Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LU Liang ¹ , LIU Bin ¹ , SHANG Xifu ¹ , ZHANG Yu ^2,3 , CHEN Weijian ¹ , LIU Shuyun ³ , HUANG Jingxiang ³ , WANG Aiyuan ³ ,  GUO Quanyi ³ , LU Shibi ³

1. Department of Orthopedics, Anhui Provincial Hospital, Hefei Anhui, 230001, P.R.China;
2. Department of Orthopedics, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing Jiangsu, 210008, P.R.China;
3. Orthopedics Research Institute, General Hospital of Chinese PLA, Beijing, 100853, P.R.China;

Corresponding author：

GUO Quanyi, Email: doctorguo_301@163.com

Keywords：

Cartilage tissue engineering; acellular cartilage extracellular matrix oriented scaffold; chondrocytes; scaffold material

DOI：

10.7507/1002-1892.201710095

Video：

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ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.

Citation： LU Liang, LIU Bin, SHANG Xifu, ZHANG Yu, CHEN Weijian, LIU Shuyun, HUANG Jingxiang, WANG Aiyuan, GUO Quanyi, LU Shibi. Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(3): 291-297. doi: 10.7507/1002-1892.201710095 Copy

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15.	郑希福. 仿生PLGA/软骨ECM复合取向支架的制备及其软骨组织工程的实验研究. 大连: 大连医科大学, 2011.
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1. Langer R, Vacanti J. Advances in tissue engineering. J Pediatr Surg, 2016, 51(1): 8-12.
2. Kalunian KC. Current advances in therapies for osteoarthritis. Curr Opin Rheumatol, 2016, 28(3): 246-250.
3. Curry AS, Pensa NW, Barlow AM, et al. Taking cues from the extracellular matrix to design bone-mimetic regenerative scaffolds. Matrix Biol, 2016, 52-54: 397-412.
4. He X, Feng B, Huang C, et al. Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering. Int J Nanomedicine, 2015, 10: 2089-2099.
5. Battiston KG, Cheung JW, Jain D, et al. Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds. Biomaterials, 2014, 35(15): 4465-4476.
6. Edwards PK, Ackland T, Ebert JR. Clinical rehabilitation guidelines for matrix-induced autologous chondrocyte implantation on the tibiofemoral joint. J Orthop Sports Phys Ther, 2014, 44(2): 102-119.
7. Kon E, Filardo G, Di Martino A, et al. ACI and MACI. J Knee Surg, 2012, 25(1): 17-22.
8. Wang YC, Meng HY, Yuan XL, et al. Fabrication and in vitro evaluation of an articular cartilage extracellular-hydroxyapatite bilayered scaffold with low permeability for interface tissue engineering. BioMedical Engineering Online, 2014, 13: 80.
9. Yang Q, Peng J, Guo Q, et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials, 2008, 29(15): 2378-2387.
10. 鹿亮, 郭全义, 杨启友, 等. 异种关节软骨脱细胞基质支架的免疫反应研究.中国矫形外科杂志, 2010, 18(1): 58-62.
11. 鹿亮, 郭全义, 杨启友, 等. 猪来源关节软骨脱细胞支架的生物安全性研究. 中国医药生物技术, 2010, 3(1): 19-23.
12. Rowland CR, Colucci LA, Guilak F. Fabrication of anatomically-shaped cartilage constructs using decellularized cartilage-derived matrix scaffolds. Biomaterials, 2016, 91: 57-72.
13. Corradetti B, Taraballi F, Minardi S, et al. Chondroitin sulfate immobilized on a biomimetic scaffold modulates inflammation while driving chondrogenesis. Stem Cells Transl Med, 2016, 5(5): 670-682.
14. Smeriglio P, Lai JH, Yang F, et al. 3D hydrogel scaffolds for articular chondrocyte culture and cartilage generation. J Vis Exp, 2015, (104): 53085.
15. 郑希福. 仿生PLGA/软骨ECM复合取向支架的制备及其软骨组织工程的实验研究. 大连: 大连医科大学, 2011.
16. Rongen JJ, van Tienen TG, van Bochove B, et al. Biomaterials in search of a meniscus substitute. Biomaterials, 2014, 35(11): 3527-3540.
17. Karimi A, Navidbakhsh M, Faghihi S. Fabrication and mechanical characterization of a polyvinyl alcohol sponge for tissue engineering applications. Perfusion, 2014, 29(3): 231-237.
18. Gao Y, Liu S, Huang J, et al. The ECM-cell interaction of cartilage extracellular matrix on chondrocytes. Biomed Res Int, 2014, 2014: 648459.
19. 姚军, 卢世壁, 彭江, 等. 关节软骨细胞外基质源性软骨组织工程取向支架的制备. 中国组织工程研究与临床康复, 2009, 13(3): 432-436.
20. Haas SJ, Bauer P, Rolfs A, et al. Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells. Acta Histochem, 2000, 102(3): 273-280.

Chinese Journal of Reparative and Reconstructive Surgery

Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes

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