Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

  • L. V. Altukhova V.N. Karazin Kharkiv National University
  • K. V. Kot V.N. Karazin Kharkiv National University
  • Y. G. Kot V.N. Karazin Kharkiv National University
  • K. S. Morozova V.N. Karazin Kharkiv National University
  • Y. E. Persky V.N. Karazin Kharkiv National University
Keywords: radiation burn, radiation ulcer, fibroblasts, keratinocytes, volumetric transplantation

Abstract

Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160)×103 fibroblasts and (130–140)×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210)×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β) increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in the burn area, and mutual stimulation of auto-fibroblasts and auto-keratinocytes to proliferate and to synthesize biologically active substances, i.e. cytokines and growth factors.

References

Altuhova, L.V., Gricenko, M.A., Kot, E.V., Kot, J.G., Perskу, E.J., 2013. Normalizacija soderzhanija soedinitel'notkannyh kletok i strukturnyh biopolimerov mezhkletochnogo matriksa v zone lokal'nogo luchevogo ozhoga kozhi obemnoj transplantaciej autofibroblastov [Normalization of connective tissue cells and extracellular matrix structural biopolymers in the area of skin local radiation burn by volumetric autofibroblasts transplantation]. Vіsnik Harkіvs'kogo Nacіonal'nogo Unіversitetu Іmenі V.N. Karazіna, Serіja Bіologіja 18(1057), 5–8 (in Russian).

Arrayit, 2015. Complete Protocol for Arrayit DNA chip. Retrieved from URL http://arrayit.com/Products/DNA_Micro-arrays/DNA_Microarrays_DCH/dna_microarrays_dch.html

Benderitter, M., Gourmelon, P., Bey, E., Chapel, A., Clairand, I., Prat, M., 2010. New emerging concepts in the medical management of local radiation injury. Health Phys. 98, 851–857. >> doi: 10.1097/HP.0b013e3181c9f79a

Birkedal-Hansen, H., 1993. Matrix metalloproteinases: A review. Crit. Rev. Oral Biol. Med. 4(2), 197–250. >> doi: 10.1002/0471143030.cb1008s17

Blomme, E.A., 1999. Parathyroid hormone-related protein is a positive regulator of keratinocyte growth factor expression by normal dermal fibroblasts. Mol. Cell Endocrinol. 152(12), 189–197. >> doi: 10.1016/S0303-7207(98)00252-4

Boilly, B., Vercoutter-Edouart, A.S., Hondermarck, H., Nurcombe, V., Le Bourhis, X., 2000. FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev. 11(4), 295–302. >> doi: 10.1016/S1359-6101(00)00014-9

Boxman, I., 1993. Modulation of IL-6 production and IL-1 activity by keratinocyte-fibroblast interaction. J. Invest. Dermatol. 101(3), 316–324. >> doi: 10.1111/1523-1747.ep12365474

Bushmanov, A.Y., Eremin, I.I., Moroz, B.B., Galstjan, I.A., Nadezhina, N.M., Slobodina, T.S., Grinakovskaja, O.S., 2012. Opyt sovremennogo lechenija luchevyh ozhogov u lic, podvergavshihsja vozdejstviju ionizirujushhego izluchenija [Experience of modern treatment of radiation burns in persons exposed to ionizing radiation]. Occupational Medicine and Industrial Ecology 10, 20–27 (in Russian).

Coffey, R., 1995. Basic actions of transforming growth factor-α and related peptides. Eur. J. Gastroen. Hepat. 7, 923–931. >> doi: 10.1097/00042737-199510000-00003

Dörr, W., Spek, K., Farre, C.L., 2002. The effect of keratinocyte growth factor on healing of manifest radiation ulcers in mouse tongue epithelium. Cell Prolif. 35(Suppl. 1), 86–92. >> doi: 10.1046/j.1365-2184.35.s1.9.x

Eldashov, S.V., 2013. Jeksperimental'noe obosnovanie sovremennyh metodov hirurgicheskogo lechenija sochetannyh luchevyh porazhenij [Experimental study of modern methods of surgical treatment of combined radiation injuries]. St. Petersburg (in Russian).

ETS No. 170, 1998. Protocol of amendment to the European convention for the protection of vertebrate animals used for experimental and other scientific purposes, Strasbourg, Retrieved from URL www.echr-base.ru/CED170.jsp

Finklestein, S., Plomaritoglou, A., 2001. Growth factors. Head trauma: Basic, preclinical, and clinical directions. New York, Wiley, 165–187.

Gabay, С., 2010. IL-1 pathways in inflammation and human diseases. Nat. Rev. Rheumatol. 6, 232–241. >> doi: 10.1038/nrrheum.2010.4

Gerasimova, L.I., Nazarenko, G.I., 2005. Termicheskie i radiatsionnie ozhogy [Thermic and radiation burn]. Medicine, Moscow (in Russian).

Glantz, S.A., 2007. Primer of Biostatistics, 4th Edition. McGraw-Hill, New York.

Groeber, F., Holeiter, M., Hampel, M., Hinderer, S., Schenke-Layland, K., 2012. Skin tissue engineering – in vivo and in vitro applications. Clin. Plast. Surg. 39, 33–58. >> doi: 10.1016/j.cps.2011.09.007

Hatzistergos, K.E., Blum, A., Ince, T.A., Grichnik, J.M., Hare, J.M., 2011. What is the oncologic risk of stem cell treatment for heart disease. Circ. Res. 108, 1300–1303. >> doi: 10.1161/CIRCRESAHA.111.246611

Haubner, F.K., Ohmann, E., Pohl, F., Strutz, J., Gassner, H.G., 2012. Wound healing after radiation therapy. Review of the literature. Radiat. Oncol. 7, 162–170. >> doi: 10.1186/1748-717X-7-162

Herberts, C.A., Kwa, M.S.G., Hermsen, H.P.H., 2011. Risk factors in the development of stem cell therapy. J. Transl. Med. 9, 29–42. >> doi: 10.1186/1479-5876-9-29

Histomix, 2008. Protokol obrabotki soedinitel'noj tkani v parafinovyh srezah dlja okrashivanija alcianovym sinim [The protocol of processing of connective tissue in paraffin sections for Alcian blue staining]. Retrieved from URL www.histomix.ru/product_4.html.

Histomix, 2010. Protokol obrabotki soedinitel'noj tkani v parafinovyh srezah dlja okrashivanija po Van-Gizonu [The protocol of processing of connective tissue in paraffin sections for Van Gieson staining]. Retrieved from URL www.histomix.ru/product_5.html.

Histomix, 2012. Protocol of processing of connective tissue in paraffin sections with hematoxylin and eosin. Retrieved from URL www.histomix.ru/product_17.html.

Histopathology, 2013. Gistohimicheskaja identifikacija uglevodov [Histochemical identification of carbohydrates]. Retrieved from URL www.histopathology.narod.ru/ documents/gistohimija_uglevodov.html.

Huang, L., Burd, A., 2012. An update review of stem cell applications in burns and wound. Сare. Indian J. Plast. Surg. 45(2), 229–236.

Imaris, 2011. Imaris for Cell Biologists. Retrieved from URL www.bitplane.com/imaris/imaris-for-cell-biologists.

Invitrogen, 2015. Tools for hybridization experiments in SecureSeal™ Hybridization Chambers. Retrieved from URL www.tools.thermofisher.com/content/sfs/manuals/mp18200.pdf.

Keller, G., 2000. Sohrannost' ineciruemyh autologichnyh chelovecheskih fibroblastov [Safety of injectable autologous human fibroblasts]. Bjul. Jeksp. Biol. Med. 130(8), 203–206.

Kligunenko, E.N., Leshhev, D.P., Slesarenko, S.V., Slinchenkov, V.V., Sorokina, E.J., 2005. Intensivnaja terapija ozhogovoj bolezni [Intensive therapy of burn disease]. MEDpress-inform, 144.

Kot, J.G., Kot, E.V., Perskij, E.J., Polikarpova, A.V., Altuhova, L.V., 2013. Tormozhenie razvitija lokal'nogo luchevogo ozhoga objomnoj autotransplantaciej fibroblastov [Inhibition of local radiation burn development by volume fibroblast autotransplantation]. Dopovіdі Nacіonal'noi Akademіi Nauk Ukraini. Matematika, Prirodoznavstvo, Tehnіchnі Nauki 4, 144–147 (in Russian).

Laboratornaja Diagnostika, 2015. Immunologija, belki ostroj fazy vospalenija [Immunology, acute phase proteins of inflammation]. Retrieved from URL www.ld.ru/IFA/ilist-14.html.

Lee, S.H., Jin, S.Y., Song, J.S., Seo, K.K., Cho, K.H., 2012. Paracrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts. Ann. Dermatol. 24, 136–143. >> doi: 10.5021/ad.2012.24.2.136

Marchese, C., 2001. Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes. J. Invest. Dermatol. 116(4), 623–628. >> doi: 10.1046/j.0022-202x.2001.01280.x

Marionnet, C., Pierrard, C., 2006. Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin. J. Invest. Dermatol. 126, 971–979. >> doi: 10.1038/sj.jid.5700230

Meyer, K., 1971. Hyaluronidases. In.: The Enzimes. Academic Press, NY. 5, 307–320. >> doi: 10.1002/9780470122587.ch6

Miyamoto, S., 1998. Fibronectin and integrins in cell adhesion, signaling, and morphogenesis. Ann. N.Y. Acad. Sci. 857, 119–129. >> doi: 10.1111/j.1749-6632.1998.tb10112.x

Paramonov, B.A., Porembskij, J.O., Jablonskij, V.G., 2000. Ozhogi: Rukovodstvo dlja vrachej [Burns: Guide for physicians]. Speclit, St. Petersburg (in Russian).

Parks, W.C., 2004. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat. Rev. Immunol. 4(8), 617–629. >> doi: 10.1111/j.1749-6632.1998.tb10112.x

Pehlivan, T., Mansour, A., Spaczynski, R.Z., Duleba, A.J., 2001. Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells. J. Endocrinol. 170(3), 639–645.

Praul, C.A., Ford, B.C., Leach, R.M., 2002. Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation. J. Cell Biochem. 84(2), 359–366. >> doi: 10.1002/jcb.1300

Qiagen, 2013. Qiagen® RNeasy FFPE Handbook. Manual for purification of total RNA from formalin-fixed, paraffin-embedded tissue sections. Retrieved from URL www.qiagen.com/ru/resources.

Qiagen, 2015. QIAGEN® OneStepRT-PCR Handbook. Manual for fast and highly sensitive one-step RT-PCR. Retrieved from URL www.qiagen.com/ru/resources.

Reed, C.C., 2002. The role of decorin in collagen fibrillogenesis and skin homeostasis. Glycoconjugate J. 19(4–5), 249–255. >> doi: 10.1023/A:1025383913444

Rennekampff, H., Kiessig, V., Griffey, S., 1997. Acellular human dermis promotes cultured keratinocyte engraftment. J. Burn Care Rehabil. 6, 535–544. >> doi: 10.1097/00004630-199711000-00012

Rittié, L., 2005. Isolation and culture of skin fibroblasts. Methods Mol. Med. 117, 83–98. >>doi: 10.1385/1-59259-940-0:083

Rowan, M.P., Cancio, L.C., Elster, E.A., Burmeister, D.M., Rose, L.F., Natesan, S., Chan, R.K., Christy, R.J., Chung, K.K., 2015. Burn wound healing and treatment: Review and advancements. Critical Care 19, 243–249. >> doi: 10.1186/s13054-015-0961-2

Ryan, J.L., 2012. Ionizing radiation: The good, the bad, and the ugly. J. Invest. Dermatol. 132, 985–993. >> doi: 10.1038/jid.2011.411

Shapovalov, S.G., 2005. Sovremennye ranevye pokrytija v kombustiologii [Modern wound cover in combustiology]. Farm Mindeks-praktik 8, 38–46 (in Russian).

Shaw, A.J., 1996. Epithelial cell culture – A practical approach. Oxford University Press, Oxford, 179–200.

Takagi, R., Yamato, M., Murakami, D., Kondo, M., Yang, J., Ohki, T., 2011. Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J. Tissue Eng. Regen. Med. 5, 63–73. >> doi: 10.1002/term.337

Tenenhaus, M., Rennekampff, H.O., 2007. Burn surgery. Clin. Plast. Surg. 34(4), 697–715. >> doi: 10.1016/j.cps.2007.08.007

Teng, M., Huang, Y., Zhang, H., 2014. Application of stems cells in wound healing. Wound Repair Regen. 22(2), 151–160.

Varani, J., Perone, P., 2007. Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage. Toxicol. Pathol. 35, 355–372. >> doi: 10.1080/01926230701481907

Vogel-Höpker, A., Momose, T., Rohrer, H., Yasuda, K., Ishihara, L., Rapaport, D.H., 2000. Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development. Mech. Dev. 94(1–2), 25–36. >> doi: 10.1016/S0925-4773(00)00320-8

Werner, S., Krieg, T., Smola, H., 2007. Keratinocyte-fibroblast interactions in wound healing. J. Invest. Dermatol. 127(5), 998–1008. >> doi: 10.1038/sj.jid.5700786

Yim, H., Cho, Y.S., Seo, C.H., Lee, B.C., Ko, J.H., Kim, D., 2010. The use of AlloDerm on major burn patients: AlloDerm prevents post-burn joint contracture. Burns 36, 322–328. >> doi: 10.1016/j.burns.2009.10.018

Published
2015-09-18
How to Cite
Altukhova, L. V., Kot, K. V., Kot, Y. G., Morozova, K. S., & Persky, Y. E. (2015). Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition. Regulatory Mechanisms in Biosystems, 6(2), 125-132. https://doi.org/10.15421/021523