Regeneration of canine tracheal cartilage by slow release of basic fibroblast growth factor from gelatin sponge

We investigated the efficiency of basic fibroblast growth factor (b-FGF) released from a gelatin sponge in the regeneration of tracheal cartilage. A 1-cm gap was made in the midventral portion of each of 10 consecutive cervical tracheal cartilages (rings 4 to 13) in 15 experimental dogs. In the control group (n = 5), the resulting gap was left blank. In the gelatin group (n = 5), a gelatin sponge alone was implanted in the gap. In the b-FGF group (n = 5), a gelatin sponge containing 100 mug b-FGF solution was implanted in the gap. We euthanatized one of the five dogs in each group at 1 month after implantation and one at 3 months and examined the implant sites macroscopically and microscopically. In the control and gelatin groups, no regenerated cartilage was observed in the tracheal cartilage gap at 1 or 3 months. The distances between the cartilage stumps had shrunk. In the b-FGF group, fibrous cartilage had started to regenerate from both host cartilage stumps at 1 month. At 3 months, regenerated fibrous cartilage filled the gap and had connected each of the stumps. The regenerated cartilage was covered with regenerated perichondrium originating from the host perichondrium. Shrinkage of the distance between the host cartilage stumps was not observed in the b-FGF group. We succeeded in inducing cartilage regeneration in the gaps in canine tracheal cartilage rings by using the slow release of b-FGF from a gelatin sponge. The regenerated cartilage induced by b-FGF was fibrous cartilage.     

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.     

Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor

A previously reported "grafting and coating" method (J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 838) was modified and used to introduce stable collagen layer and incorporate basic fibroblast growth factor (bFGF) on PLLA scaffold surface to prepare tissue engineering scaffold with improved biocompatibility. To make the modification of the 3-D porous PLLA scaffold possible, grafting of polymethacrylic acid (PMAA) onto the PLLA surface was initiated by the -OOH/Fe2+ system instead of the UV light used in the former method. Water soluble carbodimmide chemistry was applied to graft collagen onto the PLLA scaffold surface, followed by physical coating of the collagen solution with or without basic fibroblast growth factor (bFGF). Surface modification of 2-D PLLA membrane was also done for fundamental understanding of the modification. The -COOH density on/in the PMAA grafted PLLA membrane/scaffold was measured by colorimetric method and the collagen content on/in the collagen immobilized PLLA membrane/scaffold was measured by ninhydrin method. Chondrocyte culturing on the collagen immobilized PLLA surfaces showed significantly improved cell spreading and growth. Incorporation of fibroblast growth factors in the collagen layer further enhanced the cell growth. This convenient and effective method can be used to prepare bioactive scaffolds with extra cellular matrix (ECM)-mimic composition for tissue engineering.     

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.     

Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor

Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. Co-implantation with the gelatin microspheres enabled preadipocytes to induce adipose tissue formation at the implanted site. Preadipocytes isolated from human fat tissue were suspended with the gelatin microspheres containing bFGF and incorporated into a collagen sponge of cell scaffold. Following subcutaneous implantation of the collagen sponge incorporating human preadipocytes, and gelatin microspheres containing 1 microg of bFGF into the back of nude mice, adipose tissue was formed at the implanted site of collagen sponge within 6 weeks postoperatively although the extent depended on the number of preadipocytes transplanted and the bFGF dose. The formation of adipose tissue was significant compared with the implantation of collagen sponge incorporating human preadipocytes and 1 microg of free bFGF. The area of adipose tissue newly formed was increased with the number of preadipocytes transplanted until to 1.0 x 10(5) cells/site and thereafter leveled off. The maximum area was observed at the bFGF dose of 1 microg/site. The area was significantly smaller at the bFGF dose of 0.5 microg/site or larger than 1 microg/site. Immunohistochemical examination indicated that the adipose tissue newly formed was composed of human matured adipocytes. No adipogenesis was observed at the implanted site of collagen sponge incorporating either gelatin microspheres containing bFGF or human preadipocytes and the mixed gelatin microspheres containing bFGF and human preadipocytes. We conclude that combination of gelatin microspheres containing bFGF and preadipocytes with the collagen sponge is essential to achieve tissue engineering of fat tissue.     

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.     

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.     

Minimally invasive technique of auricular cartilage harvest for tissue engineering

Tissue engineered human cartilage is presently being utilized in clinical research programs in a variety of medical disciplines including otolaryngology, urology, and orthopedics. In this study, we present a new methodology for auricular cartilage harvest that can be applied to tissue engineering. Eight 16-week-old pigs were subjected to a traditional open cartilage harvest technique involving suture closure, while the other ear was subjected to the closed stitchless cartilage harvest, using a 12-gauge core biopsy needle. Surgical time was significantly (p < 0.0001) shorter (3.5 +/- 2.8 min for closed vs. 14.4 +/- 5 min for open), and no sutures where utilized in the closed technique. Sample weights were significantly (p < 0.00001) greater (0.115 +/- 0.028 g vs. 0.045 +/- 0.005 g) for the closed techniques. However, the minimally invasive closed technique had fewer incidents of bruising, hematoma, long-term stitch abscess, and scarring. Cell culture data shows no disadvantage to either technique with regards to cell growth characteristics. Final histological data from donor ears indicates favorable results with the minimally invasive technique. This technique preserves cell viability and isolation efficiency while decreasing surgical time and lessening postoperative complications.     

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.     

组织工程中控释生长因子载体的研究进展

在组织工程中，生长因子的应用是一个十分重要的组成部分，利用载体或缓释系统负载生长因子，既能保护生长因子的生物活性，又可以使生长因子缓慢释放，从而持续促进组织修复再生。由于生长因子的性质和作用方式有很大的不同，所以如何针对特定的生长因子设计缓释系统是十分重要的。对组织工程中使用较广泛的三种生长因子所使用的载体的进展做一简述。     

组织工程中控释生长因子载体的研究进展

在组织工程中,生长因子的应用是一个十分重要的组成部分,利用载体或缓释系统负载生长因子,既能保护生长因子的生物活性,又可以使生长因子缓慢释放,从而持续促进组织修复再生。由于生长因子的性质和作用方式有很大的不同,所以如何针对特定的生长因子设计缓释系统是十分重要的。对组织工程中使用较广泛的三种生长因子所使用的载体的进展做一简述。     

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.     

Binding and release of basic fibroblast growth factor from heparinized collagen matrices.

Endothelial cell seeding is a promising method to improve the performance of small-diameter vascular grafts. Growth of endothelial cells seeded on the luminal surface of synthetic vascular grafts, coated with a matrix suitable for cell seeding (e.g. collagen), can be accelerated by local, sustained release of basic fibroblast growth factor (bFGF). In this study two potential matrices for in vivo endothelial cell seeding were studied with respect to bFGF binding and release: collagen crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as heparinized EDC/NHS-crosslinked collagen. bFGF binding was determined after incubation of circular samples (10 mm diameter) with 0.25 ml bFGF solution for 90 min. Immobilization of increasing amounts of heparin, also using EDC and NHS, to crosslinked collagen containing 14 free primary amino groups per 1000 amino acid residues (E/N14C) resulted in binding of increasing amounts of bFGF. A plateau in bFGF binding was observed for heparinized E/N14C containing approximately 2.0-3.0 wt% of immobilized heparin which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 during heparin immobilization (E/N14C-H(0.4)). At concentrations up to 840 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) amounted to 22%. Both E/N14C and E/N14C-H(0.4) pre-loaded with bFGF showed sustained bFGF release. A burst release of 30% in endothelial cell culture medium (CM) was observed for E/N14C during the first 6 h, compared to 2% release from E/N14C-H(0.4). After 28 days, the bFGF release from E/N14C and E/N14C-H(0.4) in CM amounted to 100 and 65%, respectively. Combined results of binding and release of bFGF indicate that compared to E/N14C, E/N14C-H(0.4) is the substrate of choice for bFGF pre-loading and subsequent endothelial cell seeding.     

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.     

重组人表皮生长因子及碱性成纤维细胞生长因子联合应用促进内镜鼻窦手术后术腔创面上皮化的疗效观察

目的观察rhEGF凝胶及bFGF滴眼液联合应用对鼻内窥镜鼻窦相关手术后的术腔创面黏膜上皮化的效果。方法对30例（60侧）经鼻内镜全鼻窦开放术患者进行同体对照观察，左侧为观察组，鼻窦术后每周在鼻内镜下作一次详尽术腔清理，于术腔置放rhEGF凝胶及bFGF滴眼液，混合抗生素和地塞米松的明胶海绵；右侧为对照组鼻窦每天使用鼻腔冲洗器自行冲洗鼻腔，鼻内使用局部类固醇激素，每周作鼻内镜下的术腔清理1次。连续内镜随访12周，观察双侧术腔上皮化过程。结果观察组治愈（28例）93．33％，好转（2例）6．67％，平均上皮化时间4周；对照组治愈（24例）80％，好转（6例）20％，平均上皮化时间9周，平均上皮化时间差异有统计学意义（P〈0．01）。结论鼻内窥镜鼻窦手术后联合应用rhEGF及bFGF可以促进术腔上皮化，显著缩短上皮化时间，治疗效果好。     

Sequential delivery of basic fibroblast growth factor and platelet-derived growth factor for angiogenesis

An externally regulated delivery model that permits temporal separation of multiple angiogenic factors was used for the delivery of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). While bFGF plays a significant role in the sprouting of new capillaries, PDGF plays a role in the recruitment of mural cells, which stabilize neovessels. However, these two factors have been shown to inhibit each other, when presented together. Using the externally regulated model, sequential delivery of bFGF and PDGF led to not only increased endothelial cell migration, but also endothelial cell and vascular pericyte colocalization. More importantly, this delivery strategy was able to induce red blood cell-filled neovessels, suggesting integration of angiogenesis with the existing vasculature.