Changes in gene expression associated with the bone anabolic effects of basic fibroblast growth factor in aged ovariectomized rats

Basic fibroblast growth factor (bFGF) stimulates bone formation in vitro and in vivo. The purpose of this study was to determine changes in gene expression for bone matrix proteins, growth factors, and cytokines associated with the stimulatory effects of bFGF on bone formation in aged ovariectomized (ovx) rats. At 3 months of age, female Sprague-Dawley rats were sham-operated (sham) or ovariectomized (ovx), then maintained untreated for 1 year. At 15 months of age, baseline (BSL) sham and ovx rats were killed. All other rats received daily intravenous injections of bFGF (200 microg/kg) or vehicle (veh) for 14 days. Lumbar vertebrae were processed for quantitative bone histomorphometry or molecular analyses. Ovariectomy decreased vertebral cancellous bone volume by approximately 33% and increased most indices of bone turnover. Treatment of aged ovx rats with bFGF for only 14 days significantly increased cancellous bone volume compared with vehicle treatment of ovx rats, but this variable remained lower than in sham + veh rats. Osteoid volume, osteoblast surface, and osteoid surface were markedly increased, and osteoclast surface was significantly decreased in ovx + bFGF rats compared with sham + veh and ovx + veh rats. Northern analyses revealed that mRNA levels for osteocalcin and type I collagen, relative to 18S RNA, were significantly higher in ovx + bFGF rats than in ovx + veh rats by a factor of >10. RNase protection assays revealed that insulin-like growth factor (IGF-I) mRNA levels, relative to L32 housekeeping gene, were also significantly higher, by nearly a factor of 3, in ovx + bFGF rats than in ovx + veh rats. Treatment of ovx rats with bFGF did not appear to affect message levels for transforming growth factor-beta (TGF-beta), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma). These in vivo results suggest that bFGF treatment upregulates gene expression for IGF-I, which may mediate, at least in part, the increased gene expression for bone matrix proteins and the bone anabolic effects of bFGF in aged ovx rats.     

Basic fibroblast growth factor has rapid bone anabolic effects in ovariectomized rats

Basic fibroblast growth factor (bFGF) has a strong bone anabolic effect in intact and ovariectomized (OVX) rats treated for 7–14 days. Other growth factors such as IGF-I and TGF- have been implicated as potential mediators for this effect. The purpose of this study was to examine the early effects of bFGF therapy, in vivo, on bone formation and gene expression in OVX rats in order to determine whether upregulation of gene expression for IGF-I and/or TGF- precedes or coincides with the stimulatory effects of bFGF on bone formation. At 3 months of age, Sprague Dawley rats were OVX or sham-operated (SHAM), then maintained untreated for 3 months. One group of baseline OVX rats (BSL OVX) and BSL SHAM rats were then killed. Additional OVX groups were treated IV with bFGF at a daily dose of 200 g/kg and killed at 1–7 and 10 days. Another group of OVX rats was treated IV with vehicle daily for 10 days, then killed. Lumbar vertebrae were processed for cancellous bone histomorphometry or RNA isolation. Ovariectomy induced increased cancellous bone turnover and a significant decrease in vertebral bone mass. Treatment of OVX rats with bFGF resulted in a significant increase in bone formation. As early as 24 h after bFGF treatment of OVX rats, osteoblast surface, osteoid surface, and osteoid volume were more than double those in BSL OVX rats and continued to increase with time. These variables were also significantly higher in bFGF-treated OVX rats at 10 days compared with vehicle-treated OVX rats. Gene expression for IGF-I was not different between BSL OVX rats and bFGF-treated OVX rats at 1 day, but was significantly higher by approximately 50% in OVX rats treated with bFGF for 2 and 7 days, and was also significantly higher by nearly 75% in OVX rats treated for 10 days compared with OVX rats treated with vehicle. Gene expression for TGF-₁ was unchanged at early times and only significantly upregulated by a relatively modest 30% in OVX rats treated with bFGF for 10 days. The results indicate that the bone anabolic effects of bFGF in OVX rats begin as early as 24 h following the initial treatment, and increase with time. These early stages of the strong stimulatory effect of bFGF on bone formation were not associated with a large upregulation of gene expression for IGF-I and TGF-. The rapid increase in osteoblast surface in bFGF-treated OVX rats suggests that the growth factor induces conversion of bone lining cells to osteoblasts.     

Decreased bone anabolic effect of basic fibroblast growth factor at fatty marrow sites in ovariectomized rats

The purpose of the study was to compare the bone anabolic effects of basic fibroblast growth factor (bFGF) at hematopoietic (red) and fatty (yellow) marrow sites in ovariectomized (ovx) rats. Female Sprague Dawley rats were subjected to ovariectomy or sham surgery at 3 months of age and maintained untreated for 2 months after surgery. Three groups of ovx rats were then injected intravenously with bFGF for 14 days at a dose of 200 microg/kg body weight. One group of bFGF-treated OVX rats was killed at the end of the treatment period, whereas the other two groups were killed at 7 or 14 days after withdrawal of bFGF treatment. Another group of ovx rats and a group of sham-operated control rats were treated intravenously with vehicle alone for 14 days. The proximal tibia and first lumbar vertebra, bone sites with hematopoietic marrow, as well as the distal tibia and caudal vertebra, bone sites with primarily fatty marrow, were processed undecalcified for quantitative bone histomorphometry. At the hematopoietic marrow sites, bFGF treatment induced a marked accumulation of osteoid, which calcified during the withdrawal period to result in a significant increase in cancellous bone volume. Osteoblast and osteoid surfaces were increased by at least a factor of 10 at these sites in bFGF-treated ovx rats before declining rapidly during the withdrawal period. In contrast, osteoid volume was negligible in the fatty marrow sites of bFGF-treated ovx rats. Although these animals exhibited a nonsignificant trend for increased cancellous bone volume in the fatty distal tibia during the withdrawal period, no such trend was observed in the fatty caudal vertebra. Indices of bone formation (osteoblast and osteoid surfaces) were significantly increased by bFGF treatment in the fatty distal tibia, which retained some small pockets of hematopoietic cells, but not to the same great extent as in the skeletal sites with hematopoietic marrow. Furthermore, not even a trend for increased osteoblast and osteoid surfaces was observed in the fatty caudal vertebra of bFGF-treated ovx rats. These findings indicate that bFGF is a strong bone anabolic agent at skeletal sites with hematopoietic marrow, but the stimulatory effects of the growth factor on bone formation are greatly attenuated at fatty marrow sites.     

Bone anabolic effects of subcutaneous treatment with basic fibroblast growth factor alone and in combination with estrogen in osteopenic ovariectomized rats

Although basic fibroblast growth factor (bFGF) is a potent stimulator of bone formation when administered intravenously, less is known regarding the effects of this peptide on bone following subcutaneous (s.c.) administration. In addition, it is unknown whether coadministration of estrogen enhances the bone response to treatment with bFGF. Therefore, the purpose of this study was (1) to characterize the skeletal response to s.c. injection of a high dose of bFGF, and (2) to determine whether concurrent administration of estrogen affects the skeletal response to bFGF treatment. Female Sprague-Dawley rats were ovariectomized (ovx) or sham-operated (sham) at 3 months of age and left untreated for 2 months to establish cancellous osteopenia in the ovx group. The sham rats (n=10) and one group of ovx rats (n=9) were then injected s.c. with vehicle alone for 3 weeks. Two additional groups of ovx rats were injected s.c. with bFGF (n=10) or with bFGF + estrogen (n=10) for 3 weeks. bFGF was administered s.c. at a daily dose of 1 mg/kg/day and estrogen was administered s.c. 4 days per week at a dose of 10 microg/kg for the 3-week duration of treatment. Lumbar vertebrae were collected and processed undecalcified for quantitative bone histomorphometry. Cancellous bone volume was lower and cancellous bone turnover was higher in vehicle-treated ovx rats than in vehicle-treated sham rats. Subcutaneous treatment of ovx rats with bFGF for 3 weeks resulted in a 4-fold increase in osteoblast surface and an 8-fold increase in osteoid surface in comparison to vehicle treatment of ovx rats. Osteoid volume was also markedly increased in the bFGF-treated ovx rats (7 +/- 4%) in comparison to vehicle-treated ovx rats (<0.1%). Osteoblast surface, osteoid surface, and osteoid volume were nearly identical in ovx rats treated with bFGF alone and with bFGF + estrogen. Although the majority of the osteoid in bFGF- and bFGF + estrogen-treated animals was deposited along mineralized bone surfaces, osteoid spicules without any connections to preexisting bone surfaces were also detected, providing definitive proof for bone formation within bone marrow in response to bFGF administration. Osteoclast surface, an index of bone resorption, was not affected by bFGF treatment. However, cotreatment of ovx rats with bFGF + estrogen resulted in lower osteoclast surface in comparison to treatment of ovx rats with either vehicle or bFGF alone. In summary, these findings indicate that administration of a high dose of bFGF via s.c. injection markedly increases bone formation and may be a useful treatment for cancellous osteopenia in the estrogen-deplete skeleton. The anabolic effects of bFGF on bone are not enhanced by concurrent treatment with estrogen at the replacement dose used in this study.     

Dose-dependent stimulation of bone induction by basic fibroblast growth factor in rats

Implantation of demineralized bone matrix in rodents elicits a series of cellular events leading to the formation of new bone inside and adjacent to the implant. This process is believed to be initiated by an inductive protein present in bone matrix, and local growth factors may further regulate the process. We have previously shown that local application of recombinant human basic fibroblast growth factor (bFGF) in a carboxymethyl cellulose gel to demineralized bone matrix implants increases the bone yield as measured by calcium content 3 weeks after implantation in rats. We now report that this increase was seen at 3 and 4 weeks, but not earlier or later. Further, the stimulatory effect was seen with doses from 3 to 75 ng per implant. A dose of 0.6 or 380 ng did not increase the bone yield, and 1,900 ng had a marked inhibitory effect. This narrow dosage optimum may reflect the complex actions of the growth factor.     

Effects of basic fibroblast growth factor on experimental diabetic neuropathy in rats

Basic fibroblast growth factor (bFGF) stimulates angiogenesis and induces neural cell regeneration. We investigated the effects of bFGF on diabetic neuropathy in streptozotocin-induced diabetic rats. Diabetic rats were treated with human recombinant bFGF as follows: 1) intravenous administration, 2) intramuscular injection into thigh and soleus muscles with cross-linked gelatin hydrogel (CGH), and 3) intramuscular injection with saline. Ten or 30 days later, the motor nerve conduction velocity (MNCV) of the sciatic-tibial and caudal nerves, sensitivity to mechanical stimuli, sciatic nerve blood flow (SNBF), and retinal blood flow (RBF) were measured. Delayed MNCV in the sciatic-tibial and caudal nerves, hypoalgesia, and reduced SNBF in diabetic rats were all ameliorated by intravenous administration of bFGF after 10, but not 30, days. Intramuscular injection of bFGF with CGH also improved sciatic-tibial MNCV, hypoalgesia, and SNBF after 10 and 30 days, but caudal MNCV was not improved. However, intramuscular injection of bFGF with saline had no significant effects. bFGF did not significantly alter RBF in either normal or diabetic rats. These observations suggest that bFGF could have therapeutic value for diabetic neuropathy and that CGH could play important roles as a carrier of bFGF.     

增殖性肾小球肾炎中碱性成纤维细胞生长因子的表达及其作用

目的探讨碱性成纤维细胞生长因子（ｂＦＧＦ）在人类增殖性肾小球肾炎（ＰＧＮ）中的表达及作用。方法采用原位杂交、免疫组化和放免测定方法检测肾活检组织中的ｂＦＧＦｍＲＮＡ及蛋白质表达、肾小球内细胞外基质（ＥＣＭ）面积和有核细胞数、层粘蛋白（ＬＮ）和前胶原Ⅲ（ＰＣⅢ）含量。结果在以细胞增殖和ＥＣＭ积聚为特征的ＰＧＮ组中ｂＦＧＦ表达水平、ＬＮ和ＰＣⅢ含量均显著高于非增殖性肾小球肾炎（ＮＰＧＮ）组和正常组。结论过度表达的ｂＦＧＦ参与肾小球内细胞增殖和ＥＣＭ积聚，从而影响肾小球肾炎和肾小球硬化进程。     

Dose-dependent stimulation of bone induction by basic fibroblast growth factor in rats.

Implantation of demineralized bone matrix in rodents elicits a series of cellular events leading to the formation of new bone inside and adjacent to the implant. This process is believed to be initiated by an inductive protein present in bone matrix, and local growth factors may further regulate the process. We have previously shown that local application of recombinant human basic fibroblast growth factor (bFGF) in a carboxymethyl cellulose gel to demineralized bone matrix implants increases the bone yield as measured by calcium content 3 weeks after implantation in rats. We now report that this increase was seen at 3 and 4 weeks, but not earlier or later. Further, the stimulatory effect was seen with doses from 3 to 75 ng per implant. A dose of 0.6 or 380 ng did not increase the bone yield, and 1,900 ng had a marked inhibitory effect. This narrow dosage optimum may reflect the complex actions of the growth factor.     

碱性成纤维细胞生长因子对大鼠急性水肿性胰腺炎的治疗作用

目的　观察外源性碱性成纤维细胞生长因子(bFGF)对急性水肿性胰腺炎大鼠的治疗作用,并探讨其作用机制。方法　予雄性SD大鼠间隔1h皮下注射蛙皮素5 .5 μg/kg体重和7.5μg/kg体重诱导急性水肿性胰腺炎,3h后腹腔内注射bFGF 10 0 μg/kg体重。观察各组大鼠胰腺炎生化及病理学等指标的变化。结果　bFGF治疗组大鼠的血清淀粉酶,脂肪酶以及胰腺组织湿/干质量比率(13 83 .0±94.6)、(194.0±43 .6)和(4 .3 2±0 .3 2 )U /L明显低于非治疗组(P <0 .0 1) ,而与空白对照组差异无统计学意义(P >0 .0 5 )。光镜下显示其胰腺组织炎症表现显著改善。免疫组织化学结果显示该组胰腺腺泡细胞被5 溴 2 去氧胞苷标记的细胞核数[(18.9±1.4)个/显微镜视野,n =10 ]明显多于非治疗组(P <0 .0 1)。结论　早期外源性bFGF对蛙皮素诱导的大鼠急性水肿性胰腺炎有显著的治疗作用,其作用机制是通过减轻胰腺炎症和促进胰腺组织再生来实现的     

Stimulation of bone formation by intraosseous application of recombinant basic fibroblast growth factor in normal and ovariectomized rabbits

The effect on intraosseous bone formation of a single local injection of recombinant human basic fibroblast growth factor into the distal femur was examined in normal and ovariectomized rabbits. In normal rabbits, basic fibroblast growth factor increased bone mineral density around the injected site in a dose-dependent manner at 4 weeks, with significant effects at concentrations of 400 μg and greater. Doses of 400 and 1,600 μg of basic fibroblast growth factor increased bone mineral density by 8 and 9%, respectively, compared with the opposite control femur. Histological examination showed that basic fibroblast growth factor (400 μg) induced the proliferation or recruitment of undifferentiated mesenchymal cells around the existing trabeculae at 3 days after the injection. For the first 2 weeks, osteoid formation was strongly stimulated, and this was followed by mineral apposition for another 2 weeks, at which time the femurs were harvested. Consequently, basic fibroblast growth factor stimulated intraosseous bone formation at 4 weeks. We speculate that the direct action of basic fibroblast growth factor on bone formation may be to stimulate proliferation or recruitment of minimally differentiated mesenchymal cells and to initiate the cascade of events in later stages of bone formation. In ovariectomized rabbits, basic fibroblast growth factor (400 μg) also increased bone mineral density, histomorphometrical bone formation markers, and trabecular connectivity to levels similar to those in rabbits who had received sham operations.     

Expression of basic fibroblast growth factor in thyroid disorders

Morphologic and biologic studies were undertaken to clarify the biologic significance of basic fibroblast growth factor (bFGF) in human thyroid neoplasms. A total of 71 malignant tumors (50 papillary carcinomas, 14 follicular carcinomas, 7 anaplastic carcinomas), 11 follicular adenomas, 6 adenomatous goiters, and 6 Graves' disease tissues were examined employing immunohistochemical methods (avidin-biotin-peroxidase complex technique). An affinity-purified polyclonal rabbit antiserum to human bFGF was used as a primary antibody. The eluate of malignant thyroid tumor tissues from the heparin-Sepharose column was examined by Western blot analysis to elucidate the molecular weight form. With immunohistochemical staining, bFGF was frequently detected in the cytoplasm of malignant thyroid tumors compared to tissues of the benign diseases and normal controls. With anaplastic carcinoma, immunoreactivity of the tumor cells was particularly strong. In the correlative analyses between UICC TNM classification and bFGF staining in papillary carcinoma, there were significant differences when relating positive staining to the grade of nodal metastases. By Western blot analysis, the bFGF immunoreactivity was specifically detected in the two forms, with molecular weights of 18 and 33 kDa. The high-molecular-weight form was detected in only anaplastic carcinoma. The present investigations demonstrated a close correlation between the expression of bFGF and the degree of malignancy. bFGF might play an important role in promoting lymph node metastases. Moreover, the high-molecular-weight form of bFGF might have an intense influence on tumor growth.

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Resumen Se emprendió un estudio morfológico y biológico, con el propósito de clarificar la significación biológica del factor básico de crecimiento de fibroblastos (FBCF) en los neoplasmas de la glándula tiroides humana.Setenta y un tumores malignos (50 carcinomas papilares, 14 carcinomas foliculares y 7 carcinomas anaplásico), 11 adenomas foliculares, 6 bocios adenomatosos y 6 tejidos de glándula con enfermedad de Graves fueron examinados mediante métodos inmunohistoquímicos (técnica del complejo avidina-biotinaperoxidasa); se utilizó un antisuero policlonal purificado contra el FBCF humano, como anticuerpo primario. Además, se utilizó el análisis de Western blot para elucidar la forma de peso molecular.Con la coloración inmunohistoquímica, el FBCF fue detectado con frecuencia en el citoplasma de los tumores malignos de la tiroides, en comparación con lo observado en la enfermedad benigna y en los controles normales. La inmunorreactividad tumoral fue particulamente fuerte en el carcinoma anaplásico. En el análisis correlativo entre la clasificiación TNM UICC y la coloración del FBCF en el carcinoma papilar, se hallaron diferencias significativas relativas a la coloración positiva y al grado de la metástasis ganglionares.En el análisis Western blot, la inmunorreactividad FBCF fue específicamente detectada en las dos formas diferentes con pesos moleculares de 18 K y 33 K. La forma de alto peso molecular fue detectada sólamante en el carcinoma anaplásico.La presente investigación demuestra una estrecha correlación entre la expresión de FBCF y el grado de malignidad. El FBCF puede jugar un papel importante en promover las metástasis ganglionares. Además, la forma de alto peso molecular del FBCF puede tener una influencia más intensa sobre el crecimiento del tumor.

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Résumé Une étude morphologique et biologique a été effectuée pour clarifier la signification du facteur de croissance des fibroblastes de base (bFGF) dans les tumeurs de la thyroïde chez l'homme. On a étudié 71 tumeurs de la thyroïde (50 cancers papillaires, 14 cancers folliculaires, 6 adénomes solitaires, et 7 cancers anaplasiques, 11 adénomes folliculaires, 6 goitres adénomateux et 6 Maladies de Basedow) en utilisant des méthodes immunohistochimiques et notamment la technique du complexe avidinebiotine-peroxydase. On a employé un anticorps primaire fabriqué à partir d'un antisérum polyclonal de lapin purifié dirigé contre le bFGF humain. L'éluat des tissus thyroïdiens malins provenant de la colonne héparine-sepharose a été examiné selon la technique Western Blot pour déterminer son poids moléculaire. Le bFGF a été détecté plus fréquemment dans le cytoplasme des tumeurs thyroïdiennes malignes que dans les maladies bénignes et les contrôles. Dans le cancer anaplasique, l'immunoréactivité des cellules cancéreuses a été particulièrement forte. En ce qui concerne la corrélation entre la classification TNM de l'Union Internationale contre le Cancer et la coloration bFGF des cancers papillaires, il y avait des différences significatives correspondant au degré d'envahissement des ganglions lymphatiques. Dans l'analyse selon la technique du Western blot, l'immunoréactivité bFGF a été détectée spécifiquement sous les deux formes de bFGF ayant des poids moléculaires de 18 et de 33 K, respectivement. Le poids moléculaire de 33K n'a été détecté que dans le cancer anaplasique. Cette étude démontre la corrélation étroite entre l'expression bFGF et le degré de malignité. Le bFGF peut probablement jouer un rôle important dans la survenue de métastase lymphatique. Le type à poids moléculaire élevé de bFGF pourrait influencer d'advantage la croissance tumorale.

     

碱性成纤维细胞生长因子对腹部切口愈合影响的临床观察

目的观察碱性成纤维细胞生长因子(bFGF)对腹部切口愈合的影响.方法69例腹部外科手术患者随机分为bFGF治疗组(n=31)和对照组(n=38),治疗组腹壁切口全层涂抹bFGF,并用bFGF浸润纱条覆盖(4000AU/cm切口),对照组用生理盐水.以切口愈合时间、切口红肿发生率、瘢痕发生率和术后肝肾功能作观察指标.结果治疗组切口愈合天数、术后早期切口红肿及瘢痕增生发生率组间无差异,术后肝肾功能均正常且无差异.结论bFGF用于腹部外科切口,无不良反应,能否促进腹部切口愈合并提前拆线,尚待进一步研究证实.     

Recombinant basic fibroblast growth factor accelerates wound healing

Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds.     

Effects of basic fibroblast growth factor and a prostaglandin E2 receptor subtype 4 agonist on osteoblastogenesis and adipogenesis in aged ovariectomized rats.

bFGF stimulates osteo- and adipogenesis concurrently at skeletal sites with red but not with fatty marrow, whereas a PGE2 receptor subtype 4 agonist has bone anabolic effects at both skeletal sites and decreases adipose tissue within red and fatty marrow. INTRODUCTION: Basic fibroblast growth factor (bFGF) stimulates osteogenesis at skeletal sites with hematopoietic but not with fatty marrow. The prostaglandin E2 (PGE2) receptor subtype 4 agonist (EP4A) stimulates osteogenesis at the former skeletal sites, but its effects at fatty marrow sites are unknown. In addition, both bFGF and PGE2 through the EP4 receptor have also been implicated in adipogenesis. However, their specific effects on bone marrow adipogenesis and the inter-relationship with osteogenesis have never been studied in vivo. MATERIALS AND METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated and maintained for 1 yr after surgery. OVX rats were then injected daily with bFGF or with EP4A SC for 3 wk. The osteo- and adipogenic effects of these agents were assessed by histomorphometry and by determining changes in expression of genes associated with these events by real-time PCR in the lumbar and caudal vertebrae, bones with a predominance of hematopoietic and fatty marrow, respectively. Expression of FGFR1-4 and the EP4 receptor were also evaluated by real-time PCR and immunocytochemistry. RESULTS: bFGF and EP4A stimulated bone formation at skeletal sites with hematopoietic marrow, but only the later anabolic agent is also effective at fatty marrow sites. The diminished bone anabolic effect of bFGF at the fatty marrow site was not caused by a lack of cell surface receptors for the growth factor at this site. Interestingly, whereas EP4A decreased fatty marrow area and the number of adipocytes, bFGF increased osteogenesis and adipogenesis within the bone marrow. CONCLUSIONS: bFGF can stimulate osteogenesis and bone marrow adipogenesis concurrently at red marrow sites, but not at fatty marrow sites. In contrast, EP4A stimulates bone formation at skeletal sites with hematopoietic and fatty marrow and simultaneously decreased fatty marrow area and the number of adipocytes in the bone marrow, suggesting that osteogenesis occurs at the expense of adipogenesis.     

Stimulation of bone formation by intraosseous injection of basic fibroblast growth factor in ovariectomised rats

Summary. The effect on intraosseous bone formation of a single local injection of recombinant human basic fibroblast growth factor into trabecular bones was examined in ovariectomised osteoporotic rats. Fibroblast growth factor (400 μg), or the vehicle alone, was injected into the ilium at 16 weeks after ovariectomy or a simulated operation. Bone mineral density in the ovariectomised rats increased to a level similar to the latter at 2 weeks and reached a maximum at 8 weeks. After 8 weeks, BMD decreased slowly and the value at 24 weeks was still higher than that in the ovariectomised rats. Fibroblast growth factor stimulated osteoid formation in the first 2 weeks, bone volume reaching a peak at 8 weeks. From 8 to 12 weeks, bone resorption increased, resulting in decreases in bone volume to the levels of the group with simulated operations at 24 weeks. Structural analysis at 8 and 24 weeks showed that ovariectomy decreased the continuity of trabeculae and the injection of fibroblast growth factor restored it to levels higher than, or equal to, those who had the simulated operation. The present study demonstrated that intraosseous fibroblast growth factor given to ovariectomised rats restored bone volume and quality to the levels of the rats who had a simulated operation only.

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Résumé. L’effet d’une unique injection locale dans les os trabeculaires de facteur de croissance de base de fibroblaste humain recombinant (bFGF) sur la formation intra-osseuse a été examiné sur des rats ostéoporotiques ovariectomisés. L’injection bFGF (400 μg) ou véhicule seul (V) a été pratiquée dans l’ilion 16 semaines après ovariectomie (OVX) ou pseudo-opération (Sham). A 2 semaines, la densité osseuse moyenne des OVX+bFGF avait augmenté jusqu’à un niveau similaire aux Sham+V pour atteindre son maximum à 8 semaines, soit 19% de plus que les Sham+V. Au-delà de 8 semaines, la densité moyenne a décliné lentement, conservant à 24 semaines une valeur supérieure aux OVX+V. Le bFGF a stimulé la formation ostéo? de des 2 premières semaines. Le volume d’os minéralisé a atteint son sommet à 8 semaines. Entre 8 et 12 semaines, la résorption osseuse a été facilitée, conduisant à des baisses du volume osseux jusqu’aux niveaux Sham+V 24 semaines. Les analyses structurelles à 8 et 24 semaines ont montré que l’injection bFGF ramène à des niveaux égaux ou supérieurs à Sham+V la connectivité des trabeculae réduite par l’OVX. La présente étude établit qu’une application intraosseuse de bFGF après ovariectomie ramène le volume et la qualité osseuse au niveau des rats pseudo-opérés.

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Accepted: 7 July 1997     

Sequential treatment with basic fibroblast growth factor and parathyroid hormone restores lost cancellous bone mass and strength in the proximal tibia of aged ovariectomized rats.

This study was designed to determine whether sequential treatment with basic fibroblast growth factor (bFGF) and parathyroid hormone (PTH) can restore lost cancellous bone mass and strength at a severely osteopenic skeletal site in aged ovariectomized (OVX) rats. Female Sprague-Dawley rats were subjected to sham surgery or ovariectomy at 3 months of age and maintained untreated for the first year after surgery. At 15 months of age, groups of baseline control and OVX rats were killed and catheters were inserted in the jugular veins of all remaining rats. Two groups of OVX rats were injected intravenously (iv) daily with bFGF for 14 days at a dose of 200 microg/kg body weight. At the end of bFGF treatment, one group was killed whereas the other group was subjected to 8 weeks of treatment with synthetic human PTH 1-34 [hPTH(1-34)] consisting of subcutaneous (sc) injections 5 days/week at a dose of 80 microg/kg. Another group of OVX rats was treated iv with vehicle for 2 weeks followed by treatment with PTH alone for 8 weeks. Other groups of sham-operated control rats and OVX rats were treated iv and sc with vehicle alone. The right proximal tibia from each rat was processed undecalcified for quantitative bone histomorphometry and the left proximal tibia was subjected to biomechanical testing. Baseline and vehicle-treated OVX rats were severely osteopenic because their tibial cancellous bone volumes were less than 5% compared with mean values of 20.3% and 15.0% in baseline and vehicle-treated control rats, respectively. Treatment of OVX rats for 2 weeks with bFGF alone did not significantly increase tibial cancellous bone volume but induced marked increases in osteoid volume, osteoblast surface, and osteoid surface. Sequential treatment of aged OVX rats with bFGF and PTH increased tibial cancellous bone volume (15.1%) and load to failure to at least the level of vehicle-treated control rats. Tibial cancellous bone volume (10.8%) and load to failure also were significantly increased by treatment with PTH alone, and these variables were not significantly different from those of OVX rats treated with bFGF + PTH. However, tibial ash density was significantly greater in OVX rats treated sequentially with bFGF and PTH compared with OVX rats treated with PTH alone. Our findings suggest that sequential treatment with bFGF and PTH may be useful for restoration of lost cancellous bone in the severely osteopenic, estrogen-deplete skeleton, but it cannot be concluded with certainty that this sequential treatment has a greater bone restorative effect than treatment with PTH alone.     

Neuroprotective effects of basic fibroblast growth factor following spinal cord contusion injury in the rat.

Cytokines and neurotrophic factors have been implicated in the pathophysiology of injury to the central nervous system. While some cytokines are considered pro-inflammatory, other factors promote neuronal growth and survival. The present study investigated the neuroprotective effects of interleukins 1 (IL-1), 4 (IL-4), and 6 (IL-6), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF) in a contusion model of spinal cord injury. Female Sprague-Dawley rats (n = 55) sustained a 10-g weight-drop injury to the lower thoracic spinal cord (T10) from a height of 12.5 mm using the NYU impactor. A micro-infusion system (Alzet minipump) was used to continuously deliver drugs or vehicle directly into the epicenter of the contused spinal cord starting 1 or three h postinjury. At the end of 7 days, animals were perfused and the cords removed for histopathological analysis. Longitudinal serial sections were cut on a freezing microtome and stained with cresyl violet. Areas of central necrosis, partial preservation, and total zone of tissue injury were identified and traced by an independent reviewer using a computer based imaging system. The mean total zone of injury in five animals receiving vehicle infusion was 18.04+/-4.20 mm3. The mean zone of partial preservation in these animals was 16.46+/-3.32 mm. Basic fibroblast growth factor reduced the total zone of injury by 33% [p<0.01, least significant difference (LSD) of Fisher] in five animals and the zone of partial preservation by 32% (p<0.01, LSD of Fisher) when compared to controls. There were trends toward reduction in total zone of injury and zone of partial preservation in rats treated with IL-4, CNTF, and NGF versus vehicle; however, none of these reached statistical significance. No significant differences were observed between animals receiving vehicle versus bFGF treatment commencing 3 h after injury. These data demonstrate that the continuous intramedullary infusion of bFGF initiated one hour after moderate contusion injury of the spinal cord significantly reduces the total zone of injury and the zone of partial preservation. These results support the further investigation and possible future clinical application of bFGF in the treatment of acute spinal cord contusion injury.     

The effect of bovine basic fibroblast growth factor on skin flap survival in rats.

Basic fibroblast growth factor (bFGF) was prepared from bovine pituitary glands and evaluated for its effect on the viability of pedicle skin flaps in rats. Pedicle flaps measuring 3 x 7.5 cm and based cephalad were created on the backs of animals. The treatment group received bFGF in saline solution intradermally by Dermo-jet injection 30 minutes before flap elevation. Skin flaps treated with a single application of 80 U of bFGF demonstrated a significant increase in viability from 40.8% to 69.7% of the flap area (p less than 0.001); the flaps treated with 16 U of bFGF exhibited little improvement. Intradermal administration of bFGF to pedicle skin flaps produced an increase in viability that approximates the increase obtained by a surgical delay procedure; treatment of flaps with exogenous bFGF may offer advantages over surgical delay procedures.     

Role of hypoxia in growth factor responses: differential effects of basic fibroblast growth factor and platelet-derived growth factor in an ischemic wound model

Both recombinant basic fibroblast growth factor and platelet-derived growth factor-BB homodimer are potent inducers of new tissue generation in models of normal dermal repair. However, their therapeutic targets include chronic wounds, which are frequently characterized by local tissue hypoxia. To explore the potential of recombinant basic fibroblast growth factor and platelet-derived growth factor-BB homodimer to stimulate more clinically relevant repair, we created an ischemic dermal wound on the rabbit ear by ligating two of the arteries which feed the ear. Both recombinant basic fibroblast growth factor and platelet-derived growth factor-BB homodimer stimulated marked neovascularization of the wound (p < 0.0001), but only recombinant platelet-derived growth factor-BB homodimer accelerated and augmented granulation tissue formation (p = 0.01) and reepithelialization. This study is the first demonstration of a direct angiogenic effect of recombinant platelet-derived growth factor-BB homodimer in vivo. India ink perfusion coupled with endothelial cell-specific histochemistry showed that nearly all the neovessels in all wounds were functional, indicating rapid capillary morphogenesis. In the nonischemic (normal) rabbit ear, recombinant basic fibroblast growth factor and platelet-derived growth factor-BB homodimer accelerated healing comparably, as expected. Higher doses of recombinant basic fibroblast growth factor also failed to elicit stimulatory effects in ischemic wounds. These results indicate that differential responsiveness to growth factors is related to local tissue hypoxia, angiogenesis alone is an insufficient stimulus for repair. These data also suggest new therapeutic approaches for the treatment of chronic wounds.     

碱性成纤维细胞生长因子与慢性疼痛调控的研究进展

背景慢性、持续的疼痛可能是患者就医最常见的原因,因此,临床上对慢性疼痛的治疗日益重视.目的简要介绍碱性成纤维细胞生长因子(basic fibroblast growth factor,FGF-2)的生物学特性及功能,重点综述其与星形胶质细胞活化的关系及参与慢性疼痛调控的研究进展.内容星形胶质细胞在慢性疼痛的发生、发展过程中起着关键作用,FGF-2是星形胶质细胞的有效激活剂,慢性疼痛发生时FGF-2在活化的脊髓星形胶质细胞中增加.趋向FGF-2调控疼痛的信号转导通路有待进一步研究,疼痛过程是由多种细胞和因子共同调节,它们的相互作用机制有待于进一步探讨.