人根尖乳头干细胞生成牙髓牙本质复合体的实验研究

摘要

Objective To regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells. Methods SCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining. Results Round alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation. Conclusion SCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.%目的应用组织工程方法，探讨人根尖乳头干细胞（SCAP）进行牙髓牙本质复合体再生的可能性。方法从牙根未发育完全的健康阻生智齿中分离牙乳头，用组织贴块法培养SCAP。细胞分别在成骨诱导液中培养3周及普通培养基中培养60 d后，用茜素红检测钙结节形成情况，免疫荧光法和RT-PCR检测成骨细胞标志物碱性磷酸酶（ALP）、骨涎蛋白（BSP）、骨钙素（OC）和牙本质涎蛋白（DSP）的表达情况。选取5~6周雌性裸鼠6只，将SCAP与羟磷灰石/磷酸三钙（HA/TCP）复合培养作为实验组，仅有HA/TCP作为对照组，分别植入同一只裸鼠背部皮下，左右侧各1个。8周后移植物行HE染色和免疫组化染色观察新生组织情况。结果 SCAP在体外经成骨诱导3周及普通培养基培养60 d后，茜素红染色阳性，且表达成骨细胞标志物ALP、BSP、OC和DSP。实验组移植物8周后可见牙髓牙本质样结构形成，且紧靠牙本质样结构内侧的细胞DSP阳性表达，而对照组未见任何硬组织形成。结论SCAP可应用于组织工程，进行牙髓牙本质复合体再生。