由於關節軟骨缺乏血管、神經與淋巴組織，其受損後自我修復能力有限，臨床上常面臨修復組織品質不佳、組織整合不足及長期退化等問題。現有軟骨修復方式雖可部分改善缺損區域，但仍難以穩定再生具有良好組織結構、細胞外基質組成與機械功能之透明軟骨樣組織。因此，發展能同時提供結構支撐、軟骨細胞外基質微環境及生物活性訊號之支架系統，對於促進骨軟骨缺損之功能性修復具有重要意義。
本研究旨在建立一種結合去細胞軟骨層片、明膠－羥苯丙酸水膠及人類脂肪幹細胞來源外泌體之複合支架，並評估其作為骨軟骨缺損修復材料之可行性、修復效果與初步生物安全性。研究中首先以豬來源關節軟骨製備去細胞軟骨層片（acellular cartilage sheet, ACS），並評估其去細胞化效果與細胞外基質保留情形。接著，將去細胞軟骨層片與可原位交聯之明膠－羥苯丙酸水膠（gelatin–hydroxyphenyl propionic acid hydrogel, GAHPA）結合，形成具有層狀結構之複合支架，並分析其化學結構、微觀形貌與機械性質。此外，本研究亦針對人類脂肪幹細胞來源外泌體（exosomes, Exo）進行基本理化與生物性鑑定，並評估其對脂肪幹細胞分化行為之影響。最後，透過大鼠膝關節骨軟骨缺損模型，比較未處理缺損對照組（Defect）、ACS/GAHPA組與ACS/GAHPA + Exo組的骨軟骨修復表現與初步系統性生物安全性
結果顯示，去細胞化處理可有效移除軟骨層片中的細胞成分，同時保留以膠原為主之細胞外基質結構，支持ACS作為軟骨來源細胞外基質支架材料的可行性。明膠－羥苯丙酸水膠經交聯後可形成多孔且連通之三維結構，外泌體加入後未明顯改變其整體多孔形貌。當ACS與GAHPA水膠結合後，複合支架之機械支撐能力較單純水膠提升，表示ACS不僅可提供軟骨細胞外基質相關微環境，亦可作為支架內部之結構補強材料。人類脂肪幹細胞來源外泌體經鑑定後具有奈米級胞外囊泡特徵，並表現外泌體相關標記，符合外泌體之基本鑑定特徵。體外分化實驗結果顯示，外泌體處理可促進脂肪幹細胞於不同誘導條件下之分化表現，其中於軟骨分化條件下可觀察到較明顯之軟骨樣基質形成，顯示此類外泌體具有調控細胞分化與促進軟骨相關基質生成之潛力。
在大鼠骨軟骨缺損模型中，與Defect組相比，ACS/GAHPA組與ACS/GAHPA + Exo組於修復後期皆呈現較佳之缺損填補、表面連續性及周圍組織整合趨勢。組織染色結果進一步顯示，兩個支架治療組皆可改善修復組織形態，並促進蛋白多醣與第二型膠原等軟骨相關基質沉積，同時呈現軟骨下骨重建之趨勢。然而，目前結果尚未明確顯示ACS/GAHPA + Exo組之體內修復效果優於ACS/GAHPA組。主要器官組織觀察與血液生化分析結果皆未見明顯材料相關之系統性毒性或肝腎功能異常，顯示此複合支架系統具有初步系統性生物安全性。
以ACS與GAHPA水膠為基礎的層狀複合支架，並顯示其具備軟骨來源細胞外基質特性、多孔水膠結構、機械補強效果及良好之初步生物安全性。人類脂肪幹細胞來源外泌體可作為生物活性訊號來源，並於體外促進細胞分化與軟骨樣基質形成。於體內骨軟骨缺損模型中，ACS/GAHPA組與ACS/GAHPA + Exo組皆呈現促進缺損填補、軟骨樣基質沉積及軟骨下骨修復之潛力。整體而言，ACS/GAHPA複合支架可作為骨軟骨缺損修復之潛在組織工程平台，而人類脂肪幹細胞來源外泌體則具作為生物活性輔助因子的潛力；惟其於體內修復中的額外效益，仍有待進一步以較大樣本數、較長期觀察及作用機制研究加以驗證。

Articular cartilage has a limited intrinsic capacity for self-repair because it is devoid of blood vessels, nerves, and lymphatic vessels. Consequently, cartilage damage often results in poor repair tissue quality, insufficient tissue integration, and long-term degeneration. Although current cartilage repair strategies can partially improve defect filling, they do not consistently regenerate hyaline-like cartilage with appropriate tissue organization, extracellular matrix (ECM) composition, and mechanical function. Therefore, developing a scaffold system that can simultaneously provide structural support, a cartilage ECM-like microenvironment, and bioactive signals is critical to promoting functional osteochondral defect repair.
This study aimed to develop a composite scaffold consisting of acellular cartilage sheets (ACSs), gelatin-hydroxyphenyl propionic acid hydrogel (GAHPA), and human adipose-derived stem cell exosomes (Exo), and to evaluate its feasibility, regenerative potential, and preliminary biosafety as a treatment for osteochondral defects. ACSs were first prepared from porcine articular cartilage, and their decellularization efficiency and ECM preservation were evaluated. The ACSs were then combined with an in situ crosslinkable GAHPA hydrogel to form a layered composite scaffold, followed by analyses of its chemical structure, microstructure, and mechanical properties. In addition, Exo were characterized and further evaluated for their effects on the differentiation of adipose-derived stem cells. Finally, a rat knee osteochondral defect model was used to compare in vivo repair outcomes among the untreated defect control (Defect), ACS/GAHPA, and ACS/GAHPA + Exo groups and to assess the preliminary systemic biosafety of the scaffold formulations.
The results showed that the decellularization process effectively removed cellular components from the cartilage sheets while preserving the collagen-rich ECM structure, supporting the feasibility of ACSs as cartilage-derived ECM scaffolds. After crosslinking, the GAHPA hydrogel formed a porous and interconnected three-dimensional structure, and the incorporation of exosomes did not noticeably alter the hydrogel morphology. When ACSs were incorporated into the GAHPA hydrogel, the mechanical support capacity of the composite scaffold was enhanced compared with that of the hydrogel alone, suggesting that ACSs not only provide a cartilage ECM- like microenvironment but also serve as structural reinforcement within the scaffold. The characterized Exo exhibited nanoscale extracellular vesicle characteristics and expressed exosome-associated markers, supporting their suitability as components providing bioactive signals. In vitro differentiation results showed that exosome treatment enhanced differentiation-related outcomes in adipose-derived stem cells under different induction conditions. In particular, more pronounced cartilage-like matrix formation was observed under chondrogenic induction, suggesting that these exosomes have the potential to regulate cell differentiation and promote cartilage-related matrix deposition.
In the rat osteochondral defect model, compared with the Defect group, both the ACS/GAHPA and ACS/GAHPA + Exo groups showed improved defect filling, surface continuity, and integration with surrounding tissue at the later evaluation time point. Histological staining further demonstrated that both scaffold-treated groups exhibited improved repair tissue morphology and increased the deposition of cartilage-related matrix components, including proteoglycans and type II collagen. Histological examination of major organs and blood biochemical analyses revealed no evident material-related systemic toxicity or abnormalities in liver or kidney function, indicating the preliminary systemic biosafety of this composite scaffold system.
In summary, this study developed a composite scaffold composed of ACSs and GAHPA hydrogel. The resulting scaffold retained cartilage-derived ECM characteristics, exhibited a porous hydrogel structure, provided mechanical reinforcement, and demonstrated favorable preliminary systemic biosafety. Exo may serve as a source of bioactive signals to promote cell differentiation and cartilage-like matrix formation in vitro. In the in vivo osteochondral defect model, both the ACS/GAHPA and ACS/GAHPA + Exo groups showed potential for promoting defect filling and cartilage-like matrix deposition and subchondral bone repair. Overall, the ACS/GAHPA composite scaffold represents a potential tissue engineering platform for osteochondral defect repair, whereas Exo may serve as an additional bioactive component. However, the incremental in vivo benefit of exosome incorporation requires further validation through direct statistical comparison, larger sample sizes, and longer-term evaluation.

[1] Fox A. J. S., Bedi A., and Rodeo S. A., "The Basic Science of Articular Cartilage: Structure, Composition, and Function," Sports Health, vol. 1, no. 6, pp. 461–468, 2009, doi: 10.1177/1941738109350438.
[2] Vincent T. L., McClurg O., and Troeberg L., "The Extracellular Matrix of Articular Cartilage Controls the Bioavailability of Pericellular Matrix-Bound Growth Factors to Drive Tissue Homeostasis and Repair," International Journal of Molecular Sciences, vol. 23, no. 11, May 2022, doi: 10.3390/ijms23116003.
[3] Vaish A., Shanmugasundaram S., Kim S. A., Lee D. H., Shetty A. A., and Kim S. J., "Biological reconstruction of the joint: Concepts of articular cartilage regeneration and their scientific basis," Journal of Clinical Orthopaedics and Trauma, vol. 24, p. 101718, Jan 2022, doi: 10.1016/j.jcot.2021.101718.
[4] Chawla S., Mainardi A., Majumder N., Dönges L., Kumar B., Occhetta P., Martin I., Egloff C., Ghosh S., Bandyopadhyay A., and Barbero A., "Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies," Cells, vol. 11, no. 24, p. 4034, Dec 2022, doi: 10.3390/cells11244034.
[5] Hu W., Chen Y., Dou C., and Dong S., "Microenvironment in subchondral bone: predominant regulator for the treatment of osteoarthritis," Annals of the Rheumatic Diseases, vol. 80, no. 4, pp. 413–422, Apr 2021, doi: 10.1136/annrheumdis-2020-218089.
[6] Thorp H., Kim K., Kondo M., Maak T., Grainger D. W., and Okano T., "Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage," Cells, vol. 10, no. 3, p. 643, 2021, doi: 10.3390/cells10030643.
[7] GBD 2021 Osteoarthritis Collaborators, "Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021," The Lancet Rheumatology, vol. 5, no. 9, pp. e508–e522, Sep 2023, doi: 10.1016/S2665-9913(23)00163-7.
[8] Makris E. A., Gomoll A. H., Malizos K. N., Hu J. C., and Athanasiou K. A., "Repair and tissue engineering techniques for articular cartilage," Nature Reviews Rheumatology, vol. 11, no. 1, pp. 21–34, Jan 2015, doi: 10.1038/nrrheum.2014.157.
[9] Yuan X. L., Meng H. Y., Wang Y. C., Peng J., Guo Q. Y., Wang A. Y., and Lu S. B., "Bone–cartilage interface crosstalk in osteoarthritis: potential pathways and future therapeutic strategies," Osteoarthritis and Cartilage, vol. 22, no. 8, pp. 1077–1089, 2014, doi: 10.1016/j.joca.2014.05.023.
[10] Solanki K., Shanmugasundaram S., Shetty N., and Kim S. J., "Articular cartilage repair & joint preservation: A review of the current status of biological approach," Journal of Clinical Orthopaedics and Trauma, vol. 22, p. 101602, Nov 2021, doi: 10.1016/j.jcot.2021.101602.
[11] Andrade R., Vasta S., Pereira R., Pereira H., Papalia R., Karahan M., Oliveira J. M., Reis R. L., and Espregueira-Mendes J., "Knee donor-site morbidity after mosaicplasty – a systematic review," Journal of Experimental Orthopaedics, vol. 3, no. 1, p. 31, 2016, doi: 10.1186/s40634-016-0066-0.
[12] Armiento A. R., Stoddart M. J., Alini M., and Eglin D., "Biomaterials for articular cartilage tissue engineering: Learning from biology," Acta Biomaterialia, vol. 65, pp. 1–20, Jan 2018, doi: 10.1016/j.actbio.2017.11.021.
[13] Johnstone B., Alini M., Cucchiarini M., Dodge G. R., Eglin D., Guilak F., Madry H., Mata A., Mauck R. L., Semino C. E., and Stoddart M. J., "Tissue engineering for articular cartilage repair—the state of the art," European Cells and Materials, vol. 25, pp. 248–267, May 2013, doi: 10.22203/ecm.v025a18.
[14] O'Brien F. J., "Biomaterials & scaffolds for tissue engineering," Materials Today, vol. 14, no. 3, pp. 88–95, Mar 2011, doi: 10.1016/S1369-7021(11)70058-X.
[15] Zha K., Sun Z., Yang Y., Chen M., Gao C., Fu L., Li H., Sui X., Guo Q., and Liu S., "Recent Developed Strategies for Enhancing Chondrogenic Differentiation of MSC: Impact on MSC-Based Therapy for Cartilage Regeneration," Stem Cells International, vol. 2021, p. 8830834, Mar 2021, doi: 10.1155/2021/8830834.
[16] Kuppa S. S., Kim H. K., Kang J. Y., Lee S. C., and Seon J. K., "Role of Mesenchymal Stem Cells and Their Paracrine Mediators in Macrophage Polarization: An Approach to Reduce Inflammation in Osteoarthritis," International Journal of Molecular Sciences, vol. 23, no. 21, Oct 2022, doi: 10.3390/ijms232113016.
[17] Wu K. C., Chang Y. H., Ding D. C., and Lin S. Z., "Mesenchymal Stromal Cells for Aging Cartilage Regeneration: A Review," International Journal of Molecular Sciences, vol. 25, no. 23, Nov 2024, doi: 10.3390/ijms252312911.
[18] Goh D., Yang Y., Lee E. H., Hui J. H. P., and Yang Z., "Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review," Bioengineering, vol. 10, no. 3, Mar 2023, doi: 10.3390/bioengineering10030355.
[19] Chen L., Liu J., Guan M., Zhou T., Duan X., and Xiang Z., "Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering," International Journal of Nanomedicine, vol. 15, pp. 6097–6111, 2020, doi: 10.2147/IJN.S249829.
[20] Zhang S., Chuah S. J., Lai R. C., Hui J. H. P., Lim S. K., and Toh W. S., "MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity," Biomaterials, vol. 156, pp. 16–27, Feb 2018, doi: 10.1016/j.biomaterials.2017.11.028.
[21] Jelodari S., Ebrahimi Sadrabadi A., Zarei F., Jahangir S., Azami M., Sheykhhasan M., and Hosseini S., "New Insights into Cartilage Tissue Engineering: Improvement of Tissue-Scaffold Integration to Enhance Cartilage Regeneration," BioMed Research International, vol. 2022, p. 7638245, 2022, doi: 10.1155/2022/7638245.
[22] Crapo P. M., Gilbert T. W., and Badylak S. F., "An overview of tissue and whole organ decellularization processes," Biomaterials, vol. 32, no. 12, pp. 3233–3243, Apr 2011, doi: 10.1016/j.biomaterials.2011.01.057.
[23] Sun Y., Yan L., Chen S., and Pei M., "Functionality of decellularized matrix in cartilage regeneration: A comparison of tissue versus cell sources," Acta Biomaterialia, vol. 74, pp. 56–73, Jul 2018, doi: 10.1016/j.actbio.2018.04.048.
[24] Kim Y. S., Majid M., Melchiorri A. J., and Mikos A. G., "Applications of decellularized extracellular matrix in bone and cartilage tissue engineering," Bioengineering & Translational Medicine, vol. 4, no. 1, pp. 83–95, Jan 2019, doi: 10.1002/btm2.10110.
[25] Benders K. E., van Weeren P. R., Badylak S. F., Saris D. B., Dhert W. J., and Malda J., "Extracellular matrix scaffolds for cartilage and bone regeneration," Trends in Biotechnology, vol. 31, no. 3, pp. 169–176, Mar 2013, doi: 10.1016/j.tibtech.2012.12.004.
[26] Zhang Q., Hu Y., Long X., Hu L., Wu Y., Wu J., Shi X., Xie R., Bi Y., Yu F., Li P., and Yang Y., "Preparation and Application of Decellularized ECM-Based Biological Scaffolds for Articular Cartilage Repair: A Review," Frontiers in Bioengineering and Biotechnology, vol. 10, p. 908082, 2022, doi: 10.3389/fbioe.2022.908082.
[27] Santoro M., Tatara A. M., and Mikos A. G., "Gelatin carriers for drug and cell delivery in tissue engineering," Journal of Controlled Release, vol. 190, pp. 210–218, Sep 2014, doi: 10.1016/j.jconrel.2014.04.014.
[28] Wang L. S., Boulaire J., Chan P. P., Chung J. E., and Kurisawa M., "The role of stiffness of gelatin-hydroxyphenylpropionic acid hydrogels formed by enzyme-mediated crosslinking on the differentiation of human mesenchymal stem cell," Biomaterials, vol. 31, no. 33, pp. 8608–8616, Nov 2010, doi: 10.1016/j.biomaterials.2010.07.075.
[29] Mianehsaz E., Mirzaei H. R., Mahjoubin-Tehran M., Rezaee A., Sahebnasagh R., Pourhanifeh M. H., Mirzaei H., and Hamblin M. R., "Mesenchymal stem cell-derived exosomes: a new therapeutic approach to osteoarthritis?," Stem Cell Research & Therapy, vol. 10, no. 1, p. 340, Nov 2019, doi: 10.1186/s13287-019-1445-0.
[30] Kalluri R. and LeBleu V. S., "The biology, function, and biomedical applications of exosomes," Science, vol. 367, no. 6478, p. eaau6977, 2020, doi: 10.1126/science.aau6977.
[31] Cosenza S., Ruiz M., Toupet K., Jorgensen C., and Noël D., "Mesenchymal stem cells derived exosomes and microparticles protect cartilage and bone from degradation in osteoarthritis," Scientific Reports, vol. 7, no. 1, p. 16214, Nov 2017, doi: 10.1038/s41598-017-15376-8.
[32] Woo C. H., Kim H. K., Jung G. Y., Jung Y. J., Lee K. S., Yun Y. E., Han J., Lee J., Kim W. S., Choi J. S., Yang S., Park J. H., Jo D. G., and Cho Y. W., "Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration," Journal of Extracellular Vesicles, vol. 9, no. 1, p. 1735249, 2020, doi: 10.1080/20013078.2020.1735249.
[33] Cavallo C., Merli G., Borzì R. M., Zini N., D’Adamo S., Guescini M., Grigolo B., Di Martino A., Santi S., and Filardo G., “Small Extracellular Vesicles from adipose derived stromal cells significantly attenuate in vitro the NF-κB dependent inflammatory/catabolic environment of osteoarthritis,” Scientific Reports, vol. 11, no. 1, p. 1053, Jan. 2021, doi: 10.1038/s41598-020-80032-7.
[34] Tang A., Shu Q., Jia S., Lai Z., and Tian J., "Adipose Mesenchymal Stem Cell-Derived Exosomes as Nanocarriers for Treating Musculoskeletal Disorders," International Journal of Nanomedicine, vol. 19, pp. 13547–13562, Dec 2024, doi: 10.2147/IJN.S486622.
[35] Zhang Y., Yan W., Wu L., Yu Z., Quan Y., and Xie X., "Different exosomes are loaded in hydrogels for the application in the field of tissue repair," Frontiers in Bioengineering and Biotechnology, vol. 13, p. 1545636, 2025, doi: 10.3389/fbioe.2025.1545636.
[36] Singhmar R., Lamba J. K., Panwar A., and Hong C. A., "Exploring exosomes in osteoarthritis: biogenesis, functional roles, and recent hydrogel-based delivery strategies," Biomaterials Science, vol. 13, no. 23, pp. 6525–6544, 2025, doi: 10.1039/d5bm01308a.
[37] Xian Bo S., Chen W., Chang L., Hao Ran Y., Hui Hui G., Ya Kun Z., Wu Kun X., Hai Tao F., and Wen Dan C., "The Research Progress of Exosomes in Osteoarthritis, With Particular Emphasis on the Therapeutic Effect," Frontiers in Pharmacology, vol. 13, p. 731756, 2022, doi: 10.3389/fphar.2022.731756.
[38] Liu X., Yang Y., Li Y., Niu X., Zhao B., Wang Y., Bao C., Xie Z., Lin Q., and Zhu L., "Integration of stem cell-derived exosomes with in situ hydrogel glue as a promising tissue patch for articular cartilage regeneration," Nanoscale, vol. 9, no. 13, pp. 4430–4438, Mar 2017, doi: 10.1039/c7nr00352h.
[39] Sang X., Zhao X., Yan L., Jin X., Wang X., Wang J., Yin Z., Zhang Y., and Meng Z., “Thermosensitive Hydrogel Loaded with Primary Chondrocyte-Derived Exosomes Promotes Cartilage Repair by Regulating Macrophage Polarization in Osteoarthritis,” Tissue Engineering and Regenerative Medicine, vol. 19, no. 3, pp. 629–642, Jun. 2022, doi: 10.1007/s13770-022-00437-5.
[40] Hu M., Kurisawa M., Deng R., Teo C. M., Schumacher A., Thong Y. X., Wang L., Schumacher K. M., and Ying J. Y., "Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers," Biomaterials, vol. 30, no. 21, pp. 3523–3531, Jul 2009, doi: 10.1016/j.biomaterials.2009.03.004.
[41] Delwatta S. L., Gunatilake M., Baumans V., Seneviratne M. D., Dissanayaka M. L. B., Batagoda S. S., Udagedara A. H., and Walpola P. B., "Reference values for selected hematological, biochemical and physiological parameters of Sprague-Dawley rats at the Animal House, Faculty of Medicine, University of Colombo, Sri Lanka," Animal Models and Experimental Medicine, vol. 1, no. 4, pp. 250–254, Dec 2018, doi: 10.1002/ame2.12041.
[42] He Q., Su G., Liu K., Zhang F., Jiang Y., Gao J., Liu L., Jiang Z., Jin M., and Xie H., “Sex-specific reference intervals of hematologic and biochemical analytes in Sprague-Dawley rats using the nonparametric rank percentile method,” PLOS ONE, vol. 12, no. 12, p. e0189837, 2017, doi: 10.1371/journal.pone.0189837.