Center for Regenerative Medicine, Danube University Krems, 3500 Krems, Austria.
Department of Anatomy and Biomechanics, Karl Landsteiner University for Health Sciences, 3500 Krems, Austria.
Int J Mol Sci. 2021 Apr 14;22(8):4055. doi: 10.3390/ijms22084055.
Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC's) via the endochondral ossification (ECO) route. hBMSC's were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG's were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC's under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. μCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC's expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC'S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.
组织工程策略通过在工程移植物中刺激骨生成途径来促进大骨缺损的骨再生，在植入后，这些途径经常受到宿主组织血管不足的整合和功能吻合的限制。在这项研究中，我们开发了一种混合生物材料，将脱细胞软骨细胞外基质（CD-ECM）作为模板，丝素蛋白（SF）作为载体，通过软骨内骨化（ECO）途径评估骨髓间充质干细胞（hBMSC）的骨再生能力。hBMSC 经过两周的软骨形成诱导，然后在混合 CD-ECM/SF 或 SF 支架上进行六周的肥大诱导，并在体外评估矿化基质的形成。SF 和 CD-ECM/SF 构建体中的钙沉积在 4-8 周内均显著增加，并且仅在 CD-ECM/SF 构建体中观察到 sGAG 的保留。SEM/EDX 显示 hBMSC 在所有条件下均能定位钙和磷酸盐晶体。在肥大诱导 8 周后，压缩模量达到最大 40 KPa。8 周时的μCT 扫描表明，在肥大诱导后，CD-ECM/SF 构建体中的一组矿物质密度更高。通过 RT-qPCR 的基因表达表明，hBMSC 表达肥大标志物 VEGF、COL10、RUNX2，但在所有条件下均缺乏早期肥大标志物 ChM1 和晚期肥大标志物 TSBS1，以及成骨标志物 ALPL、IBSP、OSX。我们的数据表明了一种新的方法，可以在机械稳定的构建体中体外将 hBMSC 早期诱导到晚期肥大阶段，用于 ECO 介导的骨组织再生。
