conducted using next-generation sequencing (NGS).

Our data confirmed the release of EVs from all MSC strains used in the study. There were no correlations between the number of particles and the number of EVs released in the CM, and between the number of EVs released and the strain age. Nevertheless, some of the lowest concentrations of EVs were found in the CM of strains over 70 years of age, which exhibited a low/absent chondrogenic and osteogenic differentiation potential. In contrast, iPSC-MSCs, which exhibited a high growth and three-lineage differentiation potential, released a similar amount of EVs as the best performing bone marrow MSC strain. NGS analysis identified several microRNAs that were significantly enriched in EVs of young MSC strains exhibiting low senescence, and those that were enriched in EVs of strains exhibiting high differentiation potentials. Gender had no influence on microRNA profiles in EVs or releasing MSCs.

Taken together, our data provides new insights into the properties of MSC vesicular secretome and its therapeutic potential during aging.