Gomes Manuela E, Holtorf Heidi L, Reis Rui L, Mikos Antonios G
3Bs Research Group, Biomaterials, Biodegradables and Biomimetics, University of Minho, Campus de Gualtar, Braga, Portugal.
Tissue Eng. 2006 Apr;12(4):801-9. doi: 10.1089/ten.2006.12.801.
This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro.
本研究调查了由淀粉与聚(ε-己内酯)共混物制成的纤维网状支架的孔隙率对在静态和流动灌注条件下培养的骨髓基质细胞增殖和成骨分化的影响。为此,通过纤维粘结法将可生物降解支架制成具有两种不同孔隙率(50%和75%)的网状结构。然后将从Wistar大鼠收获的骨髓基质细胞接种到这些支架上,并在流动灌注生物反应器或6孔板中培养长达15天。孔隙率为75%的支架在静态和流动灌注培养条件下均显示出显著增强的细胞增殖。碱性磷酸酶活性的表达在流动培养中更高,但仅针对接种在较高孔隙率支架上的细胞。两种支架的钙沉积模式相似,与静态培养相比,流动灌注培养的细胞-支架构建体上的钙沉积显著增强。孔隙率为75%的支架中的钙沉积更高,但这种差异无统计学意义。扫描电子显微镜观察显示,在较高孔隙率支架上沉积的细胞外基质内形成了孔状结构。在流动灌注生物反应器中培养15天后,对细胞-支架构建体进行衰减全反射傅里叶变换红外光谱和薄膜X射线衍射分析,结果显示存在类似于骨的矿化基质。这些发现表明,基于淀粉的支架与流体流动生物反应器培养相结合,可将扩散限制降至最低,并为骨髓基质细胞提供机械刺激,从而促进向类骨矿化组织发育的分化增强。这些结果还表明,支架结构,即孔隙率,会影响成骨细胞的顺序发育,并且与培养条件相结合,可能会影响体外形成组织的功能。