人骨髓间充质干细胞在具有各向异性孔隙结构的海藻酸钠-明胶-羟基磷灰石支架上的增殖和成骨分化

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate-gelatine-hydroxyapatite scaffolds with anisotropic pore structure.

作者信息

Bernhardt A, Despang F, Lode A, Demmler A, Hanke T, Gelinsky M

机构信息

Institute of Materials Science, Max Bergmann Center of Biomaterials, Technische Universität Dresden, Budapester Strasse 27, D-01069 Dresden, Germany.

出版信息

J Tissue Eng Regen Med. 2009 Jan;3(1):54-62. doi: 10.1002/term.134.

Abstract

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube-like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube-like pores were generated by diffusion-controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 microm. To evaluate the potential use of alginate-gelatine-HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate-gelatine-HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10-14-fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic-induced hBMSCs. Furthermore, the expression of bone-related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic-induced hBMSCs on alginate-gelatin-HA scaffolds.

摘要

骨工程中的各种应用都需要多孔矿化支架。特别是,支架内部具有可控取向的管状孔隙可以支持细胞均匀接种以及充足的营养供应,并可能促进血管向内生长。通过藻酸盐的扩散控制离子otropic凝胶化生成了具有平行取向管状孔隙的支架。在凝胶化过程中加入羟基磷灰石(HA)得到了平均孔径约为90微米的稳定支架。为了评估藻酸盐 - 明胶 - HA支架在骨组织工程中的潜在用途,对人骨髓基质细胞(hBMSCs)进行了体外测试。我们通过显微镜方法分析了生物相容性和细胞向毛细血管孔隙的渗透情况。hBMSCs还在有和成骨补充剂存在和不存在的情况下在藻酸盐 - 明胶 - HA支架上培养3周。我们从总乳酸脱氢酶(LDH)活性、DNA含量和碱性磷酸酶(ALP)活性方面研究了增殖和成骨分化,发现在培养2周后细胞数量增加了10 - 14倍,并且对于成骨诱导的hBMSCs,特异性ALP活性也有所增加。此外,分析了骨相关基因[ALP、骨唾液蛋白II(BSPII)]的表达。我们发现,在藻酸盐 - 明胶 - HA支架上,成骨诱导的hBMSCs的ALP以及BSPII表达均增加。

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