Preliminary Study Of Dentin Matrix-Platelet-Rich Plasma Gel For Constructing Pulp Tissue Engineering Scaffold

Objective:To explore the feasibility of platelet-rich plasma (PRP) gel as a delivery system for growth factors. To evaluate the cytocompatibility of PRP gel and demineralized dentin matrix, and investigate the effect of demineralized dentin matrix on the mineralization of dental pulp cells derived from miniature pig deciduous teeth, to provide a experimental basis for constructing dentin matrix-PRP gel as pulp tissue engineering scaffold.To evaluate the performance of PRP gel as an injectable scaffold for constructing of tissue-engineered pulp by observing the growth of dental pulp cells derived from miniature pig deciduous teeth in autologous PRP gel. To investigate the effect of demineralized dentin matrix on the biological behavior of dental pulp cells derived from miniature pig deciduous teeth.Methods:1. Dental pulp cells derived from miniature pig deciduous teeth were isolated and cultured with tissue block enzyme digestion method. Vimentin and cytokeratin expression was detected with Immunohistochemical staining. 2. Venous blood samples were drawn from miniature pigs. PRP was prepared with two-step centrifugation procedure. Platelet concentration in PRP and whole blood was determined. The release of PDGF and TGF-β and VEGF from PRP gel and blood clot, which formed of activator mixed with PRP and whole blood respectively, was measured over7days by enzyme-linked immunosorbent assay.3. Dental pulp cells at passage3were seeded in50%or10%PRP gel which containing50%or10%autologous PRP and cultivated in vitro for3weeks, then evaluated by histolody.4. Root fragments of miniature pigs deciduous teeth were soaked in5.25%NaOCl following by17%EDTA then PBS, and meanwhile concussed by means of ultrasonic cleaner. Dental pulp cells derived from miniature pig deciduous teeth at passage3were seeded on the dentin surface of root canal wall. Cell growth and proliferation were observed by SEM. The demineralization ability of cells cultured with demineralized dentin matrix was determined with alizarin red staining.Results:1. Dental pulp cells derived from miniature pig deciduous teeth were polygonal or fibroblast-like. Positive immunohistochemical staining of vimentin and negative immunohistochemical staining of cytokeratin was observed.2. Platelet concentration were (1672.67±117.35)×109/L and (302.67±21.55)×109/L in PRP and whole blood respectively. Platelet concentration of PRP is (5.85±0.41) times that of whole blood (P<0.05), and the recovery rate was (66.45%±5.27%). After PRP and whole blood were activated, the release of PDGF from PRP gel were higher than blood clots at day0.5,1,3and5(P <0.05), there was no significant difference in the release of PDGF between PRP gel and blood clot at day7(P>0.05); the release of TGF-β from PRP gel were higher than blood clots at day0.5,1and3(P<0.05); there was no significant difference in the release of TGF-β between PRP gel and blood clot at day5and7(P>0.05). And no significant difference was found in the release of VEGF between PRP gel and blood clot at each time point during7days (P>0.05).3. Dental pulp cells derived from miniature pig deciduous teeth were well-distributed in the three-dimensional space of both50%and10%PRP gel. The retraction rate of cell-50%PRP gel composite at day7was61.57%±3.82%, while cell-10%PRP gel composite didn’t retract significantly. Greater collapse was observed in cell-10%PRP gel composite.4. Well-growth and proliferation of dental pulp cells derived from miniature pig deciduous teeth were observed on demineralized dentin matrix. Cells cultured with demineralized dentin matrix was positive for alizarin red staining.Conclusion:The plasticity and the characteristics of continuously release of a variety of growth factors could probably promote the application of PRP gel as a delivery system for growth factors.10%PRP gel could serve as an injectable scaffold in pulp tissue engineering. Demineralized dentin matrix could possibly provide a good microenvironment for the proliferation and mineralization of dental pulp cells.