Distraction osteogenesis (DO) is a biological process for producing new bone and overlying soft tissue by gradual controlled traction of the surgically separated bone segments. Gradual traction on living tissues creates tensile forces, which stimulate stem cells, thus promoting regeneration and active growth of the involved tissues. This process is based on the "Law of Tension Stress" [1]. The bone and its periosteum act as a guide for the newly regenerated bone. During DO, the overlying soft tissue envelope (skin, subcutaneous tissues, muscles, nerves, fascia, and periosteum) responds according to the hard tissue movements with gradual lengthening. This process is called distraction histogenesis. Simultaneous soft tissue distraction leads to decreased soft tissue resistance thus reducing bony relapse.

To achieve adequate bony elongation, preservation of blood supply, periosteum and stable fixation are required; therefore, optimal rate and rhythm of distraction is essential. Stability is an important principle, allowing for remodeling of the woven bone in the distracted gap during the consolidation period. The duration of the consolidation period may last up to several months during which the newly regenerated bone and the surrounding tissue are exposed to external forces and infections, which may interfere with the healing process and increase the complication rate [2]. In addition, due to prolonged consolidation periods, patients may experience inconvenience and psychosocial issues. Therefore, many efforts have been made to shorten the consolidation period in an attempt to overcome this major drawback of the DO procedure. This has been attempted by biophysical and biochemical strategies, including the application of growth factors, stem cells and various adjuvant modalities therapies, including extracorporeal shock wave, low level laser and low intensity pulsed ultrasound (LIPUS) [3].

Ultrasound generates acoustic energy at a high frequency (1 to 12 MHz), from a piezoelectric crystal within a transducer applied to the target tissue. Ultrasound has been widely used in medicine as a diagnostic and therapeutic tool [4]. Therapeutic ultrasound stimulates changes in cells and tissues by utilizing high energy intensities (1-3 W/cm$^{2}$). These changes may be due to the thermal effect by elevation of temperature of the local tissue [5]. In contrast, for diagnostic applications of ultrasound, low energy intensity is used and is regarded as a non-thermal mechanism producing cavitation and acoustic streaming, which stimulates biochemical events at the cellular level [6]. Duarte et al., [7] demonstrated that LIPUS enhances bone formation with minimal thermal effects [7]. This revelation generated considerable interest. The Food and Drug Administration (FDA) approved the use of LIPUS for fracture healing and for the treatment of established non-union fractures in 1994 and 2004, respectively[8,9].

In this review, we focus on the effectiveness and safety of LIPUS as an adjuvant treatment for accelerating DO, as observed in animal models.

This systematic review is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist structure. One author (JGG) searched PubMed and EMBASE electronic databases and performed a systematic search, to identify all studies from 1999 till 2017 regarding the application of LIPUS during DO in animal studies. The following keywords were used: "Distraction osteogenesis" and "low intensity ultrasound". Followed by a manual search through google scholar for additional relevant articles.

We used the PICOS format suggested by Cochrane Collaboration to describe the research question of the current review: participants -- animals of any species which underwent DO on any bone type. Intervention -- low intensity ultrasound during any period of DO. Comparison -- control group; DO without LIPUS and DO with another intervention. Outcome -- accelerated bone regeneration as evaluated by all methods reported by the authors for evaluating the effect of LIPUS during DO. Study design- animal studies. Data selection was conducted by two independent reviewers (JGG and DS). First, titles and abstracts were screened and duplicated articles were eliminated. Next, human trials, review articles, case reports and non-English publications were excluded. We then retrieved the full text of any article that was judged as potentially eligible. The reviewers independently applied eligibility criteria to the methods section of potential studies. Finally, any disagreements were resolved through discussion until consensus was reached. If there were more than 1 publication of the same experiment, the comprehensive and detailed report was selected.

Data items were extracted from each eligible study independently and included personal information, animal species and size, study design, distraction protocol, intensity and duration of the LIPUS, phase of treatment in which LIPUS was applied, assessment methods, results and whether LIPUS accelerated bone regeneration.

The Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) Risk of Bias tool, which is based on the Cochrane Collaboration Risk of Bias Tool for animal intervention studies, was adopted and applied for assessing the risk of bias in this systematic review [9]. Randomization of the study, blinding of investigators and outcome assessor, attrition bias (incomplete outcome data adequately addressed), and reporting bias (selective outcome reporting) were used to assess study quality. "Yes" indicates low risk of bias; "No" indicates high risk of bias. If one of the relevant entry is answered with "No", this indicates a high risk of bias for that specific study.

The search of PubMed and EMASE electronic databases provided a total of 86 records. After adjusting for duplicates, 50 records remained. Of which, 31 studies were eliminated after reviewing the abstracts which clearly showed these papers did not meet the criteria. Of the 19 potentially eligible articles retrieved in full text[10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28], 2 were excluded as they used high intensity ultrasound (2 W/cm$^{2}$)[27,28] and 2 studies were published in 2 articles each and were thus considered as one study for the purpose of the research[11,12,23,24], leaving 15 eligible studies included in the systematic review (Fig. 1). The results are summarized in Tables 1-3. Experimental designs for assessing the impact of LIPUS on DO were heterogeneous with respect to the animal species used, bone model and distraction protocol. The most common animal species used was rabbit (12/15, 80%) and the tibia was the bone model most frequently tested (8/15, 53%). Other studies utilized a mandible model (5/15, 33%), femur (1/15, 7%) and metatarsal (1/15, 7%).

Fig. 1.

Flow chart of the systematic search stratergy.

The DO protocols varied widely; the latency period ranged between 1-7 days, the distraction rate was between 0.3-3.0 mM/day and the consolidation period, in accordance with time of scarification, ranged from 0 to 84 days.

All studies used the pulsed technique exclusively, except for one study which compared the pulsed technique with continuous ultrasound treatment [22]. One study compared the effect of low-level laser versus LIPUS on rabbit mandible [26].

In most experimental studies, LIPUS treatment was performed with the same therapeutic parameters; 30 mW/cm$^{2}$ intensity for 20 minutes per day (14/15, 93%) and one study used 40 mW/cm$^{2}$ intensity for 10 min twice a day [24]. Whereas another study [20] used two different durations for the LIPUS treatment, 20 min and 40 min per day.

The timing of LIPUS application varied among the reviewed studies. In one study [18], LIPUS treatment was applied during the distraction, latency and consolidation period in three different groups with the intention of investigating the optimal timing of LIPUS application during DO. Nine of the 14 other studies applied LIPUS during the consolidation period. Other researchers applied the treatment during the distraction period (3/14) or during both periods (2/14).

Although different methods were used to evaluate the effects of LIPUS on DO in the reviewed studies, bone mineral density (BMD) and mechanical tests were most prevalent. BMD was used for evaluating bone regeneration in most studies (13/15, 87%). BMD was determined using conventional X-ray, Computed Tomography (CT) and Dual Energy X-Ray Absorptiometry (DEXA) 2, 5 and 6 times, respectively, out of the 13 studies. In 10/13 studies[10,11,13,15,16,18,19,22,24,26] a higher fraction of BMD (77%) was found in the experimental group as compared to the control group; the difference was significantly higher in 8/13 (62%) studies[10,11,15,18,19,22,24,26].

In addition, mechanical testing for evaluating the stiffness, torque and strength of the distracted bone was frequently conducted (12/15 of the studies). A higher mechanical structure in the LIPUS groups was reported in 8/12 studies, compared to the control groups[10,12,15,16,17,18,22,25] of which 6 showed statistical significance.

In summary, LIPUS has a positive effect on bone regeneration in 11/15 (73%) of the studies, though in a variable manner. The results of the methodological quality and risk of bias are shown in Table 3. Six studies contained information about randomization of animals. No studies reported that investigators were blinded from knowledge, which intervention each animal received during the experiment, and only 3 studies reported blinding of outcome assessor. Regarding the attrition bias, 12 studies adequately addressed the incomplete outcome data. All of the included studies were free of selective outcome reporting. Overall, all studies were considered to be a relatively high risk of performance bias, but no reporting bias.

DO is a surgical technique for bone lengthening that stimulates new bone formation and simultaneously expands the surrounding soft tissues. DO requires no donor site, thus eliminating the morbidity of a second surgical site, and is thus a safe and effective procedure that has gained widespread popularity in both orthopedic and craniofacial fields. The technique is used for treatment of congenital deformities or acquired deformities secondary to oncology, trauma, severe atrophic bone and infections. However, the prolonged consolidation period and the potential for complications during this period remain major disadvantages. Therefore, many attempts have been made to shorten the consolidation period, which would reduce costs, complications and burden on the patient. Several studies have shown the efficacy of LIPUS in stimulating bone formation during fracture repair. The current review discusses the efficacy of LIPUS during DO in animal studies for accelerating bone regeneration.

Small animals (rats and rabbits) have been extensively used, while large animal models are relatively scarce. Clearly, small animal models are easier for a large sample size and in-depth molecular and genetic analyses, but are limited in their clinical relevance to human DO patients, due to the large discrepancy in mandible size, morphology and function. Therefore, more pre-clinical studies based on large animal models are desirable.

Generally, the dosage of LIPUS treatment can be adjusted in terms of frequency, intensity and treatment duration [29]. In the reviewed studies listed in Table 1, the parameters of therapeutic LIPUS were almost identical; 20 min a day, 1-1.5 MHz sine waves repeating at 1 KHz, average intensity of 30 mW/cm$^{2}$ and a pulse width of 200 ms. In another study [24], a different intensity was applied (40 mW/cm$^{2}$ intensity for 10 min, twice a day) on dog mandibles during DO. Using this intensity, the authors also reported increased bone regeneration in comparison to the control group. These results are in accordance with other reports regarding bone fracture repair, which indicate that the appropriate intensity is generally $<$ 100 mW/cm$^{2}$ and usually in the range of 20-50 mW/cm$^{2}$ [30].

In an attempt to determine optimal duration of LIPUS treatment during rapid bone lengthening, in study [20] two different durations of treatment were selected -- 20 min and 40 min per day. The results showed that LIPUS increased the bone mineral content (BMC) and the volume of mineralized tissue of the distraction callus in a dose-dependent manner. Further studies should be conducted on conventional DO in order to determine whether or not LIPUS treatment has a dose-dependent effect.

In study [22], the effects of pulsed versus continuous ultrasound treatments on rapid DO were compared. The authors reported that earlier stages of bone formation were enhanced by pulsed ultrasound to a larger extent than continuous ultrasound. This might be explained by the additional mechanical stimulation provided in the pulsed mode, which has an effect on bone cell differentiation and bone matrix production [31].

DO consists of three periods; latency, distraction and consolidation. Each period activates various molecular and cellular events including different osteogenic factors, leading to varied regeneration potential. The timing of LIPUS application during DO varied among the currently reviewed studies; however, most of the authors applied LIPUS during the consolidation period. In study [18], LIPUS most effectively accelerated maturation of newly formed bone during DO in the distraction period, as compared to the latency and the consolidation periods, suggesting that mature mesenchymal cells, osteoblasts, or chondrocytes were affected by LIPUS, rather than inflammatory cells or immature mesenchymal cells. Furthermore, study [25] reported that LIPUS stimulated more bone formation with higher efficacy during the distraction and early consolidation periods, with no statistical difference when applied at the late consolidation period. These results can be explained by the fact that at early stages of DO, the distraction gap is filled mostly with hyper-cellular soft tissue and less mineralized tissue, thus facilitating the transmission of LIPUS. In contrast, the results in study [21] did not support the application of LIPUS at the distraction and early stages of consolidation periods.

Similarly to LIPUS, low-level laser therapy (LLLT) produces mechanical stimulation which influences bone repair and regeneration. One rabbit model compared the effects of LIPUS and LLLT on bone maturation following DO [26]. The results suggest that both LIPUS and LLLT have positive effects in terms of accelerating bone healing during DO.

The appropriate timing of removal of the distractor device following DO is still being debated. Bone strength and stiffness are the most clinically relevant variables tested in this case, because these determine whether or not a bone will deform or fracture when the distractor device is removed[1,10]. Most studies (75%) suggested that LIPUS improves the mechanical properties of the distracted bone and only 50% demonstrated significant differences. The lack of significant differences could be attributed to the discrepancy in test protocols, the time when the bones were tested and the small number of animals in each group. Generally, the BMD measurement is a quicker, cheaper and more available quantitative method. Several studies demonstrated a correlation between DEXA and CT for BMD determination and the biomechanical properties of bone after DO [32].

Generally, the most common method for the quantification of BMD is DEXA and particularly in the reviewed studies. BMD measurements in most studies increased in the LIPUS groups compared to the control groups. However, the differences were not significant in the late stage of the consolidation period as indicated in some studies[10,19,24,26], demonstrating that LIPUS is most effective at the early stage of the consolidation period.

In contrast to fracture healing, which is repaired in a process of endochondral ossification, it has been reported that bone formation during DO is generated mainly by an intramembranous process [33]. However, other researchers reported the presence of predominant endochondral ossification [34]. In addition, some authors postulated a third ossification mode called transchondroid, where they found predominance of endochondral bone formation in the early stages of distraction, but intramembranous ossification later [35]. Although the specific mechanism by which LIPUS affects bone formation is unclear, it appears that it stimulates aggrecan expression in cultured chondrocytes and in rat fracture repair, as well as upregulation of chondroitin sulfate[36,37,38]. These results are consistent with those reports showing more cartilage tissue formation during DO[12,14,17,18,20,22] and the limited data supporting the effect of LIPUS on osteoblast and intramembranous ossification[19,39]. Therefore, it is believed that LIPUS affects bone formation in DO via endochondral ossification. However, the exact cellular mechanism by which LIPUS accelerates bone regeneration in DO requires further investigation.

Although the reviewed studies showed considerable variations in study design, animal models, DO parameters, LIPUS parameters and the application and assessment methods, the conclusions suggest that LIPUS has a positive effect on accelerating bone regeneration during DO (11/15, 73%). In the articles that showed no advantage for using LIPUS (4/15), no negative effects were noted[13,14,17,21]. In addition, LIPUS showed no adverse effects or complications in any of the articles reviewed.

A quantitative analysis (meta-analysis) of the present systematic review could not be performed when considering the lack of homogeneity related to animal species, bone model, distraction protocol, LIPUS parameters, evaluation methods and measurement unites. The quality assessment found that all studies were considered as having high risk of bias. Therefore, the conclusions in the current systematic review may be hampered by the aforementioned shortcomings.

The positive effect of LIPUS on acceleration of bone regeneration in this systematic review are consistent with the results of a previous review on five human trials (four on tibia and one on a mandible) studying the effect of LIPUS during DO [40]. This review studied the optimal time for LIPUS applications, the appropriate intensity of LIPUS and the histological aspects including the ossification pattern, which could serve as a milestone for future studies investigating the biological mechanism of LIPUS on bone regeneration.

Previous studies on human subjects were performed mainly on the tibia, yet many surgeons perform DO on the mandible. No significant results were observed in the mandible. In this review on animal models, five studies performed on the mandible were included, and in all of them, accelerated bone regeneration was reported.

In conclusion, LIPUS is a safe, non-invasive, patient-friendly technique that bears no complications or adverse effects. The current comprehensive review of the literature indicates that LIPUS can accelerate bone formation and increase BMD during DO, thus shortening the consolidation period. The optimal timing of LIPUS application is during the distraction and early consolidation phases. Sporadic human trials did not show positive results in human mandibles, yet according to the current literature review, LIPUS during DO in the mandible shows promise and should be further clinically investigated. This review strengthens the preferred timing of LIPUS application during the distraction and early consolidation phases. The preferred intensity should be between 30-40 mW/cm$^{2}$. Review of the histological effects of LIPUS in these studies indicates influence via endochondral ossification (Fig. 2). Thus, the effect of LIPUS on chondrocytes should be further investigated in order to decipher the exact cellular and molecular influence of LIPUS on enhancement of bone formation. This should be the basis for better understanding and assist in establishment of protocols for accelerating DO.

Fig. 2.

Summary of the suggested effects of LIPUS on DO. The physical energy transmitted by the transducer of the LIPUS is converted into biological response, leads to up-regulation of growth factors via endochondral ossification.

None.

The authors declare that they have no financial or non-financial interests.