A prospective randomized feasibility trial comparing angiography and angiography with intravascular ultrasound for treatment of hemodialysis access failures.

OBJECTIVE: Hemodialysis accesses suffer from limited primary patency requiring frequent interventions, revisions, or even abandonment. Prolongation of access life and usability with minimization of these adverse events is paramount. Endovascular methods are established first-line interventions for failing arteriovenous access and treatment of venous outflow stenoses. The Primary goal of this feasibility study was to evaluate intravascular ultrasound (IVUS) during interventional treatments on outcomes in those undergoing angiography for failing hemodialysis access. Secondary goals were to determine differences between IVUS and angiography on vessel and lesion characteristics and impact on treatment. METHODS: In this prospective, randomized controlled trial, patients scheduled for angiography to evaluate and treat a failing hemodialysis access were randomized to use of angiography (DSA) alone or angiography plus IVUS (DSA + IVUS). Patients were treated by a standardized protocol and seen in follow-up at 2 weeks, and every 3 months for 2 years or until a study endpoint was reached. Measurement of vessel diameters, % stenosis, lesion length, and study endpoints (AV access thrombosis, re-intervention, or surgical revision) were recorded. RESULTS: A total of 55 subjects were enrolled, 27 in the DSA cohort and 28 in the DSA + IVUS cohort. There were 41 treated lesions in each group. Freedom from the composite endpoint of AV access thrombosis or re-intervention was 46.3% in the DSA cohort and 61.0% in the DSA + IVUS cohort (p = 0.27). Diameter measurements matched between the two imaging modalities only 9 times out of 41 total comparison measures. In pre-treatment lesions with >80% stenosis, IVUS had a greater tendency than DSA to underestimate the severity of stenosis, whereas in pre-treatment lesions with 50-80% stenosis, DSA was more likely than IVUS to underestimate the severity of stenosis. Post-treatment % stenosis had mean difference of -7.5% between DSA versus DSA + IVUS cohorts. In five lesions with <30% stenosis measured by angiogram, IVUS led to treatment escalation. CONCLUSION: In the interventional treatment of failing angioaccess, IVUS and angiography differ in the vast majority of cases in measurement of vessel diameter. A significant number of patients were found to have suboptimal therapeutic response by IVUS only, which led to an escalation in treatment, and in over one-third of cases, the IVUS results led to a change in treatment plan. The improved patency rates in the IVUS group was not statistically significant in this small population but should be further investigated in a larger trial.

Venous outcomes at 1 year after using the femoral vein as a conduit for passage of percutaneous femoropopliteal bypass.

OBJECTIVE: The DETOUR 1 study was performed to assess the safety of the femoral vein as a "pass through" conduit for covered stent placement during fully percutaneous femoropopliteal bypass, also known as the DETOUR procedure. METHODS: At eight participating centers in this prospective, single-arm, international trial, 78 patients (82 femoropopliteal lesions) were enrolled. All patients had patent femoral veins measuring ≥10 mm in diameter at baseline. The DETOUR procedure involved delivery of a series of TORUS stent grafts, deployed from contralateral common femoral artery access, to the ipsilateral proximal superficial femoral artery, with entry into the femoral vein and re-entry into the arterial vasculature at the above-the-knee popliteal artery. The TORUS stent grafts are deployed in an overlapping configuration as an arterial-arterial conduit. Due to this novel transvenous approach, we assessed specific considerations related to the venous system to analyze the risk of risk of venous thromboembolic complications. Symptomatic deep vein thrombosis, nonocclusive material associated with the graft such as benign endovenous graft-associated material, pulmonary embolism, Venous Clinical Severity Score (VCSS) and Villalta scores, and luminal occupancy by the stent graft were assessed as the ratio of cross-sectional areas of the stent graft to the native vein at baseline and 1 year after the procedure. RESULTS: A duplicate femoral vein was present in 20.7% of cases. The majority of patients (86.8%) had a femoral vein luminal area preservation of ≥55%. Thirty-two patients experienced an increase in the vein diameter over time after the procedure, but this pattern of venous remodeling was not uniform. The patients who had a compensatory increase in the vein diameter had a smaller average baseline vein diameter compared with the patients who did not have a compensatory increase in vein diameter (P = .0414). Only two patients (2.4%) developed ipsilateral symptomatic deep vein thrombosis) through 1 year of follow-up. There were no pulmonary embolism in any patient in the series. The overall VCSS and Villata scores did not change during follow-up. Mean VCSS and Villata were 0.8 ± 1.4 and 0.5 ± 1.1 at 1 year, compared with 0.6 ± 1.0 and 0.4 ± 0.9 at baseline, respectively. CONCLUSIONS: As a percutaneous alternative to open surgical bypass for complex femoropopliteal peripheral arterial disease, the transvenous bypass has a low rate of deep venous thrombotic and obstructive complications. Cross-sectional vein area is preserved, and in some patients, the compensatory vein diameter increases with time, supporting the feasibility and safety of using the lower extremity deep venous system as a pass-through conduit for the DETOUR percutaneous femoropopliteal bypass. TRIAL REGISTRATION: NCT02471638.

Mechanobiological modulation of in situ and in vivo osteocyte calcium oscillation by acoustic radiation force.

The biological effect of ultrasound on bone regeneration has been well documented, yet the underlying mechanotransduction mechanism is largely unknown. In relation to the mechanobiological modulation of the cytoskeleton and Ca2+ influx by short-term focused acoustic radiation force (FARF), the current study aimed to visualize and quantify Ca2+ oscillations in real-time of in situ and in vivo osteocytes in response to focused low-intensity pulsed ultrasound (FLIPUS). For in situ studies, fresh mice calvaria were subjected to FLIPUS stimulation at 0.05, 0.2, 0.3, and 0.7 W. For the in vivo study, 3-month-old C57BL/6J Ai38/Dmp1-Cre mice were subjected to FLIPUS at 0.15, 1, and 1.5 W. As observed via real-time confocal imaging, in situ FLIPUS led to more than 80% of cells exhibiting Ca2+ oscillations at 0.3-0.7 W and led to a higher number of Ca2+ spikes with larger values at >0.3 W. In vivo FLIPUS at 1-1.5 W led to more than 90% of cells exhibiting Ca2+ oscillations. Higher FLIPUS energies led to larger Ca2+ spike magnitudes. In conclusion, this study provided a pilot study of both in situ and in vivo osteocytic Ca2+ oscillations under noninvasive FARF, which aids further exploration of the mechanosensing mechanism of the controlled bone cell motility response to the stimulus.

Functional disuse initiates medullary endosteal micro-architectural impairment in cortical bone characterized by nanoindentation.

In this study, we evaluated the effect of functional disuse-induced bone remodeling on its mechanical properties, individually at periosteum and medullary endosteum regions of the cortical bone. Left middle tibiae were obtained from 5-month-old female Sprague-Dawley rats for the baseline control as well as hindlimb suspended (disuse) groups. Micro-nano-mechanical elastic moduli (at lateral region) was evaluated along axial (Z), circumferential (C) and radial (R) orientations using nanoindentation. Results indicated an anisotropic microstructure with axial orientation having the highest and radial orientation with the lowest moduli at periosteum and medullary endosteum for both baseline control as well as disuse groups. Between the groups: at periosteum, an insignificant difference was evaluated for each of the orientations (p > 0.05) and at endosteum, a significant decrease of elastic moduli in the radial (p < 0.0001), circumferential (p < 0.001) and statistically insignificant difference in axial (p > 0.05) orientation. For the moduli ratios between groups: at periosteum, only significant difference in the Z/R (p < 0.05) anisotropy ratio, whereas at endosteum, a statistically significant difference in Z/C (p < 0.001), and Z/R (p < 0.001), as well as C/R (p < 0.05) anisotropy ratios, was evaluated. The results suggested initial bone remodeling impaired bone micro-architecture predominantly at the medullary endosteum with possible alterations in the geometric orientations of collagen and mineral phases inside the bone. The findings could be significant for studying the mechanotransduction pathways involved in maintaining the bone micro-architecture and possibly have high clinical significance for drug use against impairment from functional disuse.

Retention of osteocytic micromorphology by sclerostin antibody in a concurrent ovariectomy and functional disuse model.

Prolonged mechanical unloading in bedridden patients and concurrent hormonal dysregulation represents the cause of one of the severest forms of osteoporosis, a condition for which there are very few efficacious interventions available to date. Sclerostin, a Wnt antagonist, acts as a negative regulator of bone formation. Sclerostin antibody (Scl-Ab)-mediated blockade of sclerostin can dramatically enhance bone formation and reduce bone resorption. This study was designed to investigate the therapeutic effect of the Scl-Ab on severe bone loss induced by concurrent mechanical unloading and estrogen deficiency in a hindlimb-suspended and ovariectomized rat model, and to study the cellular mechanisms underlying severe osteoporosis and Scl-Ab action. Unloading and ovariectomy resulted in severe loss of trabecular and cortical bone mass and strength; Scl-Ab can significantly counteract the deterioration of bone in unloaded and/or ovariectomized rats, with noticeably increased cortical bone formation. Scanning electron microscopy analysis revealed that unloading and ovariectomy lead to multiple morphological and structural abnormalities of osteocytes in cortical bone and the abnormalities were abolished by Scl-Ab administration. This study extends our previous conclusion that Scl-Ab represents a promising therapeutic approach for severe bone loss that occurs after being exposed to estrogen deficiency and prolonged mechanical unloading.

Mitigation of Articular Cartilage Degeneration and Subchondral Bone Sclerosis in Osteoarthritis Progression Using Low-Intensity Ultrasound Stimulation.

The purpose of this study was to evaluate the effect of low-intensity ultrasound on articular cartilage and subchondral bone alterations in joints under normal and functional disuse conditions during osteoarthritis (OA) progression. Total of thirty 5-mo-old female Sprague-Dawley rats were randomly assigned to six groups (n = 5/group): age-matched group, OA group, OA + ultrasound (US) group, hindlimb suspension (HLS) group, HLS + OA group and HLS + OA + US group. The surgical anterior cruciate ligament was used to induce OA in the right knee joints. After 2 wk of OA induction, low-intensity ultrasound generated with a 3-MHz transducer with 20% pulse duty cycle and 30 mW/cm2 acoustic intensity was delivered to the right knee joints for 20 min a day, 5 d a week for a total of 6 wk. Then, the right tibias were harvested for micro-computed tomography, histologic and mechanical analysis. Micro-computed tomography results indicated that the thickness and sulfated glycosaminoglycan content of cartilage decreased, but the thickness of the subchondral cortical bone plate and the formation of subchondral trabecular bone increased in the OA group under the normal joint use condition. Furthermore, histologic results revealed that chondrocyte density and arrangement in cartilage corrupted and the underlying subchondral bone increased during OA progression. These changes were accompanied by reductions in mechanical parameters in OA cartilage. However, fewer OA symptoms were observed in the HLS + OA group under the joint disuse condition. The cartilage degeneration and subchondral bone sclerosis were alleviated in the US treatment group, especially under normal joint use condition. In conclusion, low-intensity ultrasound could improve cartilage degeneration and subchondral sclerosis during OA progression. Also, it could provide a promising strategy for future clinical treatment for OA patients.

Sclerostin antibody prevented progressive bone loss in combined ovariectomized and concurrent functional disuse.

Osteoporosis is characterized by low bone mass and compromised trabecular architecture, and is commonly occurred in post-menopausal women with estrogen deficiency. In addition, prolonged mechanical unloading, i.e., long term bed rest, can exaggerate the bone loss. Sclerostin is a Wnt signaling antagonist and acts as a negative regulator for bone formation. A sclerostin-neutralizing antibody (Scl-Ab) increased bone mineral density in women with postmenopausal osteoporosis and healthy men. The objective of this study was to characterize the condition of bone loss in ovariectomized (OVX) rats with concurrent mechanical unloading and evaluate the effect of sclerostin antibody treatment in mitigating the prospective severe bone loss conditions in this model. Four-month-old OVX- or sham-operated female SD rats were used in this study. They were subjected to functional disuse induced by hind-limb suspension (HLS) or free ambulance after 2days of arrival. Subcutaneous injections with either vehicle or Scl-Ab at 25mg/kg were made twice per week for 5weeks from the time of HLS. µCT analyses demonstrated a significant decrease in distal metaphyseal trabecular architecture integrity with HLS, OVX and HLS+OVX (bone volume fraction decreased by 29%, 71% and 87% respectively). The significant improvements of various trabecular bone parameters (bone volume fraction increased by 111%, 229% and 297% respectively as compared with placebo group) with the administration of Scl-Ab are associated with stronger mechanical property and increased bone formation by histomorphometry. These results together indicate that Scl-Ab prevented the loss of trabecular bone mass and cortical bone strength in OVX rat model with concurrent mechanical unloading. The data suggested that monoclonal sclerostin-neutralizing antibody represents a promising therapeutic approach for severe osteoporosis induced by estrogen deficiency with concurrent mechanical unloading.

Spatial distribution and remodeling of elastic modulus of bone in micro-regime as prediction of early stage osteoporosis.

We assessed the local distribution of bone mechanical properties on a micro-nano-scale and its correlation to strain distribution. Left tibia samples were obtained from 5-month old female Sprague Dawley rats, including baseline control (n=9) and hindlimb suspended (n=9) groups. Elastic modulus was measured by nanoindentation at the dedicated locations. Three additional tibias from control rats were loaded axially to measure bone strain, with 6-10N at 1Hz on a Bose machine for strain measurements. In the control group, the difference of the elastic modulus between periosteum and endosteum was much higher at the anterior and posterior regions (2.6GPa), where higher strain differences were observed (45µÉ›). Minimal elastic modulus difference between periosteum and endosteum was observed at the medial region (0.2GPa), where neutral axis of the strain distribution was oriented with lower strain difference (5µÉ›). In the disuse group, however, the elastic modulus differences in the anterior posterior regions reduced to 1.2GPa from 2.6GPa in the control group, and increased in the medial region to 2.7GPa from 0.2GPa. It is suggested that the remodeling rate in a region of bone is possibly influenced by the strain gradient from periosteum to endosteum. Such pattern of moduli gradients was compromised in disuse osteopenia, suggesting that the remodeling in distribution of micro-nano-elastic moduli among different regions may serve as a predictor for early stage of osteoporosis.

Dynamic fluid flow induced mechanobiological modulation of in situ osteocyte calcium oscillations.

Distribution of intramedullary pressure (ImP) induced bone fluid flow has been suggested to influence the magnitude of mechanotransductory signals within bone. As osteocytes have been suggested as major mechanosensors in bone network, it is still unclear how osteocytes embedded within a mineralized bone matrix respond to the external mechanical stimuli derived from direct coupling of dynamic fluid flow stimulation (DFFS). While in vitro osteocytes show unique Ca(2+) oscillations to fluid shear, the objective of this study was to use a confocal imaging technique to visualize and quantify Ca(2+) responses in osteocytes in situ under DFFS into the marrow cavity of an intact ex vivo mouse femur. This study provided significant technical development for evaluating mechanotransduction mechanism in bone cell response by separation of mechanical strain and fluid flow factors using ImP stimulation, giving the ability for true real-time imaging and monitoring of bone cell activities during the stimulation. Loading frequency dependent Ca(2+) oscillations in osteocytes indicated the optimized loading at 10Hz, where such induced response was significantly diminished via blockage of the Wnt/ß-catenin signaling pathway. The results provided a pilot finding of the potential crosstalk or interaction between Wnt/ß-catenin signaling and Ca(2+) influx signaling of in situ osteocytes in response to mechanical signals. Findings from the present study make a valuable tool to investigate how in situ osteocytes respond and transduce mechanical signals, e.g. DFFS, as a central mechanosensor.

Mechanotransduction in musculoskeletal tissue regeneration: effects of fluid flow, loading, and cellular-molecular pathways.

While mechanotransductive signal is proven essential for tissue regeneration, it is critical to determine specific cellular responses to such mechanical signals and the underlying mechanism. Dynamic fluid flow induced by mechanical loading has been shown to have the potential to regulate bone adaptation and mitigate bone loss. Mechanotransduction pathways are of great interests in elucidating how mechanical signals produce such observed effects, including reduced bone loss, increased bone formation, and osteogenic cell differentiation. The objective of this review is to develop a molecular understanding of the mechanotransduction processes in tissue regeneration, which may provide new insights into bone physiology. We discussed the potential for mechanical loading to induce dynamic bone fluid flow, regulation of bone adaptation, and optimization of stimulation parameters in various loading regimens. The potential for mechanical loading to regulate microcirculation is also discussed. Particularly, attention is allotted to the potential cellular and molecular pathways in response to loading, including osteocytes associated with Wnt signaling, elevation of marrow stem cells, and suppression of adipotic cells, as well as the roles of LRP5 and microRNA. These data and discussions highlight the complex yet highly coordinated process of mechanotransduction in bone tissue regeneration.

Interrelation between external oscillatory muscle coupling amplitude and in vivo intramedullary pressure related bone adaptation.

Interstitial bone fluid flow (IBFF) is suggested as a communication medium that bridges external physical signals and internal cellular activities in the bone, which thus regulates bone remodeling. Intramedullary pressure (ImP) is one main regulatory factor of IBFF and bone adaptation related mechanotransduction. Our group has recently observed that dynamic hydraulic stimulation (DHS), as an external oscillatory muscle coupling, was able to induce local ImP with minimal bone strain as well as to mitigate disuse bone loss. The current study aimed to evaluate the dose dependent relationship between DHS's amplitude, i.e., 15 and 30mmHg, and in vivo ImP induction, as well as this correlation on bone's phenotypic change. Simultaneous measurements of ImP and DHS cuff pressures were obtained from rats under DHS with various magnitudes and a constant frequency of 2Hz. ImP inductions and cuff pressures upon DHS loading showed a positively proportional response over the amplitude sweep. The relationship between ImP and DHS cuff pressure was evaluated and shown to be proportional, in which ImP was raised with increases of DHS cuff pressure amplitudes (R(2)=0.98). A 4-week in vivo experiment using a rat hindlimb suspension model demonstrated that the mitigation effect of DHS on disuse trabecular bone was highly dose dependent and related to DHS's amplitude, where a higher ImP led to a higher bone volume. This study suggested that sufficient physiological DHS is needed to generate ImP. Oscillatory DHS, potentially induces local fluid flow, has shown dose dependence in attenuation of disuse osteopenia.

Dynamic fluid flow stimulation on cortical bone and alterations of the gene expressions of osteogenic growth factors and transcription factors in a rat functional disuse model.

Recently we have developed a dynamic hydraulic stimulation (DHS) as a loading modality to induce anabolic responses in bone. To further study the functional process of DHS regulated bone metabolism, the objective of this study was to evaluate the effects of DHS on cortical bone and its alterations on gene expressions of osteogenic growth factors and transcription factors as a function of time. Using a model system of 5-month-old hindlimb suspended (HLS) female Sprague-Dawley rats, DHS was applied to the right tibiae of the stimulated rats with a loading frequency of 2Hz with 30mmHg (p-p) dynamic pressure, 5days/week, for a total of 28days. Midshafts of the tibiae were analyzed using µCT and histology. Total RNA was analyzed using RT-PCR on selected osteogenic genes (RUNX2, ß-catenin, osteopontin, VEGF, BMP2, IGF-1, and TGF-ß) on 3-, 7-, 14- , and 21-day. Results showed increased Cort.Th and Ct.BV/TV as well as a time-dependent fashion of gradual changes in mRNA levels upon DHS. While DHS-driven fold changes of the mRNA levels remained low before Day-7, its fold changes started to elevate by Day-14 and then dropped by Day-21. This study further delineates the underlying molecular mechanism of DHS-derived mechanical signals, and its time-dependent optimization.

Preliminary evidence of early bone resorption in a sheep model of acute burn injury: an observational study.

Treatment with bisphosphonates within the first 10 days of severe burn injury completely prevents bone loss. We therefore postulated that bone resorption occurs early post burn and is the primary explanation for acute bone loss in these patients. Our objective was to assess bone for histological and biomechanical evidence of early resorption post burn. We designed a randomized controlled study utilizing a sheep model of burn injury. Three sheep received a 40 % total body surface area burn under isoflurane anesthesia, and three other sheep received cotton-smoke inhalation and served as control. Burned sheep were killed 5 days post procedure and controls were killed 2 days post procedure. Backscatter scanning electron microscopy was performed on iliac crests obtained immediately postmortem along with quantitative histomorphometry and compression testing to determine bone strength (Young's modulus). Blood ionized Ca was also determined in the first 24 h post procedure as was urinary CTx. Three of three sheep killed at 5 days had evidence of scalloping of the bone surface, an effect of bone resorption, whereas none of the three sheep killed at 2 days post procedure had scalloping. One of the three burned sheep killed at 5 days showed quantitative doubling of the eroded surface and halving of the bone volume compared to sham controls. Mean values of Young's modulus were approximately one third lower in the burned sheep killed at 5 days compared to controls, p = 0.08 by unpaired t test, suggesting weaker bone. These data suggest early post-burn bone resorption. Urine CTx normalized to creatinine did not differ between groups at 24 h post procedure because the large amounts of fluids received by the burned sheep may have diluted urine creatinine and CTx and because the urine volume produced by the burned sheep was threefold that of the controls. We calculated 24 h urinary CTx excretion, and with this calculation CTx excretion/24 h in the burned sheep was nearly twice that of the controls. Moreover, whole blood ionized Ca measured at 3- to 6-h intervals over the first 24 h in both burn and control sheep showed a 6 % reduction versus baseline in the burned sheep with <1 % reduction in the control animals. This sheep model was previously used to demonstrate upregulation of the parathyroid calcium-sensing receptor within the timeframe of the present study. Because both early bone resorption, supported by this study, and calcium-sensing receptor upregulation, consistent with the observed reduction in blood ionized Ca, are mediated by proinflammatory cytokines that are present as part of the post-burn systemic inflammatory response, we may postulate that post-burn upregulation of the parathyroid calcium-sensing receptor may be an adaptive response to clear the blood of excess calcium liberated by cytokine-mediated bone resorption.

Dynamic hydraulic fluid stimulation regulated intramedullary pressure.

Physical signals within the bone, i.e. generated from mechanical loading, have the potential to initiate skeletal adaptation. Strong evidence has pointed to bone fluid flow (BFF) as a media between an external load and the bone cells, in which altered velocity and pressure can ultimately initiate the mechanotransduction and the remodeling process within the bone. Load-induced BFF can be altered by factors such as intramedullary pressure (ImP) and/or bone matrix strain, mediating bone adaptation. Previous studies have shown that BFF induced by ImP alone, with minimum bone strain, can initiate bone remodeling. However, identifying induced ImP dynamics and bone strain factor in vivo using a non-invasive method still remains challenging. To apply ImP as a means for alteration of BFF, it was hypothesized that non-invasive dynamic hydraulic stimulation (DHS) can induce local ImP with minimal bone strain to potentially elicit osteogenic adaptive responses via bone-muscle coupling. The goal of this study was to evaluate the immediate effects on local and distant ImP and strain in response to a range of loading frequencies using DHS. Simultaneous femoral and tibial ImP and bone strain values were measured in three 15-month-old female Sprague Dawley rats during DHS loading on the tibia with frequencies of 1Hz to 10Hz. DHS showed noticeable effects on ImP induction in the stimulated tibia in a nonlinear fashion in response to DHS over the range of loading frequencies, where they peaked at 2Hz. DHS at various loading frequencies generated minimal bone strain in the tibiae. Maximal bone strain measured at all loading frequencies was less than 8µÎµ. No detectable induction of ImP or bone strain was observed in the femur. This study suggested that oscillatory DHS may regulate the local fluid dynamics with minimal mechanical strain in the bone, which serves critically in bone adaptation. These results clearly implied DHS's potential as an effective, non-invasive intervention for osteopenia and osteoporosis treatments.

Reversal of the detrimental effects of simulated microgravity on human osteoblasts by modified low intensity pulsed ultrasound.

Microgravity (MG) is known to induce bone loss in astronauts during long-duration space mission because of a lack of sufficient mechanical stimulation under MG. It has been demonstrated that mechanical signals are essential for maintaining cell viability and motility, and they possibly serve as a countermeasure to the catabolic effects of MG. The objective of this study was to examine the effects of high-frequency acoustic wave signals on osteoblasts in a simulated microgravity (SMG) environment (created using 1-D clinostat bioreactor) using a modified low-intensity pulsed ultrasound (mLIPUS). Specifically, we evaluated the hypothesis that osteoblasts (human fetal osteoblastic cell line) exposure to mLIPUS for 20 min/d at 30 mW/cm(2) will significantly reduce the detrimental effects of SMG. Effects of SMG with mLIPUS were analyzed using the MTS proliferation assay for proliferation, phalloidin for F-actin staining, Sirius red stain for collagen, and Alizarin red for mineralization. Our data showed that osteoblast exposure to SMG results in significant decreases in proliferation (∼ -38% and ∼ -44% on days 4 and 6, respectively; p < 0.01), collagen content (∼ -22%; p < 0.05) and mineralization (∼ -37%; p < 0.05) and actin stress fibers. In contrast, mLIPUS stimulation in SMG condition significantly increases the rate of proliferation (∼24% by day 6; p < 0.05), collagen content (∼52%; p < 0.05) and matrix mineralization (∼25%; p < 0.001) along with restoring formation of actin stress fibers in the SMG-exposed osteoblasts. These data suggest that the acoustic wave can potentially be used as a countermeasure for disuse osteopenia.

Dynamic Fluid Flow Mechanical Stimulation Modulates Bone Marrow Mesenchymal Stem Cells.

Osteoblasts are derived from mesenchymal stem cells (MSCs), which initiate and regulate bone formation. New strategies for osteoporosis treatments have aimed to control the fate of MSCs. While functional disuse decreases MSC growth and osteogenic potentials, mechanical signals enhance MSC quantity and bias their differentiation toward osteoblastogenesis. Through a non-invasive dynamic hydraulic stimulation (DHS), we have found that DHS can mitigate trabecular bone loss in a functional disuse model via rat hindlimb suspension (HLS). To further elucidate the downstream cellular effect of DHS and its potential mechanism underlying the bone quality enhancement, a longitudinal in vivo study was designed to evaluate the MSC populations in response to DHS over 3, 7, 14, and 21 days. Five-month old female Sprague Dawley rats were divided into three groups for each time point: age-matched control, HLS, and HLS+DHS. DHS was delivered to the right mid-tibiae with a daily "10 min on-5 min off-10 min on" loading regime for five days/week. At each sacrifice time point, bone marrow MSCs of the stimulated and control tibiae were isolated through specific cell surface markers and quantified by flow cytometry analysis. A strong time-dependent manner of bone marrow MSC induction was observed in response to DHS, which peaked on day 14. After 21 days, this effect of DHS was diminished. This study indicates that the MSC pool is positively influenced by the mechanical signals driven by DHS. Coinciding with our previous findings of mitigation of disuse bone loss, DHS induced changes in MSC number may bias the differentiation of the MSC population towards osteoblastogenesis, thereby promoting bone formation under disuse conditions. This study provides insights into the mechanism of time-sensitive MSC induction in response to mechanical loading, and for the optimal design of osteoporosis treatments.

Dynamic hydraulic flow stimulation on mitigation of trabecular bone loss in a rat functional disuse model.

Bone fluid flow (BFF) has been demonstrated as a critical regulator in mechanotransductive signaling and bone adaptation. Intramedullary pressure (ImP) and matrix strain have been identified as potential generators to regulate BFF. To elevate in vivo oscillatory BFF using ImP, a dynamic hydraulic stimulation (DHS) approach was developed. The objective of this study was to evaluate the effects of DHS on mitigation of bone loss and structural alteration in a rat hindlimb suspension (HLS) functional disuse model. Sixty-one 5-month old female Sprague-Dawley rats were divided into five groups: 1) baseline control, 2) age-matched control, 3) HLS, 4) HLS+static loading, and 5) HLS+DHS. Hydraulic flow stimulation was carried out daily on a "10 min on-5 min off-10 min on" loading regime, 5 days/week, for a total of 4 weeks in the tibial region. The metaphyseal trabecular regions of the proximal tibiae were analyzed using µCT and histomorphometry. Four weeks of HLS resulted in a significant loss of trabecular bone, leading to structural deterioration. HLS with static loading alone was not sufficient to attenuate the bone loss. Bone quantity and microarchitecture were significantly improved by applying DHS loading, resulting increase of 83% in bone volume fraction, 25% in trabecular number and mitigation of 26% in trabecular separation compared to HLS control. Histomorphometry analysis on trabecular mineralization coincided with the µCT analysis, in which DHS loading yielded increases of 34% in histomorphometric BV/TV, 121% in MS/BS, 190% in BFR/BS and 146% in BFR/BV, compared to the HLS control. Overall, the data demonstrated that dynamic hydraulic flow loading has potentials to provide regulatory signals for mitigating bone loss induced by functional disuse. This approach may provide a new alternative mechanical intervention for future clinical treatment for osteoporosis.

Alteration of contraction-to-rest ratio to optimize trabecular bone adaptation induced by dynamic muscle stimulation.

Disuse osteopenia has shown to decrease bone mineral density and compromise bone's integrity, i.e., in aging population and long-term functional disuse. The degree of attenuation of trabecular bone loss and deterioration of its microarchitecture is closely dependent on the mechanical loading parameters within the regimen. Dynamic muscle stimulation as a preventive countermeasure for disuse osteopenia has been shown to be effective. The objective of this study is to determine whether the contraction-to-rest ratio is a crucial parameter to affect the skeletal adaptive responses under a functional disuse environment. Fifty-six skeletally matured Sprague-Dawley rats were divided into seven groups for the 4-week experiment: baseline control, age-matched control, hindlimb suspended (HLS), and HLS plus muscle stimulation with a contraction-to-rest ratio of 1/4, 2/8, 4/6, and 2/28 s. Muscle stimulation was carried out for total of 10 min/day, 5 days/week, for 4 weeks. Trabecular bone in the distal femurs was analyzed with microcomputed tomography and histomorphometry. HLS alone for 4-week resulted in a 25-45% trabecular bone loss in the distal femur. Dynamic muscle stimulation, applied at 50 Hz frequency, with a 2/8 s contraction-to-rest ratio demonstrated significant attenuation of trabecular bone loss against the 4-week disuse, with up to +74% in bone volume fraction, +164% in connectivity, +20% in trabecular number, and -18% in spacing (p<0.05). Stimulation with 1/4 and 4/6 also showed similar effects but with lesser percentage differences when comparing to the HLS animals. Similarly, histomorphometric analysis showed partial enhancement in mineralizing surface and mineral apposition rate. The results suggested the potentials of dynamic muscle stimulation in regulating skeletal adaptive responses and illustrated the effects of optimized contraction-to-rest in mitigation of bone loss, in which 2/8 s has shown maximal adaptive response among all tested ratios.