CHONDROGENESIS OF PASSAGED CHONDROCYTES INDUCED BY DIFFERENT DYNAMIC LOADS IN BIOREACTOR_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Ning , CHEN Jiying , ZHANG Guoqiang , CHAI Wei.

Department of Orthopedics, General Hospital of Chinese PLA, Beijing, 100853, P.R.China. Corresponding author: CHEN Jiying, E-mail: chenjiying301@yahoo.com.cn;

Corresponding author：

Keywords：

Cartilage tissue engineering; Passaged chondrocytes; Biological scaffold; Bioreactor

DOI：

10.7507/1002-1892.20130174

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Objective To investigate the effect of dynamic compression and rotation motion on chondrogenesis of the 3rd passage cell-loaded three-dimensional scaffold in a joint-specific bioreactor in vitro so as to provide theoretical basis of the autologous chondrocyte transplantation in clinical practice. Methods Primary chondrocytes were isolated and cultured from the knee cartilage of 3-4 months old calves. The 3rd passage cells were seeded onto fibrin-polyurethane scaffolds (8 mm × 4 mm). Experiment included 5 groups: unloaded culture for 2 weeks (group A), direct load for 2 weeks (group B), unloaded culture for 4 weeks (group C), direct load for 4 weeks (group D), and unload for 2 weeks followed by load for 2 weeks (group E). The cell-scaffold was incubated in incubator (unload) or in a joint-specific bioreactor (load culture). At different time points, the samples were collected for DNA and glycosaminoglycan (GAG) quantification detect; mRNA expressions of chondrogenic marker genes such as collagen type I, collagen type II, Aggrecan, cartilage oligomeric matrix protein (COMP), and superficial zone protein (SZP) were detected by real-time quantitative PCR; and histology observations were done by toluidine blue staining and immunohistochemistry staining. Results No significant difference was found in DNA content, GAG content, and the ratio of GAG to DNA among 5 groups (P gt; 0.05). After load, there was a large number of GAG in the medium, and the GAG significantly increased with time (P lt; 0.05). The mRNA expression of collagen type I showed no significant difference among 5 groups (P gt; 0.05). The mRNA expression of collagen type II in group B was significantly increased when compared with group A (P lt; 0.01), and groups D and E were significantly higher than group C (P lt; 0.01); the mRNA expression of Aggrecan in groups D and E were significantly increased when compared with group C (P lt; 0.01), and group E was significantly higher than group D (P lt; 0.01); the mRNA expression of COMP in group B was significantly increased when compared with group A (P lt; 0.01), and group E was significantly higher than group C (P lt; 0.01); and the mRNA expression of SZP in group E was significantly increased when compared with groups C and D (P lt; 0.05). The toluidine blue staining and immunohistochemistry staining displayed that synthesis and secretion of GAG could be enhanced after load; no intensity changes of collagen type I and collagen type II were observed, but intensity enhancement of Agrrecan was seen in groups D and E. Conclusion Different dynamic loads can promote chondrogenesis of the 3rd passage chondrocytes. Culture by load after unload may be the best culture for chondrogenesis, while the 3rd passage chondrocytes induced by mechanical load hold less capacity of chondrogenesis.

Citation： WANG Ning,CHEN Jiying,ZHANG Guoqiang,CHAI Wei.. CHONDROGENESIS OF PASSAGED CHONDROCYTES INDUCED BY DIFFERENT DYNAMIC LOADS IN BIOREACTOR. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(7): 786-792. doi: 10.7507/1002-1892.20130174 Copy

1.	Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
2.	Marlovits S, Zeller P, Singer P, et al. Cartilage repair: generations of autologous chondrocyte transplantation. Eur J Radiol, 2006, 57(1): 24-31.
3.	Peterson L, Vasiliadis HS, Brittberg M, et al. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med, 2010, 38(6): 1117-1124.
4.	Niemeyer P, Pestka JM, Salzmann GM, et al. Influence of cell quality on clinical outcome after autologous chondrocyte implantation. Am J Sports Med, 2012, 40(3): 556-561.
5.	Brun P, Dickinson SC, Zavan B, et al. Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation. Arthritis Res Ther, 2008, 10(6): R132.
6.	Kon E, Filardo G, Di Matteo B, et al. Matrix assisted autologous chondrocyte transplantation for cartilage treatment: A systematic review. Bone Joint Res, 2013, 2(2): 18-25.
7.	Brix MO, Stelzeneder D, Trattnig S, et al. Cartilage repair of the knee with Hyalograft C: ® magnetic resonance imaging assessment of the glycosaminoglycan content at midterm. Int Orthop, 2013, 37(1): 39-43.
8.	Grad S, Gogolewski S, Alini M, et al. Effects of simple and complex motion patterns on gene expres sion of chondrocytes seeded in 3-D scaffolds. Tissue Eng, 2006, 12(11): 3171-3179.
9.	Grad S, Eglin D, Alini M, et al. Physical stimulation of chondrogenic cells in vitro: a review. Clin Orthop Relat Res, 2011, 469(10): 2764-2772.
10.	Crawford DC, Heveran CM, Cannon WD, et al. An autologous cartilage tissue implant NeoCart for treatment of grade III chondral injury to the distal femur: prospective clinical safety trial at 2 years. Am J Sports Med, 2009, 37(7): 1334-1343.
11.	Lee CR, Grad S, Gorna K, et al. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng, 2005, 11(9-10): 1562-1573.
12.	Wimmer MA, Grad S, Kaup T, et al. Tribology approach to the engineering and study of articular cartilage. Tissue Eng, 2004, 10(9-10): 1436-1445.
13.	Wimmer MA, Alini M, Grad S. The effect of sliding velocity on chondrocytes activity in 3D scaffolds. J Biomech, 2009, 42(4): 424-449.
14.	Salzmann GM, Nuernberger B, Schmitz P, et al. Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis. Tissue Eng Part A, 2009, 15(9): 2513-2524.
15.	Wysocka A, Mann K, Bursig H, et al. Chondrocyte suspension in fibrin glue. Cell Tissue Bank, 2010, 11(2): 209-215.
16.	Verdonk R, Verdonk P, Huysse W, et al. Tissue ingrowth after implantation of a novel, biodegradable polyurethane scaffold for treatment of partial meniscal lesions. Am J Sports Med, 2011, 39(4): 774-782.
17.	Oldershaw RA. Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. Int J Exp Pathol, 2012, 93(6): 389-400.
18.	Das RH, Jahr H, Verhaar JA, et al. In vitro expansion affects the response of chondrocytes to mechanical stimulation. Osteoarthritis Cartilage, 2008, 16(3): 385-391.
19.	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): 113-125.
20.	Xie J, Han Z, Kim SH, et al. Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly (L-lactide-co-epsilon-caprolactone) scaffold. Tissue Eng, 2007, 13(1): 29-40.
21.	Fehrenbacher A, Steck E, Roth W, et al. Long-term mechanical loading of chondrocyte-chitosan biocomposites in vitro enhanced their proteoglycan and collagen content. Biorheology, 2006, 43(6): 709-720.
22.	Hung BP, Hutton DL, Grayson WL. Mechanical control of tissue-engineered bone. Stem Cell Res Ther, 2013, 4(1): 10.

1. Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
2. Marlovits S, Zeller P, Singer P, et al. Cartilage repair: generations of autologous chondrocyte transplantation. Eur J Radiol, 2006, 57(1): 24-31.
3. Peterson L, Vasiliadis HS, Brittberg M, et al. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med, 2010, 38(6): 1117-1124.
4. Niemeyer P, Pestka JM, Salzmann GM, et al. Influence of cell quality on clinical outcome after autologous chondrocyte implantation. Am J Sports Med, 2012, 40(3): 556-561.
5. Brun P, Dickinson SC, Zavan B, et al. Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation. Arthritis Res Ther, 2008, 10(6): R132.
6. Kon E, Filardo G, Di Matteo B, et al. Matrix assisted autologous chondrocyte transplantation for cartilage treatment: A systematic review. Bone Joint Res, 2013, 2(2): 18-25.
7. Brix MO, Stelzeneder D, Trattnig S, et al. Cartilage repair of the knee with Hyalograft C: ® magnetic resonance imaging assessment of the glycosaminoglycan content at midterm. Int Orthop, 2013, 37(1): 39-43.
8. Grad S, Gogolewski S, Alini M, et al. Effects of simple and complex motion patterns on gene expres sion of chondrocytes seeded in 3-D scaffolds. Tissue Eng, 2006, 12(11): 3171-3179.
9. Grad S, Eglin D, Alini M, et al. Physical stimulation of chondrogenic cells in vitro: a review. Clin Orthop Relat Res, 2011, 469(10): 2764-2772.
10. Crawford DC, Heveran CM, Cannon WD, et al. An autologous cartilage tissue implant NeoCart for treatment of grade III chondral injury to the distal femur: prospective clinical safety trial at 2 years. Am J Sports Med, 2009, 37(7): 1334-1343.
11. Lee CR, Grad S, Gorna K, et al. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng, 2005, 11(9-10): 1562-1573.
12. Wimmer MA, Grad S, Kaup T, et al. Tribology approach to the engineering and study of articular cartilage. Tissue Eng, 2004, 10(9-10): 1436-1445.
13. Wimmer MA, Alini M, Grad S. The effect of sliding velocity on chondrocytes activity in 3D scaffolds. J Biomech, 2009, 42(4): 424-449.
14. Salzmann GM, Nuernberger B, Schmitz P, et al. Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis. Tissue Eng Part A, 2009, 15(9): 2513-2524.
15. Wysocka A, Mann K, Bursig H, et al. Chondrocyte suspension in fibrin glue. Cell Tissue Bank, 2010, 11(2): 209-215.
16. Verdonk R, Verdonk P, Huysse W, et al. Tissue ingrowth after implantation of a novel, biodegradable polyurethane scaffold for treatment of partial meniscal lesions. Am J Sports Med, 2011, 39(4): 774-782.
17. Oldershaw RA. Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. Int J Exp Pathol, 2012, 93(6): 389-400.
18. Das RH, Jahr H, Verhaar JA, et al. In vitro expansion affects the response of chondrocytes to mechanical stimulation. Osteoarthritis Cartilage, 2008, 16(3): 385-391.
19. 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): 113-125.
20. Xie J, Han Z, Kim SH, et al. Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly (L-lactide-co-epsilon-caprolactone) scaffold. Tissue Eng, 2007, 13(1): 29-40.
21. Fehrenbacher A, Steck E, Roth W, et al. Long-term mechanical loading of chondrocyte-chitosan biocomposites in vitro enhanced their proteoglycan and collagen content. Biorheology, 2006, 43(6): 709-720.
22. Hung BP, Hutton DL, Grayson WL. Mechanical control of tissue-engineered bone. Stem Cell Res Ther, 2013, 4(1): 10.

Chinese Journal of Reparative and Reconstructive Surgery

CHONDROGENESIS OF PASSAGED CHONDROCYTES INDUCED BY DIFFERENT DYNAMIC LOADS IN BIOREACTOR

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