Osteogenetic Differentiation Of BMSC-sheet/Col-I Scaffold Combination In Vitro

Background:Bone defect caused by trauma,inflammation,tumor or congenital malformations is a common and complicated clinical disease which results in functional disability.Autologous bone graft is regarded as the golden standard of bone repair.However,short supply and donor site morbidity are major obstacles for its clinical use.Many researchers are beginning to search artificial materials with favorable osteogenic properties that are similar or even superior to autologous bone graft.The ideal process of bone repair by artificial material concludes the degradation of scaffold and the continual proliferation and differentiation of seed cells which result in new tissue formation.However,proteolytic enzymes would deteriorate the extracellular matrix(ECM)and growth factors of seed cells which lead to a reduction of cell activity.What's more,there is a great loss of cells after cell seeding.The new technology—cell sheet technology(CST)[1]makes it possible to fabricate a sheet full of cells along with their natural extracellular environment.CST would mostly reserve the ECM and cell surface protein such as ion channel protein and growth factor receptor and could significantly improve graft-host connection and cell retention.So far,cell sheet based technology has already been applied for constructing several kinds of tissue such as myocardium,cornea,esophageal epithelium and periodontal ligament[2,3].But the application of cell sheet technology in bone repair is still not clear.Bone marrow stromal cells(BMSCs)have potential for use in bone repair owing to their general availability,great ability of self-renew and favorable osteogenic potential.Ideally,the scaffold should be biocompatible,biodegradable,osteoconductive,osteoinductive and provokes no significant inflammatory response.After it is implanted into a bone defect,the scaffold must be reabsorbed naturally as the bone grows,until finally it is completely replaced with newly formed bone.Type ? collagen(Col-I)is the major component of bone matrix which has been comfirmed to provide microenvironment for BMSC to survive by means of its porous characteristic.Col-I scaffold can also serve as a vehicle for delivering osteoinductive agents to promote the healing of bone defects.Objective:In this research,we constructed BMSC-sheet/Col-I scaffold combination as artificial bone with cell sheet technology and compared its osteogenic capacity with BMSCs/Col-I scaffold in vitro in order to discuss the advantage of cell sheet technology in bone repair.Methods:Porous Col-I scaffolds were prepared.Cell sheets comprising multilayered BMSCs were continuously cultured and flushed down with culture medium.BMSC-sheets and BMSCs were combined with Col-I scaffold and cultured in osteoblast inducing conditional media.The osteogenic capacity of BMSC-sheet/Col-I combination and BMSCs/Col-I combination were comprehensive assessed by ALP activity,steopontin(OPN)protein level,mineralized nodule formation at different points in time.Results:1.ALP activity:ALP activity of BMSC-sheet/Col-I combination on day 3 and day 7(47.03±6.37 U/L,22.61±1.95 U/L)were significantly higher than that of BMSC/Col-I combination(34.58±2.43 U/L,16.28±1.04 U/L)(P<0.05).2.OPN protein levelOPN protein level of BMSC-sheet/Col-I combination on day 7?14?21?28(1113.1±27.5ng/ml,966.5±95.2 ng/ml,1173.2±111.6 ng/ml,1157.6±47.4 ng/ml)were significantly higher than that of BMSCs/Col-I combination(943.5±18 ng/ml,735.8±101.7 ng/ml,1133.6±89.8 ng/ml,1077.2±137.6 ng/ml)(P<0.05).3.Mineralized nodule formationThe density of calcium nodule of BMSC-sheet/Col-I combination on day 28 was significantly higher than that of BMSCs/Col-I combination observed by scanning electron microscope.X-ray spectroscopy analysis indicated that the mineralized nodule consists of four elements:calcium,phosphorus,carbon,oxygen.Calcium content of calcium nodule of BMSC-sheet/Col-I combination(0.6%)on day 28 was significantly higher than that of BMSCs/Col-I combination(0.1%).Conclusion:Results demonstrated that BMSC-sheet/Col-I combination had a stronger potential in osteogenic differentiation than BMSCs/Col-I combination in vitro,indicating a promising future of cell sheet technology in the field of bone repair.