The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures

We investigated the effects of low-intensity pulsed ultrasound on distraction osteogenesis in a rabbit model. Callotasis of the right tibia was performed in 70 male Japanese white rabbits using mini-external fixators. In the first part of the study in 64 animals using normal distraction (waiting period seven days; distraction rate 0.5 mm/12 hours; distraction period ten days), we evaluated the distraction site by radiography, measurement of the bone mineral density (BMD), mechanical testing, and histology. In the second part in six rabbits using fast distraction (waiting period 0 days; distraction rate 1.5 mm/12 hours; distraction period seven days) the site was evaluated radiologically. Half of the animals (35) had received ultrasound to their right leg (30mW/cm. 2. ) for 20 minutes daily after ceasing distraction (ultrasound group), while rigid fixation only was maintained in the other half (control group). With normal distraction, the hard callus area, as shown by radiography, the BMD, and the findings on mechanical testing, were significantly greater in those receiving ultrasound than in the control group. Histological analysis showed no tissue damage attributable to exposure to ultrasound. With fast distraction, immature bone regeneration was observed radiologically in the control group, while bone maturation was achieved in the ultrasound group. We conclude that ultrasound can accelerate bone maturation in distraction osteogenesis in rabbits, even in states of poor callotasis

This study was designed to test the hypothesis that the sensory innervation of bone might play an important role in sensing and responding to low-intensity pulsed ultrasound and explain its effect in promoting fracture healing. In 112 rats a standardised mid-shaft tibial fracture was created, supported with an intramedullary needle and divided into four groups of 28. These either had a sciatic neurectomy or a patellar tendon resection as control, and received the ultrasound or not as a sham treatment. Fracture union, callus mineralisation and remodelling were assessed using plain radiography, peripheral quantitative computed tomography and histomorphology. Daily ultrasound treatment significantly increased the rate of union and the volumetric bone mineral density in the fracture callus in the neurally intact rats (p = 0.025), but this stimulating effect was absent in the rats with sciatic neurectomy. Histomorphology demonstrated faster maturation of the callus in the group treated with ultrasound when compared with the control group. The results supported the hypothesis that intact innervation plays an important role in allowing low-intensity pulsed ultrasound to promote fracture healing

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

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