Simon Kwoon-Ho Chow

Low-intensity pulsed ultrasound accelerated callus formation, angiogenesis and callus remodeling in osteoporotic fracture healing.

Osteoporotic fracture is a critical medico-social challenge leading to burdens in health care costs and hospital bed stays. Low-intensity pulsed ultrasound (LIPUS) was reported to accelerate normal fracture; however, its effect on osteoporotic fracture has not been previously addressed. We hypothesize that LIPUS can accelerate osteoporotic fracture healing and up-regulate the expression in the osteogenesis-, remodeling- and angiogenesis-related genes. Ovariectomy-induced osteoporotic fracture rat model was used to investigate the effects of LIPUS. Fractured rats were assigned to LIPUS or control group and healing was assessed by gene expression quantification, radiographic callus morphometry and histomorphometry. In the LIPUS group, Col-1 and bone morphogenetic protein-2 were up-regulated at earlier time points of week 2 to week 4 post-fracture; vascular endothelial growth factor was found to be up-regulated at week 4 to week 8; osteoprotegerin was up-regulated at week 2 post-fracture, followed by the surge of RANKL expression. Callus width and area measurements showed higher callus formation at weeks 2-4 in the LIPUS group and more rapid drop at weeks 6-8. Histomorphometry showed enhanced endochondral ossification in the callus at weeks 2-4, and lower at week 8. We conclude that LIPUS can accelerate osteoporotic fracture healing by enhancing callus formation, angiogenesis and callus remodeling.

Low Intensity Pulsed Ultrasound Enhanced Mesenchymal Stem Cell Recruitment through Stromal Derived Factor-1 Signaling in Fracture Healing

Low intensity pulsed ultrasound (LIPUS) has been proven effective in promoting fracture healing but the underlying mechanisms are not fully depicted. We examined the effect of LIPUS on the recruitment of mesenchymal stem cells (MSCs) and the pivotal role of stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) pathway in response to LIPUS stimulation, which are essential factors in bone fracture healing. For in vitro study, isolated rat MSCs were divided into control or LIPUS group. LIPUS treatment was given 20 minutes/day at 37°C for 3 days. Control group received sham LIPUS treatment. After treatment, intracellular CXCR4 mRNA, SDF-1 mRNA and secreted SDF-1 protein levels were quantified, and MSCs migration was evaluated with or without blocking SDF-1/CXCR4 pathway by AMD3100. For in vivo study, fractured 8-week-old young rats received intracardiac administration of MSCs were assigned to LIPUS treatment, LIPUS+AMD3100 treatment or vehicle control group. The migration of transplanted MSC to the fracture site was investigated by ex vivo fluorescent imaging. SDF-1 protein levels at fracture site and in serum were examined. Fracture healing parameters, including callus morphology, micro-architecture of the callus and biomechanical properties of the healing bone were investigated. The in vitro results showed that LIPUS upregulated SDF-1 and CXCR4 expressions in MSCs, and elevated SDF-1 protein level in the conditioned medium. MSCs migration was promoted by LIPUS and partially inhibited by AMD3100. In vivo study demonstrated that LIPUS promoted MSCs migration to the fracture site, which was associated with an increase of local and serum SDF-1 level, the changes in callus formation, and the improvement of callus microarchitecture and mechanical properties; whereas the blockade of SDF-1/CXCR4 signaling attenuated the LIPUS effects on the fractured bones. These results suggested SDF-1 mediated MSCs migration might be one of the crucial mechanisms through which LIPUS exerted influence on fracture healing.

The citrus flavonone hesperetin prevents letrozole-induced bone loss in a mouse model of breast cancer.

Aromatase is a key enzyme in estrogen synthesis, and aromatase inhibitors (AIs) have been developed for treating estrogen-responsive breast cancer. Because of its nondiscriminatory inhibition of estrogen synthesis, patients treated with AIs also contract diseases typically associated with estrogen deficiency, such as bone deterioration. Our laboratory found that the citrus flavonone hesperetin could inhibit aromatase, and the selective estrogen receptor modulator nature of flavonoid might counteract the undesirable effect of AIs. In the present study, we employed an established postmenopausal model for breast carcinogenesis to examine the drug interaction between hesperetin and letrozole, one of the AIs. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells (MCF-7aro). Hesperetin was administered in the diet at 5000 ppm, and letrozole was injected sc at different doses. Results showed that either hesperetin or letrozole could reduce plasma estrogen level and inhibit tumor growth. Most importantly, the letrozole-induced bone loss measured as bone volume fraction was reversed by hesperetin without compromising on the deterrence of MCF-7aro tumor growth. Taken together, the present study suggested that hesperetin could be a potential cotherapeutic agent to AI.

Muscle mass, structural and functional investigations of senescence-accelerated mouse P8 (SAMP8).

Sarcopenia is an age-related systemic syndrome with progressive deterioration in skeletal muscle functions and loss in mass. Although the senescence-accelerated mouse P8 (SAMP8) was reported valid for muscular ageing research, there was no report on the details such as sarcopenia onset time. Therefore, this study was to investigate the change of muscle mass, structure and functions during the development of sarcopenia. Besides the average life span, muscle mass, structural and functional measurements were also studied. Male SAMP8 animals were examined at month 6, 7, 8, 9, and 10, in which the right gastrocnemius was isolated and tested for ex vivo contractile properties and fatigability while the contralateral one was harvested for muscle fiber cross-sectional area (FCSA) and typing assessments. Results showed that the peak of muscle mass appeared at month 7 and the onset of contractility decline was observed from month 8. Compared with month 8, most of the functional parameters at month 10 decreased significantly. Structurally, muscle fiber type IIA made up the largest proportion of the gastrocnemius, and the fiber size was found to peak at month 8. Based on the altered muscle mass, structural and functional outcomes, it was concluded that the onset of sarcopenia in SAMP8 animals was at month 8. SAMP8 animals at month 8 should be at pre-sarcopenia stage while month 10 at sarcopenia stage. It is confirmed that SAMP8 mouse can be used in sarcopenia research with established time line in this study.

Coadministrating Luteolin Minimizes the Side Effects of the Aromatase Inhibitor Letrozole

Aromatase inhibitors (AIs) have been used as adjuvant therapeutic agents for breast cancer. Their adverse side effect on blood lipid is well documented. Some natural compounds have been shown to be potential AIs. In the present study, we compared the efficacy of the flavonoid luteolin to the clinically approved AI letrozole (Femara; Novartis Pharmaceuticals, East Hanover, NJ) in a cell and a mouse model. In the in vitro experimental results for aromatase inhibition, the Ki values of luteolin and letrozole were estimated to be 2.44 µM and 0.41 nM, respectively. Both letrozole and luteolin appeared to be competitive inhibitors. Subsequently, an animal model was used for the comparison. Aromatase-expressing MCF-7 cells were transplanted into ovariectomized athymic mice. Luteolin was given by mouth at 5, 20, and 50 mg/kg, whereas letrozole was administered by intravenous injection. Similar to letrozole, luteolin administration reduced plasma estrogen concentrations and suppressed the xenograft proliferation. The regulation of cell cycle and apoptotic proteins—such as a decrease in the expression of Bcl-xL, cyclin-A/D1/E, CDK2/4, and increase in that of Bax—was about the same in both treatments. The most significant disparity was on blood lipids. In contrast to letrozole, luteolin increased fasting plasma high-density lipoprotein concentrations and produced a desirable blood lipid profile. These results suggested that the flavonoid could be a coadjuvant therapeutic agent without impairing the action of AIs.

Three-dimensional high frequency power Doppler ultrasonography for the assessment of microvasculature during fracture healing in a rat model

We aimed to establish a novel approach with 3D high frequency power Doppler ultrasonography (3D‐HF‐PDU) to assess microvasculature at the fracture site in rat femurs by comparing with microCT‐based microangiography. Twenty‐four 9‐month‐old ovariectomized (OVX) osteoporotic rats and age‐matched sham‐ovariectomized (Sham) rats were used for establishing closed fracture models on right femora. At 2, 4, and 8 weeks post‐operatively, four rats in each group underwent in vivo 3D‐HF‐PDU scanning for evaluation of vascularization and blood flow at the fracture site. Then the fractured femora were harvested for ex vivo microangiography, and neovasculatures within the callus were reconstructed for vascular volume analysis. Correlation between the vascular volumes of the two methodologies was examined. Both 3D‐HF‐PDU and microangiography showed a decline of vascular volume at the fracture site from 2 to 8 weeks and a significantly larger volume in the Sham group than the OVX group. A significant linear positive correlation (r = 0.87, p < 0.001) was detected between the volumes measured by the two methodologies. Osteoporotic rats had a diminished angiogenic response and lower blood perfusion than Shams. We believe 3D‐HF‐PDU is feasible and reproducible for in vivo assessment of microvasculature during femoral fracture healing in rats.

Low magnitude high frequency vibration accelerated cartilage degeneration but improved epiphyseal bone formation in anterior cruciate ligament transect induced osteoarthritis rat model

OBJECTIVES To evaluate the effects of low-magnitude high-frequency vibration (LMHFV) on degenerated articular cartilage and subchondral bone in anterior cruciate ligament transection (ACLT) induced osteoarthritis (OA) rat model. METHODS 6 months old female Sprague-Dawley rats received ACLT on right knee and randomly divided into treatment and control groups. OA developed 12 weeks after surgery. LMHFV (35 Hz, 0.3 g) treatment was given 20 min/day and 5 days/week. After 6, 12 and 18 weeks, six rats of each group were sacrificed at each time point and the right knees were harvested. OA grading score, distal femur cartilage volume (CV), subchondral bone morphology, elastic modulus of cartilage and functional changes between groups were analyzed. RESULTS Increased cartilage degradation (higher OA grading score) and worse functional results (lower duty cycle, regular index and higher limb idleness index) were observed after LMHFV treatment (P = 0.011, 0.020, 0.012 and 0.005, respectively). CV increased after LMHFV treatment (P = 0.019). Subchondral bone density increased with OA progress (P < 0.01). Increased BV/TV, Tb.N and decreased Tb.Sp were observed in distal femur epiphysis in LMHFV treatment group (P = 0.006, 0.018 and 0.011, respectively). CONCLUSION LMHFV accelerated cartilage degeneration and caused further functional deterioration of OA affected limb in ACLT-induced OA rat model. In contrast, LMHFV promoted bone formation in OA affected distal femur epiphysis, but did not reverse OA progression.

Bone formation and degradation behavior of nanocrystalline hydroxyapatite with or without collagen-type 1 in osteoporotic bone defects - an experimental study in osteoporotic goats.

The intention of the current work is to assess new bone formation and degradation behavior of nanocrystalline hydroxyapatite with (HA/col-1) or without collagen-type I (HA) in osteoporotic metaphyseal bone defects in goats. After ovariectomy and special low-calcium diet for three months, 3 drill hole defects in the vertebrae of L3, L4, L5, 4 drill hole defects in the right and left iliac crest and 1 drill hole defect at the distal femur were created in three Chinese mountain goats with a total of 24 defects. The defects were either filled with one of the biomaterials or left empty (empty defect control group). After 42 days, the animals were euthanized and the samples were assessed for new bone formation using high-resolution peripheral quantitative computed tomography (HR-pQCT) and histomorphometry with 2 regions of interest. Detail histology, enzymehistochemistry and immunohistochemistry as well as connexin-43 in situ hybridization and transmission electron microscopy were carried out for evaluation of degradation behavior of the materials and cellular responses of the surrounding tissue in respect to the implants. HR-pQCT showed the highest BV/TV ratio (p = 0.008) and smallest trabecular spacing (p = 0.005) for HA compared to the other groups in the region of interest at the interface with 1mm distance to the initially created defect. The HA/col-1 yielded the highest connectivity density (Conn.D) (p = 0.034) and the highest number of trabeculae (Tb.N) (p = 0.002) compared to the HA and the control group. Histomorphometric analysis for the core region of the initially created defect revealed a statistically higher new bone formation in the HA (p = 0.001) and HA/col-1 group (p = 0.001) compared to the empty defect group including all defect sites. This result was confirmed for site specific analysis with significant higher new bone formation for the HA group for vertebral defects compared to the empty defect group (p = 0.029). For the interface region, no statistically significant differences were found between the three groups (p = 0.08). Histology revealed a good biocompatibility without inflammatory reaction for the HA- and HA/col-1 implants with a higher fragmentation of the HA-implant compared to the HA/col-1 biomaterial and formation of new bone in the region between the biomaterial fragments by osteoblasts. Fragmentation was shown by transmission electron microscopy to be caused by multinuclear osteoclast-like cells with degradation of the implant via intracellular incorporation of degraded implant material particles. In conclusion, both nanoparticulate HA with and without collagen type-1 showed better new bone formation compared to untreated drill hole defects in metaphyseal regions of this osteoporotic Chinese mountain goat model with good biocompatibility.

Ultrasound as a stimulus for musculoskeletal disorders

Summary Ultrasound is an inaudible form of acoustic sound wave at 20 kHz or above that is widely used in the medical field with applications including medical imaging and therapeutic stimulation. In therapeutic ultrasound, low-intensity pulsed ultrasound (LIPUS) is the most widely used and studied form that generally uses acoustic waves at an intensity of 30 mW/cm2, with 200 ms pulses and 1.5 MHz. In orthopaedic applications, it is used as a biophysical stimulus for musculoskeletal tissue repair to enhance tissue regeneration. LIPUS has been shown to enhance fracture healing by shortening the time to heal and reestablishment of mechanical properties through enhancing different phases of the healing process, including the inflammatory phase, callus formation, and callus remodelling phase. Reports from in vitro studies reveal insights in the mechanism through which acoustic stimulations activate cell surface integrins that, in turn, activate various mechanical transduction pathways including FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), PI3K, and Akt. It is then followed by the production of cyclooxygenase 2 and prostaglandin E2 to stimulate further downstream angiogenic, osteogenic, and chondrogenic cytokines, explaining the different enhancements observed in animal and clinical studies. Furthermore, LIPUS has also been shown to have remarkable effects on mesenchymal stem cells (MSCs) in musculoskeletal injuries and tissue regeneration. The recruitment of MSCs to injury sites by LIPUS requires the SDF-1 (stromal cell derived factor-1)/CXCR-4 signalling axis. MSCs would then differentiate differently, and this is regulated by the presence of different cytokines, which determines their fates. Other musculoskeletal applications including bone–tendon junction healing, and distraction osteogenesis are also explored, and the results are promising. However, the use of LIPUS is controversial in treating osteoporosis, with negative findings in clinical settings, which may be attributable to the absence of an injury entry point for the acoustic signal to propagate, strong attenuation effect of cortical bone and the insufficient intensity for penetration, whereas in some animal studies it has proven effective.

The effect of whole body vibration on fracture healing - a systematic review.

This systematic review examines the efficacy and safety of whole body vibration (WBV) on fracture healing. A systematic literature search was conducted with relevant keywords in PubMed and Embase, independently, by two reviewers. Original animal and clinical studies about WBV effects on fracture healing with available full-text and written in English were included. Information was extracted from the included studies for review. In total, 19 articles about pre-clinical studies were selected. Various vibration regimes are reported; of those, the frequencies of 35 Hz and 50 Hz show better results than others. Most of the studies show positive effects on fracture healing after vibration treatment and the responses to vibration are better in ovariectomised (OVX) animals than non-OVX ones. However, several studies provide insufficient evidence to support an improvement of fracture healing after vibration and one study even reports disruption of fracture healing after vibration. In three studies, vibration results in positive effects on angiogenesis at the fracture site and surrounding muscles during fracture healing. No serious complications or side effects of vibration are found in these studies. WBV is suggested to be beneficial in improving fracture healing in animals without safety problem reported. In order to apply vibration on fractured patients, more well-designed randomised controlled clinical trials are needed to examine its efficacy, regimes and safety.