Effect of different type of collagen scaffold for nucleus pulposusmesenchymal stem cell differentiation

English Keywords:Intervertebral disc  Nucleus pulposus tissue engineering  Type Ⅱ collagen  Differentiation

English Abstract:

  【Abstract】 Objectives: To investigate the cell viability, proliferation and differentiation-related gene and protein expression of nucleus pulposus mesenchymal stem cells(NPMSCs) in different types of collagen scaffolds. Methods: Type Ⅰ, type Ⅰ/Ⅱ and type Ⅱ collagen scaffolds were formed in vitro, and microstructure, porosity and degradability were detected. NPMSCs isolated from coccygeal vertebra of healthy male rats were cultured as micromass or in type Ⅰ collagen(COL-Ⅰ), type Ⅰ/Ⅱ collagen(COL-Ⅰ/Ⅱ), type Ⅱ collagen scaffold(COL-Ⅱ), and micromass served as control. Cytotoxicity and cell proliferation were detected by lactate dehydrogenase(LDH) and CCK-8 methods respectively. Differentiation related gene and protein expressions were examined by real-time quantitative PCR and western Blotting respectively, including SOX9, aggrecan, type Ⅰ collagen and type Ⅱ collagen. Alcian blue staining was used to investigate sulfate proteoglycan expression. Results: The porosity of each of collagen scaffolds was measured by more than 90%, and the degradability of COL-Ⅰ, COL-Ⅰ/Ⅱ and COL-Ⅱ was detected as (10.30±0.66)%, (9.87±0.71)%, (10.40±0.53)% respectively in 21d. Collagen scaffolds showed great biocompatibility. There was no difference of LDH among groups at the 7th day(12.24±0.65, 12.13±1.03, 12.67±1.15 and 12.50±1.32, P>0.05). Collagen scaffolds enhanced proliferation of NPMSC after 5d and 7d culture(COL-Ⅰ 0.67±0.04, 1.20±0.05; COL-Ⅰ/Ⅱ 0.62±0.05, 1.20±0.07; COL-Ⅱ 0.69±0.02, 1.34±0.04) were much higher than those of control group(CTL 0.53±0.03, 1.02±0.02, P<0.05). After 21d culture, the mRNA expressions of SOX9, type Ⅰ collagen, type Ⅱ collagen and aggrecan were increased significantly in collagen scaffold groups compared to control group. Among those, COL-Ⅱ group was the highest one and there was significant differences compared to the other groups(P<0.05). Moreover, the expression of protein in COL-Ⅰ group was up-regulated in type Ⅰ collagen and aggre?鄄can. SOX9, type Ⅰ collagen, type Ⅱ collagen and aggrecan were up-regulated in COL-Ⅰ/Ⅱ and COL-Ⅱ group, compared to control group, COL-Ⅱ had the highest protein expression. Alcian blue staining also showed that sulfate proteoglycan synthesis was up-regulated in COL-Ⅱ group. Conclusions: All of type Ⅰ, type Ⅰ/Ⅱ and type Ⅱ collagen scaffolds can promote NPMCs′ differentiation towards nucleus pulposus cell type, and the effect of type Ⅱ collagen scaffolds is most significant. Type Ⅱ collagen is the ideal biological material for nucleus pulposus tissue engineering.