Acidic Hydrogel Research Articles

008 The Effect of the Gelatin Sheet Containing bFGF on Wound Healing

Aim: It is well recognized that bFGF accelerates proliferation of almost all cells concerned with wound healing and there is a report that bFGF was well sorbed with time to the acidic gelatin hydrogel with isoelectric points of 5.0. We investigated the effect of the acidic gelatin sheet containg bFGF. Methods: Full thickness defects of skin (1.5 × 1.5 cm) were created on the backs of mice.1) The wounds were covered with gelatin sheets (2 × 2 cm)containg bFGF(100 ìg/site), (A), and without bFGF(B). The concentration of bFGF in plasma was estimated by ELISA. 2) The wounds were covered with A, B and hydrogel dressing(control group, C) and wound area was measured with computer planimeter and neoepithelium was observed using the light microscope. Results: 1) The concentration of bFGF in plasma in (A) was statistically greater than (B) by the 7th day. 2) Statistically smaller wound area was found 2 weeks postoperatively in (A) than in (C) and (D). Neoepithelium from edge of the wound was statistically longer in (A) than in (C). Conclusions: Controlled release of bFGF from the acidic gelatin sheet was fouhd and acidic gelatin sheet containg bFGF promoted neoepithelization and wound closure. The acidic gelatin sheet containg bFGF was thought to be effective on wound healing.

Towards retrievable vascularized bioartificial pancreas: induction and long-lasting stability of polymeric mesh implant vascularized with the help of acidic and basic fibroblast growth factors and hydrogel coating.

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-β1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-β1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.

Intramyocardial delivery of basic fibroblast growth factor-impregnated gelatin hydrogel microspheres enhances collateral circulation to infarcted canine myocardium.

The present study examined whether basic fibroblast growth factor (bFGF)-impregnated acidic gelatin hydrogel microspheres (AGHM) would enhance collateral development to the infarct area in dogs with coronary occlusion. Studies were conducted in 28 dogs with a 2-week occlusion of the proximal left anterior descending coronary artery (LAD). The dogs were divided into 3 groups according to treatment: Group A treated with bFGF-impregnated AGHM in the infarct area; Group B with free-form bFGF; Group C with AGHM alone. Coronary angiography (n=15; Group A, 7 dogs; Group B, 5 dogs; Group C, 3 dogs) and a regional myocardial blood flow study (n=13; Group A, 6 dogs; Group B, 4 dogs; Group C, 3 dogs) were repeated at a 2-week interval. Coronary angiography revealed that in Group A, antegrade flow in the LAD distal to the occlusion, which was assessed by Thrombolysis in Myocardial Infarction (TIMI) grade, was significantly increased after treatment. In contrast, in Groups B and C, the treatment did not change the flow grade in the LAD. In Group A, the regional myocardial blood flow in the collateral dependent area was significantly increased after treatment, and the regional myocardial blood flow reserve after adenosine injection was also significantly increased. These measurements remained after treatment in Groups B and C. The immunohistochemical study with factor VIII-related antigen revealed an increase of vascular density in the ischemic region in Group A. Intramyocardial delivery of bFGF-impregnated AGHM, but not free-form bFGF, improves the collateral circulation to the infarct area of a coronary occlusion in dogs.

Bone regeneration by transforming growth factor β1 released from a biodegradable hydrogel

This paper describes the sustained release of transforming growth factor β1 (TGF-β1) from a biodegradable hydrogel based on polyion complexation for the enhancement of bone regeneration activity. Basic TGF-β1 was adsorbed onto the biodegradable hydrogel of acidic gelatin with an isoelectric point of 5.0 by an electrostatic interaction. The TGF-β1 could not be adsorbed onto basic gelatin. When acidic gelatin hydrogels incorporating 125I-labeled TGF-β1 were implanted into the back subcutis of mice, the radioactivity decreased with time and the in vivo retention of TGF-β1 was prolonged with a decrease in the water content of hydrogels. The higher the water content of hydrogels, the faster their biodegradation. The in vivo retention of TGF-β1 correlated well with that of gelatin hydrogels, indicating that TGF-β1 was released from the gelatin hydrogel as a result of hydrogel biodegradation. The ability of TGF-β1-incorporated into acidic gelatin hydrogels to induce bone regeneration was evaluated in a rabbit calvarial defect model. Eight weeks after treatment, the gelatin hydrogels with water contents of 90 and 95 wt% induced significantly high bone regeneration compared with those with lower and higher water contents and free TGF-β1. This indicates that the sustained release of TGF-β1 from the hydrogel with suitable in vivo degradability is necessary to effectively enhance its osteoinductive function. Rapid hydrogel degradation will result in a retention time of TGF-β1 which is too short to induce bone regeneration. It is possible that the slow degradation of the hydrogel physically blocked TGF-β1-induced bone regeneration at the skull defect. It can be concluded that the gelatin hydrogel is a promising matrix of TGF-β1 release to induce skull bone regeneration.

Experimental Corneal Neovascularization by Basic Fibroblast Growth Factor Incorporated into Gelatin Hydrogel

The study was designed to investigate the feasibility of using an acidic gelatin hydrogel as a biodegradable vehicle for basic fibroblast growth factor (bFGF). bFGF was incorporated by polyion complexation into a biodegradable hydrogel prepared by cross-linking acidic gelatin with the isoelectric point of 4.9. The dried hydrogel (sized to 2 × 1 mm) was hydrated with bFGF aqueous solution including different doses of bFGF (20, 50, 125, 250 and 500 ng) and implanted into a rabbit corneal pocket (2.5 × 2 mm). As a control group, the gelatin hydrogel without bFGF or bFGF alone (500 ng) was used. Corneal angiogenesis was evaluated by biomicroscopy, corneal fluorescein angiography and histology for 21 days. Photographs were taken and corneal angiogenesis was evaluated by image analysis. The hydrogel degraded with time after its implantation into the corneal pocket. Experimental eyes receiving the hydrogel containing more than 50 ng of bFGF demonstrated significant corneal angiogenesis. Control eyes and eyes receiving the hydrogel containing 20 ng of bFGF showed no corneal angiogenesis. Corneal angiogenesis, which occurred on the 3rd or 4th day after implantation, reached maximal growth on about day 7 and regressed from day 10 after implantation. The area of angiogenesis showed a dose-dependency on bFGF. The gelatin hydrogel itself induced neither angiogenesis nor inflammation. These results suggested that acidic gelatin hydrogel releases bioactive bFGF with its biodegradation, resulting in corneal neovascularization.

Growth Factor Release from Gelatin Hydrogel for Tissue Engineering

One of the key technologies for regeneration of damaged and lost tissue is the sustained release of biologically active growth factors. The present study was undertaken to investigate sorption and desorption of various growth factors from biodegradable hydrogels prepared through glutaraldehyde crosslinking of gelatin with isoelectric points (IEPs) of 5.0 and 9.0, which are named "acidic" and "basic" gelatins, respectively, based on their overall charge. Basic bFGF and TGF-β1 were markedly sorbed with time in the acidic gelatin hydrogels, while less sorption took place in the basic gelatin hydrogels. This behavior was explained in terms of an electrostatic interaction between the basic growth factors and the acidic gelatin. However, BMP-2 was sorbed into the acidic gelatin hydrogel to a lesser extent than the other two growth factors, even though its IEP is also greater than 7.0. An in vivo experiment revealed that the acidic gelatin hydrogel was degraded with time, while growth factors were retained in the body for a longer time period as the in vitro sorption to the acidic gelatin hydrogel was larger. These findings indicate that the growth factors sonically complexes to the acidic gelatin hydrogel were released in vivo as a result of hydrogel degradation. Furthermore, animal experiments revealed that the biological performance of growth factors was enhanced by their sustaned release, in marked contrast to the growth factors administered in the solution form.

Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities.

Biodegradable gelatin hydrogels were prepared through the glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 and the basic gelatin with an IEP of 9.0. The hydrogel water content was changed by the concentration of both gelatin and glutaraldehyde, used for hydrogel preparation. An aqueous solution of basic fibroblast growth factor (bFGF) was sorbed into the gelatin hydrogel freeze-dried to obtain a bFGF-incorporating gelatin hydrogel. Irrespective of the hydrogel water content, approximately 30% of the incorporated bFGF was released from the bFGF-incorporating acidic gelatin hydrogel, within the first day into phosphate-buffered saline solution at 37 degrees C, followed by no substantial release. Probably, the basic bFGF complexed with the acidic gelatin through poly-ion complexation would not be released under the in vitro non-degradation condition of gelatin. On the contrary, almost 100% of the incorporated bFGF was initially released from all types of basic gelatin hydrogels. This is due to the simple diffusion of bFGF because of no complexation between bFGF and the basic gelatin. When implanted subcutaneously into the mouse back, bFGF-incorporating acidic and basic gelatin hydrogels with higher water contents were degraded with time faster than those with lower water contents. Significant neovascularization was induced around the implanted site of the bFGF-incorporating acidic gelatin hydrogel. The induction period prolonged with the decrease in hydrogel water content. On the other hand, such a prolonged vascularization effect was not achieved by the bFGF-incorporating basic gelatin hydrogel and the hydrogel initially exhibited less enhanced effect, irrespective of the water content. These findings indicate that the controlled release of biologically active bFGF is caused by biodegradation of the acidic gelatin hydrogel, resulting in induction of vascularization effect dependent on the water content. It is possible that only the transient vascularization by the basic gelatin hydrogel is due to the initial large burst in bFGF release, probably because of the down regulation of bFGF receptor.

Effect of hydrophobicity of a drug on its release from hydrogels with different topological structures

The effect of the topological structure; that is, the network heterogeneity, of hydrophobically modified, slightly acidic hydrogels on the binding and release of low molar mass drugs has been studied using ibuprofen and ephedrine as model compounds with varying water solubility. The difference in the heterogeneity of the gels has been produced by the choice of the hydrophobe copolymerized into the polymer network. The effect of the drug loading on the release kinetics has been investigated as well. The release of hydrophobic ibuprofen was slower from a strongly aggregated heterogeneous gel than from a more homogeneous one, because of the strong hydrophobic interaction between ibuprofen and the heterogeneous hydrogel. The release of hydrophilic ephedrine from the homogeneous gel with an initial drug content of 30 wt % of dry polymer showed negative time dependence, indicating that during and after the swelling of the gel, ephedrine started to bind to the polymer. However, the release of ephedrine from a heterogeneous hydrogel increased with time. This shows that the heterogeneous, aggregated polymer binds the hydrophobic substance more strongly than the homogeneous one does, and that the homogeneous network has higher affinity for the basic hydrophilic substance than the heterogeneous one does. The loading contents of ibuprofen and ephedrine affect the release rates in different ways because of the different binding and release mechanisms. The number of binding sites accessible for ephedrine inside the polymer network is assumed to change upon the swelling of the gel. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1031–1039, 1999

ポリイオンコンプレックスを利用したヒドロゲルからの種々の細胞増殖因子の徐放化

One of the key technologies for regenerations of damaged and lost tissues is the controlled release of biologically active growth factors. The present study was undertaken to investigate sorption and desorption of various biologically active growth factors from hydrogels prepared through glutaraldehyde crosslinking of gelatin with isoelectric points (IEPs) of 5.0 and 9.0, which are named “acidic” and “basic” gelatins, respectively, because of their electric feature. In the present study, basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-/β1), vascular endothelial growth factor (VEGF), and bone morphogenetic protein-2 (BMP-2) were employed as growth factors, because their expected biological functions seem to be closely related to clinical applications. Basic bFGF and TGF-β1 were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This can be explained in terms of the electrostatic interaction between the basic growth factors and acidic gelatin. However, VEGF and BMP-2, though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a less extent than the two other factors. An in vivo experiment revealed that the hydrogels were degraded with time. The growth factors were retained in the body for a longer time period with their increasing sorption in vitro to the acidic gelatin hydrogel. A correlation of the in vivo retention between the growth factor and hydrogel suggested that the basic growth factor ionically complexed to the acidic gelatin hydrogel was released in vivo as a result of hydrogel degradation.

In vitro sorption and desorption of basic fibroblast growth factor from biodegradable hydrogels

In vitro interaction of basic fibroblast growth factor (bFGF) with biodegradable gelatin hydrogels was investigated, focusing on its sorption into the hydrogels and desorption from them. Basic bFGF was sorbed to the hydrogel of acidic gelatin with an isoelectric point (IEP) of 5.0 over time at 4°C, in contrast to that of basic gelatin with an IEP of 9.0 and type I collagen. The bFGF sorption was almost independent of the sorption temperature except for 4°C and the hydrogel water content. Fluorescent microscopic observation revealed that bFGF was sorbed into the interior of the acidic gelatin hydrogel. The binding molar ratio of bFGF to the acidic gelatin was around 1.0. The bFGF sorption to the acidic gelatin hydrogel increased when gelatin was further carboxylated. bFGF was sorbed into the acidic gelatin hydrogel more slowly than into the poly(acrylic acid) (PAAc) hydrogel, probably because of the lower density of negative charge of gelatin. The bFGF sorption decreased with an increase in solution ionic strength, indicating that an electrostatic interaction was the main driving force for bFGF sorption to the acidic gelatin hydrogel. However, even at higher ionic strengths of solution, the sorbed bFGF was not desorbed from the acidic gelatin hydrogel, in contrast to the PAAc hydrogel.

Development of enzymatically degradable protective coatings for use in triggered drug delivery systems: derivatized starch hydrogels

Starch is a common polysaccharide which consists of glucopyranose residues in an α-D-(1–4) linkage that yields D-glucose upon hydrolysis. Saturated aqueous solutions of soluble starch are easily reacted in the presence of glycidyl methacrylate to produce methacrylate grafted starch. Grafted starch solutions were polymerized to produce hydrogels, with and without the addition of an unsaturated acid. The grafted starch solutions and the resulting hydrogels are both shown to be degraded by the enzyme α-amylase. These acidic hydrogels are potentially useful as enzymatically degradable protective coatings in self-regulated drug delivery system applications. The pH of an acidic starch hydrogel required by this self-regulated drug delivery system does indicate that an adequately acidic hydrogel can be produced.