Study on Synergistic Effects of Nanohydroxyapatite/High-Viscosity Carboxymethyl Cellulose Scaffolds Stimulated by LIPUS for Bone Defect Repair of Rats.

Despite the self-healing capacity of bone, the regeneration of critical-size bone defects remains a major clinical challenge. In this study, nanohydroxyapatite (nHAP)/high-viscosity carboxymethyl cellulose (hvCMC, 6500 mPa·s) scaffolds and low-intensity pulsed ultrasound (HA-LIPUS) were employed to repair bone defects. First, hvCMC was prepared from ramie fiber, and the degree of substitution (DS), purity, and content of NaCl of hvCMC samples were 0.91, 99.93, and 0.017%, respectively. Besides, toxic metal contents were below the permissible limits for pharmaceutically used materials. Our results demonstrated that the hvCMC is suitable for pharmaceutical use. Second, nHAP and hvCMC were employed to prepare scaffolds by freeze-drying. The results indicated that the scaffolds were porous, and the porosity was 35.63 ± 3.52%. Subsequently, the rats were divided into four groups (n = 8) randomly: normal control (NC), bone defect (BD), bone defect treated with nHAP/hvCMC scaffolds (HA), and bone defect treated with nHAP/hvCMC scaffolds and stimulated by LIPUS (HA-LIPUS). After drilling surgery, nHAP/hvCMC scaffolds were implanted in the defect region of HA and HA-LIPUS rats. Meanwhile, HA-LIPUS rats were treated by LIPUS (1.5 MHz, 80 mW cm-2) irradiation for 2 weeks. Compared with BD rats, the maximum load and bone mineral density of HA-LIPUS rats were increased by 20.85 and 51.97%, respectively. The gene and protein results indicated that nHAP/hvCMC scaffolds and LIPUS promoted the bone defect repair and regeneration of rats significantly by activating Wnt/ß-catenin and inhibiting OPG/RANKL signaling pathways. Overall, compared with BD rats, nHAP/hvCMC scaffolds and LIPUS promoted bone defect repair significantly. Furthermore, the research results also indicated that there are synergistic effects for bone defect repair between the nHAP/hvCMC scaffolds and LIPUS.

Low-intensity pulsed ultrasound mitigates cognitive impairment by inhibiting muscle atrophy in hindlimb unloaded mice.

Microgravity leads to muscle loss, usually accompanied by cognitive impairment. Muscle reduction was associated with the decline of cognitive ability. Our previous studies showed that low-intensity pulsed ultrasound (LIPUS) promoted muscle hypertrophy and prevented muscle atrophy. This study aims to verify whether LIPUS can improve cognitive impairment by preventing muscle atrophy in hindlimb unloaded mice. In this study, mice were randomly divided into normal control (NC), hindlimb unloading (HU), hindlimb unloading + LIPUS (HU+LIPUS) groups. The mice in the HU+LIPUS group received a 30 mW/cm2 LIPUS irradiation on gastrocnemius for 20 min/d. After 21 days, LIPUS significantly prevented the decrease in muscle mass and strength caused by tail suspension. The HU+LIPUS mice showed an enhanced desire to explore unfamiliar environments and their spatial learning and memory abilities, enabling them to quickly identify differences between different objects, as well as their social discrimination abilities. MSTN is a negative regulator of muscle growth and also plays a role in regulating cognition. LIPUS significantly inhibited MSTN expression in skeletal muscle and serum and its receptor ActRIIB expression in brain, upregulated AKT and BDNF expression in brain. Taken together, LIPUS may improve the cognitive dysfunction in hindlimb unloaded rats by inhibiting muscle atrophy through MSTN/AKT/BDNF pathway.

Low-intensity pulsed ultrasound enhances the positive effects of high-intensity treadmill exercise on bone in rats.

INTRODUCTION: Moderate exercise benefits bone health, but excessive loading leads to bone fatigue and a decline in mechanical properties. Low-intensity pulsed ultrasound (LIPUS) can stimulate bone formation. The purpose of this study was to explore whether LIPUS could augment the skeletal benefits of high-intensity exercise. MATERIALS AND METHODS: MC3T3-E1 osteoblasts were treated with LIPUS at 80 mW/cm2 or 30 mW/cm2 for 20 min/day. Forty rats were divided into sham treatment normal control (Sham-NC), sham treatment high-intensity exercise (Sham-HIE), 80 mW/cm2 LIPUS (LIPUS80), and high-intensity exercise combined with 80 mW/cm2 LIPUS (LIPUS80-HIE). The rats in HIE group were subjected to 30 m/min slope treadmill exercise for 90 min/day, 6 days/week for 12 weeks. The LIPUS80-HIE rats were irradiated with LIPUS (1 MHz, 80 mW/cm2) for 20 min/day at bilateral hind limb after exercise. RESULTS: LIPUS significantly accelerated the proliferation, differentiation, mineralization, and migration of MC3T3-E1 cells. Compared to 30 mW/cm2 LIPUS, 80 mW/cm2 LIPUS got better promotion effect. 12 weeks of high-intensity exercise significantly reduced the muscle force, which was significantly reversed by LIPUS. Compared with the Sham-NC group, Sham-HIE group significantly optimized bone microstructure and enhanced mechanical properties of femur, and LIPUS80-HIE further enhanced the improvement effect on bone. The mechanisms may be related to activate Wnt/ß-catenin signal pathway and then up-regulate the protein expression of Runx2 and VEGF, the key factors of osteogenesis and angiogenesis. CONCLUSION: LIPUS could augment the skeletal benefits of high-intensity exercise through Wnt/ß-catenin signal pathway.

Low intensity pulsed ultrasound reduces liver inflammation caused by fatigue exercise.

Low-intensity pulsed ultrasound (LIPUS) has been shown to have many benefits, such as inhibiting inflammation, stimulating cell proliferation and differentiation, promoting angiogenesis, and so on. So, can exercise fatigue induced liver inflammation be effectively relieved by LIPUS? If possible, what is the possible mechanism? This study first investigated the effect of different intensity exercise on liver inflammation. Rats were divided into three groups: normal control group, exercise fatigue group, and aerobic exercise group. The results showed that aerobic exercise increases both anti-inflammatory factors and pro-inflammatory factors, while fatigue exercise decreases anti-inflammatory factors and increases pro-inflammatory factors, leading to severe liver injury and fibrosis. Then, we investigated the therapeutic effect of LIPUS on liver inflammation caused by exercise fatigue. Starting from the 6th week, the liver was irradiated with LIPUS of 80 mW/cm2 for 20 min/d after daily exercise for 7 weeks. The results showed that LIPUS significantly decreased liver injury and fibrosis, significantly up-regulated the expression of STAT6, IL-13, and its receptors IL-13Rα1, and down regulated the expression of NF-κBp65 in exercise fatigue rats. These results indicate that LIPUS can reduce fatigue-induced liver inflammation, and the mechanism is related to the regulation of the IL-13/STAT6/NF-κBp65 pathway.

Study on synergistic effects of carboxymethyl cellulose and LIPUS for bone tissue engineering.

In this research, the pharmaceutical used carboxymethyl cellulose (CMC) was successfully synthesized by ramie fiber and sodium monochloroacetate. Meanwhile, this study focused on the proliferation and differentiation of osteoblast which was stimulated by ramie based CMC. Additionally, the synergistic effects between CMC and Low-intensity pulsed ultrasound (LIPUS) for the proliferation and differentiation of osteoblast was also evaluated in this study. The experimental results demonstrated that ramie based CMC were nontoxic and cytocompatible in MC3T3-E1 cells culture; the dose of 300 µg L-1 CMC applied in this work displayed better efficiency of promoting the osteogenic proliferation and differentiation. Meanwhile, the results were also exhibited that the combination of CMC and LIPUS (1.5 MHz, 80 mW cm-2) has a significant proliferation and differentiation promoting effects compared with the single factor intervention. This study provides a new pathway for bio-polymeric materials in the repair and regeneration of the bone tissues.

MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats

Weight-bearing training, as one of resistance exercises, is beneficial to bone health. Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. Animals lacking MSTN show increased bone mineral density (BMD). The aim of this study was to investigate the preventive effect of weight-bearing training on bone loss in ovariectomized rats and whether it was related to MSTN. In this study, the rats were randomly assigned to three group: Sham-ovariectomized (Sham), ovariectomized (OVX), ovariectomized and weight-bearing training (OWT). The rats in the OWT group ran at 20-m/min bearing with 35% of their body weight for 6 days/week. After 10 weeks, compared with the OVX group, weight-bearing training increased the BMD of total femur and trabecular bone by 8.13% and 57.44%, respectively. The OVX-induced destruction of bone microarchitecture including the thickness and number of trabeculae and bone volume fraction was all significantly improved (9.26%, 47.68%, 63.03%) in the OWT group. The OVX-induced degradation of bone mechanical properties was significantly enhanced in the OWT group (maximum load increased by 35.46%, stiffness increased by 89.19%, energy absorption increased by 53.4%; elastic modulus increased by 26.3%). Ten-week weight-bearing training also significantly upregulated the mRNA and protein expression of Wnt1 and β-catenin, which is crucial in bone development. Compared with the Sham group, MSTN in serum and muscle increased in the OVX group, but it decreased in the OWT group compared with the OVX group. Its receptor ActRIIB and downstream molecules Smad2/3 in the OVX group were downregulated in bone by weight-bearing training. The results indicated that MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats. (AU)

MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats.

Weight-bearing training, as one of resistance exercises, is beneficial to bone health. Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. Animals lacking MSTN show increased bone mineral density (BMD). The aim of this study was to investigate the preventive effect of weight-bearing training on bone loss in ovariectomized rats and whether it was related to MSTN. In this study, the rats were randomly assigned to three group: Sham-ovariectomized (Sham), ovariectomized (OVX), ovariectomized and weight-bearing training (OWT). The rats in the OWT group ran at 20-m/min bearing with 35% of their body weight for 6 days/week. After 10 weeks, compared with the OVX group, weight-bearing training increased the BMD of total femur and trabecular bone by 8.13% and 57.44%, respectively. The OVX-induced destruction of bone microarchitecture including the thickness and number of trabeculae and bone volume fraction was all significantly improved (9.26%, 47.68%, 63.03%) in the OWT group. The OVX-induced degradation of bone mechanical properties was significantly enhanced in the OWT group (maximum load increased by 35.46%, stiffness increased by 89.19%, energy absorption increased by 53.4%; elastic modulus increased by 26.3%). Ten-week weight-bearing training also significantly upregulated the mRNA and protein expression of Wnt1 and ß-catenin, which is crucial in bone development. Compared with the Sham group, MSTN in serum and muscle increased in the OVX group, but it decreased in the OWT group compared with the OVX group. Its receptor ActRIIB and downstream molecules Smad2/3 in the OVX group were downregulated in bone by weight-bearing training. The results indicated that MSTN is an important myokine for weight-bearing training to attenuate bone loss in ovariectomized rats.

Molecular and Metabolic Mechanism of Low-Intensity Pulsed Ultrasound Improving Muscle Atrophy in Hindlimb Unloading Rats.

Low-intensity pulsed ultrasound (LIPUS) has been proved to promote the proliferation of myoblast C2C12. However, whether LIPUS can effectively prevent muscle atrophy has not been clarified, and if so, what is the possible mechanism. The aim of this study is to evaluate the effects of LIPUS on muscle atrophy in hindlimb unloading rats, and explore the mechanisms. The rats were randomly divided into four groups: normal control group (NC), hindlimb unloading group (UL), hindlimb unloading plus 30 mW/cm2 LIPUS irradiation group (UL + 30 mW/cm2), hindlimb unloading plus 80 mW/cm2 LIPUS irradiation group (UL + 80 mW/cm2). The tails of rats in hindlimb unloading group were suspended for 28 days. The rats in the LIPUS treated group were simultaneously irradiated with LIPUS on gastrocnemius muscle in both lower legs at the sound intensity of 30 mW/cm2 or 80 mW/cm2 for 20 min/d for 28 days. C2C12 cells were exposed to LIPUS at 30 or 80 mW/cm2 for 5 days. The results showed that LIPUS significantly promoted the proliferation and differentiation of myoblast C2C12, and prevented the decrease of cross-sectional area of muscle fiber and gastrocnemius mass in hindlimb unloading rats. LIPUS also significantly down regulated the expression of MSTN and its receptors ActRIIB, and up-regulated the expression of Akt and mTOR in gastrocnemius muscle of hindlimb unloading rats. In addition, three metabolic pathways (phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism) were selected as important metabolic pathways for hindlimb unloading effect. However, LIPUS promoted the stability of alanine, aspartate and glutamate metabolism pathway. These results suggest that the key mechanism of LIPUS in preventing muscle atrophy induced by hindlimb unloading may be related to promoting protein synthesis through MSTN/Akt/mTOR signaling pathway and stabilizing alanine, aspartate and glutamate metabolism.

Weight-bearing exercise prevents skeletal muscle atrophy in ovariectomized rats.

Skeletal muscle atrophy (SMA) is a dominant symptom induced by estrogen deficiency which can lead to severe health problems of postmenopausal women. Furthermore, estrogen deficiency has severely compromised the maintenance of muscle stem cells as well as impairs self-renewal and differentiation into muscle fibers. Resistance training is commonly considered as a positive and useful intervention in accelerating the rate of muscle growth. As one of the resistance training, whether the weight-bearing exercise can alleviate SMA induced by estrogen deficiency has not been investigated. The rats were divided into 3 groups randomly: sham group, ovariectomized (OVX) group, and weight-bearing exercise (WBE) therapeutic group. The weight that rats were loaded was 35% of their body weight, and the rats were trained by treadmill training (5° slope, 20 m/min, 30 min/day, 6 days/week) for 8 weeks. After training, the quality and strength of skeletal muscle of the WBE rats were improved; meanwhile, the cross-sectional areas of the skeletal muscle were also increased. Moreover, the WBE activated Akt significantly, upregulated the expression of mTOR, and downregulated the expression of MSTN and its receptor ActRIIB and FoxO1, respectively. The SMA phenomena of rats which induced by estrogen deficiency were prevented effectively via WBE, and the MSTN/Akt/mTOR and FoxO1 signaling pathway may be the predominant way in this improvement.

MSTN is a key mediator for low-intensity pulsed ultrasound preventing bone loss in hindlimb-suspended rats.

Low-intensity pulsed ultrasound (LIPUS) has been used to accelerate bone fracture healing. However, the issue whether LIPUS is effective in preventing osteoporosis has not been clarified, and if so, what possible mechanisms might be responsible. Myostatin (MSTN) is a negative regulator of muscle growth, and its absence will trigger a positive response to bone. In this study, we examined the effects of LIPUS on bone micro-structure, mechanical properties and damage healing of hindlimb-suspended rats, and investigated whether the inhibition of MSTN plays a role in this process. The rats were randomly divided into four groups: Normal control group (NC), Hind limb suspension group (HLS), Hind limb suspension and 80 mW/cm2 LIPUS irradiation group (HLS+ 80 mW/cm2), Hind limb suspension and 30 mW/cm2 LIPUS irradiation group (HLS+ 30 mW/cm2). The HLS+ 80 mW/cm2 rats were treated with LIPUS (1 MHz, 80 mW/cm2) and the HLS+ 30 mW/cm2 rats were treated with LIPUS (1 MHz, 30 mW/cm2) on the femur for 20 min/day for 28 days. MC3T3-E1 cells were respectively cultured with the serum of wild type mouse and MSTN knockout mouse at 1% concentration for 7 days. After 28 days, LIPUS effectively prevented the destruction of bone microstructure and the decline of mechanical properties, and promoted bone defect healing in the tail-suspended rats. In addition, LIPUS effectively reduced the MSTN content in the quadriceps and serum of the tail-suspended rats, inhibited its receptor and downstream signaling molecules and activated the Wnt signaling pathway in femurs. Growth of MC-3T3-E1 cell cultured with the serum of MSTN knockout mice was superior to that with wild mice serum on day 7. These results indicate that MSTN is a key mediator in LIPUS preventing bone loss caused by hindlimb-suspension.

Inhibition of MSTN signal pathway may participate in LIPUS preventing bone loss in ovariectomized rats.

INTRODUCTION: Menopause can lead to osteoporosis, which is characterized by destruction of bone microstructure, poor mechanical properties, and prone to fracture. LIPUS can effectively promote bone formation and fracture healing. MSTN is a transforming growth factor-ß family member that acts as a negative regulator of skeletal muscle growth. A MSTN deficiency also has a positive effect on bone formation. However, whether LIPUS could inhibit bone loss and promote healing of bone injury of menopause through the inhibition of the MSTN signaling pathway has not been previously investigated. We herein investigated the effects of LIPUS on bone architecture, mechanical properties, the healing of bone defects, and its potential molecular mechanisms in ovariectomized rats. MATERIALS AND METHODS: The rats were randomly divided into three groups: sham ovariectomized group (Sham), ovariectomized model group (OVX), ovariectomized model with LIPUS therapy group (OVX + LIPUS). The OVX + LIPUS rats were treated with LIPUS (1.5 MHz, 30 mW/cm2) on the femur for 20 min/day that lasted for 19 days. RESULTS: LIPUS effectively improved the bone microstructure, increased mechanical properties and promoted the healing of bone defects in ovariectomized rats. Moreover, LIPUS effectively decreased the MSTN content in serum and quadriceps muscle in ovariectomized rats, and inhibited the expression of MSTN downstream signaling molecules and activated the Wnt signaling pathway in the femur. CONCLUSIONS: The present study shows that LIPUS improved osteoporosis and promoted bone defect healing in the ovariectomized rats may through the inhibition of the MSTN signal pathway.

Effect of exercise on bone in poorly controlled type 1 diabetes mediated by the ActRIIB/Smad signaling pathway.

Myostatin (MSTN) is not only a key negative regulator of skeletal muscle secretion, however is also an endocrine factor that is transmitted to bone. To investigate the effect and possible mechanism of weight-bearing treadmill running on bone with poorly controlled Type 1 diabetes, rats were randomly divided into three groups: Normal control (NC), diabetic mellitus (DM) and diabetic exercise training groups (DM-WTR). The DM-WTR rats were trained with weight-bearing running. The results demonstrated that the levels of serum insulin, body weight, bone mass, muscle mass, grip strength, and serum calcium in the DM-WTR rats were significantly increased, whereas the levels of blood glucose, alkaline phosphatase, and tartrate-resistant acid phosphatase were markedly reduced in the DM-WTR rats compared with the DM rats. Weight-bearing running inhibited streptozocin (STZ)-induced MSTN mRNA and protein expression in the diabetic rats. The mRNA and protein expression levels of activin type IIB receptor and mothers against decapentaplegic homolog 2/3 and its phosphorylation in femur DM-WTR rats were reduced compared with DM rats. In addition, weight-bearing running enhanced the STZ-induced Wnt and ß-catenin expression levels and reduced the STZ-induced glycogen synthase kinase (GSK)-3ß expression in diabetic rats' femora. In conclusion, the results suggested that weight-bearing running could partially ameliorate STZ-induced femur atrophy via MSTN downregulation, and this may be associated with the inactivation of Activin A Receptor Type 2B/Smad2/3 signaling pathways and the activation of the Wnt/GSK3ß/ß-catenin signaling pathway. Further studies are needed to verify these conclusions.

Pulsed electromagnetic fields alleviate streptozotocin­induced diabetic muscle atrophy.

Diabetic muscle atrophy causes a reduction of skeletal muscle size and strength, which affects normal daily activities. However, pulsed electromagnetic fields (PEMFs) can retard the atrophy of type II fibers (ActRIIB) in denervated muscles. Therefore, the purpose of the present study was to determine whether PEMFs can alleviate streptozotocin (STZ)­induced diabetic muscle atrophy. To do this, 40 Sprague­Dawley (SD) rats were randomly divided into four groups (n=10 per group): The normal control group (NC; nondiabetic rats without treatment); the diabetic mellitus group (DM; STZ­induced rats without treatment); the diabetic insulin­treated group (DT; diabetic rats on insulin treatment, 6­8 U/d twice a day for 6 weeks) as a positive control; and the diabetic PEMFs therapy group (DP; diabetic rats with PEMFs exposure treatment, 15 Hz, 1.46 mT, 30 min/day for 6 weeks). Body weight, muscle strength, muscle mass and serum insulin level were significantly increased in the DP group compared with the DM group. PEMFs also decreased the blood glucose level and altered the activity of metabolic enzymes. PEMFs significantly increased the cross­sectional area of muscle fiber. In addition, PEMFs significantly activated protein kinase B (Akt) and mammalian target of rapamycin (mTOR), and inhibited the activity of myostatin (MSTN), ActRIIB and forkhead box protein O1 (FoxO1) compared with the DM group. Thus indicating that the Akt/mTOR and Akt/FoxO1 signaling pathways may be involved in the promotion of STZ­induced diabetic muscle atrophy by PEMFs. The results of the present study suggested that PEMFs stimulation may alleviate diabetic muscle atrophy in the STZ model, and that this is associated with alterations in multiple signaling pathways in which MSTN may be an integral factor. MSTN­associated signaling pathways may provide therapeutic targets to attenuate severe diabetic muscle wasting.

[Effects of weight-bearing ladder and aerobic treadmill exercise on learning and memory ability of diabetic rats and its mechanism].

OBJECTIVE: To investigate the effects of climb ladder and aerobic treadmill exercise on learning memory ability in diabetic rats and explore its possible mechanisms. METHODS: Forty male rats were randomly divided into normal control group (NC), diabetic control group (DC), diabetic loading ladder group (DL) and diabetic aerobic treadmill group (DA), diabetes was induced by a single intraperitoneal injection of STZ. In the evening, the DL group were trained three cycle (10 times/cycle) with weight-bearing climbing ladder, 2 min intervals, 6 days/week, lasted for six weeks. The DA group was trained on a motor-driven treadmill at a speed of 20 m/min (0 incline), 30 min/day, 6 days/week, lasted for six weeks. Morris water maze was used to detect the learning and memory ability of rats after modeling success and after exercise intervention. After the last water maze test, the rats were killed to obtain the hippocampus. RT-QPCR was used to detect the gene expressions of brain derived neurophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and cAMP-response element binding protein (CREB). RESULTS: Compared with NC group, the expressions of BDNF and CREB gene in hippocampus of DC group and the learning and memory ability were significantly decreased. Compared with DC group, the expression of BDNF and CREB in hippocampus of DL and DA rats was significantly up-regulated and the learning ability was significantly increased. The TrkB gene of hippocampus in DL rats was significantly up-regulated and the spatial memory ability was significantly improved. Compared with the DA group, the TRKB and CREB genes in the hippocampus of DL group were significantly up-regulated. CONCLUSIONS: Aerobic treadmill exercise and weight-bearing ladder exercise have a positive effect on the learning ability of diabetic rats, while the weight-bearing ladder exercise improves the memory ability of diabetic rats better than aerobic exercise. These effects may be related to the up-regulation of BDNF/TrkB/CREB signaling pathway.

Preparation and application of cellulose triacetate microspheres.

Cellulose triacetate was prepared via reacting of a mixture of acetic anhydride and acetic acid containing sulfuric acid as catalyst with ramie fiber obtained from a biomass of ramie. The cellulose triacetate with a degree of substitution (DS) 2.93 of the ramie fiber was obtained. The honeycomb-like cellulose triacetate microspheres with an average diameter of 14 microm were made from the cellulose triacetate solution. The optimum conditions for preparing the microspheres were determined as cellulose triacetate/dichloromethane ratio 1:7 (w/w), and 0.75% sodium dodecylsulfonate. The cellulose triacetate microspheres were characterized using FT-IR, NMR, XRD, and SEM. Application of the microspheres as an adsorbent for removing disperse dyes in water was investigated under the temperatures from 15 to 50 degrees C, pHs from 4 to 9, and the weight of cellulose triacetate microspheres from 0.03 to 0.09 g. The cellulose triacetate microspheres exhibited a 16.5mg/g capability to remove DR dye from water at 50 degrees C and pH 7.