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In the present deliverable we present a numerical investigation of the effects on ultrasound on biophysical stimuli during bone fracture healing. A literature review has been made so as to elucidate the biophysical mechanisms that are triggered due to ultrasound and to be numerically formulated so as to be included in the dynamic model of bone fracture solidification that will be developed in C2. The deliverable is organized as follows: Chapter 2 is devoted to the description of bone structure and physiology of bone fracture healing; Chapter 3 provides an insight to biophysical mechanisms (i.e., thermal and nonthermal) that trigger various cellular processes leading to enhanced healing and Chapter 4 presents a review of clinical and animal studies that investigate the effect of US on various biophysical stimuli during the healing course and demonstrate its positive action.
Bone Structure and Physiology of bone fracture healing
Bone Structure and Composition
Bones can be categorized in five types, i.e., long, short, flat and sesamoid or irregular. The long bones include the femora, tibiae, fibulae, humeri, radii, ulnae, metacarpals, metatarsals, phalanges, clavicles, which provide skeletal mobility and are subjected to most of the load during everyday activities. The shafts of the long bones are referred to as the diaphyses, and the expanded ends as the epiphyses.
Macroscopic composition of bone
Macroscopically the bone consists of the periosteum, which protects bone from the surrounding tissues and provides provides cells for bone growth and repair, articular cartilage, which covers the ends of the epiphyses , the ossified tissue and bone marrow, which fills the medullary cavity and the spaces between the trabeculae. It serves as storage for precursor cells, which are involved in repair. The endosteum is also included in bone's composition consisting of the inner surfaces of the bone.
The periosteum is a 1-2 mm wide membrane consisting of connective tissue that encloses the whole bone apart from the articular surfaces. It is comprised of two layers, i.e., the osteogenic including progenitor cells, and the fibrous, including nerves and blood vessels. The ossified tissue is non-homogeneous, porous and anisotropic. It is divided in cortical, compact, cancellous and trabecular bone. The 80% of long bone consists of cortical bone. The epiphyses are comprised of cancellous bone whereas the diaphyses are mostly composed of compact bone which is covered with a thin layer of cancellous in the inner surface around the bone marrow.
Cortical bone includes systems of concentric lamellae as well as the Harvesian systems or osteons, which are the basic structural unit of cortical bone. Osteons are cylindrical or elliptical of 100-300 μm diameter and 10 mm long [Williams 1995, Rho et al.., 1998]. 3-8 lamellae are wrapped around each osteon. The harvesian canal of 50-100 μm long constitutes the central canal of the osteons. Harvesian canals include vessels, nerves and connective tissue. Canaliculi pass through each osteon and Volkman canals are also composed of vessels and nerves, placed in a transverse plane of that of the Harvesian canals. Volkman canals help in the communication between Harvesian canals and bone marrow.
Microscopic composition of bone
Bone is composed of organic phase, inorganic phase and water. The most important constituent of the organic phase is collagen i.e., a protein organized into strong fibers, which provide bone with flexibility and tensile strength. Proteoglycans and non-conllageneous proteins also constitute the organic phase. The inorganic phase mostly includes hydroxyl apatite crystals and provides compressive strength and rigidity to bone (Martin et al., 1998).
A very small fraction of the bone's volume is consisted of cells that are responsible for the production, resorption and maintenance of the above described bone matrix, i.e.,
Osteoblasts, which are mononuclear cells differentiated from mesenchymal stem cells. Once they are stimulated they change their shape and form new ossified organic matrix i.e., osteoid. This osteoid is produced at a rate of 1 μm/day and is then calcified to create mineralised bone. When they are deactivated they become flattened and form cells to the free bone surfaces or are self-vested with mineral matrix and become osteocytes.
Osteocytes, which are former osteoblasts sitting in cavities inside the organic matrix, i.e., the lacunae. They have oval shape and stick out in small canals i.e., the canaliculi, where they are connected with other osteocytes and osteoblasts. Their role is to sustain bone organic matrix and release calcium ions from the organic matrix when it is necessary. A small amount of osteocytes absorb mineral organic matrix, which shows that these cells play significant role on the conservation of the bone organic matrix [Lanyon 1993].
Osteoclasts, which are multinuclear cells which come from precursors of bone marrow. They are responsible for bone absorption. This process is ensured from the large number of mitochondria existing in their cytoplasm. The cytoplasm membrane creates an acid environment in which the organic matrix loses its metallic ions and is dissolved. Bone resorption takes place at a rate of tens of micrometres per day. The cooperation of osteoblasts and osteoclasts is responsible for the form, remodeling and healing of bone tissue.
Figure a) Bone structure b) orientation of collagen fibers in adjacent lamellae and c) organization of spongy bone. (Martini, 1998)
Figure Osteons structure in cortical bone
Physiology of bone fracture healing
Fracture healing is a complex regenerative process that gradually restores the functional and mechanical bone properties, such as load-bearing capacity, stiffness and strength. It includes a complex sequence of events that begin with an inflammatory reaction, lead on to the callus tissue formation, the gradual differentiation of intermediate tissues inside the callus and finally the callus resorption and bone modeling.
Two types of bone healing exist i.e., the primary or direct and the secondary or indirect healing course.
Primary bone healing
Primary healing occurs under optimal conditions i.e., when the anatomical reset of bone ends is precise without gaps and absolute mechanical stability exist in the fracture. This type involves direct healing without the formation of callus and the inflammation stage. Primary healing can be divided into contact and gap healing.
In the primary contact healing the fractured bone ends are in full contact and stable by using an internal fixation device. The osteoclasts absorb bone and pervade perpendicularly the fracture site creating conic cracks along its long axis with rate 50-80 μm per day [Williams 1995]. These cracks are used as channels for the penetration of newly formed vessels which transfer mesenchymal stem cells. The latter differentiate in osteons which eventually connect the fracture bone ends.
Primary bone formation by gap healing occurs when small gaps (i.e., 150-200 μm) exist between the fractured ends. In a first stage the gaps are filled with ossified tissue which bridges the fracture ends. The formed lamellae are not arranged in parallel with bone's long axis as in the cortical bone. In a second stage the lamellae get re-oriented across the long axis of bone.
Secondary bone healing
In most cases of either conservative of surgical treatment of bone fractures stabilisation is not adequate to permit primary bone healing and thus secondary healing takes place. This type of healing is accompanied by the formation of the external and internal fracture callus tissue. Secondary healing evolves in stages which are discussed below.
Inflammatory stage: When a fracture occurs the local blood supply is disrupted causing a hematoma and death of cells in the fracture ends. This is followed by an asceptic inflammatory response, which lasts 1-2 days. The necrotic tissue is then absorbed followed by revascularisation proliferation and differentiation of the cells in the periosteum, endosteum and marrow. Inflammatory cytokines are released which initiate angiogenesis and induce osteoclastic and macrophaginc activity (Mundi et al., 2009, Claes 2008, Tortora et al., 2003). Angiogenesis plays significant role in the healing process by supplying the fracture site with oxygen, nutrients, and cells, whereas osteoclasts and macrophages contribute in the absorption of dead tissue (Kanczler JM and Oreffo RO 2008). The hematoma stage is critical for bone healing since it stimulates molecules which commence the serial cellular mechanisms for healing (Einhorn et al., 1998, Frost et al., 1989) (Figure 4).
Figure Mesenchymal Stem Cells proliferation and differentiation (Holmes et al., 2001, Caplan 1994)
Fibrocartilaginous tissue formation: New cells are produced from progenitor cells, which further differentiate and provide new vessels, fibroblasts, intercellular materials so as to form a soft granulation tissue (Figure 4) (Frost 1989).
Bone Callus formation: Cells are further proliferated, differentiate and organised so as to create new chondrocytes and osteoblasts in the granulation tissue during the mesengenic process shown in Figure 3 (Holmes et al., 2001, Caplan 1994). According to Caplan (Caplan 1994) progenitor cells or mesenchymal stem cells are able to proliferate and differentiate into a number of different soft and hard tissue types i.e., bone, cartilage, tendon, ligament, muscle, marrow, or connective tissue lineages. The cells then form extracellular organic matrices of tissues. This is followed by mineralization, which continues for some weeks to create the fracture callus tissue. The callus tissue is divided in the hard callus, where intramembranous ossification occurs and the soft callus, where endochondral ossification takes place (Brand et al., 1990). In the interior of the initial callus and adjacent to the fracture osteochondral progenitor cells differentiate into chondrocytes. After one or two weeks, elongated proliferative chondrocytes undergo mitosis, divide and syntesize cartilage (Bailon-Plaza and Van der Meulen, 2001). Cell proliferation is then reduced and callus is mostly consisted of hypertrophic chondrocytes. Blood vessels are formed in the calcified cartilage which is then absorbed by osteoclasts. At the end of that stage, the cartilage is replaced with ossified tissue and woven bone is formed via endochondral ossification of the callus (Figure 4).
Bone Remodeling: The final stage includes bone remodeling during which the external callus is completely resorbed and in the fracture gap the disorganized osteoclasts and osteoblasts is remodeled into cortical bone (Bailon-Plaza and Van der Meulen, 2001). Any callus plugging the marrow cavity is removed, the medullary cavity is restored as well as original geometry of the bone. After completion of this stage bone gains its original strength.
Figure Stages of bone fracture healing process (2004 Pearson Education, Inc. publishing as Benjamin Cummings)
Ultrasound Mechanisms during bone fracture healing
Ultrasound (US) is defined as sound wave having a frequency greater than 20 kHz. It is a form of mechanical energy that can be transmitted into the body for therapeutic and diagnostic purposes. The ultrasound energy is produced from a piezoelectric crystal within a transducer, emitting sound waves through body tissue that cause various biological changes in tissue healing [Khan et al., 2008, Rutten et al., 2008, Mundi et al, 2009]. Diagnostic US, mostly used for medical imaging includes the transmission of pulsed waveforms of less than 1 W/cm2 intensity while therapeutic US typically uses 1 or 3 MHz frequency incident pulsed or continuous waves depending on the desired physiological effect(s).
In most relevant studies, LIPUS is used at 1.5 MHz and at 0.03 W/cm2, is pulsed, and is used with a 20% duty cycle (1:4) (Bashardoust et al., 2012). The LIPUS waves produce micromechanical stresses in the fracture site which can result in triggering various biological processes involved in bone healing at cellular and molecular level (Baker et al., 2001) and accelerate bone formation in a similar manner as bone response to mechanical stress according to Wolff's law (Wolff, 1892)
The significant effects of US on bone healing are in general due to thermal and non-thermal mechanisms.
Two non-thermal mechanisms have been proposed in the literature regarding the LIPUS induced micromechanical stress in bony tissues i.e., mechanical effects e.g. displacement of the fractured ends and cavitation.
Displacement involves the motion at both fractural ends caused by LIPUS waves. Pounder et al., 2008 suggests that this motion occurs on a nanometric scale (displacements of 0.15-0.55 nm) stimulating molecular and cellular pathways involved in healing. However Claes and Willie, 2008 suggest this motion to take place in a microscopic level i.e., 0.5-2 mm at the borders of soft and hard tissues. It is also suggested that this micromotion serves as a mechanical stimulus to the integrin mechanoreceptors included in cellular signaling and osteogenic differentiation. Local changes in pressure may create a biophysical environment that mimics Wolff's law on a microscopic scale. Pounder and Harrison (Pounder and Harrison 2008), based on Tang's (Tang et al 2004) work, suggest that ultrasound stimulates integrins on the cell surface which then promote bone healing.
Another physical mechanism by which LIPUS enhances bone healing is the acoustic streaming i.e., the creation of localized, high-velocity streams of fluid due to the absorption of the energy of the ultrasonic field. This motion of the fluid is referred as 'a sonic wind' and plays significant role in the intra- and extracellular chemical reactions (Schortinghuis et al., 2003). Acoustic streaming has been also reported to causes an increase in membrane permeability [Erdogan et al., 2009, Hadjiargiriou et al., 1998]. The ultrasound-induced increase in vascular permeability causes an increase in blood pressure at the fracture site (Watson, 2000) which results in enhanced differentiation of mesenchymal stem cells into chondroblasts (Tortora et al., 2003, Gurkan et al., 2008). This increase in local blood pressure also causes increased differentiation of osteoprogenitor cells into osteoblasts as well as decreased differentiation of progenitor cells into osteoclasts (Sena et al., 2005, Pounder et al., 2008, Yang et al., 2005, Zhou et al., 1999, Gurkan et al., 2008). Furthermore increased blood pressure induces an increase in hemodynamic shear stress which along with the subsequent increased fluid flow and fluid turbulence induced by LIPUS at the fracture site may stimulate the recruitment of osteoprogenitor cells.
Cavitation involves the interaction of gas bubbles within cells and tissues due to their exposure to LIPUS. ). It has been reported that exposure to ultrasound at an intensity of 0.5 W cm2 and at increased pressure caused augmented levels of collagen synthesis by human fibroblasts which was not observed only at a positive pressure (Webster et al., 1978). Therefore cavitation is likely to cause cellular alterations. However, the role of cavitation in vivo is an open issue and needs to be elucidated (Frizzell, 1988). Dalecki, (Dalecki, 2004) reports that cavitation is not frequent in vivo because gas inclusions cannot be formed physically in living biological tissues. According to Pounder et al., 2008 cavitation is unlikely due to the low mechanical index. Nevertheless the application of LIPUS followed by shear stress has been shown to significantly enhance osteoblastic cell alignment (McCormick et al., 2006).
Two forms of cavitation exist, i.e., stable and unstable. Stable cavitation is a phasic oscillation of the bubble within the ultrasound field supporting acoustic streaming (Mundi et al., 2009). This causes slight circular flow of tissue fluids leading to increased cell permeability and subsequently increased blood pressure at the fracture site. Unstable cavitation leads to a rapid collapse of the bubble causing high local temperatures and/or pressures. The produced energy stimulates the surrounding tissues (Mundi et al., 2009, Watson, 2000).
The temperature increase due to US depends on tissue properties, US field parameters, tissue characteristics as well as thermal conductivity and blood perfusion of tissue. In poorly vascularized tissues (e.g. tendon, fat) as well as in tissues such as bone which conduct heat, the temperature increases steeply. Bone surrounding tissues are prone to heat rise by means of thermal conduction (Srbely et al.). The thermal effects that have been reported include augmented blood flow, increased extensibility of collagenous tissues, and decreased muscle spasm (Dyson, 1987). However, thermal mechanisms of ultrasound are considered not to play significant role bone healing, due to the low intensities that are currently applied (Schortinghuis et al., 2003).
Ultrasound effects in biophysical stimuli during bone healing
Ultrasound has been reported to affect biophysical stimuli as well as bone's mechanical properties and cellular processes occurring during bone healing. It has been shown to cause an increase in hydrostatic pressure due to an increase in vascular permeability that leads to increased differentiation of mesenchymal stem cells in chondroblasts, enhancing thus the development of fibrocartilaginous callus development. Furthermore increased blood pressure induces increased hemodynamic shear stress which along with the subsequent increased fluid flow, as well as increased fluid turbulence caused by the modality sound waves at the fracture site may act as a prominent stimulant in the recruitment of osteoprogenitor cells from the bone marrow, thus enhancing bone healing and remodelling. Although the exact mechanism of LIPUS during bone healing is not clear several clinical and animal studies have been performed in the field that demonstrate the use of US as a new tool for the enhancement of fracture healing, which are described in the following parts.
The first study to report that US enhances bone healing was that of Duarte 1983 (Duarte 1983) and Xavier and Duarte (Xavier and Duarte 1987) by applying a LIPUS device on fibular osteotomy and in a femoral drill-hole defect in a rabbit model. More recent animal studies report increased stiffness, torque and strength of the fracture resulting from an earlier onset of endochondral formation due to LIUS-stimulated chondrogenesis [Tsumaki et al., 2004, Yang et al., 2005, Azuma et al., 2001]. More specifically Tsumaki et al., investigated the influence of LIPUS on callus maturation after knee surgery for osteoarthritis. Twenty-one patients were subjected to bilateral, opening-wedge, high tibial osteotomy followed by external fixation. LIPUS was applied for 20 min/d for 4 weeks randomly on one limb whereas the other was used as a control. The callus bone mineral density was measured before and after LIPUS application. A statistically significant increase in bone mineral density was found in the ultrasound-treated limb of 18 patients (0.20±0.12 g/cm2 vs 0.13±0.10 g/cm2; P=0.02).
Yang et al. (Yang et al., 1996) applied LIUS to 79 rats which were subjected to bilateral closed femoral fractures. A 200-μs burst sine US wave of 0.5 MHz was applied to the one fractured limb whereas the other served as the control. In one group the intensity of the applied US was 50 mW/cm2 US whereas in the other it was 100 mW/cm2. The healing fractures were subjected to mechanical testing 3 weeks after osteotomy. In both groups, the average maximum torque and average torsional stiffness of the LIUS treated limbs were significantly greater than the untreated ones. However only the changes in the 50 mW/cm2 group were statistically significant (average maximum torque, 223.5± 50.5 Nmm, vs 172.6±54.9 Nmm; P=0.022, paired t test).
Azuma et al. (Azuma et al. 2001) investigated the effect of LIPUS on the cellular processes during bone healing. LIPUS was applied on closed fractures of the right femur of rats, whereas the left served as control. The protocol included four groups and four phases of trials depending on the timing and duration of LIPUS application i.e., in Phase 1 LIPUS was applied in one group of animals for 8 days, for 1 to 8 after fracture; in Phase 2 a second group was LIPUS treated for 8 days, from day 9 to 16 after fracture and in Phase 3 a third group was treated for 8 days, from day 17 to 24 after fracture. Finally the T (throughout) group was treated for 24 days, from days 1 to 24 during the healing process. Rats were sacrificed on day 25 and were subjected to biomechanical testing. The maximum torque for each group is shown in Figure 5.The maximal torque and stiffness of the LIPUS treated femurs was found significantly higher than that of the control ones in all groups. Therefore fracture callus properties were enhanced both in the case of partial LIPUS treatment i.e., during Phase 1, 2, or 3 and in the case of treatment throughout the 24 days. In addition the maximal torque of the LIPUS-treated femurs in the T group was found significantly higher than that of the other three groups. These findings suggest that LIPUS affects the cellular mechanisms occurring in all phases of bone healing with the more pronounced impact to occur when applied throughout the whole process.
Figure Maximum torque of the LIPUS-treated femurs was significantly greater than the placebo controls at each phase of fracture healing. The maximum torque of the group treated throughout the repair process was significantly higher than the LIPUS groups treated for a single phase (Ph1, Ph2, Ph3) alone. (**p < 0.01, #p < 0.05) (Azuma et al. ).
Pilla et al. (Pilla et al. 1990) applied LIPUS in 139 mature New Zealand white rabbits which have been subjected to bilateral mid-shaft fibular osteotomy. LIPUS was applied to the one limb for 20 min a day. The results from biomechanical testing showed that the healing course was significantly enhanced by a factor of nearly 1.7.
In a more recent study Shakouri et al., (Shakouri et al., 2010) applied 30 mW/cm2 intensity sine waves of 1.5-MHz rabbit fractures at 5 and 8 weeks. It was found that although bone mineral density was increased in the LIPUS-treated rabbits, no significant changes in the mechanical strength were observed.
Schortinghuis et al., (Schortinghuis et al., 2003) suggest that the accelerated restoration of the mechanical strength due to LIPUS application in animals (Pilla et al., 1990; Wang et al., 1994; Yang et al., 1996) may be attributed to the fact that US triggers cellular mechanisms that lead to earlier completion of the inflammatory phase and earlier start of the reparative phase of bone healing. Indeed it has been previously reported that in the inflammatory phase, US increases mast cell degranulation (Fyfe and Chahl, 1980), which leads to increased levels of leukocyte adhesion to endothelium (Maxwell et al., 1994), stimulates collagen production (Doan et al., 1999; Reher et al., 1999), and causes augmented release of macrophage fibroblast (Young and Dyson, 1990b) and endothelial growth factors (Reher et al., 1999, Warden et al., 2000). The positive effect of LIPUS on the inflammation and soft callus phases of bone healing accompanied with augmented biomechanical strength has been also reported in the study of Rawool et al. (Rawool et al., 2003). However this was not observed in the bone remodelling phase.
Similar results were reported in the study of Wang et al. (Wang et al. 1994) by using 22 rats with bilateral closed femoral fractures. LIPUS was applied at the one limb whereas the contralateral was used as control. Sixteen rats were treated with LIPUS of either 0.5 or 1.5 MHz and six received sham treatment with the ultrasound device to account for the effects of anaesthesia and handling. LIPUS of both frequencies accelerated bone healing as shown from radiographs, histological exams and biomechanical testing. The average maximum torque and torsional stiffness were found significantly increased in the LIPUS-treated limbs as cpmared to the control ones.
Protopappas et al., (Protopappas et al., 2005) and Malizos et al., (Malizos et al., 2006) performed LIUS experiments in a sheep tibial osteotomy model treated by external fixation. Ultrasound measurements were obtained from the intact bones before the osteotomy and from the healing bones on a 4-day basis until the 100th postoperative day (endpoint of the study). It was shown that LIPUS significantly accelerated bone fracture healing course and increased cortical bone mineral density. The lateral-bending strength of the healing bones was also found improved.
Pounder and Harrison (Pounder and Harrison 2008) suggest that the increase in mechanical strength of the fracture callus is due to accelerated mineralization at the callus site. Several cellular expressions seem to be associated with this accelerated mineralization (Pounder and Harrison 2008).
McClure et al. (McClure et al. 2010) and Yang and Park (Yang and Park 2001) investigated the influence of LIPUS on bone healing by applying US on a 1-cm gap osteotomy of a horse's 4th metacarpal and on an ulna defect in a dog. It was found that new bone formation was enhanced in the ulna, while in the horse it was not affected.
Heckman et al., (Heckman et al., 1994) performed a multicenter, randomized, doubleblind, placebo-controlled study by evaluating 33 patients with fractures treated with LIPUS and 34 patients with fractures treated as a placebo. The time of clinical healing was found statistically significantly decreased in the LIPUS treatment group (86+5.8 days) as compared with that in the control group (114+10.4 days, (P ¼ 0.01)). In addition the overall healing time (clinical and radiographic) was also significantly lower in the active treatment group (96+4.9 days) compared with the control group (154+13.7 days,(P ¼ 0.0001)). Similar observations were also found from Kristiansen et al., (Kristiansen et al., 1997) in their multicenter, randomized, double-blind, placebo-controlled study by analyzing 61 closed fractures.
Malizos et al., (Malizos et al. 2006) by reviewing the clinical use of LIPUS in bone healing concluded that LIPUS accelerates the healing time of closed or grade-I open, cortical tibial fractures, and cancellous radial fractures by 38%. The authors concluded that there is evidence that ultrasound accelerates fresh fracture healing. Mayr et al. (Mayr et al., 2002) also reported enhanced bone fracture healing of scaphoid fractures.
Leung et al., (Leung et al. 2004) by applying LIPUS in open and high energy tibial fractures managed by intramedullary nailing or external fixation proved that bone healing was enhanced by 40%. Their results were based on radiograph evaluation i.e., on the callus bridging one, two or three cortices as well as on clinical measurements (i.e., 6.5 vs. 9.5 weeks, 8.5 vs. 12.5 weeks, 11.5 vs. 20 weeks in LIPUS and control groups for first, second and third cortical bridging respectively, P, 0.05).
On the contrary, Emami et al., (Emami et al., 1999) by applying LIPUS on tibial shaft fractures stabilized with intramedullary nail did not find statistical differences in healing time between the US treated and the control ones. The first callus appeared in 40+3 days in the treatment group whereas in the control group in 37+3 days. The third cortical bridging occurred in 155+22 days in the treated group whereas in the control group in 125+11 days, with the difference to be in means 3 days P: 0.05. In a meta-analysis of randomized controlled clinical trials of bone healing Busse et al. (Busse et al. 2009) found significant acceleration in healing time (about 64 days) in patients who have received LIPUS. Some recent systematic reviews of clinical studies claimed that US maybe ineffective for all fractures types and methods of fracture treatment (Watanabe et al., 2010, Mundi 2009).