三維列印水膠搭載控制釋放牙源幹細胞細胞外囊泡對骨再生之效能評估

Abstract

骨組織缺損常見於外傷、腫瘤切除、感染性壞死與先天性發育缺陷，當範圍超過臨界大小時，內源性修復機制無法橋接缺損而形成骨不癒合。現行骨移植材料分別受限於取材量與供區併發症、處理後骨誘導能力下降，或僅具骨傳導功能。間葉幹細胞之治療效益主要來自旁分泌作用，其分泌之細胞外囊泡(extracellular vesicles, EVs)可將 miRNA 與蛋白質遞送至受體細胞，已成為無細胞治療之核心；惟其體內半衰期極短，需藉載體達成局部持續釋放。 本研究以三維列印技術製備藻酸鹽／明膠複合水膠支架作為牙源幹細胞 EVs 之遞送載體。先比較 3% 與 5% 藻酸鹽配方之抗壓強度、降解速率與生物相容性以選定配方；繼以尺寸排阻層析法自牙髓幹細胞(DPSCs)與牙周韌帶幹細胞(PDLSCs)分離 EVs 並完成鑑定；最後以骨肉瘤細胞株 MG63 評估兩種來源 EVs 於不同劑量下之成骨反應。 結果顯示，3% 與 5% 配方之最大抗壓強度分別為 0.82 與 1.05 MPa，第 28 天降解率分別為 81% 與 32%，細胞存活率均高於 70%，符合 ISO 10993 標準；因降解時程與骨再生窗口相符，後續選用 3% 配方。紅外光譜佐證 EVs 已嵌入水膠網絡，釋放曲線呈兩相行為並持續七天。DPSC-EVs 與 PDLSC-EVs 之平均粒徑分別為 83.3 與 95.1 nm，均表現 CD9、CD81 與 TSG101。功能評估方面，第 14 天鹼性磷酸酶染色面積以 PDLSC-E9 組最高(約 31%)，高於 DPSC-E9 與 PDLSC-E8 組(約 15%)與對照組(約 5%)；第 21 天茜素紅吸光值亦以 PDLSC-E9 組最高(O.D. 540 nm 約 0.91)；PDLSC-EVs 組之 OCN 與 OPN 表現顯著高於對照組與 DPSC-EVs 組(p < 0.05)，且上述效果均具劑量依賴性。 綜合而言，三維列印天然水膠可有效搭載牙源幹細胞 EVs 並維持其活性與持續釋放；PDLSC-EVs 之成骨誘導效能優於 DPSC-EVs，顯示細胞來源對 EVs 功能具實質影響。惟本研究侷限於體外模式，體內效能仍待動物實驗驗證。

Bone defects arising from trauma, tumor resection, infection, or congenital disorders cannot heal spontaneously once they exceed the critical size. Current grafting materials are limited by donor availability, donor-site morbidity, or insufficient osteoinductivity. As the benefit of mesenchymal stem cells derives largely from paracrine signaling, their extracellular vesicles (EVs), which deliver miRNAs and proteins to recipient cells, have emerged as a promising cell-free alternative; their short half-life in vivo, however, necessitates a carrier for local sustained release. This study developed a three-dimensionally printed alginate/gelatin hydrogel scaffold as a carrier for dental stem cell-derived EVs. Formulations containing 3% and 5% alginate were compared for compressive strength, degradation, and biocompatibility; EVs were isolated from human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) by size-exclusion chromatography, and their osteogenic effects were evaluated in MG63 cells at two doses. Maximum compressive strengths were 0.82 and 1.05 MPa and 28-day degradation rates were 81% and 32% for the 3% and 5% formulations, respectively, with L929 viability above 70% for both, meeting ISO 10993. The 3% formulation was selected because its degradation profile better matched the window of bone regeneration. Infrared spectroscopy confirmed EV incorporation, and release was biphasic and sustained through day 7. DPSC-EVs and PDLSC-EVs showed mean diameters of 83.3 and 95.1 nm and expressed CD9, CD81, and TSG101. Alkaline phosphatase staining at day 14 was highest in the PDLSC-E9 group (approximately 31%), exceeding DPSC-E9 and PDLSC-E8 (approximately 15%) and the control (approximately 5%); alizarin red staining at day 21 was likewise highest in PDLSC-E9 (O.D. 540 nm approximately 0.91), and PDLSC-EV treatment significantly upregulated OCN and OPN (p < 0.05), all in a dose-dependent manner. In summary, this customizable hydrogel system carried dental stem cell-derived EVs while preserving bioactivity and sustaining release. PDLSC-derived EVs showed superior osteoinductive efficacy, indicating that the cellular source materially influences EV function. As the evaluation was limited to in vitro models, in vivo efficacy awaits verification.