Feasibility of prefabricated vascularized bone graft using the combination of FGF-2 and vascular bundle implantation within hydroxyapatite for osteointegration

The aim of this study was to demonstrate the feasibility of the prefabricated vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic (IP-CHA) in combination with vascular bundle implantation and basic fibroblast growth factor (FGF-2) administration in rabbit model. Thirty Japanese white rabbits were used. To make a prefabricated bone graft, the saphenous artery and vein were passed through the hole of the IP-CHA. Hundred micrograms of FGF-2 was administered into the IP-CHA before implanting the vascular bundle. First and foremost, the IP-CHA was placed subcutaneously in the medial thigh of rabbits for 4 weeks. In the experimental group, a prefabricated vascularized bone graft was used while IP-CHA alone was used in the control group. Second, the prefabricated vascularized bone graft was transplanted from the subcutaneous implanted site into the medial femoral condyle defect of the same rabbit and IP-CHA alone was implanted as the control graft in a different animal. At 4 weeks posttransplantation, bone union with host bone could be observed in the experimental group. However, the area of bone formation of the control group was significantly higher than in the experimental at 2 and 4 weeks posttransplantation. We conclude that the prefabricated vascularized bone graft when transplanted into a bone defect showed the ability for bone union with the host bone, although further studies are needed to accelerate the process of bone formation.     

Enhanced bone formation using hydroxyapatite ceramic coated with fibroblast growth factor-2

Our objective was to develop a bone substitute coated with fibroblast growth factor-2 (FGF-2) that subsequently releases FGF-2. We investigated the use of our system of bone substitutes to induce bone formation. Hydroxyapatite ceramic buttons (HAP-CBs) were coated with FGF-2 by precipitation in supersaturated calcium phosphate solution. HAP-CBs were coated with high or low doses of FGF-2, denoted as FGF-H and FGF-L. The release of FGF-2 from FGF-H and FGF-L was evaluated using its release profile and bioactivity. The efficacy of the subsequent bone formation was quantified using rats with round-shaped bone defects (5 mm in diameter) of the right parietal bone. Group 1 was treated only with HAP-CBs, group 2 with HAP-CBs and drops of FGF-2 solution, group 3 with FGF-L and group 4 with FGF-H. To detect the release of FGF-2 in vivo, the expression of bone morphogenic protein-2 (BMP-2) was measured in the defective bone tissue. FGF-2 was released in vitro from FGF-H and FGF-L, and maintained its bioactivity. Rats treated with FGF-L showed better bone formation than rats from the other groups. BMP-2 expression was detected in the defective bone tissues of group 3 at 14 days, which might indicate in vivo FGF-2 release during this period. A specific FGF-2 concentration may be needed for bone formation, and our system can release FGF-2 at adequate concentrations to induce bone formation.     

Prefabrication of vascularized bone graft using guided bone regeneration

This article describes the prefabrication of a vascularized bone graft composed of autologous particulate cancellous bone and marrow (PCBM), a vessel bundle, and a biodegradable membrane. The PCBM was placed around the saphenous vessel bundle of rats and rolled with a biodegradable membrane of L-lactide-epsilon-caprolactone copolymer to prepare the prefabricated vascularized bone graft (group A). As controls, combinations of PCBM and membrane (group B), vessel bundle and membrane (group C), and PCBM and vessel bundle (group D) were prepared. A radiographic study revealed radio-opacity in the implantation site of group A 1 week later, in contrast to the other groups. Newly formed bone in the membrane roll was histologically confirmed, and neomicrovasculature circulating from the vessel bundle through the newly formed bone tissue was observed. The increase in alkaline phosphatase activity and osteocalcin content was significant for the group A preparation compared with the other groups. We concluded that the combination of autologous PCBM, a vessel bundle, and a biodegradable membrane was promising in the prefabrication of vascularized bone with good blood circulation.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.     

Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone using interconnected porous hydroxyapatite ceramics

The aim of the present study was to investigate the possibility of integrating porous hydroxyapatite (HA) ceramics with a capillary vessel network via insertion of a vascular pedicle, and to determine whether this procedure enhances new bone formation in tissue engineering of bone. First, synthetic interconnected porous HA (IP-CHA) was implanted subcutaneously into rat groin with or without insertion of superficial inferior epigastric vessels. At 6 weeks, IP-CHA with vascular insertion contained thick fibrous connective tissue with a number of large blood vessels that seemed to derive from the inserted vascular bundle. Next, IP-CHA loaded with recombinant human bone morphogenetic protein 2 (BMP, 2 or 10 microg/block) was implanted with or without vascular insertion. At 3 weeks, IP-CHA/BMP (10 microg) composite with vascular insertion exhibited abundant new bone formation in the pores of the deep portion close to the inserted vessels. In contrast, IP-CHA/BMP (10 microg) without vascular insertion showed poor bone formation. Histomorphometric analysis demonstrated that vascular insertion significantly increased new bone formation. In IP-CHAs with a lower dose of BMP (2 microg), no bone formation was found, with or without vascular insertion. These results suggest that the present system of integrating a vascular network with IP-CHA is a useful technique for bone tissue engineering.     

Distinct role of fibroblast growth factor-2 and vascular endothelial growth factor on tumor growth and angiogenesis

Tumors express more than a single angiogenic growth factor. To investigate the relative impact of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) on tumor growth and neovascularization, we generated tumor cell transfectants differing for VEGF and/or FGF-2 expression. Human endometrial adenocarcinoma HEC-1-B-derived Tet-FGF-2 cells that express FGF-2 under the control of the tetracycline-responsive promoter (Tet-off system) were further transfected with a VEGF(121) anti-sense (AS-VEGF) cDNA. Next, Tet-FGF-2 and AS-VEGF/Tet-FGF-2 cells were transplanted subcutaneously in nude mice that received tetracycline or not in the drinking water. Simultaneous expression of FGF-2 and VEGF in Tet-FGF-2 cells resulted in fast-growing lesions characterized by high blood vessel density, patency and permeability, and limited necrosis. Blood vessels were highly heterogeneous in size and frequently associated with pericytes. Inhibition of FGF-2 production by tetracycline caused a significant decrease in tumor burden paralleled by a decrease in blood vessel density and size. AS-VEGF expression resulted in a similar reduction in blood vessel density associated with a significant decrease in pericyte organization, vascular patency, and permeability. The consequent decrease in tumor burden was paralleled by increased tumor hypoxia and necrosis. A limited additional inhibitory effect was exerted by simultaneous down-regulation of FGF-2 and VEGF expression. These findings demonstrate that FGF-2 and VEGF stimulate vascularization synergistically but with distinctive effects on vessel functionality and tumor survival. Blockade of either one of the two growth factors results in a decrease in blood vessel density and, consequently, in tumor burden. However, inhibition of the expression of VEGF, but not of FGF-2, affects also vessel maturation and functionality, leading to tumor hypoxia and necrosis. Our experimental model represents an unique tool to investigate anti-neoplastic therapies in different angiogenic environments.     

Astrocytes shed extracellular vesicles that contain fibroblast growth factor-2 and vascular endothelial growth factor

We previously set a three-cell-type coculture system in which neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological blood-brain barrier. Moreover, we recently found that neurons produce fibroblast growth factor-2 and vascular endothelial growth factor and secrete them at least in part by shedding extracellular vesicles. In this study, on the basis of immunofluorescence, scanner electron microscopy and Western blot analyses, we concluded that also astrocytes in culture shed extracellular vesicles that contain the same angiogenic factors, as well as beta1-integrin, a membrane protein that is considered a marker of shedding. Vesicles released by astrocytes are smaller than the ones produced by neurons and have an average size of 150-500 nm.     

Adsorption of bisphosphonate onto hydroxyapatite using a novel co-precipitation technique for bone growth enhancement

Premature bone resorption and remodeling by osteoclasts can limit the longevity of implant fixation and recovery time. Orally administered bisphosphonates (BPs) have been used to inhibit osteoclast action at the implant/bone interface. Ideally, these should be delivered at the interface with the osteoblast-active hydroxyapatite (HA) for maximum effect. This investigation introduces a novel BP loading technique to achieve improved BP release from a simulated body fluid-grown HA (SBF-HA) with the aim of improving implant fixation. A solution co-precipitation technique incorporates the BP (pamidronate) into a thin SBF-HA coating. Surface analysis, using X-ray photoelectron spectroscopy (XPS), of the resultant coating was employed to confirm the presence of the adsorbed BP on the surface of SBF-HA. XPS analysis was also used to determine the optimal adsorption process. Osteoclast cell culture experiments confirmed the biological effectiveness of BP adsorption and proved that the pamidronate was biologically active, causing both decreased osteoclast numbers and decreased resorption.     

预分化脂肪干细胞与纤维蛋白胶和磷酸钙骨水泥自体异位构建血管化移植物

背景：预构骨皮瓣研究启发人们构建预构血管化骨进行游离移植来替代带血管蒂游离自体骨移植修复大段骨缺损的想法。 目的：设计一种基于预分化脂肪干细胞、纤维蛋白胶和多孔磷酸钙骨水泥支架复合体的血管化移植物。 方法：将体外分离培养的大鼠脂肪干细胞在条件培养基中进行血管内皮细胞定向分化,经鉴定活性后,复合至纤维蛋白胶和多孔磷酸钙骨水泥构建血管化组织工程支架。将血管化组织工程支架、纤维蛋白胶/多孔磷酸钙骨水泥支架及多孔磷酸钙骨水泥支架分别植入SD大鼠股四头肌肌袋内,植入后2,4周进行组织学检测、血管定量分析和Western blot检测。 结果与结论：向血管内皮细胞分化的脂肪干细胞与纤维蛋白胶和多孔磷酸钙骨水泥共培养7 d,可见细胞密度适中,与支架组织结合较好。植入实验中,各组支架孔隙中充填有纤维血管组织和脂肪组织,血管化组织工程组支架孔隙中均长入大量血管,并有小动脉长入,不同时间点的血管直径和数量及血管内皮生长因子C的表达量均优于纤维蛋白胶/多孔磷酸钙骨水泥组和多孔磷酸钙骨水泥组(P < 0.01)。表明构建的血管化组织工程支架能够实现可靠迅速血管化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.     

Recombinant growth/differentiation factor-5 (GDF-5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic

To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/MSCs/HA composites could be used as bone graft substitutes.     

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.     

Repair of segmental bone defects in rabbit tibiae using a complex of beta-tricalcium phosphate, type I collagen, and fibroblast growth factor-2

The objective of this study was to evaluate the effects of a complex of beta-tricalcium phosphate (beta-TCP) granules, collagen, and fibroblast growth factor-2 (FGF-2) on cortical bone repair in rabbits. Segmental bone defects of 5 mm in length were created in the middle of the tibial shaft. The defect was stabilized with a plate and screws, and was filled with 0.3 ml of a complex of beta-TCP granules and 5% collagen, with or without 200 microg of recombinant human fibroblast growth factor-2 (rhFGF-2). Bone regeneration and beta-TCP resorption were assessed by X-ray and micro-CT scanner. A three-point bending test was also performed. The results showed that the segmental bone defect was not only radiologically, but also mechanically healed with cortical bone 12 weeks after implantation of the complex with rhFGF-2. In contrast, after implantation of the complex without rhFGF-2, most of the defect was filled with beta-TCP and only a small amount of bone formation was found. These results suggest that resorption of beta-TCP is important for bone formation and may be promoted by FGF-2 in the beta-TCP implantation site. In addition, the complex of beta-TCP granules and collagen combined with rhFGF-2 provides a paste-like material that is easy to handle. This material may be of considerable use in the treatment of cortical bone defects.     

Mineralization and calcium fixation within a porous apatitic ceramic material after implantation in the femur of rabbits

Cylinders (0.8 cm long, 1.0 cm of diameter and with an axial hole), constituted, after firing, of a ceramic mixture of hydroxylapatite (HA) and beta-tricalciumphosphate (beta-TCP) in a 10:1 ratio, were implanted into mid-diaphyseal defects of one femur of 20 rabbits and stabilized with intramedullary rods. The implantation sites were checked radiographically every month, and after 3 months (3 animals) and 6 months (17 animals) the rabbits were sacrificed and the implants with the surrounding tissue were embedded in methylmethacrylate, cut to thick sections and analyzed by scanning electron microscopy (SEM). Porosimetric and x-rays diffraction analyses were carried out before and after implantation of the cylinders, and the state of mineralization at the bone-implant interface was determined by EDAX microprobe analysis. Bony callus formation started at 1 month at the osteotomy sites, as judged by radiography, but after 3 months a not-mineralized zone had still been demonstrated between bone and the implants. At 6 months, 13 implants showed themselves firmly fixed in their implantations beds, while 4 implants were only incorporated at their proximal ends. In bone contact zones, an enrichment of Ca2+ was displayed by microanalytical techniques in the outer zone of the implanted samples which may be explained by an apparent additional phase transformation of HA into TCP, thanks to the change of the Ca/P ratio, that takes place in vivo.     

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

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

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

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

The vascular blood supply of the second metacarpal bone: anatomic basis for a new vascularized bone graft in hand surgery

Summary After traumatic loss of the index finger the second metacarpal bone is often used as a free graft to reconstruct bony defects of the thumb. Since clinical experience has shown numerous advantages of using a blood supplied bone graft, an anatomical study was performed in 104 cadaveric hands to investigate the vascularization supply of this bone. After dye injections into the arterial system the vascular situation of the second metacarpal was studied and a classification was made. Six variations in arterial vascularization were found. The nutrient vessels to the bone originate from the radial artery or from the deep palmar arch and correspond to variable metacarpal arteries. Selective injection studies via the respective pedicles showed sufficient perfusion and complete dye distribution in the bone. Further dissections could demonstrate the feasibility of raising a pedicled bone graft only partially with preservation of the index finger. The second metacarpal as a vascularized graft may be harvested entirely in serious injuries with destruction of the index finger in emergency cases of skeletal thumb reconstruction or partially as an elective procedure. All type have a useful are of rotation and their pedicles allow transpositions within the radial side of the hand, especially for applications in the thumb and carpus. The clinical application of these procedures could be shown in five cases. The results of this study therefore provide the anatomical basis for transposition or free microvascular transplantation of the second metacarpal as a vascularized bone graft for a variety of indications in hand surgery.

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Vascularisation du deuxième métacarpien. Bases anatomiques pour un nouveau greffon vascularisé en chirurgie de la main

Résumé Après une perte de substance osseuse post traumatique du pouce associée à une perte de l'index, un greffon osseux vascularisé est souvent utilisé pour reconstruire le pouce à partir du deuxième métacarpien. Un transfer osseux présente des avantages quand il est vascularisé. Pour cette raison les auteurs ont étudié, sur 104 mains prelevées sur des cadavres, les bases anatomiques et les possibilités d'une transposition du métacarpe II avec un pédicule vasculaire. Après l'injection intraartérielle, la vascularisation du 2eme métacarpien II a été étudiée et une classification a été faite. Les auteurs ont trouvé six variantes différentes. Les artères nourricières proviennent de l'a. radiale ou de l'arcade palmaire profonde et forment les aa. métacarpiennes de situation variable. Des injections sélectives avec bleu de méthylène dans les artères ont prouvé les bonnes conditions de perfusion avec une diffusion du la substance colorante dans l'os entier. La longueur des pédicules vasculaires permet une rotation correcte de ce transfert osseux sur le versant radial de la main dans chaque variante, plus particulièrement dans le cas des applications au pouce et à la racine de main. Les résultats de ces recherches constituent la base anatomique pour une nouvelle méthode reconstructive dans la chirurgie de la main.

     

A novel hydroxyapatite ceramic bone substitute transformed by ostrich cancellous bone: characterization and evaluations of bone regeneration activity

Various biomaterials have been used for bone repair and reconstruction of bone defects. Inorganic xenogenic bone substitutes have been intensively studied because they possesses favorable regenerative properties. The purpose of this study was to evaluate the properties of a novel inorganic xenogenic bone substitute, sintered ostrich cancellous bone (SOCB). Bone regeneration capability was also comparing to that of other bone substitutes in rabbit calvarial defects. Biochemical and biomechanical properties of the SOCB ceramic closely resembled those of human bone. Bone regeneration was evaluated by radiograph, histology, and histomorphometry. Bone regeneration was significantly enhanced in defects treated with SOCB when compared with other bone substitutes. The biochemical and biomechanical properties of SOCB are favorable for bone regeneration. SOCB might be a promising biomaterial for the repair of bone defects.     

成纤维细胞生长因子2、成纤维细胞生长因子受体4蛋白在人甲状腺乳头状癌中的表达及意义

目的探讨甲状腺乳头状癌(PTC)组织中成纤维细胞生长因子2(FGF-2)和成纤维细胞生长因子受体4(FGFR-4)的表达及其是否存在相关性。方法收集89例甲状腺乳头状癌及30例癌旁正常甲状腺组织标本,采用免疫组织化学和免疫印迹法(Western blotting)检测FGF-2和FGFR-4蛋白在标本中的表达,并进行统计学分析。结果免疫组织化学方法结果显示,与癌旁正常组织相比,FGF-2及FGFR-4在人类甲状腺乳头状癌组织中均高表达(P0.01,P0.01),两者差异有统计学意义;FGF-2和FGFR-4在甲状腺乳头状癌中的表达与淋巴结转移(χ2=14.798,P0.01;χ2=7.27,P0.01)和分化程度(χ2=13.824,P0.01;χ2=16.921,P0.01)相关,而与性别、年龄、肿瘤大小无关(P0.05);通过Western blotting技术分析,FGF-2和FGFR-4癌组织中的表达明显高于正常组织,随着癌组织分化程度的降低,表达明显上调(P0.05),其结果和免疫组织化学染色的检测结果一致;并且两者在甲状腺乳头状癌中的表达呈正相关(rs=0.434,P0.01)。结论 FGF-2和FGFR-4与甲状腺乳头状癌的发生、侵袭和转移有关,两者具有正协同作用,联合检测对判断甲状腺乳头状癌的恶性程度及生物学行为是一项有意义的综合性指标。