Suzuki Risako, Ide Kentaro, Tokuchi Shusuke, Kawano Toshihiro, Ntege Edward Hosea, Murahashi Makoto, Isa Mikana, Sunami Hiroshi, Shimizu Yusuke, Nakamura Hiroyuki
Department of Oral and Maxillofacial Surgery and Plastic and Reconstructive Surgery, Graduate School of Medicine Advanced Medical Research Center, Faculty of Medicine, University of the Ryukyus, 1076 Kiyuna, Ginowan, Okinawa 901-2720, Japan.
Plastic and Reconstructive Surgery, Graduate School of Medicine, 1076 Kiyuna, Ginowan, Okinawa 901-2720, Japan.
Regen Ther. 2025 Mar 18;29:162-170. doi: 10.1016/j.reth.2025.03.003. eCollection 2025 Jun.
Collagen-derived peptides demonstrate diverse biological activities, but their effects on human dental pulp stem cells (hDPSCs) remain unclear. This study investigated the influence of prolyl-hydroxyproline (Pro-Hyp) and hydroxyproline-glycine (Hyp-Gly) on stage-specific embryonic antigen 3 (SSEA3)-positive hDPSCs.
SSEA3-positive and SSEA3-negative hDPSCs were isolated and cultured with Pro-Hyp or Hyp-Gly (200 μg/ml) under stem cell medium or differentiation-inducing conditions. Cell proliferation was assessed using the MTT assay. The expression of the chondrogenic and osteogenic markers was analyzed by real-time PCR. Matrix production was evaluated using Alcian blue and Alizarin red S staining.
Pro-Hyp and Hyp-Gly enhanced the proliferation of SSEA3-positive hDPSCs compared with SSEA3-negative cells. Pro-Hyp promoted chondrogenic differentiation through early upregulation of Col II followed by sustained SOX9 expression, with enhanced matrix production after 2 weeks in chondrogenic medium. Hyp-Gly specifically enhanced the osteogenic differentiation of SSEA3-positive cells through the temporal regulation of gene expression, progressing from early transcription factors (RUNX2 and RANKL) to matrix proteins (ALPL, Col I, BSP, and OCN), resulting in increased mineralization under osteogenic conditions.
This study demonstrated that Pro-Hyp and Hyp-Gly selectively influenced SSEA3-positive hDPSC fate through the distinct temporal regulation of differentiation pathways. These findings provide new insights into the development of targeted regenerative strategies using collagen-derived peptides in dental tissue engineering.
胶原蛋白衍生肽具有多种生物活性，但其对人牙髓干细胞（hDPSCs）的影响尚不清楚。本研究调查了脯氨酰 - 羟脯氨酸（Pro - Hyp）和羟脯氨酸 - 甘氨酸（Hyp - Gly）对阶段特异性胚胎抗原3（SSEA3）阳性hDPSCs的影响。
分离SSEA3阳性和SSEA3阴性hDPSCs，并在干细胞培养基或诱导分化条件下用Pro - Hyp或Hyp - Gly（200μg/ml）进行培养。使用MTT法评估细胞增殖。通过实时PCR分析软骨生成和成骨标志物的表达。使用阿尔辛蓝和茜素红S染色评估基质产生。
与SSEA3阴性细胞相比，Pro - Hyp和Hyp - Gly增强了SSEA3阳性hDPSCs的增殖。Pro - Hyp通过早期上调II型胶原随后持续表达SOX9促进软骨生成分化，在软骨生成培养基中培养2周后基质产生增加。Hyp - Gly通过基因表达的时间调控特异性增强SSEA3阳性细胞的成骨分化，从早期转录因子（RUNX2和RANKL）发展到基质蛋白（ALPL、I型胶原、骨桥蛋白和骨钙素），导致在成骨条件下矿化增加。
本研究表明，Pro - Hyp和Hyp - Gly通过对分化途径的不同时间调控选择性地影响SSEA3阳性hDPSC的命运。这些发现为在牙组织工程中使用胶原蛋白衍生肽开发靶向再生策略提供了新的见解。
