Orthopaedic Research Center, Colorado State University, Fort Collins, Colorado 80523, USA.
J Orthop Res. 2010 Oct;28(10):1330-7. doi: 10.1002/jor.21126.
Bone marrow-derived mesenchymal stem cells (BMDMSCs) have been targeted for use in enhancement of bone healing; and their osteogenic potential may be further augmented by genes encoding bone morphogenetic proteins (BMP's). The purpose of this study was to compare the effect of genetic modification of human and equine BMDMSCs with BMP-2 or -7 or BMP-2 and -7 on their osteoblastogenic differentiation in the presence or absence of dexamethasone. The BMDMSCs were harvested from the iliac crest of three human donors and tuber coxae of three equine donors. Monolayer cells were genetically modified using adenovirus vectors encoding BMP-2, -7 or both and cultured in the presence or absence of dexamethasone. Expression of BMPs was confirmed by enzyme linked immunosorbent assay (ELISA). To evaluate osteoblastic differentiation, cellular morphology was assessed every other day and expression and secretion of alkaline phosphatase (ALP), as well as expression levels of osteonectin (OSTN), osteocalcin (OCN), and runt-related transcription factor-2 (Runx2) were measured for up to 14 days. Human and equine BMDMSCs showed a capacity for osteogenic differentiation regardless of genetic modification or dexamethasone supplementation. Dexamethasone supplementation was more important for osteoblastogenic differentiation of equine BMDMSCs than human BMDMSCs. Genetic modification of BMDMSCs increased ALP secretion with AdBMP-2 homodimer having the greatest effect in both human and equine cells compared to AdBMP 7 or AdBMP 2/7. BMP protein elution rates reached their maximal concentration between day 4 and 8 and remained relatively stable thereafter, suggesting that genetically modified BMDMSCs could be useful for cell-based delivery of BMPs to a site of bone formation.
骨髓间充质干细胞（BMDMSCs）已被用作增强骨愈合的靶细胞；其成骨潜能可以通过编码骨形态发生蛋白（BMP）的基因进一步增强。本研究旨在比较基因修饰的人源和马源 BMDMSCs 与 BMP-2 或 -7 或 BMP-2 和 -7 在有或没有地塞米松存在的情况下对其成骨细胞分化的影响。BMDMSCs 从 3 名人类供体的髂嵴和 3 名马源供体的股骨粗隆采集。单层细胞通过腺病毒载体进行基因修饰，编码 BMP-2、-7 或两者，并在有或没有地塞米松的情况下培养。通过酶联免疫吸附试验（ELISA）证实 BMP 的表达。为了评估成骨细胞分化，每隔一天评估细胞形态，并测量碱性磷酸酶（ALP）的表达和分泌，以及骨连接蛋白（OSTN）、骨钙素（OCN）和 runt 相关转录因子-2（Runx2）的表达水平，最多 14 天。无论基因修饰或地塞米松补充，人源和马源 BMDMSCs 均显示出成骨分化的能力。地塞米松补充对马源 BMDMSCs 的成骨细胞分化比人源 BMDMSCs 更为重要。BMDMSCs 的基因修饰增加了 ALP 的分泌，与 AdBMP-7 或 AdBMP-2/7 相比，AdBMP-2 同源二聚体对人源和马源细胞均具有最大的作用。BMP 蛋白洗脱率在第 4 天至第 8 天达到最高浓度，此后相对稳定，表明基因修饰的 BMDMSCs 可用于将 BMP 以细胞为基础递送至骨形成部位。
