Articular cartilage repair remains a challenge in orthopedic surgery, as none of the current clinical therapies can regenerate the functional hyaline cartilage tissue. In this study, we proposed a one-step surgery strategy that uses autologous bone marrow mesenchymal stem cells (MSCs) embedded in type II collagen (Col-II) gels to repair the full thickness chondral defects in minipig models. Briefly, 8 mm full thickness chondral defects were created in both knees separately, one knee received Col-II + MSCs transplantation, while the untreated knee served as control. At 1, 3 and 6 months postoperatively, the animals were sacrificed, regenerated tissue was evaluated by magnetic resonance imaging, macro- and microscopic observation, and histological analysis. Results showed that regenerated tissue in Col-II + MSCs transplantation group exhibited significantly better structure compared with that in control group, in terms of cell distribution, smoothness of surface, adjacent tissue integration, Col-II content, structure of calcified layer and subchondral bone. With the regeneration of hyaline-like cartilage tissue, this one step strategy has the potential to be translated into clinical application

Background. Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone marrow mesenchymal stem cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods. Thirty-two wistar rats were divided into four groups according to the type of structural bone allograft; the first: partially demineralized only (Donly), the second: partially demineralized stem cell seeded (DST), the third: partially demineralized laser-perforated (DLP), and the fourth: partially demineralized laser-perforated and stem cell seeded (DLPST). Trans-cortical holes were achieved in four rows of three holes approximated cylindrical holes 0.5 mm in diameter, with centres 2.5 mm apart. P3 MSCs were used for graft seeding. Histologic and histomorphometric analysis were performed at 12 weeks. Results. DLP grafts had the highest woven bone formation, where most parts of laser pores were completely healed by woven bone. DST and DLPST grafts surfaces had extra vessel-ingrowth-like porosities. Furthermore, in the DLPST grafts, a distinct bone formation at the interfaces was noted. Conclusion. This study indicated that surface changes induced by laser perforation, accelerated angiogenesis induction by MSCs, which resulted in endochondral bone formation at the interface. Despite non-optimal results, stem cells showed a tendency to improve osteochondrogenesis, and the process might have improved, if they could have been supplemented with the proper stipulations

INTRODUCTION. Trabecular Titanium. ™. (TT) is a novel material with a structure similar to trabecular bone, already used for prosthetic clinical applications. Being the bone-implant interface the weakest point during the initial healing period, the association of TT with a hydrogel enriched with progenitor cells and osteoinductive factors may represent a promising strategy to improve prosthesis osteointegration. In a previous in vitro study we evaluated the ability of an ammidated carboxymethylcellulose hydrogel (CMCA) and of TT enriched with CMCA to support bone marrow mesenchymal stem cells (BMSCs) viability and osteogenic differentiation [1]. The aim of this study was to evaluate in vivo if the association of TT with CMCA enriched with strontium chloride (SrCl. 2. ) and BMSCs could ameliorate TT osteointegration. METHODS. This study combines TT with CMCA, SrCl. 2. and BMSCs. To mimic prosthesis-bone implants, TT discs were seeded with human BMSCs predifferentiated in osteogenic medium, then press-fit into engineered bone. A total of 36 athymic mice were implanted subcutaneously, each animal received 2 constructs as un-seeded TT and TT+CMCA or cell seeded TT+BMSCs and TT+CMCA+BMSCs. After 4, 8 and 12 weeks, osteodeposition, bone mineral density (BMD) and osteointegration were evaluated by fluorescence imaging, micro-CT, SEM, histology and pull-out tests. RESULTS. Micro-CT analysis demonstrated the homogeneity of the engineered bone in all experimental groups, supporting the reproducibility of our novel engineered model. Macroscopic evaluation of explanted constructs after 4 weeks revealed their integration with mice subcutaneous structures. In pull-out biomechanical tests, increases in extraction energy and peak force from 4 to 12 weeks were observed in all the experimental groups, except TT+CMCA. TT+CMCA+BMSCs showed the highest value of peak force and the greatest increase in comparison to samples explanted at 4 weeks. In vivo fluorescence imaging showed osteodeposition activity inside the constructs, observation confirmed by the ex-vivo analyses revealing a higher activity in TT+BMSCs and in TT+CMCA+BMSCs in comparison to acellularized TT samples. SEM evaluation of ECM deposition at the interface between bone scaffolds and TT disks revealed a significant difference between TT+CMCA+BMSCs and the other experimental groups with the former showing an almost complete filling of the space between the integration surfaces already after 4 weeks. In histomorphometric analyses of tissue ingrowth at 8 weeks, TT+BMSCs and TT+CMCA+BMSCs showed a greater tissue ingrowth compared to TT and TT+CMCA samples. DISCUSSION. Several efforts have been made to improve osteointegration with particular attention to critical cases such as implant revision surgeries. The association of porous structures with osteoinductive factors enriched hydrogels and stem cells represents a novel and promising strategy for more effective osteointegration to reduce prosthesis mobilization risks. Our results demonstrate that the association of Trabecular Titanium. ™. with a SrCl. 2. enriched hydrogel and BMSCs increases the production of ECM and may thus represent a valid approach to accelerate prosthesis osteointegration. Further validation of these data will include construct implantation in large animal orthotopic models to better mimic surgical procedures

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

Purpose. Internal fixation of fractures in the presence of osteopenia has been associated with a failure rate as high as 25%. Enhancing bone formation and osseointegration of orthopaedic hardware is a priority when treating patients with impaired bone regenerative capacity. Fibroblast Growth Factor (FGF) 18 regulates skeletal development and could therefore have applications in implant integration. This study was designed to determine if FGF 18 promotes bone formation and osseointegration in the osteopenic FGFR3−/− mouse and to examine its effect on bone marrow derived mesenchymal stem cells (MSCs). Method. In Vivo: Intramedullary implants were fabricated from 0.4 × 10mm nylon rods coated with 300nm of titanium by physical vapour deposition. Skeletally mature, age matched female FGFR3−/− and wild type mice received bilateral intramedullary femoral implants. Left femurs received an intramedullary injection of 0.1μg of FGF 18 (Merck Serono), and right femurs received saline only. Six weeks later, femurs were harvested, radiographed, scanned by micro CT, and processed for undecalcified for histology. In Vitro: MSCs were harvested from femurs and tibiae of skeletally mature age matched FGFR3−/− and wild type mice. Cells were cultured in Alpha Modified Eagles Medium (αMEM) to monitor proliferation or αMEM supplemented with ascorbic acid and sodium beta-glycerophosphate to monitor differentiation. Proliferation was assessed through cell counts and metabolic activity at days 3, 6 and 9. Differentiation was assessed through staining for osteoblasts and mineral deposition at days 6, 9 and 12. Results. Wild type mice exhibited more peri-implant bone formation compared to FGFR3−/− mice. Peri-implant bone formation at the proximal metaphyseal-diaphyseal junction was increased in FGF18 treated femurs compared with contralateral control femurs in wild type (p = NS) and FGFR3−/− (p = 0.04) mice. Histological analysis corroborated micro CT findings, with FGF 18 treated FGFR3−/− femurs forming peri-implant bone instead of the fibrous response seen in controls. In vitro studies showed that FGF18 significantly increased MSC proliferation and metabolism in a dose dependent manner in wild type and FGFR3−/− mice. Osteoblast differentiation was inhibited by FGF18 in wild type MSCs, but was increased at physiological concentrations in cells harvested from FGFR3−/− mice. Conclusion. FGF 18 increases bone formation and osseointegration of intramedullary implants in osteopenic mice and increases MSC proliferation in both the presence and absence of FGFR3. FGF18 also promoted osteoblast differentiation in the absence of FGFR3 signalling, most likely via FGFR1 or 2. Additional work is needed to confirm the identity of the alternate FGFR and to evaluate its capacity to improve osseous healing in unfavourable in-vivo environments