Flow cytometric analysis of encapsulated chondrocytes cultured in alginate gel for cartilage…

Flow cytometric analysis of encapsulated chondrocytes cultured in alginate gel for cartilage…

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Article_Title: Flow cytometric analysis of encapsulated chondrocytes cultured in alginate gel for cartilage tissue engineering
Authors: Luminita Buia-Takacs1, Catalin Iordachel1, Ana-Maria Gheorghe1, Alexandrina Rugina1, Liana Mos2, Nicolae Efimov3, Daniela Bratosin1,4*
Affiliation: 1 National Institute of Biological Science Research and Development (INSB), Romania
2 ″Vasile Goldis″ Western University of Arad, Faculty of Medicine, Arad, Romania
3 Hospital CFR2, Bucharest, Romania
4 ”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
Abstract: In autologous cell implantation (ACI), the autologous chondrocytes recovered from the patient are amplified in tissue culture prior to re-implantation. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell therapies in coming years. The aim of this study was to investigate and to compare by flow cytometric methods, morphological changes, cellular viability and apoptosis of human chondrocytes cultured in alginate gel and conventional cell-seeding methods for cartilage tissue engineering applications. The results obtained provide the usefulness of the gel in the culture of chondrocytes for reconstructive clinical procedures.
Keywords: chondrocytes, osteoarthritic cartilage, tissue engineering, flow cytometric analysis, microencapsulation, alginat gel, apoptosis, viability test
References: Almarza AJ, Athanasiou KA, (2005) Effects of initial cell seeding density for the tissue engineering of the temporomandibular joint disc. Ann Biomed Eng., 33:943–950.
Aydelotte MB, Thonar EJ, Mollenhauer J, Flechtenmacher J, (1998) Culture of chondrocytes in alginate gel: variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes. In Vitro Cell Dev Biol Anim., 34:123–130.
Bratosin D, Palii C, Mitrofan L, Estaquier J, Montreuil J, (2005) Novel fluorescence assay using Calcein-AM for the determination of human erythrocyte viability and aging. Cytometry, 66A:78–84.
Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F, (1997) Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry, 27:1-20.
Grandolfo M, D’Andrea P, Paoletti S, Martina M, Silvestrini G, Bonucci E, Vittur F, (1993) Culture and differentiation of chondrocytes entrapped in alginate gels. Calcif Tissue Int., 52:42–48.
Green WT, (1971) Behaviour of articular chondrocytes in cell culture. Clin Orthop, 75:248-260.
Guo JF, Jourdian GW, MacCallum DK, (1989) Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res., 19:277–297.
Hamilton DW, Riehle MO, Monaghan W, Curtis AS, (2005) Articular chondrocyte passage number: Influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography. Cell Biol Int., 29: 408–421.
Koch RJ, Gorti GK, (2002) Tissue engineering with chondrocytes. Facial Plast Surg., 18:59–68.
Kuettner KE, Pauli BU, Gall G., Memoli VA, Schenk RK, (1982) Synthesis of cartilage matrix by mammalian chondrocytes in vitro. Isolation, culture characteristics and morphology. J Cell Biol., 93: 743-50.
Li Y, Ma T, Kniss DA, Lasky LC, Yang ST, (2001) Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog., 17: 935–944.
Liu X, Ma PX, (2004) Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng., 32: 477–486.
McFetridge PS, Bodamyali T, Horrocks M, Chaudhuri JB, (2004) Endothelial and smooth muscle cell seeding onto processed ex vivo arterial scaffolds using 3D vascular bioreactors. ASAIO J., 50:591–600.
Paige KT, Cima LG, Yaremchuk MJ, Vacanti JP, Vacanti CA, (1995) Injectable cartilage. Plast Reconstr Surg., 96:1390–1398.
Radisic M, Euloth M, Yang L, Langer R, Freed LE, Vunjak-Novakovic G, (2003) High-density seeding of myocyte cells for cardiac tissue engineering. Biotechnol Bioeng., 82: 403–414.
Randolph MA, Anseth K, Yaremchuk MJ, (2003) Tissue engineering of cartilage. Clin Plast Surg., 30:519–537.
Shimizu K, Ito A, Honda H, (2006) Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles. J Biomed Mater Res B Appl Biomater, 77: 265–272.
Soletti L, Nieponice A, Guan J, Stankus JJ, Wagner WR, Vorp DA, (2006) A seeding device for tissue engineered tubular structures. Biomaterials, 27: 4863–4870.
Takacs-Buia L, Iordachel C, Efimov N, Caloianu M, Montreuil J, Bratosin D, (2008) Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?, Cytometry Part B (Clinical Cytometry) 74B:356–362.
Ushida T, Furukawa K, Toita K, Tateishi T, (2002) Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant, 11: 489–494.
Vunjak-Novakovic G, Radisic M, (2004) Cell seeding of polymer scaffolds. Methods Mol Bio.,l 238:131–146.
Yang KG, Saris DB, Geuze RE, van Rijen MH, van der Helm YJ, Verbout AJ, Creemers LB, Dhert WJ, (2006) Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints. Osteoarthritis Cartilage, 14: 561–570.
Zhao F, Ma T, (2005) Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: Dynamic cell seeding and construct development. Biotechnol Bioeng., 91:482–493.
Read_full_article: http://www.jmedar.ro/pdf/vol13/iss4/JMA13-4-10Bratosin.pdf
Correspondence: Daniela Bratosin, National Institute for Biological Science Research and Development, Spl. Independentei nº296, 060031 Bucharest and ″Vasile Goldis″ Western University of Arad, Faculty of Natural Sciences, Arad, Romania, Tel/Fax: 40.21.2200881; E-mail: bratosind@yahoo.com

Read full article
Article Title: Flow cytometric analysis of encapsulated chondrocytes cultured in alginate gel for cartilage tissue engineering
Authors: Luminita Buia-Takacs1, Catalin Iordachel1, Ana-Maria Gheorghe1, Alexandrina Rugina1, Liana Mos2, Nicolae Efimov3, Daniela Bratosin1,4*
Affiliation: 1 National Institute of Biological Science Research and Development (INSB), Romania
2 ″Vasile Goldis″ Western University of Arad, Faculty of Medicine, Arad, Romania
3 Hospital CFR2, Bucharest, Romania
4 ”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
Abstract: In autologous cell implantation (ACI), the autologous chondrocytes recovered from the patient are amplified in tissue culture prior to re-implantation. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell therapies in coming years. The aim of this study was to investigate and to compare by flow cytometric methods, morphological changes, cellular viability and apoptosis of human chondrocytes cultured in alginate gel and conventional cell-seeding methods for cartilage tissue engineering applications. The results obtained provide the usefulness of the gel in the culture of chondrocytes for reconstructive clinical procedures.
Keywords: chondrocytes, osteoarthritic cartilage, tissue engineering, flow cytometric analysis, microencapsulation, alginat gel, apoptosis, viability test
References: Almarza AJ, Athanasiou KA, (2005) Effects of initial cell seeding density for the tissue engineering of the temporomandibular joint disc. Ann Biomed Eng., 33:943–950.
Aydelotte MB, Thonar EJ, Mollenhauer J, Flechtenmacher J, (1998) Culture of chondrocytes in alginate gel: variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes. In Vitro Cell Dev Biol Anim., 34:123–130.
Bratosin D, Palii C, Mitrofan L, Estaquier J, Montreuil J, (2005) Novel fluorescence assay using Calcein-AM for the determination of human erythrocyte viability and aging. Cytometry, 66A:78–84.
Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F, (1997) Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry, 27:1-20.
Grandolfo M, D’Andrea P, Paoletti S, Martina M, Silvestrini G, Bonucci E, Vittur F, (1993) Culture and differentiation of chondrocytes entrapped in alginate gels. Calcif Tissue Int., 52:42–48.
Green WT, (1971) Behaviour of articular chondrocytes in cell culture. Clin Orthop, 75:248-260.
Guo JF, Jourdian GW, MacCallum DK, (1989) Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res., 19:277–297.
Hamilton DW, Riehle MO, Monaghan W, Curtis AS, (2005) Articular chondrocyte passage number: Influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography. Cell Biol Int., 29: 408–421.
Koch RJ, Gorti GK, (2002) Tissue engineering with chondrocytes. Facial Plast Surg., 18:59–68.
Kuettner KE, Pauli BU, Gall G., Memoli VA, Schenk RK, (1982) Synthesis of cartilage matrix by mammalian chondrocytes in vitro. Isolation, culture characteristics and morphology. J Cell Biol., 93: 743-50.
Li Y, Ma T, Kniss DA, Lasky LC, Yang ST, (2001) Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog., 17: 935–944.
Liu X, Ma PX, (2004) Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng., 32: 477–486.
McFetridge PS, Bodamyali T, Horrocks M, Chaudhuri JB, (2004) Endothelial and smooth muscle cell seeding onto processed ex vivo arterial scaffolds using 3D vascular bioreactors. ASAIO J., 50:591–600.
Paige KT, Cima LG, Yaremchuk MJ, Vacanti JP, Vacanti CA, (1995) Injectable cartilage. Plast Reconstr Surg., 96:1390–1398.
Radisic M, Euloth M, Yang L, Langer R, Freed LE, Vunjak-Novakovic G, (2003) High-density seeding of myocyte cells for cardiac tissue engineering. Biotechnol Bioeng., 82: 403–414.
Randolph MA, Anseth K, Yaremchuk MJ, (2003) Tissue engineering of cartilage. Clin Plast Surg., 30:519–537.
Shimizu K, Ito A, Honda H, (2006) Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles. J Biomed Mater Res B Appl Biomater, 77: 265–272.
Soletti L, Nieponice A, Guan J, Stankus JJ, Wagner WR, Vorp DA, (2006) A seeding device for tissue engineered tubular structures. Biomaterials, 27: 4863–4870.
Takacs-Buia L, Iordachel C, Efimov N, Caloianu M, Montreuil J, Bratosin D, (2008) Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?, Cytometry Part B (Clinical Cytometry) 74B:356–362.
Ushida T, Furukawa K, Toita K, Tateishi T, (2002) Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant, 11: 489–494.
Vunjak-Novakovic G, Radisic M, (2004) Cell seeding of polymer scaffolds. Methods Mol Bio.,l 238:131–146.
Yang KG, Saris DB, Geuze RE, van Rijen MH, van der Helm YJ, Verbout AJ, Creemers LB, Dhert WJ, (2006) Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints. Osteoarthritis Cartilage, 14: 561–570.
Zhao F, Ma T, (2005) Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: Dynamic cell seeding and construct development. Biotechnol Bioeng., 91:482–493.
*Correspondence: Daniela Bratosin, National Institute for Biological Science Research and Development, Spl. Independentei nº296, 060031 Bucharest and ″Vasile Goldis″ Western University of Arad, Faculty of Natural Sciences, Arad, Romania, Tel/Fax: 40.21.2200881; E-mail: bratosind@yahoo.com