Epidermal growth factor: layered silicate nanocomposites for tissue regeneration.

Wound healing is a complex, multistep process that can be summarized into three stages, namely, hemostasis and inflammation, proliferation, and finally, tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we functionalized Na-MMT with epidermal growth factor (EGF) via ion exchange reaction to create a nanocomposite (MMT-EGF) with approximately 0.004 EGF molecules per Na(+) exchange site and conduct biochemical analyses of keratinocytes after treatment with MMT-EGF. Our results demonstrate that EGF immobilized on MMT retains the ability to activate the epidermal growth factor receptor (EGRF), causing phosphorylation of the AKT and MEK1 pathways, as well as upregulation of its downstream target gene expression involved in cell growth and migration. This study also shows that like EGF, MMT-EGF treatment can stimulate cell migration in vitro, which is dependent on ERK1/2 phosphorylation.

[1]  N. Kotov,et al.  Polyelectrolyte‐Clay‐Protein Layer Films on Microfluidic PDMS Bioreactor Surfaces for Primary Murine Bone Marrow Culture , 2007 .

[2]  Yiling Lu,et al.  Exploiting the PI3K/AKT Pathway for Cancer Drug Discovery , 2005, Nature Reviews Drug Discovery.

[3]  E. Krebs,et al.  The MAPK signaling cascade , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  K. Madison,et al.  Barrier function of the skin: "la raison d'être" of the epidermis. , 2003, The Journal of investigative dermatology.

[5]  Hasmukh A. Patel,et al.  Montmorillonite intercalated with vitamin B1 as drug carrier , 2009 .

[6]  F. Arnaud,et al.  Exothermic Reaction in Zeolite Hemostatic Dressings: QuikClot ACS and ACS+® , 2008, Annals of Biomedical Engineering.

[7]  Kam W Leong,et al.  In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF). , 2008, Biomaterials.

[8]  Peter Rhee,et al.  Hemorrhage control in the battlefield: role of new hemostatic agents. , 2005, Military medicine.

[9]  G R Tobin,et al.  Physiology and healing dynamics of chronic cutaneous wounds. , 1998, American journal of surgery.

[10]  Yongjin Li,et al.  Insights into Montmorillonite Nanoclay Based ex Situ Nanocomposites from SEBS and Modified SEBS by Small-Angle X-ray Scattering and Modulated DSC Studies , 2008 .

[11]  Kwok-Fai So,et al.  Nano hemostat solution: immediate hemostasis at the nanoscale. , 2006, Nanomedicine : nanotechnology, biology, and medicine.

[12]  David L Kaplan,et al.  In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth. , 2008, Biomaterials.

[13]  G. Thiel,et al.  Epidermal growth factor and thrombin induced proliferation of immortalized human keratinocytes is coupled to the synthesis of Egr‐1, a zinc finger transcriptional regulator , 2002, Journal of cellular biochemistry.

[14]  Timothy J. Griffin,et al.  Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40 , 2007, Nature Cell Biology.

[15]  K. Ting,et al.  Skin repair and scar formation: the central role of TGF-β , 2003, Expert Reviews in Molecular Medicine.

[16]  James T. Elder,et al.  Differential regulation of EGF-like growth factor genes in human keratinocytes. , 1999, Biochemical and biophysical research communications.

[17]  H. Larjava,et al.  HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha. , 2006, Experimental cell research.

[18]  C. Caramella,et al.  Use of clays as drug delivery systems: Possibilities and limitations , 2007 .

[19]  D. Schmaljohann,et al.  Epidermal growth factor therapy and wound healing--past, present and future perspectives. , 2008, The surgeon : journal of the Royal Colleges of Surgeons of Edinburgh and Ireland.

[20]  N. Zheng,et al.  Controlling bioprocesses with inorganic surfaces: Layered clay hemostatic agents , 2007 .

[21]  A. Alonso,et al.  EGFR family-mediated signal transduction in the human keratinocyte cell line HaCaT. , 1999, Experimental cell research.

[22]  James T. Elder,et al.  EGF stimulates transcription of CaN19 (S100A2) in HaCaT keratinocytes. , 1998, The Journal of investigative dermatology.

[23]  Miki Ebisuya,et al.  ERK Activation Propagates in Epithelial Cell Sheets and Regulates Their Migration during Wound Healing , 2004, Current Biology.

[24]  M. Kadakia,et al.  p73 is essential for vitamin D mediated osteoblastic differentiation , 2009, Cell Death and Differentiation.

[25]  Xianzhong Xiao,et al.  Anti‐apoptotic role of EGF in HaCaT keratinocytes via a PPARβ‐dependent mechanism , 2008, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[26]  I. Marenholz,et al.  Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21. , 1996, The Journal of investigative dermatology.

[27]  P B Deasy,et al.  Characterisation of halloysite for use as a microtubular drug delivery system. , 2002, International journal of pharmaceutics.

[28]  Jun Liang,et al.  Tramadol hydrochloride/montmorillonite composite: Preparation and controlled drug release , 2010 .

[29]  G. Sharma,et al.  p38 and ERK1/2 Coordinate Cellular Migration and Proliferation in Epithelial Wound Healing , 2003, Journal of Biological Chemistry.

[30]  L. Calamai,et al.  Interaction of horseradish peroxidase with montmorillonite homoionic to Na+ and Ca2+: effects on enzymatic activity and microbial degradation. , 2001 .

[31]  R. Vaia,et al.  Flexible Silk–Inorganic Nanocomposites: From Transparent to Highly Reflective , 2010 .

[32]  Xiaobing Fu,et al.  Engineered growth factors and cutaneous wound healing: Success and possible questions in the past 10 years , 2005, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[33]  Paul Martin,et al.  Wound Healing--Aiming for Perfect Skin Regeneration , 1997, Science.

[34]  M. Shieh,et al.  A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier. , 2002, Biomaterials.

[35]  L. Hudson,et al.  Contributions of the epidermal growth factor receptor to keratinocyte motility , 1998, Microscopy research and technique.

[36]  S. Sugunan,et al.  Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10 , 2006 .

[37]  Chuanshu Huang,et al.  VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway , 2006, Respiratory research.

[38]  J. Kennedy,et al.  Cytotoxic effects induced by unmodified and organically modified nanoclays in the human hepatic HepG2 cell line , 2011, Journal of applied toxicology : JAT.

[39]  Jared G Clay,et al.  Comparative testing of new hemostatic agents in a swine model of extremity arterial and venous hemorrhage. , 2010, Military medicine.

[40]  S. D'souza,et al.  Signalling In The Epidermis: The E2f Cell Cycle Regulatory Pathway In Epidermal Morphogenesis, Regeneration And Transformation , 2005, International journal of biological sciences.

[41]  Laura A. Sowards,et al.  Patterned silk films cast from ionic liquid solubilized fibroin as scaffolds for cell growth. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[42]  T. Kealey,et al.  Id proteins are dynamically expressed in normal epidermis and dysregulated in squamous cell carcinoma. , 2000, Cancer research.

[43]  R. Jope,et al.  The glamour and gloom of glycogen synthase kinase-3. , 2004, Trends in biochemical sciences.

[44]  A. Singer,et al.  Cutaneous wound healing. , 1999, The New England journal of medicine.