title: Controlled release of fibroblast growth factors and heparin from photocrosslinked chitosan hydrogels and subsequent effect on in vivo vascularization.
abstract: Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.
authors.affiliation: National Defense Medical College, Research Institute, Division of Biomedical Engineering, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. ishihara@res.ndmc.ac.jp
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishihara
authors.forename: Kiyohaya
authors.initials: K
authors.lastname: Obara
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Masanori
authors.initials: M
authors.lastname: Fujita
authors.forename: Masato
authors.initials: M
authors.lastname: Sato
authors.forename: Kazunori
authors.initials: K
authors.lastname: Masuoka
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Saito
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yura
authors.forename: Takemi
authors.initials: T
authors.lastname: Matsui
authors.forename: Hidemi
authors.initials: H
authors.lastname: Hattori
authors.forename: Makoto
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Akira
authors.initials: A
authors.lastname: Kurita
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D048271:Chitosan; D004396:Coloring Agents / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005260:Female; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D020100:Hydrogels / Q000737:chemistry / Q000378:metabolism*; D020007:Implants, Experimental; D008422:Materials Testing; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D001672:Biocompatible Materials; D001704:Biopolymers; D004396:Coloring Agents; D003432:Cross-Linking Reagents; D020100:Hydrogels; D002686:Chitin; D012710:Serum Albumin, Bovine; D005346:Fibroblast Growth Factors; D006493:Heparin; D048271:Chitosan; D014343:Trypan Blue