负载牛磺熊去氧胆酸的光交联明胶水凝胶支架在兔膝关节软骨缺损修复中的效能

doi:10.3969/j.issn.1674-8115.2025.07.004

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (7): 829-837.doi: 10.3969/j.issn.1674-8115.2025.07.004

负载牛磺熊去氧胆酸的光交联明胶水凝胶支架在兔膝关节软骨缺损修复中的效能

魏祥¹^,², 魏凌飞¹^,³, 徐纯峰², 高玉洁⁴^,⁵, 聂萍⁴(), 于德栋²()

^1.滨州医学院，烟台 264003
^2.上海交通大学医学院附属第九人民医院口腔第二门诊部，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 201999
^3.滨州医学院附属烟台口腔医院口腔种植科，烟台 264003
^4.上海交通大学医学院附属第九人民医院口腔颅颌面科，上海交通大学口腔医学院，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^5.新疆维吾尔自治区克拉玛依市中心医院口腔科，克拉玛依 834000

收稿日期:2025-01-14 接受日期:2025-03-25 出版日期:2025-07-28 发布日期:2025-07-18
通讯作者: 聂　萍，副主任医师，博士；电子信箱：nieping1011@sina.com。
于德栋，主任医师，博士；电子信箱：yudedong@sjtu.edu.cn。
基金资助:
国家自然科学基金(32101094)

Efficacy of photo-crosslinked gelatin hydrogel scaffolds loaded with tauroursodeoxycholic acid on knee cartilage defect repair in a rabbit model

WEI Xiang¹^,², WEI Lingfei¹^,³, XU Chunfeng², GAO Yujie⁴^,⁵, NIE Ping⁴(), YU Dedong²()

^1.Binzhou Medical University, Yantai 264003, China
^2.Department of Second Dental Center, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 201999, China
^3.Department of Oral Implantology, Yantai Stomatology Hospital, Binzhou Medical College, Yantai 264003, China
^4.Department of Oral and Craniomaxillofacial Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
^5.Department of Stomatology, Central Hospital of Karamay, Xinjiang Uygur Autonomous Region, Karamay 834000, China

Received:2025-01-14 Accepted:2025-03-25 Online:2025-07-28 Published:2025-07-18
Contact: NIE Ping, E-mail: nieping1011@sina.com.
YU Dedong, E-mail: yudedong@sjtu.edu.cn.
Supported by:
National Natural Science Foundation of China(32101094)

摘要/Abstract

摘要：

目的·探讨负载牛磺熊去氧胆酸（tauroursodeoxycholic acid，TUDCA）的光交联甲基丙烯酸酐化明胶（gelatin methylacrylated，GelMA）水凝胶支架对兔膝关节软骨缺损的修复作用。方法·采用紫外光照射法制备负载TUDCA的光交联GelMA水凝胶支架，对其进行理化性能表征，并测定GelMA水凝胶支架中TUDCA的累积释放率。构建兔膝关节软骨缺损模型，随机分为3组：对照组（n=6，软骨缺损处不进行处理）、GelMA组（n=6，软骨缺损处填充GelMA水凝胶支架）、GelMA+TUDCA组（n=6，软骨缺损处填充负载TUDCA的GelMA水凝胶支架）。术后第12周，采用酶联免疫吸附分析（enzyme-linked immunosorbent assay，ELISA）检测膝关节滑液内炎症因子肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和白细胞介素-1β（interleukin-1β，IL-1β）的浓度。对兔实施安乐死后，分离膝关节软骨，行大体观察、番红-固绿染色和甲苯胺蓝染色，并按照国际软骨修复协会（International Cartilage Repair Society，ICRS）和改良O'Driscoll评分（Modified O'Driscoll Score，MODS）标准对软骨缺损处修复情况进行评分。免疫组织化学染色检测膝关节软骨组织Ⅱ型胶原蛋白（typeⅡ collagen，COL-Ⅱ）的表达，Western blotting检测聚集蛋白聚糖（aggrecan，ACAN）及COL-Ⅱ蛋白表达水平。结果·GelMA水凝胶支架在紫外光照射后微观结构更加紧凑，且具备适宜的质量溶胀比和压缩模量。负载TUDCA的光交联GelMA水凝胶支架能有效、持续释放TUDCA，在28 d内累积释放率可达90.2%±2.5%。与对照组和GelMA组相比，GelMA+TUDCA组关节滑液中TNF-α和IL-1β浓度显著降低（P<0.001）。GelMA+TUDCA组膝关节软骨缺损基本修复，表面平整且与周围组织整合良好；新生软骨细胞数量增加、排列整齐，新生软骨形成良好；ICRS评分和MODS评分均明显高于对照组和GelMA组（P<0.001）。相比对照组和GelMA组，GelMA+TUDCA组ACAN和COL-Ⅱ蛋白的表达水平显著增加（P<0.001）。结论·负载TUDCA的光交联GelMA水凝胶支架可有效促进兔膝关节软骨缺损的修复。

关键词: 软骨缺损, 骨关节炎, 牛磺熊去氧胆酸, 甲基丙烯酸酐化明胶, 软骨细胞, 组织工程

Abstract:

Objective ·To investigate the reparative effects of photo-crosslinked gelatin methylacrylated (GelMA) hydrogel scaffolds loaded with tauroursodeoxycholic acid (TUDCA) on cartilage defects in rabbit knee joints. Methods ·Photo-crosslinked GelMA hydrogel scaffolds loaded with TUDCA were prepared by the ultraviolet light irradiation method. The physicochemical properties of GelMA hydrogel scaffolds were characterized, and the cumulative release rate of TUDCA was determined. A rabbit knee cartilage defect model was established, and 18 rabbits were randomly assigned into three groups, with six rabbits in each group: the control group (no treatment was applied to the cartilage defect), the GelMA group (the cartilage defect was filled with GelMA hydrogel scaffold), and the GelMA+TUDCA group (the cartilage defect was filled with the GelMA hydrogel scaffold loaded with TUDCA). At 12 weeks postoperatively, the concentrations of two inflammatory factors in synovial fluid, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were detected by enzyme-linked immunosorbent assay (ELISA). After euthanasia, knee cartilage samples were harvested for gross observation, safranin O-fast green staining, and toluidine blue staining. The repair of the cartilage defects was evaluated according to the International Cartilage Repair Society (ICRS) and Modified O′Driscoll Score (MODS) systems. Immunohistochemical staining was performed to detect type Ⅱ collagen (COL-Ⅱ) protein in the cartilage tissue, and Western blotting was used to assess the protein levels of aggrecan (ACAN) and COL-Ⅱ. Results ·GelMA hydrogel scaffolds exhibited a more compact microstructure after ultraviolet light irradiation, along with an suitable mass swelling ratio and compressive modulus. The TUDCA-loaded photo-crosslinked GelMA hydrogel scaffolds demonstrated effective and sustained TUDCA release, achieving a cumulative release rate of 90.2%±2.5% within 28 d. ELISA results showed that compared to the control and GelMA groups, the concentrations of TNF-α and IL-1β in the synovial fluid of the GelMA+TUDCA group were significantly reduced (P<0.001). In the GelMA+TUDCA group, the cartilage defects were nearly fully repaired, with a smooth surface and good integration with the surrounding tissue. The number of newly formed chondrocytes increased, displaying orderly alignment, and the neocartilage exhibited excellent formation. Both ICRS and MODS scores were significantly higher in the GelMA+TUDCA group than those in the control and GelMA groups (P<0.001). Additionally, the expression levels of ACAN and COL-Ⅱ proteins were significantly elevated in the GelMA+TUDCA group compared to the control and GelMA groups (P<0.001). Conclusion ·Photo-crosslinked GelMA hydrogel scaffolds loaded with TUDCA can effectively promote the repair of cartilage defects in rabbit knee joints.

Key words: cartilage defect, osteoarthritis, tauroursodeoxycholic acid, gelatin methylacrylated, chondrocyte, tissue engineering

中图分类号:

R782

魏祥, 魏凌飞, 徐纯峰, 高玉洁, 聂萍, 于德栋. 负载牛磺熊去氧胆酸的光交联明胶水凝胶支架在兔膝关节软骨缺损修复中的效能[J]. 上海交通大学学报（医学版）, 2025, 45(7): 829-837.

WEI Xiang, WEI Lingfei, XU Chunfeng, GAO Yujie, NIE Ping, YU Dedong. Efficacy of photo-crosslinked gelatin hydrogel scaffolds loaded with tauroursodeoxycholic acid on knee cartilage defect repair in a rabbit model[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(7): 829-837.

图/表 7

图1 GelMA水凝胶支架的合成与理化性能表征Note： A. Schematic diagram of GelMA hydrogel scaffold synthesis. B. Photos and scanning electron microscope images of GelMA hydrogel scaffolds before and after ultraviolet light irradiation. C. Measurement results of remaining mass of GelMA hydrogel scaffolds after 24-h incubation in type Ⅱ collagenase.

Fig 1 Synthesis and physicochemical characterization of GelMA hydrogel scaffolds

图2 GelMA/TUDCA水凝胶支架的制备及TUDCA释放动力学Note： A. Preparation process of TUDCA-loaded photo-crosslinked GelMA hydrogel scaffolds. B. The cumulative release rate of sustained-release TUDCA from the TUDCA-loaded photo-crosslinked GelMA hydrogel scaffolds, as determined by HPLC.

Fig 2 Fabrication of GelMA/TUDCA hydrogel scaffolds and the release kinetics of TUCDA from the scaffolds

图3 ELISA检测膝关节滑液中IL-1β、TNF-α的浓度

Fig 3 Concentrations of IL-1β and TNF-α in knee synovial fluid detected by ELISA

图4 各组膝关节软骨缺损处修复后的形态学观察及ICRS评分Note： A. Gross view and Micro-CT images of knee cartilage defects in each group at 12 weeks postoperatively. B. ICRS scores of each group.

Fig 4 Morphological observation and ICRS scores of repaired knee articular cartilage defects in each group

图5 膝关节软骨缺损处修复后的组织学观察及MODS评分Note： A/B. Safranin O-fast green staining (A) and toluidine blue staining (B) of knee joint tissues of each group at 12 weeks postoperatively. The red boxes indicate areas of joint cartilage damage in each group and the red arrows indicate the ACAN-stained areas. C. MODS scores of each group.

Fig 5 Histological observation and MODS scores of repaired knee articular cartilage defects

图6 免疫组织化学染色检测膝关节软骨缺损处COL-Ⅱ的表达Note： A. Immunohistochemical staining results of COL-Ⅱ in knee cartilage defects of each group at the 12th week. The red boxes indicate areas of joint cartilage damage in each group. B. Relative expression of COL-Ⅱ in each group, as detected by immunohistochemical staining.

Fig 6 Immunohistochemical staining of COL-Ⅱ in repaired knee articular cartilage defects

图7 Western blotting检测膝关节软骨缺损处ACAN、COL-Ⅱ的蛋白水平

Fig 7 ACAN and COL-Ⅱ protein levels in repaired knee articular cartilage defects detected by Western blotting

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负载牛磺熊去氧胆酸的光交联明胶水凝胶支架在兔膝关节软骨缺损修复中的效能

Efficacy of photo-crosslinked gelatin hydrogel scaffolds loaded with tauroursodeoxycholic acid on knee cartilage defect repair in a rabbit model

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