Department of Oral & Maxillofacial Surgery, School of Dentistry, Seoul National University, Seoul, South Korea.
Regen Med. 2011 Jan;6(1):67-79. doi: 10.2217/rme.10.96.
Platelet-rich plasma (PRP) is fabricated from autologous blood and extensively used to promote soft and hard tissue healing. In the dental field, autologous PRP is widely used combined with dental implant installation and bone graft. This study will evaluate the biologic effect of PRP on the proliferation and the differentiation of human dental stem cells, and find the key cytokines inducing these effects to estimate the clinical feasibility of PRP for dental tissue engineering.
MATERIALS & METHODS: Venous blood was obtained from four individuals and each PRP was fabricated. The human dental stem cells were obtained from the periodontal ligament (PDL) and dental pulp of the surgically extracted human third molars and expanded in vitro. Immunocytochemical staining and flow cytometry with STRO-1 and CD146 confirmed existence of mesenchymal stem cells in the PDL and dental pulp. The effect of PRP on the proliferation of PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) was assessed by colony-forming ability measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay. Alkaline phosphatase activity and calcium deposit were measured to evaluate the mineralization effect of PRP PDLSCs and DPSCs. Alizarin red S staining was used to detect mineral nodules. Odontogenic and osteogenic gene expressions were evaluated in the PRP-treated PDLSCs and DPSCs by real-time quantitative PCR. A protein array was performed to detect the key cytokines that have an important role in the tissue regenerative effect of PRP.
Flow cytometry cell sorting showed that the cells from human PDL and dental pulp contained mesenchymal stem cell populations. Colony-forming ability and cellular proliferation of the dental stem cells were increased at 0.5 and 1% PRP concentration but decreased at 5% concentration. Long-term treatment with 1% PRP enhanced proliferation of the human dental stem cells PDLSCs and DPSCs by 120 h and showed the most significant enhancement at 96 h. PRP also promoted mineralization differentiation of the two kinds of dental stem cells as shown by measurement of alkaline phosphatase activity and calcium deposit under mineralization conditioned media. Increased formation of mineral nodules stained with alizarin red was observed in both PDLSCs and DPSCs after treatment with 1% PRP. Real-time quantitative PCR showed higher odontogenic and osteogenic gene expressions in PRP-treated PDLSCs and DPSCs. RANTES/CCL5 and ICAM-1 were the two key cytokines that were detected in human cytokine array with PRP.
The appropriate concentration of the PRP treatment enhanced proliferation and mineralization differentiation of human dental stem cells. RANTES/CCL5 and ICAM-1 might play an important role in PRP-induced tissue regeneration but further study is needed to investigate the whole mechanism.
富血小板血浆（PRP）由自体血液制成，广泛用于促进软组织和硬组织愈合。在牙科领域，自体 PRP 广泛用于与种植牙安装和骨移植结合使用。本研究将评估 PRP 对人牙髓干细胞增殖和分化的生物学影响，并找到诱导这些作用的关键细胞因子，以评估 PRP 在牙科组织工程中的临床可行性。
从 4 个人中采集静脉血，并制备每份 PRP。从外科拔出的人第三磨牙的牙周韧带（PDL）和牙髓中获得牙髓干细胞，并在体外扩增。用 STRO-1 和 CD146 的免疫细胞化学染色和流式细胞术证实 PDL 和牙髓中存在间充质干细胞。通过集落形成能力测量、3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）比色法和溴脱氧尿苷掺入试验评估 PRP 对牙周膜干细胞（PDLSCs）和牙髓干细胞（DPSCs）增殖的影响。碱性磷酸酶活性和钙沉积的测量用于评估 PRP PDLSCs 和 DPSCs 的矿化作用。茜素红 S 染色用于检测矿化结节。通过实时定量 PCR 评估 PRP 处理的 PDLSCs 和 DPSCs 的成牙和成骨基因表达。进行蛋白质芯片分析以检测在 PRP 组织再生作用中起重要作用的关键细胞因子。
流式细胞术细胞分选显示，来自人牙周和牙髓的细胞含有间充质干细胞群体。在 0.5%和 1% PRP 浓度下，牙源性干细胞的集落形成能力和细胞增殖增加，但在 5%浓度下减少。长期用 1% PRP 处理可使 PDLSCs 和 DPSCs 的人牙髓干细胞增殖增加 120 小时，并在 96 小时时表现出最显著的增强。PRP 还促进了两种牙源性干细胞的矿化分化，这可通过在矿化条件培养基中测量碱性磷酸酶活性和钙沉积来证明。用 1% PRP 处理后，在 PDLSCs 和 DPSCs 中观察到用茜素红染色的矿化结节形成增加。实时定量 PCR 显示，PRP 处理的 PDLSCs 和 DPSCs 中成牙和成骨基因表达更高。人细胞因子芯片检测到 RANTES/CCL5 和 ICAM-1 是 PRP 诱导组织再生中起重要作用的两种关键细胞因子。
适当浓度的 PRP 处理可增强人牙髓干细胞的增殖和矿化分化。RANTES/CCL5 和 ICAM-1 可能在 PRP 诱导的组织再生中起重要作用，但需要进一步研究以探讨整个机制。
