Abstract
Summary
Human amniotic membrane has interesting properties for regenerative medicine. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating: the necessity of its osteodifferentiation and the impact on immunogenicity; its optimal condition for storage.
Introduction
The human Amniotic Membrane (hAM) is known to have a good potential to help the regeneration of tissues. It has been used for 100 years in many medical disciplines because of its properties: a flexible scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Previous published data showed the possibility of in vitro osteodifferentiation of the whole tissue. We aim to use this «boosted membrane» as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis, so, we are studying:
The necessity to osteodifferentiate the tissue and its consequence on the immunogenicity; Its in vivo osteogenic potential; The effects of the cryopreservation on cell viability and function.
Materials and Methods
HAM from cesarean delivery were provided by a local bank of tissue.
4 groups of hAM were performed:
Group 1: non treated (fresh hAM);
Group 2: cryopreserved hAM;
Group 3: cultured hAM in osteogenic medium 3 weeks;
Group 4: cultured hAM in osteogenic medium 3 weeks and then cryopreserved.
Cell viability was measured and the osteodifferentiation was revealed by Alizarin Red and von Kossa staining on whole tissue and on histological sections.
We grafted these 4 groups of hAM in an ectopic murine model (subcutaneously) either alone (to evaluate its resorption and its immunological tolerance) or wrapped around bone substitute (to observe its osteogenic potential). We explanted grafted tissues after 1, 2, 4 and 8 weeks and performed histological and immunological studies.
Results
To date, in vitro studies showed: A macroscopic mineralization of hAM in group 3. Different cell viability according to the groups that apparently doesn't alter the osteogenic potential. A tensile strength dependent on the time of cryopreservation and the type of cyropreservant used. In vivo macroscopic studies showed: no immunological reaction neither resorption of hAM after 4 weeks of implantation in all the groups and a tissue calcification in group 3. In vivo microscopic studies showed: the presence of hAM in a vascularised fibrous capsule; the bilayer hAM structure is still observed without sign of inflammation.
Discussion
We confirmed the in vitro osteodifferentiation of hAM reported by others. First in vivo experiments let us think that the tissue keeps its low immunogenicity after its osteodifferentiation; the histological results will confirm it. The definition of optimal conditions for cryopreservation is in progress.
At the end of this proof of concept, we will able to conclude if hAM can be used as an allograft for bone repair and if its preliminary osteodifferentiation is necessary. Next step will consist to test it in an orthotopic critical size bone defect model.
