A Study Of The Adhesion Of Chondrocytes Co-cultivated Porous Tantalum By Rgd Peptide Coated In Vitro

Objective To investigate the influence on the hydrophilicity and compatibility of Tanmodified with different materials （RGD、ColⅡand RGD/ColⅡ）as cell scaffold, byobserving the adhesion, proliferation and matrix secretion of chondrocytes.Methods1The morphology of porous tantalum modified by RGD, ColⅡ and mixturecontaining1mg/mlRGD and1mg/ml ColⅡ were recorded under the SEM; Thehydrophilicity of these porous tantalum scaffolds were detected and observed.2Theporous tantalum were modified with different materials,0.2mg/ml RGD/ColⅡ,1mg/mlRGD/ColⅡ,5mg/ml RGD/ColⅡ,10mg/ml RGD/ColⅡ,1mg/ml RGD and1mg/ml ColⅡ, pure porous tantalum as control group,24-well plate as a blank control group.Chondrocytes were seeded onto these porous tantalum scaffolds, and incubated for2hand4h at standard condition, the number of adhesive cells was counted to evaluate theefficiency of cell adhesion, and the photoes of adhesive cells cultured for4h were takenby SEM.3The second generation chondrocytes were cultured on these scaffolds invitro.The morphological changes and growth feature were recorded under the SEM. Theproliferation of chondrocytes on scaffolds were evaluated by MTT assay. The contenttype Ⅱcollagen were estimated from the determination of hydroxyproline content.Results1The observed result of canning electron microscope showed that the poresizes of porous tantalum ranged from400μm to600μm, which had a similar three-dimensional connected hole morphology to cancellous bone. The three-dimensionalconnected hole morphology of modified tantalum was not changed. There are spots onthe surface of RGD coated tantalum and film-like coating on the surface of ColⅡorRGD/ColⅡcoated tantalum.2The water absorption rates of pure Ta, Ta-RGD, Ta-ColⅡand Ta-RGD/ColⅡ1.0mg/ml were1.3%±0.1%,1.5%±0.3%,4.1%±0.6%,4.3%±0.5%.The result showed that the water absorption rates of modified tantalum were significantlyincreased, and the difference was statistically significant (P<0.05).3After2h and4hculture, the efficiency of cell adhesion in the modified tantalum was higher than theunmodified tantalum, Ta-RGD/ColⅡgroup＞Ta-ColⅡgroup＞Ta-RGD group＞pure Tagroup, and the difference was statistically significant (P<0.05). The porous tantalum wasmodified by different concentrations of RGD mixed with Col Ⅱ, the adhesion rateincreased with increasing concentrations of RGD, but when RGD concentration increased to a certain extent, its ability to promote cell adhesion exists saturation.4Cellproliferation was assessed by an MTT assay. After1-day cultivation, Ta-RGD group＞pure Ta group, Ta-ColⅡgroup＞pure Ta group, Ta-RGD/ColⅡ1.0mg/ml group＞Ta-RGD group, Ta-RGD/ColⅡ1.0mg/ml group＞Ta-ColⅡgroup, and the difference wasstatistically significant (P<0.05). The OD value was higher in the ColⅡgroup than in theRGD group, but the difference was not statistically significant. After3,5,7,10and14days of cell cultivation, Ta-RGD/ColⅡ1.0mg/ml group＞Ta-ColⅡgroup＞Ta-RGDgroup＞pure Ta group, and the difference was statistically significant (P<0.05).5Thecontent type Ⅱcollagen were estimated from the determination of hydroxyprolinecontent, Ta-RGD/ColⅡ1.0mg/ml group＞Ta-ColⅡgroup＞Ta-RGD group＞pure Tagroup, and the difference was statistically significant (P<0.05).Conclusion1The three-dimensional connected hole morphology of modifiedtantalum was not changed, but the water absorption rates of modified tantalum weresignificantly increased.2RGD peptide-immobilized tantalum layer has a positive effecton chondrocyte early adhesion and proliferation．Porous tantalum coating with collagenhas a better biocompatibility for the attatchment and proliferation of the chondrocyte thanpure tantalum.3Porous tantalum was modified by the mixture of RGD and ColⅡasartificial cartilage extracellular matrix, can significantly promote the tantalum surfaceadhesion and proliferation of chondrocytes, and the best group is Ta-RGD/ColⅡ1mg/ml,It is expected to become an ideal scaffold for cartilage tissue engineering.