Low intensity mechanical signals promote proliferation in a cell-specific manner: Tailoring a non-drug strategy to enhance biomanufacturing yields.

Biomanufacturing relies on living cells to produce biotechnology-based therapeutics, tissue engineering constructs, vaccines, and a vast range of agricultural and industrial products. With the escalating demand for these bio-based products, any process that could improve yields and shorten outcome timelines by accelerating cell proliferation would have a significant impact across the discipline. While these goals are primarily achieved using biological or chemical strategies, harnessing cell mechanosensitivity represents a promising - albeit less studied - physical pathway to promote bioprocessing endpoints, yet identifying which mechanical parameters influence cell activities has remained elusive. We tested the hypothesis that mechanical signals, delivered non-invasively using low-intensity vibration (LIV; <1g, 10-500Hz), will enhance cell expansion, and determined that any unique signal configuration was not equally influential across a range of cell types. Varying frequency, intensity, duration, refractory period, and daily doses of LIV increased proliferation in CHO-adherent cells (+79% in 96h) using a particular set of LIV parameters (0.2g, 500Hz, 3x30 min/d, 2h refractory period), yet this same mechanical input suppressed proliferation in CHO-suspension cells (-13%). Exposing these same CHO-suspension cells to distinct LIV parameters (30Hz, 0.7g, 2x60 min/d, 2h refractory period) increased proliferation by 210%. Particle image velocimetry combined with finite element modeling showed high transmissibility of these signals across fluids (>90%), and LIV effectively scaled up to T75 flasks. Ultimately, when LIV is tailored to the target cell population, its highly efficient transmission across media represents a means to non-invasively augment biomanufacturing endpoints for both adherent and suspended cells, and holds immediate applications, ranging from small-scale, patient-specific personalized medicine to large-scale commercial bio-centric production challenges.

Postural Stability in Obese Preoperative Bariatric Patients Using Static and Dynamic Evaluation.

INTRODUCTION: Globally, 300 million adults have clinical obesity. Heightened adiposity and inadequate musculature secondary to obesity alter bipedal stance and gait, diminish musculoskeletal tissue quality, and compromise neuromuscular feedback; these physiological changes alter stability and increase injury risk from falls. Studies in the field focus on obese patients across a broad range of body mass indices (BMI >30 kg/m2) but without isolating the most morbidly obese subset (BMI ≥40 kg/m2). We investigated the impact of obesity in perturbing postural stability in morbidly obese subjects elected for bariatric intervention, harboring a higher-spectrum BMI. SUBJECTS AND METHODS: Traditional force plate measurements and stabilograms are gold standards employed when measuring center of pressure (COP) and postural sway. To quantify the extent of postural instability in subjects with obesity before bariatric surgery, we assessed 17 obese subjects with an average BMI of 40 kg/m2 in contrast to 13 nonobese subjects with an average BMI of 30 kg/m2. COP and postural sway were measured from static and dynamic tasks. Involuntary movements were measured when patients performed static stances, with eyes either opened or closed. Two additional voluntary movements were measured when subjects performed dynamic, upper torso tasks with eyes opened. RESULTS: Mean body weight was 85% (p < 0.001) greater in obese than nonobese subjects. Following static balance assessments, we observed greater sway displacement in the anteroposterior (AP) direction in obese subjects with eyes open (87%, p < 0.002) and eyes closed (76%, p = 0.04) versus nonobese subjects. Obese subjects also exhibited a higher COP velocity in static tests when subjects' eyes were open (47%, p = 0.04). Dynamic tests demonstrated no differences between groups in sway displacement in either direction; however, COP velocity in the mediolateral (ML) direction was reduced (31%, p < 0.02) in obese subjects while voluntarily swaying in the AP direction, but increased in the same cohort when swaying in the ML direction (40%, p < 0.04). DISCUSSION AND CONCLUSION: Importantly, these data highlight obesity's contribution towards increased postural instability. Obese subjects exhibited greater COP displacement at higher AP velocities versus nonobese subjects, suggesting that clinically obese individuals show greater instability than nonobese subjects. Identifying factors contributory to instability could encourage patient-specific physical therapies and presurgical measures to mitigate instability and monitor postsurgical balance improvements.

Mechanical signals protect stem cell lineage selection, preserving the bone and muscle phenotypes in obesity.

The incidence of obesity is rapidly rising, increasing morbidity and mortality rates worldwide. Associated comorbidities include type 2 diabetes, heart disease, fatty liver disease, and cancer. The impact of excess fat on musculoskeletal health is still unclear, although it is associated with increased fracture risk and a decline in muscular function. The complexity of obesity makes understanding the etiology of bone and muscle abnormalities difficult. Exercise is an effective and commonly prescribed nonpharmacological treatment option, but it can be difficult or unsafe for the frail, elderly, and morbidly obese. Exercise alternatives, such as low-intensity vibration (LIV), have potential for improving musculoskeletal health, particularly in conditions with excess fat. LIV has been shown to influence bone marrow mesenchymal stem cell differentiation toward higher-order tissues (i.e., bone) and away from fat. While the exact mechanisms are not fully understood, recent studies utilizing LIV both at the bench and in the clinic have demonstrated some efficacy. Here, we discuss the current literature investigating the effects of obesity on bone, muscle, and bone marrow and how exercise and LIV can be used as effective treatments for combating the negative effects in the presence of excess fat.

Incorporating Refractory Period in Mechanical Stimulation Mitigates Obesity-Induced Adipose Tissue Dysfunction in Adult Mice.

OBJECTIVE: The aim of this study was to determine whether inclusion of a refractory period between bouts of low-magnitude mechanical stimulation (LMMS) can curb obesity-induced adipose tissue dysfunction and sequelae in adult mice. METHODS: A diet-induced obesity model that included a diet with 45% of kilocalories from fat was employed with intention to treat. C57BL/6J mice were weight matched into four groups: low-fat diet (LFD, n = 8), high-fat diet (HFD, n = 8), HFD with one bout of 30-minute LMMS (HFDv, n = 9), and HFD with two bouts of 15-minute LMMS with a 5-hour separation (refractory period, RHFDv, n = 9). Two weeks of diet was followed by 6 weeks of diet plus LMMS. RESULTS: HFD and HFDv mice continued gaining body weight and visceral adiposity throughout the experiment, which was mitigated in RHFDv mice. Compared with LFD mice, HFD and HFDv mice had increased rates of adipocyte hypertrophy, increased immune cell infiltration (B cells, T cells, and macrophages) into adipose tissue, increased adipose tissue inflammation (tumor necrosis factor alpha gene expression), and a decreased proportion of mesenchymal stem cells in adipose tissue, all of which were rescued in RHFDv mice. Glucose intolerance and insulin resistance were elevated in HFD and HFDv mice, but not in RHFDv mice, as compared with LFD mice. CONCLUSIONS: Incorporating a 5-hour refractory period between bouts of LMMS attenuates obesity-induced adipose tissue dysfunction and improves glucose metabolism.

Low intensity vibration mitigates tumor progression and protects bone quantity and quality in a murine model of myeloma.

Myeloma facilitates destruction of bone and marrow. Since physical activity encourages musculoskeletal preservation we evaluated whether low-intensity vibration (LIV), a means to deliver mechanical signals, could protect bone and marrow during myeloma progression. Immunocompromised-mice (n=25) were injected with human-myeloma cells, while 8 (AC) were saline-injected. Myeloma-injected mice (LIV; n=13) were subjected to daily-mechanical loading (15min/d; 0.3g @ 90Hz) while 12 (MM) were sham-handled. At 8w, femurs had 86% less trabecular bone volume fraction (BV/TV) in MM than in AC, yet only a 21% decrease in LIV was observed in comparison to AC, reflecting a 76% increase versus MM. Cortical BV was 21% and 15% lower in MM and LIV, respectively, than in AC; LIV showing 30% improvement over MM. Similar outcomes were observed in the axial skeleton, showing a 35% loss in MM with a 27% improved retention of bone in the L5 of LIV-treated mice as compared to MM. Transcortical-perforations in the femur from myeloma-induced osteolysis were 9× higher in MM versus AC, reduced by 57% in LIV. Serum-TRACP5b, 61% greater in MM versus AC, rose by 33% in LIV compared to AC, a 45% reduction in activity when compared to MM. Histomorphometric analyses of femoral trabecular bone demonstrated a 70% elevation in eroded surfaces of MM versus AC, while measures in LIV were 58% below those in MM. 72% of marrow in the femur of MM mice contained tumor, contrasted by a 31% lower burden in LIV. MM mice (42%) presented advanced-stage necrosis of tibial marrow while present in just 8% of LIV. Myeloma infiltration inversely correlated to measures of bone quality, while LIV slowed the systemic, myeloma-associated decline in bone quality and inhibited tumor progression through the hindlimbs.

Diminished satellite cells and elevated adipogenic gene expression in muscle as caused by ovariectomy are averted by low-magnitude mechanical signals.

Age-related degeneration of the musculoskeletal system, accelerated by menopause, is further complicated by increased systemic and muscular adiposity. The purpose of this study was to identify at the molecular, cellular, and tissue levels the impact of ovariectomy on adiposity and satellite cell populations in mice and whether mechanical signals could influence any outcomes. Eight-week-old C57BL/6 mice were ovariectomized, with one half subjected to low-intensity vibration (LIV; 0.3 g/90 Hz, 15 min/day, 5 day/wk; n = 10) for 6 wk and the others sham vibrated (OVX; n = 10). Data are compared with age-matched, intact controls (AC; n = 10). In vivo µCT analysis showed that OVX mice gained 43% total (P < 0.001) and 125% visceral adiposity (P < 0.001) compared with their baseline after 6 wk, whereas LIV gained only 21% total (P = 0.01) and 70% visceral adiposity (P < 0.01). Relative to AC, expression of adipogenic genes (PPARγ, FABP4, PPARδ, and FoxO1) was upregulated in OVX muscle (P < 0.05), whereas LIV reduced these levels (P < 0.05). Adipogenic gene expression was inversely related to the percentage of total and reserve satellite cell populations in the muscle, with both declining in OVX compared with AC (-21 and -28%, respectively, P < 0.01). LIV mitigated these declines (-11 and -17%, respectively). These results provide further evidence of the negative consequences of estrogen depletion and demonstrate that mechanical signals have the potential to interrupt subsequent adipogenic gene expression and satellite cell suppression, emphasizing the importance of physical signals in protecting musculoskeletal integrity and slowing the fat phenotype.

High fat diet rapidly suppresses B lymphopoiesis by disrupting the supportive capacity of the bone marrow niche.

The bone marrow (BM) niche is the primary site of hematopoiesis, and cues from this microenvironment are critical to maintain hematopoiesis. Obesity increases lifetime susceptibility to a host of chronic diseases, and has been linked to defective leukogenesis. The pressures obesity exerts on hematopoietic tissues led us to study the effects of a high fat diet (HFD: 60% Kcal from fat) on B cell development in BM. Seven week old male C57Bl/6J mice were fed either a high fat (HFD) or regular chow (RD) diet for periods of 2 days, 1 week and 6 weeks. B-cell populations (B220+) were not altered after 2 d of HFD, within 1 w B-cell proportions were reduced by -10%, and by 6 w by -25% as compared to RD (p<0.05). BM RNA was extracted to track the expression of B-cell development markers Il-7, Ebf-1 and Pax-5. At 2 d, the expression of Il-7 and Ebf-1 were reduced by -20% (p = 0.08) and -11% (p = 0.06) whereas Pax-5 was not significantly impacted. At one week, however, the expressions of Il-7, Ebf-1, and Pax-5 in HFD mice fell by -19%, -20% and -16%, and by six weeks were further reduced to -23%, -29% and -34% as compared to RD (p<0.05 for all), a suppression paralleled by a +363% increase in adipose encroachment within the marrow space (p<0.01). Il-7 is a critical factor in the early B-cell lineage which is secreted by supportive cells in the BM niche, and is necessary for B-cell commitment. These data indicate that BM Il-7 expression, and by extension B-cell differentiation, are rapidly impaired by HFD. The trend towards suppressed expression of Il-7 following only 2 d of HFD demonstrates how susceptible the BM niche, and the cells which rely on it, are to diet, which ultimately could contribute to disease susceptibility in metabolic disorders such as obesity.

The potential benefits and inherent risks of vibration as a non-drug therapy for the prevention and treatment of osteoporosis.

The delivery of mechanical signals to the skeleton using vibration is being considered as a non-drug treatment of osteoporosis. Delivered over a range of magnitudes and frequencies, vibration has been shown to be both anabolic and anti-catabolic to the musculoskeletal tissues, yet caution must be emphasized as these mechanical signals, particularly chronic exposure to higher intensities, is a known pathogen to many physiological systems. In contrast, accumulating preclinical and clinical evidence indicates that low intensity vibration (LIV) improves bone quality through regulating the activity of cells responsible for bone remodeling, as well as biasing the differentiation fate of their mesenchymal and hematopoietic stem cell progenitors. In vitro studies provide insights into the biologic mechanisms of LIV, and indicate that cells respond to these low magnitude signals through a distinct mechanism driven not by matrix strain but acceleration. These cell, animal, and human studies may represent the foundation of a safe, non-drug means to protect and improve the musculoskeletal system of the elderly, injured, and infirmed.

Separating Fluid Shear Stress from Acceleration during Vibrations in Vitro: Identification of Mechanical Signals Modulating the Cellular Response.

The identification of the physical mechanism(s) by which cells can sense vibrations requires the determination of the cellular mechanical environment. Here, we quantified vibration-induced fluid shear stresses in vitro and tested whether this system allows for the separation of two mechanical parameters previously proposed to drive the cellular response to vibration - fluid shear and peak accelerations. When peak accelerations of the oscillatory horizontal motions were set at 1g and 60Hz, peak fluid shear stresses acting on the cell layer reached 0.5Pa. A 3.5-fold increase in fluid viscosity increased peak fluid shear stresses 2.6-fold while doubling fluid volume in the well caused a 2-fold decrease in fluid shear. Fluid shear was positively related to peak acceleration magnitude and inversely related to vibration frequency. These data demonstrated that peak shear stress can be effectively separated from peak acceleration by controlling specific levels of vibration frequency, acceleration, and/or fluid viscosity. As an example for exploiting these relations, we tested the relevance of shear stress in promoting COX-2 expression in osteoblast like cells. Across different vibration frequencies and fluid viscosities, neither the level of generated fluid shear nor the frequency of the signal were able to consistently account for differences in the relative increase in COX-2 expression between groups, emphasizing that the eventual identification of the physical mechanism(s) requires a detailed quantification of the cellular mechanical environment.

Bone structure and B-cell populations, crippled by obesity, are partially rescued by brief daily exposure to low-magnitude mechanical signals.

Deterioration of the immune and skeletal systems, each of which parallel obesity, reflects a fragile interrelationship between adiposity and osteoimmunology. Using a murine model of diet-induced obesity, this study investigated the ability of mechanical signals to protect the skeletal-immune systems at the tissue, cellular, and molecular level. A long-term (7 mo) high-fat diet increased total adiposity (+62%), accelerated age-related loss of trabecular bone (-61%), and markedly reduced B-cell number in the marrow (-52%) and blood (-36%) compared to mice fed a regular diet. In the final 4 mo of the protocol, the application of low-magnitude mechanical signals (0.2 g at 90 Hz, 15 min/d, 5 d/wk) restored both bone structure and B cells to those levels measured in control mice fed a regular diet. These phenotypic outcomes were achieved, in part, by reductions in osteoclastic activity and a biasing of hematopoietic stem cell differentiation toward the lymphoid B-cell lineage and away from a myeloid fate. These results emphasize that obesity undermines both the skeletal and immune systems, yet brief exposure to mechanical signals, perhaps as a surrogate to the salutary influence of exercise, diminishes the consequences of diabetes and obesity, restoring bone structure and normalizing B-cell populations by biasing of the fate of stem cells through mechanosensitive pathways.

Low magnitude mechanical signals mitigate osteopenia without compromising longevity in an aged murine model of spontaneous granulosa cell ovarian cancer.

Cancer progression is often paralleled by a decline in bone mass, raising risk of fracture. Concerns persist regarding anabolic interventions for skeletal protection, as these may inadvertently exacerbate neoplastic tissue expansion. Given bone's inherent mechanosensitivity, low intensity vibration (LIV), a mechanical signal that encourages osteoblastogenesis, could possibly slow cancer-associated bone loss, but this goal must be achieved without fostering disease progression. Seventy 12w female F1-SWRxSWXJ-9 mice, a strain prone to developing granulosa cell tumors, were randomized into baseline control (BC: n=10), age-matched control (AC: n=30), and LIV (n=30), which received mechanical signals (90Hz @ 0.3g) for 15m/day, 5 day/w over the course of 1 year. Survival curves for AC (10 died) and LIV (8 died) followed similar trends (p=0.62), indicating longevity was unperturbed by LIV. At 1 year, bone volume of proximal tibiae in LIV mice was 25% greater than AC (p<0.02), while bone volume of L5 vertebrae was 16% higher in LIV over AC (p<0.02). Primary lesions and peripheral metastases were apparent in both LIV and AC; however, overall tumor incidence was approximately 30% less in LIV (p=0.27) and, when disease was evident, involved fewer organ systems (p=0.09). Marrow-derived mesenchymal stem cells (MSC) were 52% lower (p<0.01) in LIV, and 31% lower (p=0.08) in mice lacking pathology, suggesting higher MSC levels in this model of cancer susceptibility may have contributed to tumor progression. These experiments indicate that LIV helps protect bone mass in mice inherently susceptible to cancer without compromising life expectancy, perhaps through mechanical control of stem cell fate. Further, these data reflect the numerous system-level benefits of exercise in general, and mechanical signals in particular, in the preservation of bone density and the suppression of cancer progression.

Engineering anatomically shaped human bone grafts.

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.