ABSTRACT
Objective To study the biological behavior of peripheral blood mesenchymal stem cells (PBMSCs) in 3D composite scaffolds.Methods The proliferation and chondrogenesis of rabbit PBMSCs seeded on porcine cancellous bone (DCB) scaffolds were evaluated,and bone marrow mesenchymal stem cells (BMMSCs) and articular chondrocytes (ACCs) were used as controls.Cell morphology and distribution in scaffolds were observed using scanning electron microscopy (SEM).Live/Dead staining was employed to detect cell viability,Hoechst 33258 method to measure DNA content,dimethylmethylene blue (DMMB) assay to detect glycosaminoglycan (GAG),enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to detect the content of type 2 collagen (COL 2),and RT-PCR to analyze chondrogenesis-related gene expression.Results SEM showed that three kinds of cells uniformly adhered and evenly distributed in DCB scaffolds.Live/Dead staining observed the similar viability of the three kinds of cells three days after seeding (P＞0.05).There was no significant difference in the proliferation ability and DNA content among three kinds of cells after seven days of in vitro culture.After 21 days of chondrogenic culture,both PBMSCs and BMMSCs secreted more GAGs than ACCs,while the secretion of COL 2 was similar to that of ACCs.Moreover,the gene expression of AGC,COL 2 and alkaline phosphatase (ALP) were significantly up-regulated (P＜0.05) in PBMSCs and BMMSCs but significantly down-regulated in ACCs (P＜0.05).The expression of COL 1 in MSCs groups displayed an increasing trend but a decreasing trend in ACCs group (P＞0.05).The gene expression of COL 2 and ALP,but not of AGC and COL 1,in PBMSCs and BMMSCs was higher than those in ACCs (P＜0.05).Conclusions PBMSCs and BMMSCs have similarly excellent proliferation and chondrogenesis potential in 3D porous DCB scaffolds.However,hypertrophic gene expression is still observed under in vitro culturing condition,suggesting the need to further optimize the culture system.