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Investigation on reconstruction of skin for autologous transplantation
Investigation on Reconstruction of Skin for Autologous Transplantation C. Johnen, A. Kage, A. Oechel, P. Neuhaus, and J.C. Gerlach
T
HE LONG-TERM aim is the availability of functional proliferated autologous skin transplants for burn injured patients. Human epidermal basal keratinocytes and dermal fibroblasts should be considered to enable clinically valuable results. Biodegradable membranes are the actual topic enabling in vitro proliferation in bioreactors and for the transfer of the cells to the wound. To choose appropriate material we grew keratinocytes and fibroblasts separately onto different membranes. Keratinocytes were cultured in different media with various concentrations of growth factors to avoid the established use of bovine pituitary extract (BPE). MATERIAL AND METHODS Human keratinocytes and dermal fibroblasts were isolated from the same specimen. The attachment and proliferation of keratinocytes in different media were compared as well as the influence of the adhesion matrix. Keratinocytes were cultivated according the method of Rheinwald and Green1–3 with minor modifications, and fibroblasts obtained from outgrowth of the same piece of skin were grown in DMEM containing 10% human serum. Keratinocytes were plated in secondary cultures at a density of 2 ⫻ 104 cells/cm2 into standard tissue culture flasks and onto biodegradable collagen membranes and digestible cellulose-sulfate membranes. In addition, different culture media were tested. MCDB with BPE and Gibco SFM served as controls. Biochrome complete medium without epidermal growth factor (EGF) and kerantinocytes growth factor (KGF) versus various concentrations of EGF and KGF (5 ng/mL EGF ⫹ 5 ng/mL KGF, 10 ng/mL EGF, 5 ng/mL EGF ⫹ 10 ng/mL KGF, 10 ng/mL EGF ⫹ 10 ng/mL KGF, and 10 ng/mL EGF ⫹ 50 ng/mL KGF) were examined. Cell spreading and density was determined by light microscopy and proliferation rates by a colorimetric cell proliferation assay every 24 hours (end of observation period: 96 hours). To determine the proliferation rate of human keratinocytes, the MTT-based (thiazolyl blue) cytotoxicity test was performed according to Denizot and Lang4 with minor modifications. The test is based on mitochondria viability, that is, only functional mitochondria can oxidize an MTT solution, giving a typical blue-violet product. This assay is an indirect method to assess cell growth and proliferation since the optical density (OD) values can be correlated to the cell number.
RESULTS
Fifty human keratinocytes isolations were performed. In all cases, isolation of fibroblasts from the same donor was successful. The use of BPE and serum could be avoided 0041-1345/01/$–see front matter PII S0041-1345(00)02169-2
using EGF and KGF. Human keratinocytes cultured in media with various concentrations of EGF and KGF showed different proliferation rates. This experiment indicated that cell growth is related to concentration of EGF and KGF, and the concentration of the factors are dependent on each other. The proliferation rates are also dependent on time of cultivation. Culture media with 10 ng/mL EGF and 5 ng/mL KGF showed sufficient results for up to 72 hours, and culture media with 10 ng/mL EGF and 50 ng/mL KGF showed better results in long-term experiments of more than 72 hours. Morphology confirmed the proliferation rates for the different points of time (24, 48, 72, and 96 hours). Human fibroblasts and keratinocytes seeded onto a biodegradable collagen membrane were able to adhere and proliferate. For points of time 24, 48, 72, and 96 hours cell growth on this material was compared to a control culture on tissue culture plastic. The results of the MTT-test exhibited that there is no marked difference in cell proliferation and cell density between collagen membrane and the control. Cellulose-sulfate membranes showed a more unphysiological behaviour. DISCUSSION AND CONCLUSION
Our study demonstrates that the cultivation and proliferation of human keratinocytes and fibroblasts from the same specimen was possible, enabling the construction of bilayer transplants on membranes. Cells were adherent to the membrane surface and did not show any visible signs of undesired differentiation. The use of bovine pituitary extract can be avoided by utilizing defined growth factors. Collagen membranes represent a suitable substrate for human keratinocytes under serum free conditions. It is well known that keratinocytes produce a series of soluble proteins in vivo and in vitro that modulate both their own growth and that of other cell types within the epidermis and dermis.5 Recently published data indicated a regulation of keratinocytes growth by double paracrine mechanism From the Department of Surgery, Institute of Laboratory Medicine, Charite´, Campus Virchow-Clinic, Humboldt-University of Berlin, University of Potsdam, Potsdam, Germany. Address reprint requests to Jo¨rg Gerlach, PD, MD, Department of Surgery and Transplantation, Charite´, Campus VirchowClinic, Augustenburger Platz 1, 13353 Berlin, Germany. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
618
Transplantation Proceedings, 33, 618–619 (2001)
RECONSTRUCTION OF SKIN FOR AUTOLOGOUS TRANSPLANT
through release of IL-1, which induces KGF in cocultured fibroblasts.6 Thus data exhibited that in vitro cultivation of human keratinocytes and fibroblasts from the same specimen could be an advantage for both cells. REFERENCES 1. Rheinwald JG, Green H: Cell 6:317, 1975
619 2. Rheinwald JG, Green H: Cell 6:331, 1975 3. Rheinwald JG, Green H: Nature 265:421, 1977 4. Denizot F, Lang R: J Immunol Meth 89:271, 1986 5. Luger TA, Schwarz T: Epidermal Growth Factors and Cytokines. New York: Marcel Dekker; 1996 6. Maas-Szabowski N, Shimotoyodome A, Fusenig NE: J Cell Sci 112:1843, 1999
T
HE LONG-TERM aim is the availability of functional proliferated autologous skin transplants for burn injured patients. Human epidermal basal keratinocytes and dermal fibroblasts should be considered to enable clinically valuable results. Biodegradable membranes are the actual topic enabling in vitro proliferation in bioreactors and for the transfer of the cells to the wound. To choose appropriate material we grew keratinocytes and fibroblasts separately onto different membranes. Keratinocytes were cultured in different media with various concentrations of growth factors to avoid the established use of bovine pituitary extract (BPE). MATERIAL AND METHODS Human keratinocytes and dermal fibroblasts were isolated from the same specimen. The attachment and proliferation of keratinocytes in different media were compared as well as the influence of the adhesion matrix. Keratinocytes were cultivated according the method of Rheinwald and Green1–3 with minor modifications, and fibroblasts obtained from outgrowth of the same piece of skin were grown in DMEM containing 10% human serum. Keratinocytes were plated in secondary cultures at a density of 2 ⫻ 104 cells/cm2 into standard tissue culture flasks and onto biodegradable collagen membranes and digestible cellulose-sulfate membranes. In addition, different culture media were tested. MCDB with BPE and Gibco SFM served as controls. Biochrome complete medium without epidermal growth factor (EGF) and kerantinocytes growth factor (KGF) versus various concentrations of EGF and KGF (5 ng/mL EGF ⫹ 5 ng/mL KGF, 10 ng/mL EGF, 5 ng/mL EGF ⫹ 10 ng/mL KGF, 10 ng/mL EGF ⫹ 10 ng/mL KGF, and 10 ng/mL EGF ⫹ 50 ng/mL KGF) were examined. Cell spreading and density was determined by light microscopy and proliferation rates by a colorimetric cell proliferation assay every 24 hours (end of observation period: 96 hours). To determine the proliferation rate of human keratinocytes, the MTT-based (thiazolyl blue) cytotoxicity test was performed according to Denizot and Lang4 with minor modifications. The test is based on mitochondria viability, that is, only functional mitochondria can oxidize an MTT solution, giving a typical blue-violet product. This assay is an indirect method to assess cell growth and proliferation since the optical density (OD) values can be correlated to the cell number.
RESULTS
Fifty human keratinocytes isolations were performed. In all cases, isolation of fibroblasts from the same donor was successful. The use of BPE and serum could be avoided 0041-1345/01/$–see front matter PII S0041-1345(00)02169-2
using EGF and KGF. Human keratinocytes cultured in media with various concentrations of EGF and KGF showed different proliferation rates. This experiment indicated that cell growth is related to concentration of EGF and KGF, and the concentration of the factors are dependent on each other. The proliferation rates are also dependent on time of cultivation. Culture media with 10 ng/mL EGF and 5 ng/mL KGF showed sufficient results for up to 72 hours, and culture media with 10 ng/mL EGF and 50 ng/mL KGF showed better results in long-term experiments of more than 72 hours. Morphology confirmed the proliferation rates for the different points of time (24, 48, 72, and 96 hours). Human fibroblasts and keratinocytes seeded onto a biodegradable collagen membrane were able to adhere and proliferate. For points of time 24, 48, 72, and 96 hours cell growth on this material was compared to a control culture on tissue culture plastic. The results of the MTT-test exhibited that there is no marked difference in cell proliferation and cell density between collagen membrane and the control. Cellulose-sulfate membranes showed a more unphysiological behaviour. DISCUSSION AND CONCLUSION
Our study demonstrates that the cultivation and proliferation of human keratinocytes and fibroblasts from the same specimen was possible, enabling the construction of bilayer transplants on membranes. Cells were adherent to the membrane surface and did not show any visible signs of undesired differentiation. The use of bovine pituitary extract can be avoided by utilizing defined growth factors. Collagen membranes represent a suitable substrate for human keratinocytes under serum free conditions. It is well known that keratinocytes produce a series of soluble proteins in vivo and in vitro that modulate both their own growth and that of other cell types within the epidermis and dermis.5 Recently published data indicated a regulation of keratinocytes growth by double paracrine mechanism From the Department of Surgery, Institute of Laboratory Medicine, Charite´, Campus Virchow-Clinic, Humboldt-University of Berlin, University of Potsdam, Potsdam, Germany. Address reprint requests to Jo¨rg Gerlach, PD, MD, Department of Surgery and Transplantation, Charite´, Campus VirchowClinic, Augustenburger Platz 1, 13353 Berlin, Germany. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
618
Transplantation Proceedings, 33, 618–619 (2001)
RECONSTRUCTION OF SKIN FOR AUTOLOGOUS TRANSPLANT
through release of IL-1, which induces KGF in cocultured fibroblasts.6 Thus data exhibited that in vitro cultivation of human keratinocytes and fibroblasts from the same specimen could be an advantage for both cells. REFERENCES 1. Rheinwald JG, Green H: Cell 6:317, 1975
619 2. Rheinwald JG, Green H: Cell 6:331, 1975 3. Rheinwald JG, Green H: Nature 265:421, 1977 4. Denizot F, Lang R: J Immunol Meth 89:271, 1986 5. Luger TA, Schwarz T: Epidermal Growth Factors and Cytokines. New York: Marcel Dekker; 1996 6. Maas-Szabowski N, Shimotoyodome A, Fusenig NE: J Cell Sci 112:1843, 1999