**4. Discussion**

#### **4.1 Introduction of marginal bone loss around dental implant**

Marginal bone loss is considered to be an inevitable risk factor in implant therapy. The reduction in height and width of marginal bone level affects the success rate of implant treatment in terms of esthetic and function.

The majority of marginal bone loss occurs in the first year after implant placement [67]. Thus, the clinical crown-to-implant ratio rises with time to become more unfavorable as years go by. However, the etiology of long-term marginal bone loss or late implant failure seems to be of different origin and prone to peri-implantitis or occlusal overload [68]. It is important to consider multiple factors together in assessing implant failure rates as interactive effects may be observed in the establishment and maintenance of osseointegration [69, 70]. Thus, in the present study, attempts were made to control the relevant confounding variables (patient gender and age, implant location, implant diameter and neck design, insertion torque, insertion depth, and crown-to-implant ratios).

In this study project, we tried to measure the marginal bone level around the implant and its stability both at the time of implant placement and at the time of loading. For this reason we chose the 3- and 6-month intervals to examine the marginal bone level and implant stability after soft and hard tissue maturation and early bone remodeling [71].

Ultrasound is the generation of sound waves with a frequency above the limit of human audibility of 20 kHz that transfers mechanical energy into the tissues; it is used extensively in sports medicine and physiotherapy. Therapeutic ultrasound can induce angiogenic and bone morphogenetic factors and bone formation in vitro [72].

Dinno et al. [73] demonstrated that intensities of ultrasound of less than 100 mW/cm2 spatial average and temporal average were nonthermal. Duarte [63] and Pilla et al. [74] reported that low-intensity ultrasound treatment in the range of 30–57 mW/cm<sup>2</sup> yielded minimal temperature changes when applied to the site of a bone fracture. Application of low-intensity pulsed ultrasound (30 mW/cm<sup>2</sup> ) was considered to have little thermal effect.

**19**

*Marginal Bone Changes around Dental Implants after LIPUS Application: CBCT Study*

All patients in the ultrasound group tolerated the ultrasound therapy very well. The therapy was conducted over 20 minutes comfortably without any rejection from the patients. The results showed that the ultrasound therapy with the intensity set at

Furthermore, the color of the gingival soft tissue remains pink and did not change to erythematic state at the end of the procedure which further proves there was no inflammation and untoward tissue response following the therapy. Therefore, the pain symptoms from patients were minimal as shown by minimal need for analgesia, and healing of the soft tissue wound in the ultrasound group was excellent. These clinical findings demonstrate wide acceptance of patients toward postoperative ultrasound therapy. Kamath et al. [75] in his study on the effect of LIPUS on healing of femur fracture revealed that there was more significant callous formation at the early stage of femur fracture in the LIPUS group than in the control group. Therefore, even in other parts of the body like femur, there are good results when LIPUS is applied. In view of the increasing use of high-intensity and low-frequency ultrasonic technology, in medicine and in surgery, better understanding of the benefits or side effects of US application is significant in order to establish appropriate clinical studies. LIPUS has disadvantages besides the advantages as mentioned. Erdogan and Esen [76] showed that the effects of ultrasound therapy on growing bones and brain tissues are unclear. Thus, its use in children and in skull bones should be avoided. Its use in sites with suspected neoplasia and acute infections is contraindicated because of possible accelerated disease progression. Patients should be evaluated for allergic reactions to the coupling gel, and patients with cardiac pacemakers should avoid ultrasound treatment because of possible interaction with the ultrasound signals specially when using US with both high-intensity and high-frequency waves. Miller et al. [77] mentioned that the induced heat by US is the result of the absorption of US energy in biological tissue and the heat can be concentrated by focused beams until tissue is coagulated for the purpose of tissue ablation. Unlike ultrasound for medical imaging (which transmits ultrasonic waves and processes a returning echo to generate an image), therapeutic ultrasound is a one-way energy delivery that might cause harmful effect in a cumulative way into the tissue, which utilizes a crystal sound head to transmit acoustic waves at 1–3 MHz and at amplitude

generated minimum heat that did not cause discomfort for the patients.

[78]. US heating, which can lead to irreversible

is known to promote

, and

tissue changes, follows an inverse time-temperature relationship. Depending on the temperature gradients, the effects from ultrasound exposure can include mild heating, coagulative or liquefactive necrosis, tissue vaporization, or all three [77]. Angle et al. [79] demonstrated that the therapeutic ultrasound with frequencies varying

healing, bone deposition, and growth. Nevertheless, therapeutic ultrasound is proposed to deliver energy to deep tissue sites through ultrasonic waves, to produce

the duration of application was only for 20 minutes, and this treatment was commenced 2 weeks after the acute inflammatory phase has subsided. We

Ebadi et al. [80] explained that ultrasonic energy causes soft tissue molecules to vibrate from exposure to the acoustic wave. This increased molecular motion generates frictional heat, thus increasing tissue temperature. The thermal effects of ultrasound are proposed to increase collagen extensibility, increase nerve conduction velocity, alter local vascular perfusion, increase enzymatic activity, alter contractile activity of skeletal muscle, and increase nociceptive threshold [78]. However, in our study, the intensity of LIPUS used was 30 mW/cm<sup>2</sup>

increases in tissue temperature or nonthermal physiologic changes [78].

**4.2 Clinical evaluation of application of ultrasound therapy**

*DOI: http://dx.doi.org/10.5772/intechopen.87220*

densities between 0.1 and 3 W/cm2

between 0.5 and 1.5 MHz and intensities 30–200 mW/cm2

30 mW/cm2

*Marginal Bone Changes around Dental Implants after LIPUS Application: CBCT Study DOI: http://dx.doi.org/10.5772/intechopen.87220*

#### **4.2 Clinical evaluation of application of ultrasound therapy**

*Clinical Implementation of Bone Regeneration and Maintenance*

thickness is contributed by ultrasound therapy.

insertion depth, and crown-to-implant ratios).

early bone remodeling [71].

in vitro [72].

100 mW/cm2

of 30–57 mW/cm<sup>2</sup>

considered to have little thermal effect.

**4.1 Introduction of marginal bone loss around dental implant**

cess rate of implant treatment in terms of esthetic and function.

as p value was less than 0.05. Thus, this increase in bone plate width is contributed to ultrasound therapy. There was no statistically significant increase in buccal and palatal bone height at 3 months between ultrasound and control groups, but there was statistically significant increase in buccal and palatal bone height at 6 months. Thus, this increase in bone plate height is contributed by ultrasound therapy. In the sagittal view, there was statistically significant increase in mesial and distal bone plates' width between two groups (ultrasound and control) at 3 and 6 months as p value was less than 0.05. Thus, this increase in bone plate thickness is contributed by ultrasound therapy. There was no statistically significant increase in mesial and distal bone height at 3 months between ultrasound and control groups, but there was statistically significant increase in mesial and distal bone height at 6 months. Thus, this increase in bone plate height is contributed by ultrasound

In the axial view, there was statistically significant increase in buccal, palatal, mesial, and distal bone plates' width between two groups (ultrasound and control) at 3 and 6 months as p value was less than 0.05. Thus, this increase in bone plate

Marginal bone loss is considered to be an inevitable risk factor in implant therapy. The reduction in height and width of marginal bone level affects the suc-

The majority of marginal bone loss occurs in the first year after implant placement [67]. Thus, the clinical crown-to-implant ratio rises with time to become more unfavorable as years go by. However, the etiology of long-term marginal bone loss or late implant failure seems to be of different origin and prone to peri-implantitis or occlusal overload [68]. It is important to consider multiple factors together in assessing implant failure rates as interactive effects may be observed in the establishment and maintenance of osseointegration [69, 70]. Thus, in the present study, attempts were made to control the relevant confounding variables (patient gender and age, implant location, implant diameter and neck design, insertion torque,

In this study project, we tried to measure the marginal bone level around the implant and its stability both at the time of implant placement and at the time of loading. For this reason we chose the 3- and 6-month intervals to examine the marginal bone level and implant stability after soft and hard tissue maturation and

Ultrasound is the generation of sound waves with a frequency above the limit of human audibility of 20 kHz that transfers mechanical energy into the tissues; it is used extensively in sports medicine and physiotherapy. Therapeutic ultrasound can induce angiogenic and bone morphogenetic factors and bone formation

spatial average and temporal average were nonthermal. Duarte [63]

yielded minimal temperature changes when applied to the site of

) was

Dinno et al. [73] demonstrated that intensities of ultrasound of less than

and Pilla et al. [74] reported that low-intensity ultrasound treatment in the range

a bone fracture. Application of low-intensity pulsed ultrasound (30 mW/cm<sup>2</sup>

**18**

therapy.

**4. Discussion**

All patients in the ultrasound group tolerated the ultrasound therapy very well. The therapy was conducted over 20 minutes comfortably without any rejection from the patients. The results showed that the ultrasound therapy with the intensity set at 30 mW/cm2 generated minimum heat that did not cause discomfort for the patients. Furthermore, the color of the gingival soft tissue remains pink and did not change to erythematic state at the end of the procedure which further proves there was no inflammation and untoward tissue response following the therapy. Therefore, the pain symptoms from patients were minimal as shown by minimal need for analgesia, and healing of the soft tissue wound in the ultrasound group was excellent. These clinical findings demonstrate wide acceptance of patients toward postoperative ultrasound therapy. Kamath et al. [75] in his study on the effect of LIPUS on healing of femur fracture revealed that there was more significant callous formation at the early stage of femur fracture in the LIPUS group than in the control group. Therefore, even in other parts of the body like femur, there are good results when LIPUS is applied.

In view of the increasing use of high-intensity and low-frequency ultrasonic technology, in medicine and in surgery, better understanding of the benefits or side effects of US application is significant in order to establish appropriate clinical studies. LIPUS has disadvantages besides the advantages as mentioned. Erdogan and Esen [76] showed that the effects of ultrasound therapy on growing bones and brain tissues are unclear. Thus, its use in children and in skull bones should be avoided. Its use in sites with suspected neoplasia and acute infections is contraindicated because of possible accelerated disease progression. Patients should be evaluated for allergic reactions to the coupling gel, and patients with cardiac pacemakers should avoid ultrasound treatment because of possible interaction with the ultrasound signals specially when using US with both high-intensity and high-frequency waves.

Miller et al. [77] mentioned that the induced heat by US is the result of the absorption of US energy in biological tissue and the heat can be concentrated by focused beams until tissue is coagulated for the purpose of tissue ablation. Unlike ultrasound for medical imaging (which transmits ultrasonic waves and processes a returning echo to generate an image), therapeutic ultrasound is a one-way energy delivery that might cause harmful effect in a cumulative way into the tissue, which utilizes a crystal sound head to transmit acoustic waves at 1–3 MHz and at amplitude densities between 0.1 and 3 W/cm2 [78]. US heating, which can lead to irreversible tissue changes, follows an inverse time-temperature relationship. Depending on the temperature gradients, the effects from ultrasound exposure can include mild heating, coagulative or liquefactive necrosis, tissue vaporization, or all three [77]. Angle et al. [79] demonstrated that the therapeutic ultrasound with frequencies varying between 0.5 and 1.5 MHz and intensities 30–200 mW/cm2 is known to promote healing, bone deposition, and growth. Nevertheless, therapeutic ultrasound is proposed to deliver energy to deep tissue sites through ultrasonic waves, to produce increases in tissue temperature or nonthermal physiologic changes [78].

Ebadi et al. [80] explained that ultrasonic energy causes soft tissue molecules to vibrate from exposure to the acoustic wave. This increased molecular motion generates frictional heat, thus increasing tissue temperature. The thermal effects of ultrasound are proposed to increase collagen extensibility, increase nerve conduction velocity, alter local vascular perfusion, increase enzymatic activity, alter contractile activity of skeletal muscle, and increase nociceptive threshold [78].

However, in our study, the intensity of LIPUS used was 30 mW/cm<sup>2</sup> , and the duration of application was only for 20 minutes, and this treatment was commenced 2 weeks after the acute inflammatory phase has subsided. We

feel that this dosage of US therapy is harmless to the active cells in the healing wound which was in the proliferative phase. The dose recommended may be harmful to the cells in the healing wound because they are vulnerable to damage from heat generation or prolonged treatment duration. Therefore, although the mechanotransduction mechanism for cell stimulation following US therapy is an acceptable phenomenon, it may only work favorably within certain limitations of the delivered energy.

#### **4.3 Evaluation of marginal bone level**

CBCT images showed adequate availability of bone height and width at the dental implant platform at day 0 for both groups at the time of implant placement. In this study, results obtained using CBCT images were reliable for linear measurements of bone thickness in height and width for both ultrasound-treated group and control group. CBCT enables us to expose the patient to low radiation doses, giving more comfort, and it is an economical procedure [81]. At 3 months, there was an increase of the mean difference of buccal bone plate width of 0.19 mm in the ultrasound group compared to the control group. At 6 months, there was a mean difference marginal bone loss of 0.58 mm in width of the buccal bone plate around the dental implant platform in the control group, while there was 0.38 mm increase in the mean difference of buccal bone width in the ultrasound group. These findings were consistent with the previous study by Chen who investigated the effect of LIPUS on bone regeneration in the rat parietal bone defects [26]. In Chen study, the defects were analyzed with micro-CT (μCT) and then histologically, which demonstrated new bone formation with the newly formed thick and matured bone compared to the one of the control group.

The justification of using LIPUS in this study is to accelerate the bone wound healing processes within the region of interest (ROI) which is the region replacing single missing maxillary premolar following trauma to the bone as implant placement surgery is considered to be a traumatic procedure even though the surgery is minimally invasive to the bone. Our aim in this study is to mimic what happened in natural tissue repair by inducing, triggering, and provocation of the cells related to bone formation by encouragement of mechanotransduction pathways involved in cell responses. These responses include integrin/mitogen-activated protein kinase (MAPK) and other kinase signaling pathways, gap-junctional intercellular communication, upregulation and clustering of integrins, involvement of the COX-2/PGE2 and iNOS/NO pathways, and activation of mechanoreceptor [44]. Mechanotransduction involves various signal transduction pathways, including the activation of ion channels and other mechanoreceptors in the membrane of the bone cell, resulting in gene regulation in the nucleus [42].

Based on time intensity and period of exposure of cells to waves of ultrasound, LIPUS can recruit mesenchymal stem cells from neighboring tissues and other sites in the body in attractive processes (chemotactic) with other biomedical pro-inflammatory mediators (growth factors) that are considered necessary in bone wound healing processes and trigger it from inactive form to active phase when LIPUS is used. This suggests that LIPUS is able to enhance osteogenesis and angiogenesis in vivo and in vitro as was well documented by literature review that angiogenesis precede osteogenesis process [54]. Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration [55].

In our study, the marginal bone level was assessed and measured at three different views (coronal, sagittal, and axial) in which four points were located and

**21**

*Marginal Bone Changes around Dental Implants after LIPUS Application: CBCT Study*

measured per implant site (corono-buccal, corono-palatal, apico-buccal, apicopalatal, sagitto-mesial, sagitto-distal, apico-mesial, apico-distal, axio-buccal, axio-palatal, axio-mesial, and axio-distal), respectively, and at three different time intervals postoperatively at day 0, 3, and 6 months. The results of this study showed an increase in buccal bone width from 1.43 mm at day 0 to 1.81 mm at 6 months which revealed that the mean difference of buccal bone plate width increased by 0.38 mm, while the palatal bone mean difference width was also increased by 0.18 mm at 6 months. In the sagittal view, there was an increase of mean difference of 0.26 mm at the mesial aspect of the dental implant at 6 months in the ultrasound group compared to the control group that had marginal bone loss from 1.43 mm at day 0 to 0.85 mm at 6 months at the buccal bone plate in the coronal view. The reason why the height and width of bone thickness had increased in the ultrasound compared to the control is that LIPUS can promote bone healing and repair by inducing osteogenesis and angiogenesis. Earlier work has shown that the therapeutic range of US stimulates bone formation, osteoblast proliferation, and the synthesis of angiogenic vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and interleukin 8 [72, 82]. Ramli et al. [83] have proven that ultrasound should be considered to have angiogenic and osteogenic values in their in vivo study looking at ultrasound effects on angiogenesis using the chick chorioallantoic membrane. In vitro ultrasound has also been shown to upregulate the release of the osteogenic cytokine OPG and downregulate RANKL, the ligand of the receptor activator nuclear factor kappa B, which recruits and

In this study, the transducer was applied on the buccal aspect of the dental implant very close to the buccal bone plate and showed clinically that at 1.5 MHz frequency, a penetration of up to 2 cm is possible, thus influencing the palatal plate. Ramli et al. [83] demonstrated that the traditional 1- to 3-MHz frequency of ultrasound therapy has a penetration of up to 2 cm. Doan et al. [72] reported that the best effect of therapeutic ultrasound on angiogenesis occurs with intensities

Results of the control group showed increased loss of bone height from 1.20 mm

implant and increased marginal bone loss (MBL) in width from 1.43 mm at day 0 to 0.85 mm at 6 months as compared with LIPUS-treated group. It reveals that LIPUS has a positive effect on the healing of bone, and the loss of marginal bone in the control group was contributed by not using US therapy. This finding is consistent

Angle et al. [79] explained in his vitro study, using rat bone marrow stromal

), and then studied them at early (cell activation), middle (differentiation into osteogenic cells), and late (biological mineralization) stages of osteogenic differentiation. They concluded that LIPUS with intensities of 2, 15,

showed a positive effect on osteogenic differentiation of rat bone

responses in bone cells. They cultured bone cells under defined conditions with

marrow stromal cells in early stage compared with the control group. Monden et al. [87] also suggested that the injured bone may be treated with LIPUS, as LIPUS has the capability to induce the cellular as well as molecular pathways of bone healing. LIPUS treatment matures the newly formed bone in the cortical bone area producing bone differentiation markers, osteocalcin (OCN) and osteopontin (OPN), and reduces the depression by enhancing the periosteal cellular

at 3 months to 0.88 mm at 6 months at the apico-buccal aspect of the dental

has a theoretical advantage of penetrating tissues up to 10 cm.

cells that the LIPUS intensities below 30 mW/cm<sup>2</sup>

and a frequency of 45 kHz, as the long wave machine

, compared them with the control group

are able to provoke phenotypic

*DOI: http://dx.doi.org/10.5772/intechopen.87220*

activates osteoclasts [83].

between 15 and 30 mW/cm2

with those of [26, 84–86].

(0 mW/cm<sup>2</sup>

and 30 mW/cm<sup>2</sup>

intensities of 2, 15, and 30 mW/cm<sup>2</sup>

#### *Marginal Bone Changes around Dental Implants after LIPUS Application: CBCT Study DOI: http://dx.doi.org/10.5772/intechopen.87220*

measured per implant site (corono-buccal, corono-palatal, apico-buccal, apicopalatal, sagitto-mesial, sagitto-distal, apico-mesial, apico-distal, axio-buccal, axio-palatal, axio-mesial, and axio-distal), respectively, and at three different time intervals postoperatively at day 0, 3, and 6 months. The results of this study showed an increase in buccal bone width from 1.43 mm at day 0 to 1.81 mm at 6 months which revealed that the mean difference of buccal bone plate width increased by 0.38 mm, while the palatal bone mean difference width was also increased by 0.18 mm at 6 months. In the sagittal view, there was an increase of mean difference of 0.26 mm at the mesial aspect of the dental implant at 6 months in the ultrasound group compared to the control group that had marginal bone loss from 1.43 mm at day 0 to 0.85 mm at 6 months at the buccal bone plate in the coronal view. The reason why the height and width of bone thickness had increased in the ultrasound compared to the control is that LIPUS can promote bone healing and repair by inducing osteogenesis and angiogenesis. Earlier work has shown that the therapeutic range of US stimulates bone formation, osteoblast proliferation, and the synthesis of angiogenic vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and interleukin 8 [72, 82]. Ramli et al. [83] have proven that ultrasound should be considered to have angiogenic and osteogenic values in their in vivo study looking at ultrasound effects on angiogenesis using the chick chorioallantoic membrane. In vitro ultrasound has also been shown to upregulate the release of the osteogenic cytokine OPG and downregulate RANKL, the ligand of the receptor activator nuclear factor kappa B, which recruits and activates osteoclasts [83].

In this study, the transducer was applied on the buccal aspect of the dental implant very close to the buccal bone plate and showed clinically that at 1.5 MHz frequency, a penetration of up to 2 cm is possible, thus influencing the palatal plate. Ramli et al. [83] demonstrated that the traditional 1- to 3-MHz frequency of ultrasound therapy has a penetration of up to 2 cm. Doan et al. [72] reported that the best effect of therapeutic ultrasound on angiogenesis occurs with intensities between 15 and 30 mW/cm2 and a frequency of 45 kHz, as the long wave machine has a theoretical advantage of penetrating tissues up to 10 cm.

Results of the control group showed increased loss of bone height from 1.20 mm at 3 months to 0.88 mm at 6 months at the apico-buccal aspect of the dental implant and increased marginal bone loss (MBL) in width from 1.43 mm at day 0 to 0.85 mm at 6 months as compared with LIPUS-treated group. It reveals that LIPUS has a positive effect on the healing of bone, and the loss of marginal bone in the control group was contributed by not using US therapy. This finding is consistent with those of [26, 84–86].

Angle et al. [79] explained in his vitro study, using rat bone marrow stromal cells that the LIPUS intensities below 30 mW/cm<sup>2</sup> are able to provoke phenotypic responses in bone cells. They cultured bone cells under defined conditions with intensities of 2, 15, and 30 mW/cm<sup>2</sup> , compared them with the control group (0 mW/cm<sup>2</sup> ), and then studied them at early (cell activation), middle (differentiation into osteogenic cells), and late (biological mineralization) stages of osteogenic differentiation. They concluded that LIPUS with intensities of 2, 15, and 30 mW/cm<sup>2</sup> showed a positive effect on osteogenic differentiation of rat bone marrow stromal cells in early stage compared with the control group. Monden et al. [87] also suggested that the injured bone may be treated with LIPUS, as LIPUS has the capability to induce the cellular as well as molecular pathways of bone healing. LIPUS treatment matures the newly formed bone in the cortical bone area producing bone differentiation markers, osteocalcin (OCN) and osteopontin (OPN), and reduces the depression by enhancing the periosteal cellular

*Clinical Implementation of Bone Regeneration and Maintenance*

the delivered energy.

**4.3 Evaluation of marginal bone level**

compared to the one of the control group.

bone cell, resulting in gene regulation in the nucleus [42].

cells play an important role in bone regeneration [55].

feel that this dosage of US therapy is harmless to the active cells in the healing wound which was in the proliferative phase. The dose recommended may be harmful to the cells in the healing wound because they are vulnerable to damage from heat generation or prolonged treatment duration. Therefore, although the mechanotransduction mechanism for cell stimulation following US therapy is an acceptable phenomenon, it may only work favorably within certain limitations of

CBCT images showed adequate availability of bone height and width at the dental implant platform at day 0 for both groups at the time of implant placement. In this study, results obtained using CBCT images were reliable for linear measurements of bone thickness in height and width for both ultrasound-treated group and control group. CBCT enables us to expose the patient to low radiation doses, giving more comfort, and it is an economical procedure [81]. At 3 months, there was an increase of the mean difference of buccal bone plate width of 0.19 mm in the ultrasound group compared to the control group. At 6 months, there was a mean difference marginal bone loss of 0.58 mm in width of the buccal bone plate around the dental implant platform in the control group, while there was 0.38 mm increase in the mean difference of buccal bone width in the ultrasound group. These findings were consistent with the previous study by Chen who investigated the effect of LIPUS on bone regeneration in the rat parietal bone defects [26]. In Chen study, the defects were analyzed with micro-CT (μCT) and then histologically, which demonstrated new bone formation with the newly formed thick and matured bone

The justification of using LIPUS in this study is to accelerate the bone wound healing processes within the region of interest (ROI) which is the region replacing single missing maxillary premolar following trauma to the bone as implant placement surgery is considered to be a traumatic procedure even though the surgery is minimally invasive to the bone. Our aim in this study is to mimic what happened in natural tissue repair by inducing, triggering, and provocation of the cells related to bone formation by encouragement of mechanotransduction pathways involved in cell responses. These responses include integrin/mitogen-activated protein kinase (MAPK) and other kinase signaling pathways, gap-junctional intercellular communication, upregulation and clustering of integrins, involvement of the COX-2/PGE2 and iNOS/NO pathways, and activation of mechanoreceptor [44]. Mechanotransduction involves various signal transduction pathways, including the activation of ion channels and other mechanoreceptors in the membrane of the

Based on time intensity and period of exposure of cells to waves of ultrasound, LIPUS can recruit mesenchymal stem cells from neighboring tissues and other sites in the body in attractive processes (chemotactic) with other biomedical pro-inflammatory mediators (growth factors) that are considered necessary in bone wound healing processes and trigger it from inactive form to active phase when LIPUS is used. This suggests that LIPUS is able to enhance osteogenesis and angiogenesis in vivo and in vitro as was well documented by literature review that angiogenesis precede osteogenesis process [54]. Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast

In our study, the marginal bone level was assessed and measured at three different views (coronal, sagittal, and axial) in which four points were located and

**20**

differentiation. In vitro studies have shown that LIPUS leads to the increased expression of genes related to the bone formation. These genes include osteocalcin, aggrecan, bone sialoprotein, insulin-like growth factor-I, collagen types I and X, transforming growth factor beta, alkaline phosphatase, and runt-related gene-2 [88, 89].

Additionally, LIPUS treatment also promotes the synthesis of protein and uptake of calcium by osteoblasts. LIPUS treatment also plays an important role in the remodeling of the bone by stimulating the cyclooxygenase pathway. LIPUS increases the expression of COX-2 gene that promotes the synthesis of prostaglandin E2 (PGE2) in the osteoblasts [88, 89].

Huang et al. [90] concluded in his recent study in vitro that the LIPUS stimulates the expression of BMP-2 which means positive effects of LIPUS on osteogenesis. In vitro study by Sun et al. [91] showed that LIPUS upregulated osteoblasts and downregulated osteoclasts in the rat alveolar mononuclear cells. Lu et al. [92] explained that the mechanical signals from LIPUS could stimulate osteoblasts by means of gene expression and stimulated proteins that were translated by these genes causing activation of apoptotic genes and osteogenesis in acceleration of the tissue remodeling and expedite clinical outcomes as we have seen in our current study.

Iwanabe et al. [93] demonstrated in his recent study in vitro that the number of cells at 5 days after LIPUS exposure was significantly higher than that of the control, while that at 7 days was about 35% higher than that of the control. This means that LIPUS has the potential to be an effective agent in inducing migration, proliferation, and cell differentiation.

In vitro as well as in vivo studies, using animal models showed that LIPUS has stimulatory effect on cellular activity, release of cytokines, and bone healing [94]. Cell physiology is directly affected by LIPUS. It increases the uptake of calcium by the developing cartilage and bone cells in the culture. It also stimulates a large number of genes that help in the process of healing [62]. Barzelai et al. [95] reported that LIPUS not only modulates the expression of genes, but it also enhances the process of angiogenesis and increases the flow of blood at the site of fracture.
