Department of Otolaryngology, Chonnam National University Medical School, Gwangju, Republic of Korea
Department of Biology, College of Natural Science, Chosun University, Gwangju, Republic of Korea.
In Vivo. 2020 May-Jun;34(3):1103-1110. doi: 10.21873/invivo.11881.
BACKGROUND/AIM: This study aimed to prospectively compare the osteogenesis of bone powder (BP) substances with and without mesenchymal stem cells (MSCs) and evaluate the synergistic effect of topically applied recombinant human bone morphogenic protein-2 (BMP2) on MSC-loaded BP using fibrin glue in a mastoid obliteration model.
To determine the expression of osteocyte-specific genes, total RNA was isolated from three MSC groups: Untreated MSCs, MSCs cultured with BP, and MSCs cultured with BP and BMP2. Real-time polymerase chain reaction was carried out with specific primers of osteogenesis-related genes runt-related transcription factor 2, osteocalcin, osteoprotegerin, osterix, alkaline phosphatase, transforming growth factor beta, and type I collagen. Live/dead staining was also performed. To observe the adhesion of MSCs to the BP, MSCs were treated with BP for 2 days and the surface was observed by scanning electron microscopy (SEM). Under general anesthesia, mastoid obliteration was performed in rats using three groups: treated with BP alone, BP/MSCs, and BP/MSC/BMP2. Before decapitation at 8 weeks post operation, in vivo micro computed tomography (micro CT) was performed. The bullae were dissected, fixed, and decalcified. followed by dehydration, paraffin embedding, and staining by hematoxylin and eosin and Masson's trichrome.
SEM showed the MSCs to be well-attached to the superficial area of the BP. The expression of osteocyte-specific genes was the highest in the MSCs cultured with BP and BMP2, followed by cultured with BP only, and untreated MSCs. The BP/MSC/BMP2 group showed the highest radiodensity of bullae in microCT analysis. The microCT findings revealed that the BP/MSC/BMP2 group showed the most enhanced osteogenesis of the scaffold compared to the other two groups. No significant difference was found in osteoconductive osteogenesis between the control and BP/MSC groups. However, the BP/MSC/BMP2 group showed significantly enhanced osteoconductive osteogenesis and osteoinductive change of the BP as shown by hematoxylin and eosin staining. Histomorphometry of osteogenesis revealed that the difference between the BP/MSC/BMP2 group and the other two groups was statistically significant.
A small amount of BMP2 is necessary during MSC loading to enhance the osteogenesis of BP and avoid complications associated with high doses of BMP2. These results may be applicable to mastoid obliteration in clinical practice.
背景/目的：本研究旨在前瞻性比较载骨髓间充质干细胞（MSCs）和未载 MSC 的骨粉（BP）物质的成骨作用，并评估在乳突填塞模型中使用纤维蛋白胶局部应用重组人骨形态发生蛋白 2（BMP2）对负载 MSC 的 BP 的协同作用。
为了确定成骨细胞特异性基因的表达，从未处理的 MSC、培养有 BP 的 MSC 和培养有 BP 和 BMP2 的 MSC 三个 MSC 组中分离总 RNA。使用成骨相关基因 runt 相关转录因子 2、骨钙素、骨保护素、osterix、碱性磷酸酶、转化生长因子β和 I 型胶原的特异性引物进行实时聚合酶链反应。还进行了死活染色。为了观察 MSC 对 BP 的黏附，将 MSC 用 BP 处理 2 天，并用扫描电子显微镜（SEM）观察表面。在全身麻醉下，使用三组大鼠进行乳突填塞：单独用 BP 处理、BP/MSCs 和 BP/MSC/BMP2。在术后 8 周断头前进行体内微计算机断层扫描（micro CT）。将鼓室解剖、固定、脱钙，然后进行脱水、石蜡包埋和苏木精和伊红以及 Masson 三色染色。
SEM 显示 MSC 很好地黏附在 BP 的表面区域。在培养有 BP 和 BMP2 的 MSC 中，成骨细胞特异性基因的表达最高，其次是仅培养有 BP 的 MSC 和未处理的 MSC。micro CT 分析显示，BP/MSC/BMP2 组的鼓室密度最高。micro CT 结果显示，与其他两组相比，BP/MSC/BMP2 组的支架成骨增强最为明显。在骨传导成骨方面，对照组和 BP/MSC 组之间没有明显差异。然而，BP/MSC/BMP2 组的 BP 具有明显增强的骨传导和成骨诱导变化，通过苏木精和伊红染色显示。成骨组织形态计量学显示，BP/MSC/BMP2 组与其他两组之间的差异具有统计学意义。
在 MSC 加载过程中，少量的 BMP2 是增强 BP 成骨作用所必需的，可避免与高剂量 BMP2 相关的并发症。这些结果可能适用于临床实践中的乳突填塞。
