Introduction: Repair of large bone defects remains a significant clinical challenge. Bone marrow stromal cells (BMSCs), a subset of which is known as bone marrow-derived mesenchymal stem cells, show therapeutic potential for bone regeneration. However, their isolation, expansion and implantation will need to be conducted under good manufacturing practices (GMP) at separate locations. An investigation which mimics this clinical scenario where large bone defects shall be regenerated is required before clinical trials can be initiated. Methods: Seven batches of 100 million human ex-vivo expanded BMSCs from five donors were transported fresh in syringes from a GMP facility in Germany to France. BMSCs were mixed with biphasic calcium phosphate (BCP) biomaterial prior to subcutaneous implantation in nude mice. The capacity of BMSCs in unison with BCP to regenerate critical sized cranial bone defects was also evaluated. BMSCs expressing luciferase were used to assess the viability and bio-distribution of implanted cells. In situ hybridization, using the human-specific repetitive Alu sequence, was performed for the identification of human cells in explants. Results: Eight weeks after implantation of BMSCs, mineralized bone containing mature bone marrow territories was formed in ectopic sites and in calvaria defects. Significant loss of cell viability was observed by bioluminescence imaging and only 1.5 percent of the initial number of transplanted cells remained after 37 days. After eight weeks, while explants were comprised primarily of host cells, there were also human cells attached along the periphery of BCP and embedded in osteocyte lacunae dispersed throughout the newly formed bone matrix. Conclusions: This study demonstrates the safety and efficacy of BMSC/BCP combinations and provides crucial information for the implementation of BMSC therapy for bone regeneration.

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.

Introduction: Osteonecrosis of the femoral head (ONFH) is a degenerative disease progressing to a femoral head (FH) collapse. Injection of osteoprogenitor cells like bone marrow mesenchymal stromal cells (BMSCs) into the FH appears to be a good therapeutic treatment. However, safety and efficacy of BMSCs to treat bone defect are the main preclinical data required for clinical application. Efficacy and the lack of risk of cell transformation after amplification of BMSCs have been extensively described. The main objectives of this study were to develop a simple and usable procedure for clinicians and control its feasibility by evaluating the biodistribution of BMSCs after injection into the FH in a large animal model. The impact of this approach was evaluated on one natural pig ONFH. Methods: BMSCs were directly injected in the pig FH, and then the biodistribution of grafted cells was detected by quantitative real-time polymerase chain reaction, cytometry, or a combination of classic histology analysis and in situ hybridization (ISH). BMSC efficacy on bone regeneration was evaluated by magnetic resonance imaging (MRI) and histology. Results: After 30-minute and 24-hour follow-up, grafted cells were detected at the injection site and no BMSCs were detected in filter organs or body fluids. The combination of classic histology analysis and ISH showed a good homogeneity of cell distribution in FH. Local delivery of BMSCs onto a bone scaffold associated with bone formation in vivo confirmed the preferential tropism of BMSCs to the bone tissue as well as their efficacy to form bone. Treatment of a natural pig ONFH by autologous BMSCs indicated a beginning of bone healing as early as 2 weeks with a complete healing after 9 weeks. At this stage, MRI and histological analysis were similar to those of a normal FH. Conclusions: Intra-osseous injection of BMSCs in FH seems to be a good strategy for ONFH treatment as the safety concerning the biodistribution of BMSCs is ensured. Moreover, the efficacy of BMSCs in natural ONFH seems to indicate that this is a promising approach. Altogether, these results constitute the preclinical data necessary for the setup of a clinical application with expanded BMSCs in the context of advanced therapy medicinal products.

Background: Ewing’s sarcoma (ES) is the second most frequent primitive malignant bone tumor in adolescents with a very poor prognosis for high risk patients, mainly when lung metastases are detected (overall survival <15% at 5 years). Zoledronic acid (ZA) is a potent inhibitor of bone resorption which induces osteoclast apoptosis. Our previous studies showed a strong therapeutic potential of ZA as it inhibits ES cell growth in vitro and ES primary tumor growth in vivo in a mouse model developed in bone site. However, no data are available on lung metastasis. Therefore, the aim of this study was to determine the effect of ZA on ES cell invasion and metastatic properties. Methods: Invasion assays were performed in vitro in Boyden’s chambers covered with Matrigel. Matrix Metalloproteinase (MMP) activity was analyzed by zymography in ES cell culture supernatant. In vivo, a relevant model of spontaneous lung metastases which disseminate from primary ES tumor was induced by the orthotopic injection of 10⁶ human ES cells in the tibia medullar cavity of nude mice. The effect of ZA (50 μg/kg, 3x/week) was studied over a 4-week period. Lung metastases were observed macroscopically at autopsy and analysed by histology. Results: ZA induced a strong inhibition of ES cell invasion, probably due to down regulation of MMP-2 and −9 activities as analyzed by zymography. In vivo, ZA inhibits the dissemination of spontaneous lung metastases from a primary ES tumor but had no effect on the growth of established lung metastases. Conclusion: These results suggest that ZA could be used early in the treatment of ES to inhibit bone tumor growth but also to prevent the early metastatic events to the lungs.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan–Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

The effect of amino-bisphosphonates on osteoblastic lineage and its potential contribution to the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BONJ) remain controversial. We assessed the effects of zoledronic acid (ZOL) on bone and vascular cells of the alveolar socket using a mouse model of BONJ. Thirty-two mice were treated twice a week with either 100 μg/kg of ZOL or saline for 12 weeks. The first left maxillary molar was extracted at the third week. Alveolar sockets were assessed at both 3 weeks (intermediate) and 9 weeks (long-term) after molar extraction by semi-quantitative histomorphometry for empty lacunae, preosteoblasts (Osterix), osteoclasts (TRAP), and pericyte-like cells (CD146). Also, the bone microarchitecture was assessed by micro-CT. Osteonecrotic-like lesions were observed in 21% of mice. Moreover, a decreased number of preosteoblasts contrasted with the increased number of osteoclasts at both time points. In addition, osteoclasts display multinucleation and detachment from the endosteal surface. Furthermore, the number of pericyte-like cells increased at the intermediate time point. The alveolar bone mass increased exclusively with long-term ZOL treatment. The severe imbalance between bone-forming cells and bone-resorbing cells shown in this study could contribute to the pathogenesis of BONJ.