Neovascularization effect of biodegradable gelatin microspheres incorporating basic fibroblast growth factor

Biodegradable microspheres were prepared through glutaraldehyde cross-linking of gelatin without using any surfactants as a carrier matrix of basic fibroblast growth factor (bFGF). In the in vitro system, bFGF was sorbed to microspheres of acidic gelatin with an isoelectric point (IEP) of 5.0, but not to those of basic gelatin with an IEP of 9.0. The rate of bFGF sorption to the acidic gelatin microsphere in phosphate-buffered saline solution (pH 7.4) was smaller than that in water. Following incorporation of bFGF into the microspheres at 4 degrees C for 12 h, bFGF release from the bFGF-incorporating microspheres was studied. Approximately 30% of incorporated bFGF was released from the acidic gelatin microsphere within the initial 3 h, followed by no substantial release, whereas the basic gelatin microsphere released almost completely the incorporated bFGF within 1 day. It is likely that when basic bFGF molecules were immobilized to the acidic gelatin constituting microspheres through polyion complexation, they were not readily released under the in vitro nondegradation condition of gelatin. Incorporation of anionic carboxylmethyl cellulose (CMC) into the acidic gelatin microspheres reduced the amount of bFGF desorbed initially. This indicates that the initial burst is ascribed to free bFGF which is not ionically interacted with the acidic gelatin. CMC will function as a bFGF sorbent to suppress the initial leakage from the microspheres. When injected subcutaneously into the mouse back, bFGF-incorporating acidic gelatin microspheres were degraded over time and induced neovascularization around the injection site, in marked contrast to bFGF in the solution form. CMC incorporation slowed down the biodegradation and vascularization effect of bFGF-incorporating gelatin microspheres. It was concluded that the gelatin microsphere was a promising carrier matrix of bFGF to enhance the vascularization effect.     

Vascularization into a porous sponge by sustained release of basic fibroblast growth factor

Vascularization into a poly(vinyl alcohol) (PVA) sponge was investigated using basic fibroblast growth factor (bFGF). This growth factor was impregnated into biodegradable gelatin microspheres for its sustained release and then the bFGF-containing microspheres or free bFGF were incorporated into PVA sponges. Following subcutaneous implantation into the back of mice, the bFGF-containing gelatin microspheres induced vascularization in and around the sponge to a significantly greater extent than that of free bFGF from 3 days after implantation. Significant ingrowth of fibrous tissue into the sponge was also observed when bFGF-containing microspheres were added to the sponge in contrast to free bFGF. Tissue ingrowth occurred into the deeper portion of the sponge over time while it accompanied formation of new capillaries. Empty gelatin microspheres had no effect on vascularization and the level of fibrous tissue ingrowth into the sponge was similar to that of the control group. It was concluded that incorporation of gelatin microspheres containing bFGF into the PVA sponge was effective in prevascularization of the sponge pores.     

Vascularization into a porous sponge by sustained release of basic fibroblast growth factor

Vascularization into a poly(vinyl alcohol) (PVA) sponge was investigated using basic fibroblast growth factor (bFGF). This growth factor was impregnated into biodegradable gelatin microspheres for its sustained release and then the bFGF-containing microspheres or free bFGF were incorporated into PVA sponges. Following subcutaneous implantation into the back of mice, the bFGF-containing gelatin microspheres induced vascularization in and around the sponge to a significantly greater extent than that of free bFGF from 3 days after implantation. Significant ingrowth of fibrous tissue into the sponge was also observed when bFGF-containing microspheres were added to the sponge in contrast to free bFGF. Tissue ingrowth occurred into the deeper portion of the sponge over time while it accompanied formation of new capillaries. Empty gelatin microspheres had no effect on vascularization and the level of fibrous tissue ingrowth into the sponge was similar to that of the control group. It was concluded that incorporation of gelatin microspheres containing bFGF into the PVA sponge was effective in prevascularization of the sponge pores.     

Time course of de novo adipogenesis in matrigel by gelatin microspheres incorporating basic fibroblast growth factor

Controlled release of basic fibroblast growth factor (bFGF) from gelatin microspheres achieved de novo adipogenesis at the implanted site of a basement membrane extract (Matrigel). Following subcutaneous co-implantation of Matrigel and gelatin microspheres incorporating 0.1 microg of bFGF into the back of mice, adipose tissue was formed at the implanted site after 4 weeks postoperatively although the extent increased with implantation time. Formation of adipose tissue was significantly faster than the co-implantation of Matrigel, and 0.1 microg of free bFGF while a larger volume of the adipose tissue formed was retained 15 weeks later. When measured in Matrigel co-implanted with the gelatin microspheres incorporating bFGF, the number of cells infiltrated into Matrigel increased to a significantly high extent compared with the bFGF co-implantation. Matrigel alone was much less effective in inducing formation of adipose tissue. We conclude that gelatin microspheres incorporating bFGF enable Matrigel to efficiently induce de novo adipogenesis at the implanted site in respect to the formation rate and volume of adipose tissue.     

De novo formation of adipose tissue by controlled release of basic fibroblast growth factor

De novo adipogenesis at the implanted site of a basement membrane extract (Matrigel) was induced through controlled release of basic fibroblast growth factor (bFGF). bFGF was incorporated into biodegradable gelatin microspheres for its controlled release. When the mixture of Matrigel and bFGF-incorporated gelatin microspheres was implanted subcutaneously into the back of mice, a clearly visible fat pad was formed at the implanted site 6 weeks later. Histologic examination revealed that the de novo formation of adipose tissue accompanied with angiogenesis was observed in the implanted Matrigel at bFGF doses of 0.01, 0.1, and 1 microg/site, the lower and higher doses being less effective. The de novo formation induced by the bFGF-incorporated microspheres was significantly higher than that induced by free bFGF of the same dose. The mRNA of a lipogenesis marker protein, glycerophosphate dehydrogenase, was detected in the formed adipose tissues, biochemically indicating de novo adipogenesis. Free bFGF, the bFGF-incorporated gelatin microspheres, or Marigel alone and bFGF-free gelatin microspheres with or without Matrigel did not induce formation of adipose tissue. This de novo adipogenesis by mixture of Matrigel and the bFGF-incorporated gelatin microspheres will provide a new idea for tissue engineering of adipose tissue.     

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.     

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.     

Incorporation of growth factor containing Matrigel promotes vascularization of porous PLGA scaffolds

In tissue engineering, rapid ingrowth of blood vessels into scaffolds is a major prerequisite for the survival of three-dimensional tissue constructs. In the present study, we investigated whether the vascularization of implanted poly-D,L-lactic-co-glycolic acid (PLGA) scaffolds may be accelerated by incorporation of Matrigel. For this purpose, we investigated in the aortic ring assay the proangiogenic properties of growth factor reduced Matrigel (GFRM) and growth factor containing Matrigel (GFCM), which were then incorporated into the pores of PLGA scaffolds. Subsequently, we analyzed vascularization, biocompatibility, and incorporation of these scaffolds during 14 days after implantation into dorsal skinfold chambers of balb/c mice by means of intravital microscopy, histology, and immunohistochemistry. Matrigel-free scaffolds served as controls. In the aortic ring assay, GFCM stimulated the development of a network of tubular vessel structures with a significantly increased sprout area and density when compared with GFRM. Accordingly, GFCM accelerated and improved in vivo the ingrowth of new blood vessels into scaffolds, resulting in the formation of a pericyte-coated vascular network with an increased functional capillary density in comparison to the GFRM and control group. Besides, analysis of leukocyte-endothelial cell interaction in host tissue venules located in close vicinity to the scaffolds showed no marked differences in numbers of rolling and adherent leukocytes between the observation groups, indicating that incorporation of Matrigel did not affect biocompatibility of PLGA scaffolds. These findings demonstrate that the combination of proangiogenic extracellular matrices with solid scaffold biomaterials may represent a novel approach to accelerate adequate vascularization of tissue engineering constructs.     

Sustained release and activation of the growth factor basic fibroblast growth factor from loaded scaffolds in heart valve tissue engineering

Objectives. Loading of biological matrices offers an opportunity to induce specific cell behaviour. We previously reported the use of growth factors to promote cell invasion and proliferation in tissue valve engineering.

We investigated biological matrices preloaded with heparin as an ionically attractive template for the binding, activation and sustained release of basic fibroblast growth factor (bFGF).

Methods. Heparin loading concentrations were evaluated and different incubation times were tested. Heparin and heparin-bound bFGF uptake and release were evaluated by ¹²³I radio-labelling. Biological activity of bFGF was evaluated in vitro.

Results. Maximum heparin uptake was observed for 2000 μg/ml at 2 h and stabilized thereafter. bFGF-loaded matrices showed an initial burst release of 15% within 4 h and thereafter sustained release reaching 21% at 24 h. Released bFGF was bioactive.

Conclusions. This model would be useful in tissue engineering using porcine aortic matrices and could be applied using other growth factors or combinations.     

Effect of basic fibroblast growth factor on vascularization in esophagus tissue engineering

We carried out an experimental study to evaluate the effect of basic fibroblast growth factor (bFGF)-containing collagen gel on vascularization in esophageal tissue engineering. We compared an acellular collagen sponge scaffold and an acellular collagen gel scaffold in combination with bFGF using a canine model. The construct was implanted in the cervical esophagus and the regenerated tissue was evaluated one month after surgery. Histological analysis confirmed a significantly large amount of blood vessels in the bFGF-containing collagen gel group as compared to the collagen gel group without bFGF (bFGF (-)). However, in the collagen sponge groups, no difference was observed between the bFGF (+) group and the bFGF (-) group. These results showed that bFGF-containing collagen gel is suitable not only for an acellular scaffold for tissue engineering but also for an effective tropic factor vehicle in vivo.     

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor

In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.     

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-l-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.     

Controlled and modulated release of basic fibroblast growth factor.

Basic fibroblast growth factor has multivariate effects in stimulating cell growth and the processes that surround tissue repair. Pathophysiologic studies have been hampered by the stability of the compound. Though very potent, basic fibroblast growth factor is rapidly degraded when injected or ingested. Controlled release of basic fibroblast growth factor would allow for examination of the chronic effects of this compound. Conventional matrix polymer-based release devices were fabricated and basic fibroblast growth factor released in a sustained fashion, but 99% of basic fibroblast growth factor mitogenic activity was lost. The source of these losses was identified and preventative measures examined. Preservation and stabilization of basic fibroblast growth factor was accomplished by binding the factor to heparin-Sepharose beads. This permitted prolonged storage, repeated handling, and the encapsulation of basic fibroblast growth factor within a microspherical controlled-release device using a naturally occurring polymer material, alginate. Encapsulation was accomplished with 77% efficiency and 87.5 +/- 12% of the basic fibroblast growth factor was released in a biologically active form. Release activation and regulation was achieved when cleavage of the basic fibroblast growth factor-heparin bonds was enhanced (e.g. by enzymatic bond cleavage with heparinase). Kinetic profiles were identified for a variety of experimental conditions and the effects of the controlled release of basic fibroblast growth factor on BALBc/3T3 fibroblasts examined.     

Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration

In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three-dimensional (3-D) scaffolds with encapsulated growth factor that enhance the sustained release of growth factor and induce the angiogenesis. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with basic fibroblast growth factor (bFGF) suspension. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. A 3-D network of nanofibers was formed by mixing bFGF suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vitro and in vivo release profile of bFGF from 3-D network of nanofibers was investigated while angiogenesis induced by the released bFGF was assessed. When aqueous solution of PA was subcutaneously injected together with bFGF suspension into the back of mice, a transparent 3-D hydrogel was formed at the injected site and induced significant angiogenesis around the injected site, in marked contrast to bFGF injection alone or PA injection alone. The combination of bFGF-induced angiogenesis is a promising procedure to improve tissue regeneration.     

Sustained release of basic fibroblast growth factor and angiogenesis in a novel covalently crosslinked gel of heparin and alginate

The construction of organs by tissue engineering and regenerative engineering, using an artificial extracellular matrix, is an innovative method that is expected to replace artificial organs and organ transplantation. We have produced an artificial extracellular matrix of alginate and demonstrated that the matrix stimulated the regeneration of skin, nerve, and bone. In this report, the new matrix, which consists of heparin and alginate covalently crosslinked with ethylenediamine, was produced to stabilize and control the release of growth factors. Heparin content of the new matrix was confirmed by toluidine blue absorption, elementary analysis, and Fourier transform infrared spectrum. In vitro experiments showed that the new matrix significantly suppressed the initial burst of basic fibroblast growth factor, which is a representative member of heparin-binding growth factors, and released biologically active basic fibroblast growth factor for 1 month under physiological conditions. Obvious cellular infiltration and angiogenesis were shown to occur in the new matrix which was implanted subcutaneously in the dorsal area of rat with 1 microg of basic fibroblast growth factor for 2 weeks. This new matrix may be useful for not only the construction of transplantable blood vessels of small diameter, but also the induction of angiogenesis in regenerated skin constructed by tissue engineering.     

Delivery of basic fibroblast growth factor (bFGF) from photoresponsive hydrogel scaffolds

Exogenous growth factor therapy has shown a notable promise in accelerating the healing of acute and chronic wounds. However, their susceptibility to enzymatic degradation and short contact time with the wound bed warrant the use of sophisticated delivery vehicles that stabilize the encapsulated peptides and control their rate of release. Herein, we describe the synthesis of a nitrocinnamate-derived polyethylene glycol (PEG-NC) hydrogel system and study the release kinetics of basic fibroblast growth factor (bFGF) as a function of hydrogel properties. Long-wave ultraviolet irradiation (365 nm) was used to alter the physical properties of the gel scaffold (i.e. degree of swelling) and consequently control the release rates of the encapsulated bFGF. The degree of swelling (DS) decreased from 10.7 to 8 as the length of irradiation increased from 5 to 30 min. Similarly, the DS decreased from 17.5 to 11.5 by increasing the initial PEG-NC concentration from 10 to 30 w/v% while keeping the crosslinking irradiation at 10 min. Radiolabeled I(125) studies were used to monitor the release of bFGF from PEG-NC hydrogels with variable swellabilities. By increasing the length of irradiation from 2 to 10 min the rate of bFGF release from PEG-NC gel scaffolds was decreased by 29% due to the enhanced crosslinking density. The bFGF-releasing PEG-NC hydrogels were not cytotoxic to human neonatal fibroblast cells and the released growth factor maintained its activity and induced fibroblast proliferation and collagen production in vitro. The addition of heparin within the gel scaffolds further increased the growth factor's activity.     

Magnetic alginate microspheres: system for the position controlled delivery of nerve growth factor

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles for targeted delivery of drugs in clinical practice has attracted the interest of the scientific community. In this paper a system comprised of alginate microparticles with a core of magnetite and carrying nerve growth factor (NGF) is described. The magnetic properties of these microspheres, typical of superparamagnetic materials, allow precise and controlled delivery to the intended tissue environment. Experiments carried out on PC12 cells with magnetic alginate microspheres loaded with NGF have confirmed the induction of cell differentiation which is strongly dependent on the distance from the microsphere cluster. In addition, finite element modelling (FEM) of the release profile from the microspheres in culture, indicated the possibility of creating defined and predictable NGF gradients from the loaded microspheres. These observations on the carriage and release of growth factors by the proposed microparticles open new therapeutic options for both neuronal regeneration and of the development of effective neuronal interfaces.     

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor

Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.     

Preparation and drug controlled release of porous octyl-dextran microspheres

In this work, porous octyl-dextran microspheres with excellent properties were prepared by two steps. Firstly, dextran microspheres were synthesized by reversed-phase suspension polymerization. Secondly, octyl-dextran microspheres were prepared by the reaction between dextran microspheres and ethylhexyl glycidyl ether and freezing-drying method. Porous structure of microspheres was formed through the interaction between octyl groups and organic solvents. The structure, morphology, dry density, porosity and equilibrium water content of porous octyl-dextran microspheres were systematically investigated. The octyl content affected the properties of microspheres. The results showed that the dry density of microspheres decreased from 2.35 to 1.21 g/ml, porosity increased from 80.68 to 95.05% with the octyl content increasing from 0.49 to 2.28 mmol/g. Meanwhile, the equilibrium water content presented a peak value (90.18%) when the octyl content was 2.25 mmol/g. Octyl-dextran microspheres showed high capacity. Naturally drug carriers play an important role in drug-delivery systems for their biodegradability, wide raw materials sources and nontoxicity. Doxorubicin (DOX) was used as a drug model to examine the drug-loading capacity of porous octyl-dextran microspheres. The drug-loading efficiency increased with the increase in microspheres/drug ratio, while the encapsulation efficiency decreased. When microspheres/drug mass ratio was 4/1, the drug-loading efficiency and encapsulation efficiency were 10.20 and 51.00%, respectively. The release rate of DOX increased as drug content and porosity increased. In conclusion, porous octyl-dextran microspheres were synthesized successfully and have the potential to serve as an effective delivery system in drug controlled release.