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 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.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of 125I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

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.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of ¹²⁵I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

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.     

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein

The objective of this study is to develop a carrier for the controlled release of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of the bone regeneration activity. Hydrogels with different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Following subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled BMP-2 into the back of mice, the in vivo retention period of BMP-2 prolonged with a decrease in the water content of hydrogels used, although every time period was much longer than that of BMP-2 solution injection. Ectopic bone formation studies demonstrated that the alkaline phosphatase (ALP) activity and osteocalcin content around the implanted site of BMP-2-incorporated gelatin hydrogels were significantly high compared with those around the injected site of BMP-2 solution. The values became maximum for the gelatin hydrogel incorporating BMP-2 with a middle period of BMP-2 retention, while bone formation was histologically observed around the hydrogel incorporating BMP-2. The ALP activity was significantly higher than that of the collagen sponge incorporating BMP-2. We concluded that the controlled release technology of BMP-2 for a certain time period was essential to induce the potential activity for bone formation.     

Promoted growth of murine hair follicles through controlled release of basic fibroblast growth factor

This study is an investigation to evaluate how the controlled release of basic fibroblast growth factor (bFGF) affects the hair follicle growth of mice in different hair cycle stages: second anagen and second telogen. bFGF was incorporated into biodegradable gelatin hydrogels for its controlled release. After subcutaneous implantation of gelatin hydrogels incorporating 0, 0.7, 7, and 70 microg of bFGF or injection of 0 and 70 microg of free bFGF into the backs of mice, hair follicle growth was evaluated photometrically and histologically on the basis of three parameters: skin color of the reverse side of the implanted or injected site, skin thickness, and area occupied by hair follicle tissue. For mice in second anagen, the darkness of the reverse side of skin implanted with gelatin hydrogel incorporating 7 microg of bFGF was significantly higher than that of skin injected with 70 microg of bFGF 10 days after bFGF application. Implantation of gelatin hydrogel incorporating bFGF enabled the hair follicles to increase the area occupied in skin tissue to a significantly greater extent than in other groups, whereas no effect on skin thickness was observed. bFGF-free, empty gelatin hydrogels did not affect hair follicle growth. Moreover, hair shaft length was significantly elongated by gelatin hydrogel incorporating 7 microg of bFGF, in marked contrast to other agents. The skin of telogen mice receiving gelatin hydrogel incorporating 7 microg of bFGF did not show any change in darkness of reverse skin side or skin thickness, but a significant increase in the size of hair follicles 10 days later. These results indicate that the controlled release of bFGF positively affects the hair growth cycle of mice.     

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.     

Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor

Synthetic hydrogel mimics of the extracellular matrix (ECM) were created by crosslinking a thiol-modified analog of heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol) diacrylate (PEGDA). The covalently bound heparin provided a crosslinkable analog of a heparan sulfate proteoglycan, thus providing a multivalent biomaterial capable of controlled release of basic fibroblast growth factor (bFGF). Hydrogels contained >97% water and formed rapidly in <10min. With as little as 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content), the rate of release of bFGF in vitro was substantially reduced. Total bFGF released increased with lower percentages of heparin; essentially quantitative release of bFGF was observed from heparin-free hydrogels. Moreover, the hydrogel-released bFGF retained 55% of its biological activity for up to 28 days as determined by a cell proliferation assay. Finally, when these hydrogels were implanted into subcutaneous pockets in Balb/c mice, neovascularization increased dramatically with HA and CS hydrogels that contained both bFGF and crosslinked heparin. In contrast, hydrogels lacking bFGF or crosslinked heparin showed little increase in neovascularization. Thus, covalently linked, heparin-containing glycosaminoglycan hydrogels that can be injected and crosslinked in situ constitute highly promising new materials for controlled release of heparin-binding growth factors in vivo.     

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.     

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by l-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo.     

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.     

Injectable extracellular matrix derived hydrogel provides a platform for enhanced retention and delivery of a heparin-binding growth factor

Injectable hydrogels derived from the extracellular matrix (ECM) of decellularized tissues have recently emerged as scaffolds for tissue-engineering applications. Here, we introduce the potential for using a decellularized ECM-derived hydrogel for the improved delivery of heparin-binding growth factors. Immobilization of growth factors on a scaffold has been shown to increase their stability and activity. This can be done via chemical crosslinking, covalent bonding, or by incorporating natural or synthetic growth factor-binding domains similar to those found in vivo in sulfated glycosaminoglycans (GAGs). Many decellularized ECM-derived hydrogels retain native sulfated GAGs, and these materials may therefore provide an excellent delivery platform for heparin-binding growth factors. In this study, the sulfated GAG content of an ECM hydrogel derived from decellularized pericardial ECM was confirmed by Fourier transform infrared spectroscopy and its ability to bind basic fibroblast growth factor (bFGF) was established. Delivery in the pericardial matrix hydrogel increased retention of bFGF both in vitro and in vivo in ischemic myocardium compared to delivery in collagen. In a rodent infarct model, intramyocardial injection of bFGF in pericardial matrix enhanced neovascularization by approximately 112% compared to delivery in collagen. Importantly, the newly formed vasculature was anastomosed with existing vasculature. Thus, the sulfated GAG content of the decellularized ECM hydrogel provides a platform for incorporation of heparin-binding growth factors for prolonged retention and delivery.     

Use of collagen sponge incorporating transforming growth factor-beta1 to promote bone repair in skull defects in rabbits.

The objective of this study was to evaluate the potential of collagen sponge incorporating transforming growth factor-beta1 (TGF-beta1) to enhance bone repair. The collagen sponge was prepared by freeze-drying aqueous foamed collagen solution. Thermal cross-linking was performed in a vacuum at 140 degrees C for periods ranging from 1 to 48 h to prepare a number of fine collagen sponges. When collagen sponges incorporating 125I-labeled TGF-beta1 were placed in phosphate-buffered saline (PBS) solution at 37 degrees C, a small amount of TGF-beta1 was released for the first hour, but no further release was observed thereafter, irrespective of the amount of cross-linking time the sponges had received. Collagen sponges incorporating 125I-labeled TGF-beta1 or simply labeled with 125I were implanted into the skin on the backs of mice. The radioactivity of the 125I-labeled TGF-beta1 in the collagen sponges decreased with time; the amount of TGF-beta1 remaining dependent on the cross-linking time. The in vivo retention of TGF-beta1 was longer in those sponges that had been subjected to longer cross-linking times. The in vivo release profile of the TGF-beta1 was matched with the degradation profile of the sponges. Scanning electron microscopic observation revealed no difference in structure among sponges subjected to different cross-linking times. The TGF-beta1 immobilized in the sponges was probably released in vivo as a result of sponge biodegradation because TGF-beta1 release did not occur in in vitro conditions in which sponges did not degrade. We applied collagen sponges incorporating 0.1 microg of TGF-beta1 to skull defects in rabbits in stress-unloaded bone situations. Six weeks later, the skull defects were covered by newly formed bone, in marked contrast to the results obtained with a TGF-beta1 free empty collagen sponge and 0.1 microg of free TGF-beta1. We concluded that the collagen sponges were able to release biologically active TGF-beta1 and were a promising material for bone repair.     

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.     

Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells

We examined whether transplantation of autologous bone marrow mononuclear cells (BM-MNCs) can augment neovascularization and bone regeneration of bone marrow in femoral bone defects of rabbits. Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. To evaluate the in vivo effect of implanted BM-MNCs, we created bone defects in the rabbit medial femoral condyle, and implanted into them 5 x 10(6) fluorescent-labeled autologous BM-MNCs together with gelatin microspheres containing 10 microg bFGF on an atelocollagen gel scaffold. The four experimental groups, which were Atelocollagen gel (Col), Col + 5 x 10(6) BM-MNCs, Col + 10 microg bFGF, and Col + 5 x 10(6) BM-MNCs + 10 microg bFGF, were implanted into the sites of the prepared defects using Atelocollagen gel as a scaffold. The autologous BM-MNCs expressed CD31, an endothelial lineage cell marker, and induced efficient neovascularization at the implanted site 2 weeks after implantation. Capillary density in Col + BM-MNCs + bFGF was significantly large compared with other groups. This combination also enhanced regeneration of the bone defect after 8 weeks to a significantly greater extent than either BM-MNCs or bFGF on their own. In summary, these findings demonstrate that a combination of BM-MNCs and bFGF gelatin hydrogel enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration.     

Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF

Controlled release of human vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate growth factor (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (15 kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel growth at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.     

Enhanced bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel

The objective of this study is to investigate the feasibility of a biodegradable hydrogel of gelatin as the controlled release carrier of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of bone regeneration at a segmental bone defect. Hydrogels with three different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Segmental critical-sized defects (20 mm) were created at the ulnar bone of skeletally mature New Zealand white rabbits, and gelatin hydrogels incorporating BMP-2 (17 microg/hydrogel) were implanted into the defects. When bone regeneration was evaluated by soft x-ray observation and bone mineral density (BMD) measurement, the gelatin hydrogels incorporating BMP- 2 exhibited significantly high osteoinduction activity compared with that of free BMP-2, although the activity depended on the water content of the hydrogels. Significantly higher BMD enhancement was observed in the gelatin hydrogel with a water content of 97.8 wt% than that with the lower or higher water content. We concluded that the biodegradable gelatin hydrogel is a promising controlled release carrier of BMP-2 for bone regeneration at the segmental bone defect.