软骨脱细胞基质/丝素蛋白活性支架的构建及其软骨组织工程研究

doi:10.3969/j.issn.1674-8115.2023.07.001

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (7): 795-803.doi: 10.3969/j.issn.1674-8115.2023.07.001

• 生物材料与再生医学专题 •

软骨脱细胞基质/丝素蛋白活性支架的构建及其软骨组织工程研究

王千懿¹^,²(), 冉欣悦¹^,², 张沛灵², 慈政², 雷东²(), 周广东¹^,²()

^1.潍坊医学院整形外科研究所，潍坊 261042
^2.上海市组织工程研究重点实验室，上海交通大学医学院附属第九人民医院整复外科，上海 200125

收稿日期:2022-11-30 接受日期:2023-03-31 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 雷东,周广东 E-mail:wangqianyi400@163.com;370309803@qq.com;guangdongzhou@126.com
作者简介:王千懿（1996—），男，硕士生；电子信箱：wangqianyi400@163.com。
基金资助:
国家重点研发计划(2018YFC1105800);国家自然科学基金(82102211)

Construction of acellular cartilage matrix/silk fibroin scaffold and its cartilage tissue engineering study

WANG Qianyi¹^,²(), RAN Xinyue¹^,², ZHANG Peiling², CI Zheng², LEI Dong²(), ZHOU Guangdong¹^,²()

^1.Research Institute of Plastic Surgery, Weifang Medical University, Weifang 261042, China
^2.Department of Plastic and Reconstructive Surgery, Shanghai Key Lab of Tissue Engineering, Shanghai 9th People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China

Received:2022-11-30 Accepted:2023-03-31 Online:2023-07-28 Published:2023-07-28
Contact: LEI Dong,ZHOU Guangdong E-mail:wangqianyi400@163.com;370309803@qq.com;guangdongzhou@126.com
Supported by:
National Key Research and Development Program of China(2018YFC1105800);National Natural Science Foundation of China(82102211)

摘要/Abstract

摘要：

目的·利用软骨脱细胞基质（acellular cartilage matrix，ACM）与复合天然丝素蛋白（silk fibroin，SF）生物材料，构建具有双网络交联的生物活性组织工程支架，用于软骨组织再生。方法·用核酸酶消化法消化掉软骨组织中细胞相关的免疫原性成分，并将细胞外基质相关糖蛋白、胶原结构保留，用DNA、组织糖胺多糖、胶原定量试剂盒通过分光光度仪检测软骨组织脱细胞效率。将ACM和SF配置成混合溶液，通过加入乙二醇二缩水甘油醚与两者所含羟基、氨基发生亲核交联反应，冷冻干燥制成多孔仿生支架（n=5），同时以相同方法制备仅含有ACM或SF的多孔支架（n=5）。扫描电子显微镜（scanning electron microscope，SEM）观察支架微观孔隙结构，并通过力学测试对不同组支架的力学强度、弹性模量以及回弹性进行评估，以吸水率反映支架的内外物质交换能力。分离并培养兔耳软骨细胞，接种于ACM-SF支架上，SEM观察培养1、4、7 d后细胞在支架上的黏附、分布与基质分泌情况，活/死细胞双染色观察细胞活力状况，通过CCK-8检测支架的细胞毒性，并于裸鼠皮下植入细胞支架复合物，体内培养4周、8周后，行组织学检测。各组间比较采用单因素方差分析。P<0.05为差异具有统计学意义。结果·经酶消化后的软骨脱细胞基质几乎无细胞残留，并保留了软骨细胞外基质活性成分。ACM-SF制备的复合支架具有相互连通的微孔结构和良好的弹性，湿态下多次压缩后能恢复原状，ACM-SF的吸水率达到了近20倍，为细胞黏附环境提供了有效的物质交换条件。此外，该支架无生物毒性，具有促进软骨细胞增殖的能力；组织学检测显示，ACM-SF支架可在体内再生均质、典型的软骨组织。结论·ACM-SF复合多孔支架具有良好的仿生微环境，可应用于组织工程软骨再生。

关键词: 软骨脱细胞外基质, 丝素蛋白, 多孔支架, 软骨再生

Abstract:

Objective ·To construct a bioactivity tissue engineering scaffold with double network cross-linking for cartilage tissue regeneration using an acellular cartilage matrix (ACM) with a natural silk fibroin (SF) biomaterial. Methods ·The cell-associated immunogenic components were removed by nuclease digestion, and the extracellular matrix-associated glycoproteins and collagen structures were retained, The efficiency of cartilage tissue decellularization was measured by spectrophotometry by using DNA, histoglycosaminoglycan and collagen quantification kits. ACM and SF were configured into a mixed solution, and the nucleophilic cross-linking reaction with the hydroxyl and carboxyl groups contained in both was carried out by adding ethylene glycol diglycidyl ether. Then it was freeze-dried to make porous bionic scaffolds (n=5). At the same time, porous scaffolds containing only ACM or SF were prepared by the same method (n=5). The microstructure of the scaffolds was observed by scanning electron microscopy (SEM), and the mechanical strength, elastic modulus and resilience of different groups of scaffolds were evaluated by mechanical tests. The internal and external nutrient exchange capacity of the scaffolds was reacted by water absorption rate. Chondrocytes from rabbit ears were isolated, cultured, and seeded on ACM-SF scaffolds. After 1, 4, and 7 days of culture, the adhesion, distribution, and matrix secretion of the cells on the scaffolds were observed by SEM, and the viability status of the cells was determined by double-staining of live and dead cells. CCK-8 method was used to determine the cytotoxicity of the scaffolds. The cells were implanted subcutaneously in nude mice, cultured in vivo for 4 and 8 weeks, and finally removed for histological testing. Differences between groups were tested by One-Way ANOVA. Statistical significance was accepted at a value of P<0.05. Results ·After enzymatic digestion, almost no cells remained in the acellular matrix, and the active components of the extracellular matrix were retained. The composite scaffold prepared by ACM-SF has interconnected microporous structure and good elasticity, and could recover its original shape after repeated compression in the wet state. The water absorption rate of ACM-SF reached nearly 20 times, which provided an effective material exchange condition for the cell adhesion environment. Histological tests showed that the ACM-SF scaffold regenerated homogeneous, typical cartilage tissue in vivo. Conclusion ·ACM-SF composite porous scaffold has a good bionic microenvironment and can be applied to tissue engineering cartilage regeneration.

Key words: acellular cartilage matrix, silk fibroin, porous scaffold, cartilage regeneration

中图分类号:

Q813.1⁺2

王千懿, 冉欣悦, 张沛灵, 慈政, 雷东, 周广东. 软骨脱细胞基质/丝素蛋白活性支架的构建及其软骨组织工程研究[J]. 上海交通大学学报（医学版）, 2023, 43(7): 795-803.

WANG Qianyi, RAN Xinyue, ZHANG Peiling, CI Zheng, LEI Dong, ZHOU Guangdong. Construction of acellular cartilage matrix/silk fibroin scaffold and its cartilage tissue engineering study[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 795-803.

图/表 7

图1 软骨脱细胞效率评估Note： A. H-E staining of decellularized cartilage (black arrows indicate the lumen-like structures remaining after decellularization). B. DNA content. C. Total GAG content. D. Collagen content. ①P=0.000, compared with the native group.

Fig1 Evaluation of cartilage decellularization efficiency

图2 冰冻条件下混合溶液中冰晶致孔及ACM-SF双网络交联机制

Fig2 Mechanism of ice crystal pore-forming and ACM-SF cross-linking in mixed solution under freezing conditions

图3 不同组ACM-SF多孔支架的整体外观和SEM观察Note： A. Gross appearances (above) and SEM images (below) of ACM, ACM-SF and SF porous stents. B. Pore size distribution of each group of stents. ①P=0.000, compared with the ACM-SF group; ②P=0.000, compared with the SF group.

Fig3 Gross appearances and SEM images of different groups of ACM-SF porous scaffolds

图4 不同组别ACM-SF多孔支架力学分析Note： A. Stress-strain curves of ACM, ACM-SF and SF stents in the wet state. B. Comparison of elastic modulus. C. Ten cycles of compression with a maximum strain of 60% for each group of stents in the wet state. D. Comparison of dynamic compressive strength at 30% strain for the 2nd?10th cycles with the 1st compression strength. ①P=0.000, compared with the ACM-SF group; ②P=0.000, compared with the SF group.

Fig4 Mechanical analysis of ACM-SF porous scaffolds of different groups

图5 ACM-SF多孔支架的吸水能力Note： A. Water absorption efficiency of ACM-SF scaffolds. B. Water absorption of the scaffold under different compressive strains (strain=0 and 50%) for multiple cycles.

Fig5 Water absorption capacity of ACM-SF porous scaffolds

图6 ACM-SF支架的生物相容性Note： A. SEM images (above) and live-dead staining analysis (below) of chondrocytes after inoculation on ACM-SF porous scaffold for 1, 4 and 7 days (live and dead cells were stained green and red, respectively). C. Toxicity analysis of scaffold cytotoxicity. ①P=0.004, compared with the control group.

Fig6 Biocompatibility of ACM-SF scaffolds

图7 体内培养后组织学观察Note： A/B. H-E, safranin-O, and COL-Ⅱ immunohistochemical staining in regenerated cartilage tissues of ACM-SF (A) and SF (B) groups after 4 and 8 weeks of in vivo implantation.

Fig7 Histological observation after in vivo culture

参考文献 25

1	WIGGENHAUSER P S, SCHWARZ S, KOERBER L, et al. Addition of decellularized extracellular matrix of porcine nasal cartilage improves cartilage regenerative capacities of PCL-based scaffolds in vitro[J]. J Mater Sci Mater Med, 2019, 30(11): 121.
2	HUA Y J, HUO Y Y, BAI B S, et al. Fabrication of biphasic cartilage-bone integrated scaffolds based on tissue-specific photo-crosslinkable acellular matrix hydrogels[J]. Mater Today Bio, 2022, 17: 100489.
3	LIU J, WANG X Y, LU G G, et al. Bionic cartilage acellular matrix microspheres as a scaffold for engineering cartilage[J]. J Mater Chem B, 2019, 7(4): 640-650.
4	JIA L T, HUA Y J, ZENG J S, et al. Bioprinting and regeneration of auricular cartilage using a bioactive bioink based on microporous photocrosslinkable acellular cartilage matrix[J]. Bioact Mater, 2022, 16: 66-81.
5	GONG M, SUN J C, LIU G M, et al. Graphene oxide-modified 3D acellular cartilage extracellular matrix scaffold for cartilage regeneration[J]. Mater Sci Eng C Mater Biol Appl, 2021, 119: 111603.
6	LU Q, ZHU H S, ZHANG C C, et al. Silk self-assembly mechanisms and control from thermodynamics to kinetics[J]. Biomacromolecules, 2012, 13(3): 826-832.
7	MAO Z N, BI X W, YE F, et al. Controlled cryogelation and catalytic cross-linking yields highly elastic and robust silk fibroin scaffolds[J]. ACS Biomater Sci Eng, 2020, 6(8): 4512-4522.
8	MORI H, TSUKADA M. New silk protein: modification of silk protein by gene engineering for production of biomaterials[J]. Rev Mol Biotechnol, 2000, 74(2): 95-103.
9	SETAYESHMEHR M, ESFANDIARI E, RAFIEINIA M, et al. Hybrid and composite scaffolds based on extracellular matrices for cartilage tissue engineering[J]. Tissue Eng Part B Rev, 2019, 25(3): 202-224.
10	CRAPO P M, GILBERT T W, BADYLAK S F. An overview of tissue and whole organ decellularization processes[J]. Biomaterials, 2011, 32(12): 3233-3243.
11	LAI Y X, LI Y, CAO H J, et al. Osteogenic magnesium incorporated into PLGA/TCP porous scaffold by 3D printing for repairing challenging bone defect[J]. Biomaterials, 2019, 197: 207-219.
12	贾立涛, 姚琳, 刘延群, 等. 软骨脱细胞基质仿生支架体外构建组织工程软骨[J]. 组织工程与重建外科杂志, 2019, 15(4): 222-226, 244.
	JIA L T, YAO L, LIU Y Q, et al. Cartilage decellularized matrix bionic scaffold for in vitro construction of tissue-engineered cartilage[J]. Journal of Tissue Engineering and Reconstructive Surgery, 2019, 15(4): 222-226, 244.
13	GOMEZ M, WITTIG O, DIAZ-SOLANO D, et al. Mesenchymal stromal cell transplantation induces regeneration of large and full-thickness cartilage defect of the temporomandibular joint[J]. Cartilage, 2021, 13(1_suppl): 1814S-1821S.
14	MARDONES R, GIAI VIA A, PIPINO G, et al. BM-MSCs differentiated to chondrocytes for treatment of full-thickness cartilage defect of the knee[J]. J Orthop Surg Res, 2020, 15(1): 455.
15	PATTAPPA G, KRUECKEL J, SCHEWIOR R, et al. Physioxia expanded bone marrow derived mesenchymal stem cells have improved cartilage repair in an early osteoarthritic focal defect model[J]. Biology (Basel), 2020, 9(8): 230.
16	ZHENG R, DUAN H C, XUE J X, et al. The influence of gelatin/PCL ratio and 3-D construct shape of electrospun membranes on cartilage regeneration[J]. Biomaterials, 2014, 35(1): 152-164.
17	HE A J, XIA H T, XIAO K Y, et al. Cell yield, chondrogenic potential, and regenerated cartilage type of chondrocytes derived from ear, nasoseptal, and costal cartilage[J]. J Tissue Eng Regen Med, 2018, 12(4): 1123-1132.
18	ZHOU G D, JIANG H Y, YIN Z Q, et al. In vitro regeneration of patient-specific ear-shaped cartilage and its first clinical application for auricular reconstruction[J]. EBioMedicine, 2018, 28: 287-302.
19	REID J A, DWYER K D, SCHMITT P R, et al. Architected fibrous scaffolds for engineering anisotropic tissues[J]. Biofabrication, 2021, 13(4): 234.
20	JU X J, LIU X Z, ZHANG Y, et al. A photo-crosslinked proteinogenic hydrogel enabling self-recruitment of endogenous TGF-β1 for cartilage regeneration[J]. Smart Mater Med, 2022, 3: 85-93.
21	CHEN Y C, CHEN R N, JHAN H J, et al. Development and characterization of acellular extracellular matrix scaffolds from porcine menisci for use in cartilage tissue engineering[J]. Tissue Eng Part C Methods, 2015, 21(9): 971-986.
22	张沛灵, 慈政, 贾立涛, 等. 软骨脱细胞基质仿生支架的制备及其对骨髓基质干细胞成软骨分化的影响[J]. 组织工程与重建外科, 2021, 17(1): 19-24.
	ZHANG P L, CI Z, JIA L T, et al. Preparation of cartilage decellularized matrix bionic scaffold and its effect on chondrogenic differentiation of bone marrow stromal stem cells[J]. Journal of Tissue Engineering and Reconstructive Surgery, 2021, 17(1): 19-24.
23	JIA L T, ZHANG Y, YAO L, et al. Regeneration of human-ear-shaped cartilage with acellular cartilage matrix-based biomimetic scaffolds[J]. Appl Mater Today, 2020, 20: 100639.
24	AK F, OZTOPRAK Z, KARAKUTUK I, et al. Macroporous silk fibroin cryogels[J]. Biomacromolecules, 2013, 14(3): 719-727.
25	MAO Z N, BI X W, YE F, et al. The relationship between crosslinking structure and silk fibroin scaffold performance for soft tissue engineering[J]. Int J Biol Macromol, 2021, 182: 1268-1277.

[1]	刘宏强, 陆艳青, 高宇轩, 王一云, 王传东, 张晓玲. 构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究[J]. 上海交通大学学报（医学版）, 2022, 42(7): 846-857.
[2]	冯超，李喆，吕向国，等. 改良型人发角蛋白/丝素蛋白复合材料体外理化及生物学特性评估[J]. 上海交通大学学报（医学版）, 2015, 35(1): 29-.
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软骨脱细胞基质/丝素蛋白活性支架的构建及其软骨组织工程研究

Construction of acellular cartilage matrix/silk fibroin scaffold and its cartilage tissue engineering study

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