Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with ¹²⁵I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.     

PEG-cross-linked heparin is an affinity hydrogel for sustained release of vascular endothelial growth factor

An affinity-based controlled release system for growth factors having heparin-binding domains was prepared using a cross-linked heparin gel. The heparin gel was made by reacting hydrazide-functionalized heparin (Hep-ADH) with the N-hydroxysuccinimidyl ester of poly(ethylene glycol)-bis-butanoic acid (SBA-PEG-SBA). The degree of cross-linking could be controlled by defining the stoichiometry of hydrazide modification and the PEG cross-linker addition. The release of vascular endothelial growth factor (VEGF) was characterized as a heparin-binding growth factor. VEGF was directly injected into the heparin gel and the loaded VEGF displayed a slow, controlled release over 3 weeks with little initial burst phase. The biological activity of the released VEGF was measured with a proliferation assay utilizing human umbilical vein endothelial cells. The released VEGF maintained its biological activity at all time points investigated. The heparin gel with loaded VEGF was implanted sub-cutaneously in the dorsal region of mice. A significantly increased density of the endothelial cell marker platelet endothelial adhesion molecule (PECAM-1) was observed in histological specimens of the tissues surrounding the implanted gel.     

Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When ¹²⁵I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating ¹²⁵I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically active VEGF was released as a result of in vivo degradation of the hydrogel.     

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-beta1 (TGF-beta1) 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-beta1 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-beta1 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.     

Controlled Release of Stromal-Cell-Derived Factor-1 from Gelatin Hydrogels Enhances Angiogenesis

Controlled release of a chemokine, stromal-cell-derived factor-1 (SDF-1), could be achieved with gelatin hydrogels of release carrier. Gelatin was chemically derivatized to give it different electric charge and hydrophobicity. Among the derivatives, succinylated gelatin (Succ) of an anionic charge was the most suitable for preparation of the hydrogel in terms of SDF-1 release. The time profile of SDF-1 release from the hydrogel of succinylated gelatin could be controlled by changing the water content of hydrogel which could be modified by changing the conditions of hydrogel preparation. When evaluated after the subcutaneous implantation of Succ hydrogels incorporating SDF-1 or injection of SDF-1 solution, significantly stronger angiogenesis by the hydrogel was observed. The hydrogel implantation also enhanced the mRNA level of SDF-1 receptor at the site implanted. It is possible that the gelatin hydrogel enabled SDF-1 to be released locally, resulting in an enhanced angiogenesis at the site implanted.     

Interaction of hepatocyte growth factor with gelatin as the carrier material

The objective of this study was to physicochemically investigate the interaction between hepatocyte growth factor (HGF) and acidic gelatin compared with that between HGF and basic gelatin or heparin. Gelatin- or heparin-immobilized agarose beads were prepared and HGF interaction with them was evaluated by Scatchard binding assay. The dissociation constant of HGF with the acidic gelatin was about 2–3 orders of magnitude higher than that of heparin. The cell proliferation assay revealed that the proliferation promotion activity of HGF complexed with the acidic gelatin was detected, although it was lower than that of original HGF. The ability of HGF to enhance the cell proliferation was reduced by the trypsin treatment, although the extent of the reduction was significantly suppressed by HGF complexation with acidic gelatin. Electrophoresis experimentally confirmed enhanced resistance to the molecular mass loss of HGF by gelatin complexation. Moreover, the recognized level of an antibody to HGF was reduced by the complexation with the acidic gelatin, indicating that the acidic gelatin is present around HGF molecules. It is possible that the HGF molecule is covered with the acidic gelatin, resulting in protection from enzymatic digestion.     

Controlled release of lysozyme from succinylated gelatin microspheres

Gelatin was anionized to increase the carboxylic acid groups through succinylation. Succinylation of gelatin was performed using varying amounts of succinic anhydride. This gave various percentages of substitution. Lysozyme, a cationic antibacterial enzyme, which has important applications in the reduction of prosthetic valve endocarditis, was chosen as a model protein drug. Microspheres were prepared using unmodified gelatin and succinylated gelatin (SG) and lysozyme was incorporated into them. The percentage loading and release profiles of lysozyme for gelatin and SG microspheres were evaluated and compared. It was found that the SG microspheres exhibited higher loading efficiency for lysozyme (50%) than the unmodified gelatin microspheres. The in vitro release of lysozyme from SG microspheres occurred up to 122 h, compared to 96 h for gelatin microspheres, for the release of most of the lysozyme incorporated. This prolonged release of lysozyme from SG microspheres was attributed to the electrostatic interaction between the cationic lysozyme and the anionic SG microsphere carrier.     

Vascularization Around Poly(tetrafluoroethylene) Mesh with Coating of Gelatin Hydrogel Incorporating Basic Fibroblast Growth Factor

For successful mesh hernia treatment with medical meshes, it is important to induce angiogenesis and fibroplasia around the site of the mesh implanted. The objective of this study is to combine a mesh with a gelatin hydrogel for basic fibroblast growth factor (bFGF) release and evaluate the angiogenic activity in vivo. The MotifMesh^® (MM) of poly(tetrafluoroethylene) was treated with corona discharge to make the surface hydrophilic. This corona discharge treatment increased the bonding strength between the gelatin hydrogel coated and the mesh surface. When implanted into the back subcutis of mice, the MM coated with the gelatin hydrogel incorporating bFGF showed significant angiogenesis around the implanted site, in contrast to the MM alone and that without gelatin hydrogel or bFGF incorporation. It is concluded that the coating of hydrogel incorporating bFGF is a promising technology to give the mesh angiogenic properties.     

Recombinant growth hormone delivery systems based on vinylpyrrolidone-hydroxyethyl methacrylate copolymer matrices: Monitoring optimization by capillary zone electrophoresis

Experimental conditions for the fabrication of two new polymeric devices (i.e. films and slabs) useful for the controlled release of recombinant growth hormone (GH) are given. The release rate is controlled by the resorption profile of the vinylpyrrolidone-hydroxyethyl methacrylate (VPHEMA) tested systems which is related to the copolymer composition. The suitability of capillary electrophoresis (CE) for following the complete preparation of the different VP-HEMA devices is shown. Moreover, CE allows simultaneous monitoring of the controlled release of GH and dissolved polymer during in vitro experiments. From these results, guidelines are given for the fabrication of polymeric devices containing protein as active drug as well as for the correct selection of conditions during in vitro experiments.     

Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue

A gelatin/hyaluronic acid (GH) sponge has been fabricated by freeze-drying and cross-linking. The GH sponge was insoluble when cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The morphologies of sponges were investigated using a field emission scanning electron microscope. The porosity of the GH sponge increased with hyaluronic acid content. The GH sponge was biodegradable, as evidenced by implantation in Wistar rat subcutaneous connective tissue. Fibroblasts infiltrated into the sponge matrix, and regenerated collagen in the matrix to a level of 25% by 15 days after surgery. The GH73 sponge induced an acute inflammatory response compared with the GH91 sponge. This inflammatory response could have been stimulated by the presence of hyaluronic acid up to Day 10, as it decreased afterwards. The C-reactive protein of blood samples also indicated the same result. The blood tests and histological results show that GH sponges have good biocompatibility and low antigenicity for tissue engineering scaffolds.     

Bioadhesion and biocompatibility evaluations of gelatin and polyacrylic acid as a crosslinked hydrogel in vitro

This study describes the potentiality of crosslinked hydrogels comprised of gelatin and polyacrylic acid (CHGP) as a biological glue for soft tissues and compares its bonding strength with that of fibrin glue. Water-soluble carbodimide (WSC) was used to crosslink the mixture of gelatin and polyacrylic acid (PAA). An addition of PAA to gelatin increases bonding strength and reduces the gelation time and WSC concentration. Increasing the gelatin, WSC and PAA concentration increases the bonding strength. There is a critical concentration to have a maximum bonding strength. The cured hydrogel exhibited sufficient adhesion to mouse skin with a higher bonding strength than fibrin glue. The in vitro test has been done for evaluating CHGP toxicity.     

Influence of gelatin complexation on cell proliferation activity and proteolytic resistance of basic fibroblast growth factor

The objective of this study is to investigate the influence of gelatin complexation on the biological activity of basic fibroblast growth factor (bFGF) and its resistance to trypsin digestion. When bFGF was mixed at 37°C with acidic gelatin with an isoelectric point(IEP)of 5.0, the activity to promote in vitro proliferation of BHK cells became lower compared with that of free bFGF,in contrast to mixing with the basic gelatin with an IEP of 9.0. A maximum reduction in the bFGF activity was observed for the bFGF-gelatin complex prepared at a mixing molar ratio of 1/1. The bFGF activity of cell proliferation reduced at the initial period after mixing with the acidic gelatin at 37°C, followed by no substantial change. Complexation with the acidic gelatin at 4°C had no influence on the bFGF activity, irrespective of the bFGF/gelatin ratio and complexation time. The biological activity of bFGF was reduced by the trypsin treatment, but the reduced extent was suppressed through gelatin complexation at 37°C. In an electrophoresis study, the protective effect of gelatin complexation on the trypsin digestion was also confirmed in terms of the molecular weight loss. It is possible that the complexing gelatin covers bFGF molecules, resulting in suppression of their interaction with the cell surface receptor as well as protection from their enzymatic attack.     

Photo-cross-linkable and thermo-responsive hydrogels containing chitosan and Pluronic for sustained release of human growth hormone (hGH)

A Pluronic/chitosan hydrogel was prepared by employing di-acrylated Pluronic and acrylated chitosan for thermo-responsive and photo-cross-linkable in situ gelation. Mixtures of diacrylated Pluronic and acrylated chitosan were transformed to physical gels at elevated temperatures and the gelation temperature of the hydrogels gradually increased by increasing chitosan content in the hydrogels from 0% to 15%. Photo-cross-linked Pluronic/chitosan hydrogels were prepared by UV irradiation of the physical gels above their gelation temperatures. Hydrogels with a long photo-cross-linking time showed low degradation rates and chitosan contents in the hydrogels also impeded the degradation rates of the hydrogels, which was caused by a high degree of inter-connected polymer networks between acrylated Pluronic and acrylated chitosan. Human growth hormone (hGH), mixed with the mixture of Pluronic and chitosan, was photo-cross-linked to prepare biodegradable hGH hydrogels. The hydrogels containing hGH showed sustained release profiles for those with long photo-cross-linking times and high chitosan contents in the hydrogel. The hydrogels with a long cross-linking time showed impeded release of the protein and high content of chitosan in the hydrogels also decreased burst release of hGH from the hydrogels while hGH was rapidly released out for the hydrogels with low content of chitosan.     

Multilayer nanocapsules of polysaccharide chitosan and alginate through layer-by-layer assembly directly on PS nanoparticles for release

Polysaccharide multilayer nanocapsules have been fabricated in aqueous media by the layer-by-layer self-assembly of chitosan (CHI) and sodium alginate (ALG) on monodisperse polystyrene (PS) nanoparticles with a diameter of 180 nm as template, followed by removal of the templates through dissolving in THF. The pH and added salt concentration of the polyelectrolyte deposition solutions were optimized to ensure the alternating deposition. Consequently, the most suitable pH values were found to be 6.0–8.0 for ALG and 3.5 for CHI and were used in the deposition. The concentration of added NaCl used in the adsorption solutions was 0.5 M, which led to an average thickness of about 13 nm for 5 bilayers of CHI/ALG shell-wall.ζ-potential indicated the stepwise and alternating adsorption of CHI and ALG to form multilayer film on the PS nanoparticles. The characteristic bands of PS residue almost disappeared in the IR spectrum of the nanocapsule after dipped in THF, confirming thorough removal of PS templates from the core-shell particles. TEM, SEM and AFM were utilized to observe the nanocapsules of about 225 nm in diameter (by TEM). A hydrophilic drug model, acridine hydrochloride (AH), was chosen to investigate the loading and release properties of the nanocapsules. The positively charged AH spontaneously deposited into the capsule due to the electrostatic interaction with the negatively charged styrene sulfonate residues from the PS template inside the capsule. The rate of AH release became slightly slower when the capsule wall was cross-linked with glutaraldehyde, but the accumulative released amount for the cross-linked capsule was obviously reduced. These nanocapsules made from nature polysaccharides have a potential application in controlled drug release.     

Effect of growth factors and extracellular matrix materials on the proliferation and differentiation of microencapsulated myoblasts

An alternative approach to gene therapy via non-autologous somatic gene therapy is to implant genetically-engineered cells protected from immune rejection with microcapsules to deliver a therapeutic gene product. This delivery system may be optimized by using myoblast cell lines which can undergo terminal differentiation into myotubes, thus removing the potential problems of tumorigenesis and space restriction. However, once encapsulated, myoblasts do not proliferate or differentiate well. We now report the use of extracellular matrix components and growth factors to improve these properties. Addition of matrix material collagen, merosin or laminin all stimulated myoblast proliferation, particularly when merosin and laminin were combined; however, none, except collagen, stimulated differentiation. Inclusion of basic fibroblast growth factor (bFGF) within the microcapsules in the presence of collagen stimulated proliferation of C₂C₁₂ myoblasts, as well as differentiation into myotubes. Inclusion of insulin growth factor (IGF-II) in the microcapsules had no effect on proliferation but accelerated myoblasts differentiation. When the above matrix material and growth factors were provided in combination, the use of merosin and laminin together with bFGF and IGF-II stimulated myoblast proliferation but had no effect on differentiation. In contrast, the cocktail containing bFGF, IGF-II and collagen induced increased myoblasts proliferation and subsequent differentiation. Hence, the combination of bFGF, IGF-II and collagen appears optimal in improving proliferation and differentiation in encapsulated myoblasts.     

Controlled release systems for proteins based on gelatin microspheres

The preparation and characterization of biodegradable gelatin microspheres for the controlled release of peptides and proteins has been investigated. Bovine serum albumin (BSA) was chosen for incorporation into the gelatin microspheres and the spheres were characterized for the in vitro release of BSA and other properties. BSA was labelled with fluorescein isothiocyanate (FITC) for easy analysis. FITC-BSA was entrapped into the gelatin microspheres using a polymer dispersion technique developed in our earlier studies. The morphological characteristics of microspheres were analysed by optical and scanning electron microscopy (SEM). The optical and SEM photographs of FITC-BSA microspheres showed the solid spherical nature of the spheres. The entrapment efficiency of FITC-BSA was about 62%. The in vitro release pattern of FITC-BSA showed that 51 % of the entrapped drug was released during the first day and the release followed approximate zero order kinetics from day 2 onwards. The total release of FITC-BSA lasted for about 8 days. SDS-PAGE analysis revealed that BSA was not degraded by this preparation of microspheres.     

Preparation of gelatin hydrogel sponges incorporating bioactive glasses capable for the controlled release of fibroblast growth factor-2

Gelatin hydrogel sponges incorporating bioactive glasses (Gel-BG) were fabricated. We evaluated the characteristics of Gel-BG as scaffolds from the perspective of their mechanical properties and the formation of hydroxyapatite by the incorporation of bioactive glasses (BG). In addition, the Gel-BG degradation and the profile of fibroblast growth factor-2 (FGF-2) release from the Gel-BG were examined. Every Gel-BG showed an interconnected pore structure with the pore size range of 180–200?µm. The compression modulus of sponges incorporating BG increased. The time profiles of degradation for the 72-h crosslinked gelatin hydrogel sponges incorporating 10?wt% BG (Gel-BG(10)) and 50?wt% BG (Gel-BG(50)) were analogous to that of the 24-h crosslinked gelatin hydrogel sponge without BG (Gel-BG(0)). In measuring the release of FGF-2 from Gel-BG, the Gel-BG(10) and Gel-BG(50) showed almost analogous 100% cumulative release within 28?days in vivo. Additionally, a bioactivity evaluation showed that the presence of gelatin does not affect the in vitro bioactivity of Gel-BG. These sponges showed mechanical and chemical functionality as scaffolds, featuring both the controlled release of FGF-2 and the induction of hydroxyapatite crystallization.     

Controlled release of fibroblast growth factor-2 from an injectable 6-O-desulfated heparin hydrogel and subsequent effect on in vivo vascularization

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo.     

Studies on biodegradation and release of gentamicin sulphate from interpenetrating network hydrogels based on poly(acrylic acid) and gelatin: in vitro and in vivo

Interpenetrating network hydrogels (IPNs) based on poly(acrylic acid) and gelatin (Ge) were evaluated for in vitro and in vivo biodegradation and in vivo release of gentamicin sulphate. In vitro and in vivo degradation studies demonstrated that with the increase of acrylic acid content in the polymer, the rate of degradation decreases, and a reverse phenomenon was observed with increasing Ge content in the hydrogel. The rate of in vivo degradation was much lower than in vitro degradation. Incorporation of gentamicin sulphate in hydrogel further reduces their degradation. In vitro and in vivo drug release profile showed a burst effect, followed by controlled release. Drug concentration was measured in the local skin tissue, blood serum, kidney, liver and spleen. The local skin tissue concentration of 50% and 100% gentamicin sulphate, loaded full IPNs (i.e., Ax-1 and Ax-2), was found to be higher (20+/-2mug/g) than the minimum bactericidal concentration for Staphylococcus aureus (1.2mug/g) and Pseudomonas aeruginosa (10mug/g), respectively, for a study time of 60 days.     

扶肾降浊方含药血清对肾小管间质损害大鼠成纤维细胞抗纤维化因子HGF和BMP-7的影响

目的:探讨扶肾降浊方对系膜增生性肾小球肾炎(Ms PGN)大鼠肾小管间质损害的疗效及机制。方法:采用扶肾降浊方水溶液灌胃Wistar大鼠常规制备含药血清,在Ms PGN动物模型的基础上,延长造模时间至20周,使其自然发展为肾小管间质损害模型,体外培养造模12、16和20周末大鼠间质成纤维细胞,采用real-time PCR和Western blotting法检测扶肾降浊方含药血清对病理状态间质成纤维细胞中抗纤维化因子肝细胞生长因子(HGF)、骨形态发生蛋白-7(BMP-7)mRNA及蛋白表达的影响。结果:病理状态间质成纤维细胞中抗纤维化因子HGF、BMP-7 mRNA和蛋白表达下调,扶肾降浊方含药血清随着给药周期的延长可部分逆转间质损害造成的上述mRNA和蛋白表达异常。结论:扶肾降浊方含药血清对Ms PGN大鼠间质成纤维细胞的保护作用可能与调节抗纤维化因子HGF、BMP-7 mRNA和蛋白表达有关。