自愈合可注射性透明质酸水凝胶的构建及促进血管生成的研究

doi:10.3969/j.issn.1674-8115.2023.12.003

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (12): 1480-1492.doi: 10.3969/j.issn.1674-8115.2023.12.003

• 论著 · 基础研究 • 上一篇

自愈合可注射性透明质酸水凝胶的构建及促进血管生成的研究

杨淑¹^,²(), 崔文国², 魏杰¹(), 蔡正伟²()

^1.华东理工大学材料科学与工程学院，上海 200237
^2.上海交通大学医学院附属瑞金医院，上海市伤骨科研究所，上海市中西医结合防治骨与关节病损重点实验室，上海 200025

收稿日期:2023-11-08 接受日期:2023-12-11 出版日期:2023-12-28 发布日期:2024-02-01
通讯作者: 魏杰,蔡正伟 E-mail:Yangshu12101999@163.com;jiewei7860@sina.com;caizhengwei@shsmu.edu.cn
作者简介:杨淑（1999—），女，硕士生；电子信箱：Yangshu12101999@163.com。
基金资助:
国家自然科学基金(32101104);上海市卫生健康委员会基金(202140127)

Fabrication of self-healing injectable hyaluronic acid hydrogel for promoting angiogenesis

YANG Shu¹^,²(), CUI Wenguo², WEI Jie¹(), CAI Zhengwei²()

^1.School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, 200237, China
^2.Shanghai Key laboratory for Prevention and Treatment of Bone and Joint Disease, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2023-11-08 Accepted:2023-12-11 Online:2023-12-28 Published:2024-02-01
Contact: WEI Jie,CAI Zhengwei E-mail:Yangshu12101999@163.com;jiewei7860@sina.com;caizhengwei@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(32101104);Foundation of Shanghai Municipal Health Commission(202140127)

摘要/Abstract

摘要：

目的·构建一种基于透明质酸（hyaluronic acid，HA）的自愈合可注射性水凝胶，探究不同浓度铜离子对水凝胶性能及其促成血管功效的影响，评估其应用于临床上伤口愈合的可行性。方法·在光引发剂2959的存在下，通过蓝光诱发了巯基化透明质酸（thiolated hyaluronic acid，HASH）和丙烯酸化双膦酸盐（acrylated bisphosphonate，Ac-PD）之间的巯基-烯点击反应，制备了双膦酸盐化透明质酸（bisphosphonated hyaluronic acid，HAPD）；基于HAPD和Cu²⁺的金属配位作用，构建了不同Cu²⁺浓度的HAPD-Cu水凝胶，即HAPD-Cu1、HAPD-Cu2、HAPD-Cu3以及HAPD-Cu4。采用核磁氢谱以及傅里叶红外光谱验证HASH、Ac-PD、HAPD和HAPD-Cu的分子结构；采用扫描电镜观察HAPD-Cu的微观形貌；采用流变仪验证HAPD-Cu的剪切变稀和自修复特性；采用液相质谱仪测定HAPD-Cu的离子释放；通过活/死细胞染色和CCK-8评价HAPD-Cu的生物相容性；通过人脐静脉血管内皮细胞的成小管实验测定HAPD-Cu的体外促成血管活性；通过CD31组织染色评估HAPD-Cu的体内促成血管活性并建立体外大鼠伤口缺陷模型评价其实际修复效果。结果·化学定性和定量分析手段证明材料的成功制备；体外研究表明，HAPD-Cu均具有疏松多孔的内部结构，且具有优异的自愈性、可注射性和可降解性，降解周期为7d并具有突释行为，满足伤口愈合周期的需求；HAPD-Cu具有良好的生物相容性，但HAPD-Cu4因Cu²⁺浓度高而具有细胞毒性。此外，在允许的Cu²⁺浓度范围内，其体外或体内的成血管效果随着Cu²⁺浓度的增加而增强；且体外伤口模型实验表明，与对照组相比，HAPD-Cu水凝胶显著促进了伤口的愈合。结论·基于金属配位制备的HAPD-Cu水凝胶具有优异的形状可塑性，允许以微创的形式填充缺陷部位，并释放Cu²⁺以促进早期血管网络的建立，在用于临床上不规则的伤口修复方面具有良好的应用潜力。

关键词: 自愈合, 可注射水凝胶, 血管生成, 伤口愈合, 软组织修复

Abstract:

Objective ·To construct a self-healing injectable hyaluronic acid (HA)-based hydrogel (HAPD-Cu) and investigate the effects of different copper ions on the properties of the hydrogel and its vasogenic efficacy to evaluate its feasibility for clinical wound healing. Methods ·Bisphosphonated hyaluronic acid (HAPD) was prepared via a blue-light mediated thiol-ene click reaction between thiolated hyaluronic acid (HASH) and acrylated bisphosphonate (Ac-PD) in the presence of photoinitiator 2959. Then, HAPD was further interacted with Cu²⁺ through metal coordination to prepare HAPD-Cu hydrogels with different Cu²⁺ concentrations, i.e. HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4. The molecular structures of HASH, Ac-PD, HAPD and HAPD-Cu were verified with ¹HNMR and FTIR. Microscopic morphology of HAPD-Cu was observed under SEM. The shear-thinning and self-healing properties of HAPD-Cu were verified by rheometer. The Cu²⁺ release from HAPD-Cu was determined with ICP. Live-dead staining and CCK-8 assay were applied to evaluate the biocompatibility of HAPD-Cu. The in vitro vasculogenic activity of HAPD-Cu was determinedby a tubule-forming assay with human umbilical vein vascular endothelial cells and the in vivo vasculogenic activity of HAPD-Cu was assessed by CD31 tissue staining. A rat wound defect model was established in vitro to evaluate its actual repair effect. Results ·The preparation of the materials was demonstrated through chemical qualitative and quantitative analytical means. In vitro studies showed that all HAPD-Cu with a loose porous internal structure exhibited outstanding self-healing, injectability and degradability, with a one-week degradation cycle and abrupt release behavior, which can meet the needs of wound healing cycle. All HAPD-Cu showed good biocompatibility except HAPD-Cu4, due to its high Cu²⁺ concentrations. Moreover, its angiogenic effect in vitro or in vivo was enhanced with increasing Cu²⁺ concentrations within the permissible Cu²⁺ concentration range. In vitro wound model experiments also showed that the HAPD-Cu hydrogel significantly promoted wound healing compared with the control group. Conclusion ·HAPD-Cu hydrogel constructed via the metal coordination shows excellent shape plasticity, allowing the filling of defective sites in a minimally invasive form, and the release of Cu²⁺ greatly facilitates the establishment of early vascular networks, with giant potential for use in the repair of clinically irregular wounds.

Key words: self-healing, injectable hydrogel, angiogenesis, would healing, soft tissue repair

中图分类号:

R318.08

杨淑, 崔文国, 魏杰, 蔡正伟. 自愈合可注射性透明质酸水凝胶的构建及促进血管生成的研究[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1480-1492.

YANG Shu, CUI Wenguo, WEI Jie, CAI Zhengwei. Fabrication of self-healing injectable hyaluronic acid hydrogel for promoting angiogenesis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(12): 1480-1492.

图/表 10

图1 Ac-PD、HASH和HAPD的化学反应方程式Note： A. Reaction equations of Ac-PD. B. Reaction equations of HASH and HAPD.

Fig 1 Chemical reaction equations of Ac-PD, HASH, and HAPD

图2 Ac-PD、HASH、HAPD和HAPD-Cu的化学表征分析Note： A. 1HNMR of Ac-PD. B. 1HNMR of HASH. C. 1HNMR of HAPD. D. FTIR of samples (HA, HAPD, HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4).

Fig 2 Chemical characterization analysis of Ac-PD, HASH, HAPD, and HAPD-Cu

图3 HAPD-Cu的微观结构Note：A. ×150 SEM images of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4). B. ×400 SEM images of the local areas of A. C. Elemental mapping images of HAPD-Cu1 hydrogel samples. D. Semi-quantitative analysis of the pore diameter of hydrogels. ①P = 0.004, ②P = 0.000;compared with the HAPD-Cu1 group.

Fig 3 Microstructure of HAPD-Cu

图4 HAPD-Cu的自愈合和可注射性能Note：A. Self-healing photographs of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4) at various time points (0, 5, 15 min). B. Injectable photographs of samples from each group (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4). C. Self-healing mechanism of HAPD-Cu hydrogels.

Fig 4 Self-healing and injectable properties of HAPD-Cu

图5 HAPD-Cu的流变力学Note：A. Strain scan curves of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4) with amplitudes of oscillatory strain scanned from 0.1% to 800% at constant frequency (0 Hz). B. Strain cycling curves of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4): high strain (800%) for 60 s and low strain (10%) for 60 s, cycled 4 times.

Fig 5 Rheology of HAPD-Cu

图6 HAPD-Cu的溶胀、降解和离子释放Note：A. Swelling-time curves of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4). B. Equilibrium swelling rate of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4). C. Degradation-time curves of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4). D. Cumulative Cu2+ release curves of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4).

Fig 6 Swelling, degradation and ion release of HAPD-Cu

图7 HAPD-Cu的生物相容性Note：A. Images of live/dead staining of BMSCs co-cultured with extracts from each group of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4) for 1, 3 and 5 days. B. Cell viability of BMSCs co-cultured with extracts from each group of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4) for 1, 3 and 5 days (①P = 0.000, compared with the control group). C. CCK-8 results of BMSCs co-cultured with extracts from each group of samples (HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4) for 1, 3 and 5 days (①P = 0.000, compared with the control group). Controls were cells cultured in normal medium.

Fig 7 Biocompatibility of HAPD-Cu

图8 HAPD-Cu的管形成实验Note：A. Tube formation images of HUVECs co-cultured with the extracts of each group of samples (HAPD-Cu1, HAPD-Cu2 and HAPD-Cu3) for 6h. B. Junctions of HUVECs co-cultured with the extracts of each group of samples (HAPD-Cu1, HAPD-Cu2 and HAPD-Cu3) for 6h (①P = 0.018, ②P = 0.010, ③P = 0.034). C. Total length of HUVECs co-cultured with the extracts of each group of samples (HAPD-Cu1, HAPD-Cu2 and HAPD-Cu3) for 6h (①P = 0.000, ②P = 0.004, ③P = 0.001); controls were cells cultured in normal medium.

Fig 8 Tube formation experiments of HAPD-Cu

图9 HAPD-Cu的CD31免疫荧光染色Note：A. CD31 staining image of Control. B. CD31 staining image of HAPD-Cu1. C. CD31 staining image of HAPD-Cu2. D. CD31 staining image of HAPD-Cu3.

Fig 9 CD31 immunofluorescence staining and optical photographs of wound repair of HAPD-Cu

图10 HAPD-Cu的创面愈合观察实验 (n=3)Note：A. Original wound healing model. B. Wound healing at day 14.

Fig 10 Observation of diabetic wound healing of HAPD-Cu (n=3)

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自愈合可注射性透明质酸水凝胶的构建及促进血管生成的研究

Fabrication of self-healing injectable hyaluronic acid hydrogel for promoting angiogenesis

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