Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities.

Cite this article: Bone Joint Res 2015;4:105–116

Purpose. Vitamin D is a key regulator of bone homeostasis. The enzyme CYP24A1 is responsible for transforming vitamin D into 24,25(OH)2vitD. The putative biological activity of 24,25(OH)2vitD remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following a fracture in chicks. Our laboratory has engineered a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25(OH)2vitD. We set out to study the role of 24,25(OH)2vitD in endochondral and intramembranous bone formation in fracture repair in this mouse model based on the results of the chick fracture repair study. Method. Wild-type and mutant Cyp24a1 gene deficient mice were subjected to two different surgical procedures to simulate bone development and fracture repair. To mimic endochondral ossification, we devised a modified technique to perform intramedullary nailing of a mouse tibia followed by an induced fracture. To evaluate intramembranous ossification, we applied distraction osteogenesis to a mouse tibia using a mini Ilizarov external fixator apparatus. Histomorphometric parameters and gene expression differences in fracture repair between the mutant mice and the wild-type controls were measured using micro computed tomography, histology and reverse-transcription quantitative PCR (RT-qPCR) respectively. Results. Quantitative histomorphometric results showed a delay in endochondral fracture repair in the mutant mice calluses as compared to the wild-type mice calluses. In the same model, gene expression of type X collagen in the callus was higher in the wild-type mice. These significant differences were fully rescued by injecting the mutant mice with exogenous 24,25(OH)2vitD. In the intramembranous bone formation model, we found a trend towards reduced bone formation in the gap created by the distraction process in the mutant mice as compared to the wild-type mice. However, the differences did not reach statistical significance. Conclusion. Our results support a role for 24,25(OH)2vitD in fracture repair which is more dominant in a chondrocyte-mediated bone formation pathway like endochondral ossification. Although our results did not reach statistical significance in the intramembranous ossification model, the observed trend suggests a potential role as well. Further study of the role of 24,25(OH)2vitD in bone healing has the potential to support novel approaches in accelerating bone formation and fracture repair

It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation

Aim. This study describes the histologic changes seen with a gentamicin-eluting synthetic bone graft substitute (BGS)(1) in managing bone defects after resection of chronic osteomyelitis (cOM). Method. 154 patients with mean follow-up of 21.8 months (12–56) underwent treatment of cOM with an antibiotic-loaded BGS for defect filling. Nine patients had subsequent surgery, not related to infection recurrence, allowing biopsy of the implanted material. These biopsies were harvested between 19 days and two years after implantation, allowing a description of the material's remodelling over time. Samples were fixed in formalin and stained with haematoxylin-eosin. Immunohistochemistry, using an indirect immunoperoxidase technique, identified the osteocyte markers Dentine Matrix Protein-1 (DMP-1) and Podoplanin, the macrophage/osteoclast marker CD68, and the macrophage marker CD14. Results. The material was actively remodelled and was osteoconductive. There was evidence of osteoblast recruitment, leading to osteoid and intramembranous formation of woven and lamellar bone on the material's surface, seen most prominently in areas of well-vascularised fibrous tissue. Osteocytes in woven bone expressed the markers DMP-1 and Podoplanin. No cartilage or endochondral ossification was seen. There was a prominent (CD14+/ CD68+) macrophage response to the BSG and macrophages within reparative cellular and collagenous fibrous tissue. In biopsies taken between 4 and 5 months, there were bone trabeculae containing BGS of mainly woven but partly lamellar type. Giant cells on the surface of newly formed mineralised osteoid and woven bone expressed an osteoclast phenotype (CD68+/CD14-). In later biopsies (up to 2 years), larger bone trabeculae were seen more frequently within well-vascularised reparative fibrous tissue. The BGS was replaced with predominantly lamellar bone. One biopsy was taken from an extraosseous leak of BGS into the soft tissues, behind the distal tibia. The histology showed a heavy macrophage infiltrate, but notably no evidence of osteoid or bone formation in the material or surrounding soft tissues. Conclusion. There was clear evidence that this BGS is osteoconductive with first osteoid then woven and lamellar bone being formed. DMP-1 and podoplanin-expressing osteocytes present in woven and lamellar bone demonstrate osteoclastic bone remodelling. Increased lamellar bone was noted in later samples and bone formation was most prominent in well-vascularised areas. There was on-going remodelling of the material beyond one year. The BGS did not ossify in soft tissue. The hydroxyapatite scaffold in this material is probably responsible for its high osteoconductivity and potential to be transformed into bone

To elucidate the molecular biology of fracture healing, murine models are preferred. We performed a study with the first internal fixation system that allows studying murine fracture healing in a controlled mechanical environment, to characterise the timing of the fracture healing cascade with this model, based on a histological evaluation. Femoral osteotomies were performed in 68 male C57BL/six mice and stabilised with locking internal fixation plates in either stiff, or defined, flexible configurations. Healing progression was studied at 10 time points between 3 and 42 days post- surgery. After surgery, mice were radiographed to confirm the correct implant positioning. After sacrifice, the extracted femora were processed for decalcified histology. Thin sections were taken as serial transverse sections and stained for subsequent histomorphometric analysis and three-dimensional reconstruction of the different fracture callus tissues. The surgery was successful in 62 animals. Only six6 (8.8%) animals had to be sacrificed due to complications during surgery. The post-operative radiographs demonstrated a high reproducibility of implant positioning and no implant failure or screw loosening occurred during the experimental period. The improved consistency in surgical technique leading to more uniform results represents a key advantage of this system over other mouse fracture healing models. As such, it may allow a reduction in the sample size needed in future murine fracture healing studies. The histological evaluation confirmed the lack of a periosteal callus, and exclusively endosteal, intramembraneous bone formation in the bones stabilised with the stiff implants. The bones that were stabilised with the more flexible internal fixation plates showed additional endochondral ossification with extensive, highly asymmetrical, periosteal callus formation. Our results demonstrate that this murine fracture model leads to different healing patterns depending on the flexibility of the chosen plate system. This allows researchers to investigate the molecular biology of fracture healing in different ossification modes by selection of the appropriate fixation

Fibrocartilaginous entheses are formed through endochondral ossification and characterized by four zones morphologically separated into tendon, uncalcified fibrocartilage, calcified fibrocartilage and bone [1]. These zones are not successfully regenerated following surgical repair. Demineralized Bone (DBM) presented at the tendon bone interface may improve healing between tendon and bone. Fifty six female nude rats were randomly allocated into either a control reconstruction or treatment group (DBM at the tendon-bone healing site). A modified rodent model of anterior cruciate ligament reconstruction was adopted [2]. Animals were sacrificed at 2, 4 and 6 weeks following surgery. Four rats per group were prepared for histology at each time point while eight rats were culled for biomechanical testing at 4 and 6 week time points. ANOVA and post hoc tests were used to examine differences which were considered significant at p < 0.05. The surgical procedure was well tolerated. Macroscopic dissection did not reveal any infection and all joint surfaces appeared normal. An intra-articular graft between the femur and tibia was present in all specimens. Mechanical differences were noted between groups. Peak loads were significantly higher in treatment group at 4 and 6 weeks (6.0 ± 3.6N and 9.1 ± 2.6 N, respectively) compared to controls (2.9 ± 1.9 N and 5.8 ± 2.7 N). No statistical differences were found in graft stiffness between the groups at 4 or 6 week time points. Histology showed an initial influx of inflammatory cells coupled with formation of a loose disorganized fibrovascular interface layer between tendon and bone in both groups. By the 6 weeks the interface layer in the DBM group fused into the newly formed bone to create a continuum between the tendon and bone, in an interdigitated fashion, containing Sharpy's like fibres. In the control group the continuum was less apparent with evidence of large areas of discontinuity between the two zones. A thicker region of newly formed woven bone with increased osteoblast activity along the bone tunnel was evident in the DBM group. DBM has the potential to increase the quality of repair following surgical procedures involving reattachment of tendon to bone

Introduction and aims. Growth plate cartilage is responsible for bone growth in children. Injury to growth plate can often lead to faulty bony repair and bone growth deformities, which represents a significant clinical problem. This work aims to develop a biological treatment. Methods. Recent studies using rabbit models to investigate the efficacy of bone marrow mesenchymal stem cells (MSC) to promote cartilage regeneration and prevent bone defects following growth plate injury have shown promise. However, translational studies in large animal models (such as lambs), which more closely resemble the human condition, are lacking. Results. Very recently, our labs have shown that ovine bone marrow MSC are multipotential and can form cartilage-like tissue when transplanted into mice. However, using a growth plate injury model in lambs, analogous to those described in the rabbit, autologous marrow MSC seeded into gelatine scaffold containing chondrogenic factor TGF-1, failed to promote growth plate regeneration. T o date, no large animal studies have reported successful regeneration of injured growth plate cartilage using MSC highlighting the possibility that ex vivo expanded MSC may not represent a viable cellular therapy for growth plate injury repair. In addition, using a growth plate injury repair model in young rats, our studies have also focused on understanding mechanisms of the faulty repair and identifying potential targets for enhancing growth plate regeneration using endogenous progenitor cells. We have observed that bony repair of injured growth plate is preceded sequentially by inflammatory, fibrogenic, chondrogenic and osteogenic responses involving both intramembranous and endochondral ossification mechanisms. We have observed infiltration of mesenchymal progenitor cells into the injury site, some of which have the potential to differentiate to osteoblasts or chondrocytes and contribute to the bony repair of the injured growth plate. Conclusion. This presentation will focus on our studies examining the efficacy of ex vivo expanded autologous MSC to enhance growth plate regeneration in the ovine model and work using a rat model aimed at identifying potential targets for enhancing cartilage regeneration by mobilising endogenous stromal progenitor cells

Intracapsular and para-articular osteochondromas are a rare subtype of soft tissue chondroma occurring in and around joints. We report a giant 5.5cm × 5.5cm × 3.0cm mass occurring in the knee of a 45 years old lady and examine previous cases to update our understanding of para-articular soft tissue osteochondromas. Clinicopathological data were obtained from medical records for the case report whilst a multi-database literature search was conduction for the literature review. 27 articles containing 39 cases were identified in the English literature under our strict inclusion criteria. Along with our data, 40 cases were collated and analysed to provide a set of reference characteristics. These included: age, male-female ratio, spatial location, time of onset, tumour size, clinical symptoms, mechanism of injury, investigations used, treatment received, histopathology features, follow-up and recurrence characteristics. Statistical analysis was performed on data to elicit any discernable pattern of tumour formation. Median age of patients was 50 years old with a male to female ratio of 1:1.11. Most commonly occurs in the 40s, 50s and 60s accounting for two-thirds of all cases. Majority of tumours were located within or adjacent to a fat pad structure. 33 were located in the infra-patellar region, 3 in the suprapatella/pre-femoral region and 4 in other para-articular locations. Average time of onset to diagnosis was 5.81 yrs with a mean volume of 87.5 cubic centimetres. No discernable correlation between time of onset to diagnosis and tumour size was found (spearman correlation co-efficient 0.534, p=0.007). The main symptom reported was pain in 29 cases, whilst 5 were pain free, 6 cases were unspecified. X-Rays, CT and MRI have become the core imaging modalities in investigation. En bloc excision is the choice of treatment, whilst arthroscopic techniques have also been used with similar success. Histologically, 35 cases had a typical description of a cartilage capped lesion with central trabecular bone and areas of endochondral ossification. 3 cases had a histological appearance of predominantly bone whilst 2 cases had predominantly cartilage. All tumours analysed were benign. No recurrences were reported with an average follow up period of 1.91 years. We have provided the latest set of data for the characterisation of para-articular and intracapsular soft tissue osteochondromas. These tumours are benign entity with an invariably good outcome following simple excision. Recognition of this entity is important to prevent over investigation and the performance unnecessarily invasive and radical procedures

INTRODUCTION. Appropriate, well characterized animal models remain essential for preclinical research. This study investigated a relevant animal model for cancellous bone defect healing. Three different defect diameters of fixed depth were compared in both skeletally immature and mature sheep. This ovine model allows for the placement of four confined cancellous defects per animal. METHODS. Defects were surgically created and placed in the cancellous bone of the medial distal femoral and proximal tibial epiphyses (See Figure 1). All defects were 25 mm deep, with defect diameters of 8, 11, and 14 mm selected for comparison. Defects sites were flushed with saline to remove any residual bone particulate. The skeletally immature and mature animals corresponded to 18 month old and 5 year old sheep respectively. Animals were euthanized at 4 weeks post-operatively to assess early healing. Harvested sites were graded radiographically. The percentage of new bone volume within the total defect volume (BV/TV) was quantified through histomorphometry and μ-CT bone morphometry. Separate regions of interest were constructed within the defect to assess differences in BV/TV between periosteal and deep bone healing. Defect sites were PMMA embedded, sectioned, and stained with basic fuschin and methylene blue for histological evaluation. RESULTS. The animals tolerated the surgery well, with no incidence of fractures within the four weeks. Healing of the defects progressed via endochondral ossification, with none of the defects being completely healed within the 4 week time point. Bone volume fraction (BV/TV) significantly decreased with an increasing defect diameter. Actual bone volume (BV), however, increased with defect diameter, suggesting a correlation between biological response and severity of injury. Three distinct healing regions were found to exist within the defect and along the axis of the defect, with significant differences detected in the BV/TV between adjacent regions. Histologically, the 5 year old animals appeared to have decreased osteoblast activity, and lower osteocyte density within the newly formed woven bone. On occasion, the defects were found to intersect the tibial growth plate in the 18 month old animals, with bone replacing the proliferating chondrocyte zone (See Figure 2). Additionally, the 14 mm defect was not able to be placed in the tibia of sheep due to the possibility of the defect entering the tibial intramedullary (IM) canal, and the lack of cancellous bone between the tibial plateau and IM canal. Both these issues considerably affect this model and should be avoided. CONCLUSION. The surgical placement of 11 mm diameter defects in the proximal tibial and distal femoral epiphyses of skeletally mature sheep presents a suitable large animal model to study early healing of cancellous bone defects. This refined model allows for the placement of four separate non-healing defects within a single sheep, and allows for the possibility to reduce animal numbers required to obtain information

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

Currently, there is no animal model in which to evaluate the underlying physiological processes leading to the heterotopic ossification (HO) which forms in most combat-related and blast wounds. We sought to reproduce the ossification that forms under these circumstances in a rat by emulating patterns of injury seen in patients with severe injuries resulting from blasts. We investigated whether exposure to blast overpressure increased the prevalence of HO after transfemoral amputation performed within the zone of injury. We exposed rats to a blast overpressure alone (BOP-CTL), crush injury and femoral fracture followed by amputation through the zone of injury (AMP-CTL) or a combination of these (BOP-AMP). The presence of HO was evaluated using radiographs, micro-CT and histology. HO developed in none of nine BOP-CTL, six of nine AMP-CTL, and in all 20 BOP-AMP rats. Exposure to blast overpressure increased the prevalence of HO.

This model may thus be used to elucidate cellular and molecular pathways of HO, the effect of varying intensities of blast overpressure, and to evaluate new means of prophylaxis and treatment of heterotopic ossification.

Cite this article: Bone Joint J 2015;97-B:572–6

Nanotechnology is the study, production and controlled manipulation of materials with a grain size < 100 nm. At this level, the laws of classical mechanics fall away and those of quantum mechanics take over, resulting in unique behaviour of matter in terms of melting point, conductivity and reactivity. Additionally, and likely more significant, as grain size decreases, the ratio of surface area to volume drastically increases, allowing for greater interaction between implants and the surrounding cellular environment. This favourable increase in surface area plays an important role in mesenchymal cell differentiation and ultimately bone–implant interactions.

Basic science and translational research have revealed important potential applications for nanotechnology in orthopaedic surgery, particularly with regard to improving the interaction between implants and host bone. Nanophase materials more closely match the architecture of native trabecular bone, thereby greatly improving the osseo-integration of orthopaedic implants. Nanophase-coated prostheses can also reduce bacterial adhesion more than conventionally surfaced prostheses. Nanophase selenium has shown great promise when used for tumour reconstructions, as has nanophase silver in the management of traumatic wounds. Nanophase silver may significantly improve healing of peripheral nerve injuries, and nanophase gold has powerful anti-inflammatory effects on tendon inflammation.

Considerable advances must be made in our understanding of the potential health risks of production, implantation and wear patterns of nanophase devices before they are approved for clinical use. Their potential, however, is considerable, and is likely to benefit us all in the future.

Cite this article: Bone Joint J 2014; 96-B: 569–73.