A Xenogeneic Acellular Nerve Scaffold Via Myroilysin Processing Seeded With Human Dental Pulp Precursor Cells

The quality of one’s life will suffer dramatically if he/she suffers from peripheral nerve injury, partial or total loss of sensation and motion. Tissue engineering approaches provide potential strategies to develop biological substitutes which can restore and maintain normal functional anatomy of peripheral nerve. This research is to fabricate a novel tissue-engineered nerve graft and evaluate its biological performance.Part I The preparation and structural observation of the xenogeneic acellular nerve scaffold via MyroilysinObjective:To prepare a novel acellular nerve scaffold and observe its structure.Methods:Sciatic nerves form rabbits were processed by Myroilysin or Sondell method. Tissue sections stained by HE and Masson were observed to evaluate decellularised effect and reservation of basement membrane and collagen in acellular nerve. Surface structure of acellular nerve was observed by a scanning electron microscope.Results:The diameter of the nerves processed by Sondell method was unchanged or slightly less than the natural nerves, while those processed by Myroilysin went swelling and looked more transparent. With histological evaluation, cells, axons and myelin disappeared in acellular nerve processed by Myroilysin. nerve basement membrane and collagen fibers appear orderly arrangement. The acellular nerve scaffold processed by Myroilysin showed porous and longitudinal arrangement with scanning electron microscopy.Conclusion:Acellular nerve scaffold processed by Myroilysin possesses good decellularised effect and excellent basement membrane structure. This nerve scaffold can also have special structure to induce the growth of seeded cells topographically. Part Ⅱ Human dental pulp precursor cells cultivation and their expression of S-100Objective:To culture and identify human dental pulp precursor cells and to evaluate the feasibility to take these cells as seed cells in the tissue-engineered nerve graft.Methods:The third molars of patients aged15-18years were extracted to get dental pulp tissue. Human dental pulp precursor cells were identified by mesenchymal stem cell marker STRO-1after these cells were obtained from those dental pulp tissue by Gronthos’ method. These cells also expressed S-100.Results:The obtained cells showed high proliferation, STRO-1+and S-100+.Conclusion:Dental pulp precursor cells with STRO-1+and S-100+may have have some neurotrophic activities. The acellular nerve scaffold seeded with dental pulp precursor cells with STRO-1+and S-100+may work with more neurotrophic function. Part Ⅲ The biological properties of xenogeneic acellular nerve scaffold via Myroilysin processingObjective:To evaluate the biological properties of xenogeneic acellular nerve scaffold processed by Myroilysin and the suitability between the Myroilysin scaffold and human dental pulp precursor cells.Methods:CCK-8cell proliferation assay was done to evaluate the compatibility of the acellular nerve scaffolds and human dental pulp precursor cells. The Myroilysin acellular nerve was implanted subcutaneously in Wistar rats, after which tissue sections were observed to evaluate the response of tissue to the implanted scaffold. Collagenase I was used to test the degradation of the acellular nerves processed by Myroilysin. The EdU test was done to observe how human dental pulp progenitor cells to grow into the nerve scaffolds and their arrangement pattern. The dental pulp precursor cells were cultured with the leach liquor of the Myroilysin acellular nerve for4days, after which RT-PCR was performed to analyse the relative mRNA expression of NGF and BDNF.Results:The human dental pulp precursor cells showed higher proliferation than they were cultured alone. Adverse reactions were not observed after the acellular scaffolds were implanted subcutaneously in rats although acute inflammation happened in the early period. The degradation of Myroilysin acellular nerve scaffolds was consistent with the natural nerves. Human dental pulp precursor cells showed oriented growth after they were seeded into the xenogeneic acellular nerve scaffolds via Myroilysin processing. These precursor cells showed more mRNA expression of NGF and BDNF after they were cultured with the leach liquor of the acellular nerve scaffold.Conclusion:Xenogeneic acellular nerve scaffold processed by Myroilysin has good biocompatibility and degradation. Its porous and longitudinal structure can induce dental pulp precursor cells to align orderly. This kind of nerve scaffold can also play a central role by creating a three-dimensional microenvironment that supports attachment, proliferation, migration and differentiation of transplanted or regenerating resident cells and promotes cell-cell interactions and new tissue development.