Cytokine expression and ultrastructural alteration

Authors: Adolfo Paolin Diletta Trojan Antonio Leonardi Stefano Mellone Antonio Volpe Augusto Orlandi Elisa Cogliati

Publish Date: 2016/04/12

Volume: 17, Issue: 3, Pages: 399-406

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Abstract

The aim of this work was to compare the effects on human amniotic membrane of freezedrying and γirradiation at doses of 10 20 and 30 kGy with freezing For this purpose nine cytokines interleukin 10 plateletderived growth factorAA plateletderived growth factorBB basic fibroblast growth factor epidermal growth factor transforming growth factor beta 1 and tissue inhibitors of metalloproteinase1 2 and 4 were titrated in 5 different preparations for each of 3 amniotic membranes included in the study In addition the extracellular matrix structure of each sample was assessed by transmission electron microscopy While freezedrying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples γirradiation led to a significant decrease in the tissue inhibitors of metalloproteinase4 basic fibroblast growth factor and epidermal growth factor and induced structural damage to the epithelium basement membrane and lamina densa The higher the irradiation dose the more severe the damage to the amniotic membrane structure In conclusion the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γirradiation thereby avoiding alteration to the biological characteristics of the amniotic membraneHuman amniotic membrane HAM has been used in a variety of surgical procedures First employed in skin transplantation by Davis 1910 HAM was subsequently found to be useful as a biological wound dressing for burns Ramakrishnan and Jayaraman 1997 Branski et al 2008 acute Tekin et al 2008 and chronic wounds Gajiwala and Lobo 2003 Insausti et al 2010 and in the reconstruction of the dura mater Tomita et al 2012 De Weerd et al 2013 oral cavity Lawson 1985 vaginal vault Ashworth et al 1986 tendons Ozbölük et al 2010 and nerves O’Neill et al 2009 HAM has also long been used in ophthalmic surgery the earliest reported application being in 1940 when De Rötth used fetal membranes to correct symblepharon De Rötth 1940 Today HAM is widely used for ocular surface reconstruction and treating several important ocular diseases Paolin et al 2016 All these applications are possible because HAM has antiinflammatory antifibrotic properties Solomon et al 2001 Tseng et al 1999Hao et al have shown that human amniotic epithelial and mesenchymal cells both express interleukin1 receptor antagonist all the four tissue inhibitors of metalloproteinase TIMPs collagen XVIII and interleukin10 Hao 2000 Moreover reverse transcriptase–polymerase chain reaction RTPCR has shown that HAM expresses several additional cytokines such as transforming growth factor TGFα β1 β2 epidermal growth factor EGF keratinocyte growth factor KGF basic fibroblast growth factor bFGF keratinocyte growth factor receptor KGFR hepatocyte growth factor HGF and hepatocyte growth factor receptor HGFR Koizumi et al 2000 Li et al 2005 Gicquel et al 2009In 2001 Adds et al reported no differences in terms of clinical results between fresh and frozen HAM both preparations resulting in improved visual acuity Furthermore fresh tissue performed no better than frozen tissue in promoting reepithelialization Adds et al 2001 It has however been demonstrated that different processing storage and sterilization methods do affect HAM properties von VersenHöynck et al 2004RodriguezAres et al studied the effects of freezedrying and cryopreservation on HAM histological characteristics and protein levels The authors found that although lyophilization does not affect the histological structure of HAM it seems to reduce growth factor concentration more than cryopreservation RodriguezAres et al 2009 Ricci et al demonstrated that cryopreservation maintains the antifibrotic properties of HAM when used as a patch to reduce the severity of liver fibrosis Ricci et al 2013 Freezedried HAM does however have the advantage of allowing storage and shipment at room temperature making handling much easier

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