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Chitosan-alginate Gel Modified Poly (L-Lactic-co-ε-Caprolactone) (PLCL) as a Scaffold for Cartilage Tissue Engineering

변형된 키토산 알지네이트 겔 poly (L-Lactic-co-ε-Caprolactone) 지지체의 연골 조직 재생 평가

  • Sutradhar, Bibek Chandra (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Hwang, Yawon (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Choi, Seokhwa (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Kim, Gonhyung (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University)
  • ;
  • 황야원 (충북대학교 수의과대학 수의외과학 교실) ;
  • 최석화 (충북대학교 수의과대학 수의외과학 교실) ;
  • 김근형 (충북대학교 수의과대학 수의외과학 교실)
  • Accepted : 2015.04.09
  • Published : 2015.06.30

Abstract

This study was designed in the fabricated poly (L-Lactic-co-${\varepsilon}$-Caprolactone) (PLCL) scaffold using chitosan-alginate hydrogel, which would be more suitable to maintain the biological and physiological functions continuing three dimensional spatial organizations for chondrocytes. As a scaffold, hydrogels alone is weak at endure complex loading within the body. In this study, we made cell hybrid scaffold constructs with poly (L-Lactic-co-${\varepsilon}$-Caprolactone) (PLCL) scaffold and hydrogels to make a three-dimensional composition of cells and extracellular matrix, which would be a mimic of a native cartilage. Using a particle leaching technique with NaCl, we fabricated a highly-elastic scaffold from PLCL with 85% porosity and $300-500{\mu}m$ pore size. A mixture of bovine chondrocytes and chitosan-alginate gel was seeded and compared with alginate as a control on the PLCL scaffold. The cell maturation, proliferation, extracellular matrix synthesis, glycosaminoglycans (sGAG) production and collagen type-II expressions were better in chondrocytes seeded in chitosan-alginate hydrogel than in alginate only. These results indicate that chondrocytes with chitosan-alginate gel on PLCL scaffolds provide an appropriate biomimetic environment for cell proliferation and matrix synthesis, which could successfully be used for cartilage repair and regeneration.

본 연구는 키토산 알지네이트 수화겔을 사용하여 제작된 연골세포의 3차원 구조를 유지하며 생물학적, 생리학적인 기능을 유지하는데 적합한 poly (L-Lactic-co-${\varepsilon}$-Caprolactone) (PLCL) 지지체의 효과에 대한 연구이다. 체내에서 수화겔은 단독으로 지지체 역할을 하기에는 부하를 견디기에 약하다. 이에 본 연구에서는 연골세포와 유사한 세포, 세포외 기질의 3차원적 구성을 만들기 위해 PLCL 지지체와 수화겔을 사용하여 합성 지지체를 제작하였다. 염화나트륨을 사용한 입자 침출 기법으로 85%의 다공성, $300-500{\mu}m$ 크기의 구멍을 가진 탄성력 높은 지지체를 제작하였다. 소의 연골세포와 키토산 알지네이트 겔 혼합물이 PLCL 지지체에 적용되었고 대조군의 알지네이트와 비교 연구하였다. 키토산 알지네이트 수화겔과 연골세포가 혼합된 경우에 알지네이트 단독 사용에 비해 세포 성숙, 증식, 세포외 기질의 합성, sGAG 생성과 II 형 콜라겐의 발현 등의 효과가 좋은 것으로 확인되었다. 본 연구 결과를 통해 PLCL 지지체에 연골세포와 키토산 알지네이트 겔 혼합물을 적용할 경우 세포 증식과 기질의 합성에 적합한 환경을 만들 수 있으며 연골의 복구와 재생에 효과적으로 사용될 수 있을 것으로 기대된다.

Keywords

References

  1. Abarrategi A, Lopiz-Morales Y, Ramos V, Civantos A, Lopez- Duran L, Marco F, Lopez-Lacomba JL. Chitosan scaffolds for osteochondral tissue regeneration. J Biomed Mater Res A 2010; 95: 1132-1141.
  2. Almqvist KF, Wang L, Wang J, Baeten D, Cornelissen M, Verdonk R, Veys EM, Verbruggen G. Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks. Ann Rheum Dis 2001; 60: 781-790. https://doi.org/10.1136/ard.60.8.781
  3. Amaral IF, Sampaio P, Barbosa MA. Three-dimensional culture of human osteoblastic cells in chitosan sponges: the effect of the degree of acetylation. J Biomed Mater Res A 2006; 76: 335-346.
  4. Ayala R, Zhang C, Yang D, Hwang Y, Aung A, Shroff SS, Arce FT, Lal R, Arya G, Varghese S. Engineering the cellmaterial interface for controlling stem cell adhesion, migration, and differentiation. Biomaterials 2011; 32: 3700-3711. https://doi.org/10.1016/j.biomaterials.2011.02.004
  5. Bentley G, Biant LC, Vijayan S, Macmull S, Skinner JA, Carrington RW. Minimum ten-year results of a prospective randomised study of autologous chondrocyte implantation versus mosaicplasty for symptomatic articular cartilage lesions of the knee. J Bone Joint Surg Br 2012; 94: 504-509.
  6. Beekman B, Verzijl N, Bank RA, von der Mark K, TeKoppele JM. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell matrix interaction. Exp Cell Res 1997; 237: 135-141. https://doi.org/10.1006/excr.1997.3771
  7. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C, Freisinger P. Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res 1994; 212: 97-104. https://doi.org/10.1006/excr.1994.1123
  8. Boyan BD, Bonewald LF, Paschalis EP, Lohmann CH, Rosser J, Cochran DL, Dean DD, Schwartz Z, Boskey AL. Osteoblast-mediated mineral deposition in culture is dependent on surface microtopography. Calcif Tissue Int 2002; 71: 519-529. https://doi.org/10.1007/s00223-001-1114-y
  9. Cao Y, Vacanti JP, Paige KT, Upton J, Vacanti CA. Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg 1997; 100: 297-302. https://doi.org/10.1097/00006534-199708000-00001
  10. Chang SH, Hsiao YW, Lin HY. Low-frequency electromagnetic field exposure accelerates chondrocytic phenotype expression on chitosan substrate. Orthopedics 2011; 34: 20-26.
  11. Chatelet C, Damour O, Domard A. Influence of the degree of acetylation on some biological properties of chitosan films. Biomaterials 2001; 22: 261-268. https://doi.org/10.1016/S0142-9612(00)00183-6
  12. Chouzouri G, Xanthos M. In vitro bioactivity and degradation of polycaprolactone composites containing silicate fillers. Acta Biomater 2007; 3: 745-756. https://doi.org/10.1016/j.actbio.2007.01.005
  13. Dang JM, Sun DD, Shin-Ya Y, Sieber AN, Kostuik JP, Leong KW. Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells. Biomaterials 2006; 27: 406-418. https://doi.org/10.1016/j.biomaterials.2005.07.033
  14. Dare EV, Griffith M, Poitras P, Kaupp JA, Waldman SD, Carlsson DJ, Dervin G, Mayoux C, Hincke MT. Genipin cross-linked fibrin hydrogels for in vitro human articular cartilage tissue-engineered regeneration. Cells Tissues Organs 2009; 190: 313-325. https://doi.org/10.1159/000209230
  15. Dhiman HK, Ray AR, Panda AK. Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines. Biomaterials 2004; 25: 5147-5154. https://doi.org/10.1016/j.biomaterials.2003.12.025
  16. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science 2005; 310: 1139-1143. https://doi.org/10.1126/science.1116995
  17. Docherty S, Engstrand T. Bone morphogenetic proteins in cranial reconstructions: clinical evaluation of heparin-chitosan as a carrier for BMP-2. Plast Reconstr Surg 2009; 123: 192-193. https://doi.org/10.1097/PRS.0b013e3181934812
  18. Donati I, Stredanska S, Silvestrini G, Vetere A, Marcon P, Marsich E, Mozetic P, Gamini A, Paoletti S, Vittur F. The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan. Biomaterials 2005; 26: 987-998. https://doi.org/10.1016/j.biomaterials.2004.04.015
  19. Hsu SH, Whu SW, Hsieh SC, Tsai CL, Chen DC, Tan TS. Evaluation of chitosan-alginate-hyaluronate complexes modified by an RGD-containing protein as tissue-engineering scaffolds for cartilage regeneration. Artif Organs 2004; 28: 693-703. https://doi.org/10.1111/j.1525-1594.2004.00046.x
  20. Jung Y, Kim SH, Kim YH. The effect of hybridization of hydrogels and poly (L-lactide-co-epsilon-caprolactone) scaffolds on cartilage tissue engineering. J Biomater Sci Polym Ed 2010; 21: 581-592. https://doi.org/10.1163/156856209X430579
  21. Kang Y, Yang J, Khan S, Anissian L, Ameer GA. A new biodegradable polyester elastomer for cartilage tissue engineering. J Biomed Mater Res A 2006; 77: 331-339.
  22. Khetan S and Burdick J. Cellular encapsulation in 3D hydrogels for tissue engineering. J Vis Exp 2009; 26: 32.
  23. Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC, Cho CS. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv 2008; 26: 1-21. https://doi.org/10.1016/j.biotechadv.2007.07.009
  24. Lahiji A, Sohrabi A, Hungerford DS, Frondoza CG. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J Biomed Mater Res 2000; 51: 586-595. https://doi.org/10.1002/1097-4636(20000915)51:4<586::AID-JBM6>3.0.CO;2-S
  25. Li XM, Peng WJ, Wu H, Kacher D, Xia LM, Ai F, Li F, Xiong W. MRI findings in injured articular cartilage of the knee correlated with surgical findings. Chin Med J 2009; 122: 2624-2630.
  26. Lu JX, Prudhommeaux F, Meunier A, Sedel L, Guillemin G. Effects of chitosan on rat knee cartilages. Biomaterials 1999; 20: 1937-1944. https://doi.org/10.1016/S0142-9612(99)00097-6
  27. Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001; 22: 331-336. https://doi.org/10.1016/S0142-9612(00)00188-5
  28. Mandl EW, van der Veen SW, Verhaar JA, van Osch GJ. Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity. Tissue Eng 2004; 10: 109-118. https://doi.org/10.1089/107632704322791754
  29. Marler JJ, Guha A, Rowley J, Koka R, Mooney D, Upton J, Vacanti JP. Soft-tissue augmentation with injectable alginate and syngeneic fibroblasts. Plast Reconstr Surg 2000; 105: 2049-2058. https://doi.org/10.1097/00006534-200005000-00020
  30. Mei Y, Saha K, Bogatyrev SR, Yang J, Hook AL, Kalcioglu ZI, Cho SW, Mitalipova M, Pyzocha N, Rojas F, Van Vliet KJ, Davies MC, Alexander MR, Langer R, Jaenisch R, Anderson DG. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat Mater 2010; 9: 768-778. https://doi.org/10.1038/nmat2812
  31. Mobasheri A, Barrett-Jolley R, Carter SD, Martin-Vasallo P, Schulze-Tanzil G, Shakibaei M. 2005. Functional roles of mechanosensitive ion channels, ss1 integrins and kinase cascades in chondrocyte mechanotransduction. In: Kamkin A, Kiseleva I, editors. Mechanosensitivity in cells and tissues. Moscow: Academia 2005: 1-21.
  32. Montembault A, Tahiri K, Korwin-Zmijowska C, Chevalier X, Corvol MT, Domard A. A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering. Biochimie 2006; 88: 551-564. https://doi.org/10.1016/j.biochi.2006.03.002
  33. Nettles DL, Elder SH, Gilbert JA. Potential use of chitosan as a cell scaffold material for cartilage tissue engineering. Tissue Eng 2002; 8: 1009-1016. https://doi.org/10.1089/107632702320934100
  34. Perka C, Schultz O, Spitzer RS, Lindenhayn K. The influence of transforming growth factor beta1 on mesenchymal cell repair of full-thickness cartilage defects. J Biomed Mater Res 2000; 52: 543-552. https://doi.org/10.1002/1097-4636(20001205)52:3<543::AID-JBM13>3.0.CO;2-2
  35. Poon YF, Cao Y, Liu Y, Chan V, Chan-Park MB. Hydrogels based on dual curable chitosan-graft-polyethylene glycolgraft- methacrylate: application to layer-by-layer cell encapsulation. ACS Appl Mater Interfaces 2010; 2: 2012-2025. https://doi.org/10.1021/am1002876
  36. Prasad BR, Brook MA, Smith T, Zhao S, Chen Y, Sheardown H, D'Souza R, Rochev Y. Controlling cellular activity by manipulating silicone surface roughness. Colloids Surf B Biointerfaces 2010; 78: 237-242. https://doi.org/10.1016/j.colsurfb.2010.03.006
  37. Rui YF, Du L, Wang Y, Lui PP, Tang TT, Chan KM, Dai KR. Bone morphogenetic protein 2 promotes transforming growth factor beta3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells. Chin Med J 2010; 123: 3040-3048.
  38. Shi C, Zhu Y, Ran X, Wang M, Su Y, Cheng T. Therapeutic potential of chitosan and its derivatives in regenerative medicine. J Surg Res 2006; 133: 185-192. https://doi.org/10.1016/j.jss.2005.12.013
  39. Shim IK, Suh WH, Lee SY, Lee SH, Heo SJ, Lee MC, Lee SJ. Chitosan nano-/microfibrous double-layered membrane with rolled-up three-dimensional structures for chondrocyte cultivation. J Biomed Mater Res 2009; 90: 595-602.
  40. Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Noth U. Major biological obstacles for persistent cellbased regeneration of articular cartilage. Arthritis Res Ther 2007; 9: 213. https://doi.org/10.1186/ar2195
  41. Sutradhar BC, Hong G, Ge Z, Kim G. Coculture of bovine chondrocytes with demineralized bone matrix in chitosanalginate beads enhances chondrogenesis. J Med Biol Eng 2012; 33: 518-525.
  42. Swieszkowski W, Tuan BH, Kurzydlowski KJ, Hutmacher DW. Repair and regeneration of osteochondral defects in the articular joints. Biomol Eng 2007; 24: 489-495. https://doi.org/10.1016/j.bioeng.2007.07.014
  43. Tan H, Chu CR, Payne KA, Marra KG. Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials 2009; 30: 2499-2506. https://doi.org/10.1016/j.biomaterials.2008.12.080
  44. Widuchowski W, Lukasik P, Kwiatkowski G, Faltus R, Szyluk K, Widuchowski J, Koczy B. Isolated full thickness chondral injuries. Prevalance and outcome of treatment. A retrospective study of 5233 knee arthroscopies. Acta Chir Orthop Traumatol Cech 2008; 75: 382-386.
  45. Xie J, Ihara M, Jung Y, Kwon IK, Kim SH, Kim YH, Matsuda T. Mechano-active scaffold design based on microporous poly (L-lactide-co-epsilon-caprolactone) for articular cartilage tissue engineering: dependence of porosity on compression force-applied mechanical behaviors. Tissue Eng 2006; 12: 449-458. https://doi.org/10.1089/ten.2006.12.449