Preparation Of The Nanosized And Release-controlled RhBMP-7 Microsphere And Its Effect On Proliferation And Differentiation Of Pdlscs And SCAP.

BMP-7, also known as osteogenic protein-1 , is a member of the transforming growth factor-βsuperfamily.BMP-7 plays an important role in regulating root and reparative dentin formation. With a short half-life and low specificity, it is prone to deactivation because of deliquation and metabolism if the growth factor is used directly in vivo. Microsphere encapsulating growth factors which can slow-release factors were prepared by the double emulsion solvent evaporation technique. By means of isolation, culture and identification of human PDLSCs (periodontal ligament stem cells) and SCAP (stem cells from the apical papilla), the present study prepares the nanosized and controlled rhBMP-7 microsphere and explores its effects on the proliferation and differentiation of PDLSCs and SCAP. The results of the study provided experimental evidences for further research on continuous formation of the root to its completion and bioroot engineering.This study includes three parts:1. Cell culture and identification: Normal human impacted third molars with immature roots or premolares were collected from healthy patients. The root apical papilla and periodontium were digested in a solution of collagenase and dispase and the stem cells were isolated by limited dilution of cultured cell for single cell clone. Their morphology and growth condition were observed under inverted light microscope. Immunohistochemistry procedure was employed to detect surface marker of the cloning cell. Results: PDLSCs and SCAP exhibited self-renewal and clonogenic cell population. The growth velocity of SCAP was slower than PDLSCs. Immunohistochemical analysis showed that cultured cells expressed vimentin and STRO-1 while keratin were absent in PDLSCs and SCAP.2. Preparation of the nanosized and controlled rhBMP-7 microsphere and its relative determine: The rhBMP-7 loaded microspheres with biodegradable poly (3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) were prepared by a double-emulsion, solvent-extraction [(water-in-oil)-in-water] technique and assess the microspheres'morphology, size and size distribution by electron microscope. Determine the encapsulated ratio of the microsphere by using Elisa kit and collect the releasing liquid from the microsphere after 1d, 3d, 7d, 14d, 28d and 56d. Results: The diameter of PHBV microsphere was 50μm±12μm,the load density of rhBMP-7 was 0.67%±0.27%,In the first day, the microsphere release 17.22%±0.94% and raise to 52.75%±4.67% in the 56th day. 3. Effects of the rhBMP-7 loaded microspheres with PHBV on proliferation and differentiation of PDLSCs and SCAP: Cells between the fourth and sixth passages were used in this study. The optical density was obtained by MTT assay in cells stimulated by microspheres controlling and releasing BMP-7 in different date. Effect of microsphere on differentiation of PDLSCs and SCAP was indirectly measured by the activity of alkaline phosphatase of the cells. Result: When compared with control group, the releasing liquid from the microsphere stimulated the cell proliferation significantly and the ALP activities (P<0.05).Moreover,the cell stimulated by the releasing liquid(14d,28d,56d) proliferation and ALP activities increased gradually. Group of the microspheres without BMP-7 had no significant difference comparing with control group(P>0.05). When comparing the group of PDLSCs and SCAP, the effect on proliferation of PDLSCs was slightly greater than SCAP, but the difference is not very significant. However, the ALP activity of SCAP was greater than PDLSCs(P<0.05).It is concluded that with good osteogenetic activity, the biodegradable PHBV microphere system can be served as carrier for sustained release of rhBMP-7.