骨關節炎為造成全球多數人口行動不便之主要病因，由於關節軟骨再生能力不佳，若受損早期未能及時治療，最終將走向骨關節炎；而現行臨床治療多生成纖維軟骨，與關節覆蓋之透明軟骨相異。為解決此臨床修復困境，軟骨組織工程日益興盛，細胞、支架、訊號三要素構成主體研究軟骨再生。本研究基於軟骨組織工程概念，透過結合去細胞軟骨層片與包埋細胞之原位成型水膠形成三明治模型，希望透過整合富含原有軟骨基質結構之去細胞軟骨層片，增強注射型水膠一直以來所缺乏的機械性質，期待能發展具潛力之創新複合支架應用於軟骨修復。
本研究成功製備去細胞程度完整且無毒性之軟骨層片，與可注射性酵素交聯原位成型水膠，其前導水膠分別含有辣根過氧化物酶與過氧化氫，研究中固定酵素比例並分三組過氧化氫濃度 (0.01、0.03、0.05 wt%) 進行實驗。各組皆具備高吸水能力且未發生劇烈體積變化；中高濃度過氧化氫水膠於降解表現較佳；高濃度過氧化氫水膠之細胞活性不理想，僅中低濃度過氧化氫水膠具良好生物相容性。確立兩項材料製程後，透過注射水膠並堆疊去細胞軟骨層片製備複合支架，在微觀結構中發現層片與水膠間鍵結完整；複合支架於機械性質測試比純水膠支架突出；在包埋細胞進行體外培養測試中，雖無顯著基質分泌，但有軟骨細胞簇出現在中高濃度過氧化氫水膠組別。
綜上所述，去細胞軟骨層片與0.03 wt% 過氧化氫水膠之複合支架為最佳組別，具良好水膠特性、生物相容性、細胞團聚表現與提升機械性質，為擁有發展潛力之生醫材料應用於軟骨修復。期望透過改善臨床現有骨軟骨自體移植系統 (OATS) ，於軟骨缺陷早期進行軟骨修復，不僅能避免軟骨於手術中受到二次傷害，亦能避免患者走向骨關節炎。

Osteoarthritis (OA) is the leading impaired mobility disease for most global populations. Due to the inferior regeneration ability of articular cartilage, once the cartilage defect is not treated in time, it will eventually turn into OA. Current clinical treatments still cannot get satisfactory long-term results because fibrocartilage different from the hyaline cartilage covering the joints is generated in those treatments. To overcome this clinical challenge, cartilage tissue engineering has been increasingly prosperous. Cells, scaffolds, and signals constitute the main elements in studying cartilage regeneration. An innovative composite scaffold was established based on the concept of cartilage tissue engineering in this study. The sandwich model scaffolds were formed by combining acellular cartilage sheets (ACS) and in-situ hydrogels to explore the feasibility of cartilage applications.
This study fabricated the ACSs with non-toxicity properties and successfully prepared the injectable enzyme-catalyzed in-situ hydrogels. The precursory hydrogels contained horseradish peroxidase and hydrogen peroxide (H2O2), respectively. The experimental groups in this research were divided into three different H2O2 concentrations (0.01, 0.03, and 0.05 wt%) with fixed enzyme ratios. All groups owned excellent swelling ratios with no drastic volume change, and the groups with 0.03 and 0.05 wt% H2O2 had better degradation properties. The cell viability of 0.05 wt% H2O2 hydrogel was not qualified; only the groups with 0.01 and 0.03 wt% H2O2 possessed good biocompatibility. After the manufacturing processes of ACSs and in-situ hydrogels were set up, sandwich model scaffolds were created by injecting hydrogels and stacking ACSs. From the results of the microstructure, it was found that the crosslinking between ACSs and the hydrogel was complete; the mechanical properties of the composite scaffolds were better than that of the pure hydrogel scaffolds; in vitro analyses, though there was no significant ECM formation, chondrocytes clusters appeared in the groups with 0.03 and 0.05 wt% H2O2.
In conclusion, the composite scaffolds formed by acellular cartilage sheets and 0.03 wt% H2O2 hydrogel was the most optimal group. This sandwich model scaffold had appropriate hydrogel properties, biocompatibility, cell aggregation performance, and enhanced mechanical properties, which was a promising biomaterial for cartilage repair. Comparing to the current clinical osteochondral autologous transplantation system (OATS), this novel system can promote cartilage regeneration in the early stage of cartilage defects, which can not only prevent the cartilage from secondary damage during the operation but also prevent the patient from developing osteoarthritis.

[1] Vyas C., Mishbak H., Cooper G., Peach C., Pereira R. F., and Bartolo P., "Biological perspectives and current biofabrication strategies in osteochondral tissue engineering," Biomanufacturing Reviews, vol. 5, no. 1, pp. 1-24, 2020.
[2] Statham P., Jones E., Jennings L. M., and Fermor H. L., "Reproducing the biomechanical environment of the chondrocyte for cartilage tissue engineering," Tissue Engineering Part B: Reviews, 2021.
[3] Sophia Fox A. J., 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.
[4] Ulrich-Vinther M., Maloney M. D., Schwarz E. M., Rosier R., and O'Keefe R. J., "Articular cartilage biology," JAAOS-Journal of the American Academy of Orthopaedic Surgeons, vol. 11, no. 6, pp. 421-430, 2003.
[5] Eyre D., "Articular cartilage and changes in arthritis: collagen of articular cartilage," Arthritis Research & Therapy, vol. 4, no. 1, pp. 1-6, 2001.
[6] Wei W. and Dai H., "Articular cartilage and osteochondral tissue engineering techniques: Recent advances and challenges," Bioactive materials, vol. 6, no. 12, pp. 4830-4855, 2021.
[7] Poole A. R., Kojima T., Yasuda T., Mwale F., Kobayashi M., and Laverty S., "Composition and structure of articular cartilage: a template for tissue repair," Clinical Orthopaedics and Related Research®, vol. 391, pp. S26-S33, 2001.
[8] 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.
[9] Zheng L., Zhang Z., Sheng P., and Mobasheri A., "The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis," Ageing research reviews, vol. 66, p. 101249, 2021.
[10] Hunter D. J., Schofield D., and Callander E., "The individual and socioeconomic impact of osteoarthritis," Nature Reviews Rheumatology, vol. 10, no. 7, pp. 437-441, 2014.
[11] Cui A., Li H., Wang D., Zhong J., Chen Y., and Lu H., "Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies," EClinicalMedicine, vol. 29, p. 100587, 2020.
[12] Kwon H., Brown W. E., Lee C. A., Wang D., Paschos N., Hu J. C. et al., "Surgical and tissue engineering strategies for articular cartilage and meniscus repair," Nature Reviews Rheumatology, vol. 15, no. 9, pp. 550-570, 2019.
[13] Medvedeva E. V., Grebenik E. A., Gornostaeva S. N., Telpuhov V. I., Lychagin A. V., Timashev P. S. et al., "Repair of damaged articular cartilage: current approaches and future directions," International journal of molecular sciences, vol. 19, no. 8, p. 2366, 2018.
[14] Roseti L., Desando G., Cavallo C., Petretta M., and Grigolo B., "Articular cartilage regeneration in osteoarthritis," Cells, vol. 8, no. 11, p. 1305, 2019.
[15] Khademhosseini A. and Langer R., "A decade of progress in tissue engineering," Nature protocols, vol. 11, no. 10, pp. 1775-1781, 2016.
[16] Qasim M., Chae D. S., and Lee N. Y., "Bioengineering strategies for bone and cartilage tissue regeneration using growth factors and stem cells," Journal of Biomedical Materials Research Part A, vol. 108, no. 3, pp. 394-411, 2020.
[17] 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, p. 6097, 2020.
[18] Choi J. R., Yong K. W., and Choi J. Y., "Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering," Journal of cellular physiology, vol. 233, no. 3, pp. 1913-1928, 2018.
[19] Baugé C. and Boumédiene K., "Use of adult stem cells for cartilage tissue engineering: current status and future developments," Stem Cells International, vol. 2015, 2015.
[20] Kim Y. S. and Mikos A. G., "Emerging strategies in reprogramming and enhancing the fate of mesenchymal stem cells for bone and cartilage tissue engineering," Journal of Controlled Release, vol. 330, pp. 565-574, 2021.
[21] Vinatier C. and Guicheux J., "Cartilage tissue engineering: From biomaterials and stem cells to osteoarthritis treatments," Annals of physical and rehabilitation medicine, vol. 59, no. 3, pp. 139-144, 2016.
[22] Zhao Z., Fan C., Chen F., Sun Y., Xia Y., Ji A. et al., "Progress in articular cartilage tissue engineering: a review on therapeutic cells and macromolecular scaffolds," Macromolecular bioscience, vol. 20, no. 2, p. 1900278, 2020.
[23] Zhao X., Hu D. A., Wu D., He F., Wang H., Huang L. et al., "Applications of biocompatible scaffold materials in stem cell-based cartilage tissue engineering," Frontiers in Bioengineering and Biotechnology, vol. 9, p. 603444, 2021.
[24] Zhang Y., Liu X., Zeng L., Zhang J., Zuo J., Zou J. et al., "Polymer fiber scaffolds for bone and cartilage tissue engineering," Advanced Functional Materials, vol. 29, no. 36, p. 1903279, 2019.
[25] Wasyłeczko M., Sikorska W., and Chwojnowski A., "Review of synthetic and hybrid scaffolds in cartilage tissue engineering," Membranes, vol. 10, no. 11, p. 348, 2020.
[26] Zhang X., Chen X., Hong H., Hu R., Liu J., and Liu C., "Decellularized extracellular matrix scaffolds: Recent trends and emerging strategies in tissue engineering," Bioactive materials, vol. 10, pp. 15-31, 2022.
[27] Massaro M. S., Pálek R., Rosendorf J., Červenková L., Liška V., and Moulisová V., "Decellularized xenogeneic scaffolds in transplantation and tissue engineering: Immunogenicity versus positive cell stimulation," Materials Science and Engineering: C, vol. 127, p. 112203, 2021.
[28] Yao Q., Zheng Y.-W., Lan Q.-H., Kou L., Xu H.-L., and Zhao Y.-Z., "Recent development and biomedical applications of decellularized extracellular matrix biomaterials," Materials Science and Engineering: C, vol. 104, p. 109942, 2019.
[29] Xia C., Mei S., Gu C., Zheng L., Fang C., Shi Y. et al., "Decellularized cartilage as a prospective scaffold for cartilage repair," Materials Science and Engineering: C, vol. 101, pp. 588-595, 2019.
[30] Yang J., Dang H., and Xu Y., "Recent advancement of decellularization extracellular matrix for tissue engineering and biomedical application," Artificial Organs, vol. 46, no. 4, pp. 549-567, 2022.
[31] Chang C. H., Chen C. C., Liao C. H., Lin F. H., Hsu Y. M., and Fang H. W., "Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium‐derived mesenchymal stem cells," Journal of biomedical materials research Part A, vol. 102, no. 7, pp. 2248-2257, 2014.
[32] Zhang Y., Feng G., Xu G., and Qi Y., "Microporous acellular extracellular matrix combined with adipose-derived stem cell sheets as a promising tissue patch promoting articular cartilage regeneration and interface integration," Cytotherapy, vol. 21, no. 8, pp. 856-869, 2019.
[33] Zahiri S., Masaeli E., Poorazizi E., and Nasr‐Esfahani M. H., "Chondrogenic response in presence of cartilage extracellular matrix nanoparticles," Journal of Biomedical Materials Research Part A, vol. 106, no. 9, pp. 2463-2471, 2018.
[34] Xue J. X., Gong Y. Y., Zhou G. D., Liu W., Cao Y., and Zhang W. J., "Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells induced by acellular cartilage sheets," Biomaterials, vol. 33, no. 24, pp. 5832-5840, 2012.
[35] Gong Y. Y., Xue J. X., Zhang W. J., Zhou G. D., Liu W., and Cao Y., "A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes," Biomaterials, vol. 32, no. 9, pp. 2265-2273, 2011.
[36] Zhou L., Guo P., D'Este M., Tong W., Xu J., Yao H. et al., "Functionalized hydrogels for articular cartilage tissue engineering," Engineering, 2022.
[37] Akindoyo J., Mariatti M., Hamid Z. A., Nurul A., and Teramoto N., "Injectable hydrogel scaffold from natural biomaterials-An overview of recent studies," in AIP Conference Proceedings, 2020, vol. 2267, no. 1: AIP Publishing LLC, p. 020068.
[38] Bello A. B., Kim D., Kim D., Park H., and Lee S.-H., "Engineering and functionalization of gelatin biomaterials: From cell culture to medical applications," Tissue Engineering Part B: Reviews, vol. 26, no. 2, pp. 164-180, 2020.
[39] Ngadimin K. D., Stokes A., Gentile P., and Ferreira A. M., "Biomimetic hydrogels designed for cartilage tissue engineering," Biomaterials Science, vol. 9, no. 12, pp. 4246-4259, 2021.
[40] Campiglio C. E., Contessi Negrini N., Farè S., and Draghi L., "Cross-linking strategies for electrospun gelatin scaffolds," Materials, vol. 12, no. 15, p. 2476, 2019.
[41] Bao W., Li M., Yang Y., Wan Y., Wang X., Bi N. et al., "Advancements and frontiers in the high performance of natural hydrogels for cartilage tissue engineering," Frontiers in chemistry, vol. 8, p. 53, 2020.
[42] Zhao W., Jin X., Cong Y., Liu Y., and Fu J., "Degradable natural polymer hydrogels for articular cartilage tissue engineering," Journal of Chemical Technology & Biotechnology, vol. 88, no. 3, pp. 327-339, 2013.
[43] Wang L.-S., Du C., Toh W. S., Wan A. C., Gao S. J., and Kurisawa M., "Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties," Biomaterials, vol. 35, no. 7, pp. 2207-2217, 2014.
[44] Bae J. W., Choi J. H., Lee Y., and Park K. D., "Horseradish peroxidase‐catalysed in situ‐forming hydrogels for tissue‐engineering applications," Journal of tissue engineering and regenerative medicine, vol. 9, no. 11, pp. 1225-1232, 2015.
[45] Teixeira L. S. M., Bijl S., Pully V. V., Otto C., Jin R., Feijen J. et al., "Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair," Biomaterials, vol. 33, no. 11, pp. 3164-3174, 2012.
[46] Lee Y., Bae J. W., Oh D. H., Park K. M., Chun Y. W., Sung H.-J. et al., "In situ forming gelatin-based tissue adhesives and their phenolic content-driven properties," Journal of Materials Chemistry B, vol. 1, no. 18, pp. 2407-2414, 2013.
[47] Le Thi P., Lee Y., Nguyen D. H., and Park K. D., "In situ forming gelatin hydrogels by dual-enzymatic cross-linking for enhanced tissue adhesiveness," Journal of Materials Chemistry B, vol. 5, no. 4, pp. 757-764, 2017.
[48] Jung H. Y., Le Thi P., HwangBo K.-H., Bae J. W., and Park K. D., "Tunable and high tissue adhesive properties of injectable chitosan based hydrogels through polymer architecture modulation," Carbohydrate Polymers, vol. 261, p. 117810, 2021.
[49] Tran H. D., Park K. D., Ching Y. C., Huynh C., and Nguyen D. H., "A comprehensive review on polymeric hydrogel and its composite: Matrices of choice for bone and cartilage tissue engineering," Journal of Industrial and Engineering Chemistry, vol. 89, pp. 58-82, 2020.
[50] Antunes F. and Cadenas E., "Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar levels of H2O2 induce apoptosis through Fenton chemistry independent of the cellular thiol state," Free Radical Biology and Medicine, vol. 30, no. 9, pp. 1008-1018, 2001.
[51] Akkiraju H. and Nohe A., "Role of chondrocytes in cartilage formation, progression of osteoarthritis and cartilage regeneration," Journal of developmental biology, vol. 3, no. 4, pp. 177-192, 2015.