Abstract
Summary
Properties of human amniotic membrane are particularly interesting. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating its association with a potentially osteoinductive scaffold.
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 membrane containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Moreover, previous published data showed the possibility of in vitro osteodifferenciation of the whole tissue.
We aim to use hAM as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis. So we are studying the association of hAM with nanofiber jet sprayed polycaprolactone (PCL) scaffolds and the possibility to induce its osteodifferenciation.
Materials and Methods
HAM from cesarean delivery were provided by a local bank of tissue. A biodegradable microfiber PCL scaffold (∼500 μm thick) was produced using a novel jet spraying technique and provided by Biomedical Tissues society (Nantes, France).
We cultured hAM in contact with PCL scaffolds either in MSCs expansion medium or in MSCs osteogenic medium. Then we grafted these montages in an ectopic murin model (in subcutaneous implantation) and we explanted grafted tissues after 1, 2, 4 and 8 weeks. Osteogenic potential was evaluated by immunological studies immediately after in vitro cell studies and after explantations from the mice. Control studies were performed with human mesenchymal stromal cells (hMSCs) seeded on PCL scaffold in MSCs osteogenic medium and cultured with/ without dynamic culture, via an orbital rotator at 150 rpm for 28 days. Cell viability, proliferation and osteoblastic differentiation were evaluated at different times of culture until 28 days.
Results
To date, in vitro studies showed a macroscopic mineralization of hAM cultured in MSCs osteogenic medium. Osteogenic potential evaluated by immunological investigations are currently underway.
Control studies showed that:
- PCL jet sprayed scaffolds supported proliferation and maintained viability of hMSCs.
- Scanning electron microscopy analysis and confocal imaging showed cell attachment with a spread cell morphology after just 1.5 hours. Cells attached along the PCL nanofibres.?
- Entire scaffold depth was infiltrated with cells at days 7 and 28, as seen by DAPI and hematoxylin and eosin staining.
- Minimal collagen deposition was evident after 7 days but was observed in significant amounts after 28 days. Static conditions had the greatest collagen matrix production.
- Alkaline phosphatse gene expression increased in dynamic conditions compared to 2D cultures. Osteocalcin gene expression increased on PCL scaffolds compared to 2D plastic.
- Dynamic loading did not appear necessary for proliferation, infiltration or collagen production.
Discussion
The association of the hAM with an osteoinductive scaffold could have 2 benefits:
- The handling of the hAM
- The osteodifferentiation of the hAM without the employment of chemical products (osteoinductive supplements that would need to be use with a “good manufacturing products” validation).
These preliminary data showed that PCL scaffolds presented osteoinduction properties.
The association of hAM with PCL scaffolds seems to be a good compromise answering both our needs.
