Nishiguchi Yusuke, Ueda Mamoru, Kubo Hirohito, Jo Jun-Ichiro, Hashimoto Yoshiya, Takenobu Toshihiko
Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka, Japan.
Department of Biomaterials, Osaka Dental University, Osaka, Japan.
J Dent Sci. 2025 Jan;20(1):278-285. doi: 10.1016/j.jds.2024.07.028. Epub 2024 Aug 6.
BACKGROUND/PURPOSE: Bone reconstruction in the maxillofacial region typically relies on autologous bone grafting, which presents challenges, including donor site complications and graft limitations. Recent advances in tissue engineering have identified highly pure and proliferative dedifferentiated fat cells (DFATs) as promising alternatives. Herein, we explored the capacity for osteoblast differentiation and the osteoinductive characteristics of extracellular vesicles derived from DFATs (DFAT-EVs).
DFATs were isolated from human buccal fat pads, cultured to confluency, and placed in either a standard or osteogenic induction medium. After culturing for 3 days, the conditioned medium was used to generate EVs using the size-exclusion chromatography and concentration filter method.
Characterization of DFAT-EVs revealed typical EV morphology and positive markers (CD9 and CD63), with no differences between the two groups. assays demonstrated that EVs derived from the osteogenic induction medium (OI-EVs) significantly increased alkaline phosphatase activity and osteogenesis-related genes (Runx2 and collagen type I) compared to control EVs. Next-generation sequencing identified differentially expressed miRNAs, and gene ontology analysis suggested pathways involved in osteoblast differentiation.
Isolating DFATs from buccal fat pads under osteogenic induction conditions offers a procedure confined to the oral cavity, eliminating the need for harvesting from other sites. Thus, DFAT-EVs hold promise for promoting bone regeneration in maxillofacial applications.
背景/目的：颌面部骨重建通常依赖自体骨移植，这存在一些挑战，包括供区并发症和移植物局限性。组织工程学的最新进展已确定高纯度且具有增殖能力的去分化脂肪细胞（DFATs）是有前景的替代物。在此，我们探究了DFATs来源的细胞外囊泡（DFAT-EVs）的成骨细胞分化能力和骨诱导特性。
从人颊脂垫中分离DFATs，培养至汇合，然后置于标准培养基或成骨诱导培养基中。培养3天后，使用尺寸排阻色谱法和浓缩过滤法，用条件培养基生成细胞外囊泡。
DFAT-EVs的表征显示出典型的细胞外囊泡形态和阳性标志物（CD9和CD63），两组之间无差异。 分析表明，与对照细胞外囊泡相比，成骨诱导培养基来源的细胞外囊泡（OI-EVs）显著增加了碱性磷酸酶活性和成骨相关基因（Runx2和I型胶原）。下一代测序确定了差异表达的微小RNA，基因本体分析提示了参与成骨细胞分化的途径。
在成骨诱导条件下从颊脂垫中分离DFATs提供了一种局限于口腔的操作方法，无需从其他部位获取。因此，DFAT-EVs在颌面部应用中促进骨再生方面具有前景。
