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.

Manipulation of hydrogel assembly and growth factor delivery via the use of peptide–polysaccharide interactions

“…In addition to the rheological properties of these materials, the reported bFGF release rates from these hydrogels are also suggested to be useful, as they are comparable to those previously reported to be useful for neovascularization in vivo [50,[67][68][69]. The increased rate of bFGF release relative to that observed for the previously reported PEG-LWMH/PEG-HIP hydrogel [11] is directly related to variations in the mechanical properties between these two classes of noncovalently assembled hydrogels and demonstrates the versatility of these compositions for mediating growth factor release.…”

“…Following Eqs. (A2) and (A3), the dissociation constant K d can be calculated from the following equation [68]:…”

Approaches for Functional Modification or Cross‐Linking of Chitosan

“…Radiation polymerization has been utilized by researchers to obtain IPNs for drug delivery applications [80][81][82][83]. Also, photo-cross-linkable chitosan was developed by introducing azide and lactose moieties on chitosan through a condensation reaction [84], and these hydrogels found applications in the release of fibroblast growth factors and heparin [85].…”

Attenuation of Limb Loss in an Experimentally Induced Hindlimb Ischemic Model by Fibroblast Growth Factor-2/Fragmin/Protamine Microparticles as a Delivery System

“…22,23 Heparin and low-molecular-weight heparin (LMWH) can interact with a variety of functional proteins such as growth factors, cytokines, extracellular matrix components, and cell adhesion molecules. 24 Therefore, heparin has been used as a therapeutic agent for treating diseases caused by either a deficiency or an overexpression of these proteins, although the administration of high-dose heparin may increase the risk of bleeding. 25 The LMWH (fragmin) has several pharmacological and practical advantages compared with heparin.…”