Flow cytometric analysis of human chondrocytes cultured in a new medium for autologous therapie and tissue engineering cartilage

Flow cytometric analysis of human chondrocytes cultured in a new medium for autologous therapie and tissue engineering cartilage

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Title: Flow cytometric analysis of human chondrocytes cultured in a new medium for autologous therapie and tissue engineering cartilage
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Article_Title: Flow cytometric analysis of human chondrocytes cultured in a new medium for autologous therapie and tissue engineering cartilage
Authors: Daniela Bratosin, Ana-Maria Gheorghe, Alexandrina Rugina, Liana Mos, Nicolae Efimov, Catalin Iordachel, Manuela Sidoroff, Jerome Estaguier
Affiliation: National Institute for Biological Science Research and Development, Bucharest, Romania
”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
”Vasile Goldis” Western University of Arad, Faculty of General Medicine, Pharmacy and Dental Medicine, Arad, Romania
CFR 2 Hospital, Bucharest, Romania
Faculté Créteil Henri Mondor, Institut National de la Sante et de la Recherche Médicale, U955, Créteil, France.
Abstract: Autologous Cell Implantation (ACI) is a currently practiced cell-based therapy to repair cartilage defects. Several strategies have been explored to expand the number of chondrocytes ex vivo. However, these methods are unable to provide sufficient quantity of chondrocytes with unaltered phenotype. To maintain the original phenotype in monolayer culture and to expand cell proliferation, primary human chondrocytes isolated by enzymatic digestion were cultured in a DMEM medium supplemented with Ac-Gly-Gly-OH dipeptide The aim of our study was to investigate and compare by flow cytometry the viability and the cell proliferation of chondrocytes obtained by culture in medium containing the dipeptide with the cells cultured in a classical system. The results we obtained provide that proliferation and viability of chondrocytes cultured in presence of DMEM medium containing Ac-Gly-Gly-OH were higher and thus can be used in the culture of chondrocytes devoted to reconstructive clinical procedures.
Keywords: chondrocytes, osteoartritic cartilage, flow cytometric analysis, viability test, cell proliferation, PKH-26, tissue engineering
References: Arevalo-Silva C.A., Cao Y., Vacanti M., Weng Y., Vacanti C. A., Eavey R. D., Influence of growth factors on tissue-engineered pediatric elastic cartilage. Arch. Otolaryngol. Head Neck Surg. 126, 1234, 2000.
Aroen A., Loken S., Heir S., Alvik E., Ekeland A., Granlund O. G., Engebretsen L., Articular cartilage lesions in 993 consecutive knee arthroscopies. Am. J. Sports Med., 32, 211-215, 2004.
Bratosin D., Palii C., Mitrofan L., Estaquier J., Montreuil J., Novel fluorescence assay using Calcein- AM for the determination of human erythrocyte viability and aging, Cytometry 66A, 78–84, 2005.
Brittberg M., Lindahl A., Nilsson A., Ohlsson C., Isaksson O., Peterson L., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N. Engl. J. Med., 331(14), 889–895, 1994.
Buia-Takacs L., Iordachel C., Gheorghe A-M., Rugina A., Mos L., Efimov N., Bratosin D., Flow cytometric alalysis of encapsulated chondrocytes cultured in alginate gel for cartilage tissue engineering, Jurnal Medical Aradean (Arad Medical Journal), 13 4, 25-33, 2010.
Cancedda R., Dozin B., Giannoni P., Quarto R., Tissue engineering and cell therapy of cartilage and bone, Matrix Biol., 22, 81-91, 2003.
Caplan A. I., Stem cell delivery vehicle, Biomaterials, 11, 44-46, 1990.
Darzynkiewicz Z., Juan G., Li X., Gorczyca W., Murakami T., Traganos F, Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis), Cytometry, 27,1-20, 1997.
Delise A.M., Archer C.W., Benjamin M., Ralphs J.R., Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture, J. Anat. 194, 343–335, 1999.
Dowthwaite G. P., Bishop J. C., Redman S. N, Khan I. M., Rooney P., Evans D. J., Haughton L, Bayram Z., Boyer S., Thomson B., Wolfe M. S., Archer C. W., The surface of articular cartilage contains a progenitor cell population, J.Cell Sci., 117, 889-897, 2004.
Green W.T., Behaviour of articular chondrocytes in cell culture, Clin. Orthop., 75, 248-260, 1971.
Huang J. I., Beanes S. R., Zhu M., Lorenz H. P., Hedrick M. H., Benhaim P., Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells, Plast. Reconstr. Surg., 109, 1033-1041, 2002.
Kuettner K.E., Pauli B.U., Gall G., Memoli V.A., Schenk R.K., Synthesis of cartilage matrix by mammalian chondrocytes in vitro, Isolation, culture characteristics and morphology, J. Cell. Biol., 93,743–50, 1982.
Lebaron R.G., Athanasiou K.A., Ex vivo synthesis of articular cartilage, Biomaterials, 21, 2575–2587, 2000.
Levy M.M., Joyner C. J., Virdi A. S., Reed A., Triffitt J. T., Simpson A. H., Kenwright J., Stein H., Francis M. J., Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study, Bone, 29, 317-322, 2001.
Marijnissen W. J., Van Osch G.J., Aigner J., Verwoerd-Verhoef H. L., Verhaar J. A.,Tissue-engineered cartilage using serially passaged articular chondrocytes: Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier(DBM), Biomaterials, 21, 571, 2000.
Martin I., Suetterlin R., Baschong W., Heberer M., Vunjak-Novakovic G., Freed L. E., Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation,J.Cell. Biochem., 83, 121-128, 2001.
Mizuno S., Glowacki J., Three-dimensional composite of demineralized bone powder and collagen for in vitro analysis of chondroinduction of human dermal fibroblasts, Biomaterials, 17, 1819-1825, 1996.
Muir H., The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules, bioessays, 17, 1039–1048, 1995.
Takács-Buia L., Iordachel C., Efimov N., Caloianu M., Montreuil J., Bratosin D., Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?, Clinical Cytometry, 74B, 356-362, 2008.
U.S. Markets for Current and Emerging Orthopedic Biomaterials Products and Technologies; Medtech Insight: Newport Beach, CA, 2002; Report A320
Von der Mark K., Gauss V., von der Mark H., Muller P., Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture, Nature, 267, 531, 1977.
Watt F.M., Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture, J. Cell. Sci., 89, 373, 1988.
Zuk P. A., Zhu M., Mizuno H., Huang J., Futrell J. W., Katz A. J., Benhaim P., Lorenz H. P., Hedrick M. H., Multilineage cells from human adipose tissue: implications for cellbased therapies, Tissue Eng., 7, 211-228,2001.
Read_full_article: pdf/22-2012/22-2-2012/SU22-2-2012-Bratosin.pdf
Correspondence: Bratosin Daniela, National Institute for Biological Science Research and Development, Bucharest, Romania; Splaiul Independentei no. 296, district 6, Bucharest, Romania, Tel/Fax +40-(021)-2200881, email: bratosind@yahoo.com.

Read full article
Article Title: Flow cytometric analysis of human chondrocytes cultured in a new medium for autologous therapie and tissue engineering cartilage
Authors: Daniela Bratosin, Ana-Maria Gheorghe, Alexandrina Rugina, Liana Mos, Nicolae Efimov, Catalin Iordachel, Manuela Sidoroff, Jerome Estaquier
Affiliation: National Institute for Biological Science Research and Development, Bucharest, Romania
”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
”Vasile Goldis” Western University of Arad, Faculty of General Medicine, Pharmacy and Dental Medicine, Arad, Romania
CFR 2 Hospital, Bucharest, Romania
Faculté Créteil Henri Mondor, Institut National de la Sante et de la Recherche Médicale, U955, Créteil, France.
Abstract: Autologous Cell Implantation (ACI) is a currently practiced cell-based therapy to repair cartilage defects. Several strategies have been explored to expand the number of chondrocytes ex vivo. However, these methods are unable to provide sufficient quantity of chondrocytes with unaltered phenotype. To maintain the original phenotype in monolayer culture and to expand cell proliferation, primary human chondrocytes isolated by enzymatic digestion were cultured in a DMEM medium supplemented with Ac-Gly-Gly-OH dipeptide The aim of our study was to investigate and compare by flow cytometry the viability and the cell proliferation of chondrocytes obtained by culture in medium containing the dipeptide with the cells cultured in a classical system. The results we obtained provide that proliferation and viability of chondrocytes cultured in presence of DMEM medium containing Ac-Gly-Gly-OH were higher and thus can be used in the culture of chondrocytes devoted to reconstructive clinical procedures.
Keywords: chondrocytes, osteoartritic cartilage, flow cytometric analysis, viability test, cell proliferation, PKH-26, tissue engineering
References: Arevalo-Silva C.A., Cao Y., Vacanti M., Weng Y., Vacanti C. A., Eavey R. D., Influence of growth factors on tissue-engineered pediatric elastic cartilage. Arch. Otolaryngol. Head Neck Surg. 126, 1234, 2000.
Aroen A., Loken S., Heir S., Alvik E., Ekeland A., Granlund O. G., Engebretsen L., Articular cartilage lesions in 993 consecutive knee arthroscopies. Am. J. Sports Med., 32, 211-215, 2004.
Bratosin D., Palii C., Mitrofan L., Estaquier J., Montreuil J., Novel fluorescence assay using Calcein- AM for the determination of human erythrocyte viability and aging, Cytometry 66A, 78–84, 2005.
Brittberg M., Lindahl A., Nilsson A., Ohlsson C., Isaksson O., Peterson L., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N. Engl. J. Med., 331(14), 889–895, 1994.
Buia-Takacs L., Iordachel C., Gheorghe A-M., Rugina A., Mos L., Efimov N., Bratosin D., Flow cytometric alalysis of encapsulated chondrocytes cultured in alginate gel for cartilage tissue engineering, Jurnal Medical Aradean (Arad Medical Journal), 13 4, 25-33, 2010.
Cancedda R., Dozin B., Giannoni P., Quarto R., Tissue engineering and cell therapy of cartilage and bone, Matrix Biol., 22, 81-91, 2003.
Caplan A. I., Stem cell delivery vehicle, Biomaterials, 11, 44-46, 1990.
Darzynkiewicz Z., Juan G., Li X., Gorczyca W., Murakami T., Traganos F, Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis), Cytometry, 27,1-20, 1997.
Delise A.M., Archer C.W., Benjamin M., Ralphs J.R., Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture, J. Anat. 194, 343–335, 1999.
Dowthwaite G. P., Bishop J. C., Redman S. N, Khan I. M., Rooney P., Evans D. J., Haughton L, Bayram Z., Boyer S., Thomson B., Wolfe M. S., Archer C. W., The surface of articular cartilage contains a progenitor cell population, J.Cell Sci., 117, 889-897, 2004.
Green W.T., Behaviour of articular chondrocytes in cell culture, Clin. Orthop., 75, 248-260, 1971.
Huang J. I., Beanes S. R., Zhu M., Lorenz H. P., Hedrick M. H., Benhaim P., Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells, Plast. Reconstr. Surg., 109, 1033-1041, 2002.
Kuettner K.E., Pauli B.U., Gall G., Memoli V.A., Schenk R.K., Synthesis of cartilage matrix by mammalian chondrocytes in vitro, Isolation, culture characteristics and morphology, J. Cell. Biol., 93,743–50, 1982.
Lebaron R.G., Athanasiou K.A., Ex vivo synthesis of articular cartilage, Biomaterials, 21, 2575–2587, 2000.
Levy M.M., Joyner C. J., Virdi A. S., Reed A., Triffitt J. T., Simpson A. H., Kenwright J., Stein H., Francis M. J., Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study, Bone, 29, 317-322, 2001.
Marijnissen W. J., Van Osch G.J., Aigner J., Verwoerd-Verhoef H. L., Verhaar J. A.,Tissue-engineered cartilage using serially passaged articular chondrocytes: Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier(DBM), Biomaterials, 21, 571, 2000.
Martin I., Suetterlin R., Baschong W., Heberer M., Vunjak-Novakovic G., Freed L. E., Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation,J.Cell. Biochem., 83, 121-128, 2001.
Mizuno S., Glowacki J., Three-dimensional composite of demineralized bone powder and collagen for in vitro analysis of chondroinduction of human dermal fibroblasts, Biomaterials, 17, 1819-1825, 1996.
Muir H., The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules, bioessays, 17, 1039–1048, 1995.
Takács-Buia L., Iordachel C., Efimov N., Caloianu M., Montreuil J., Bratosin D., Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?, Clinical Cytometry, 74B, 356-362, 2008.
U.S. Markets for Current and Emerging Orthopedic Biomaterials Products and Technologies; Medtech Insight: Newport Beach, CA, 2002; Report A320
Von der Mark K., Gauss V., von der Mark H., Muller P., Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture, Nature, 267, 531, 1977.
Watt F.M., Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture, J. Cell. Sci., 89, 373, 1988.
Zuk P. A., Zhu M., Mizuno H., Huang J., Futrell J. W., Katz A. J., Benhaim P., Lorenz H. P., Hedrick M. H., Multilineage cells from human adipose tissue: implications for cellbased therapies, Tissue Eng., 7, 211-228,2001.
*Correspondence: Bratosin Daniela, National Institute for Biological Science Research and Development, Bucharest, Romania; Splaiul Independentei no. 296, district 6, Bucharest, Romania, Tel/Fax +40-(021)-2200881, email: bratosind@yahoo.com.