Low-intensity pulsed ultrasound in dentofacial tissue engineering

Review paper: Low-intensity pulsed ultrasound in dentofacial tissue engineering Eiji Tanaka1, Shingo Kuroda1, Shinya Horiuchi1, Akira Tabata2, Tarek El-Bialy3

1

Department of Orthodontics and Dentofacial Orthopedics, Institute of Health

Biosciences, Tokushima University Graduate School, Tokushima, Japan. 2

ITO Co., LTD., Tokyo, Japan.

3

Division of Orthodontics, School of Dentistry, Faculty of Medicine and Dentistry,

University of Alberta, Edmonton, Alberta, Canada.

Corresponding author: Eiji Tanaka, DDS, PhD Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, Tokushima University Graduate School 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan E-mail address: [email protected]

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Abstract Oral and maxillofacial diseases affect millions of people worldwide and hence tissue engineering can be considered an interesting and clinically relevant approach to regenerate orofacial tissues after being affected by different diseases. Among several innovations for tissue regeneration, low-intensity pulsed ultrasound (LIPUS) has been used extensively in medicine as a therapeutic, operative, and diagnostic tool. LIPUS is accepted to promote bone fracture repair and regeneration. Furthermore, the effect of LIPUS on soft tissues regeneration has been paid much attention, and many studies have performed to evaluate the potential use of LIPUS to tissue engineering soft tissues. The present article provides an overview about the status of LIPUS stimulation as a tool to be used to enhance regeneration/tissue engineering. This review consists of five parts. Part 1 is a brief introduction of the acoustic description of LIPUS and mechanical action. In Part 2, biological problems in dentofacial tissue engineering are proposed. Part 3 explores biologic mechanisms of LIPUS to cells and tissues in living body. In Part 4, the effectiveness of LIPUS on cell metabolism and tissue regeneration in dentistry are summarized. Finally, Part 5 relates the possibility of clinical application of LIPUS in orthodontics. The present review brings out better understanding of the bioeffect of LIPUS therapy on orofacial tissues which is essential to the successful integration of management remedies for tissue regeneration/engineering. To develop an evidence-based approach to clinical management and treatment of orofacial degenerative diseases using LIPUS, we would like to be in full pursuit of LIPUS biotherapy. Still, there are many challenges for this relatively new strategy, but the up to date achievements using it promises to go far beyond the present possibilities.

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INTRODUCTION Tissue engineering is defined as “the reconstitution of tissues and organs, in vitro, for use as model systems in basic and applied research or for use as grafts to replace damaged, or diseased body parts or body functions”.50 While this definition of tissue engineering covers a broad range of applications, in practice the term has come to represent applications that repair or replace different tissues. Nevertheless tissue engineering can be described by actions taken to improve biological functions. Several methods have been described to enhance cellular performance and tissue regeneration, and low-intensity pulsed ultrasound (LIPUS) has shown to play an important role in cell metabolism and tissue repair.38 The present article provides an overview about the status of LIPUS stimulation as a tool to be used to enhance regeneration /tissue engineering of hard and soft tissues in oral and maxillofacial regions. This review is divided into five parts. Part 1 will introduce acoustic description of LIPUS and mechanical action. In Part 2, biological problems in dentofacial tissue engineering will be proposed on the anatomical basis. Part 3 will relate biologic mechanisms of LIPUS to cells and tissues in living body. Part 4 will present the effectiveness of LIPUS on cell metabolism and tissue regeneration in dentistry. In part 5, the possibility of clinical application of LIPUS in orthodontics will be discussed. Finally, the requirement and possibility of tissue engineering through LIPUS for treatment of oral and maxillofacial diseases will be concluded.

1. Acoustic description of LIPUS and mechanical action Ultrasound is acoustic mechanical waves with frequencies above the human hearing. Ultrasound has been used in medicine extensively as a therapeutic, operative, and diagnostic technique. Therapeutic ultrasound intensity ranges from 30 mW/cm2 to 70 W/cm2, operative ultrasound (shock waves) intensity ranges (0.05 -27000 W/cm2)66,92 while diagnostic ultrasound intensity ranges from 5-50 mW/cm2 to avoid excessive heating of the tissues.38 Most of the studies evaluated the stimulatory effect of LIPUS on different cells, tissues or organs used the following LIPUS parameters: The most reported studies about LIPUS stimulation presented the following parameters: pulse frequency of 1.5 MHz, pulse repetition frequency of 1 kHz, spatial average temporal average intensity of 30 mW/cm2 of the LIPUS transducer‟s surface area. It is widely known that LIPUS has no deleterious effects. In addition, LIPUS treatment has no thermal effects on the treated tissues. Also, LIPUS can be considered as a non3

invasive fractures.

and

safe

therapeutic

6,32,37,40,54,83,110,113

technique

for

the

treatment

of

bone

Furthermore, various cell types have been reported to be

stimulated by ultrasound exposure, including gingival cells,71,101 periodontal ligament cells,38,43,46,90

cementoblastic

chondrocytes,49,72,98,105 cells,

4,67,72,87,101

bone

cells,15,16,47,88

cells,67,78

muscular

and synovial membrane cells.

74

odontoblast-like cells,73

mesenchymal

cells,97 stem

Because of the aforementioned, many

studies have performed to evaluate the potential of LIPUS to tissue engineering in dentistry. A recent study evaluated the mechanical stresses induced by LIPUS propagation in cancellous bone.111 In addition, it seems that each tissue has its own optimum LIPUS parameters for regenerative stimulation. For example, Tsai et al.110 reported that optimum LIPUS parameters for bone formation/healing are 0.5 mW/cm2, while intensity of 1 W/cm2 showed deleterious effect on bone healing. Chondrocytes treatment showed increased stimulation by increasing LIPUS treatment from 20 minutes per day to 40 minutes per day.98 Moreover, it has been recently reported that 30mW/cm2 daily LIPUS application to calvarial bone defects in rats enhances regeneration of these defects when compared to controls at 2(7% compared to controls at 3.6%),3 (12.0% vs. 5.8%) and 4 weeks (18.1% vs. 9.8%) of LIPUS application.39 Although many clinical studies reported that the optimum frequency for bone healing and ossification is 30mW/cm2 of the LIPUS transducer‟s surface area, a recent in vitro study by Angle, et al.4 on the effect of different LIPUS intensities showed that lower intensities (2 and 15 mW/cm2) are better than 30 mW/cm2 in inducing osteogenic differentiation of rat bone marrow stem cells as well as in mineralization products by these cells. Therefore, it is a prerequisite to evaluate the biological effects of the different phenomena with respect to particular parameters of LIPUS, like intensity, frequency, or duty cycle on different cells and tissues.4,83 Future studies would be needed to explore optimum LIPUS parameters required for each tissue stimulation that beyond this level, LIPUS may be deleterious to this tissue. Absolutely, we should notice the limits of LIPUS that LIPUS is not efficient in all cases and its effectiveness depends on the individual characteristic and condition.

2. Biological problems in dentofacial bioengineering Orofacial structures are very unique in their development and function. For instance, orofacial bones are derived from both neural crest and paraxial mesoderm; however, the skeletal bones are derived from mesoderm. Furthermore, orofacial tissues have limited and variable capacity for regeneration. For example, cementum has a very slow and weak regenerative capacity,108 and dental pulp has a limited capacity for 4

regeneration because of limited apical blood supply.108 Oral and maxillofacial diseases affect millions of people worldwide. Tooth decay, periodontal disease, dental pulp infection, and inflammatory root resorption can result in tooth loss and can seriously compromise human health and quality of life. In this context, tissue engineering can be an interesting and clinically relevant approach to regenerate dental tissues as well as the whole tooth.46 The successful identification and combination of tissue engineering, scaffold, progenitor cells, and physiologic signaling molecules have enabled to design, recreate the missing tissue in its near natural form, compared to the restorative and prosthodontic approaches. Some limitations to MSCs based therapy include scarceness of their numbers and extended time is need in the lab to differentiate these cells into chondrogenic and osteogenic lineages. LIPUS therapy stimulates stem cell growth and differentiation.4,6,31 LIPUS can be an effective tool to enhance tissueengineering of mandibular condyles for many reasons. Importantly, LIPUS is the preferred method of mechanical stimulation, also reported as “preferred bioreactor” 75 as it enhances angiogenesis.3,22,120 This is especially relevant because vasculature is required to integrate the engineered tissue with the native surrounding tissue.93 Recent studies showed that LIPUS enhances cell expansion and differentiation in tissue culture.4,74,77,97,119

3. Biologic Mechanisms of LIPUS LIPUS is mechanical vibration with frequencies above the limit of human sound detection, and can be transmitted into the body as high-frequency acoustic pressure waves.3,6 It has been recognized that such waves produce mechanical stimuli in living tissues, resulting in biochemical events promoting tissue healing.11,12-13 The biologic mechanisms involved in LIPUS-stimulated tissue repair have not been sufficiently uncovered. However, it is recognized that the anabolic biophysical effects caused by LIPUS are most likely to be caused by mechanical stress and/or fluid micro-streaming. These events when impacting the cell plasma membrane, focal adhesion and cytoskeletal structures, they trigger intracellular signal transduction and subsequent gene transcription.49,54,56,90,94 It has been postulated that LIPUS may transmit signals into the cell through an integrin that may act as a mechanoreceptor on the cell membrane.56 When LIPUS signals are transmitted to integrin molecules, the attachment of various focal adhesion adaptor proteins are stimulated. Focal adhesion kinase (FAK) is then phosphorylated by LIPUS exposure which then initiates this signal transduction. Sato et al.96 reported that LIPUS induces up-regulation of phosphorylated FAK in the synovial cells. They 5

also reported that FAK phosphorylation inhibition led significant downregulation of mitogen-activated protein kinase (MAPK) phosphorylation. In addition, it has been reported that activation of integrins and the downstream signaling pathway in vitro study of LIPUS effect on human skin fibroblasts.122 In addition, it has been reported that integrins act as a link between extracellular matrix, cytoskeletal proteins, and actin filaments. Treatment with anti-Integrin β1 and β3 antibodies or transfection with siRNA against Integrin β1 and β3 showed antagonizing effect of ultrasound stimulation on cyclooxygenase (COX)-2 expression. This indicates that Integrin β1 and β3 are very important in the effect of ultrasound in chondrocytes.72 Furthermore, it is reported that LIPUS treatment to cementoblasts stimulates cell metabolism through MAPK pathway as LIPUS enhanced extracellular signal-regulated kinase (ERK) 1/2 expression. Also, this effect is evidenced by the fact that methyl ethyl ketone (MEK) 1/2 inhibitor treatment suppressed the up-regulation of Cox-2 mRNA expression induced by LIPUS.49 The integrin/Ras/MAPK pathway is well accepted a general pathway that modulates cell proliferation. Based on the evidences presented here, it can be concluded that the bioeffect of LIPUS exposure to various cells might be promoted via integrin/FAK/MAPK pathway particularly (Figure 1). Various cells proliferation is mediated by growth factor or cytokine-induced MAPK, which is a family of serine-threonine proteins.116 It is recognized that regardless the fact that the three MAPK modules102, JNK, ERK and p38, run in parallel, there is a significant degree of cross-talk between them. This cross talking may create multiple chances for modulating or fine-tuning responses to different signals.117 Moreover, it has been reported that activation of ERK signaling pathway has a role in mediating cell division, migration and survival.116 Activation of the JNK signaling pathway results in apoptosis regardless that it has also been shown to promote cell survival under different conditions.20 p38 subfamily is known to be involved in cell motility, transcription and chromatin remodeling.62 It has also been reported that cyclic mechanical stimulation of human patellar tendon fibroblasts stimulates JNK and is involved in apoptosis.102 Kanbe et al.51 reported that ERK and JNK expression without p38 in the synovium of the rotator interval with positive β1-integrin. In addition, JNK expression about blood vessels showed epithelial cells extended more widely than ERK and β1-integrin expression. Because of these findings, it can be concluded that ERK can be considered as specific factor for expression in epithelial cells, and fibroblastic cells and epithelial cells expressed JNK in response to mechanical stimuli. It is widely known that apoptosis and proliferation are important in controlling cell number and viability.5 Taken together, it can be suggested that LIPUS upregulates 6

phosphorylated FAK and that FAK phosphorylation inhibition led significant downregulation of ERK, JNK, and p38 phosphorylation (Figure 1). Meanwhile, it has been suggested that the non-thermal mechanisms induced by LIPUS that can produce beneficial changes in living tissue may be cyclic or non-cyclic in nature.103 This is presumably thought to be an effect due to the periodic nature of the sound pressure. The main non-cyclic effect thought to be involved in ultrasound therapy is acoustic streaming, which may be caused by stable and oscillative cavities or radiation forces in intracellular and/or extracellular fluids. Acoustic streaming may act to modify the local environment of a cell, leading to altered concentration gradients in the vicinity of an extracellular membrane. The concentration gradient affects the diffusion of ions and molecules across a membrane, and thus streaming may account for the reported changes in the potassium and calcium content of cells following ultrasonic exposure.11,70

4. Effectiveness of LIPUS on cell metabolism and tissue regeneration (Table) 4.1. Application of LIPUS to bone injuries Bone fracture healing has traditionally been thought to be a naturally optimized process with predetermined time-course for cellular recruitment, gene expression, and synthesis of reparative compounds. Fracture healing follows the same inflammatory, proliferative and remodeling phases.6,17 The first is inflammatory as the aftermath of the physiological consequences of injured tissue. Following a week of this acute phase, a longer second step initiates fracture repair. At the site of the hematoma, a callus forms. The formation of callus requires that osteoblasts be recruited into the region. This process is the step requiring osteoanabolic input both for reparative cytokines and the cells that participate in this process. Finally, the remodeling phase replaces the calcified callus with lamellar bone. This final reparative stage helps to account for slow strengthening of the fractured bone, a process that may take months, and perhaps even years, to complete. The duration of fracture healing is highly variable, depending in part on individual reparative capabilities, the location of the fracture and the degree to which mechanical loading takes place.10 4.1.1. In vitro studies Mechanical stimulus to bone is of a great importance for maintaining the bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement 7

of fluid within the spaces surrounding bone cells generates fluid shear stress that stimulates osteoclasts and osteoblasts, resulting in enhanced anabolic activity for bone remodeling with appropriate bone resorption and the subsequent new bone formation.83 The mechanisms such as mechanotransduction process, by which osteoclasts or osteoblasts convert the external stimulation from fluid shear stress into biochemical changes, remain unclear.121 It has also been shown in in vitro studies that LIPUS stimulation can enhance expression of bone formation-related genes such as collagen type I and X, aggrecan, TGF-β,71 Runx-2, osteocalcin,12 IGF-1, bone sialoprotein78,80and alkaline phosphatase.71,114 In addition, LIPUS has been reported to promote protein synthesis and calcium uptake in various osteoblastic cell lines. 78 Moreover, LIPUS stimulation has been reported to enhance COX-2 gene expression and subsequently enhance endogenous prostaglandin E2 (PGE2) synthesis in various osteoblastic cell lineages, playing an important role in bone remodeling.56,85,103 A recent in vitro study on the effect of LIPUS on osteogenic differentiation of rat bone marrow stem cells and mineralization showed that lower LIPUS intensities (2 and 15 mW/cm2) are better than 30 mW/cm2. Therefore, the clinically applied LIPUS parameters are different from those ones to be applied to the in-vitro stings. This may be due to the fact that LIPUS power attenuates while propagates through living tissues when it is applied in vivo.

4.1.2. In vivo studies In vivo studies have demonstrated that therapeutic LIPUS can promote bone repair and regeneration, accelerate bone fracture healing, and enhance osteogenesis at the distraction site.

25,28

In addition, it has been reported that LIPUS enhances bone

regeneration in sinus lift.55 Previous experimental studies using fractures in rat fibulae have found that ultrasound treatment during the inflammatory and early proliferative phases of repair enhanced healing, but that exposure during late proliferative phase proved disadvantageous, leading to a delay in bone union.23,93 Heckman et al.40 demonstrated similar acceleration of healing in human tibial fractures. Meanwhile, orofacial bones have a different origin from skeletal bone, and El-Bialy et al.25 demonstrated that during mandibular osteodistraction, earlier stages of bone healing were enhanced more by continuous ultrasound, whereas late stages were enhanced more by pulsed ultrasound.

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In addition, daily application of 30mW/cm2 LIPUS to calvarial bone defects in rats promotes regeneration of these defects when compared to controls Moreover, it has been recently reported that 30mW/cm2 daily LIPUS application to calvarial bone defects in rats enhances regeneration of these defects compared to controls at 2(7% compared to controls at 3.6%),3 (12.0% vs. 5.8%) and 4 weeks (18.1% vs. 9.8%).39 Therefore, the protocol and time-course of LIPUS therapy for bone fracture healing should be determined considering bone fracture site (orofacial region and skeletal region).

4.2. Application of LIPUS to periodontal diseases Physiologically, the periodontal ligament is continuously subjected to mechanical stress caused by occlusal forces. Furthermore, remodeling of the ligament and alveolar bone occurs in response to orthodontic forces. Taken together, these clearly indicate that responses of the ligament to mechanical stress are involved in its cell proliferation and differentiation. Periodontal ligament includes precursor cells of cementoblasts at the perivascular area in the middle portion and shows greater differentiation toward the surface of the root.108 Repair of soft tissues injuries consists of three phases (inflammatory, proliferative and remodeling phases) as well as hard tissues healing. It has been demonstrated both in the laboratory and in clinical trials that ultrasound can stimulate tissue repair and wound healing if correctly applied.22,24 It appears that exposure to ultrasound during the inflammatory phase of tissue repair can lead to an acceleration of this phase, which may eventually lead to an anti-inflammatory effect by LIPUS exposure.62,73, The second phase of healing is the „proliferative‟ stage. This is the stage at which cells migrate to the site of injury and start to divide, granulation tissue is formed, and fibroblasts begin to synthesize collagen. Ultrasound has been shown to enhance collagen synthesis by fibroblasts.70 Cementum is a thin mineralized tissue covering the tooth root surface and assists in anchoring teeth to surrounding alveolar bone, maintaining the structural stability and physiological function of the dentition.108 Resorption of the dental root surface can be a relevant adverse outcome of orthodontic treatment. A certain degree of root resorption occurs in most treatment cases, ranging from just a slight apical resorption to a complete tooth root loss.41,59,64 It is well accepted that the cementum layer covering the root surface plays a crucial role in preventing resorption during orthodontic tooth movement. In addition, the damaged areas are also repaired in part by cementoblasts lining the root surface. Root resorption during orthodontic treatment is a multifactorial 9

event and several biological and mechanical factors have been identified to increase its susceptibility, however, the exact mechanism still remains unclear.91,95 Cementoblasts share many characteristics with osteoblasts, including similar molecular properties and the ability to promote mineralization.69 Previous studies have shown that, as in bone, cementum metabolism is also controlled by mechanical stimulus. 4.2.1. In vitro studies In a previous study, it has been shown that cyclic stretch stimulation mediated periodontal ligament cells differentiation, thus regulating the function of the periodontal ligament as a source of cementoblasts and osteoblasts through the EGF/EGF-R system.68

In addition, It has been reported that LIPUS is effective in releasing

fibroblast growth factors from a macrophage-like cell line.114 Inubushi et al.47 reported that LIPUS induced early cementoblastic differentiation of human immature cementoblasts from the periodontal ligament by promoting the formation of substrate and increasing alkaline phosphatase (ALP) activity, enabling the regeneration of periodontal tissue destroyed by periodontal disease and the acceleration of the repair of root resorption. Mostafa et al.72 demonstrated that ALP and osteopontin expressions were also induced in human gingival fibroblasts treated with LIPUS, confirming that after 3 weeks of 5 min/day exposure the osteogenic differentiation potential was enhanced. Therefore, it can be hypothesized that LIPUS might promote the differentiation of immature cementoblasts in the periodontal ligament to mature cementoblasts, leading to periodontal ligament regeneration and repair. This implies that LIPUS may be a candidate of treatment remedies for periodontal diseases. Using an immortalized mouse cementoblast cell line, OCCM-30, studies performed by our group showed that LIPUS up-regulated the expression of several genes related to mineral metabolism15 and enhanced PGE2 production inducing cementoblastic differentiation and matrix mineralization through EP2/EP4 prostaglandin receptors pathway.88 The immune-expression of tumor necrosis factor (TNF) -α was not observed in LIPUS treated sample as was evident as in the control sample. It was shown in vitro that LIPUS may contribute to the reduction of the trauma-induced inflammatory reaction through impairment of the TNF-α signaling pathway. These indicate that LIPUS shows potential as a therapeutic tool to optimize the regenerative potential of periodontal tissues on replanted teeth.89 4.2.2. In vivo studies

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It has been reported that mechanical loading enhances the expression of phenotypic markers such as osteocalcin and bone sialoprotein in cementoblast in vivo; however, the expression was just moderately stimulated compared to osteoblasts.80 Regarding ultrasound stimulation, a pioneer study published by El-Bialy et al.27 showed that LIPUS prevented root resorption during experimental tooth movement in humans. Furthermore, in vivo study using an orthodontically-induced root resorption model revealed that LIPUS exposure significantly reduced root resorption by the suppression of cementoclastogenesis during orthodontic tooth movement without interfering with tooth movement, suggestively by altering OPG/RANKL expression ratio (Figure 2).48 Furthermore, Rego et al.88 assessed the inhibitory effect of a 21-day LIPUS application on root resorption using an experimental model of tooth replantation involving luxation and immediate replacement of maxillary first molars in rats. The results showed that the area of root resorption lacunae was statistically decreased in LIPUS treated sample. Taking all together, it is anticipated that LIPUS can be a clinically effective therapy to prevent orthodontically-induced root resorption in the future. Recent studies also have shown that LIPUS can enhance periodontal tissue regeneration after injury.112 In addition, it has been reported that LIPUS enhances periodontal healing after periodontal surgery.36,45

4.3. Application of LIPUS to degenerative joint diseases Synovial joints allow various degrees of relative motion of the bones produced by surrounding muscle forces.116 The bone ends come together within a fibrous joint capsule. The inner lining of this joint capsule is a metabolically active tissue, known as the synovium. The ends of the bones are covered by a thin and highly deformable layer of dense connective tissue known as articular cartilage.115 Ligaments, tendons, and other soft tissues inside and outside the joint cavity give stability to the joint and maintain the proper alignment of the articulating bone ends during motion.115 Daily activity accompanies joint motion, resulting in joint loads. The temporomandibular joint (TMJ) is only one synovial joint in orofacial region. Like other synovial joints, the TMJ enables

large

relative

movements

between

separate

bones.87

A

dense

fibrocartilaginous articular disc is located between the bones in each TMJ. The TMJ disc divides the joint cavity into two compartments (superior and inferior) and is a structure with an important functional role. The disc provides a largely passive movable 11

articular surface accommodating the translatory movement made by the condyle. Osteoarthritis of the TMJ (TMJ-OA) is characterized by mandibular condylar cartilage degradation due to mechanical overloading.63 Mechanical overloading of the mandibular condylar cartilage induces the expression of IL-1β,118 an inflammatory cytokine closely related to the progression of TMJ-OA.35,57,58 Thus, a large amount of IL-1β has been detected in the synovial fluid of patients with TMJ-OA.53 Since the fibrocartilage covering both the TMJ condyle and articular eminence is avascular, intraarticular synovial fluid provides nourishment to these fibrocartilage cells, which also have limited ability for self-repair.96,97 Therefore, once mandibular condylar cartilage degradation occurs in the TMJ, this pathology can be crippling, leading to a variety of morphological and functional deformities. This suggests the importance of suppression of cartilage degradation during the early stage of TMJ-OA. COX-2 is induced in human joint tissues including articular cartilage and synovium by various

inflammatory

stimuli

such

as

IL-1β,

IL-17,

and

TNF-α.33,57

These

proinflammatory cytokines appear to play a major role in regulation of COX-2 expression in joint disease and subsequent production of PGE2 resulting in cartilage degradation, inflammation, and angiogenesis.106 Therefore, agents that suppress COX2 activity have been promoted as potential drug targets to suppress the inflammatory conditions involving most tissues.33 It is widely accepted that synovitis is associated with clinical symptoms and reflects joint degradation in OA and Rheumatoid arthritis (RA),99 and several studies have focused attention on synovial hyperplasia.5 RA is a systemic, chronic inflammatory disease of the joints that is characterized by synovial hyperplasia, cartilage destruction, and infiltration of inflammatory cells into synovial tissue.79 Synovial hyperplasia is a major pathophysiologic feature of RA and appears to be associated with proinflammatory cytokines, notably TNF-α and IL-1β.99 Therefore, the importance of synovitis in the pathophysiology of RA has been increasingly recognized, particularly in early stages of the disease. Furthermore, synovial fibroblasts in the synovial intimal lining play a key role in producing cytokines and proteases.44 Since targeting synovial fibroblasts may improve clinical outcomes in inflammatory arthritis, it is thought that the control of proliferation and viability of synovial fibroblasts is an important consideration for treatment strategies. 4.3.1. In vitro studies Previously, LIPUS has been reported to affect cartilage matrix metabolism through 12

integrin β1, a possible cell surface receptor for LIPUS in chondrocytes.105 However, chondrocytes derived from hyaline cartilages were used in these studies. Therefore, study by Iwabuchi et al.49 is the first attempt to investigate the effects of LIPUS on mandibular condylar chondrocytes metabolism. Iwabuchi et al.49 elucidated the effect of LIPUS on COX-2 expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with IL-1β. As the result, COX-2 mRNA level was upregulated by the treatment with IL-1β and was suppressed significantly by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin β, and it inhibited the expression of pERK1/2. It was concluded that LIPUS exposure inhibited IL-1β-induced COX-2 expression through the integrin β1 receptor followed by the phosphorylation of ERK1/2. This indicates that LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in TMJ-OA, leading to tissue engineering in the mandibular condylar cartilage. In this line, El-Bialy et al.28 reported that LIPUS enhanced chondrogenic and osteogenic differentiation of bone marrow stromal cells, resulting in ultrasound-assisted tissue-engineered mandibular condyles in vivo. Nakamura et al.74 examined the effects of LIPUS exposure on metabolism of hyaluronan (HA) in synovial membrane cells stimulated by IL-1β. The results showed that LIPUS significantly up-regulated the expression of HA synthase (HAS) 2 and 3, and down-regulated the hyaluronidase (HYAL) 2 expression in the IL-1β-stimulated synovial membrane cells. This suggests that LIPUS enhances synthesis of highmolecular-weight HA, indicating anti-inflammatory response. 4.3.2. In vivo studies El-Bialy et al.26 firstly evaluated the effect of LIPUS on condylar and mandibular growth in the rabbit model with wear of functional appliance, and demonstrated that the daily use of LIPUS for four weeks stimulates mandibular condylar growth and increases the mandibular condylar, ramal, and total mandibular heights in growing rabbits. This indicates that LIPUS accelerates condylar and mandibular growth during orthopedic treatment. Oyonarte et al.81 also investigated the morphological effects of LIPUS stimulation on the mandibular condyles of growing rats, and demonstrated that LIPUS application may affect mandibular growth pattern in rats acting at the cartilage and bone level. They also indicateed that the effect of LIPUS on the growing condyle is expressed through a variation in trabecular shape and perimeter. Furthermore, recent study by Oyonarte et al.82 showed that LIPUS and mesenchymal stem cells (MSC) 13

application to the TMJ region of growing rats favoured transverse condylar growth, while LIPUS application alone may enhance sagittal condylar development. These studies may give an insight regarding the utility of LIPUS as a novel treatment tool for patients with mandibular growth deficient. In addition, Kaur et al.52 showed increased mandibular condylar growth in either bFGF pDNA gene therapy or LIPUS groups compared to the combined group (bFGF pDNA + LIPUS) that showed only increased bone volume fraction. They also concluded that It appears that there is an additive effect of bFGF + LIPUS on the mandibular growth. Furthermore, Nakamura et al.74 conducted in vivo study to examine the effectiveness of LIPUS treatment of synovitis in the knee joints of RA animal models. As the result, histological lesions of RA were significantly reduced in the joints treated with LIPUS for 3 weeks. Cox-2-positive cells in the knee joints treated with LIPUS were markedly decreased compared to the control joints. Considering these findings, LIPUS stimulation may also be a better candidate as medical remedy to treat inflammatory joint diseases accompanied with HA degradation in synovial fluid, such as synovitis.

4.4. LIPUS and pulp cells differentiation Pulp tissue contains precursor cells which have the potential to differentiate into odontoblasts. It is well understood that after physiologic or pathologic stimulation pulp stem cells may be recruited to proliferate and differentiate into odontoblasts which can produce dentin as a protection mechanism. It is also well documented that LIPUS has the potential to induce bone and periodontal cells differentiation. In this context, the effect of LIPUS on pulp cells differentiation has risen as a subject of investigation. 4.4.1. In vitro studies A previous study showed that LIPUS (30 mW/cm2) promoted odontoblast-like cells differentiation through stimulation of vascular endothelial growth factor (VEGF) secretion.97 It has also been shown that ultrasound stimulation (0.5 W/cm2) induced pulp stem cells to form reparative dentin formation in vivo by optimizing gene transfer of growth/differentiation factor 11 plasmid DNA and subsequently up-regulation of dentin sialoprotein gene expression.76 A recent study also showed that gingival multipotent cells can be differentiated into neural cells and this technique may be used in the future for dental pulp tissue engineering (Figures 3, 4).31 Moreover, it has been reported that LIPUS stimulates odontoblasts to secrete predentine in rat mandible and in human tooth slice organ cultures.1,2,30 14

4.4.2. In vivo studies Taken together, the current in-vitro studies suggest that LIPUS may be a future treatment modality in dental pulp therapy with or without gene therapy or growth factors. However, up till this point, there are no known in-vivo studies of using LIPUS for dental pulp tissue engineering. Future in vivo studies may be conducted in animal and possible pilot studies in human to study the effect of LIPUS in dental pulp therapy.

5. Clinical application of LIPUS in Orthodontics At this time, it is expected that the near future clinical application of LIPUS would be in minimizing orthodontically induced tooth-root resorption (OITRR) and enhancement of tooth movement during orthodontic treatment. Al-Daghreer et al.3 have shown that LIPUS can minimize OITRR in Beagle dogs at the same time tooth movement was enhanced by LIPUS. It is expected that this intraoral LIPUS devices will be universally available for human use for prevention of OITRR and enhancement of orthodontic tooth movement in year 2015. Other clinical applications of LIPUS in orthodontics may be to enhance periodontal ligament regeneration in cases with periodontal infraboney defect or gingival recession due to alveolar bone resorption, before commencing orthodontic treatment in such cases. Mandibular growth stimulation by LIPUS is still under laboratory experimental animal investigations to optimize this technique before standard clinical human use may be recommended. Based on the previous studies, it seems that daily application of LIPUS is agreed on to be 20 minutes per day throughout the treatment time. Different LIPUS devices may be introduced to the market with the aim of precisely applying LIPUS to specific teeth, ensure accurate LIPUS penetration to tissues, monitor patient compliance through internal micro-chip that can register each intraoral use of the patient and also can produce an average use of each patient over a period of time which can reflects patient‟s compliance. It is expected that orthodontists who will be using this technology will need to use a special software that can also remotely monitor each patient compliance and send the treating orthodontist a warning signal once the patient‟s compliance falls below acceptable values. Best device choice will be dependent on the device‟s capacity to produce consistent treatment over specific period of time and efficiency of reporting patient‟s compliance and any possible error/defect in the device that might happen during treatment period.

15

Conclusions Non-invasive modality such as LIPUS therapy has been given increased attention and raised as promising therapeutic tool for the regeneration of orofacial tissues. LIPUS presents low toxicity, low immunogenicity, non-invasiveness, highly targeted selectivity, and repeated applicability. Although LIPUS therapy has been widely used in the medical fields such as orthopedic surgery and rehabilitation, its availability and potential by dental professionals are still on initial stage. The effects of LIPUS in bony tissue seem to be well understood, but the literature has still lacked for available information about its effects on temporomandibular joint components and periodontal tissues. The present review provides current evidences that LIPUS has a positive effect on dental and periodontal cells metabolism and tissues repair, suggesting that LIPUS can be a promising therapeutic tool for the regeneration of tooth tissues. An understanding of the bioeffect of LIPUS therapy on orofacial tissues as described in this review is essential to the successful integration of management remedies for tissue engineering. To develop an evidence-based approach to clinical trials for treatment for orofacial degenerative diseases, we would like to be in full pursuit of LIPUS biotherapy. Still, there are many challenges for this relatively new strategy, but its achievements promise to go far beyond the present possibilities. Although there is a consensus in the literature about the stimulatory effect of LIPUS on different cell type, the optimum LIPUS parameter for each cell type has not yet been identified. Future comparative studies that can identify these optimum LIPUS treatment condition for each cell type is recommended.

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Figure legends Figure 1 Schematic illustration of signal transduction pathways activated by lowintensity pulsed ultrasound.

Figure 2 (A) Schematic representation of the appliance for tooth movement. The maxillary first molar was protracted using 50 gf activated Ni-Ti coil spring. (B) Micro-CT and histological observation. Representive histological sections (40x) of the upper first molar distobuccal root from 2-week time period stained with H.E. Resorption lacunae considerably occurred along the root in the control group, while in the LIPUS group root resorption was not obvious. White arrows indicate resorption lacnae. Red arrows indicate new bone apposition, white dotted line indicates border of root surface. (Modified from Inubushi et al.41.) Figure 3 Immunohistochemistry of human gingival progenitor cells (HGPCs) stained with anti-Beta-tubulin; Glial Fibrillary; MAPK2; nucleostemin; neurofilament and synaptophysin antibodies in the negative control group; after LIPUS treatment; after neuroinductive medium (NIM) treatment and after NIM and LIPUS. It can be seen that control group show no staining to any of the antibodies used. However HGPCs show increased staining with most of the antibodies used. LIPUS alone slightly increased staining than the control group. Most of the HGPCs show deep staining after differentiation using NIM or NIM + LIPUS. Bar = 100 µm. (Reprinted from El-Biary et al.28, Copyright (2014), with permission from Springer).

Figure 4 qPCR data showing significant up-regulation of Neurofilament (NF) gene by NIM and by LIPUS+NIM. Neucleostemin (NCT) is down-regulated by LIPUS and by LIPUS+NIM (non-significant). Vementin is significantly up-regulated by LIPUS+NIM than the control. * p