AN IN VITRO STUDY ON THREE-DIMENSIONAL CULTIVATION WITH DYNAMIC COMPRESSIVE STIMULATION FOR CARTILAGE TISSUE ENGINEERING_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGYongcheng , MENGHaoye , YUANXueling , PENGJiang , GUOQuanyi , LUShibi ,  WANGAiyuan

Key Laboratory of Chinese PLA, Institute of Orthopedics, General Hospital of Chinese PLA, Beijing, 100853, P. R. China;

Corresponding?author：

WANGAiyuan, Email: aiyuanwang301@126.com

Keywords：

Tissue engineered cartilage; Bioreactor; Mechanical load; Dynamic culture; Rabbit

DOI：

10.7507/1002-1892.20140248

Video：

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Objective To investigate the effect of three-dimensional cultivation with dynamic compressive stimulation on promotion of cartilage growth in vitro, by constructing tissue engineered cartilage with three-dimensional porous articular cartilage extracellular matrix (ECM) scaffolds laden with rabbit chondrocytes and performing mechanical stimulation by compressive stress in bioreactor. Methods Chondrocytes of healthy adult New Zealand rabbits were isolated, and passage 2 chondrocytes were seeded onto three-dimensional porous articular cartilage ECM scaffolds for 5 days pre-cultivation, and then were divided into 2 groups:Group A continued static culture as control; group B (dynamic culture condition) underwent dynamic compressive strain stimulation (compressive strain of 15%, frequence of 1 Hz) in a bioreactor. Cell viability and distribution in scaffolds were observed; the glycosaminoglycan (GAG) content, collagen content, and total DNA content were measured after 3 weeks of culturing; and elastic modulus was evaluated by mechanical test. Results Laser scanning confocal microscopy indicated that cells grew well and evenly distributed in the scaffold of group B, while poor cells growth and loss of staining in the central region of the scaffolds were observed in group A. Scanning electron microscopy showed that chondrocytes possessed good adhesion, proliferation, and growth on the scaffolds of group B; while the number of chondrocytes was significantly reduced, and cells scattered in group A. Biochemical composition analysis showed that collagen, GAG, and DNA contents of cell-scaffold constructs were (675.85±27.93) μg/mg, (621.72±26.75) μg/mg, and (16.98±3.23) μg/sample in group B, and were (438.72±6.35) μg/mg, (301.63±30.51) μg/mg, and (10.18±4.39) μg/sample in group A respectively, which were significantly higher in group B than in group A (t=18.512, P=0.000;t=17.640, P=0.000;t=2.790, P=0.024). Mechanical testing indicated that the elastic modulus of group B[(0.67±0.09) MPa] was significantly higher than that of group A[(0.49±0.16) MPa] and cell-free scaffolds[(0.43±0.12) MPa] (P < 0.05). Conclusion Mimetic compressive stress with three-dimensional dynamic conditions created in the bioreactor is superior to the ordinary static three-dimensional cultivation, it can provide the optimal environment for chondrocytes on the ECM scaffolds, which may be a good way to construct tissue engineered cartilage in vitro.

Citation： WANGYongcheng, MENGHaoye, YUANXueling, PENGJiang, GUOQuanyi, LUShibi, WANGAiyuan. AN IN VITRO STUDY ON THREE-DIMENSIONAL CULTIVATION WITH DYNAMIC COMPRESSIVE STIMULATION FOR CARTILAGE TISSUE ENGINEERING. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(9): 1145-1149. doi: 10.7507/1002-1892.20140248 Copy

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2.	DiMicco MA, Sah RL.Integrative cartilage repair:adhesive strength is correlated with collagen deposition.J Orthop Res, 2001, 19(6):1105-1112.
3.	Liu Y, Chen F, Liu W, et al.Repairing large porcine full-thickness defects of articular cartilage using autologous chondrocyte-engineered cartilage.Tissue Eng, 2002, 8(4):709-721.
4.	Harris JD, Siston RA, Pan X, et al.Autologous chondrocyte implantation:a systematic review.J Bone Joint Surg (Am), 2010, 92(12):2220-2233.
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7.	楊強, 彭江, 盧世璧, 等.新型脫細胞軟骨基質三維多孔支架的制備.中國修復重建外科雜志, 2008, 22(3):359-363.
8.	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.
9.	Kang H, Peng J, Lu S, et al.In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds.J Tissue Eng Regen Med, 2014, 8(6):442-453.
10.	Park S, Hung CT, Ateshian GA.Mechanical response of bovine articular cartilage under dynamic unconfined compression loading at physiological stress levels.Osteoarthritis Cartilage, 2004, 12(1):65-73.
11.	Appelman TP, Mizrahi J, Elisseeff JH, et al.The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation.Biomaterials, 2009, 30(4):518-525.
12.	Kock L, van Donkelaar CC, Ito K.Tissue engineering of functional articular cartilage:the current status.Cell Tissue Res, 2012, 347(3):613-627.
13.	Kisiday JD, Jin M, DiMicco MA, et al.Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.J Biomech, 2004, 37(5):595-604.
14.	Kino-Oka M, Maeda Y, Yamamoto T, et al.A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.J Biosci Bioeng, 2005, 99(3):197-207.
15.	Pelttari K, Wixmerten A, Martin I.Do we really need cartilage tissue engineering? Swiss Med Wkly, 2009, 139(41-42):602-609.
16.	Xie J, Han ZY, Matsuda T.Mechanical compressive loading stimulates the activity of proximal region of human COL2A1 gene promoter in transfected chondrocytes.Biochem Biophys Res Commun, 2006, 344(4):1192-1199.
17.	Wang PY, Chow HH, Tsai WB, et al.Modulation of gene expression of rabbit chondrocytes by dynamic compression in polyurethane scaffolds with collagen gel encapsulation.J Biomater Appl, 2009, 23(4):347-366.
18.	Mauck RL, Byers BA, Yuan X, et al.Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.Biomech Model Mechanobiol, 2007, 6(1-2):113-125.
19.	Gemmiti CV, Guldberg RE.Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue.Biotechnol Bioeng, 2009, 104(4):809-820.
20.	Chen T, Buckley M, Cohen I, et al.Insights into interstitial flow, shear stress, and mass transport effects on ECM heterogeneity in bioreactor-cultivated engineered cartilage hydrogels.Biomech Model Mechanobiol, 2012, 11(5):689-702.

1. Schinhan M, Gruber M, Vavken P, et al.Critical-size defect induces unicompartmental osteoarthritis in a stable ovine knee.J Orthop Res, 2012, 30(2):214-220.
2. DiMicco MA, Sah RL.Integrative cartilage repair:adhesive strength is correlated with collagen deposition.J Orthop Res, 2001, 19(6):1105-1112.
3. Liu Y, Chen F, Liu W, et al.Repairing large porcine full-thickness defects of articular cartilage using autologous chondrocyte-engineered cartilage.Tissue Eng, 2002, 8(4):709-721.
4. Harris JD, Siston RA, Pan X, et al.Autologous chondrocyte implantation:a systematic review.J Bone Joint Surg (Am), 2010, 92(12):2220-2233.
5. 葉鋼, 章方彪, 史宏燦.生物反應器生物物理因素在軟骨組織工程中的研究進展.中國修復重建外科雜志, 2013, 27(7):810-813.
6. Plunkett N, O'Brien FJ.Bioreactors in tissue engineering.Technol Health Care, 2011, 19(1):55-69.
7. 楊強, 彭江, 盧世璧, 等.新型脫細胞軟骨基質三維多孔支架的制備.中國修復重建外科雜志, 2008, 22(3):359-363.
8. 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.
9. Kang H, Peng J, Lu S, et al.In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds.J Tissue Eng Regen Med, 2014, 8(6):442-453.
10. Park S, Hung CT, Ateshian GA.Mechanical response of bovine articular cartilage under dynamic unconfined compression loading at physiological stress levels.Osteoarthritis Cartilage, 2004, 12(1):65-73.
11. Appelman TP, Mizrahi J, Elisseeff JH, et al.The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation.Biomaterials, 2009, 30(4):518-525.
12. Kock L, van Donkelaar CC, Ito K.Tissue engineering of functional articular cartilage:the current status.Cell Tissue Res, 2012, 347(3):613-627.
13. Kisiday JD, Jin M, DiMicco MA, et al.Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.J Biomech, 2004, 37(5):595-604.
14. Kino-Oka M, Maeda Y, Yamamoto T, et al.A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.J Biosci Bioeng, 2005, 99(3):197-207.
15. Pelttari K, Wixmerten A, Martin I.Do we really need cartilage tissue engineering? Swiss Med Wkly, 2009, 139(41-42):602-609.
16. Xie J, Han ZY, Matsuda T.Mechanical compressive loading stimulates the activity of proximal region of human COL2A1 gene promoter in transfected chondrocytes.Biochem Biophys Res Commun, 2006, 344(4):1192-1199.
17. Wang PY, Chow HH, Tsai WB, et al.Modulation of gene expression of rabbit chondrocytes by dynamic compression in polyurethane scaffolds with collagen gel encapsulation.J Biomater Appl, 2009, 23(4):347-366.
18. Mauck RL, Byers BA, Yuan X, et al.Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.Biomech Model Mechanobiol, 2007, 6(1-2):113-125.
19. Gemmiti CV, Guldberg RE.Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue.Biotechnol Bioeng, 2009, 104(4):809-820.
20. Chen T, Buckley M, Cohen I, et al.Insights into interstitial flow, shear stress, and mass transport effects on ECM heterogeneity in bioreactor-cultivated engineered cartilage hydrogels.Biomech Model Mechanobiol, 2012, 11(5):689-702.

Chinese Journal of Reparative and Reconstructive Surgery

AN IN VITRO STUDY ON THREE-DIMENSIONAL CULTIVATION WITH DYNAMIC COMPRESSIVE STIMULATION FOR CARTILAGE TISSUE ENGINEERING

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