VEGF-promoted surgical angiogenesis in necrotic bone

The ability of vascular endothelial growth factor (VEGF) to accelerate neoangiogenesis from implanted arterovenous (AV) bundles in necrotic bone was evaluated. A saphenous AV bundle was placed in a necrotic segment of rabbit ilium. In group II, VEGF (100 ng/h x 3 days) was administered by continuous infusion. Bone blood flow was measured with radioactive-labeled microspheres, and capillary density was determined by microangiography combined with Sp?lteholtz bone clearing at 1, 2, and 4 weeks. Neovascularization was observed along the implanted vascular bundle in both groups. One week after surgery, bone blood flow and vessel area were significantly higher in VEGF-treated animals (P < 0.05). No significant difference was observed at later times. Direct VEGF administration increased surgical angiogenesis and improved blood flow and neovascularization in necrotic bone 1 week after AV bundle implantation. Thereafter, a robust angiogenic response from the AV bundle was seen in both groups.     

Revascularization and bone remodeling of frozen allografts stimulated by intramedullary sustained delivery of FGF‐2 and VEGF

Frozen bone allografts are susceptible to nonunion and fracture due to limited revascularization and incomplete bone remodeling. We aim to revascularize bone allografts by combining angiogenesis from implanted arteriovenous (AV) bundles with delivery of fibroblast growth factor (FGF‐2) and/or vascular endothelial growth factor (VEGF) via biodegradable microspheres. Rat femoral diaphyseal allografts were frozen at ?80°C, and heterotopically transplanted over a major histocompatibility mismatch. A saphenous AV bundle was inserted into the intramedullary canal. Growth factor was encapsulated into microspheres and inserted into the graft, providing localized and sustained drug release. Forty rats were included in four groups: (I) phosphate‐buffered saline, (II) FGF‐2, (III) VEGF, and (IV) FGF‐2 + VEGF. At 4 weeks, angiogenesis was measured by the hydrogen washout method and microangiography. Bone remodeling was evaluated by quantitative histomorphometry and histology. Bone blood flow was significantly higher in groups III and IV compared to control (p < 0.05). Similarly, bone remodeling was higher in VEGF groups. FGF‐2 had little effect on allograft revascularization. No synergistic effect was observed with use of both cytokines. Delivered in microspheres, VEGF proved to be a potent angiogenic cytokine, increasing cortical bone blood flow and new bone formation in frozen allografts revascularized with an implanted AV bundle. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1431–1436, 2011     

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF‐2 and VEGF

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF‐2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF‐2, VEGF, or FGF‐2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF‐2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF‐2 + VEGF was observed. VEGF delivered in microspheres had superior long‐term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF‐2 or FGF‐2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1556–1562, 2012     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

Contrasting roles of leukemia inhibitory factor in murine bone development and remodeling involve region-specific changes in vascularization

We describe here distinct functions of leukemia inhibitory factor (LIF) in bone development/growth and adult skeletal homeostasis. In the growth plate and developing neonate bones, LIF deficiency enhanced vascular endothelial growth factor (VEGF) levels, enlarged blood vessel formation, and increased the formation of "giant" osteoclasts/chondroclasts that rapidly destroyed the mineralized regions of the growth plate and developing neonatal bone. Below this region, osteoblasts formed large quantities of woven bone. In contrast, in adult bone undergoing remodeling osteoclast formation was unaffected by LIF deficiency, whereas osteoblast formation and function were both significantly impaired, resulting in osteopenia. Consistent with LIF promoting osteoblast commitment, enhanced marrow adipocyte formation was also observed in adult LIF null mice, and adipocytic differentiation of murine stromal cells was delayed by LIF treatment. LIF, therefore, controls vascular size and osteoclast differentiation during the transition of cartilage to bone, whereas an anatomically separate LIF-dependent pathway regulates osteoblast and adipocyte commitment in bone remodeling.     

Vascular endothelial growth factor promotion of neoangiogenesis in conventional nerve grafts

The effect of vascular endothelial growth factor (VEGF) on angiogenesis and neovascularization of conventional nerve grafts was evaluated in 48 rabbits. A 2.5-cm segment of right sciatic nerve was removed and orthotopically repaired. This graft was wrapped in dialysis tubing to prevent vessel ingrowth from adjacent tissue. An osmotic pump delivered either VEGF (100 ng/h for 3 days) or control solution. Evaluation methods included angiography, vessel density, and nerve blood flow measurement at 3, 7, and 14 days. On day 3, 42% of the control nerves and 100% of VEGF-treated nerves had partial longitudinal neovascularization. Vessel density (0.84 +/- 0.22 vessel/mm(2)) and nerve blood flow [25.34 +/- 7.62 mL/(min.100 g)] in VEGF-treated nerves were significantly higher than control group values [0.23 +/- 0.13 vessel/mm(2) and 5.35 +/- 0.99 mL/(min.100 g)]. Progressive improvement in parameters was seen at 7 and 14 days. Vascular endothelial growth factor infusion accelerates longitudinal neoangiogenesis and shortens the nerve ischemic time to 3 days in this model. The revascularization of VEGF-treated conventional nerve grafts in a poorly vascularized bed is identical to that of grafts in a healthy bed.     

骨形态发生蛋白2基因治疗在修复骨缺损中对血管化影响的实验研究

目的观察骨形态发生蛋白-2（BMP-2）基因治疗在修复骨缺损中对血管化的影响。方法分离培养兔骨髓基质干细胞（MSCs），经BMP-2腺病毒载体转染后复合异种骨支架移植修复1．5cm桡骨缺损。分为5组：①BMP-2基因转染细胞＋去抗原牛松质骨支架（BCB）；②未转染细胞＋重组BMP-2＋BCB；③对照基因转染细胞＋BCB；④未转染细胞＋BCB；⑤BCB。术后4、8、12周行微血管墨汁灌注、血管内皮生长因子（VEGF）免疫组化染色、组织学等检测。结果术后4周，基因治疗组骨间血管的密度在周边区域较高，通常每一个骨小梁孔隙中都有一支新形成的小血管；透射电镜见成骨细胞总是毗邻血管内皮细胞而存在，并随着微血管的增生而逐渐向骨细胞发展；间质细胞VEGF表达明显增强。结论BMP-2基因治疗可通过上调VEGF表达，间接诱导移植骨血管化，对骨不连、骨缺损的治疗具有重要意义。     

Interactions between osteopontin and vascular endothelial growth factor: Implications for skeletal disorders

Osteopontin (OPN) and vascular endothelial growth factor (VEGF) are characterized by a convergence in function for maintaining the homeostasis of the skeletal and renal systems (the bone–renal–vascular axis regulates bone metabolism). The two cytokines contribute to bone remodeling, dental healing, kidney function, and the adjustment to microgravity. Often, they are co-expressed or one molecule induces the other, however, in some settings OPN-associated pathways and VEGF-associated pathways are distinct. In bone remodeling, OPN and VEGF are regulated under the influence of growth factors and hormones, hypoxia and inflammation, the micro-environment, and various physical forces. Their abundance can be affected by drug treatment. OPN and VEGF are variably associated with kidney disease. Their balanced levels are critical for restoring endothelial cell function and ameliorating the adverse effects of microgravity. Here, we review the relevant 83 papers of 257 articles published, and listed in PubMed under the key words OPN and VEGF.     

Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model

This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 microg), BMP-2 (2 mug), both VEGF (12 microg) and BMP-2 (2 microg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n=8-9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1+/-9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7+/-14.1% and 37.4+/-18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3+/-4.8% and 7.8+/-7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone.     

Regulation of the Bone Vascular Network is Sexually Dimorphic

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone–derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.     

Changes in innervation of long bones after insertion of an implant: Immunocytochemical study in goats with antibodies to calcitonin gene-related peptide and B-50/GAP-43

In this study, we describe the distribution of fibres that contain calcitonin gene-related peptide in normal bones and in bones that are remodeling after insertion of an implant. With routine histology and antibodies to calcitonin gene-related peptide, small neural and free-running fibres staining positively for calcitonin gene-related peptide were found in the periosteum, endosteum, and cortical bone of the tibia in the goat. In many cases, the free-running fibres were associated with blood vessels that entered the bone through Volkmann's canals. The endosteal blood supply was destroyed as a result of insertion of the implant. The necrotic bone was no longer innervated, as shown by the lack of staining for the antibodies. At 6 weeks, a repair phase started with revascularization and remodeling of the necrotic endosteal bone. During this repair phase, there was increased innervation with fibres containing calcitonin gene-related peptide in the remodeling cavities at the interface between living and necrotic bone. These fibres ended blindly, with many large varicosities, and could be demonstrated by immunostaining with monoclonal antibodies to B-50/growth associated protein-43, an antibody to outgrowing neuronal fibres. The correlative occurrence between extensive sprouting of fibres containing calcitonin gene-related peptide and the remodeling of necrotic endosteal bone suggests that sensory fibres with calcitonin gene-related peptide have a regulatory role in the control of angiogenesis or of bone remodeling associated with the insertion of an implant, or with both processes.     

Intermittent PTH(1–84) is osteoanabolic but not osteoangiogenic and relocates bone marrow blood vessels closer to bone‐forming sites

Intermittent parathyroid hormone (PTH) is anabolic for bone. Our aims were to determine (1) whether PTH stimulates bone angiogenesis and (2) whether vascular endothelial growth factor (VEGF A) mediates PTH‐induced bone accrual. Male Wistar rats were given PTH(1–84) daily, and trabecular bone mass increased 150% and 92% after 30 and 15 days, respectively. The vascular system was contrasted to image and quantify bone vessels with synchrotron radiation microtomography and histology. Surprisingly, bone vessel number was reduced by approximately 25% and approximately 40% on days 30 and 15, respectively. PTH redistributed the smaller vessels closer to bone‐formation sites. VEGF A mRNA expression in bone was increased 2 and 6 hours after a single dose of PTH and returned to baseline by 24 hours. Moreover, anti‐VEGF antibody administration (1) blunted the PTH‐induced increase in bone mass and remodeling parameters, (2) prevented the relocation of bone vessels closer to bone‐forming sites, and (3) inhibited the PTH‐induced increase in mRNA of neuropilin 1 and 2, two VEGF coreceptors associated with vascular development and function. In conclusion, PTH(1–84) is osteoanabolic through VEGF‐related mechanism(s). Further, PTH spatially relocates blood vessels closer to sites of new bone formation, which may provide a microenvironment favorable for growth. © 2011 American Society for Bone and Mineral Research     

AACB/BMP/bFGF复合物修复犬股骨头中再血管化与新骨形成的作用

目的观察吸附有骨形态发生蛋白（BMP）及碱性成纤维细胞生长因子（bFGF）的异体脱抗原松质骨（AACB）植入股骨头坏死（FHN）病灶清除区骨缺损后的修复过程,评价bFGF对FHN的再血管化作用与新骨形成的关系. 方法建立液氮冷冻诱导性犬FHN模型,以AACB/BMP/bFGF作为材料充填骨缺损处.术后行组织学大体和光镜观察,组织切片免疫组化血管染色,进行血管计数和新生骨面积图像分析,并分析再血管化作用与新骨形成之间的关系.结果①免疫组化血管计数：术后第3周移植孔隙内大量血管增生;第6、12周时血管数量进一步增加.②组织切片光镜观察：术后第3周移植物周围间充质细胞大量增生,新骨开始形成;第12周充填区广布成熟骨小梁.术后第3周新骨面积占视野的7.73%;第6、12周时新骨面积进一步加大.③血管形成与新骨形成的关系：术后所有时间点血管数与新骨形成面积呈正相关关系. 结论 AACB是生长因子的良好吸附载体,适宜新生血管长入.吸附有bFGF及BMP的AACB具有较强的再血管化能力,bFGF促进再血管化的同时,加速新骨形成.这一疗法有望成为FHN治疗的一种手段.     

肝门部胆管癌组织内血管形成与细胞凋亡相关性的研究

目的　研究肝门部胆管癌中细胞凋亡和血管形成的相互关系。方法　采用原位末端标记技术和抗因子Ⅷ的抗体分别检测肝门部胆管癌细胞凋亡指数和瘤内微血管的密度,同时用免疫组化法检测血管内皮细胞生长因子（ＶＥＧＦ） 和胆管癌细胞凋亡调控基因ｂｃｌ２ 的表达。结果　肝门部胆管癌组织ＶＥＧＦ阳性表达率５５－５６％ （２０／３６）,ＶＥＧＦ阳性表达与ＭＶＤ呈正相关（Ｐ＜ ０－０１） 。高血管组的ｂｃｌ２ 阳性表达明显高于低血管组（ Ｐ＜ ０－０５）,而低血管组的细胞凋亡指数高于高血管组（ Ｐ＜０－０１）。ｂｃｌ２ 表达丰富的区域,ＶＥＧＦ的表达率高。结论　肝门部胆管癌血管形成影响细胞凋亡的敏感性和发生率。     

VEGF165基因修饰脂肪干细胞对糖尿病大鼠骨缺损的修复研究

目的 :探讨采用基因转染大鼠脂肪干细胞构建血管化组织工程的方法对糖尿病骨质疏松性骨缺损的修复效果。方法:选取雄性Wistar大鼠78只,体重180~220 g,其中72只通过化学药物(STZ)诱导法建立糖尿病动物模型,成模大鼠血糖值均≥16.7 mmol/L。将实验动物随机分为5组,正常对照组6只,其他实验组各18只。正常对照组:在正常大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞;糖尿病组:单纯糖尿病骨缺损大鼠;生长因子组:在糖尿病大鼠骨缺损内单纯植入VEGF生长因子;干细胞组:在糖尿病大鼠骨缺损内单纯植入脂肪干细胞;实验组:在糖尿病大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞。将5×106个VEGF165-ADSCs细胞与凝胶海绵结合后,植入到糖尿病大鼠骨缺损模型中,在植入后第4周时,采用光学显微镜观察缺损修复组织大体形态;采用免疫组化SP法测定骨缺损区修复后局部微血管密度;应用美国IRIS IntrepidⅡXSP电感耦合等离子体发射光谱仪对修复骨痂内钙/磷含量和碱性磷酸酶(ALP)含量测定;统计分析上述测量结果验证VEGF165-ADSCs对糖尿病大鼠骨缺损的修复作用。结果:荧光染色结果显示,VEGF165表达定位于ADSCs的细胞浆,表达率在87﹪以上;大体组织学观察结果显示:实验组修复区内骨痂生成范围和质量接近正常组,糖尿病组、生长因子组、干细胞组修复效果欠佳。植入后第4周,实验组单位体积的修复组织钙、磷含量和ALP含量明显高于生长因子组、干细胞组(P0.05),与正常对照组组比较差异无统计学意义(P0.05);第4周时,实验组修复局部的血管密度低于正常对照组(P0.05),而显著高于其他组(P0.05)。结论 :VEGF165基因修饰的脂肪干细胞在糖尿病大鼠体内具有良好的成骨及成血管作用,有望成为修复糖尿病特定骨质条件下骨缺损的一种有效手段。