To create a comprehensive, user-friendly, database that facilitates selection of optimized animal models for fracture research. Preclinical testing using research animal models can expedite effective and safe interventions for clinical fracture patients but ethical considerations (e.g., adherence to 3R humane principles) and failure to meet critical review (e.g., clinical translation, reproducibility) currently complicate the model selection process. English language publications (1980-2021) were derived from PubMed® using the search-term ‘bone and fracture and animal’. Clinical cases, reviews, and cadaver studies were excluded. Qualifying papers reporting use of fracture models had the following data transcribed: Author, journal, abstract, summary data, animal data, bone, focus (e.g., allograft) and model (e.g., articular fracture). Publications were quantitatively scored (1 star [very poor] – 5 stars [excellent]) for reproducibility, clinical translation and animal welfare. 4602 papers were derived from 677 journals from 177 publishers. Number of annual publications progressively increased from 18 (1980), peaking in 2015 (250) before substantially declining in 2020 (121) and 2021 (51). Descriptors (low to high) included 15 species (frog [1]–rat [1586]), 24 bones (phalanx [1]–femur [1646]), 134 research foci (bioprinting [4]–fracture healing [3533]), and 37 fracture models (avulsion [4]–diaphyseal [2113]). Percent of total publications scoring 1 or more stars for reproducibility, clinical translation and animal welfare ranged from: 1.0–5.8% (1 star), 5.9–30.6% (2 star), 21.3–42.8% (3 star), 19.2–44.4% (4 stars), and 1.3–26.7% (5 stars). FRAMD provides a dedicated resource that enhances selection of animal models that pertain to researchers’ fracture focus while being clinically relevant, reproducible and humane. FRAMD will help improve scientific data, reduce unnecessary use of animals, heighten workplace efficiency, and reduce cost by avoiding ill-suited or outdated models. FRAMD may particularly benefit grant writers and organizations seeking ‘best-practice’ assurance (e.g., funding agencies, academic research societies, CROs)

Aim. To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 10. 4. CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 10. 4. CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results. The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomyelitis, i.e., osteolysis, while only one animal in group B did, and none in group C. Histopathological examination of the bones showed more extensive inflammation in group A animals compared to those in group B, which showed more osteoid formation. Conclusions. Formation and separation of low metabolism bacterial aggregates into different size fractions was possible. The aggregates can be used as inoculum in the porcine IAO model, with microbiological re-isolation from both implants and tissue. Furthermore, the aggregates caused a less aggressive IAO, than the planktonic counterparts. Using aggregated bacteria as inoculum appears to be more relevant to the clinical situation of infecting bacteria

Introduction. Degenerative meniscal tears are the most common meniscal lesions, representing huge clinical and socio-economic burdens. Their role in knee osteoarthritis (OA) onset and progression is well established and demonstrated by several retrospective studies. Effective preventive measures and non-surgical treatments for degenerative meniscal lesions are still lacking, also because of the lack of specific and accurate animal models in which test them. Thus, we aim to develop and validate an accurate animal model of meniscus degeneration. Method. Three different surgical techniques to induce medial meniscus degenerative changes in ovine model were performed and compared. A total of 32 sheep (stifle joints) were subjected to either one of the following surgical procedures: a) direct arthroscopic mechanical meniscal injury; b) peripheral devascularization and denervation of medial meniscus; c) full thickness medial femoral condyle cartilage lesion. In all the 3 groups, the contralateral joint served as a control. Result. From a visual examination of the knee joint emerged a clear difference between control and operated groups, in the menisci but also in the cartilage, indicating the onset of OA-related cartilage degeneration. The meniscal and cartilaginous lesions were characterized by different severity and location in the different groups. For instance, a direct meniscal injury caused cartilaginous lesions especially in the medial part of the condyles, and the other approaches presented specific signature. Evaluation of scoring scales (e.g. ICRS score) allowed the quantification of the damage and the identification of differences among the four groups. Conclusion. We were effectively able to develop and validate a sheep model of meniscal degeneration which led to the onset of OA. This innovative model will allow to test in a pre-clinical relevant setting innovative approaches to prevent meniscal-related OA. Funding. Project PNRR-MAD-2022-12375978 funded by Italian Ministry of Health

Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:. Source – while MSCs can be recovered from many tissues, the predominant sources for autologous MSCs have been bone and fat. Other sources, including blood, amnion, synovium, and dental pulp have also been commercialised for allogenic treatments. Preparation – ex vivo culture requires transport from a licensed laboratory while ‘minimally manipulated’ preparations can be prepared patient-side. Cells also need a vehicle for transport and implantation. Delivery – transport of cells from the laboratory to the clinic for autologous ex vivo culture techniques; implantation technique (usually by ultrasound-guided injection to minimise damage to the cells (or, more rarely, incorporated into a scaffold). They can also be delivered by regional perfusion via venous or arterial routes. Retention – relatively poor although small numbers of cells do survive for at least 5 months. Immediate loss to the lungs if the cells are administered via vascular routes. Synovially administered cells do not engraft into tendon. Adverse effects – very safe although needle tracts often visible (but do not seen to adversely affect the outcome). Allogenic cells require careful characterisation for MHC Class II antigens to avoid anaphylaxis or reduced efficacy. Appropriate injuries to treat – requires a contained lesion when administered via intra-lesional injection. Intrasynovial tendon lesions are more often associated with surface defects and are therefore less appropriate for treatment. Earlier treatment appears to be more effective than delayed, when implantation by injection is more challenging. Efficacy - beneficial effects shown at both tissue and whole animal (clinical outcome) level in naturally-occurring equine tendinopathy using bone marrow-derived autologous MSCs Recent (licenced) allogenic MSC treatment has shown equivalent efficacy while intra-synovial administration of MSCs is ineffective for open intra-synovial tendon lesions. Regulatory hurdles – these have been lighter for veterinary treatments which has facilitated their development. There has been greater regulation of commercial allogenic MSC preparations which have required EMA marketing authorisation

Introduction. Femoral head osteonecrosis (FHO) is a condition in which the inadequate blood supply disrupts osteogenic-angiogenic coupling that results in diminishment of femoral perfusion and ends up with FHO. The insufficient knowledge on molecular background and progression pattern of FHO and the restrictions in obtaining human samples bring out the need for a small animal trauma model to research FHO aetiology. Hence, this study aims to develop a mouse trauma model to elucidate the molecular mechanisms behind FHO. Method. Left femoral head was dislocated from the hip joint, ligamentum teres was cut, and a slight circular incision was done around the femoral neck of 8-week-old male C57BL/6J mice to disrupt the blood supply to femoral head. Right hip joint was left unoperated as control. Animals (n=5 per time point) were sacrificed on 2-3-4-6-8-10-12 weeks, and ex-vivo µCT was taken to assess bone structural parameters. Haematoxylin/eosin (HE)- and immunohistochemical-staining (IHCS) for CD31 and EMCN were done to observe histology and marrow-specific H-type vascular structures, respectively. Result. μCT assessment showed trabecular bone loss and decreased BV/TV from 2 to 8 weeks in FHO side. HE staining displayed the increased number of empty lacunae was observed in FHO side as early as 24h after operation. By 4. th. week, IHCS results displayed the invasion of the epiphyseal plate by H-type blood vessels in FHO side, while the epiphyseal plate was observed intact in control side. Also, by 6. th. week the HE-staining showed the presence of bone marrow necrosis and bone fat accumulation in FHO side. Conclusion. Trabecular bone loss, increased number of empty lacunae, bone fat imbalance and bone marrow necrosis are reported as the signs of osteonecrosis. Thus, our results are coherent with the literature and indicated that we were able to effectively generate a trauma model for FHO in mice for the first time in literature

Aims. Periprosthetic joint infections (PJIs) are rare, but represent a great burden for the patient. In addition, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing. The aim of this rat experiment was therefore to compare the antibiotics commonly used in the treatment of PJIs caused by MRSA. Methods. For this purpose, sterilized steel implants were implanted into the femur of 77 rats. The metal devices were inoculated with suspensions of two different MRSA strains. The animals were divided into groups and treated with vancomycin, linezolid, cotrimoxazole, or rifampin as monotherapy, or with combination of antibiotics over a period of 14 days. After a two-day antibiotic-free interval, the implant was explanted, and bone, muscle, and periarticular tissue were microbiologically analyzed. Results. Vancomycin and linezolid were able to significantly (p < 0.05) reduce the MRSA bacterial count at implants. No significant effect was found at the bone. Rifampin was the only monotherapy that significantly reduced the bacterial count on implant and bone. The combination with vancomycin or linezolid showed significant efficacy. Treatment with cotrimoxazole alone did not achieve a significant bacterial count reduction. The combination of linezolid plus rifampin was significantly more effective on implant and bone than the control group in both trials. Conclusion. Although rifampicin is effective as a monotherapy, it should not be used because of the high rate of resistance development. Our animal experiments showed the great importance of combination antibiotic therapies. In the future, investigations with higher case numbers, varied bacterial concentrations, and changes in individual drug dosages will be necessary to be able to draw an exact comparison, possibly within a clinical trial. Cite this article: Bone Joint Res 2022;11(3):143–151

Objectives. The treatment of osteoporotic fractures is a major challenge, and the enhancement of healing is critical as a major goal in modern fracture management. Most osteoporotic fractures occur at the metaphyseal bone region but few models exist and the healing is still poorly understood. A systematic review was conducted to identify and analyse the appropriateness of current osteoporotic metaphyseal fracture animal models. Materials and Methods. A literature search was performed on the Pubmed, Embase, and Web of Science databases, and relevant articles were selected. A total of 19 studies were included. Information on the animal, induction of osteoporosis, fracture technique, site and fixation, healing results, and utility of the model were extracted. Results. Fracture techniques included drill hole defects (3 of 19), bone defects (3 of 19), partial osteotomy (1 of 19), and complete osteotomies (12 of 19). Drill hole models and incomplete osteotomy models are easy to perform and allow the study of therapeutic agents but do not represent the usual clinical setting. Additionally, biomaterials can be filled into drill hole defects for analysis. Complete osteotomy models are most commonly used and are best suited for the investigation of therapeutic drugs or noninvasive interventions. The metaphyseal defect models allow the study of biomaterials, which are associated with complex and comminuted osteoporotic fractures. Conclusion. For a clinically relevant model, we propose that an animal model should satisfy the following criteria to study osteoporotic fracture healing: 1) induction of osteoporosis, 2) complete osteotomy or defect at the metaphysis unilaterally, and 3) internal fixation. Cite this article: R. M. Y. Wong, M. H. V. Choy, M. C. M. Li, K-S. Leung, S. K-H. Chow, W-H. Cheung, J. C. Y. Cheng. A systematic review of current osteoporotic metaphyseal fracture animal models. Bone Joint Res 2018;7:6–11. DOI: 10.1302/2046-3758.71.BJR-2016-0334.R2

Surgeons treating fractures with many small osteochondral fragments have often expressed the clinical need for an adhesive to join such fragments, as an adjunct to standard implants. If an adhesive would maintain alignment of the articular surfaces and subsequently heal it could result in improved clinical outcomes. However, there are no bone adhesives available for clinical indications and few pre-clinical models to assess safety and efficacy of adhesive biomaterial candidates. A bone adhesive candidate based on water, α-TCP and an amino acid phosphoserine was evaluated in-vivo in a novel murine bone core model (preliminary results presented EORS 2019) in which excised bone cores were glued back in place and harvested @ 0, 3, 7, 14, 28 and 42days. Adhesive pull-out strength was demonstrated 0–28 days, with a dip at 14 days increasing to 11.3N maximum. Histology 0–42 days showed the adhesive progressively remodelling to bone in both cancellous and cortical compartments with no signs of either undesirable inflammation or peripheral ectopic bone formation. These favourable results suggested translation to a large animal model. A porcine dental extraction socket model was subsequently developed where dental implants were affixed only with the adhesive. Biomechanical data was collected @ 1, 14, 28 and 56 days, and histology at 1,14,28 and 56 days. Adhesive strength assessed by implant pull-out force increased out to 28 days and maintained out to 56 days (282N maximum) with failure only occurring at the adhesive bone interface. Histology confirmed the adhesive's biocompatibility and osteoconductive behavior. Additionally, remodelling was demonstrated at the adhesive-bone interface with resorption by osteoclast-like cells and followed by new bone apposition and substitution by bone. Whilst the in-vivo dental implant data is encouraging, a large animal preclinical model is needed (under development) to confirm the adhesive is capable of healing, for example, loaded osteochondral bone fragments. Acknowledgements: The murine study was supported, in part, by the Swedish Foundation for Strategic Research (#RMA15-0110)

Aims. Periprosthetic joint infections (PJIs) and osteomyelitis are clinical challenges that are difficult to eradicate. Well-characterized large animal models necessary for testing and validating new treatment strategies for these conditions are lacking. The purpose of this study was to develop a rabbit model of chronic PJI in the distal femur. Methods. Fresh suspensions of Staphylococcus aureus (ATCC 25923) were prepared in phosphate-buffered saline (PBS) (1 × 10. 9. colony-forming units (CFUs)/ml). Periprosthetic osteomyelitis in female New Zealand white rabbits was induced by intraosseous injection of planktonic bacterial suspension into a predrilled bone tunnel prior to implant screw placement, examined at five and 28 days (n = 5/group) after surgery, and compared to a control aseptic screw group. Radiographs were obtained weekly, and blood was collected to measure ESR, CRP, and white blood cell (WBC) counts. Bone samples and implanted screws were harvested on day 28, and processed for histological analysis and viability assay of bacteria, respectively. Results. Intraosseous periprosthetic introduction of planktonic bacteria induced an acute rise in ESR and CRP that subsided by day 14, and resulted in radiologically evident periprosthetic osteolysis by day 28 accompanied by elevated WBC counts and histological evidence of bacteria in the bone tunnels after screw removal. The aseptic screw group induced no increase in ESR, and no lysis developed around the implants. Bacterial viability was confirmed by implant sonication fluid culture. Conclusion. Intraosseous periprosthetic introduction of planktonic bacteria reliably induces survivable chronic PJI in rabbits. Cite this article: Bone Joint Res 2021;10(3):156–165

Objectives. The primary purpose of this meta-analysis was to determine whether statin usage could reduce the risk of glucocorticoid-related osteonecrosis in animal models. Methods. A systematic literature search up to May 2015 was carried out using the PubMed, Ovid, EBM reviews, ISI Web of Science, EBSCO, CBM, CNKI databases with the term and boolean operators: statins and osteonecrosis in all fields. Risk ratio (RR), as the risk estimate of specific outcome, was calculated along with 95% confidence intervals (CI). The methodological quality of individual studies was assessed using a quantitative tool based on the updated Stroke Therapy Academic Industry Roundtable (STAIR) recommendations. Results. A total of 11 eligible studies were included according to predetermined criteria. The pooled data demonstrated that animals with statin usage, either alone or combined with other treatments, were at a decreased risk of developing glucocorticoid-related osteonecrosis (RR = 2.06, 95% confidence interval (CI) 1.71 to 2.50). Moreover, subgroup analysis revealed that compared with statins alone, statins combined with other treatments significantly decreased the risk of osteonecrosis (RR = 1.23, 95% CI 1.02 to 1.47). However, we could find no significant risk difference for different gender, or for different time points. Conclusions. The present study suggests that statins combined with other treatments are efficient in preventing the development of glucocorticoid-related osteonecrosis in animals. These results might shed light on clinical practice when glucocorticoids are prescribed, and could be further investigated in high-quality clinical trials. Cite this article: Z. Yang, H. Liu, D. Li, X. Xie, T. Qin, J. Ma, P. Kang. The efficacy of statins in preventing glucocorticoid-related osteonecrosis in animal models: A meta-analysis. Bone Joint Res 2016;5:393–402. DOI: 10.1302/2046-3758.59.2000500

Cartilage repair in terms of replacement, or regeneration of damaged or diseased articular cartilage with functional tissue, is the ‘holy grail’ of joint surgery. A wide spectrum of strategies for cartilage repair currently exists and several of these techniques have been reported to be associated with successful clinical outcomes for appropriately selected indications. However, based on respective advantages, disadvantages, and limitations, no single strategy, or even combination of strategies, provides surgeons with viable options for attaining successful long-term outcomes in the majority of patients. As such, development of novel techniques and optimisation of current techniques need to be, and are, the focus of a great deal of research from the basic science level to clinical trials. Translational research that bridges scientific discoveries to clinical application involves the use of animal models in order to assess safety and efficacy for regulatory approval for human use. This review article provides an overview of animal models for cartilage repair. Cite this article: Bone Joint Res 2014;4:89–94

Cite this article: Bone Joint Res 2022;11(8):514–517.

Aim. In trauma surgery, the development of biomaterial-associated infections (BAI) is one of the most common complications affecting trauma patients, requiring prolonged hospitalization and the intensive use of antibiotics. Following the attachment of bacteria on the surface of the biomaterial, the biofilm-forming bacteria could initiate a chronic implant-related infection. Despite the use of conventional local and systemic antibiotic therapies, persistent biofilms involve various resistance mechanisms that contribute to therapeutic failures. The development of in vivo chronic BAI models to optimize antibiofilm treatments is a major challenge. Indeed, the biofilm pathogenicity and the host response need to be finely regulated, and compatible with the animal lifestyle. Previously, a Galleria mellonella larvae model for the formation of an early-stage biofilm on the surface of a Kirschner (K)-wire was established. In the present study, two models of mature biofilm using clinical Staphylococcus aureus strains were assessed: one related to contaminated K-wires (in vitro biofilm maturation) and the second to hematogenous infections (in vivo biofilm maturation). Rifampicin was used as a standard drug for antibiofilm treatment. Method. In the first model, biofilms were formed following an incubation period (up to 7 days) in the CDC Biofilm Reactor (CBR, BioSurface Technologies). Then, after implantation of the pre-incubated K-wire in the larvae, rifampicin (80 mg/kg) was injected and the survival of the larvae was monitored. In the second model, biofilm formation was achieved after an incubation period (up to 7 days) inside the larvae and then, after removing the K-wires from the host, in vitro rifampicin susceptibility assays were performed (according to EUCAST). Results. The first model indicate that in vitro biofilm maturation affects the bacterial pathogenicity in the host, depending on the S. aureus strain used. Furthermore, the more the biofilm is matured, the more the rifampicin treatment efficiency is compromised. The second model shows that, despite the fast in vivo biofilm formation in the host, the number of bacteria, either attached to the surface of the K-wire surface or in surrounding tissue of the larvae, was not increased over time. Conclusions. Altogether, these results allow the establishment of biofilm models using G. mellonella larvae in order to understand the impact of biofilm maturation on both the bacterial pathogenicity and the efficiency of antibiofilm treatments

Aim. Treatment of infected and non-infected non-unions remain a major challenge after orthopedic fracture-related surgery. In clinical practice, several revision surgeries are usually required, including a radical debridement and exchange of implants, to control or even eradicate the infection to finally achieve bone healing. However, a clear treatment algorithm in clinical practice may be difficult to follow due to the heterogeneous patient population. Thus, so controlled settings for research purposes is better achieved in standardized animal studies. So far, there exists no multi-stage animal model that can be realistically transferred to the clinical situation in humans. The importance of such a model is obvious in order to be able to investigate different therapy concepts for infected and non-infected non unions. Methods. In 20 female Sprague-Dawley rats, a critical size defect by a femur osteotomy with 5 mm width was done. The periosteum at the fracture zone was cauterized proximal and distal to the osteotomy to achieve an hypovascularized situation. After randomization, 10 animals were intramedullary infected with a multisensible Staph. aureus strain (10. 3. CFU). After 5 weeks, a second surgery was performed with removing the K-wire, debridement of the osteotomy-gap and re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and underwent biomechanical testing to evaluate bone consolidation or delayed union, respectively. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, by the score of Lane and Sandhu and to quantify callus formation and the mineralized area of the callus. Results. 5 weeks after the first surgery a non-union had formed in all septic and aseptic animals. According to the Lane and Sandhu score a significantly higher callus formation was found in the infected group. In all infected animals, the inoculated Staph. aureus strain was detected during the revision surgery. 8 weeks after the second surgery no bone healing could be detected in the µ-CT analysis in both groups and biomechanical testing showed a significant lower maximum torque in both groups as compared to the untreated contralateral femura. Conclusion. Here we show first results of a new two-stage pseudarthrosis animal model, which reflects a very realistic clinical situation of an infection-related non-union model. Based on this model, various therapeutic strategies in the treatment of infectious and non-infectious pseudarthrosis, such as the use of bone substitutes, can be evaluated in further studies

Tendinopathy is a debilitating musculoskeletal condition which can cause significant pain and lead to complete rupture of the tendon, which often requires surgical repair. Due in part to the large spectrum of tendon pathologies, these disorders continue to be a clinical challenge. Animal models are often used in this field of research as they offer an attractive framework to examine the cascade of processes that occur throughout both tendon pathology and repair. This review discusses the structural, mechanical, and biological changes that occur throughout tendon pathology in animal models, as well as strategies for the improvement of tendon healing. Cite this article: Bone Joint Res 2014;3:193–202

Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications

Objectives. We studied subchondral intraosseous pressure (IOP) in an animal model during loading, and with vascular occlusion. We explored bone compartmentalization by saline injection. Materials and Methods. Needles were placed in the femoral condyle and proximal tibia of five anaesthetized rabbits and connected to pressure recorders. The limb was loaded with and without proximal vascular occlusion. An additional subject had simultaneous triple recordings at the femoral head, femoral condyle and proximal tibia. In a further subject, saline injections at three sites were carried out in turn. Results. Loading alone caused a rise in subchondral IOP from 11.7 mmHg (. sd. 7.1) to 17.9 mmHg (. sd. 8.1; p < 0.0002). During arterial occlusion, IOP fell to 5.3 mmHg (. sd. 4.1), then with loading there was a small rise to 7.6 mmHg (. sd. 4.5; p < 0.002). During venous occlusion, IOP rose to 20.2 mmHg (. sd. 5.8), and with loading there was a further rise to 26.3 mmHg (. sd. 6.3; p < 0.003). The effects were present at three different sites along the limb simultaneously. Saline injections showed pressure transmitted throughout the length of the femur but not across the knee joint. Conclusion. This is the first study to report changes in IOP in vivo during loading and with combinations of vascular occlusion and loading. Intraosseous pressure is not a constant. It is reduced during proximal arterial occlusion and increased with proximal venous occlusion. Whatever the perfusion state, in vivo load is transferred partly by hydraulic pressure. We propose that joints act as hydraulic pressure barriers. An understanding of subchondral physiology may be important in understanding osteoarthritis and other bone diseases. Cite this article: M. Beverly, S. Mellon, J. A. Kennedy, D. W. Murray. Intraosseous pressure during loading and with vascular occlusion in an animal model. Bone Joint Res 2018;7:511–516. DOI: 10.1302/2046-3758.78.BJR-2017-0343.R2

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

INTRODUCTION. In the treatment of nonunions, and other complications of bone repair, an attractive alternative to bone autografts would be the use of a combination of autologous mesenchymal progenitors cells (MSCs), biomaterials and growth factors. Our goal was to determine the therapeutic potential and contribution to the repair process of different sources of mesenchymal stem cells for the treatment of nonunions. METHODS. The right femur of Sprague-Dawley (SD) rats was stabilized with an aluminum plate (20 mm long, 4 mm wide, 2 mm thick) and four screws (1.5 mm diameter, 8 mm long). A diaphyseal critical size defect was performed (5 mm). Six groups (n=6–8 animals each) were created. A nonunion group (Control group, empty defect); LBA group, live bone allograft; BMP2 group, rhBMP-2 (2 μg) in collagen sponge; PCL group, polycaprolactone scaffold; PMSCs group, PCL scaffold loaded with 5×10. 6. periosteum-derived MSCs; and BMSCs group, PCL scaffold loaded with 5×10. 6. bone marrow-derived MSCs. For cell tracking purposes, LBA and MSCs were derived from SD-GFP transgenic rats. The repair process was followed up by x-rays up to sacrifice, week 10. After sacrifice, femurs were analyzed by micro computed tomography (μCT), histology and immunohistochemistry. For multiple comparisons one-way ANOVA followed by Dunnett”s test for single comparisons was used. Statistical significance was established for p<0.05. RESULTS. Control group did not show healing during follow up or by μCT and histological analysis. Treatment groups BLA and BMP2 showed full healing by week 10 (LBA, 6 out of 6 animals; BMP2, 4 out of 6 animals). The repair callus was quantified by mCT, Control group showed limited formation of bone (11.47±2.01 mm. 3. ) while both LBA and BMP2 groups showed increased bone formation by week 10 when compared with control group (LBA, 35.36±2.24 mm. 3. , p=0.0022; BMP2, 33.32±1.84 mm. 3. , p=0.0022). Histological and μCT analysis confirmed the experimental nonunion model. In PCL treated groups a low number of animals showed radiographic healing: PCL group 1 out of 8 animals; PMSCs group, 2 out of 6 animals; BMSCs group, 0 out of 6 animals. Interestingly, quantification of the repaired callus showed that only PMSCs group produced a significant volume of bone when compared with the Control group (PMSCs, 24.97±6.03 mm. 3. , p=0.0411). PCL and BMSCs groups do not produced significant amount of bone in the repair callus (PCL, 19.00±4.25 mm. 3. , p=0.3095; BMSCs, 12.88±2.38 mm. 3. , p=0.9372). Healing was confirmed by histology and μCT analysis. Finally, the engraftment of transplanted cells was analysed by immunohistochemistry (anti-GFP antibody). Of the three groups receiving cells only the LBA group showed positive signal for GFP at week 10-post surgery. CONCLUSIONS. In conclusion, periosteum-derived progenitor cells are suitable for mimetic autograft design although integration is not yet achieved

The Masquelet or induced membrane technique (IMT) is a two-stage surgical procedure used for the treatment of segmental bone defects. In this technique, the defect is first filled with a polymethyl methacrylate (PMMA) spacer, which triggers the formation of a membrane that will encapsulate the defect. During the second surgery, the spacer is carefully removed and replaced by autologous bone graft while preserving the membrane. This membrane is vascularized, contains growth factors, and provides mechanical stability to the graft, all of which are assumed to prevent graft resorption and promote bone healing. The technique is gaining in popularity and several variations have been introduced in the clinical practice. For instance, orthopaedic surgeons now often include antibiotics in the spacer to treat or prevent infection. However, the consequences of this approach on the properties of the induce membrane are not fully understood. Accordingly, in a small animal model, this study aimed to determine the impact on the induced membrane of impregnating spacers with antibiotics frequently used in the IMT. We surgically created a five-mm segmental defect in the right femur of 25 adult male Sprague Dawley rats. The bone was stabilized with a plate and screws before filling the defect with a PMMA spacer. Animals were divided into five equal groups according to the type and dose of antibiotics impregnated in the spacer: A) no antibiotic (control), B) low-dose tobramycin (1.2 g/40 g of PMMA), C) low-dose vancomycin (1 g/40 g of PMMA), D) high-dose tobramycin (3.6 g/40 g of PMMA), E) high-dose vancomycin (3 g/40 g of PMMA). The animals were euthanized three weeks after surgery and the induced membranes were collected and divided for analysis. We assessed the expression of selected genes (Alpl, Ctgf, Runx2, Tgfb1, Vegfa) within the membrane by quantitative real-time PCR. Moreover, frozen sections of the specimens were used to quantify vascularity by immunohistochemistry (CD31 antigen), proliferative cells by immunofluorescence (Ki-67 antigen), and membrane thickness. Microscopic images of the entire tissue sections were taken and analyzed using FIJI software. Finally, we measured the concentration of vascular endothelial growth factor (VEGF) in the membranes by ELISA. No significant difference was found among the groups regarding the expression of genes related to osteogenesis (Alpl, Runx2), angiogenesis (Vegfa), or synthesis of extracellular matrix (Ctgf, Tgfb1) (n = four or five). Similarly, the density of proliferative cells and blood vessels within the membrane, as well as the membrane thickness, did not vary substantially between the control, low-dose, or high-dose antibiotic groups (n = four or five). The concentration of VEGF was also not significantly influenced by the treatment received (n = four or five). The addition of tobramycin or vancomycin to the spacer, at the defined low and high doses, does not significantly alter the bioactive characteristics of the membrane. These results suggest that orthopaedic surgeons could use antibiotic-impregnated spacers for the IMT without compromising the induced membrane and potentially bone healing