Introduction and Objective. Heterotopic ossification is the formation of extraskeletal mineralized tissue commonly associated with either trauma or surgery. While several mouse models have been developed to better characterize the pathologic progression of HO, no model currently exists to study HO of the hip, the most common location of acquired HO in patients. Owing to the unique biological mechanisms underpinning the formation of HO in different tissues, we sought to develop a model to study the post-surgical HO of the hip. Materials and Methods. Wild-type mice C57BL/6J mice were used to study the procedure outcomes, while Pdgfra-CreERT2;mT/mG and Scx-GFP reporter animals were used for the lineage tracing experiments (total n=16 animals, male, 12 weeks old). An anterolateral approach to the hip was performed. Briefly, a 2 cm incision was made centered on the great trochanter and directed proximal to the iliac crest and distally over the lateral shaft of the femur. The joint was then reached following the intermuscular plane between the rectus femoris and gluteus medius muscles. After the joint was exposed, the articular cartilage was removed using a micropower drill with a 1.2 mm reamer. The medius gluteus and superficial fascia were then re-approximated with Vicryl 5-0 suture (Ethicon Inc, Somerville, NJ) and skin was then closed with Ethilon 5-0 suture (Ethicon Inc). Live high resolution XR imaging was performed every 2 wks to assess the skeletal tissues (Faxitron Bioptics, Tucson, AZ). The images were then scored using the Brooker classification. Ex-vivo microCT was conducted using a Skyscan 1275 scanner (Bruker-MicroCT, Kontich, Belgium). 3D reconstruction and analysis was performed using Dragonfly (ORS Inc., Montreal, Canada). For the histological analysis of specimens, Hematoxylin and Eosin (H&E), modified Goldner's Trichrome (GMT) stainings were performed. Reporter activity was assessed using fluorescent imaging. Results. Substantial periarticular heterotopic bone was seen in all cases. A periosteal reaction and an initial formation of calcified tissue within the soft tissue was apparent starting from 4 wks after surgery. By XR, progressive bone formation was observed within the periosteum and intermuscular planes during the subsequent 8 weeks. Stage 1 HO was observed in 12.5% of cases, stage 2 in 62.5% of cases, and stage 3 HO in 25% of cases. 3D microCT reconstructions of the treated hip joints demonstrated significant de novo heterotopic bone in several location which phenocopy human disease. Heterotopic bone was observed in an intracapsular location, periosteal location involving the iliac bone and proximal femur, and intermuscular locations. Histological analyses further confirmed these findings. To assess the cells which gave rise to HO in this model, an inducible PDGFRα and constitutive Scx-GFP reporter mice were used. A dramatic increase in mGFP reporter activity was noted PDGFRα within the HO injury site, including in areas of new cartilage and bone formation. Scx-associated reporter activity increased in the soft tissue and periosteal periacetabular areas of injured hips. Conclusions. HO has a diverse set of pathologies, of which joint associated HO after elective surgery is the most common. Here, we present the first mouse model of hip dislocation and acetabular reaming that mimics elements of human periarticular HO. The diverse locations of HO after acetabular reaming (intracapsular, intermuscular and periosteal) suggests the activation of different and specific HO program after surgery. Such a field effect would be consistent with local trauma and inflammation, which is a well-studied contributor to HO genesis. Not surprisingly, joint-associated HO significantly derives from PDGFRα-expressing cells, which has been shown to similarly give rise to intramuscular and intratendinous HO

Osteoarthritis (OA) is a disease that affects both bone and cartilage. Typically, this disease leads to cartilage degradation and subchondral bone sclerosis but the link between the two is unknown. Also, while OA was traditionally thought of as non-inflammatory condition, it now seems that low levels of inflammation may be involved in the link between these responses. This is particularly relevant in the case of Post-Traumatic OA (PTOA), where an initial phase of synovial inflammation occurs after injury. The inflammatory mediator interleukin 1 beta (IL-1B) is central to this response and contributes to cartilage degradation. However, whether there is a secondary effect of this mediator on subchondral bone, via bone-cartilage crosstalk, is not known. To address this question, we developed a novel patellar explant model, to study bone cartilage crosstalk which may be more suitable than commonly used femoral head explants. The specific aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response after joint injury and the subsequent development of PTOA. Female Sprague Dawley rats (n=48) were used to obtain patellar explants, under an institutional ethical approval license. Patellae were maintained in high glucose media, under sterile culture conditions, with or without IL-1B (10ng/ml), for 7 days. Contralateral patellae served as controls. One group (n= 12) of patellae were assessed for active metabolism, using two both Live and Dead (L/D) staining and an Alamar Blue assay (AB). A second group (n=12) was used for tissue specific biochemical assays for both bone (Alkaline Phosphatase) and cartilage (sulfated proteoglycan and glycosaminoglycan (sGaG)). Finally, a third group (n=28) of explants were used for histologically analysis. Samples were decalcified, embedded in paraffin and sectioned to 7µm thickness, and then stained using H&E; and Safranin O with fast green. Additionally, toluidine blue and alkaline phosphatase staining were also performed. Our results demonstrate that our system can maintain good explant viability for at least 7 days, but that IL-1B reduces cell viability in patellar cartilage, as measured by both L/D and AB assays after 0, 2, 4 and 7 days in culture. In contrast, sGaG content in cartilage were increased by this treatment. Additionally, ALP, a marker of osteoblastic activity, was increased in IL-1B treated group 4 and 7 days, but was also showed some increase in control groups. Histological analyses showed that IL-1B treatment resulted in reduced proteoglycan staining, demonstrating the powerful effect of this factor in injury response over time. Thus, we conclude that IL-1B affects both bone and cartilage tissues independently in this system, which may have relevance in understanding bone-cartilage crosstalk after injury and how this is involved in PTOA development

Mesenchymal Stromal Cells (MSC) are promising therapies for fracture healing. However, undifferentiated MSC may act only through an inductive paracrine effect without direct bone formation. Here, we developed an injectable product constituted of human bone-forming cells derived from bone marrow (BM)-MSC (ALLO-P2) that display more potent bone repair properties not only by stimulating host osteoinduction but also by direct bone formation. In vitro, ALLO-P2 overexpressed markers such as ALP compared to BM-MSC isolated from the same donors, suggesting their engagement into the osteogenic lineage. In vivo, a single dose of ALLO-P2 significantly enhanced bone neoformation 14 days post-administration over the calvaria of NMRI-Nude mice compared to the control excipient. Histological analyses and mouse/human type I collagen double-immunolabelling revealed the presence of mineralized bone nodules of mixed host and donor origins in mice administered with ALLO-P2. Together, these results show that ALLO-P2 is a potential promising clinical candidate to promote bone repair, since it can be produced at high yields, is injectable and boosts ossification mechanisms involved in the physiological repair process

Skeletal sequels of traumatisms, diseases or surgery often lead to bone defects that fail to self-repair. Although the gold standard for bone reconstruction remains the autologous bone graft (ABG), it however exhibits some drawbacks and bone substitutes developed to replace ABG are still far for having its bone regeneration capacity. Herein, we aim to assess a new injectable allogeneic bone substitute (AlloBS) for bone reconstruction. Decellularized and viro-inactivated human femoral heads were crushed then sifted to obtain cortico-spongious powders (CSP). CSP were then partly demineralized and heated, resulting in AlloBS composed of particles consisting in a mineralized core surrounded by demineralized bone matrix, engulfed in a collagen I gelatin. Calvarial defects (5mm in diameter, n=6/condition) in syngeneic Lewis1A rats were filled with CSP, AlloBS±TBM (total bone marrow), BCP (biphasic calcium phosphate)±TBM or left unfilled (control). After 7 weeks, the mineral volume/total volume (MV/TV) ratios were measured by µCT and Movat's pentachrome staining were performed on undemineralized frontal sections. The MV/TV ratios in defects filled with CSP, AlloBS or BCP were equivalent, whereas the MV/TV ratio was higher in AlloBS+TBM compared to CSP, AlloBS or BCP (p<0.01; Mann-Whitney). Histological analyses exhibited a collagen-rich matrix in all the defects, and osteoid at the surface of all implanted biomaterials. Our data indicates that AlloBS is a promising candidate for bone reconstruction, with ease of manipulation, injectability and substantial osteogenic capacity. Further experiments in larger animal models are under consideration to assess whether AlloBS may be a relevant clinical alternative to ABG

Background. Administration of Botulinum toxin type A (BTX-A) in patients with spastic cerebral palsy aims to improve mobility by increasing joint range of motion and decreasing passive resistance. However, our recent animal experiments indicated that BTX-A can decrease muscle”s length range of force exertion (Lrange), and increase its passive forces and extracellular matrix (ECM) collagen content. Moreover, BTX-A injected into the tibialis anterior (TA) was shown to spread into non-injected synergistic muscles in the whole anterior crural compartment. These effects that contradict the treatment aims deserve further investigation. Aim. To test in a rat model if: (1) BTX-A injected into the medial and lateral gastrocnemius (GM&GL) muscles spreads into the synergistic soleus (SOL) as well as antagonistic TA and extensor digitorum longus (EDL). (2) The muscles exposed show a wider Lrange, decreased muscle passive force and reduced ECM collagen. Methods. 2×0.1U/20µl of BTX-A (BTX-A group, n=6) or only 2×20µl of saline (Control group, n=6) were prepared and each was injected into the mid-belly of the GM and GL separately. 5 days post injection, forces of all muscles were measured in passive state and also on activation. The GM&GL length was changed whereas; all other muscles were kept at constant length. After biomechanical testing, the muscles were histologically analyzed using Gomori trichrome stain to detect ECM collagen. Two-way ANOVA (factors: GM&GL length and animal group) was used to assess BTX-A effects on forces, and the Kruskal-Wallis test was used to test the change in proportion of collagenous tissue for each muscle. Differences were considered significant at p<0.05. Results. Injected muscles: ANOVA showed significant main effects of both factors on GM&GL total forces and a significant interaction. Force reductions are more pronounced at shorter lengths (increase from 80.8% to 88.4% with decreasing length). Lrange decreased (by 24.1%). ANOVA showed significant main effects of only muscle length on GM&GL passive forces and no significant interaction. Non-injected muscles: ANOVA showed significant main effects of both factors (for SOL), or only of BTX-A (for TA and EDL) only on muscle total forces, but no significant interaction. Force drops for the SOL (89.8%) and anterior crural muscles (57.0% and 51.0% for TA and EDL) do indicate spread of BTX-A intra- and extra-compartmentally. Histological analyses showed increased ECM collagen contents of BTX-A group for the GM&GL, TA, and EDL. Conclusion. Narrowed Lrange and increased ECM collagen content are not in accord with the clinical purpose of the treatment. BTX-A did not reduce passive forces, but did not cause an increase either. Remarkably, the results show that BTX-A leakage is a major issue that can affect muscles of even antagonistic muscle compartments. Hence, our animal experiments indicate much more complex BTX-A effects than considered, which requires further testing in patients

Summary. A novel in vivo animal model to establish new surgical interventions for patients with ACL insufficiency. Introduction. After ACL reconstruction, recruited cells from surrounding tissues play crucial roles in ligamentization to obtain adequate structural properties. To allow athletes to return sports activity sooner, these remodeling processes should be elucidated and be accelerated. However, in conventional animal models, it has been difficult to differentiate donor and recipient cells. Here we introduce the transgenic Kusabira-Orange pigs, in which cells produce fluorescence systemically, as in vivo model to trace cell recruitment after ACL reconstruction. Methods. After the approval by the Institutional Animal Care and Used Committee, a transgenic pig that carries and produces red fluorescent Kusabira-Orange (KO) was established. Skeletally immature transgenic pigs (n=12) (20 wks old, 76.0 ± 17.5 kg) and wild type (WT) pigs were used as recipient and donor, respectively. For validation of the pigs as in vivo model, the ACL histological structure, cell shape, mitogenic activity, and migration activity were assessed and were compared to those of wild type pigs. The sensitivity and specificity of KO fluorescence under microscopy were analyzed. Histological analyses were conducted with HE, Masson trichrome (MT), and DAPI staining. The length change pattern in our ACL reconstruction was evaluated to validate the surgical procedure. After allograft ACL reconstruction with fresh-frozen flexor digital tendon of WT pigs, pigs were euthanised at 3, 6, 12, and 24 weeks postoperatively (3 pigs each) for the histological analyses. Results. The histological analyses, and mitogenic/migration assays did not show any apparent differences between KO and WT pigs. The sensitivity and specificity of KO fluorescence revealed to be 98%. Maximal length change of the reconstructed ACL was less than 3.5 mm. Three weeks postoperatively, host cells producing KO fluorescence repopulated mainly at the peripheral part of the graft, while, interestingly, cells also located in inter-territorial space of collagen fascicles. More cells migrated towards the mid of the graft in 6–12 week. Cell distribution became homogeneous in parallel to matrix remodeling in 12–24 week. Discussion. As far as we know, this is the first study to apply the genetically engineered pig producing a fluorescent protein as in vivo model to analyze biological remodeling processes after ACL reconstruction. ACL fibroblasts in KO pigs could be detected under fluorescence with high sensitivity and specificity. In addition, structural organization, mitogenic and migration activity were not different from those in WT. As for histological experiments, the recipient cells could be easily and effectively differentiated from donor cells especially 3 weeks postoperatively. Cells migrated in the inter-territorial region among collagen fascicles earlier than we expected. We are going to investigate angiogenesis, matrix remodeling, and structural properties in parallel to the cell migration

Titanium is a popular orthopaedic implant material, but it requires surface modification techniques to improve osseointegration and long term functionality. This project compares a new method of modifying surface topography (nano-patterning) with an existing clinical technology (grit-blasting and acid-etching (GAE)). Titanium discs were blasted with aluminium oxide and etched in sulphuric and acetic acid. Injection moulded discs (with two different nano-patterns) were coated in titanium by evaporation. The topography and chemistry of the discs was assessed using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle measurements, and X-ray photo-electron spectroscopy (XPS). Two discs were plated bilaterally onto a flattened area of the tibiae of 12 rabbits. Tibiae were removed after 4 and 8 weeks for histological assessment of the bone-implant contact (BIC) ratio. AFM and SEM demonstrated a difference in pattern between the square array of nano-pits (SQ) and the randomly positioned nano-pits (RAND). The GAE implants exhibited increased surface roughness (Ra = 570nm) compared to the titanium coated SQ and RAND implants (Ra = 12nm). Water contact angle measurements showed the surface had comparable wettability and XPS demonstrated similar chemical compositions, except GAE surfaces contained 6.8% aluminium. Histological samples analysed at 4 weeks showed a BIC ratio of 36% for GAE, 56% for SQ, and 48% for RAND. At 8 weeks, the BIC ratio was 52% for GAE, 80% for SQ, and 72% for RAND implants. This increase in BIC at 8 weeks for both SQ and RAND implants compared to GAE was statistically significant (P < 0.05). This project demonstrated there was an increase in interfacial bone to implant contact when using a nano-scale topography incorporating nano-pits compared to conventional grit-blasted acid-etched micro-scale topographies. This enhancement of BIC may reduce long term loosening of orthopaedic implants due to mechanical and biological attrition at the interface