Ruckh Timothy T, Carroll Derek A, Weaver Justin R, Popat Ketul C
School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA.
Department of Mechanical Engineering, Colorado State University, Fort Collins, CO 80523, USA.
J Funct Biomater. 2012 Nov 14;3(4):776-98. doi: 10.3390/jfb3040776.
Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations-1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.
合成组织支架对患有成骨不全症的患者具有很高的潜在影响。利用静电纺丝技术，制备了由羟基磷灰石/聚己内酯（HAp/PCL）复合纳米纤维组成的组织支架，其中HAp的浓度有两种，分别为固体支架重量的1%和10%。在对支架进行物理化学表征后，将大鼠骨髓基质细胞在维持培养基中培养于复合支架上，然后再置于成骨培养基中培养。通过定量PCR、比色测定、免疫荧光标记和电子显微镜观察来测量成骨细胞对HAp/PCL支架的反应。在维持培养条件下，HAp/PCL支架和对照支架在七天内均支持细胞定植，差异较小。在成骨条件下，10% HAp支架在第3周时碱性磷酸酶测定水平显著升高，这与先前的报道一致。然而，qPCR分析表明，HAp/PCL支架上与骨基质相关的基因总体上有所减少。骨桥蛋白和骨钙素免疫荧光显微镜检查显示出一种趋势，即两种矿化支架中这两种蛋白质的含量都更高，尽管qPCR结果显示骨桥蛋白的趋势相反。此外，I型胶原蛋白在HAp支架上的表达下降。这些结果表明，即使在仅1% w/w的情况下，细胞对纳米纤维中矿物质含量的微小变化也很敏感，阐明这种传感机制可能会带来优化的成骨支架设计。
