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

Purpose: To investigate the effect of pressurizing vertebral bodies during vertebroplasty using different materials in the development of fat embolism (FE) and any associated cardiovascular changes. Polymethylmethacrylate (PMMA) is the material of choice for vertebroplasty (VP). However, PMMA has several disadvantages such as exothermic curing, uncertain long-term biomechanical effects and biocompatibility. As a result alternative materials are being developed to overcome these problems. In order to determine the role of PMMA in the generation of cardiovascular changes following vertebroplasty we compared injection of cement with wax in an animal model. Method: In twenty sheep, four vertebral bodies were augmented either with PMMA or bone wax. Heart rate, arterial, central venous and pulmonary artery pressure, cardiac output and blood gas values were recorded. At postmortem the lungs were subjected to histological evaluation. Results: The consecutive augmentation of four vertebral bodies with PMMA induced cumulative fat embolism causing significant deterioration of baseline mean arterial blood pressure (MABP) and blood gas values. Injection of bone wax resulted in similar cardiovascular changes and amount of intravascular fat in the lungs. Conclusion: In this animal model cardiovascular complications during multiple VP happen regardless of the augmentation material used. The deteriorating baseline MABP during VP is associated with the pressurization and displacement of bone marrow/fat into the circulation rather than caused by polymethylmethacrylate

To investigate the effect of pressurizing vertebral bodies during vertebroplasty using different materials in the development of fat embolism (FE) and any associated cardiovascular changes. Polymethylmethacrylate (PMMA) is the material of choice for vertebroplasty (VP). However, PMMA has several disadvantages such as exothermic curing, uncertain long-term biomechanical effects and biocompatibility. As a result alternative materials are being developed to overcome these problems. In order to determine the role of PMMA in the generation of cardiovascular changes following vertebroplasty we compared injection of cement with wax in an animal model. In twenty sheep, four vertebral bodies were augmented either with PMMA or bone wax. Heart rate, arterial, central venous and pulmonary artery pressure, cardiac output and blood gas values were recorded. At postmortem the lungs were subjected to histological evaluation. The consecutive augmentation of four vertebral bodies with PMMA induced cumulative fat embolism causing significant deterioration of baseline mean arterial blood pressure (MABP) and blood gas values. Injection of bone wax resulted in similar cardiovascular changes and amount of intravascular fat in the lungs. Conclusion: In this animal model cardiovascular complications during multiple VP happen regardless of the augmentation material used. The deteriorating baseline MABP during VP is associated with the pressurization and displacement of bone marrow/fat into the circulation rather than caused by polymethylmethacrylate

Costoplasty remains useful in the treatment of adolescent idiopathic scoliosis, rib hump and associated chest wall deformities. However traditional costoplasty increases morbidity and blood loss. We examine the feasibility and possible effectiveness of a more conservative costoplasty using an animal model. 4 fresh half Ovine rib cages from separate animals were obtained, stored at +4 °C and warmed to room temperature before testing. Each rib cage was randomly assigned to group 1, 2, 3 or 4. Ribs 2–10 were dissected out for testing. The ribs then underwent stepwise deconstruction according to their group. Beginning at the convexity, removing first the convex cortex, then the cancellous, then the cranial and caudal cortices to leave just the concave cortex. Testing for stiffness was by three-point bending on the concave side of each rib with the rib fixed at the head of the rib and 5 cm from the resected area. The ribs were deformed at a constant rate of 0.5 mm.sec . −. 1 up to a maximum load of 9.99 kg or until fracturing. Then stress was plotted against strain to find the Young's modulus of each group and statistics carried out with an ANOVA test. The ribs in each group were as follows: Group 1= control, group 2= 30 mm long convex side cortical bone removed 10 mm from lateral tubercle, group 3= convex, cortical and cancellous bone removal and group 4= removal of convex, caudal and cranial cortices with cancellous removal. The Young's Modulus of the groups were: 1= 3.38 N-m (+/− 0.84), 2= 2.65 N-m (+/− 1.58), 3= 1.55 N-m (+/− 0.55) and 4= 0.74 N-m (+/− 0.55). Groups 3 and 4 were significantly less stiff than group 1 (p< 0.01.) No ribs in groups 1, 2 and 4 fractured under the maximum load. 5/8 ribs in group 3 fractured before the maximum load was administered. By deconstructing the rib down to only the concave side it becomes significantly more flexible by approximately 4.5 times than the control Ribs. Coupled with its increase in flexibility it still retains its ability to withstand up to 10 kg of load without fracture. It may be possible to perform a costoplasty whilst preserving ventilatory integrity. This may improve rib hump correction, and curve correction due to increased flexibility of the stiff thoracic cage

Objectives: Development a giant cell tumor model arising from the mutated mesenchymal cells present in its stroma. This establishes the pathogenic mechanism of giant cell tumor, and allows the evaluation of the possible role of biphosphonates and retinoic acid in medical therapy of giant cell tumor of bone. Introduction: In previous studies our group has shown that mesenchymal stroma contains mesenchymal cells capable of recruiting osteoclasts, and lacking capacity to undergo osteoblastic differentiation. These cells represent the actual neoplastic component of the tumor. In the current study, an attempt was made to establish a giant cell tumor in an animal model by injection of these cells. Methods: 6 Balb/C named mice were used. The mice were kept in a laminar flow hood and injected when they were 4 weeks old. The injection was in an intra-osseous location into the distal femur. The cell inoculum consisted of 1 million stromal cells. The cells were derived from a grade III giant cell tumor occurring in the hip joint of a 30 years old woman. The mice were kept for 2 months and than sacrificed. Results: A lytic lesion similar to that occurring in humans developed. The tumor consisted of stromal cells with interspersed osteoclasts. These were identified as being of host origin by mice-specific monoclonal antibodies. The tumor penetrated the cortex but did not infiltrate the articular cartilage. Metastases were not observed. Discussion: Giant cell tumor of bone is typified by osteolytic bone destruction mediated by osteoclasts. In previous studies, our group has shown that the proliferation rate of the stromal component correlates closely with prognosis and grade of the tumor. The stromal component was shown to consist of pre-osteoblasts that fail to differentiate into osteoblasts, but instead recruit giant cells (osteoclasts), mediating bone destruction. Addition of retinoic acid in culture induces osteoblastogenesis cells by blocking AP-1. The current study confirms in an animal model that indeed the stromal cells are capable of osteoclast recruitment and bone destruction. This animal model might allow development of medical remedies to this tumor

Background: Aseptic loosening of total joint arthroplasty is characterised by osteolysis caused by osteoclasts and macrophages. Osteolysis occurs by acidification and dissolution of hydroxyapatite crystals then proteolysis of the bone collagen matrix. N-Telopeptide (NTx) and deoxypyridinolone (DPD) represent highly specific markers for bone resorption. Aim: To investigate whether urinary NTx and DPD generated in-vivo can be used as bone markers in a small animal model of wear debris induced osteolysis. Materials and Methods: 41 and 38 urinary samples were collected from mice at autopsy four weeks following either the implantation of clinically relevant ceramic particles or sham surgery into their femora and assayed for NTx and DPD respectively. Bone markers were corrected for urinary creatinine. Results: The mean urinary NTx concentration for mice that underwent the implantation of clinically relevant ceramic particles was 95.0 nM BCE/mM creatinine compared to 85.3 nM BCE/mM creatinine for mice who had sham surgery (p = 0.8, 95%CI: −29.0 to 30.7). The mean urinary DPD concentration for mice that underwent the implantation of clinically relevant ceramic particles was 5.3 nM DPD/mM creatinine compared to 4.0 nM DPD/ mM creatinine for mice who had sham surgery (p = 0.07, 95%CI: −2.8 to 1.4). Conclusion: The absolute values of NTx and DPD increased in mice that underwent the implantation of clinically relevant ceramic particles compared to sham surgery even though this was not statistically significant. Extending the post operative interval might allow both NTx and DPD to be utilised as bone markers of osteolysis in our small animal model of aseptic loosening

Summary. Osteoporosis reduces particle-induced osteolysis in rat model. Introduction. Wear particle induced osteolysis is considered to be a vital factor that reduces the life span of joint prosthesis. Osteoporosis is not rare in patients with indication for arthroplasty. However, the influence of osteoporosis on wear particles induced osteolysis is not clear. This study is aimed to explore on this issue by using animal model. Methods. 42 female Sprague-Dawley (SD) rats aged 6 months were randomly divided into 3 groups: A, B and C group. Group A and B contained 18 rats each, and group C contained 6 rats. The rats in group A underwent bilateral ovariectomy. Group B was normal control, and group C was sham control. After 3 months, 6 rats in group A, 6 rats in group B and all the rats of group C were sacrificed. Bone mineral density (BMD), μCT and bone histomorphometry were conducted. The rest of rats in group A were randomly divided into 2 groups: group A1 and group A2, and so were the rats in group B. 5mg titanium particles were implanted onto the calvaria of groups A1 and B1, and isometric PBS solution were injected to group A2 and B2. Calvaria were harvested after 14 days. Calvaria were analyzed by μCT and histomorphometry to measure the osteolysis area of calvarial sagittal suture. Results. Compared with B and C group, BMD and bone histomorphometry index of group A was significantly reduced (P<0.05), and tibial trabeculae of group A was slimmer. Area of calvarial sagittal suture osteolysis were 0.262±0.009mm. 2. , 0.130±0.013mm. 2. , 0.307±0.013mm. 2. and 0.178±0.011mm. 2. in A1, A2, B1and B2 groups, respectively. There was significant difference among the groups. Conclusions. Osteoporosis may reduce particle-induced osteolysis in rat model

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

Aim. A gentamicin-eluting biocomposite consisting of hydroxyapatite and calcium sulfate. 1. can provide effective dead space management in chronic osteomyelitis. However, radiographic follow-up after implantation of this novel material has consistently shown evidence of several unique imaging features previously not described with other comparable bone graft substitutes. Conclusive interpretation of these newly described imaging features is difficult as long term follow-up and histological correlation is not yet available. The aim of this study was to establish a large animal model, closely simulating the clinical situation in order to permit further analysis of imaging features in correlation with histological progression of bone remodelling. Method. Standardised bone defects were created in ten Merino-wool sheep (age: two to four years). Large drill holes (diameter 2.5cm, depth 2cm, volume approx. 10ml) were placed in the medial femoral condyles of both hind legs and filled with a gentamicin antibiotic eluting bone graft substitute. *. Initially surgery was carried out on the right hind leg. Three months later, an identical intervention was performed on the contralateral side. With sacrifice planned after six or twelve months, bone voids three, six, nine and twelve months post-implantation are obtained for evaluation. The study was approved by the Animal Care Committee of Thuringia, Germany. Results. We present our preliminary radiographic results after a follow-up of six months. The bio-composite was clearly visible on all initial post-operative radiographs, showing intimate contact to the surrounding cancellous bone of the distal femur. At one month, a radio-dense ring around the bone void (the so called “halo sign”) was found in four of six bone voids treated with the biocomposite. From 2 months onwards this “halo” typically appeared to progress towards the centre of the treated defects, where spherical remnants of the composite often become increasingly apparent. This pattern has been termed “marble sign” and often appears in combination with the halo-sign. Between three to six months bone remodelling appears to continue, halo- and marble sign increasingly disappear and the composite becomes more and more indistinct from surrounding cancellous bone. Conclusions. We have established a large animal model, which appears to mimic the clinical situation very well and reproduces comparable radiographic post implantation features previously observed and described in clinical cases (including the “halo” and the “marble” sign). We expect that this model will provide valuable information regarding the correlation between histological and basic & advanced imaging features (including MRI, CT and Dexa scans) in the future

Background: The commonest cause of long term failure of total joint arthroplasty is aseptic loosening. As a result, many patients will require complex revision surgery that is not only technically challenging but associated with poorer results. Revisions procedures are also associated with higher morbidity and costs. Aim: To quantify osteolysis in a small animal model of aseptic loosening. This model can then be utilised for screening therapeutic agents to inhibit aseptic loosening. Materials and Methods: 7 time mated female mice were injected with radioactive calcium 45 on day 14 of gestation. The 52 offsprings were divided into 2 equal groups and subjected to either the implantation of clinically relevant ceramic particles or sham surgery into their femora. The non-operated femora were used as control. Animals were killed 4 weeks following surgery. Femora were retrieved, dissolved and radioactivity measured as outcome (CPM/mg = Counts Per Minute per milligram). A Linear mixed effects model was utilised to examine the difference in outcome between the 2 groups. Results: The mean scintillation count for sham surgery was 388 CPM/mg compared to 449 CPM/mg in the control femora. The mean scintillation count for ceramic particles was 351 CPM/mg compared to 420 CPM/mg in the control femora. The mean effect on outcome of surgery with ceramic particles relative to sham surgery was estimated at 16.7 CPM/mg (95CI%: 0.9 to 32.5 CPM/mg; p = 0.025). Conclusion: We have successfully shown that this model can quantify osteolysis. However, the difference detected between sham surgery and ceramic particles was biologically small displaying the inert properties of ceramic. Extending the post surgery interval might show a larger difference between sham surgery and ceramic particles and permit quantitative analysis of therapeutic agents to be screened to inhibit aseptic loosening

Aim. A gentamicin-eluting biocomposite consisting of hydroxyapatite (HA) and calcium sulphate (CaS)*1 can provide effective dead space management and bone formation in chronic osteomyelitis. However, radiographic follow-up after implantation of this biomaterial has shown imaging features previously not described with other comparable bone graft substitutes. Last year we presented preliminary results with a follow-up of 6 months. Now we present the radiographic, µCT and histological one-year follow-up of the critical-size bone defect model in sheep. The aim of this study was to simulate the clinical situation in a large animal model to correlate different imaging techniques used in the clinic (Radiography, CT and MRI scans) with histological finding. Methods. Standardised bone defects were created in ten Merino-wool sheep (age two to four years). Large drill holes (diameter 2.5cm, depth 2cm, volume approx. 10ml) were placed in the medial femoral condyles of both hind legs and filled with gentamicin-eluting biocomposite. Initially surgery was carried out on the right hind leg. Three months later, an identical intervention was performed on the contralateral side. Animals were sacrificed at three and six weeks and 4.5, six and twelve months. Radiographs and MRI scans were taken immediately after sacrifice. Filled bone voids were harvested en-block and analysed using µCT, and histology. Results. We present our radiographic, µCT and histological results after a follow-up of twelve months. The bio-composite was clearly visible on all post-operative radiographs and resorbed over the next four months following the before described pattern of “halo sign” and “marble sign”. µCT images of the “halo sign” show degradation of the biocomposite starting at its surface, with the degradation products CaS and HA carried into the periphery of the bone void. µCT images of the “marble sign” showed the further degradation of the biocomposite from the surface to its core, leaving a “marble shaped” remnant of the biocomposite behind. These remnants are completely resorbed at 4.5 months. µCT scans at twelve and six months' reveal progression of trabecula bone formation. The histological results confirm the µCT findings. Conclusion. We have established a large animal model, which mimics the clinical situation and reproduces comparable radiographic post implantation features previously observed in clinical cases (including the “halo” and the “marble” sign). Using µCT imaging and histology we can describe and understand the biodegradation process and the bone formation capacity of the biocomposite in detail. *1 CERAMENTTM|G, BONESUPPORT, Lund, Sweden. *2 CERAMENTTM|G

Introduction: Currently used small animal models of a critical size defect do not sufficiently simulate the biologically unreactive situation in an atrophic non-union. Furthermore, models using intramedullary nails are of little, and poorly standardised, biomechanical stability. This is a characteristic known to promote callus formation though, rather leading to a hypertrophic non-union. The aim of this study was to establish an atrophic non-union model in the rat femur under well defined biomechanical conditions and with minimised interactions between the processes in the healing zone and the implant by using external fixation. MATERIALS AND METHODS: 80 male Sprague Dawley rats were randomly divided into two groups (non-union vs. control). All animals received an osteotomy (app. 0.5 mm gap) of the left femur, stabilised with a custom made external fixator. In the non-union group the periosteum was cauterised 2mm distal and proximal of the osteotomy, and the bone marrow was removed. X-rays were performed once weekly. Animals were sacrificed at 14 or 56 days post-operation. At both time points the femurs of 16 animals of each group underwent histological/histomorphometrical and immunhis-tochemical analyses (PMMA or paraffin embedding). Additionally at 56 days 8 animals of each group were tested biomechanically. The maximum torsional failure moment and the torsional stiffness were determined in relation to the intact femur. Post-mortem x-rays were evaluated in a descriptive manner. RESULTS: At 14 days the histology and radiology showed considerable mineralised periosteal callus in the control group, while the non-union group only showed very little periosteal callus, distant to the osteotomy. At 56 days the control group was completely, or at least partially, bridged by mineralised callus. The non-union group did not show a bridging of the osteotomy gap in any of the animals, moreover the bone ends were resorbed and the gap widened. The relative mean torsional stiffness was significantly larger (p< 0.001) in the control group compared to the non-union group (136.2±34.5% vs. 2.3±1.2%). In the non-union group no maximal torsional failure moment could be detected for the osteotomised femurs. In the control group it was 134.2±79.1%, relative to the intact femur. DISCUSSION: The cauterisation of the periosteum and the removal of the bone marrow, in combination with a high stiffness of the external fixator may create an atrophic non-union under well defined biomechanical conditions and with minimised interactions between the healing zone and the implant. This model will allow better standardised investigations on the subject of atrophic non-unions

Introduction: Atrophic nonunion is a well recognised complication of long bone fractures. Clinical trials show that BMP-2 accelerates healing and reduces nonunion in open tibial fractures. We are interested in a natural small molecule that has been previously demonstrated to stimulate angiogenesis in vivo. Our aim is to assess the two treatments in the prevention of nonunion. The small animal model we used is a non-critical size defect of the tibia deprived it of its blood supply by surgical stripping of the periosteum and curetting of the local endosteum thus closely reflecting the clinical situation. The outcomes were measured by radiographic assessment and histology. Methods: Wistar rats were treated with either the angiogenic molecule (0.1% or 0.003%), BMP-2 or vehicle alone (PBS) soaked in a type I collagen sponge. All animals underwent a 2mm osteotomy, stripping of the periosteum and endosteum proximally and distally for the length of the diameter of the tibia. Fluorescent markers were injected at 2 weekly intervals. The rats were sacrificed at 8 weeks. Both tibiae were disarticulated; fixator and soft tissues were removed and AP and lateral X-rays were taken. Subjective assessment of the healing on X-ray was carried out in two ways; using a radiographic scoring system and by grey scale analysis. The samples were embedded, sectioned and stained for new bone formation. Results: Bridging or potential to bridge was seen in a number of animals on x-ray. Bridging or potential to bridge was judged to be present in 72.22% of the BMP-2 group and 66.67% of the high dose group compared to 22.22% of the control group. Histological analysis is being performed to confirm these findings. Discussion: Atrophic nonunion is a serious clinical complication, unfortunately BMP-2 is a highly costly treatment option and therefore alternative molecular therapies are much sought after. We describe here an angiogenic molecule has some potential in preventing formation of nonunion

Purpose: Regeneration of skeletal tissue for fracture repair or during morphogenesis involves common phases of cell proliferation and differentiation. Mesenchymatous precursor cells have multiple origins. These cells can be identified in the bone marrow, in the deep layer of the periosteum and in the endosteum. More recently, the presence of circulating multipotent stem cells has been demonstrated in the general circulation. Their contribution to skeletal regeneration processes is suspected. The experiments we report allow visualisation of the multidirectional differentiation phenomena involving mesenchymatous precursor cells in an animal model of skeletal tissue regeneration. Material and methods: An experimental surgical protocol was developed to study the regeneration of skeletal tissue in New Zealand rabbits. Eighteen animals were used. A vascularised periosteum flap was transferred onto the medial aspect of the knee. The flap was fixed in order to be exposed to flexion and extension stress during spontaneous ambulation. The joint was not damaged in any way and the adjacent bone segments were left intact. The animal was allowed to move freely postoperatively. The animal was sacrificed two days to eight weeks later to study standard histological slices taken from the regenerate region and the recipient knee joint. Results: The zone of influence of the flap was recognised early in the environment where it was apposed. This zone involved the marrow of the metaphyseal regions, the neighbouring muscles, the joint cavity, and the menisci. Cell proliferation was noted in each of these sites. It was associated with differentiation of the precursor elements in multiple directions of the mesenchymatous lines. This led to production of cartilaginous, bony, fibrous, and even muscle tissue in the medullary cavity, in the menisci, and in the open joint space. Immunohistochemistry demonstrated the contribution of the mesenchymatous stem cells whose circulating pool was visualised. Discussion: This work is in agreement with the recent demonstration of the contribution of stem cells to general healing phenomena, and the physiological turnover of healthy tissue. Conclusion: The strong potential of multipotent stem cells for tissue reparation and regeneration processes opens important perspectives for cell therapy and tissue engineering. The demonstration of physiological processes operating in vivo which involve participation of the endogenous cell pool is of importance for all fields of medicine and surgery for the treatment of the musculoskeletal system

Aim. Implant-associated osteomyelitis is a devastating complication with poor outcomes following treatment, especially when caused by antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). A large animal model of a two-stage revision to treat MRSA implant-associated osteomyelitis has been developed to assess novel treatments. A bioresorbable, thermo-responsive hyaluronan hydrogel (THH) loaded with antibiotics has been developed and our aim was to investigate it´s in vivo efficacy as a local antibiotic carrier compared to the current standard of care i.e. antibiotic-loaded polymethylmethacrylate (PMMA) bone cement. Method. 12 female, 2 to 4 year old, Swiss Alpine Sheep were inoculated with MRSA at the time of intramedullary nail insertion in the tibia to develop chronic osteomyelitis. After 8 weeks sheep received a 2-stage revision protocol, with local and systemic antibiotics. Group 1 received the gold standard clinical treatment: systemic vancomycin (2 weeks) followed by rifampicin plus trimethoprim/sulfamethoxazole (4 weeks), and local gentamicin/vancomycin via PMMA. Group 2 received local gentamicin/vancomycin delivered via THH at both revision surgeries and identical systemic therapy to group 1. Sheep were euthanized 2 weeks following completion of antibiotic therapy. At euthanasia, soft tissue, bone, and sonicate fluid from the hardware was collected for quantitative bacteriology. Results. Sheep tolerated the surgeries and both local and systemic antibiotics well. Gold standard of care successfully treated 3/6 sheep with a total of 10/30 culture-positive samples. All 6 sheep receiving antibiotic-loaded THH were successfully treated with 0/30 culture-positive samples, p=0.0008 gold-standard vs. hydrogel (Fisher's Exact). Conclusions. The clinical gold standard treatment was successful in 50% of sheep, consistent with outcomes reported in the literature treating MRSA infection. The antibiotic-loaded THH clearly outperformed the gold standard in this model. Superior efficacy of the THH is likely due to 1) the ability to administer local antibiotics at the both revision surgies due to the bioresorbable nature of the hydrogel, and 2) complete antibiotic release compared to bone cement, which is known to retain antibiotics. Our results highlight the potential of local delivered, biodegradable systems for antibiotics for eradicating implant-related infection caused by antibiotic-resistant pathogens. Acknowledgement. Funding provided by AO Trauma

Aims: Little is known about effects of extracorporeal shock wave application (ESWA) on normal bone physiology. Therefore, we investigated ESWA effects on intact distal rabbit femura as an in vivo animal model. Methods: Animals received 1,500 SW pulses each of different energy ßux densities (EFD) on either left or right femur or remained untreated. ESWA effects were investigated by bone scintigraphy, MRI and histopathological examination. Results: Ten days after ESWA, local blood ßow and bone metabolism were decreased (0.5 mJ/mm2 and 0.9 mJ/mm2 EFD), but were increased 28 days after ESWA (0.9 mJ/mm2). ESWA with 0.9 mJ/mm2 EFD (but not with 0.5 mJ/mm2 ) resulted in MRI signs of soft-tissue-edema, epiperiosteal ßuid and bone marrow edema one day after ESWA, as well as in hemosiderin deposits found epiperiosteally and within the marrow cavity ten days after ESWA. Conclusions: ESWA with both 0.5 mJ/mm2 and 0.9 mJ/mm2 EFD had effects on normal bone physiology in the distal rabbit femur, with considerable damaging side effects of ESWA with 0.9 mJ/mm2 EFD on periosteal soft tissue and tissue within the bone marrow

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined. Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4). After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated. The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models

Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model. Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time. Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology. Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level. Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species. Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant

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:

Introduction. Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism. Methods. 140 male Sprague-Dawley rats were fed with selenium-deficient or normal diet for 4 weeks to produce a low selenium or normal nutrition status. The rats were then fed for a further 4 weeks with low selenium or normal diets with or without T-2 toxin (100ng per gram body weight per day). The rat knee joints were fixed and paraffin embedded and histological and immunohistochemical staining was performed to analyse the metabolism of articular cartilage. Results. There was increased cell cluster formation in the middle and/or deep zones in rats fed with both diets. However, an apparent cell loss was observed in the low selenium + T-2 toxin group with an apparent increase in caspase-3 staining, indicating the increased cell apoptosis. Moreover, toluidine blue staining was reduced in the low selenium + T-2 toxin group, suggesting a loss of sulphated glycosaminoglycans. Similarly, there was reduced 2B6 and 6C3 staining in the territorial matrix of chondrocytes, indicating a reduced synthesis in 4-sulhated and native CS motifs. In contrast, increased 1B5 staining was observed in the articular cartilage from the low selenium + T-2 toxin group, suggesting a lack of CS sulphatransferase activity. Interestingly, there was increased 7D4 staining in the superficial zone of articular cartilage from low selenium + T-2 toxin group, suggesting an initiation of an osteoarthritis-like lesion. Discussion. These results indicated that low selenium nutrition and T-2 toxin could promote cell apoptosis and disrupt CS-GAG metabolism in ECM of rat articular cartilage in this animal model, which is similar to that observed in KBD patients. Collectively, our results support the hypothesis that low selenium and T-2 toxin are the cause of KBD