İnsülin, transferin ve selenöz asit preparatı kondrosit hücre sayısında artışa yol açar mı?

Year 2014, , 313 - 319, 30.05.2014

Alper Gokce İbrahim Yilmaz Nevzat Selim Gokay Levent Can Cigdem Gokce

https://doi.org/10.3944/AOTT.2014.2635

Abstract

Amaç:Bu çalışmanın amacı insülin, insan transferin, selenöz asit (ITS) preparatının kondrosit çoğalması ve morfolojisi üzerine olumlu etkisi olduğu varsayımını test etmek ve bu eklenen maddelerin farklı hücre kültürü ortamlarındaki etkilerini biyokimyasal ve histolojik olarak araştırmak idi.

Çalışma planı: Gonartroz tanısı koyulan 57 yaşındaki bayan hastanın kıkırdak dokusundan insan kaynaklı kıkırdak hücreleri (human cartilage-derived cell, hCDC) ayrıştırıldı. Bu doku örnekleri DMEM (Dulbecco’s modified Eagle’s medium) ve RPMI-1640 içerisinde kültüre alındı. Hücrelerin kondrojenik aktiviteleri gözlendi. Hücreler, yapılan pasajlamalar sonrası, 6. hafta sonunda dört gruba ayrıldılar. 14. gün sonunda sayıları artan hücrelerin x4, x10, x20 ve x40 büyütmede mikro fotoğrafları çekildi ve invert mikroskop altında incelendiler. Yaşayabilen hücre sayısı 1. ve 14. günde MTS-ELISA hücre proliferasyonu testi ile belirlendi.

Bulgular: ITS premiks ilave edilmeyen DMEM ve ITS premiks ilave edilen RPMI-1640 kültür ortamlarında kondrojenik hücrelerin sayısının arttığı olduğu görüldü. Bu çalışmada kondrositlerin canlılığı ve sayı artışının sadece DMEM içeren Grup 3’te daha fazla olduğu gözlemlendi.

Çıkarımlar: Kültür ortamı içeriklerinin osteokondral dokudan elde edilen hücre kültürlerindeki kondrosit hücre sayısının artmasında önemli rol oynadığını düşünüyoruz.

Keywords

DMEM; hücre kültürü; ITS premiks solüsyon; kondrojenik farklılaşma; RPMI-1640.

Does insulin, transferrin and selenous acid preparation effect chondrocyte proliferation?

Abstract

Objective:The aim of this study was to test the hypothesis that insulin, human transferrin, and selenous acid (ITS) preparation have positive effects on chondrocyte proliferation and morphology and investigate the biochemical and histological effects of these additive substances in different cell culture media.

Methods: Human cartilage-derived cells (hCDCs) were isolated from the cartilage tissue of a 57-year-old woman diagnosed with gonarthrosis. Tissue samples were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640. The cells’ chondrogenic activities were observed. After serial passagings, cells were divided into 4 groups at the end of the 6th week. On the 14th day, proliferated cells were examined using an inverted microscope with x4, x10, x20 and x40 magnification and microphotographs were taken. Living cell quantity was determined on the first and 14th days using MTS-ELISA cell proliferation assay.

Results: DMEM (without adding ITS premix solution) and RPMI-1640 containing ITS premix solution provide proliferation of the chondrogenic cells. The proliferation and viability of chondrocytes were revealed in this study in the 3rd group (DMEM solution without additives).

Conclusion: It is suggested that the culture medium ingredients play crucial roles on chondrogenic proliferation in osteochondral tissue cultures.

Keywords

Cell culture, chondrogenic differentiation, DMEM, ITS premix solution, RPMI-1640

References

• Bilir A, Ceyhan T, Altinoz MA, Guneri AD, Bayrak I, Altug T. Culturability of osteoblast cells extracted from mature and fetal BALB/c mice calvaria. [Article in Turkish] Acta Orthop Traumatol Turc 2000;34:389-95.
• Bellows CG, Ciaccia A, Heersche JN. Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone. Bone 1998;23:119-25.
• Wang Q, Zhong S, Ouyang J, Jiang L, Zhang Z, Xie Y, et al. Osteogenesis of electrically stimulated bone cells mediated in part by calcium ions. Clin Orthop Relat Res 1998;348:259-68.
• Ishida Y, Bellows CG, Tertinegg I, Heersche JN. Progesterone-mediated stimulation of osteoprogenitor proliferation and differentiation in cell populations derived from adult or fetal rat bone tissue depends on the serum component of the culture media. Osteoporos Int 1997;7:323
• Dontchos BN, Coyle CH, Izzo NJ, Didiano DM, Karpie JC, Logar A, et al. Optimizing CO2 normalizes pH and enhances chondrocyte viability during cold storage. J Orthop Res 2008;26:643-50.
• Ben-Galim P, Rand N, Giladi M, Schwartz D, Ashkenazi E, Millgram M, et al. Association between sciatica and microbial infection: true infection or culture contamination? Spine 2006;31:2507-9.
• Tay LY, Herrera DR, Quishida CC, Carlos IZ, Jorge JH. Effect of water storage and heat treatment on the cytotoxicity of soft liners. Gerodontology 2012;29:e275-80.
• Yang C, Hornicek FJ, Wood KB, Schwab JH, Choy E, Iafrate J, et al. Characterization and analysis of human chordoma cell lines. Spine 2010;35:1257-64.
• Sastre S, Suso S, Segur JM, Bori G, Carbonell JA, Agustí E, et al. Cryopreserved and frozen hyaline cartilage imaged by environmental scanning electron microscope. An experimental and prospective study. J Rheumatol 2008;35:1639-44.
• Fedewa MM, Oegema TR Jr, Schwartz MH, MacLeod A, Lewis JL. Chondrocytes in culture produce a mechanically functional tissue. J Orthop Res 1998;16:227-36.
• Wu X, Lin M, Li Y, Zhao X, Yan F. Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteumderived cells. Cytotechnology 2009;59:103-11.
• Zhang C, Hu YY, Cui FZ, Zhang SM, Ruan DK. A study on a tissue-engineered bone using rhBMP-2 induced periosteal cells with a porous nano-hydroxyapatite/collagen/ poly(L-lactic acid) scaffold. Biomed Mater 2006;1:56-62.
• Agata H, Asahina I, Yamazaki Y, Uchida M, Shinohara Y, Honda MJ, et al. Effective bone engineering with periosteum-derived cells. J Dent Res 2007;86:79-83.
• Perka C, Schultz O, Spitzer RS, Lindenhayn K, Burmester GR, Sittinger M. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials 2000;21:1145-53.
• Mizuno H, Hata K, Kojima K, Bonassar LJ, Vacanti CA, Ueda M. A novel approach to regenerating periodontal tissue by grafting autologous cultured periosteum. Tissue Eng 2006;12:1227-335.
• Kim WS, Kim HK. Tissue engineered vascularized bone formation using in vivo implanted osteoblast-polyglycolic acid scaffold. J Korean Med Sci 2005;20:479-82.
• Cirpar M, Korkusuz F. The future of treatment for chondral and osteochondral lesions. Acta Orthop Traumatol Turc 2007;41 Suppl 2:153-9.
• Sabatini M, Pastoureau P, De Ceuninck F. Cartilage and Osteoarthritis: Cellular and molecular tools. 1st ed. Vol. 1, New Jersey: Humana Press; 2004.
• Leibovitz A. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am J Hyg 1963;78:173-80.
• Trehan K. Biotechnology. 2nd ed. New Age International (P) Ltd.; 2002.
• Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg Am 2004;86-A:1541-58.
• Lynn AK, Brooks RA, Bonfield W, Rushton N. Repair of defects in articular joints. Prospects for material-based solutions in tissue engineering. J Bone Joint Surg Br 2004;86:1093-9.
• Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science 2002;295:1009-14.
• Köse GT, Korkusuz F, Korkusuz P, Hasirci V. In vivo tissue engineering of bone using poly(3-hydroxybutyric acidco-3-hydroxyvaleric acid) and collagen scaffolds. Tissue Eng 2004;10:1234-50.
• Risbud MV, Sittinger M. Tissue engineering: advances in in vitro cartilage generation. Trends Biotechnol 2002;20:351-6.
• Raghunath J, Salacinski HJ, Sales KM, Butler PE, Seifalian AM. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol 2005;16:503-9.
• Gomez-Camarillo MA, Almonte-Becerril M, Vasquez Tort M, Tapia-Ramirez J, Kouri Flores JB. Chondrocyte proliferation in a new culture system. Cell Prolif 2009;42:207-18.
• Ceyhan T, Bilir A, Karaca C. Culturability of rat bone marrow stromal cells and evaluation for osteoblastic formation. [Article in Turkish] Acta Orthop Traumatol Turc 2006;40:67-71.
• Yu FY, Lu SB, Huang LH, Xu WJ, Peng J, Zhao B, et al. Mechanisms of autologous chondrocytes mass transplantation in the repair of cartilage defects of rabbits’ knee. Zhongguo Gu Shang 2010;23:683-7.
• Priddy NH 2nd, Cook JL, Kreeger JM, Tomlinson JL, Steffen DJ. Effect of ascorbate and two different media on canine chondrocytes in three-dimensional culture. Vet Ther 2001;2:70-7.
• Zhang XQ, Li X, Wu T, Li JW, DU H, Pei GX. [Isolation, culture and chondrogenic differentiation of goat bone marrow mesenchymal stem cells]. [Article in Chinese] Nan Fang Yi Ke Da Xue Xue Bao 2009;29:419-22. [Abstract]
• Kiliç E, Ceyhan T, Cetinkaya DU. Evaluation of differentiation potential of human bone marrow-derived mesenchymal stromal cells to cartilage and bone cells. [Article in Turkish] Acta Orthop Traumatol Turc 2007;41:29530