Effects Of Surface Modification Of Biogegradable Polymers PLGA On The Adhesion, Proliferation And Differentiation Of Rabbit Bone Marrow Stromal Cells In Vitro And In Vivo

Cell adhesion to surface of the substrate is essential to development of the anchorage-dependent cells.Only after adhering to surface followed by spreading can cells develop and proliferate.Most synthetic polymers used as orthopaedic matrix substitute present hydrophobicity,which may correlates to the low degree of cell attachment.Modification with cell adhesion protein/peptides can be benificial to the cell adhesion on polymers and then affect the cell proliferation and differentiation.Cell attachment to substrate is primarily mediated by integrins,a widely expressed family of heterodimeric surface receptors.Most extrcellular matrix proteins such as fibronectin,osteopontin,collagen type I , bone sialoprotein and vitronectin contain an Arg-Gly-Asp (RGD) sequence which is specific to the fixation of cell membrane receptors like integrin.The main aim of this research is to measure,assess adhesion,proliferation of rabbit marrow stromal cells(MSCs) on the polymers coated by fibronectin, collagen type I or biotiegen,which includes:(1)Biologic characteristics of rabbit MSCs were observed by two types of separating method in primary culture.(2)Adhesion,proliferation and differentiation of MSCs cultured on polymers coated with biotiegen were assessed.(3)also, adhesion,proliferation and differentiation of MSCs were assessed on PLGA film orporous PLGA substrates coated with fibronectin, or collagen type I respectively.(4)Bone formation was observed on the porous PLGA substrates coated with collagen type I in vivo.This research aims to give new way to make novel synthetic bone with cell adhesion and high bone induction capabilities.Results obtained included:(1) MSCs began to attach to culture flask after 24h cell inoculation by centrifugation method.Cell proliferation was active after 3d.and MSCs began to attach to culture flask only after 36h cell inoculation by erythrocyte splitting method. Cell morphology was similar no matter what method was used.Percentage of live cells was significantly higher in erythrocyte splitting method that was (91 ±4)%than in centrifugation method that was (83±5) % .Proliferative ability of MSCs seperated by erythrocyte splitting method at 2 to 6 generation was much higher than by another method.The maximal cell number was 4.67 vs 4.10 times that of the initial cell number at cell inoculation. There was no difference in other biologic characteristic of MSCs between the two separation method,such as cell anchorage ratio and clone formation ratio.(2)PLGA film presented uniformity frame with no protuberance and fissure under scanning electron microscopy(SEM).Big aperture with smooth wall and average 400 μ m in size running-through each other was observed in porous PLGA substrate,around the big aperture there were many round micropores about 5μm size.All of the structure were equal and uniform,which satisfied the further research work.(3)MSCs adhesion at earlier time was promoted by biotiegenrAfter 3h ,cell number was (1.5 ±0.18) × 105 in the PLGA film coated with biotiegen group,which was significantly higher than that in PLGA film group (P<0.01) and higher than that in coverslip group (P<0.05) ,which cell number was (1.04± 0.21) × 105 .After 6h and 12h biotiegen could not promote cell adhesion,and cell proliferation and alkaline phosphatase(ALP) activity were not promoteddramatically during 9 days.(4) Cell adhesion was promoted by fibronectin or collagen type I . Cell adhesion dramatically increased after 6h by fibronectin and after 12h by collagen type I in monolayer culture. Fibronectin or collagen type I promoted cell adhesion after 6h under three dimension condition. Fibronectin had more ability to promote cell adhsion than collagen type I under such circumstances,SEM micrographs showed that MSCs all had a spread-out appearance with uniform distribution on polymer substrates precoated with fibronectin or collagen type I .(5) Fibronectin or collagen type I also promoted cell proliferation within 9 days culture under three dimension condi...