ALBERT

Description: Bone is one of the most frequently transplanted tissues, with allografts and autografts accounting for more than 80% of total grafts. Synthetic biomaterials in association with autologous or allogeneic mesenchymal stromal cells (MSCs) represent a valid alternative in orthopaedic and maxillofacial surgery. Aim of REBORNE consortium is to perform clinical trials using standardized protocols based on advanced biomaterials and MSCs. Aim of our Unit was to assess MSC immune modulatory properties in presence of a novel biomaterial consisting of hydroxyapatite and tricalcium-phosphate (HA/TCP, Biomatlante, France) as scaffold for MSC delivery. We assessed immune modulatory properties, in terms of immunophenotype, inhibitory and anti-apoptotic effects, of MSCs of different origin when associated with the HA/TCP biomaterial, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), adipose-derived MSCs (ASCs) and cord blood-derived MSCs (CB-MSCs). The culture of all MSCs with HA/TCP, did not modulate the anti-apoptotic and suppressive features of all MSCs toward sorted-T, B and NK lymphocytes as compared to standard culture setting. When exposed to inflammatory cytokines, such as IFN-γ and TNF-α, all MSCs were induced to acquire the suppressive phenotype, characterized by MHC-1 and CAM molecules up-regulation, and de novo expression of HLA-DR molecules. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype, as indicated by the up-regulation of osterix and osteocalcin transcription. The differentiation process is promoted in all MSC types, but especially in BM-MSCs, by dexamethasone and, to a higher extent, BMP-4 treatment. In view of using MSCs for advanced therapies in allogeneic setting, we evaluated the immunogenicity and immune modulatory features of pre-differentiated MSCs in association with HA/TCP towards both innate and adaptive immune cells, as well as the molecular pathways involved in MSC-mediated immune regulation. MSC-derived osteoblasts did not induce immune cell activation, as demonstrated by co-culture of unstimulated immune effector cells with pre-differentiated MSCs. We found a lower suppressive capability of pre-differentiated MSCs towards stimulated T and NK cells. However, some inhibitory effects could be exerted by BMP-4 treated-MSCs, and could be related to the presence of undifferentiated MSCs in culture, which could inhibit lymphocyte proliferation. The balance between the number of bone depositing osteocyte-like MSCs and immunosuppressive undifferentiated MSCs could be useful, in vivo, to promote bone healing and to reduce local inflammation, respectively. We then investigated in pre-differentiated MSCs the molecular mechanisms involved in the inhibition of T cell proliferation, by interfering with IDO and COX-2 activation. We found that pre-differentiated MSCs switched on their suppressive machinery by activating both IDO, which plays a central role in immune regulation, and COX-2, whose role is not significant in undifferentiated MSCs. COX-2 is produced rapidly as inflammation mediator after fracture, and recent data highlighted the role of COX-2 in improving fracture healing. In conclusion, this study increases the knowledge on MSC biology and gives pre-clinical data concerning the use of allogeneic MSC in combination with ceramic scaffolds to treat bone defects. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1924 Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries. Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante). Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results: We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC. Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix. These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures: No relevant conflicts of interest to declare.