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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Building predictive signaling models by perturbing yeast cells with time-varying stimulations resulting in distinct signaling responses

This protocol provides a step-by-step approach to perturb single cells with time-varying stimulation profiles, collect distinct signaling responses, and use these to infer a system of ordinary differential equations to capture and predict dynamics of protein-protein regulation in signal transduction pathways. The models are validated by predicting the signaling activation upon new cell stimulation conditions. In comparison to using standard step-like stimulations, application of diverse time-varying cell stimulations results in better inference of model parameters and substantially improves model predictions. For complete details on the use and results of this protocol, please refer to Jashnsaz et al. (2020).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Looking in the axion mirror: An all-sky analysis of stimulated decay

Axion dark matter (DM) produces echo images of bright radio sources via stimulated decay. These images appear as a faint radio line centered at half the axion mass, with the line width set by the DM velocity dispersion. Due to the kinematics of the decay, the echo can be emitted in the direction nearly opposite to the incoming source of stimulating radiation, meaning that axions effectively behave as imperfect monochromatic mirrors. We present an all-sky analysis of axion DM-induced echo images using extragalactic radio point sources, Galactic supernova remnants (SNRs), and Galactic synchrotron radiation (GSR) as sources of stimulating radiation. The aggregate signal strength is not significantly affected by unknown properties of individual sources of stimulating radiation, which we sample from an empirical distribution to generate an ensemble of realizations for the all-sky signal template. We perform forecasts for CHIME, HERA, CHORD, HIRAX, and BURSTT, finding that they can run as competitive axion experiments simultaneously with other objectives, requiring no new hardware.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2022

Foam Fracturing Study for Stimulation Development of Enhanced Geothermal Systems

The large thermal gradients and high subsurface temperatures of the western region of the U.S. hold great potential for the implementation of enhanced geothermal systems (EGS). The development of these potential EGS resources requires stimulation of the reservoir to enhance permeability and it has been widely reported that a substantial amount of water will be required should conventional hydraulic stimulation be used. This presents a huge challenge and a high risk to the geothermal development because the water stress1 in these areas is already high or extremely high. The use of foam, a gas/liquid mixture predominantly composed of gas, in fracturing is considered and explored in this project as a potential approach to address water concerns with hydraulic stimulation in the development of EGS. This project, led by Oak Ridge National Laboratory (ORNL) in collaboration with Temple University, was awarded in an open lab call in 2018, and was part of the DOE GTO waterless stimulation initiative. The goal of the project was to demonstrate the feasibility of foam fracturing for EGS development through two primary tasks: Task 1: Laboratory study of the effectiveness of foam fracturing for representative geological materials, including cyclic pressurization using foam (led by ORNL) and Task 2: High temperature foam material selection and characterization (led by Temple University). In FY19, ORNL finished the critical review on serval issues associated with foam fracturing and the implementation of the proposed tasks in a lab study (Wang, et al., 2019), and completed the foam fracturing testing using cement as a model material (Wang, et al, 2020a). The work at ORNL was geared up to develop a brand-new foam testing system in FY20. The purchase of main components for the new system was finished in the first half of the FY20. The assembly of the foam testing system and foam fracturing testing were completed in the second half of the FY20 (Wang, et al., 2021a). Task 1 required the development of a test system which can be used to perform hydraulic fracturing of geological specimens with both water and foamed liquids at pressure up to 6,000 psi (41.4 MPa). The system possesses several capabilities that conventional injection systems lack for hydraulic fracturing. In addition to its ability to generate foam with controlled quality, it is capable of cycling pressure levels between specified values up to frequencies of 50 Hz. The latter capability was developed to evaluate the hypothesis that cyclic loading of samples would produce enhanced fracturing. The system consists of two sections: one for foam generation and another for foam injection. The foam is generated through separate control and pressurization of liquid and gas phases with controlled flow rates. The injection section is equipped with a low-flow Coriolis flow that monitors the density of foam to ensure the injection is in the range of target foam quality2. Experimental results of foam fracturing are reported for cylindrical granite specimens using water and aqueous N2 foam as the fracturing fluids. All experiments were performed for unconfined conditions. The effects of injection mode (i.e., monotonic vs cyclic pressurization) on breakdown pressure and failure response sample were investigated using water alone as a fracturing fluid and foams with a range of compositions. It was found that in the case of monotonic injection, the breakdown pressure of granite specimens tended to be slightly higher when fracturing with foam. Additionally, with a foam quality of 90%, the water use can be reduced by 50 to 84%, depending on hole size. On the other hand, it was observed that the breakdown pressure can be brought down to 70% of the monotonic breakdown pressure by using low cycle fatigue. Finally, discussions are presented regarding injectivity and water use reduction.

15 GEOTHERMAL ENERGY↗
NASA NTRS1980

Technique of electrical stimulation of the vestibular analyzer under clinical conditions

Vestibular reactions appear under the action of direct current (dc) on the labyrinth of man and animals. A decrease of the stimulation effect of dc on the extralabyrinthine nervous formations in the suggested method is achieved by the use of electric pulses with steep front and back parts, as well as by previous anesthetization of the skin in the electrode application area by means of novocain solution electrophoresis. For this purpose a pulse producer giving trapezoid pulses with smoothly changing fronts and duration was constructed. With the help of an interrupter it is possible to stop the current increase instantly, and stimulation is performed at the level of the pulse 'plateau'. To induce vestibular reactions under monopolar stimulation, it is necessary to apply the current twice as high as that with bipolar electrode position. The use of short pulses with steep front and back parts for electrode stimulation of the vestibular analyzer is considered to be inexpedient.

Khechinashvili, S. N.↗
NASA NTRS1986

The effective intensity of Coriolis, cross-coupling stimulation is gravitoinertial force dependent - Implications for space motion sickness

The effect of gravity on the severity of the Coriolis-induced motion sickness was investigated in ten individuals subjected to high and low G-force phases of parabolic flight maneuvers using constant level Coriolis, cross-coupled angular acceleration stimulation. Using seven levels of severity in the diagnosis of motion sickness, it was found that the subjects were less susceptible at 0 G than at +2 Gz, and that the perceived intensity and provocativeness of Coriolis stimulation decreased in 0 G and increased in +2 Gz relative to the +1 Gz baseline values. The changes in the apparent intensity of Coriolis stimulation occur virtually immediately when the background gravitatioinertial force level is varied. These findings explain why the Skylab astronauts were refractory to motion sickness during Coriolis stimulation in-flight.

Lackner, J. R.↗
NASA NTRS1991

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins E2 and F2(alpha) which regulate protein turnover rates and muscle cell growth. Mechnical stimulation significantly increases the breakdown rate of (3)H-arachidonic acid labelled phospholipids, releasing free (3)H-arachidonic acid, and the rate-limiting precursor of prostaglandin synthesis. Mechanical stimulation also significantly increases (3)H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-2-(3)H inositol labelled phospholipids. Phospholipase A2, phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are activated by stretch. The lipase inhibitors bromophenacylbromide and RHC80267 together reduce stretch-induced prostaglandin production by 73-83 percent. The stretch-induced increases in prostaglandin production, (3)H-arachidonic acid labelled phospholipid breakdown, and (3)H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-2-(3)H inositol labelled phospholipids are dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and prostaglandins through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

Vandenburgh, Herman H.↗
NASA NTRS1992

Magnetized stimulated scattering in pulsar winds

The effects of stimulated scattering on a collimated high brightness temperature beam of photons traversing a relativistically streaming magnetized plasma are studied. Under the assumption that the center of the photon beam is parallel to the bulk motion, we calculate the scattering rate as a function of the angular spread of the beam and the Lorentz factor gamma. Magnetization changes the photon recoil, without which stimulated scattering has no effect. It also introduces a strong dependence on frequency and polarization: if the photon frequency matches the electron cyclotron frequency, the scattering rate of photons polarized perpendicular to the magnetic field can be substantially enhanced relative to Thomson, and if the photon frequency is much less than the cyclotron frequency, the scattering is suppressed. Applying these calculations to pulsars, we find that stimulated scattering of the radio beam in the magnetized wind believed to exist outside the light cylinder can substantially alter the spectrum and polarization state of the radio signal. We suggest that the scattering rate is so high in some pulsars that the ability of the radio signal to penetrate the pulsar magnetosphere requires modification of either the conventional model of the magnetosphere or assumptions about the effects of stimulated scattering upon a beam.

Sincell, Mark W.↗
NASA NTRS1993

Mechanical stimulation of skeletal muscle mitigates glucocorticoid induced decreases in prostaglandin synthesis

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content of tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the role of prostaglandins as growth modulators in these processes was examined. Dex at 10(exp -8) M reduced PGF(sub 2(alpha)) production 55 percent - 65 percent and PGE(sub 2) production 84 - 90 percent after 24 - 72 h of incubation in static cultures. Repetitive 10 percent stretch-relaxations of the non-Dex treated cultures increased PGF(sub 2(alpha)) efflux 41 percent at 24 h and 276 percent at 72 h and increased PGE(sub 2) production 51 percent at 24 h and 236 percent at 72 h. Mechanical stimulation of Dex treated cultures increased PGF(sub 2(alpha)) production 162 percent after 24 h, thus returning PGF(sub 2(alpha)) efflux to the level of non-Dex treated cultures. At 72 h, stretch increased PGF(sub 2(alpha)) efflux 65 percent in Dex treated cultures, but PGF(sub 2(alpha)) production was 45-84 percent less than non-Dex treated cultures. Mechanical stimulation of Dex treated cultures increased PGE(sub 2) production at 24 h, but not at 72 h. Dex reduced prostaglandin H synthase (PGHS) activity in the muscle cultures by 70 percent after 8 - 24 h of incubation, and mechanical stimulation increased PGHS activity of the Dex treated cultures by 98 percent. It is concluded that repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by reversing the Dex-induced declines in PGHS activity and prostaglandin production.

Chromiak, Joseph A.↗
NASA NTRS1988

A precision mechanical nerve stimulator

An electromechanical device, used to apply and monitor stimulating pulses to a mammalian motor nerve, has been successfully developed at NASA Langley Research Center. Two existing force transducers, a flight skin friction balance and a miniature skin friction balance which were designed for making aerodynamic drag measurements, were modified and incorporated to form this precision instrument. The nerve stimulator is a type one servomechanism capable of applying and monitoring stimulating pulses of 0 to 10 grams with a precision of better than +/- 0.05 grams. Additionally, the device can be independently used to apply stimulating pulses by displacing the nerve from 0 to 0.25 mm with a precision of better than +/- 0.001 mm while measuring the level of the load applied.

Tcheng, Ping↗
NASA NTRS2003

Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

Vercoutere, W.↗
NASA NTRS1990

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.

NASA Discipline Number 29-20↗
NASA NTRS1999

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

NASA Discipline Regulatory Physiology↗
NASA NTRS1994

An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

NASA Discipline Musculoskeletal↗
NASA NTRS2005

Concept Developed for an Implanted Stimulated Muscle-Powered Piezoelectric Generator

Implanted electronic devices are typically powered by batteries or transcutaneous power transmission. Batteries must be replaced or recharged, and transcutaneous power sources burden the patient or subject with external equipment prone to failure. A completely self-sustaining implanted power source would alleviate these limitations. Skeletal muscle provides an available autologous power source containing native chemical energy that produces power in excess of the requirements for muscle activation by motor nerve stimulation. A concept has been developed to convert stimulated skeletal muscle power into electrical energy (see the preceding illustration). We propose to connect a piezoelectric generator between a muscle tendon and bone. Electrically stimulated muscle contractions would exert force on the piezoelectric generator, charging a storage circuit that would be used to power the stimulator and other devices.

Lewandowski, Beth↗
NASA NTRS2009

Cytotoxic Effects of Re-Activated Lunar Dust Stimulant on Human Lung Cells

Lunar dust has been of significant concern due to various problems observed on the Apollo missions. Reports from astronauts have shown that the dust may have caused eye and nasal irritation as well as possible hay fever like symptoms. As NASA hopes to go to the Moon within the next few years, we hope to understand the possible toxic effects the dust might have. In these studies, we are looking at the effect of "re-activated" lunar dust stimulant on human bronchial cells. A simple grinding analog as a method of simulating micrometeorite crushing on the moon is used to "activate" the dust stimulant, i.e. capable of producing hydroxyl radicals. These radicals could then interact with human cells and may lead to a loss in membrane integrity and cell death. (Castranova, 1994) Cells are exposed to the dust for 6 and 24 hour intervals to assess cytotoxicity. Cytotoxicity is measured by looking at the production of inflammatory cytokines. Cells are exposed to ground and unground stimulant and compared to cytokine production from cells exposed to quartz which have a known toxicity. Here we look at the cytotoxicity of the lunar dust stimulant relative to quartz by measuring the production of inflammatory cytokines.

Upadhyaya, Krishna↗
NASA NTRS2016

Estimation of Optimum Stimulus Amplitude for Balance Training using Electrical Stimulation of the Vestibular System

Sensorimotor changes such as posture and gait instabilities can affect the functional performance of astronauts after gravitational transitions. Sensorimotor Adaptability (SA) training can help alleviate decrements on exposure to novel sensorimotor environments based on the concept of 'learning to learn' by exposure to varying sensory challenges during posture and locomotion tasks (Bloomberg 2015). Supra-threshold Stochastic Vestibular Stimulation (SVS) can be used to provide one of many challenges by disrupting vestibular inputs. In this scenario, the central nervous system can be trained to utilize veridical information from other sensory inputs, such as vision and somatosensory inputs, for posture and locomotion control. The minimum amplitude of SVS to simulate the effect of deterioration in vestibular inputs for preflight training or for evaluating vestibular contribution in functional tests in general, however, has not yet been identified. Few studies (MacDougall 2006; Dilda 2014) have used arbitrary but fixed maximum current amplitudes from 3 to 5 mA in the medio-lateral (ML) direction to disrupt balance function in healthy adults. Giving this high level of current amplitude to all the individuals has a risk of invoking side effects such as nausea and discomfort. The goal of this study was to determine the minimum SVS level that yields an equivalently degraded balance performance. Thirteen subjects stood on a compliant foam surface with their eyes closed and were instructed to maintain a stable upright stance. Measures of stability of the head, trunk, and whole body were quantified in the ML direction. Duration of time they could stand on the foam surface was also measured. The minimum SVS dosage was defined to be that level which significantly degraded balance performance such that any further increase in stimulation level did not lead to further balance degradation. The minimum SVS level was determined by performing linear fits on the performance variable at different stimulation levels. Results from the balance task suggest that there are inter-individual differences and the minimum SVS amplitude was found to be in the range of 1 mA to 2.5 mA across subjects. SVS resulted in an average decrement of balance task performance in the range of 62%-73% across different measured variables at the minimum SVS amplitude in comparison to the control trial (no stimulus). Training using supra-threshold SVS stimulation is one of the sensory challenges used for preflight SA training designed to improve adaptability to novel gravitational environments. Inter-individual differences in response to SVS can help customize the SA training paradigms using minimal dosage required. Another application of using SVS is to simulate acute deterioration of vestibular sensory inputs in the evaluation of tests for assessing vestibular function.

Goel, R.↗
NASA NTRS2017

Brain Activations for Vestibular Stimulation and Dual Tasking Change with Spaceflight

Previous studies have documented the effects of spaceflight on human physiology and behavior, including muscle mass, cardiovascular function, gait, balance, manual motor control, and cognitive performance. An understanding of spaceflight-related changes provides important information about human adaptive plasticity and facilitates future space travel. In the current study, we evaluated how brain activations associated with vestibular stimulation and dual tasking change as a function of spaceflight. Five crewmembers were included in this study. The durations of their spaceflight missions ranged from 3 months to 7 months. All of them completed at least two preflight assessments and at least one postflight assessment. The preflight sessions occurred, on average, about 198 days and 51 days before launch; the first postflight sessions were scheduled 5 days after return. Functional MRI was acquired during vestibular stimulation and dual tasking, at each session. Vestibular stimulation was administered via skull taps delivered by a pneumatic tactile pulse system placed over the lateral cheekbones. The magnitude of brain activations for vestibular stimulation increased with spaceflight relative to the preflight levels, in frontal areas and the precuneus. In addition, longer flight duration was associated with greater preflight-to-postflight increases in vestibular activation in frontal regions. Functional MRI for finger tapping was acquired during both single-task (finger tapping only) and dual-task (simultaneously performing finger tapping and a secondary counting task) conditions. Preflight-to-post-spaceflight decreases in brain activations for dual tasking were observed in the right postcentral cortex. An association between flight duration and amplitude of flight-related change in activations for dual tasking was observed in the parietal cortex. The spaceflight-related increase in vestibular brain activations suggests that after a long-term spaceflight, more neural resources are required to process vestibular input.

Yuan, Peng↗
NASA NTRS2017

Modulation of Bone Marrow Primary Cell Osteoblastogenesis and Cell Senescence by Mechanical Stimulation

Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. Specifically CDKN1A/p-21 a cell senescence protein, was found to be upregulated in osteoprecursor cells of the femur during 15-day spaceflight, leading to the working hypothesis that CDKN1A/p-21 plays a role in inhibition of bone formation via mechanical regulation. To evaluate this hypothesis, utilizing a p-21 knockout mouse-line and relevant wildtype control, we cultured femoral bone marrow primary cells under unloaded (static) and cyclically stretched loading through a 30 day osteoblastogenesis protocol. Morphologic evaluation of the cultures demonstrated that mechanical stretching aligned the cells and increased the presence of defined focal adhesion expressing talin, integrin αvβ3, and PTK2 protein tyrosine kinase 2, also known as focal adhesion kinase (FAK) in both mouse strains. In corroboration with previous investigations of cell survival signals relation to FAK, our study found that with greater concentration of focal adhesions via stretch stimulation the live cell percentage was significantly higher than the unloaded controls (p-21 knockout line: +49.70%, p*=0.009, wildtype control: +18.14%, p*=.01). Also evaluated was the mineralization and ECM secretion capability of the differentiating cells. Von Kossa staining has shown that in the p-21 knockout cells unloaded cells produce more matrix that the stretch stimulated, however the matrix is unorganized presenting in sporadic nodules covering approximately 30% of the culture area at day 14 (n=6 wells) while the stretch stimulated cultures have less mineralization content the surface area containing mineralized matrix is greater (~68% at day 14). Q-PCR evaluation of the p-21 knockout cells revealed that canonical (β-catenin cascade) and non-canonical wnt11 and downstream planar cell polarity (wnt/PCP) pathway molecule RAC1 are prevalently upregulated with mechanical stimulation. Immunofluorescence for β-catenin and RAC1 showed co-localization at the nuclear membrane of the p-21 knockout cells but not the wildtype (n=1) suggesting that molecular communication via the canonical and wnt/PCP pathway are initiated by mechanical loading and experience regulation along the signaling cascade by CDKN1A/p-21. Future investigations will further elucidate this relationship and provide causal data demonstrating mechanical loading’s modulatory effect on p-21 expression change.

Juran, Cassandra M.↗