Aims. To evaluate whether low-intensity pulsed ultrasound (LIPUS) accelerates bone healing at osteotomy sites and promotes functional recovery after open-wedge high tibial osteotomy (OWHTO). Methods. Overall, 90 patients who underwent OWHTO without bone grafting were enrolled in this nonrandomized retrospective study, and 45 patients treated with LIPUS were compared with 45 patients without LIPUS treatment in terms of bone healing and functional recovery postoperatively. Clinical evaluations, including the pain visual analogue scale (VAS) and Japanese Orthopaedic Association (JOA) score, were performed preoperatively as well as six weeks and three, six, and 12 months postoperatively. The progression rate of gap filling was evaluated using anteroposterior radiographs at six weeks and three, six, and 12 months postoperatively. Results. The pain VAS and JOA scores significantly improved after OWHTO in both groups. Although the LIPUS group had better pain scores at six weeks and three months postoperatively, there were no significant differences in JOA score between the groups. The lateral hinge united at six weeks postoperatively in 34 (75.6%) knees in the control group and in 33 (73.3%) knees in the LIPUS group. The progression rates of gap filling in the LIPUS group were 8.0%, 15.0%, 27.2%, and 46.0% at six weeks and three, six, and 12 months postoperatively, respectively, whereas in the control group at the same time points they were 7.7%, 15.2%, 26.3%, and 44.0%, respectively. There were no significant differences in the progression rate of gap filling between the groups. Conclusion. The present study demonstrated that LIPUS did not promote bone healing and functional recovery after OWHTO with a locking plate. The routine use of LIPUS after OWHTO was not recommended from the results of our study. Cite this article: Bone Jt Open 2022;3(11):885–893

Aims. Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods. A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results. The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion. In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue. Cite this article: Bone Joint Res 2024;13(7):332–341

Introduction and Objective. Nonunion is incomplete healing of fracture and fracture that lacks potential to heal without further intervention. Nonunion commonly presents with persistent pain, swelling, or instability. Those symptoms affect patient quality of life. It is known that using low intensity pulsed ultrasound (LIPUS) for fresh fractures promotes healing. However, effectiveness of LIPUS for nonunion is still controversial. If LIPUS is prove to be effective for healing nonunion, it can potentially provide an alternative to surgery. In addition, we can reduce costs by treating nonunion with LIPUS than performing revision surgery. Materials and Methods. The two authors carried out a systematic search of PubMed, Ovid MEDLINE, and the Cochrane Library. Meta-analysis of healing rate in nonunion and delayed union patients who underwent LIPUS was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) instruction method using a random effects model. Results. The initial search identified 652 articles. Of these, 541 were excluded on the basis of the title because they were either a review paper or covered an unrelated topic. The abstracts of the remaining 111 articles were examined further. That review resulted in a sample of 12 articles. We performed a meta-analysis with a random effects model using Open Meta Analyst software. The result of pooled effect size of healing rate was 73.4% (95%CI: 65.3–81.6%). Due to the fact that nonunion lacks potential to heal without further intervention, we suggest that the therapeutic effect of 73.4% from LIPUS is sufficiently effective. As far as we know, there are no trials comparing the therapeutic effectiveness of surgery and LIPUS, so it cannot be said which is more advantageous. However, the healing rate of revision surgery was reported between 68–96%; therefore, our result is within that range. Thus, if surgery is difficult due to complications, we can recommend LIPUS. Conclusions. Meta-analysis of healing rate of nonunion treated by low-intensity pulsed ultrasound is 73.4%, which suggests sufficient therapeutic effectiveness. Furthermore, we can say that LIPUS may provide an alternative treatment for nonunion patients who cannot tolerate revision surgery due to complications

Introduction. Articular cartilage injuries have a limited potential to heal and, over time, may lead to osteoarthritis, an inflammatory and degenerative joint disease associated with activity-related pain, swelling, and impaired mobility. Regeneration and restoration of the joint tissue functionality remain unmet challenges. Stem cell-based tissue engineering is a promising paradigm to treat cartilage degeneration. In this context, hydrogels have emerged as promising biomaterials, due to their biocompatibility, ability to mimic the tissue extracellular matrix and excellent permeability. Different stimulation strategies have been investigated to guarantee proper conditions for mesenchymal stem cell differentiation into chondrocytes, including growth factors, cell-cell interactions, and biomaterials. An interesting tool to facilitate chondrogenesis is external ultrasound stimulation. In particular, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to have a role in regulating the differentiation of adipose mesenchymal stromal cells (ASCs). However, chondrogenic differentiation of ASCs has been never associated to a precisely measured ultrasound dose. In this study, we aimed to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D hydrogel. Materials and Methods. Human adipose mesenchymal stromal cells at 2∗10. 6. cells/mL were embedded in a hydrogel ratio 1:2 (VitroGel RGD®) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm. 2. , duty cycle: 20%, pulse repetition frequency: 1 kHz, stimulation time: 5 min) in order to assess its influence on cell differentiation. Hydrogel-loaded ASCs were cultured and differentiated for 2, 7, 10 and 28 days. At each time point cell viability (Live&Dead), metabolic activity (Alamar Blue), cytotoxicity (LDH), gene expression (COL2, aggrecan, SOX9, and COL1), histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated respect to a non-stimulated control. Results. Histological analysis evidenced a uniform distribution of ASCs both at the periphery and at the center of the hydrogel. Live & Dead test evidenced that the encapsulated ASCs were viable, with no signs of cytotoxicity. We found that LIPUS induced chondrogenesis of ASCs embedded in the hydrogel, as demonstrated by increased expression of COL2, aggrecan and SOX9 genes and proteins, and decreased expression of COL1 respect to the non-stimulated control. Conclusions. These results suggest that the LIPUS treatment could be a valuable tool in cartilage tissue engineering, to push the differentiation of ASCs encapsulated in a 3D hydrogel

Background. Fractures of the metatarsal bones are the most frequent fracture of the foot. Up to 70% involve the fifth metatarsal bone, of which approximately eighty percent are located proximally. Low-intensity pulsed ultrasound (LIPUS) has been shown to be a useful adjunct in the treatment of delayed fractures and non unions. However, there is no study looking at the success rate of LIPUS in fifth metatarsal fracture delayed unions. Objectives. The aim of our study was to investigate the use of LIPUS treatment for delayed union of fifth metatarsal fractures. Study Design & Methods. A retrospective review of patients who were treated with LIPUS following a delayed union of fifth metatarsal fracture was conducted over a three-year period (2013 – 2015). Delayed union was defined as lack of clinical and radiological evidence of union, bony continuity or bone reaction at the fracture site if 3 months has elapsed from the initial injury. Results. There were thirty patients (9 males, 21 females) in our cohort. The average age was 39.3 years. Type 2 fractures made up 43% of our cohort. Twenty-seven (90%) patients went on to progress to union clinically and radiologically following LIPUS treatment. Smoking (p=0.014) and size of fracture gap (p=0.045) were predictive of non-union. Conclusions. This is the first study looking at the use of LIPUS in the treatment of delayed union of fifth metatarsal fractures. We report a success rate of 90%. There is a role in the use of LIPUS in delayed union of fifth metatarsal fractures and can serve as an adjunct prior to consideration of surgery

Introduction. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance healing of fracture and nonunion. Bone morphogenetic protein-7 (BMP-7) has also been reported to promote bone formation. Recently, we demonstrated progenitor cells with osteogenic/chondrogenic differentiation potential existed in human fracture hematoma and nonunion tissue. Hypothesis. We hypothesised the combined application of LIPUS and BMP-7 would cause major effect on osteogenesis of hematoma-derived cells (HCs) and nonunion tissue-derived cells (NCs). Materials & Methods. HCs and NCs were isolated, and cultured. The cells were divided into two groups: (1) BMP-7 group: cells cultured in osteogenic medium (OM), and (2) BMP-7 + LIPUS group: cells cultured in OM with LIPUS treatment. LIPUS (30 mW/cm2, intensity at 1.5 MHz) was given for 20 minutes daily. Osteogenic differentiation potential and proliferation were analysed. Results. ALP activity, the gene expression of osteogenic genes, and mineralisation of HCs and NCs were shown to be higher in BMP-7 + LIPUS group than in BMP-7 group. There was no significant difference in cell proliferation between the two groups. Discussion. Our findings demonstrated the significant effect of LIPUS on the osteogenic differentiation of HCs and NCs induced by BMP-7. This study may provide significant evidence for the clinical combined application of BMP-7 and LIPUS for the treatment of severe bone fracture and nonunion

Objective. To determine what factors affect fracture healing with low-intensity pulsed ultrasound (LIPUS) for delayed unions and nonunions. Patients. A consecutive cohort of 101 delayed unions and 50 nonunions after long bone fractures treated with LIPUS between May 1998 and April 2007. Main Outcome and Measurements. Radiographic determination of osseous bone union within one year after start of LIPUS therapy. Recognition of predictable factors that affect treatment results of LIPUS. Main Results. 1) Delayed union group (n=101): Seventy-five delayed union (74.3%) united without an additional major surgical intervention. Failure of LIPUS therapy was associated with types of nonunion (atrophic vs. hypertrophic, RR 23.72 [95%CI: 1.20–11.5], p<0.01), instability at fracture site (unstable vs. stable, RR 3.03 [1.67–5.49], p<0.001), and maximum fracture gap size not less than 9 mm (RR 3.30 [1.68–6.45]). 2) Nonunion group (n=50): Thirty-four nonunions (68.0%) united without an additional major surgical intervention. Failure of LIPUS therapy was associated with method of fixation (IM nail vs. others, RR 4.50 [95%CI: 1.69–12.00], p<0.001), instability at fracture site (unstable vs. stable, RR 4.56 [2.20–9.43], p<0.0001), and maximum fracture gap size not less than 8 mm (RR 5.09 [1.65–15.67]). Conclusions. LIPUS should be applied as an adjuvant therapy in combination with surgical intervention for an established atrophic nonunion with instability and/or with larger fracture gap

Background: Low-intensity pulsed ultrasound (LIPUS) was shown to accelerate fracture healing with delayed unions and non-unions as well as fresh fractures. Objective: To know the factors which affected clinical results of LIPUS treatment for delayed unions and non-unions. Design: Prospective Cohort Study. Setting: University Hospital. Intervention: 192 delayed or non-unions of long bone or clavicle were treated by LIPUS from May 1998 to April 2007. Background factors (age and gender of patient, history of smoking, personality of each fracture, intervals from injury to application of LIPUS treatment) were prospectively investigated. All patients were followed up at the outpatient clinic and AP and lateral view of radiographs were taken usually every 4 weeks. Main outcome of this study was set as “bone union” and it was defined as cortical continuity in a minimum of three cortices on two views on radiographs and without pain at the fracture site on palpation. Main Outcome Measurement: The overall success rate was 75%, and the success rate of subcutaneous bones were higher (tibia: 81%, radius and ulna: 80%)than that of deeper bone (femur: 64%, humerus 58%). Logistic multi-variant regression showed that the greatest gap size between the main bone fragments (p< 0.0001), instability of a fracture site (p< 0.0001), and the intervals between injury to the application of LIPUS (p< 0.05) were independent predictors for the success of LIPUS treatment for delayed and non-unions. Conclusion: We believe that the greatest gap size of main fragments, instability of a fracture site, and the age of non-union are the factors that affected LIPUS clinical results

Aims: Low-intensity pulsed ultrasound has shown acceleration of bone healing in fresh fractures. The goal of this study is to assess the effect of low-intensity ultrasound on bone healing in established nonunion cases and following osteotomy. Methods: A non randomized trial on 29 cases, located in the tibia, femur, radius/ulna, scaphoid, humerus, metatarsal and clavicle, met the criteria for established nonunions. On average, the post-fracture period prior to the start of ultrasound treatment was 61 weeks. Daily, twenty-minute applications of low-intensity ultrasound at the site of the non-union were performed by the patients at home. In a placebo-controlled, randomized clinical trial double-blinded trial, 97 adults, who had undergone an osteotomy of the lower extremity were randomly allocated an active- or placebo ultrasound device. Every two weeks the patients were examined both clinical and radiological. Results: Twenty-five of the twenty-nine non-union cases (86%) healed in an average treatment time of 22 weeks. Forty-six patients (75 bones) were treated with an active ultrasound device and 44 patients (78 bones) with a placebo device were eligible for analysis. An overall reduction of time to consolidation of 32% was established. A nonunion, which had to be treated operatively, occurred in four cases in the placebo group and in none in the active group. No other prognostic variables were found. Conclusions: Low-intensity ultrasound can stimulate bone healing in osteotomies and nonunions. In patients with a fracture or osteotomy, who are at high risk of developing nonunion, low-intensity ultrasound can be valuable as additional therapy

Introduction: Goal of study to demonstrate that treatment with low-intensity pulsed ultrasound [LIPUS] results in greater increases in bone density and greater reductions in bone gap area as compared to sham control in tibia fractures with delayed union (no progression of healing for at least four months). Methods: Two primary effectiveness variables, change of bone density and gap area during treatment, were selected as surrogates for bone healing. Abbreviated treatment period was maximum that sham treatment could be administered ethically. Both variables measured by blinded central reviewers from CT-scans taken before/after termination of treatment. All adverse events recorded, evaluated. Treatment duration was 16wks. Patients instructed to apply device once daily for 20 minutes. Control devices were visually identical but did not transmit ultrasound waves. Neither patients nor physicians could recognize shams. Results: 101 patients enrolled (51 LIPUS, 50 sham), mean age 42.6 (active) versus 45.1 years (sham). Based on log-transformed data, mean improvement in bone density was 1.34 (90% CI 1.14 to 1.57) times greater for patients randomized to LIPUS compared to sham (p=0.002). A mean reduction in bone gap area also favored LIPUS treatment (p=0.014). Conclusion: Double-blind, intent-to-treat analyses demonstrated statistically significant superior effectiveness for LIPUS device compared to sham in terms of both endpoints over 16wks of treatment. Estimated increase in bone density among patients randomized to LIPUS treatment was 34% greater than among patients randomized to sham. A significantly greater mean reduction in bone gap area after LIPUS treatment was also shown. Evaluation of adverse events showed that ultrasound therapy is safe

Ultrasound has been shown to have positive biological effects, including increased angiogenic, chondrogenic, and osteogenic activities. The aim of our study was to evaluate the evidence available in the scientific literature for the ultrasound treatment for tendon healing. To identify “best evidence” published research a computerized literature search of Medline, Cochrane, PEDro, IME, IBECS and ENFISPO. Keywords used to identify the study population and interventions were: ultrasound, low intensity pulsed ultrasound, physiotherapy, clinical trial, meta-analysis, practice guideline, randomized controlled trial, repair tendon and tendon healing. The scientific evidence of the group of selected documents were measured using the scale described by the US Preventive Task Force. The assignment of the evidence level to each study was evaluated independently by two reviewers without communication among them. To determine inter-rather reliability Kappa index it was used (K) with a value of CI of 95%. The study populations were 39 pertinent recovered documents. The findings suggest that therapeutic ultrasound can increase in collagen synthesis and enhance the maturation of collagen fibrils of repairing tendons. Researchers have reported that therapeutic ultrasound could facilitate tissue recovery and US with dosages between 0.125–3 W/cm2 have been used in the treatment of tendon ruptures reported an improvement in both strength and energy absorption capacity of repairing rabbit or rat tendons with 1-MHz continuous US. Best results were: continuous US at 1 MHz, 0.5w/cm2 starting from day 5 after injury, 20 treatment sessions, 4 mi each session. There is not a general consensus on the choice of parameters for US treatment and the evidence for efficacy of therapeutic. Limits of studies: The time needed to develop such an interface in humans was reported to be much longer than that reported in animal models. Continuous and low-intensity pulsed ultrasound was able to accelerate tendon healing and facilitating earlier physiotherapy

Over the past 100 years, experimental and clinical studies have tried to accelerate fracture healing and to bring ununited fractures to union . Besides advances in surgical management, non-surgical means have been investigated. Mechanical enhancement of fracture healing using controlled micromotion has been used with some success but does not seem to have been applied to nonunions. Electrical stimulation has been found effective in hypertrophic nonunions, but less so in atrophic nonunions and in the presence of a gap; the various devices available have never gained wide acceptance for various reasons. Low-intensity pulsed ultrasound has been found effective to heal non-unions, especially hypertrophic, with a success rate around 85 % . High-energy extracorporeal shock wave therapy (ESWT) has also been found effective in non-union management, but this is still controversial and there is a need for prospective controlled studies. Biological action has also been attempted for a long time. All attempts to stimulate fracture healing using systemic drugs, diet supplementations, vitamins or hormones have been essentially unsuccessful unless when correcting a pre-existing deficiency . More recently, several molecules have demonstrated an osteoinductive capacity in animal studies; human recombinant BMP-2 is currently under investigation in clinical trials. Percutaneous injection of bone marrow into a non-union has also proved of interest, particularly following centrifugation to increase the number of osteoprogenitor cells; current research aims at selecting these cells prior to injection. To conclude, a number of non-surgical means are currently available which may be of interest to accelerate fracture healing or to heal nonunions. Some are totally non-invasive, others are minimally invasive; early results have been encouraging for several of them, but there is still a need for clinical validation using prospective controlled studies. Some of those methods may well turn into alternate solutions to surgery in the future . Cost is currently a limiting factor, as long as it is not reimbursed by national health systems in most countries

INTRODUCTION. Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. 1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix. 2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated. EXPERIMENTAL METHODS. Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain. 3). The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 10. 5. cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm. 2. and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method. 4). and the LDH method. 5). , respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test. RESULTS and DISCUSSION. In the transgenic group, the number of chondrocytes and GAG production were increased by the LIPUS stimulation in 1 day of culture (Fig. 1,2), and the mRNA expression levels of agrrecan (Fig. 3) and Sox 9 were increased in 2 days of culture. However the mRNA expression level of aggrecan was decreased after 3 days of culture. These LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS

Introduction: Low-intensity pulsed ultrasound stimulation (LIPUS) can enhance bone regeneration and callus healing during fracture repair. However, whether a certain phase of the healing process in fracture repair in particular is infiuenced by LIPUS treatment remains unclear. In this investigation, the effect of LIPUS on callus remodeling in a gap healing model was evaluated by bone morphometric analyses using 3-dimensional (3D) quantitative micro computed tomography (μCT) at the healing site, providing information on the temporal sequence of mineralized remodeling events that characterize the gap healing. Materials and Methods: The rabbit osteotomy model with 2-mm gap for the right tibia was immobilized with four pins fixed to an external fixator with double side bars. LIPUS was continued for both the treatment group (n=7/group/time point) and the control group (n=7/group/time point), for 20 min, six times/week, for 4, 6, or 8 weeks. The control group also received a sham inactive transducer under exactly the same condition as the LIPUS group. After the harvested tibia was scanned by μCT, region of interest was set at the callus healing area. It defined as a center of the osteotomy gap with a width of 1 mm. Morphometric parameters used for evaluation were mineralized callus volume (BV, cm. 3. ) and volumetric bone mineral density of mineralized tissue comprising the callus (mBMD, mBMD = BMC/ BV, mgHA/cm. 3. ). The whole ROI was measured and was subdivided into three zones. The periosteal callus zone (External), the medullary callus zone (Endosteal) and the remaining zone was the cortical gap zone (Intercortical). For each zone, BV and mBMD were measured. Data of the μCT evaluations were analyzed using a one-way ANOVA test. Statistically significant difference was set at p < 0.05. Results: In the LIPUS groups, BV for the Endosteal zone was significantly lower for the 8-week group than for the 4-week group. Comparing results at the same time point, the LIPUS group at 8 weeks was significantly higher than that of the control group in the Intercortical zone. As for mBMD, in the LIPUS group, the 8-week group was significantly higher than the 4-week group for Total, External, Internal, and Endosteal zones, respectively. Comparing results at the same time point, mBMD was significantly higher for the LIPUS group at 8 weeks than for the control group in both External and Intercortical zones. Discussion: The most striking finding in our study was that LIPUS accelerated bone formation in the Intercortical zone and callus resorption in the Endosteal zone. This suggests that LIPUS could shorten the time required for remodeling. However, the results of this study do not clarify whether an early phase in callus formation in particular is infiuenced by LIPUS

INTRODUCTION: The process of ligamentization includes the histological and structural remodelling of the tendons graft to ligamentous tissue. There is little information documenting the mechanism of ligamentization process in molecular level. A number of essential genes are involved in this process and their expression can be regulated through complex biochemical pathways. Animal studies have shown that transcutaneous application of low intensity pulsed ultrasound (LiUS) accelerate the tendon and ligament healing process and recent reports have proven the efficacy of the transosseous application of LiUS for both enhancement and monitoring of the bone healing. The purpose of this study is to investigate the effect of transosseous low-intensity pulsed ultrasound (LiUS) during lingamentization process on the healing at tendon graft-bone interface in rabbits, by examining the expression levels of TGF-β1, biglycan and collagen I using semi-quantitive RT-PCR. MATERIALS AND METHODS: Twenty-eight New Zealand rabbits were used in this study. The anterior cruciate ligament was excised and replaced with the long digital extensor. Custom-made ultrasound transducers were implanted onto the bone fragment and along the surface of the bone tunnel at the right knees of the rabbits (study group). The LiUS-treated animals received 200-μsec bursts of 1 MHz sine waves with pulse repetition rates of 1 KHz and average intensity of 30 mW/cm2, for 20 minutes daily, while the left knee received no LiUS (control group). Semi-quantitative RT-PCR was performed from RNA samples representing both study and control groups at 1, 2, 3, 5, 7, 8, 9, 12, 14 and 21 days, using specific primers. RESULTS: Analysis of the RT-PCR products showed that there is significant up-regulation of biglycan and collagen-encoding genes in the study group compared to the control group. In addition, TGFb1-encoding gene exhibits a bimodal profile. In the study group, it represses its mRNA levels from day 1 until day 9 and then the initial expression levels are restored. The control group showed no essential alteration of expression levels for TGFb1. DISCUSSION: Transosseous LiUS treatment affects the expression levels of significant genes like TGF-β1, big-lycan and collagen type I. All the above studied genes mediate important biochemical pathways in lingamentization process and possibly enhance the healing rate of the tendon graft-bone interface in a bone tunnel in rabbits. The present report is supportive of the hypothesis that transosseous application of LiUS enhances tendon graft healing to bone through effects on molecular level. These present findings suggest that indeed ultrasound treatment after joint ligament reconstruction may facilitate earlier rehabilitation

Summary Statement. Low-intensity pulsed ultrasound (LIPUS) enhanced osteogenic differentiation of osteoprogenitor cells derived from mouse induced pluripotent cells (iPSCs) without embryoid body formation. Our findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs. Introduction. iPSCs represent a promising cell source for regenerative medicine such as bone regeneration because of their unlimited self-renewal property and ability of differentiation into all somatic cell types. Recently, we developed an efficient protocol for generating a highly homogeneous population of osteoprogenitor cells from embryonic stem cells by using a direct-plating method without EB formation step. It is well-recognised that LIPUS accelerates the fracture healing. There have been several reports showing that LIPUS stimulates the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. To date, effect of LIPUS on iPSCs remains unknown. In this study, we investigated in vitro effect of LIPUS on osteogenic differentiation of osteoprogenitor cells derived from mouse iPS cells via a direct-plating method. Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates without feeders in MSC medium and FGF-2. Adherent fibroblastic cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. For osteogenic differentiation, DPCs were divided into two groups: (1) control group: DPCs cultured in osteogenic medium (OM) without LIPUS, and (2) LIPUS group: DPCs cultured in OM with LIPUS treatment. LIPUS was given through the bottom of the culture plates for 20 minutes daily. After 14-day culture, osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin red S staining. Expression of osteoblast-related genes, Rnux2 and ALP was also analyzed by real-time PCR. Results. Flow cytometry analysis revealed DPCs had similar characteristics to MSCs. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs, suggesting DPCs lost pluripotency. After 14-day osteogenic induction, ALP activity was shown to be higher in LIPUS group than control group on days 3 and 7. Real-time PCR analysis revealed that in LIPUS group, expression level of Runx2 on day 1 and that of ALP on days 3 and 5 were significantly up-regulated compared to control group. The quantity of calcium deposition measured by Alizarin red staining on day 14 was shown to be higher in LIPUS group than control group. Conclusion. The novel direct-plating method described here provides a significant technical advance over conventional methods of isolating iPSC-induced osteoprogenitor cells by avoiding the embryonic body formation that often leads to heterogeneous, variable, and unpredictable osteogenic differentiation. Our results demonstrated that osteogenic differentiation of osteoprogenitor cells from iPSCs was robustly increased by LIPUS treatment. LIPUS may be a promising enhancer of osteogenesis of iPSCs. These findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs

Introduction: Low-intensity pulsed ultrasound stimulation (LIPUS) reportedly enhances restoration of strength at fracture healing sites. However, evaluation of strength by mechanical testing was limited to only one direction, with either bending or torsion. Quantitative micro computed tomography (μCT) scans allow us to calculate strength-related parameters such as cross-sectional moment (CSM) and cross-sectional moment of inertia (CSMI). Previous studies have performed 2-dimensional (2D) analyses, and 3-dimensional (3D) evaluations have not been described. The purpose of this study was thus to investigate the effects of LIPUS on osteotomy healing using 3D analyses of CSM and CSMI. Materials and Methods: Bilateral, transverse, mid-tibial osteotomies with a 2-mm gap were performed in 42 rabbits. LIPUS was continued for both the treatment group (n=7/group/time point) and the control group (n=7/ group/time point), for 20 min, six times/week, for 4, 6, or 8 weeks. The control group also received a sham inactive transducer under the same condition as the LIPUS group. After the tibia was scanned by μCT, region of interest (ROI) was set at the center of the osteotomy gap with a width of 1 mm. Center of gravity for the ROI and the XYZ coordinate was calculated. An optional line (I) can be drawn in this coordinate. The angle of the Z axis (𝛉) was measured, and also the degree of angle of the X axis (φ) was measured. The 3D CSM [I (φ, 𝛉)] around this line was calculated using the following equation: I (φ, 𝛉) = ∫ r. 2. dV (mm5), where r is the distance of a voxel to the center of gravity (mm) and dV is the area of a voxel (mm3). The axial CSM was defined as CSMx: I (0, 90), CSMy: I (90, 90), whereas the polar CSM was also defined as CSMp: I (any, 0). 3D CSMI weighted by density distribution was calculated using the following equation: I’ (φ, 𝛉) = ∫ r. 2. dm = ∫ ρr. 2. dV (mg.mm. 2. ), ρ is the measured volumetric callus mineral density. Likewise CSMIx, CSMIy and CSMIp were calculated. These data of the μCT evaluations were analyzed using a one-way ANOVA test (p< 0.05). Results: When 3D CSMs at the same time point were compared, values for the LIPUS groups were significantly higher than those for control groups for CSMx at 6 weeks and CSMp at 8 weeks. As for comparison of 3D CSMIs at the same time point, values for the LIPUS groups were significantly higher than those of the control groups for CSMIx, CSMIy, and CSMIp at 6 and 8 weeks. Discussion: Bone healing by 3D CSM and CSMI has not been described before. Our results demonstrate that these bone strength parameters improved with LIPUS during the early phases. However, whether the late phase of callus formation is infiuenced remains unclear

Bone is one of the most highly adaptive tissues in the body, possessing the capability to alter its morphology and function in response to stimuli in its surrounding environment. The ability of bone to sense and convert external mechanical stimuli into a biochemical response, which ultimately alters the phenotype and function of the cell, is described as mechanotransduction. This review aims to describe the fundamental physiology and biomechanisms that occur to induce osteogenic adaptation of a cell following application of a physical stimulus. Considerable developments have been made in recent years in our understanding of how cells orchestrate this complex interplay of processes, and have become the focus of research in osteogenesis. We will discuss current areas of preclinical and clinical research exploring the harnessing of mechanotransductive properties of cells and applying them therapeutically, both in the context of fracture healing and de novo bone formation in situations such as nonunion.

Cite this article: Bone Joint Res 2019;9(1):1–14.

Objectives

The aim of this study was to review the current evidence and future application for the role of diagnostic and therapeutic ultrasound in fracture management.