Angiogenesis is a vital part of fracture healing, since it re-establishes the circulation at the injury site, limiting ischaemic necrosis and permitting repair. The present deliverable presents an investigation of the effect of US on angiogenesis during bone healing…. The deliverable is organized as follows: Chapter 2 is devoted to the description of angiogenesis' physiology Chapter 3 describes the effect of US in blood vessel formation and Chapter 4 is devoted to the mathematical formulation of the fundamental events that occur during angiogenesis endowed with equations that describe the effect of US.
Long bones receive blood from several groups of arteries: proximal/distal metaphyseal arteries, proximal/ distal epiphyseal arteries, diaphyseal nutrient arteries and periosteal arteries (Glowacki 1998). Bone fracture results in disruption of the marrow architecture and blood vessels within and around the fracture site (Glowacki et al., 1998, Rhinelander 1974, Brighton 1991).
After a bone fracture, blood vessels are disrupted and a hematoma is formed. Stem cells are then recruited to the site and new blood vessels are formed from pre-existing ones through the migration and proliferation of endothelial cells in a process known as angiogenesis. Vascular endothelial cells are then activated, form pseudopodia and begin to degrade their surrounding vessel membrane. Then the endothelial cells migrate into the interstitium, resulting in the formation of capillary sprouts. These sprouts create a central lumen and eventually connect to another sprout or capillary, forming capillary loops, which later either disappear or develop into larger vessels (Hudlicka and Tyler 1986). The newly developing blood vessels are lengthened due to a chemotactic response to growth factors (Harper 1991). Osteogenesis takes place near newly formed vessels, which mediate delivery of osteoprogenitor cells, secrete mitogens for osteoblasts, and transport nutrients and oxygen (Grundnes 1993). In regions of poor vascularity it has been reported that osteogenic cells follow a chondrogenic rather than an osteogenic pathway (Street et al., 2000, Peng et al., 2002). In addition, animal studies have shown that if new blood vessel formation does not occur (Hausman et al., 2001 6) fracture healing is blocked. Eckardt et al., (Eckardt et al., 2005) have also shown that exogenous application of vascular endothelial growth factor (VEGF) significantly accelerates fracture healing (Checa et Prendergast, 2009).
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The newly generated blood supply to the callus and cortical bone appears to persist until the medullary blood supply is fully regenerated (Rhinelander 19741). Marsh et al., (Marsh et al., 1998) report that local alterations in normal, delayed, and mal- unions may be attributed to the inhomogeneity of vascularity in the fracture site. Many growth factors/cytokines, who are active during fracture healing, play a key role in the process, having direct or indirect osteogenic and angiogenic actions (Carano and Filvaroff, 2003).
These factors include members of the fibroblast growth factor (FGF), transforming growth factor (TGF), bone morphogenetic protein (BMP) and vascular endothelial growth factor (VEGF) families (Geris et al., 2007). These factors are produced by and/or responded to by many cell types present at the fracture site (Carano and Filvaroff, 2003).
Endogenous VEGF is a keyplayer in bone repair, where its temporal and spatial expression pattern corresponds to the one observed during long bone development (Street et al., 2002). It is currently believed that blood vessel incursion plays a significant role in tissue healing and VEGF production combines angiogenesis with osteogenesis during fracture healing (Street et al., 2002). VEGF is expressed by various stimuli and regulates proliferation and action of endothelial cells, osteoblasts and osteoclasts (Carano and Filvaroff 2003, Street et al., 2002, Mayr-Wohlfart et al., 2002, Midy and Plouet, 1994, Deckers et al., 2000).
It has been proved that impaired angiogenesis and inhibition of the action of VEGF or its homologues cause a disruption in the healing process and lead to the occurrence of nonunions and mal-unions. Maes et al., (Maes et al., 2006) in their experimental study on fractures in PlGF (i.e., a placental growth factor)-knock-out mice found decreased levels of osteoblastic differentiation of mesenchymal progenitor cells. In addition a persistent cartilaginous matrix was created without any sign of endochondral ossification. Street et al., (Street et al., 2002) by using a mouse model of fracture healing also found reduced levels of angiogenesis, bone formation and callus mineralisation when VEGF activity is blocked. In addition vascular damage that occurs after injury, also results in delayed union or nonunion with low levels of cartilage or bone formation (Lu et al., 2007). Fang et al., (Fang et al., 2005) by using a distraction osteogenesis set-up found that the angiogenic process is disrupted by severe distraction (causing severe mechanical overload). In addition normal osteogenesis was prevented leading to fibrous nonunion. Fibrous nonunion also occured when proliferation of endothelial cells was inhibited by an anti-angiogenic agent.
Effect of Ultrasound on new blood vessel formation during bone healing
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The close relationship between blood vessel formation, i.e., angiogenesis and bone formation has long been recognized. Trueta (Tuerta, 1963) reports that Albrecht von Haller had suggested that the main responsible for bone formationis the vascular system (Haller 1763).More specifically Haller stated that 'the origin of bone is the artery carrying the blood and in it the mineral elements' (Haller 1763). From that time, the activity of osteoblasts in bone formation as well as the role of blood vessels, have gained significant interest.
The rate of angiogenesis is one of the basic elements in the bone healing process (Cohen 2006). It has been shown that ultrasound application increases synthesis of angiogenesis-related cytokines such as interleukin, fibroblast growth factor, and vascular endothelial growth factor (VEGF).
Reher et al., (Reher et al., 1999) and Doan et al., (Doan et al., 1999) performed in vitro studies to identify which cytokines and angiogenesis factors are induced by ultrasound. Ultrasound was applied to human mandibular osteoblasts, gingival fibroblasts and peripheral blood mononuclear cells (monocytes) in two different ways (i.e., 1 MHz, pulsed 1:4, tested at four intensities and 45 kHz, continuous, also tested at four intensities). In both cases the angiogenesis-related cytokines, i.e., IL-8 and bFGF, were found significantly stimulated in osteoblasts, and VEGF levels were increased in all cell types.
Rawool et al., (Rawool et al., 2003) by using power Doppler ultrasound showed that low-intensity ultrasound applied on ulnar osteotomies in dogs resulted in increased vascularity around the fracture site. While these investigators originally hypothesized that ultrasound would increase blood flow during treatment, increased blood flow was evident at the fracture site for an extended period after removal of the stimulus. This increased vascularity to the fracture site enhances the delivery of growth factors and cytokines which are necessary for the healing process. Capillary and new blood vessel formation takes place in the fracture site during the inflammatory phase of bone healing (Erdogan et al., 2009, Martinez et al., 2011).
Figure 1 Power Doppler images of the fracture site. On postoperative day 1 (POD1) minimal blood flow exists in both treated (A) and control limbs (B).On POD7 increased flow is shown in treated limb (C) but not in control one (D). On POD11 blood flow is again greater in treated limb (E) but not in control one (F) (Rawool et al., 2003).
In vitro studies on human osteoblasts (Reher et al., 2002, Wang et al., 2004) have also shown that LIPUS increases such as nitric oxide production and hypoxia-inducible factor-1a activation, which are crucial mediators for bone healing. This increase resulted in augmented levels of VEGF-A expression in osteoblasts stimulating angiogenesis in the early healing phase.
Young et Dyson (Young et Dyson, 1990) investigated the effect of ultrasound on new blood vessel formation in excised wounds on the flank skin of rats. The wounds were examined either in 5 days post fracture (i.e., during the late inflammatory phase) or in 7 days post-injury, (i.e., during the proliferative phase of repair). Ultrasound was applied for 5 min daily at intensity of 0.1 W/cm2 SATA (frequency either 0.75 MHz or 3.0 MHz). It was found that 5 days after injury blood vessel formation in the granulation tissue was increased in the US treated wounds than in the control ones. This result suggested that US has a significant effect on angiogenesis during the inflammatory stage. However, no significant differences in blood vessel number between the three groups were found 7 days post-injury (Figure 1). In this study it was also found that US frequency plays a significant role in angiogenesis since US application at 0.75 MHz the effect was more pronounced that at 3.0 MHz. The authors also suggested that the main US receptive components of the granulation tissue during the inflammatory stage are the macrophages. Macrophages have been proved to induce angiogenesis in vivo by producing angiogenic factors, and also to enhance endothelial cell proliferation in vitro (Povlerini et al., 1977, Leibovich 1984).
Figure 2 Average number of blood vessels formed at day 5 and day 7 in the three groups. *significantly different to control group
Cheung et al., (Cheung et al., 2011) investigated the effect of LIPUS on osteoporotic fracture rat models. By using quantification of gene expression, radiographic callus morphometry and histomorphometry, they found that LIPUS cause an upregulation in VGEF expression at week 4 post-fracture, which indicates increased amounts of new blood vessel formation. As the callus formation genes' expression reduced (Col-1 and BMP-2), the expression of bone remodelling gene of RANKL rapidly increased; new blood vessel formation was initiated by the significant increase in VEGF expression. Vascular endothelial growth factor was also found to be upregulated at week 4 to8. The authors suggested that LIPUS affects angiogenesis during the callus remodeling phase of the osteoporotic fracture healing process.
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By applying US on chick chorioallantoic membrane (CAM) (Ramli et al., 2009) also showed that ultrasound can induce angiogenesis in-vivo. The most pronounced angiogenic effect was found when US was directly applied at 45-kHz with intensity of 15 mW/cm2 and when idirectly applied through ultrasonicated fibroblasts at 1 MHz with intensity of 0.4 W/cm2 (Figure 2).
Figure 3 Angiogenesis after application of Ultrasound. There is a difference in the values between 45 kHz and 1MHz frequency treatments. S-US=sham treatment
Mathematical models describing angiogenesis during bone healing
Several ultrasonic parameters have been reported to influence the effect of LIPUS on fracture healing including temperature changes, intensity, dose, duration and timing of LIUS application.