An In Vitro Assessment of a Cell-Containing Collagenous Extracellular Matrix-like Scaffold for Bone Tissue Engineering
An In Vitro Assessment of a Cell-Containing Collagenous Extracellular Matrix-like Scaffold for Bone Tissue Engineering
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DOI：
10.1089/ten.tea.2009.0351
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发表时间：
2010-03-01
影响因子：
4.1
通讯作者：
Nazhat, Showan N.
中科院分区：
文献类型：
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作者：
Pedraza, Claudio E.;Marelli, Benedetto;Nazhat, Showan N.
细胞外基质（ECM）由胶原、蛋白多糖、糖蛋白和弹性纤维等大分子组成。ECM对于保存组织结构、向细胞发出信号以及调节矿化组织中的钙化是必不可少的。培养的成骨细胞分泌并组装大量的ECM，富含I型胶原和其他可矿化的非胶原蛋白。三维基质模型可以在体外使用，以最适当地模拟天然ECM的几何形状和生物化学。在本研究中，MC 3 T3-E1小鼠颅骨前成骨细胞培养在致密的三维胶原ECM样支架通过塑料压缩的方法产生。塑料压缩快速产生胶原密度接近天然组织水平的支架，具有增强的生物力学性能，同时保持驻留细胞的活力。在培养中研究了长达7周的接种MC 3 T3的增殖、形态和基因表达，以及胶原蛋白的产生和基质矿化。可溶性胶原分泌的浓度范围为5至30 μ g/mL，在24小时内，伴随着稳定的胶原mRNA表达率。成骨标志物，如组织非特异性碱性磷酸酶（Alpl），骨唾液酸蛋白（Bsp），骨桥蛋白（Opn）的表达，通过生化分析和逆转录聚合酶链反应检测证明细胞分化。细胞周围ECM的细胞周空隙，连同MMP 13 mRNA表达的证据，表明基质重塑。超微结构分析，X射线显微分析，显微计算机断层扫描，以及傅立叶变换红外和成像都证实了纤维胶原基质内的钙磷酸盐矿物相的形成。总之，前成骨细胞MC 3 T3细胞接种在ECM样致密胶原支架内分泌基质蛋白，并以潜在适用于骨组织工程用途的方式诱导支架矿化。
Extracellular matrix (ECM) consists of a complex mixture of macromolecules such as collagens, proteoglycans, glycoproteins, and elastic fibers. ECM is essential to preserving tissue architecture, signaling to cells, and regulating calcification in mineralized tissues. Osteoblasts in culture secrete and assemble an extensive ECM rich in type I collagen, and other noncollagenous proteins that can be mineralized. Three-dimensional matrix models can be used in vitro to most appropriately resemble the geometry and biochemistry of natural ECMs. In the present study, MC3T3-E1 mouse calvarial preosteoblasts were cultured within a dense three-dimensional collagenous ECM-like scaffold produced through the method of plastic compression. Plastic compression rapidly produces scaffolds of collagen density approaching native tissue levels with enhanced biomechanical properties while maintaining the viability of resident cells. The proliferation, morphology, and gene expression of seeded MC3T3s, as well as collagen production and matrix mineralization, were investigated for up to 7 weeks in culture. Soluble collagen secretion ranged in concentration from 5 to 30 mu g/mL over a 24-h period, concomitant with a steady rate of collagen mRNA expression. Expression of osteogenic markers such as tissue-nonspecific alkaline phosphatase (Alpl), bone sialoprotein (Bsp), and osteopontin (Opn) examined by biochemical assay and reverse transcription-polymerase chain reaction demonstrated cell differentiation. Pericellular voids of ECM around cells, together with evidence of MMP13 mRNA expression, suggested matrix remodeling. Ultrastructural analyses, X-ray micro-analysis, micro-computed tomography, as well as Fourier-transform infrared and imaging all confirmed the formation of a calcium-phosphate mineral phase within the fibrillar collagen matrix. In conclusion, preosteoblastic MC3T3 cells seeded within an ECM-like dense collagen scaffold secrete matrix proteins and induce scaffold mineralization in a manner potentially appropriate for bone tissue engineering uses.