骨髓基质干细胞复合改性的聚羟基丁酸-羟基异戊酯体外构建组织工程化软骨

In vitro cartilage tissue engineering with bone marrow stromal cells and photografting modified copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate

目的探讨应用骨髓基质干细胞（BMSCs）复合光接枝改性的聚羟基丁酸-羟基异戊酯（PHBV）构建组织工程化软骨的可行性。方法将3代绵羊骨髓基质细胞接种于光接枝改性的三维PHBV支架材料上，24h后以成软骨诱导液培养，3周后，复合培养物行扫描电镜观察，组织学观察，测定糖胺聚糖（GAG）含量。并将复合物埋植于绵羊腹部皮下，4周后取出，行大体及组织学观察。结果经成软骨诱导后，扫描电镜下，BMSCs的突触逐渐变短，由长梭形向扁平形转变，分泌基质量亦明显增多。组织学观察见细胞支架复合物阿利新蓝、番红O、Ⅱ型胶原免疫组化染色均呈阳性。GAG含量测定示诱导3周后，细胞分泌的GAG量（1306．7±192．3）明显高于未经诱导的BMSCs（205．0±26．2）（P〈0．001），但仍低于正常软骨细胞（1969．2±235．3）（P〈0．001）。复合物埋植于皮下4周后，其内炎症细胞浸润明显，但番红O、Ⅱ型胶原免疫组化染色均呈阳性。结论以BMSCs为种子细胞．复合改性的PHBV．可于体外构建出软骨样组织．但该组织的理化特点与正常软骨仍有筹距．

Objective To explore the feasibility of building tissue engineered cartilage by bone marrow stromal cells and photografting modified copolymers of 3-hydroxybutyrate and 3- hydroxyvalerate. Methods Sheep BMSCs were seeded in three-dimensional photografting modified PHBV scaffolds, Twenty-four hours later, composites were cultured with chondrogenically inductive medium （DMEM） containing TGF-β （10 ng/ml）, IGF-I （150 ng/ml） and 20% fetal bovine serum. Three weeks later, the constructs were evaluated by scanning electron microscopy （SEM） and light microscopy with alcian blue, gafrine O and type Ⅱ collage immunohistochemical staining. GAG contents of constructs were determined by DMB（1,9-dimethylmethylene blue） binding assay at weekly intervals up to 3 weeks. The composites were implanted subcutaneously in sheep abodoml and were evaluated macroscopically and histologically at 4 weeks postoperatively. Results SEM photograph showed, after one week culture, cell morphology changed from fibroblast-like elongated spindle to the fiat rounded like chondrocytes, and the extra cellular matrix also increased obviously, Furthmore, with the culture time extension, this change were more evident. HE staining showed that cells filled all the inter-connected pores in the constructs, And more cells were observed in the outer layer of the constructs. ECM （extracellular matrix） was strongly positive by Alcian blue, Saltine O staining and type Ⅱ collage immunohistochemical staining. DMB binding assay revealed that the induced BMSCs GAG secretion（ 1306.7 ± 192.3） was significantly higher than BMSCs （205.0 ± 26.2）（P 〈 0.001 ）, but it was significantly lower than passage 2 chondrocytes（ 1969.2 ± 235.3） （P 〈 0.001 ）, Safrine O and type Ⅱ collage immunohistochemical staining were positive in constructs implanted subcutaneously, Conclusion Tissue engineered cartilage could be obtained using BMSCs and photografting modified PHBV, but there are still gaps physiologically between the constructs and the nature cartilage.