Acute Inflammatory Response in Osteoporotic Fracture Healing Augmented with Mechanical Stimulation is Regulated In Vivo through the p38-MAPK Pathway.

Low-magnitude high-frequency vibration (LMHFV) has previously been reported to modulate the acute inflammatory response of ovariectomy-induced osteoporotic fracture healing. However, the underlying mechanisms are not clear. In the present study, we investigated the effect of LMHFV on the inflammatory response and the role of the p38 MAPK mechanical signaling pathway in macrophages during the healing process. A closed femoral fracture SD rat model was used. In vivo results showed that LMHFV enhanced activation of the p38 MAPK pathway at the fracture site. The acute inflammatory response, expression of inflammatory cytokines, and callus formation were suppressed in vivo by p38 MAPK inhibition. However, LMHFV did not show direct in vitro enhancement effects on the polarization of RAW264.7 macrophage from the M1 to M2 phenotype, but instead promoted macrophage enlargement and transformation to dendritic monocytes. The present study demonstrated that p38 MAPK modulated the enhancement effects of mechanical stimulation in vivo only. LMHFV may not have exerted its enhancement effects directly on macrophage, but the exact mechanism may have taken a different pathway that requires further investigation in the various subsets of immune cells.

Fibrinolysis as a target to enhance osteoporotic fracture healing by vibration therapy in a metaphyseal fracture model.

AIMS: Fibrinolysis plays a key transition step from haematoma formation to angiogenesis and fracture healing. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical modality proven to enhance fibrinolytic factors. This study investigates the effect of LMHFV on fibrinolysis in a clinically relevant animal model to accelerate osteoporotic fracture healing. METHODS: A total of 144 rats were randomized to four groups: sham control; sham and LMHFV; ovariectomized (OVX); and ovariectomized and LMHFV (OVX-VT). Fibrinolytic potential was evaluated by quantifying fibrin, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) along with healing outcomes at three days, one week, two weeks, and six weeks post-fracture. RESULTS: All rats achieved healing, and x-ray relative radiopacity for OVX-VT was significantly higher compared to OVX at week 2. Martius Scarlet Blue (MSB) staining revealed a significant decrease of fibrin content in the callus in OVX-VT compared with OVX on day 3 (p = 0.020). Mean tPA from muscle was significantly higher for OVX-VT compared to OVX (p = 0.020) on day 3. Mechanical testing revealed the mean energy to failure was significantly higher for OVX-VT at 37.6 N mm (SD 8.4) and 71.9 N mm (SD 30.7) compared with OVX at 5.76 N mm (SD 7.1) (p = 0.010) and 17.7 N mm (SD 11.5) (p = 0.030) at week 2 and week 6, respectively. CONCLUSION: Metaphyseal fracture healing is enhanced by LMHFV, and one of the important molecular pathways it acts on is fibrinolysis. LMHFV is a promising intervention for osteoporotic metaphyseal fracture healing. The improved mechanical properties, acceleration of fracture healing, and safety justify its role into translation to future clinical studies. Cite this article: Bone Joint Res 2021;10(1):41-50.

Efficacy of low-magnitude high-frequency vibration (LMHFV) on musculoskeletal health of participants on wheelchair: a study protocol for a single-blinded randomised controlled study.

BACKGROUND: Osteoporosis is an age-related disease with progressive loss of bone, leading to fragile bone. It is one of the major health issues in older adults and causes medical, social and economic impacts globally. Patients with osteoporosis have high risk of osteoporotic fractures. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical intervention providing whole-body mechanical stimulation. Previous studies showed that LMHFV is beneficial to muscle strength, postural control, balancing ability, new bone formation, spinal bone mineral density (BMD) and blood circulation. During the LMHFV treatment, older adults need to stand upright on the platform for 20 min/day. However, some physically weak elderlies with poor musculoskeletal ability cannot stand for a long period. Therefore, the design of vibration platform is modified for the disabled patients to treat at sitting position and the efficacy of LMHFV on this group of elderlies will be verified. It is hypothesised that new design of LMHFV is beneficial to wheelchair users in terms of vertebral BMD, muscle health and musculoskeletal functions. METHODS: This study is a single-blinded randomised controlled trial to investigate the effect of LMHFV on vertebral BMD, muscle health, balancing ability and functional ability in wheelchair users (mainly on wheelchair for outdoor activities). Healthy elderlies aged 65 years or above with walking difficulties and using wheelchair are eligible. Exclusion criteria are those: (1) who cannot stand and walk independently, (2) who have vibration treatment before, (3) with malignancy, (4) with acute fractures or severe osteoarthritis, (5) with cardiovascular concern such as with pacemaker in situ, (6) with chronic inflammatory conditions known to affect muscle metabolism such as rheumatoid arthritis and (7) with high frequency of physical activities, such as participants who participated in regular exercise five times a week or more. Recruited participants will be randomised to either LMHFV or control group. Participant assigned to LMHFV group will receive LMHFV (35 Hz, 0.3g (g=gravitational acceleration), 20 min/day, at least three times/week) for 6 months. The primary outcome is BMD at the lumbar spine to be assessed by dual-energy X-ray absorptiometry that is clinically recommended for the diagnosis of osteoporosis. All primary and secondary outcome assessments for all groups will be performed in the investigators' institute at baseline and 6 months post treatment. DISCUSSION: This study aims to investigate the effects of LMHFV on wheelchair users. The findings of this study will help to confirm the efficacy of LMHFV on vertebral BMD, muscle health, balancing ability and functional outcomes in wheelchair using elderlies. LMHFV therapy is an intervention strategy that is easy to implement at the community healthcare level or individually at home that has previously been proven to reduce fall risk and muscle strength at the lower limb. The ultimate goal is to improve their bone and muscle quality of wheelchair users, as well as enhancing their quality of life. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT04180267).

Inflammatory response in postmenopausal osteoporotic fracture healing.

A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing. Cite this article: Bone Joint Res 2020;9(7):368-385.

Elastic-band resistance exercise or vibration treatment in combination with hydroxymethylbutyrate (HMB) supplement for management of sarcopenia in older people: a study protocol for a single-blinded randomised controlled trial in Hong Kong.

INTRODUCTION: Sarcopenia is a geriatric syndrome characterised by progressive loss of skeletal muscle mass and function with risks of adverse outcomes and becomes more prevalent due to ageing population. Elastic-band exercise, vibration treatment and hydroxymethylbutyrate (HMB) supplementation were previously proven to have positive effects on the control of sarcopenia. The purpose of this study is to evaluate the effectiveness of elastic-band exercise or vibration treatment with HMB supplementation in managing sarcopenia. Our findings will provide a safe and efficient strategy to mitigate the progression of sarcopenia in older people and contribute to higher quality of life as well as improved long-term health outcomes of elderly people. METHODS AND ANALYSIS: In this single-blinded, randomised controlled trial (RCT), subjects will be screened for sarcopenia based on the Asian Working Group for Sarcopenia (AWGS) definition and 144 sarcopenic subjects aged 65 or above will be recruited. This RCT will have three groups evaluated at two time points to measure changes over 3 months-the control and the groups with combined HMB supplement and elastic-band resistance exercise or vibration treatment. Changes in muscle strength in lower extremity will be the primary outcome. Muscle strength in the upper extremity, gait speed, muscle mass (based on AWGS definition), functional performance in terms of balancing ability and time-up-and-go test and quality of life will be taken as secondary outcomes. In addition, each participant's daily activity will be monitored by a wrist-worn activity tracker. Repeated-measures analysis of variance will be performed to compare within-subject changes between control and treatment groups at two time points of pretreatments and post-treatments. ETHICS AND DISSEMINATION: The procedures have been approved by the Joint CUHK-NTEC Clinical Research Management Office (Ref. CREC 2018.602) and conformed to the Declaration of Helsinki. Results will be disseminated through peer-reviewed publications, conferences and workshops. TRIAL REGISTRATION NUMBER: NCT04028206.

How much do we know about the role of osteocytes in different phases of fracture healing? A systematic review.

BACKGROUND: Although emerging studies have provided evidence that osteocytes are actively involved in fracture healing, there is a general lack of a detailed understanding of the mechanistic pathway, cellular events and expression of markers at different phases of healing. METHODS: This systematic review describes the role of osteocytes in fracture healing from early to late phase. Literature search was performed in PubMed and Embase. Original animal and clinical studies with available English full-text were included. Information was retrieved from the selected studies. RESULTS: A total of 23 articles were selected in this systematic review. Most of the studies investigated changes of various genes and proteins expression patterns related to osteocytes. Several studies have described a constant expression of osteocyte-specific marker genes throughout the fracture healing cascade followed by decline phase with the progress of healing, denoting the important physiological role of the osteocyte and the osteocyte lacuno-canalicular network in fracture healing. The reports of various markers suggested that osteocytes could trigger coordinated bone healing responses from cell death and expression of proinflammatory markers cyclooxygenase-2 and interleukin 6 at early phase of fracture healing. This is followed by the expression of growth factors bone morphogenetic protein-2 and cysteine-rich angiogenic inducer 61 that matched with the neo-angiogenesis, chondrogenesis and callus formation during the intermediate phase. Tightly controlled regulation of osteocyte-specific markers E11/Podoplanin (E11), dentin matrix protein 1 and sclerostin modulate and promote osteogenesis, mineralisation and remodelling across different phases of fracture healing. Stabilised fixation was associated with the finding of higher number of osteocytes with little detectable bone morphogenetic proteins expressions in osteocytes. Sclerostin-antibody treatment was found to result in improvement in bone mass, bone strength and mineralisation. CONCLUSION: To further illustrate the function of osteocytes, additional longitudinal studies with appropriate clinically relevant model to study osteoporotic fractures are crucial. Future investigations on the morphological changes of osteocyte lacuno-canalicular network during healing, osteocyte-mediated signalling molecules in the transforming growth factor-beta-Smad3 pathway, perilacunar remodelling, type of fixation and putative biomarkers to monitor fracture healing are highly desirable to bridge the current gaps of knowledge.The translational potential of this article: This systematic review provides an up-to-date chronological overview and highlights the osteocyte-regulated events at gene, protein, cellular and tissue levels throughout the fracture healing cascade, with the hope of informing and developing potential new therapeutic strategies that could improve the timing and quality of fracture healing in the future.

Vibration and ß-hydroxy-ß-methylbutyrate treatment suppresses intramuscular fat infiltration and adipogenic differentiation in sarcopenic mice.

BACKGROUND: Sarcopenia is an aging-induced deterioration of skeletal muscle mass and function. Low-magnitude high-frequency vibration (LMHFV) was shown to improve muscle functions and ß-hydroxy-ß-methylbutyrate (HMB) to increase muscle mass and strength. Muscle-derived stem cells (MDSCs) are progenitor cells important for muscle regeneration. We hypothesized that LMHFV and HMB could retard sarcopenia by reducing fat infiltration through inhibiting adipogenesis in MDSCs. METHODS: Senescence-accelerated mouse P8 male mice were randomized into control (CTL), HMB, LMHFV (VIB), and combined (COM) groups. Interventions started at age of month 7 and assessed at 1, 2, and 3 months post-intervention by densitometry, histology, and functional tests. In vitro, MDSCs isolated from gastrocnemius of senescence-accelerated mouse P8 mice were characterized, randomized into CTL, VIB, HMB, and COM groups, and assessed by oil red O staining, mRNA, and protein expression. RESULTS: At 2 months post-intervention, percentage lean mass of HMB, VIB, and COM groups were significantly higher than CTL group. Twitch, tetanic, and specific tetanic forces of COM group were higher, while specific twitch force of both VIB and COM groups were higher. Grip strength of HMB, VIB, and COM groups were higher. Histologically, both VIB and COM groups presented lower oil red O area than CTL group. Type I muscle fibre in CTL group was higher than HMB, VIB, and COM groups. MDSC were detected in situ by immunofluorescence stain with stem cell antigen-1 signals confirmed with higher ß-catenin expression in the COM group. The observations were also confirmed in vitro, MDSCs in the HMB, VIB, and COM groups presented lower adipogenesis vs. the CTL group. ß-Catenin mRNA and protein expressions were lower in the CTL group while their relationship was further validated through ß-catenin knock-down approach. CONCLUSIONS: Our results showed that combined LMHFV and HMB interventions enhanced muscle strength and decreased percentage fat mass and intramuscular fat infiltration as compared with either treatment alone. Additive effect of LMHFV and HMB was demonstrated in ß-catenin expression than either treatment in MDSCs and altered cell fate from adipogenesis to myogenesis, leading to inhibition of intramuscular lipid accumulation. Wnt/ß-catenin signalling pathway was found to be the predominant regulatory mechanism through which LMHFV and HMB combined treatment suppressed MDSCs adipogenesis.

Impaired Fracture Healing in Sarco-Osteoporotic Mice Can Be Rescued by Vibration Treatment Through Myostatin Suppression.

Sarcopenia is highly prevalent in fragility fracture patients and is associated with delayed healing. In this study, we investigated the effect of low-magnitude high-frequency vibration (LMHFV) on osteoporotic fracture with sarcopenia and the potential role of myostatin. Osteoporotic fractures created in sarcopenic SAMP8, non-sarcopenic SAMR1 were randomized to control or LMHFV (SAMP8, SAMR1, SAMP8-V, or SAMR1-V) groups. Healing and myostatin expression were evaluated at 2, 4, and 6 weeks post-fracture. In vitro, conditioned-media were collected from myofibers isolated from aged and young SAMP8 or C2C12 myoblasts with or without LMHFV. Osteoblastic MC3T3-E1 under osteogenic differentiation were treated with plain or conditioned-medium (±myostatin propeptide). LMHFV significantly enhanced callus formation was in non-sarcopenic SAMR1 mice; but the enhancement effect was not significant in SAMP8 mice at week 2. Myostatin expressions in callus and biceps femoris of SAMP8 group were significantly higher all groups with significant negative correlation with callus size (R2 = 0.7256; p = 0.0004). Mechanical properties (week 4) and callus remodeling (week 6) were inferior in SAMP8 versus SAMR1 and were significantly enhanced by LMHFV. Alkaline Phosphatase (ALP) and Runx2 expression of MC3T3-E1 was lower in aged myofiber compared with young, but upregulated by LMHFV or myostatin inhibition; also confirmed with C2C12. LMHFV enhanced early callus formation, microarchitecture, callus remodeling and mechanical properties of fracture healing in both SAMP8 and SAMR1; however, more effective in non-sarcopenic SAMR1 mice. Impaired fracture healing in sarcopenic SAMP8 mice is attributed by elevated myostatin expression in callus and muscle, which correlated negatively with callus formation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:277-287, 2020.