Handschel Jörg, Berr Karin, Depprich Rita A, Kübler Norbert R, Naujoks Christian, Wiesmann Hans-Peter, Ommerborn Michelle A, Meyer Ulrich
Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University Düsseldorf, Moorenstr, 5, 40225 Düsseldorf, Germany.
Head Face Med. 2008 Jun 10;4:10. doi: 10.1186/1746-160X-4-10.
Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.
Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and beta-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.
ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.
Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.
头面部的面部创伤或肿瘤手术常常导致面部骨骼的大规模破坏。基于细胞的骨重建疗法有望为修复因疾病或损伤而受损的骨骼提供新的治疗机会。目前，胚胎干细胞（ESCs）被认为是骨组织工程的潜在细胞来源。本研究的目的是研究培养基中的各种补充剂对成骨分化诱导的影响。
将小鼠胚胎干细胞在白血病抑制因子（LIF）、DAG（地塞米松、抗坏血酸和β-甘油磷酸）或骨形态发生蛋白-2（BMP-2）存在的条件下培养。使用冯·科萨染色进行显微镜分析，并通过实时PCR测定成骨标记基因的表达。
用DAG培养的胚胎干细胞显示出迄今为止最大量的含磷酸钙矿物质沉积。从培养第9天开始，在DAG处理的细胞中检测到I型胶原蛋白mRNA表达强烈增加。在BMP-2处理的胚胎干细胞中，I型胶原蛋白mRNA的诱导增加较少。骨钙素是成骨分化的高度特异性标志物，其表达在DAG处理的细胞中呈双峰曲线。在DAG存在下培养的胚胎干细胞在第9天骨钙素mRNA强烈增加，随后在第17天开始出现第二个峰值。
在胚胎干细胞培养中添加DAG可有效诱导成骨分化，且似乎比单独用BMP-2刺激更有效。因此，对于生成用于骨组织工程的具有成骨分化能力的胚胎干细胞群体，推荐使用DAG处理。
