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.     

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.     

Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres

Site-specific delivery of angiogenic growth factors from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.     

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.     

生物可降解聚合物结合碱性成纤维细胞生长因子与内皮细胞的黏附

背景：可降解材料的应用是体外构建小口径组织工程血管的重要研究部分。如何对可降解材料进行改性,以利于材料本身体外抗凝与促进内皮细胞黏附,是目前血管组织工程研究的热点之一。 目的：利用可降解聚己内酯接枝肝素材料体外负载碱性成纤维细胞生长因子,观察其对于内皮细胞黏附的影响。 方法：应用聚己内酯可降解材料,将肝素活化后并与聚己内酯的端羟基发生酯化反应从而被锚定在聚己内酯两端。经过电纺丝技术,制备血管支架。同时利用肝素和生长因子间的静电吸附作用,使支架负载碱性成纤维细胞生长因子。采用低密度内皮细胞短期静态种植,观察负载细胞生长因子的可降解聚己内酯材料对内皮细胞生长黏附情况的影响。 结果与结论：成功地制备了负载成纤维细胞生长因子的可降解聚己内酯接枝肝素支架。内皮黏附实验证实,该支架利于内皮细胞黏附。提示可降解聚己内酯接枝肝素材料负荷碱性细胞生长因子支架对内皮细胞有很好的体外黏附性。     

Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis

The objective of this study was to evaluate the effect of incorporation of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres into an artificial dermis on the regeneration of dermis-like tissues. When used in the free form in vivo, bFGF cannot induce sufficient wound healing activity, because of its short half-life. Therefore, sustained release of bFGF was achieved by impregnation into biodegradable gelatin microspheres. A radioisotope study revealed that incorporation of bFGF-impregnated gelatin microspheres significantly prolonged in vivo retention of bFGF in the artificial dermis. Artificial dermis with incorporated bFGF-impregnated gelatin microspheres or bFGF in solution was implanted into full-thickness skin defects on the back of guinea pigs (1.5 cm x 1.5 cm) (n = 4). Incorporation of bFGF into the artificial dermis accelerated fibroblast proliferation and capillary formation in a dose-dependent manner. However, the accelerated effects were more significant with the incorporation of bFGF-impregnated gelatin microspheres than with free bFGF at doses of 50 microg or higher. We conclude that the gelatin microsphere is a promising tool to accelerate bFGF-induced tissue regeneration in artificial dermis.     

In situ regeneration of adipose tissue in rat fat pad by combining a collagen scaffold with gelatin microspheres containing basic fibroblast growth factor

This study is an investigation to evaluate in situ adipose tissue regeneration in fat pads. Gelatin microspheres with different water contents were prepared for the controlled release of basic fibroblast growth factor (bFGF). After a collagen sponge scaffold was incorporated by the microspheres containing 0, 0.01, 0.1, 1, and 10 microg of bFGF with or without syngeneic rat preadipocytes (1 x 10(5) cells/site) into a defect of rat fat pad, adipogenesis at the implanted site of scaffold was evaluated histologically. in situ formation of adipose tissue accompanied with angiogenesis was observed in the scaffold implanted with the microspheres containing 1.0 microg of bFGF, although the extent was less at the lower and higher bFGF doses. The in situ formation induced by the microspheres containing bFGF was significantly higher than that induced by free bFGF of the same dose. Adipogenesis was enhanced with time after implantation up to 4 weeks and thereafter leveled off. Such in situ adipogenesis was reproducibly induced by implantation of collagen scaffold incorporating gelatin microspheres containing 1 microg of bFGF, whereas addition of rat syngeneic preadipocytes did not promote the adipogenesis. The degradation of microspheres and the consequent FGF release became faster with an increase in the water content of gelatin microspheres. Less in situ formation of adipose tissue was observed at the lower water content of microspheres, which showed longer-term bFGF release. We conclude that combination of scaffold collagen with an appropriate controlled release of bFGF was essential to achieve the in situ formation of adipose tissue even without preadipocytes.     

碱性成纤维细胞生长因子与卵巢癌的关系

目的　探讨碱性成纤维细胞生长因子 (basic fibroblast growth factor,b FGF)对卵巢癌细胞增殖、浸润和肿瘤血管生成的影响 ,及 b FGF单克隆抗体 (b FGF monoclonal antibody,b FGF- MAb)的治疗作用。　方法　将人卵巢癌细胞株 SKOV3接种于 2 4孔板 ,加入不同浓度的 b FGF,每日行结晶紫染色后测定光密度 (D4 90 )值 ,绘制细胞生长曲线 ;将 SKOV3细胞团接种于铺设有细胞外基质凝胶的 4孔板 ,每日测定癌细胞在凝胶中的浸润距离 ;建立 SKOV3细胞裸鼠皮下移植瘤模型 ,每周两次分别将 b FGF、b FGF-MAb和生理盐水注射于移植瘤周围 ,8周后测量肿瘤体积 ;对移植瘤组织切片行 因子的免疫组化染色、测定肿瘤内微血管密度 (microvessel density,MVD)。　结果　 b FGF能促进 SKOV3细胞增殖并呈浓度依赖 ,实验第 5天 ,5 ng/ml、10 ng/ml组细胞 D4 90 值是对照组的 1.0 9倍和 1.2 1倍 ;b FGF能促进 SKOV3细胞浸润并呈浓度依赖 (P<0 .0 5 ) ,第 7天 ,5 ng/ml、10 ng/ml组细胞浸润距离分别是对照组的 1.5 3倍和2 .4 5倍 ;b FGF组移植瘤体积和 MVD分别是对照组的 1.80倍和 1.4 6倍 (P<0 .0 5 ) ,b FGF- MAb组移植瘤体积和 MVD分别是对照组的 6 3.7%和 6 2 .8% (P<0 .0 5 )。　结论　b FGF能明显促进卵巢癌细胞的增殖、     

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.     

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 degrees 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 degrees C, followed by no substantial change. Complexation with the acidic gelatin at 4 degrees 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 degrees 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.     

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.     

Effect of gelatin hydrogel incorporating fibroblast growth factor 2 on human meniscal cells in an organ culture model

Efforts to use growth factors to enhance the healing potential of the meniscus have been impeded because their half-lives are too short to maintain the biological activity. The thread was coated with gelatin hydrogel and fibroblast growth factor 2 (FGF 2) was biologically stabilized by incorporating in a gelatin hydrogel-coated thread. The purpose of this study is to investigate the effect of gelatin hydrogel-coated thread incorporating FGF 2 on human meniscal cells in an organ culture. Twenty-five menisci were cut into small pieces, and selected pieces were sutured with gelatin hydrogel-coated thread incorporating FGF 2 (FGF(+) group) or physiologic saline (FGF(−) group), followed by organ culture. The meniscal samples histologically evaluated 4, 7, and 14 days later. The cell density and the number of PCNA-positive cells for the FGF(+) group were higher than those of the FGF(−) group, while the number of TUNEL-positive cells was lower. These results suggest that FGF 2 stimulates the proliferation of meniscal cells and inhibits meniscal cell death. Gelatin hydrogel-coated threads releasable FGF 2 may be useful to promote repairing of human meniscus.     

Biodegradation of hydrogel carrier incorporating fibroblast growth factor.

In vivo release of basic fibroblast growth factor (bFGF) from a biodegradable gelatin hydrogel carrier was compared with the in vivo degradation of hydrogel. When gelatin hydrogels incorporating 125I-labeled bFGF were implanted into the back subcutis of mice, the bFGF radioactivity remaining decreased with time and the retention period was prolonged with a decrease in the water content of the hydrogels. The lower the water content of 125I-labeled gelatin hydrogels, the faster both the weight of the hydrogels and the gelatin radioactivity remaining decreased with time. The decrement profile of bFGF remaining in hydrogels was correlated with that of hydrogel weight and gelatin radioactivity, irrespective of the water content. Subcutaneous implantation of bFGF-incorporating gelatin hydrogels into the mice induced significant neovascularization. The retention period of neovascularization became longer as the water content of the hydrogels decreased. To study the decrease of activity of bFGF when implanted, bFGF-incorporating hydrogels were placed in diffusion chamber and implanted in the mouse subcutis for certain periods of time. When hydrogels explanted from the mice were again implanted, significant neovascularization was still observed, indicating that most of the biological activity of bFGF was retained in the hydrogels. It was concluded that, in our hydrogel system, biologically active bFGF was released as a result of in vivo degradation of the hydrogel. The release profile was controllable by changing the water content of hydrogels.     

Delivery of basic fibroblast growth factor from gelatin microsphere scaffold for the growth of human umbilical vein endothelial cells

One of the major obstacles for engineering large tissue or organs such as the liver in vitro is the insufficient supply of nutrients and oxygen to the cells growing inside the scaffold, which reduces cell viability significantly. Therefore, vascularization of the scaffolding system is necessary for successful engineering of such tissues. In this study, we investigated the use of gelatin microsphere as scaffold to culture human umbilical vein endothelial cells, which is considered to be the basis and premise for the formation of blood vessels. The gelatin microspheres were crosslinked with different concentrations of glutaraldehyde to study the effects of crosslinking extent on the growth of endothelial cells. The swelling ratios of the gelatin microspheres decreased from 5.9 +/- 0.8 to 3.9 +/- 0.6 with the increase of the crosslinking extent. Basic fibroblast growth factors (bFGFs), which can improve endothelial cell proliferation as well as stimulate the formation of capillary vessels, were incorporated into the gelatin microspheres through ionic complexation. Sustained delivery of the growth factors was achieved for at least 2 weeks. The proliferation of the cells cultured on the bFGF-encapsulated microspheres was improved by about two times as compared to control and about 1.3 times as compared to blank microspheres, which indicated that the bioactivity of bFGF was well maintained, and the delivery of the growth factors directly to the cells significantly improved the success of this tissue engineering system.     

Enhanced angiogenesis by multiple release of platelet-rich plasma contents and basic fibroblast growth factor from gelatin hydrogels

The objective of this study is to evaluate the angiogenic effects induced by biodegradable gelatin hydrogel granules incorporating mixed platelet-rich plasma (PRP) growth factor mixture (PGFM) and bioactive basic fibroblast growth factor (bFGF). The PRP was prepared by a double-spinning technique for isolating animal bloods, followed by treatment with different concentrations of calcium chloride (CaCl(2)) solution. The CaCl(2) solution treatment activated the platelets of PRP, allowing the release of various growth factors, such as platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β(1), and epithelial growth factor (EGF). In the PRP treated with different CaCl(2) solutions, high amounts of representative platelet growth factor, PDGF-BB, VEGF, EGF, and TGF-β(1) were detected in the CaCl(2) concentrations of 1, 2, and 4 wt.% compared with higher or lower ones. The PRP treated was impregnated into gelatin hydrogel granules freeze-dried at 37°C for 1h, and then the percentage of PGFM desorbed from the gelatin hydrogel granules was evaluated. The percentages of PDGF-BB, VEGF, EGF, and TGF-β(1) desorbed tended to decrease with decreasing CaCl(2) concentration. Taken together, the CaCl(2) concentration to activate PRP for PGFM release was fixed at 2 wt.%. In vitro release tests demonstrated that the PGFM was released from the gelatin hydrogel granules with time. For the gelatin hydrogels incorporating PGFM and bFGF, the time profile of PDGF-BB or bFGF release was in good correspondence with that of gelatin hydrogel degradation. The gelatin hydrogel granules incorporating mixed PGFM and bFGF were prepared and intramuscularly injected to a mouse leg ischemia model to evaluate the angiogenic effects in terms of histological and laser Doppler perfusion imaging examinations. As controls, hydrogel granules incorporating bFGF, PGFM, and platelet-poor plasma were used for the angiogenic evaluation. The number of blood vessels newly formed and the percentage of anti-α-smooth muscle actin antibody-positive cells increased around ischemic sites injected with the gelatin hydrogel granules incorporating mixed PGFM and bFGF, in marked contrast to other control groups. The blood reperfusion level of ischemic tissues was enhanced by the hydrogel granules incorporating mixed PGFM and bFGF, whereas no enhancement was observed for other groups. It is concluded that the dual-release system of PGFM and bFGF from gelatin hydrogel granules shows promise as a method to enhance angiogenic effects.     

碱性成纤维细胞生长因子骨诱导作用的研究进展

介绍碱性成纤维细胞生长因子对骨干细胞、成骨细胞、破骨细胞和血管形成的可能作用机理,及其与其它生长因子之间的相互作用。对碱性成纤维细胞生长因子骨诱导作用的研究进展进行综述。     

肠道缺血再灌流对肾内源性碱性成纤维细胞生长因子和 …

目的和方法：研究肠道缺血再灌流过程对肾脏内源性碱性成纤维细胞生长因子和转化生长因子β基因表达的影响，采用肠系膜上动脉夹闭４５ｍｉｎ与再灌流６ｈ和２４ｈ动物模型，用原位杂交与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）技术研究两种生长因子基因在正常，缺血以及再灌流肾组织中的表达。     

碱性成纤维细胞生长因子共聚物缓释微球与兔跨区轴型皮瓣的成活

背景：研究表明使用生长因子直接或间接刺激新生血管形成可以促进皮瓣远端缺血部分的成活。 目的：观察碱性成纤维细胞生长因子-聚乳酸-聚羟基乙酸共聚物缓释微球对家兔侧腹制作跨区轴型皮瓣成活的影响。 方法：取24只健康家兔制作侧腹壁跨区轴型皮瓣,随机分组：实验组术前5 d皮内注入碱性成纤维细胞生长因子-聚乳酸-聚羟基乙酸共聚物微球,对照组术前5 d皮内注入碱性成纤维细胞生长因子+空微球悬浊液,空白对照组术前5 d皮内注入生理盐水。5 d后掀起皮瓣原位缝合。 结果与结论：①皮瓣成活率：实验组显著高于对照组和空白对照组(P < 0.01),②皮瓣组织学变化：实验组新生血管增生明显,以毛细血管为主。③CD34+免疫组织化学结果：实验组新生血管大量形成,平均血管数目高于对照组和空白对照组(P < 0.05)。表明术前5 d局部注射碱性成纤维细胞生长因子-聚乳酸-聚羟基乙酸共聚物缓释球可促进皮瓣新生血管形成,增加皮瓣血运,促进皮瓣成活。     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.