煅烧骨与水凝胶复合对骨髓基质细胞贴附和生长的影响

EFFECT OF COMPOUND PATTERN OF CERAMIC BOVINE BONE AND HYDROGEL ON ATTACHMENT AND GROWTH OF BONE MARROW STOMAL CELL

目的 观察煅烧骨 (CBB)与聚氧化乙烯 -聚氧化丙烯 -聚氧化乙烯三聚体 (PEO- PPO- PEO)聚合物水凝胶 (HG)复合对骨髓基质细胞 (MSC)的贴附、增殖及分化的影响 ,探讨组织工程骨构建的最佳方法。方法 CBB、HG、MSC按不同方式和顺序复合 ,形成三种复合物 CBB/HG/MSC为 A组、HG/MSC/CBB为 B组、CBB/MSC/HG为 C组 ,CBB/MSC作为对照进行体外培养。 5、10天取材 ,扫描电镜观察细胞形态以及与材料的贴附情况 ,细胞计数观察其增殖情况 ,组织化学方法检测 MSC的碱性磷酸酶 (AL P)活性及免疫组织化学方法检测其 型胶原基因表达情况。结果 扫描电镜见 A、C组与对照组细胞完全伸展 ,但 A组细胞数量明显增多 ,重叠生长 ,细胞外基质分泌增加 ,优于 C组和对照组 ,而 B组细胞大多为圆形 ,轻微伸展 ,无细胞外基质分泌。A组与 C组的 AL P活性无差异 (P>0 .0 5 ) ,但显著高于 B组和对照组 (P<0 .0 1)。5天时四组细胞 型胶原合成情况无明显差异 ,但 10天时 B组细胞的 型胶原平均灰度值明显高于其它三组 (P<0 .0 1)。结论 CBB、HG与细胞不同的复合对 MSC的贴附、增殖、分化均有影响 ,CBB/HG/MSC顺序复合较好。

Objective To investigate the effect of compound pattern of ceramic bovine bone (CBB) and hydrogel(HG) on attachment, proliferation and differentiation of bone marrow stromal cell (MSC), and to find out the best way of constructing tissue engineered bone. Methods CBB, HG and MSC was compounded in different patterns and sequences to form CBB/HG/MSC (group A), HG/MSC/CBB(group B), CBB/MSC/HA(group C) and CBB/MSC (control group). Attachment and morphology of MSC were observed by scanning electronic microscope; the proliferation of MSC was evaluated by cell count; alkaline phosphatase(ALP) activity was examined by histochemistry and type I collagen synthesis was examined by immunohistochemistry staining 5 and 10 days later. Results In group A, MSC spread better, and ALP activity of group A was significantly higher than that of group B and control group( P< 0.01);but there was no significant difference between group A and group C( P >0.05). There was no significant difference in type I collagen synthesis between four groups on the 5th day; but mean gray scale of type I collagen in group B was significantly higher than that in the other groups on the 10th day( P<0.01) .Conclusion Different compound patterns of CBB, HG and MSC affect attachment, proliferation, differentiation of MSC. The compound pattern of CBB/HG/MSC is better than the others.