Ultrathin chitosan-poly(ethylene glycol) hydrogel films for corneal tissue engineering.

Due to the high demand for donor corneas and their low supply, autologous corneal endothelial cell (CEC) culture and transplantation for treatment of corneal endothelial dysfunction would be highly desirable. Many studies have shown the possibility of culturing CECs in vitro, but lack potential robust substrates for transplantation into the cornea. In this study, we investigate the properties of novel ultrathin chitosan-poly(ethylene glycol) (PEG) hydrogel films (CPHFs) for corneal tissue engineering applications. Cross-linking of chitosan films with diepoxy-PEG and cystamine was employed to prepare ~50 μm (hydrated) hydrogel films. Through variation of the PEG content (1.5-5.9 wt.%) it was possible to tailor the CPHFs to have tensile strains and ultimate stresses identical to or greater than those of human corneal tissue while retaining similar tensile moduli. Light transmission measurements in the visible spectrum (400-700 nm) revealed that the films were >95% optically transparent, above that of the human cornea (maximum ~90%), whilst in vitro degradation studies with lysozyme revealed that the CPHFs maintained the biodegradable characteristics of chitosan. Cell culture studies demonstrated the ability of the CPHFs to support the attachment and proliferation of sheep CECs. Ex vivo surgical trials on ovine eyes demonstrated that the CPHFs displayed excellent characteristics for physical manipulation and implantation purposes. The ultrathin CPHFs display desirable mechanical, optical and degradation properties whilst allowing attachment and proliferation of ovine CECs, and as such are attractive candidates for the regeneration and transplantation of CECs, as well as other corneal tissue engineering applications.

[1]  G. Hsiue,et al.  Carbodiimide cross-linked hyaluronic acid hydrogels as cell sheet delivery vehicles: characterization and interaction with corneal endothelial cells , 2008, Journal of biomaterials science. Polymer edition.

[2]  C. Ladavière,et al.  Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers. , 2008, Biomacromolecules.

[3]  Anton Blencowe,et al.  Epoxy-amine synthesised hydrogel scaffolds for soft-tissue engineering. , 2010, Biomaterials.

[4]  P. Mahadevan,et al.  An overview , 2007, Journal of Biosciences.

[5]  T. Okano,et al.  Functional human corneal endothelial cell sheets harvested from temperature‐responsive culture surfaces , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  K. Shakesheff,et al.  Controlling Biological Interactions with Poly(lactic acid) by Surface Entrapment Modification , 2001 .

[7]  J. Vacanti,et al.  Ocular cell monolayers cultured on biodegradable substrates. , 1999, Tissue engineering.

[8]  Changsheng Liu,et al.  Preparation and characterization of chitosan/PEG/gelatin composites for tissue engineering , 2009 .

[9]  M. Araie,et al.  Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. , 2004, Investigative ophthalmology & visual science.

[10]  D. Larkin,et al.  The first successful full-thickness corneal transplant: a commentary on Eduard Zirm’s landmark paper of 1906 , 2006, British Journal of Ophthalmology.

[11]  K. Healy,et al.  Synthesis and characterization of injectable poly(N-isopropylacrylamide-co-acrylic acid) hydrogels with proteolytically degradable cross-links. , 2003, Biomacromolecules.

[12]  S. Pasternak,et al.  Pharmaceutical applications , 1993, Current Opinion in Biotechnology.

[13]  M. Riley.  Anion-sensitive ATPase in rabbit corneal endothelium and its relation to corneal hydration. , 1977, Experimental eye research.

[14]  Renato Ambrósio,et al.  Pentacam characterization of corneas with Fuchs dystrophy treated with Descemet membrane endothelial keratoplasty. , 2010, Journal of refractive surgery.

[15]  A. Quantock,et al.  Ultraviolet light transmission through the human corneal stroma is reduced in the periphery. , 2012, Biophysical journal.

[16]  P. Arratia,et al.  Absence of filamin A prevents cells from responding to stiffness gradients on gels coated with collagen but not fibronectin. , 2009, Biophysical journal.

[17]  E. Pels,et al.  A surgical technique for posterior lamellar keratoplasty. , 1998, Cornea.

[18]  Ratana Jiraratananon,et al.  Modification of PVDF membrane by chitosan solution for reducing protein fouling , 2009 .

[19]  M. Macsai,et al.  Suture Technique for Descemet Stripping and Endothelial Keratoplasty , 2007, Cornea.

[20]  J. V. van Best,et al.  Light transmission of the cornea in whole human eyes. , 1990, Experimental eye research.

[21]  J. Rubin,et al.  Novel multiarm PEG-based hydrogels for tissue engineering. , 2009, Journal of biomedical materials research. Part A.

[22]  M. Taha,et al.  Epoxy-Diamine Thermoset/Thermoplastic Blends. 1. Rates of Reactions before and after Phase Separation , 1999 .

[23]  J. Dart,et al.  Amniotic membrane transplantation for ocular disease: a review of the first 233 cases from the UK user group , 2007, British Journal of Ophthalmology.

[24]  Jian-wen Wang,et al.  Sugar-mediated chitosan/poly(ethylene glycol)-beta-dicalcium pyrophosphate composite: mechanical and microstructural properties. , 2003, Journal of biomedical materials research. Part A.

[25]  E. Wylęgała,et al.  Monitoring Cornea and Graft Morphometric Dynamics After Descemet Stripping and Endothelial Keratoplasty With Anterior Segment Optical Coherence Tomography , 2010, Cornea.

[26]  P. Supaphol,et al.  Dilute solution properties of hexanoyl chitosan in chloroform, dichloromethane, and tetrahydrofuran , 2006 .

[27]  Kinam Park,et al.  In vitro and in vivo test of PEG/PCL-based hydrogel scaffold for cell delivery application. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[28]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  F. Price,et al.  Descemet's stripping with endothelial keratoplasty in 50 eyes: a refractive neutral corneal transplant. , 2005, Journal of refractive surgery.

[30]  J. Hubbell,et al.  Surface characteristics and biocompatibility of lactide-based poly(ethylene glycol) scaffolds for tissue engineering. , 1998, Journal of biomaterials science. Polymer edition.

[31]  Riccardo A.A. Muzzarelli,et al.  Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids , 2009 .

[32]  C. Viappiani,et al.  Ligand reactivity and allosteric regulation of hemoglobin-based oxygen carriers. , 2008, Biochimica et biophysica acta.

[33]  W. Berthold,et al.  Polyethylene glycol-conjugated pharmaceutical proteins , 1998 .

[34]  N. Joyce.  Proliferative capacity of the corneal endothelium , 2003, Progress in Retinal and Eye Research.

[35]  P. Eggli,et al.  Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope. , 1999, Cornea.

[36]  C. Hunt,et al.  Amniotic membrane grafts, “fresh” or frozen? A clinical and in vitro comparison , 2001, The British journal of ophthalmology.

[37]  X. D. Wang,et al.  The controlling biodegradation of chitosan fibers by N-acetylation in vitro and in vivo , 2007, Journal of materials science. Materials in medicine.

[38]  C. Jordan,et al.  Graft rejection episodes after Descemet stripping with endothelial keratoplasty: part one: clinical signs and symptoms , 2008, British Journal of Ophthalmology.

[39]  Z. Ranjbar,et al.  Preparation and stability behavior of the colloidal epoxy-1,1-iminodi-2-propanol adducts , 2011 .

[40]  S. Dravida,et al.  Recombinant human collagen for tissue engineered corneal substitutes. , 2008, Biomaterials.

[41]  Micah Dembo,et al.  Cell-cell mechanical communication through compliant substrates. , 2008, Biophysical journal.

[42]  R. Muzzarelli.  Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone , 2009 .

[43]  G. Hsiue,et al.  Bioengineered human corneal endothelium for transplantation. , 2006, Archives of ophthalmology.

[44]  G. Betageri,et al.  Water Soluble Polymers for Pharmaceutical Applications , 2011 .

[45]  J. Rubin,et al.  Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration , 2010, Organogenesis.

[46]  I. Bahar,et al.  Busin Guide vs Forceps for the Insertion of the Donor Lenticule in Descemet Stripping Automated Endothelial Keratoplasty. , 2009, American journal of ophthalmology.

[47]  Fengfu Li,et al.  PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering. , 2008, Biomaterials.

[48]  Alan Colman,et al.  Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. , 2011, Transplantation.

[49]  Alain Domard,et al.  The use of physical hydrogels of chitosan for skin regeneration following third-degree burns. , 2007, Biomaterials.

[50]  M. Qureshi,et al.  Experience and 12-Month Results of Descemet-Stripping Endothelial Keratoplasty (DSEK) with a Small-Incision Technique , 2007, Cornea.

[51]  A. Quantock,et al.  Light transmission in the human cornea as a function of position across the ocular surface: theoretical and experimental aspects. , 2008, Biophysical journal.

[52]  N. Fullwood,et al.  Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. , 2004, Investigative ophthalmology & visual science.

[53]  J. Mehta,et al.  The variation in transparency of amniotic membrane used in ocular surface regeneration , 2009, British Journal of Ophthalmology.

[54]  Y. Inoue,et al.  Preparation and characterization of PEG-cross-linked chitosan hydrogel films with controllable swelling and enzymatic degradation behavior , 2010 .

[55]  G. Melles,et al.  Descemet membrane endothelial keratoplasty (DMEK). , 2006, Cornea.

[56]  Xiaojun Ma,et al.  The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation. , 2005, Carbohydrate research.

[57]  Bochu Wang,et al.  Biodegradation of Silk Biomaterials , 2009, International journal of molecular sciences.

[58]  C. Pillai,et al.  Chitin and chitosan polymers: Chemistry, solubility and fiber formation , 2009 .

[59]  Xun Yan,et al.  Chitosan analysis using acid hydrolysis and HPLC/UV , 2012 .

[60]  S. Neau,et al.  In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation. , 2001, Biomaterials.

[61]  S. Klebe,et al.  PROLONGATION OF SHEEP CORNEAL ALLOGRAFT SURVIVAL BY EX VIVO TRANSFER OF THE GENE ENCODING INTERLEUKIN-101 , 2001, Transplantation.

[62]  Victor H Barocas,et al.  Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent. , 2006, Tissue engineering.

[63]  Y. Inoue,et al.  Characterization and enzymatic degradation of PEG‐cross‐linked chitosan hydrogel films , 2009 .

[64]  Baoqin Han,et al.  Fabrication and characters of a corneal endothelial cells scaffold based on chitosan , 2011, Journal of materials science. Materials in medicine.

[65]  Walter Steurbaut,et al.  Chitosan as antimicrobial agent: applications and mode of action. , 2003, Biomacromolecules.

[66]  J. Feijen,et al.  Injectable chitosan-based hydrogels for cartilage tissue engineering. , 2009, Biomaterials.

[67]  T. Wood,et al.  ATPase pump site density in human dysfunctional corneal endothelium. , 1987, Investigative ophthalmology & visual science.

[68]  J. Pascault,et al.  Do epoxy-amine networks become inhomogeneous at the nanometric scale? , 2003 .

[69]  H. Klassen,et al.  Aqueous Humor Enhances the Proliferation of Rat Retinal Precursor Cells in Culture, and This Effect Is Partially Reproduced by Ascorbic Acid , 2006, Stem cells.

[70]  Traian V Chirila,et al.  Human corneal endothelial cell growth on a silk fibroin membrane. , 2011, Biomaterials.

[71]  Y. Tabata,et al.  Effect of charge and molecular weight on the functionality of gelatin carriers for corneal endothelial cell therapy. , 2006, Biomacromolecules.

[72]  C. Laurencin,et al.  Biologically active chitosan systems for tissue engineering and regenerative medicine. , 2008, Current topics in medicinal chemistry.

[73]  K. Yurdakoç,et al.  Synthesis, characterization, and enzymatic degradation of chitosan/PEG hydrogel films , 2011 .

[74]  S. Richer,et al.  Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide , 1998, Vision Research.

[75]  Won-Gun Koh,et al.  Preparation of protein microarrays on non‐fouling and hydrated poly(ethylene glycol) hydrogel substrates using photochemical surface modification , 2009 .

[76]  S. Dravida,et al.  Tissue-engineered recombinant human collagen-based corneal substitutes for implantation: performance of type I versus type III collagen. , 2008, Investigative ophthalmology & visual science.

[77]  S. Oh,et al.  In vitro and in vivo degradation behavior of acetylated chitosan porous beads , 2008, Journal of biomaterials science. Polymer edition.

[78]  I. Dapena,et al.  Endothelial keratoplasty: DSEK/DSAEK or DMEK - the thinner the better? , 2009, Current opinion in ophthalmology.

[79]  Sanjay V. Patel,et al.  Host and graft thickness after Descemet stripping endothelial keratoplasty for Fuchs endothelial dystrophy. , 2010, American journal of ophthalmology.

[80]  T. Cheng,et al.  Therapeutic potential of chitosan and its derivatives in regenerative medicine. , 2006, The Journal of surgical research.

[81]  H. Sheardown,et al.  Glucose permeable poly (dimethyl siloxane) poly (N-isopropyl acrylamide) interpenetrating networks as ophthalmic biomaterials. , 2005, Biomaterials.

[82]  Miqin Zhang,et al.  PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[83]  Peng Zhao,et al.  In-situ forming biodegradable glycol chitosan-based hydrogels: Synthesis, characterization, and chondrocyte culture. , 2012, Materials science & engineering. C, Materials for biological applications.

[84]  Kwideok Park,et al.  Peptide-grafted lactide-based poly(ethylene glycol) porous scaffolds for specific cell adhesion , 2010 .

[85]  Gary L. Bowlin,et al.  The Use of Natural Polymers in Tissue Engineering: A Focus on Electrospun Extracellular Matrix Analogues , 2010 .

[86]  K. Engelmann,et al.  Prospects for endothelial transplantation. , 2004, Experimental eye research.

[87]  D. Drexler,et al.  Transplantation of adult human or porcine corneal endothelial cells onto human recipients in vitro. Part I: Cell culturing and transplantation procedure. , 1999, Cornea.

[88]  T. Wood,et al.  Transplantation of Corneal Endothelial Cells Using a Cell Carrier Device , 1994, Cornea.

[89]  F. Price,et al.  Descemet Membrane Endothelial Keratoplasty , 2010, International ophthalmology clinics.