Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors

OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro. In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined. METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes. In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage. RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number. Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen. Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization. The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2. Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix. To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used. Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18. Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation. Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization. CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.     

Fibroblast growth factor-2 promotes the repair of partial thickness defects of articular cartilage in immature rabbits but not in mature rabbits

OBJECTIVE: To investigate cartilage response to fibroblast growth factor-2 (FGF-2) with increasing age in vivo, we examined the effect of FGF-2 on partial thickness defects of immature and mature rabbits. DESIGN: Sixty-nine Japanese white rabbits (34 immature rabbits, 35 mature rabbits) were examined. We made experimental partial thickness defects in articular cartilage of the knees. Then, we injected FGF-2 into the knees eight times, immediately after surgery and every 2 days for 2 weeks. A single dose of FGF-2 was 10 ng/0.1 ml or 100 ng/0.1 ml. In the control group, 0.1 ml saline was injected on the same time schedule. The rabbits were sacrificed at intervals following surgery that ranged from 2 to 48 weeks. The specimens were stained with toluidine blue and examined microscopically. We used a modified semiquantitative scale for evaluating the histological appearance of repair. RESULTS: In immature rabbits, the cartilage repair in the FGF-2 (100 ng)-treated group was significantly better than that of the other groups. The defects were almost completely repaired with chondrocytes that showed a round to polygonal morphology, and large amounts of extracellular matrix with intense metachromatic staining. In mature rabbits, however, there was apparently no effect from FGF-2 in either group. CONCLUSIONS: Application of FGF-2 facilitated cartilage repair in partial thickness defects in immature rabbits, but not in mature ones.     

成纤维细胞生长因子-23与肿瘤源性骨软化症

肿瘤源性骨软化症是一种少见的副肿瘤综合征,其病理机制尚不清楚.近年研究发现,肿瘤源性骨软化症患者血磷水平降低而成纤维细胞生长因子(FGF)-23水平显著增高,两者呈负相关性.FGF-23既可抑制肾脏对磷的重吸收及维生素D3活化,又可负性调节成骨细胞分化和基质矿物化.肿瘤源性骨软化症患者肿瘤切除后血FGF-23水平可恢复正常,低磷血症即获纠正,表明FGF-23与肿瘤源性骨软化症的病理生成密切相关.该文就FGF-23表达特点及其在肿瘤源性骨软化症发病机制中的作用等方面的研究进展作一综述.     

Fibroblast and vascular endothelial growth factor coating of decellularized vascular grafts stimulates undesired giant cells and graft encapsulation in a rat model

Replacing an infected prosthesis with a bioimplant provides a hopeful alternative in septic vascular surgery. The objective of this study was to determine the effect of fibroblast endothelial growth factors (FGF) and vascular endothelial growth factors (VEGF) coating on a decellularized vascular graft in a rat model and the possible impact on recellularization processes. Rat aortas were decellularized, crosslinked with genipin, and coated with poly-(D, L) lactide containing either FGF or VEGF. Observation periods were 6 and 12 weeks. Surprisingly, we found moderate accumulation of giant cells around the grafts that contained poly-(D, L) lactide acid. FGF and VEGF grafts showed massive stimulation of giant cells and eosinophils leading to complete graft encapsulation (P < 0.05). Pseudointmal hyperplasia was significantly increased in the FGF group (P < 0.05). Both results can only be interpreted as very negative. We achieved a situation in diametric opposition to that which we had hoped for. These data demonstrate that the use of growth factors may produce harmful side effects.     

Fibroblast growth factor-10 serves a regulatory role in duodenal development

Purpose

Duodenal obstruction occurs in 1 of 6000 live births and requires urgent surgical intervention. Duodenal atresia previously has been ascribed to a developmental failure of luminal recanalization; however, the cause of duodenal atresia remains incompletely understood. Although familial intestinal atresias have been described and syndromic associations are known, no specific genetic link has been established. Fibroblast growth factor-10 (Fgf10) is a known regulatory molecule relevant to mesenchymal-epithelial interactions, and mice deficient in Fgf10 demonstrate congenital anomalies in several organ systems including the gastrointestinal tract. The authors hypothesized that Fgf10 could serve a regulatory role in establishing normal duodenal development.

Methods

Wild-type mice with β-galactosidase under the control of the Fgf10 promoter were harvested from timed-pregnancy mothers. The expression of Fgf10 in the duodenum during development was evaluated by developing the embryos in X-Gal solution. Wild-type and mutant Fgf10^−/− embryos were harvested from timed-pregnancy mothers at 18.5 days postconception (near term) and were analyzed for duodenal morphology (Institutional Animal Care and Use Committee-approved protocol 32-02). Photomicrographs were reviewed.

Results

Fibroblast growth factor-10 is active in the duodenum at a late stage of development. The Fgf10^−/− mutants demonstrate duodenal atresia with a variable phenotype similar to clinical findings. The duodenum fails to develop luminal continuity and has proximal dilation. The phenotype occurs in an autosomal recessive pattern with incomplete penetrance (38%).

Conclusions

Fibroblast growth factor-10 serves as a regulator in normal duodenal growth and development. Its deletion leads to duodenal atresia and challenges traditionally accepted theories of pathogenesis. This novel, genetically mediated duodenal malformation reflects an animal model that will allow further evaluation of the pathogenesis of this surgically correctable disease. By studying the mechanism of Fgf10 function in foregut development, the authors hope to better understand these anomalies and to explore possible therapeutic alternatives.     

大鼠膝关节不稳定骨性关节炎软骨与软骨下骨组织微观结构的变化

[目的]观察大鼠膝关节经前交叉韧带切断术(anterior cruciate ligament transection,ACLT)造成关节不稳定情况下,其关节软骨与软骨下骨骨组织微结构的变化.[方法] 20只3个月龄雄性SD大鼠按体重随机分为对照组与实验组,每组10只动物.实验组切断右膝关节前交叉韧带,对照组只暴露前交叉韧带而不切断,术后12周处死所有动物并取材.取右肢股骨远端膝关节于70％酒精固定后脱钙6周,石蜡包埋切片,经HE染色及Masson染色,普通光学显微镜观察摄片,采用图像分析系统做定量分析;取右肢胫骨膝关节于70％酒精固定后不脱钙做硬组织包埋切片,行Van Kossa染色后,定量分析软骨下骨组织微观结构变化.[结果]术后12周后大体观察发现,实验组关节面较对照组明显失去光泽,呈颗粒感,部分软骨面破溃,关节面周围可见骨赘形成;实验组关节软骨HE及Masson染色着色不均,软骨细胞排列紊乱,且实验组Mankin评分结果显著高于对照组,差异具有统计学意义(P＜0.05);实验组软骨下骨组织微结构较对照组改变明显,差异具有统计学意义(P＜0.05).[结论]ACLT术导致膝关节不稳定,在此情况下对大鼠膝关节软骨形态及软骨下骨组织微结构造成显著破坏,若不能及时采取干预措施,将加速膝关节功能退变进程.     

Joint instability and cartilage compression in a mouse model of posttraumatic osteoarthritis

Joint instability and cartilage trauma have been previously studied and identified as key mediators in the development of posttraumatic osteoarthritis (PTOA). The purpose of this study was to use an in vivo model to compare the effect of joint instability, caused by the rupture of the anterior cruciate ligament (ACL), versus cartilage compression. In this study, mice were subjected to cyclical axial loads of twelve Newtons (N) for 240 cycles or until the ACL ruptured. One and eight weeks after this procedure, knees were sectioned coronally and evaluated for osteoarthritis by histology. Using a scoring scale established by [Pritzker K, Gay S, Jimenez S, et al. (2006): Osteoarthritis Cartilage 14:13–29], the articular cartilage across each surface was scored and combined to produce a total degeneration score. The ACL‐ruptured group had a significantly greater total degeneration score than either control or compression treated joints at 1 and 8 weeks. Additionally, only sections from ACL‐ruptured knees consistently showed synovitis after 1 week and osteophyte formation after 8 weeks. Thus, it appears using that ACL rupture consistently creates a severe osteoarthritis phenotype, while axial cartilage compression alone does not appear to be an appropriate method of inducing PTOA in vivo. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:318–323, 2014.     

兔骨关节炎软骨缺损动物模型的建立

目的探讨建立一种便捷实用的兔骨关节炎软骨缺损动物模型的方法，以适应软骨组织工程技术修复骨关节炎软骨缺损研究的要求。方法5-7月龄新西兰大白兔22只，雌雄不限，体重2．5～3．0kg。根据侧别，分为改良侧（左侧膝）及对照侧（右侧膝）。改良侧分别切除兔内侧半月板、前十字韧带并在股骨髌沟部制造一直径4mm，深3mm的软骨缺损，对照侧仅切除内侧半月板和前十字韧带。分别在术后第3周和第6周在双侧股骨髁部和髌沟部取材，比较两种骨关节炎动物模型的大体形态及病理变化，进行Mankin评分及统计学分析。结果术后6周，改良侧股骨髌沟软骨缺损仍明显存在，但缺损面直径减小，股骨髌沟墨汁染色均达Ⅳ级，光学显微镜下示股骨髌沟软骨缺损达钙化层以下；而对照侧股骨髌沟墨汁染色均未达Ⅳ级。术后3周，改良侧股骨髌沟部Mankin法OA评分（11．82±1．07）分，对照侧（2．37±0．62）分；术后6周，改良侧股骨髌沟部Mankin法OA评分（13．29±1．15）分，对照侧（5．65±1．03）分；改良侧与对照侧股骨髌沟部Mankin评分比较差异有统计学意义（P〈0．01），但股骨髁部Mankin评分两组比较，差异无统计学意义（P〉0．05）。改良侧关节软骨退变进行性加重。结论改良侧和对照侧均能获得满意的骨关节炎动物模型。改良侧在股骨髌沟处形成一个明显的陈旧性软骨缺损，为应用软骨组织工程技术研究骨关节炎软骨缺损修复提供了一种便捷实用的动物模型。     

Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing

The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type II procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and III procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type II procollagen and proteoglycan core protein.     

Acceleration of articular cartilage repair by combined gene transfer of human insulin-like growth factor I and fibroblast growth factor-2 in vivo

Introduction

Improving the biochemical and structural qualities of the new tissue that fills deep osteochondral defects is critical to enhance articular cartilage repair. We developed a novel molecular therapy to increase articular cartilage repair based on a combined strategy to stimulate chondrogenesis by co-transfection of the human insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) in a xenogenic transplantation model.

Materials and methods

NIH 3T3 cells were transfected with expression plasmid vectors containing a cDNA for the E. coli lacZ gene (lacZ implants), the human IGF-I gene (IGF-I implants) or both the human IGF-I and FGF-2 genes (IGF-I/FGF-2 implants). The expression patterns of the transgenes were monitored in vitro for 21 days. LacZ, IGF-I and IGF-I/FGF-2 implants were transplanted into osteochondral defects in the trochlear groove of rabbits. At 3 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively.

Results

Both IGF-I and IGF-I/FGF-2 implants secreted increased levels of the corresponding recombinant proteins in vitro. In vivo, transplantation of the co-transfected IGF-I/FGF-2 implants increased the DNA content of the repair tissue, accelerated the formation of the subchondral bone and improved articular cartilage repair in a magnitude that was larger than with IGF-I alone or when compared to lacZ implants.

Conclusion

These results suggest that gene delivery of a combination of IGF-I and FGF-2 to cartilage defects may be more beneficial than application of IGF-I alone.     

帕瑞昔布对膝骨关节炎大鼠关节软骨及软骨下骨的影响

[目的]观察帕瑞昔布对膝骨关节炎大鼠关节软骨、软骨下骨及自噬标志物Beclin-1表达的影响。[方法] 30只雄性SD大鼠随机分为假手术组、模型组、治疗组。假手术组仅切开膝关节皮肤,模型组与治疗组应用Hulth法复制OA模型。治疗组自术后首天起腹腔注射帕瑞昔布[10 mg/(kg·d)],每周5次,连续12周。12周后处死大鼠,ELISA法检测血清IL-1、IL-6、CTX-Ⅰ、CTX-Ⅱ、TRACP-5b;关节软骨行HE、番红O染色及Mankin评分;Micro-CT扫描软骨下骨;RT-QPCR技术检测Beclin-1 mRNA表达量。[结果]模型组IL-1、IL-6、CTX-Ⅰ、CTX-Ⅱ、TRACP-5b水平高于假手术组(P0.05);治疗组IL-1、IL-6、CTX-Ⅱ水平低于模型组(P0.05)。治疗组Mankin评分低于模型组(P0.05)。与假手术组比较,模型组骨体积分数(BV/TV)、骨小梁量(Tb.N)显著降低(P0.05),而骨小梁分离度(Tb.Sp)显著增高(P0.05)。与模型组比较,治疗组骨体积分数(BV/TV)、骨小梁量(Tb.N)降低(P0.05),而骨小梁分离度Tb.Sp增高(P0.05)。治疗组Beclin-1 mRNA表达量高于假手术组与模型组(P0.05)。[结论]帕瑞昔布可改善OA大鼠关节软骨退变,其机制可能是通过增强细胞自噬来完成。并能促进大鼠软骨下骨骨量丢失,可能给OA治疗带来负面影响。