Disulfide-crosslinked hyaluronan-gelatin hydrogel films: a covalent mimic of the extracellular matrix for in vitro cell growth.

A new disulfide crosslinking method was developed for the preparation of blended hyaluronan (HA)-gelatin hydrogels to form a synthetic, covalently linked mimic of the extracellular matrix (ECM). The HA and gelatin were chemically modified using 3,3'-dithiobis(propionic hydrazide) (DTP). After reduction with dithiothreitol (DTT), the thiol derivatives of HA (HA-DTPH) and gelatin (gelatin-DTPH) were obtained and characterized. To minimize interference with biological function, the degree of substitution of HA-DTPH and gelatin-DTPH was kept below 50%. Solutions of HA-DTPH and gelatin-DTPH in varying blends (20%, 40%, 60%, 80% gelatin) were prepared in 1% w/v NaCl and crosslinked by disulfide bond formation in air. Hydrogel films were dried and further crosslinked with dilute hydrogen peroxide. Disulfide crosslinked HA-DTPH, gelatin-DTPH, and blends thereof, were degradable enzymatically by collagenase and by hyaluronidase (HAse). The rapid digestion of the crosslinked 100% gelatin-DTPH film by collagenase was significantly retarded by the presence of 20% or 40% HA-DTPH. Addition of at least 40% w/v gelatin into the 100% HA-DTPH films significantly improved the attachment and spreading of Balb/c 3T3 murine fibroblasts seeded on the surface of the hydrogel. These results demonstrate that disulfide-crosslinked HA-gelatin hydrogels, a new type of covalent synthetic ECM, constitute biocompatible and biodegradable substrata for cell culture in vitro.

[1]  D. Piacquadio,et al.  Evaluation of hylan b gel as a soft-tissue augmentation implant material. , 1997, Journal of the American Academy of Dermatology.

[2]  D. Mooney,et al.  Hydrogels for tissue engineering. , 2001, Chemical Reviews.

[3]  F. Nicolas,et al.  Denatured thiolated collagen. I. Synthesis and characterization. , 1997, Biomaterials.

[4]  A. Kenchington.  Chemical modification of the side chains of gelatin. , 1958, The Biochemical journal.

[5]  N. Yui,et al.  Hyaluronic acid grafted with poly(ethylene glycol) as a novel peptide formulation. , 1999, Journal of controlled release : official journal of the Controlled Release Society.

[6]  P. Band.  Hyaluronan Derivatives: Chemistry and Clinical Applications , 1998 .

[7]  D. Nance,et al.  Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility [published erratum appears in J Cell Biol 1992 Aug;118(3):753] , 1992, The Journal of cell biology.

[8]  S. Shalaby,et al.  Efficacy of hyaluronic acid/nonsteroidal anti-inflammatory drug systems in preventing postsurgical tendon adhesions. , 1997, Journal of biomedical materials research.

[9]  D. Aeschlimann,et al.  New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels. , 1999, Journal of biomedical materials research.

[10]  S. Patai.  The chemistry of the thiol group. , 1974 .

[11]  G. Prestwich,et al.  Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[12]  V. Hentz,et al.  Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine. , 2000, Tissue engineering.

[13]  E. Turley,et al.  HA receptors: Regulators of signalling to the cytoskeleton , 1996, Journal of cellular biochemistry.

[14]  W. Knudson,et al.  Cartilage proteoglycans. , 2001, Seminars in cell & developmental biology.

[15]  Yoshito Ikada,et al.  Crosslinking of hyaluronic acid with glutaraldehyde , 1997 .

[16]  Andrew A. Pitsillides,et al.  An Essential Role for the Interaction Between Hyaluronan and Hyaluronan Binding Proteins During Joint Development , 1998, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[17]  G. Prestwich,et al.  Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing. , 2002, Biomaterials.

[18]  S. Aiba,et al.  Cell growth on poly(vinyl alcohol) hydrogel membranes containing biguanido groups. , 1994, Journal of biomedical materials research.

[19]  M. Radice,et al.  Semisynthetic resorbable materials from hyaluronan esterification. , 1998, Biomaterials.

[20]  Y. Nakayama,et al.  Thermoresponsive artificial extracellular matrix for tissue engineering: hyaluronic acid bioconjugated with poly(N-isopropylacrylamide) grafts. , 2001, Biomacromolecules.

[21]  Y. M. Lee,et al.  Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. , 1999, Journal of biomedical materials research.

[22]  E. Balazs,et al.  The chemistry, biology and medical applications of hyaluronan and its derivatives , 1998 .

[23]  G. Prestwich,et al.  Hyaluronate derivatives in drug delivery. , 1998, Critical reviews in therapeutic drug carrier systems.

[24]  T. Yoldemir,et al.  Comparison of the reduction of postoperative adhesions by two barriers, one solution, and two pharmacologic agents in the rat uterine model. , 2002, Fertility and sterility.

[25]  G. Prestwich,et al.  Functionalized derivatives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials. , 1994, Bioconjugate chemistry.

[26]  K. Zhu,et al.  Polyanion/gelatin complexes as pH‐sensitive gels for controlled protein release , 2001 .

[27]  Ikada,et al.  Protein release from gelatin matrices. , 1998, Advanced drug delivery reviews.

[28]  K. Zhu,et al.  Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release. , 2001, International journal of pharmaceutics.

[29]  F. Nicolas,et al.  Denatured thiolated collagen. II. Cross-linking by oxidation. , 1997, Biomaterials.

[30]  Y. Ikada,et al.  Crosslinking of hyaluronic acid with water-soluble carbodiimide. , 1997, Journal of biomedical materials research.

[31]  T. Tanabe,et al.  Films of collagen crosslinked by S-S bonds: preparation and characterization. , 2001, Biomaterials.

[32]  R. Benesch.  THIOLATION OF PROTEINS. , 1958, Proceedings of the National Academy of Sciences of the United States of America.

[33]  N. Minoura,et al.  Attachment and growth of cultured fibroblast cells on PVA/chitosan-blended hydrogels. , 1998, Journal of biomedical materials research.

[34]  J. Fraser,et al.  Hyaluronan: its nature, distribution, functions and turnover , 1997, Journal of internal medicine.

[35]  H. Scheraga,et al.  Analysis for disulfide bonds in peptides and proteins. , 1987, Methods in enzymology.

[36]  J. Porter,et al.  A modification of the Ellman procedure for the estimation of protein sulfhydryl groups. , 1967, Archives of biochemistry and biophysics.

[37]  P. Dubin,et al.  Effects of protein charge heterogeneity in protein-polyelectrolyte complexation , 1992 .

[38]  A. Ramamurthi,et al.  Smooth muscle cell adhesion on crosslinked hyaluronan gels. , 2002, Journal of biomedical materials research.

[39]  J. Hubbell,et al.  Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids. , 2001, Bioconjugate chemistry.

[40]  Glenn D Prestwich,et al.  Disulfide cross-linked hyaluronan hydrogels. , 2002, Biomacromolecules.

[41]  G. Prestwich,et al.  Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid. , 1997, Bioconjugate chemistry.

[42]  G. Prestwich,et al.  Profiles Therapeutic applications of hyaluronic acid and hyaluronan derivatives , 1998 .

[43]  G. Ellman.  A colorimetric method for determining low concentrations of mercaptans. , 1958, Archives of biochemistry and biophysics.

[44]  M. Diamond,et al.  Reduction of postsurgical adhesion formation in the rabbit uterine horn model with use of hyaluronate/carboxymethylcellulose gel. , 1998, Fertility and sterility.

[45]  Jeffrey A. Hubbell,et al.  Biomaterials in Tissue Engineering , 1995, Bio/Technology.

[46]  S. Avrameas,et al.  Properties of protein polymers as substratum for cell growth in vitro , 1974, Journal of cellular physiology.

[47]  W. Hunter,et al.  Paclitaxel-Loaded Crosslinked Hyaluronic Acid Films for the Prevention of Postsurgical Adhesions , 2002, Pharmaceutical Research.

[48]  T. Cruz,et al.  Receptor for hyaluronan-mediated motility (RHAMM), a hyaladherin that regulates cell responses to growth factors. , 1999, Biochemical Society transactions.