In vitro osteodifferentiation of intact human amniotic membrane is not beneficial in the context of bone repair.

The human amniotic membrane (hAM) is an attractive biomaterial for regenerative medicine, as it contains amniotic mesenchymal stromal cells (hAMSC), epithelial cells (hAEC) and growth factors. We examined the potential use of hAM in orthopaedic and maxillofacial bone surgery, integrating the requirements of current regulations regarding advanced therapy medicinal products (ATMP) in the European Union. Previous studies have described the potential osteodifferentiation of intact hAM during whole-tissue culture in osteogenic conditions. The present study aims to determine whether in vitro osteodifferentiation of hAM is needed in the context bone repair, and the influence of this process on tissue structure, cell phenotype and cell function. Different conditions (fresh or cultured hAM; intact or hAM-derived cells) were tested. Phenotypic and functional analyses were performed with standard approaches (cell culture and staining, histological and immunolabelling) as well as original approaches (tissue staining, energy dispersive X-ray and X-ray diffraction). In our study, non-osteodifferentiated hAM (i.e., fresh or native hAM) exhibited innate pre-osteoblastic potential. Osteodifferentiation of fresh hAM induced a change in tissue structure, cell phenotype and function. Therefore, we hypothesize that pre-osteodifferentiation may not be necessary, especially if it induces unwanted changes. To our surprise, in these osteogenic conditions, hAEC had a mesenchymal phenotype with osteocyte function, and even native synthesis of hydroxyapatite, focusing osteogenic potential mainly in this epithelial layer. In conclusion, in vitro osteodifferentiation by tissue culture does not appear to be necessary for hAM to be used as an innovative ATMP for bone repair.

Fresh and in vitro osteodifferentiated human amniotic membrane, alone or associated with an additional scaffold, does not induce ectopic bone formation in Balb/c mice.

The human amniotic membrane (hAM) has been successfully used as a natural carrier containing amniotic mesenchymal stromal cells, epithelial cells and growth factors. It has a little or no immunogenicity, and possesses useful anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. It has been used for many years in several indications for soft tissue repair. We previously reported that hAM represents a natural and preformed sheet containing highly potent stem cells, and could thus be used for bone repair. Indeed, native hAM possesses pre-osteoblastic potential that can easily be stimulated, even as far as mineralization, by means of in vitro osteogenic culture. However, cell culture induces damage to the tissue, as well as to cell phenotype and function. The aim of this study was to evaluate new bone formation by fresh and in vitro osteodifferentiated hAM, alone or associated with an additional scaffold presenting osteoinductive properties. Moreover, we also aimed to determine the effect of in vitro hAM pre-osteodifferentiation on its in vivo biocompatibility/tissue degradation. Results showed that neither fresh nor osteodifferentiated hAM induced ectopic bone formation, whether or not it was associated with the osteoinductive scaffold. Secondly, fresh and osteodifferentiated hAM presented similar in vivo tissue degradation, suggesting that in vitro hAM pre-osteodifferentiation did not influence its in vivo biocompatibility.

Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank.

Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this "boosted membrane" as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are "materially and easily transferable" to a tissue bank.