Vascular inflammation and endothelial dysfunction in fracture healing

Angiogenesis is an important step in bone fracture healing. In this article, we report on the healing of long bone fractures, and the involvement of the vascular and the inflammatory systems in the process. We conducted a prospective study of 20 healthy adults with traumatic long bone fracture. One week after fracture, and then 1 month later, we evaluated markers of inflammation: vascular responsiveness (brachial endothelial function and ankle brachial index) and inflammatory and cytokine levels osteopontin [OPN], E-selectin, and vascular endothelial growth factor [VEGF]). Long bone fractures caused intense vascular and inflammatory responses, represented by high levels of OPN, Eselectin, and VEGF. In vivo measurements demonstrated severe endothelial dysfunction, which could support the idea that the vascular system is recruited to build new blood vessels that support bone regeneration.     

Bringing new life to damaged bone: The importance of angiogenesis in bone repair and regeneration

Bone has the unique capacity to heal without the formation of a fibrous scar, likely because several of the cellular and molecular processes governing bone healing recapitulate the events during skeletal development. A critical component in bone healing is the timely appearance of blood vessels in the fracture callus. Angiogenesis, the formation of new blood vessels from pre-existing ones, is stimulated after fracture by the local production of numerous angiogenic growth factors. The fracture vasculature not only supplies oxygen and nutrients, but also stem cells able to differentiate into osteoblasts and in a later phase also the ions necessary for mineralization.This review provides a concise report of the regulation of angiogenesis by bone cells, its importance during bone healing and its possible therapeutic applications in bone tissue engineering. This article is part of a Special Issue entitled “Stem Cells and Bone”.     

The role of pleiotrophin in bone repair

Bone has an enormous capacity for growth, regeneration, and remodelling, largely due to induction of osteoblasts that are recruited to the site of bone formation. Although the pathways involved have not been fully elucidated, it is well accepted that the immediate environment of the cells is likely to play a role via cell–matrix interactions, mediated by several growth factors. Formation of new blood vessels is also significant and interdependent to bone formation, suggesting that enhancement of angiogenesis could be beneficial during the process of bone repair. Pleiotrophin (PTN), also called osteoblast-specific factor 1, is a heparin-binding angiogenic growth factor, with a well-defined and significant role in both physiological and pathological angiogenesis. In this review we summarise the existing evidence on the role of PTN in bone repair.     

血管内皮细胞生长因子165及骨形态发生蛋白2促进兔骨缺损部位再血管化的研究

目的为大段骨缺损修复过程的血管再生探索一条可行的途径。方法成年大耳白兔30只,随机分为五组。右前肢为实验肢体,缺损长度约15 mm。第1～4组都采用胶原膜引导,结合纳米羟基磷灰石／聚酰胺骨水泥,不同的是在第1组中,复合血管内皮生长因子165(VEGF165)及骨形态发生蛋白2(BMP2)质粒;第2组复合VEGF165质粒;第3组复合BMP2质粒;第4组不复合任何质粒;第5组仅制作动物模型而不做任何处理,作为空白对照组。结果术后2周,核素断层摄影(ECT)结果显示第1组骨缺损修复后的局部血流量高于第2、3、4组(P<0．05);术后4周,X线片示第1组骨缺损处的骨痂明显增多;SEM显示在正常骨与工程骨交界处可见新生的骨小梁结构以及成骨细胞的附着;ECT结果照示第1组骨缺损的局部血流量高于第2、3、4组(P<0．01);第2组骨缺损的局部血流量高于第3、4组(P<0．01);术后8周,SEM显示第1组工程骨表面成骨细胞的附着多于其它各组, ECT结果显示与术后4周相同。结论在骨缺损的局部,联合应用表达VEGF165和BMP2质粒可以促进骨缺损局部的血液供应;附着质粒DNA的纳米羟基磷灰石／聚酰胺及引导性胶原膜在大段骨缺损局部的联合应用有助于新骨的形成。     

血管内皮生长因子与治疗性血管生成研究进展

血管内皮生长因子(vascular endothelial growtll factor,VEGF)是内皮细胞特异的有丝分裂原,有促进内皮细胞增生、增强血管通透性、加速新血管形成的作用.血管生成是一个具有重要生理、病理意义的过程.在人体的创伤愈合、炎症反应、器官再生过程以及肿瘤生长转移、血管增生性疾病中,血管生成有重要作用.治疗性血管生成是指利用成血管诱导因子或内皮祖细胞,模拟体内血管生成机制,促进新生血管形成,改善侧支循环.本文就VEGF和治疗性血管生成研究进展做一综述.     

Antiangiogenic treatment of cancer

The process of formation of new vessels from pre-existing capillaries is called angiogenesis. Angiogenesis is a complex process which involves distinct cells, soluble components and factors related to the extra-cellular matrix and which is highly important in a large variety of physiological and pathological processes in the body. Angiogenesis regulation takes place through a perfect equilibrium between the production and release of different stimulatory and inhibitory factors which vary in relation to needs and tissue types. A large number of diseases are characterized by alterations in the angiogenic process, either by an insufficiency or by excessive angiogenesis. The requirement of blood vessel proliferation for tumor growth was observed more than a century ago. Angiogenic treatment would have an indirect antitumoral action, inhibiting tumor vascularization and impairing the supply of essential nutrients for tumoral growth and development.     

Role of mesenchymal stem cells in bone regeneration and fracture repair: a review

Mesenchymal stem cells (MSCs) are non-haematopoietic stromal stem cells that have many sources, such as bone marrow, periosteum, vessel walls, adipose, muscle, tendon, peripheral circulation, umbilical cord blood, skin and dental tissues. They are capable of self-replication and of differentiating into, and contributing to the regeneration of, mesenchymal tissues, such as bone, cartilage, ligament, tendon, muscle and adipose tissue. The homing of MSCs may play an important role in the repair of bone fractures. As a composite material, the formation and growth of bone tissue is a complex process, including molecular, cell and biochemical metabolic changes. The recruitment of factors with an adequate number of MSCs and the micro-environment around the fracture are effective for fracture repair. Several studies have investigated the functional expression of various chemokine receptors, trophic factors and adhesion molecules in human MSCs. Many external factors affect MSC homing. MSCs have been used as seed cells in building tissue-engineered bone grafts. Scaffolds seeded with MSCs are most often used in tissue engineering and include biotic and abiotic materials. This knowledge provides a platform for the development of novel therapies for bone regeneration with endogenous MSCs.     

Dual-color imaging of angiogenesis and its inhibition in bone and soft tissue sarcoma

BACKGROUND: Angiogenesis is a critical step in tumor growth, progression, and metastasis. Soft tissue and bone sarcoma are resistant to most therapeutic approaches. Angiogenesis of these tumors may be an effective target. We hypothesized that we could inhibit tumor growth by targeting angiogenesis in a mouse model of sarcoma. We demonstrate in this report, using powerful color-coded fluorescent imageable tumor-host models, the onset of angiogenesis of these sarcomas and its inhibition. MATERIALS AND METHODS: Transgenic mice were used as the host in which green fluorescent protein (GFP) is driven by a regulatory element of the stem cell marker nestin (ND-GFP). Nascent blood vessels express ND-GFP in this model. We visualized, by dual-color fluorescence imaging, angiogenesis of sarcoma formed by the HT-1080 human fibrosarcoma cell line expressing red fluorescent protein (RFP) in the ND-GFP mice. Tumor cells were injected into either the muscle or the bone. RESULTS: Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, including the surrounding tissues. Immunohistochemical staining showed that CD31 colocalized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. The mice were given daily i.p. injections of 5 mg/kg of doxorubicin after implantation of tumor cells. Doxorubicin significantly decreased the mean nascent blood vessel density in the tumors as well as decreased tumor volume. CONCLUSION: The dual-color model of the ND-GFP nude mouse and RFP sarcoma cells is useful for the visualization and quantitation of bone and soft tissue tumor angiogenesis and evaluation of angiogenic inhibitors for such tumors. These data suggest targeting angiogenesis of sarcomas as a promising clinical approach.     

Prevention of fracture healing in rats by an inhibitor of angiogenesis

Angiogenesis is considered essential to fracture healing, but its role in the healing process remains poorly understood. Angiogenesis inhibitors, which block new blood vessel formation by specifically targeting vascular cells, are currently under development for use in cancer chemotherapy, and are potentially powerful tools for defining the consequences of angiogenic impairment on fracture healing. In this study, we directly tested the effects of the angiogenesis inhibitor TNP-470 on the healing of closed femoral fractures in an established rat model system. Beginning 1 day after fracture, animals received either angiogenesis inhibitor at a therapeutically effective antitumor dose, or a weight-adjusted amount of carrier vehicle. The progress of fracture healing was assessed at weekly intervals for 21 days by radiography and histology; functional assessment was carried out at day 24 by biomechanical testing. By all three criteria, treatment with the angiogenesis inhibitor completely prevented fracture healing. Formation of both callus and periosteal woven bone were suppressed, indicating that both the intramembranous and endochondral pathways of osteogenesis were affected. The resulting tissue resembled “atrophic nonunions” often seen clinically in cases of failed fracture healing, but rarelly achieved in animal models. These results show that angiogenesis is essential to very early stages of fracture healing, and suggest this model system may be useful for understanding the mechanisms underlying fracture nonunions due to vascular impairment. Finally, the data raise the possibility that impairment of fracture healing may be an adverse effect of clinical treatments with antiangiogenic drugs.     

Bone Morphogenetic Protein‐2 and Vascular Endothelial Growth Factor in Bone Tissue Regeneration: New Insight and Perspectives

The study of bone tissue regeneration in orthopaedic diseases has stimulated great interest among bone tissue engineering specialists and orthopaedic surgeons. Combinations of biomaterials, growth factors and stem cells for repairing bone have been much studied and researched, yet remain a challenge for both scientists and clinicians pursuing regenerative medicine. The purpose of this review was to elucidate the role of sequential release of bone morphogenetic protein‐2 and vascular endothelial growth factor in producing better outcomes in the field of bone tissue regeneration.     

The angiogenic response to skeletal injury is preserved in the elderly.

Angiogenesis is essential for normal bone formation and repair. Avascularity characterizes aberrant fracture union in the elderly, while angiogenic mechanisms during cutaneous wound repair are attenuated in aged humans. We hypothesized that skeletal injury results in local (circulating) and systemic (fracture site) 'angiogenic' responses and that these reparative mechanisms are attenuated with advanced patient age. This prospective study examined peripheral blood and fracture hematoma from 32 patients, 16 under 40 years and 16 over the age of 75, undergoing emergent surgery for isolated fracture. The angiogenic cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) were assayed. Endothelial cell cultures were supplemented with patient plasma and fracture hematoma and angiogenesis determined in vitro by measuring cell proliferation and blood vessel tube formation. Angiogenesis was determined in vivo using a murine dorsal wound pocket model and quantification of new blood vessel formation after 7 days. We found that all injured patients, irrespective of age, have elevated plasma and fracture hematoma levels of VEGF and PDGF. These elevated cytokine concentrations translate into biologically significant angiogenic effects, in vitro and in vivo. These effects are primarily VEGF mediated and are not dependent on patient age. The biological activity of these growth factors does not diminish with advanced age. Thus skeletal injury does result in local and systemic angiogenic responses whereby angiogenic cytokine availability and activity is preserved in the aged suggesting alternative mechanisms for the development of avascularity in delayed and fracture non-union in the elderly.     

AMPK在组织工程骨促骨再生研究中的应用及进展

组织工程骨(tissue engineered bone,TEB)由于其材料范围广泛,骨传导性及骨诱导能力极佳,在大段骨缺损的治疗及骨再生领域显示出广阔的前景。TEB与骨缺损处的血管再生过程极其复杂,需要种子细胞及细胞产生的各种生长因子的参与,而间充质干细胞(mesenchymal stem cells,MSCs)取材方便,伦理争议及免疫原性低,且具有成骨及成血管分化等能力,已作为骨缺损修复治疗中的新方式,可改善目前现有治疗方法的缺点及提高临床治疗效果。AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)是真核生物中调节细胞能量稳态及多种代谢相关激素的重要分子,可被多种途径激活而发挥生理调控作用。近年来,研究发现它与骨再生及骨微环境形成等生理过程密切相关,通过作用于MSCs、成骨细胞、破骨细胞及血管再生等发挥促成骨效应,也有不少研究者利用AMPK与MSCs联合治疗骨损伤的再生修复,在TEB促骨再生中产生积极影响。另外,该文也总结出AMPK参与骨再生作用机制可能与活性氧、细胞自噬及沉默信息调节因子1等信号通路有关。笔者就AMPK在TEB促骨再生中的研究及机制作一综述,旨在为骨缺损修复、骨再生靶向治疗及相关分子机制研究提供理论依据和新的思路。     

The biology of fracture healing

The biology of fracture healing is a complex biological process that follows specific regenerative patterns and involves changes in the expression of several thousand genes. Although there is still much to be learned to fully comprehend the pathways of bone regeneration, the over-all pathways of both the anatomical and biochemical events have been thoroughly investigated. These efforts have provided a general understanding of how fracture healing occurs. Following the initial trauma, bone heals by either direct intramembranous or indirect fracture healing, which consists of both intramembranous and endochondral bone formation. The most common pathway is indirect healing, since direct bone healing requires an anatomical reduction and rigidly stable conditions, commonly only obtained by open reduction and internal fixation. However, when such conditions are achieved, the direct healing cascade allows the bone structure to immediately regenerate anatomical lamellar bone and the Haversian systems without any remodelling steps necessary. In all other non-stable conditions, bone healing follows a specific biological pathway. It involves an acute inflammatory response including the production and release of several important molecules, and the recruitment of mesenchymal stem cells in order to generate a primary cartilaginous callus. This primary callus later undergoes revascularisation and calcification, and is finally remodelled to fully restore a normal bone structure. In this article we summarise the basic biology of fracture healing.     

Expression of angiogenic factors during distraction osteogenesis

Distraction osteogenesis is a unique and effective way to treat limb length inequality resulting from congenital and posttraumatic skeletal defects. However, despite its widespread clinical use, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation are not well understood. Previous studies in distraction osteogenesis have noted increased blood flow and vessel formation within the zone of distraction. These observations suggest that distraction osteogenesis may be driven in part by an angiogenic process. Using immunohistological analysis, the expression of two different angiogenic factors (VEGF and bFGF) was shown to localize at the leading edge of the distraction gap, where nascent osteogenesis was occurring. These cells were spatially adjacent to new vessels that were identified by staining for factor VIII. Microarray analysis detected maximal mRNA expression for a wide variety of angiogenic factors including angiopoietin 1 and 2, both Tie receptors, VEGF-A and -D, VEGFR2, and neuropilin 1. Expression of these factors was found to be maximal during the phase of active distraction. Expression of mRNA for extracellular matrix proteins and BMPs was also maximal during this period. A comparison between the patterns of gene expression in fracture healing and distraction osteogenesis revealed similarities; however, the expression of a number of genes showed selective expression in these two types of bone healing. These data suggest that bone formation during distraction osteogenesis is accompanied by the robust induction of factors associated with angiogenesis and support further investigations to elucidate the mechanisms by which angiogenic events promote bone repair and regeneration.     

Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF-inhibitor to the rabbit tibia during distraction osteogenesis.

INTRODUCTION: Therapeutic angiogenesis, a novel concept in tissue engineering, is neo-formation of blood vessels in a tissue upon delivery of an angiogenic growth factor to the tissue. We hypothesised that therapeutic angiogenesis could enhance bone formation and challenged the hypothesis in an experimental model of distraction osteogenesis. METHODS: Rabbits, divided into three equal groups of 12, had their right tibia lengthened by distraction osteogenesis. A mini-osmotic pump delivered to the osteotomy gap either recombinant human vascular endothelial growth factor (VEGF), VEGF-inhibitor, or vehicle alone during the latency and distraction phase. After consolidation, we assessed bone blood flow by radioactive microsphere entrapment, measured torsional stiffness and bone mineral content, and did histomorphometry. RESULTS: VEGF and VEGF-inhibitor treatment failed to influence bone blood flow, torsional stiffness, bone mineral content and histomorphometric indices of the bone regenerate. However, VEGF treatment increased the blood flow in bone of the distracted limb and VEGF-inhibitor treatment decreased bone blood flow. CONCLUSION: The regenerate was unresponsive to VEGF and VEGF-inhibitor treatment in contrast to the neighbouring bone, which implies different biological properties of the vasculature in native and regenerating bone. VEGF is not recommended for enhancement of bone formation in this setting.     

骨组织工程中促进血管化策略的研究进展

随着骨组织工程的不断发展,各种新型的骨移植材料为骨缺损的治疗提供了新的选择。目前骨组织工程技术面临的主要难点是保证骨移植材料在植入后早期、快速地实现材料内部的血运重建,即血管化问题。血管长入为骨组织的再生和重建提供必要的营养支持,因此血管化一直是骨组织工程领域研究的焦点。然而目前还没有一种促血管化的金标准策略。支架材料、种子细胞和生长因子作为组织工程3个要素仍是目前各种促血管化策略努力的基本方向,其中多种生长因子、多种细胞复合支架材料联合构建组织工程骨等方法取得了良好的血管化效果,是近来研究的热点。     

Angiogenesis: general mechanisms and implications for rheumatoid arthritis

In rheumatoid arthritis, the vascular endothelium is among the key targets for circulating mediators of inflammation and controls the trafficking of cells and molecules from the bloodstream toward the synovial tissue. Local blood vessel proliferation allows the pannus to develop and grow, thereby promoting cartilage and bone destruction and joint remodeling. Angiogenesis, the production of new capillaries from preexisting blood vessels, is a key process in rheumatoid arthritis that involves multiple substances such as cytokines, chemokines, growth factors, cell adhesion molecules, proteinases, proteinase inhibitors, and matrix proteins. In animal models of arthritis, angiogenesis inhibitors have been found to improve clinical and radiological outcomes, opening up the possibility of therapeutic applications in humans. Before this possibility is realized, the steady accumulation of data on the mechanisms that regulate angiogenesis will have to continue until a clear picture of angiogenesis is formed.     

Mechanisms and targets of angiogenesis and nerve growth in osteoarthritis

During osteoarthritis (OA), angiogenesis is increased in the synovium, osteophytes and menisci and leads to ossification in osteophytes and the deep layers of articular cartilage. Angiogenic and antiangiogenic factors might both be upregulated in the osteoarthritic joint; however, vascular growth predominates, and the articular cartilage loses its resistance to vascularization. In addition, blood vessel growth is increased at--and disrupts--the osteochondral junction. Angiogenesis in this location is dependent on the creation of channels from subchondral bone spaces into noncalcified articular cartilage. Inflammation drives synovial angiogenesis through macrophage activation. Blood vessel and nerve growth are linked by common pathways that involve the release of proangiogenic factors, such as vascular endothelial growth factor, β-nerve growth factor and neuropeptides. Proangiogenic factors might also stimulate nerve growth, and molecules produced by vascular cells could both stimulate and guide nerve growth. As sensory nerves grow along new blood vessels in osteoarthritic joints, they eventually penetrate noncalcified articular cartilage, osteophytes and the inner regions of menisci. Angiogenesis could, therefore, contribute to structural damage and pain in OA and provide potential targets for new treatments.     

Role of angiogenesis in the development and growth of liver metastasis

Cancer metastasis is a highly complex process that involves aberrations in gene expression by cancer cells leading to transformation, growth, angiogenesis, invasion, dissemination, survival in the circulation, and subsequent attachment and growth in the organ of metastasis. Angiogenesis facilitates metastasis formation by providing a mechanism to (1) increase the likelihood of tumor cells entering the blood circulation and (2) provide nutrients and oxygen for growth at the metastatic site. The formation and establishment of metastatic lesions depend on the activation of multiple angiogenic pathways at both primary and metastatic sites. A variety of factors involved in the angiogenesis of liver metastasis have been identified and may serve as prognostic markers and targets for therapy. Vascular endothelial growth factor, interleukin-8, and platelet-derived endothelial cell growth factor are all proangiogenic factors that have been associated with liver metastasis from various primary tumor types. Inhibition of the activity of these factors is a promising therapeutic approach for patients with liver metastases. In addition, inhibition of integrins that mediate endothelial cell survival may also serve as a component of therapeutic regimens for liver metastases. This review focuses on the biology of angiogenesis in liver metastasis formation and growth. Because colorectal carcinoma is the most tumor to metastasize to the liver, this disease will serve as a paradigm for the study of angiogenesis in liver metastases.     

Vascular Endothelial Growth Factor: An Essential Component of Angiogenesis and Fracture Healing

Fractures require adequate stability and blood supply to heal. The vascular supply to long bones is compromised in a fracture, and the ability to heal hinges on the ability of new blood vessels to proliferate from surrounding vessels in a process known as angiogenesis. This process is largely driven by the growth factor, vascular endothelial growth factor (VEGF), whose levels are increased locally and systemically during fracture healing. VEGF is involved in many steps throughout the fracture healing cascade, from initially being concentrated in fracture hematoma, to the promotion of bone turnover during the final remodeling phase. This article reviews the current literature surrounding the role of VEGF and other growth factors in reestablishing vascular supply to fractured bone, as well as medications and surgical techniques that may inhibit this process.