The vacuolated morphology of chordoma cells is dependent on cytokeratin intermediate filaments.

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.

A computational model to describe the regional interlamellar shear of the annulus fibrosus.

Interlamellar shear may play an important role in the homeostasis and degeneration of the intervertebral disk. Accurately modeling the shear behavior of the interlamellar compartment would enhance the study of its mechanobiology. In this study, physical experiments were utilized to describe interlamellar shear and define a constitutive model, which was implemented into a finite element analysis. Ovine annulus fibrosus (AF) specimens from three locations within the intervertebral disk (lateral, outer anterior, and inner anterior) were subjected to in vitro mechanical shear testing. The local shear stress-stretch relationship was described for the lamellae and across the interlamellar layer of the AF. A hyperelastic constitutive model was defined for interlamellar and lamellar materials at each location tested. The constitutive models were incorporated into a finite element model of a block of AF, which modeled the interlamellar and lamellar layers using a continuum description. The global shear behavior of the AF was compared between the finite element model and physical experiments. The shear moduli at the initial and final regions of the stress-strain curve were greater within the lamellae than across the interlamellar layer. The difference between interlamellar and lamellar shear was greater at the outer anterior AF than at the inner anterior region. The finite element model was shown to accurately predict the global shear behavior or the AF. Future studies incorporating finite element analysis of the interlamellar compartment may be useful for predicting its physiological mechanical behavior to inform the study of its mechanobiology.

Enhancing biocompatibility of D-oligopeptide hydrogels by negative charges.

Oligopeptide hydrogels are emerging as useful matrices for cell culture with commercial products on the market, but L-oligopeptides are labile to proteases. An obvious solution is to create D-oligopeptide hydrogels, which lack enzymatic recognition. However, D-oligopeptide matrices do not support cell growth as well as L-oligopeptide matrices. In addition to chiral interactions, many cellular activities are strongly governed by charge-charge interactions. In this work, the effects of chirality and charge on human mesenchymal stem cell (hMSC) behavior were studied using hydrogels assembled from oppositely charged oligopeptides. It was found that negative charges significantly improved hMSC viability and proliferation in D-oligopeptide gels but had little effect on their interactions with L-oligopeptide gels. This result points to the possibility of using charge and other factors to engineer biomaterials whose chirality is distinct from that of natural biomaterials, but whose performance is close to that of natural biomaterials.

A biomechanical comparison of multidirectional nail and locking plate fixation in unstable olecranon fractures.

BACKGROUND: The main theoretic advantage of proximal olecranon fracture intramedullary fixation is decreased soft-tissue irritation and, potentially, less subsequent hardware removal. Despite this possible benefit, questions remain as to whether intramedullary devices are capable of controlling olecranon fractures to the same extent as locking plates. This study evaluates the ability of a novel multidirectional locking nail to stabilize comminuted fractures and directly compares its biomechanical performance with that of locking olecranon plates. MATERIALS AND METHODS: We implanted 8 stainless steel locking plates and stainless steel intramedullary nails to stabilize a simulated comminuted fracture in 16 fresh-frozen cadaveric elbows. Flexion-extension, varus-valgus, gap distance, and rotational 3-dimensional angular displacement analysis was conducted over a 60° motion arc (30° to 90°) to assess fragment motion through physiologic cyclic arcs of motion and failure loading. Displacements in all planes were compared. RESULTS: Both implants showed less than 1° of motion in all measured planes and allowed less than 1 mm of gapping through all loads tested until ultimate failure. All failures occurred by sudden, catastrophic means. The mean failure weight for the nail was 14.4 kg compared with 8.7 kg for the plate (P = .02). The nail survived 1102 cycles, whereas the plate survived 831 cycles (P = .06). CONCLUSION: In simulated comminuted olecranon fractures, the multidirectional locking intramedullary nails sustained significantly higher maximum loads than the locking plates. The two implants showed no significant differences in fragment control or number of cycles survived. Surgeons can expect the multidirectional locking nails to stabilize comminuted fractures at least as well as locking plates.

The effect of distal radius locking plates on articular contact pressures.

PURPOSE: Fractures of the distal radius are among the most common injuries treated in hand surgery practice, and distal radius locking plates have become an increasingly popular method of fixation. Despite widespread use of this technology, it is unknown whether the subchondral placement of locking screws affects the loading profile of the distal radius. Our study was designed to determine whether subchondral locking screws change the articular contact pressures in the distal radius. METHODS: Twelve cadaveric forearms underwent a previously described axial loading protocol in a materials testing machine. We used an intra-articular, real-time computerized force sensor to measure peak contact pressure, total pressure, and contact area in the distal radius. Internal validation of sensor placement and reproducibility was conducted. Each specimen was tested before fixation (control), after application of a palmar distal radius locking plate, and after simulation of a metaphyseal fracture. RESULTS: We identified no statistically significant differences in maximum pressure, total pressure, and contact area among control, plated, and plated and fractured specimens. However, the contact footprint-represented by squared differences in force across the sensor-were significantly different between the control group and both plated groups. CONCLUSIONS: The technique for measuring contact pressures produces highly repeatable values. Distal radius locking plates with subchondral hardware placement do not seem to significantly change articular contact pressures.

Environmental regulation of notochordal gene expression in nucleus pulposus cells.

Cells of the nucleus pulposus (NP) in the intervertebral disc are derived directly from the embryonic notochord. In humans, a shift in NP cell population coincides with the beginning of age-related changes in the extracellular matrix that can lead to spinal disorders. To begin identifying the bases of these changes, the manner by which relevant environmental factors impact cell function must be understood. This study investigated the roles of biochemical, nutritional, and physical factors in regulating immature NP cells. Specifically, we examined cell morphology, attachment, proliferation, and expression of genes associated with the notochord and immature NP (Sox9, CD24, and type IIA procollagen). Primary cells isolated from rat caudal discs were exposed to different media formulations and physical culture configurations either in 21% (ambient) or 2% (hypoxic) O2. As expected, cells in alginate beads retained a vacuolated morphology similar to chordocytes, with little change in gene expression. Interestingly, NP tissues not enzymatically digested were more profoundly influenced by oxygen. In monolayer, alpha-MEM preserved vacuolated morphology, produced the highest efficiency of attachment, and best maintained gene expression. DMEM and Opti-MEM cultures resulted in high levels of proliferation, but these appeared to involve small non-vacuolated cells. Gene expression patterns for cells in DMEM monolayer cultures were consistent with chondrocyte de-differentiation, with the response being delayed by hypoxia. Overall, results indicate that certain environmental conditions induce cellular changes that compromise the notochordal phenotype in immature NP. These results form the foundation on which the mechanisms of such changes can be elucidated.

Miniature fiber optic pressure sensor with composite polymer-metal diaphragm for intradiscal pressure measurements.

We developed a miniature fiber optic pressure sensor system and utilized it for in vitro intradiscal pressure measurements for rodents. One of the unique features of this work is the design and fabrication of a sensor element with a multilayer polymer-metal diaphragm. This diaphragm consists of a base polyimide layer (150 nm thick), a metal reflective layer (1 microm thick), and another polyimide layer for protection and isolation (150 nm thick). The sensor element is biocompatible and can be fabricated by simple, batch-fabrication methods in a non-cleanroom environment with good device-to-device uniformity. The fabricated sensor element has an outer diameter of only 366 microm, which is small enough to be inserted into the rodent discs without disrupting the structure or altering the intradiscal pressures. In the calibration and in vitro rodent intradiscal pressure measurements, the sensor element exhibits a linear response to the applied pressure over the range of 0-70 kPa, with a sensitivity of 0.0206 microm/kPa and a resolution of 0.17 kPa. To our best knowledge, this work is the first successful demonstration of rodent intradiscal pressure measurements.

Anulus fibrosus tension inhibits degenerative structural changes in lamellar collagen.

Mechanical stress is one of the risk factors believed to influence intervertebral disc degeneration. Animal models have shown that certain regimes of compressive loading can induce a cascade of biological effects that ultimately results in cellular and structural changes in the disc. It has been proposed that both cell-mediated breakdown of collagen and the compromised stability of collagen with loss of anular tension could result in degradation of lamellae in the anulus fibrosus (AF). To determine whether this may be important in the AF, we subjected entire rings of de-cellularized AF tissue to MMP-1 digestion with or without tension. Biomechanical testing found trends of decreasing strength and stiffness when tissues were digested without tension compared with those with tension. To determine the physiologic significance of tissue level tension in the AF, we used an established in vivo murine model to apply a disc compression insult known to cause degeneration. Afterward, that motion segment was placed in fixed-angle bending to impose tissue level tension on part of the AF and compression on the contralateral side. We found that the AF on the convex side of bending retained a healthy lamellar appearance, while the AF on the concave side resembled tissues that had undergone degeneration by loading alone. Varying the time of onset and duration of bending revealed that even a brief duration applied immediately after cessation of compression was beneficial to AF structure on the convex side of bending. Our results suggest that both cell-mediated events and cell-independent mechanisms may contribute to the protective effect of tissue level tension in the AF.

Correction to: Deformability of Human Mesenchymal Stem Cells Is Dependent on Vimentin Intermediate Filaments.

This erratum is to correct the following: (1) in the Western Blotting subsection under the Materials and Methods section, the concentration of protein from each sample loaded into Criterion Tris-HCl gels was incorrectly stated as 155 µg of protein. The correct value is 9.7 µg; (2) in Fig. 1b, the bar graph showed incorrect values for semi-quantitation of Western blots. Figure 1 has been updated with a corrected graph in Fig. 1b only.

Deformability of Human Mesenchymal Stem Cells Is Dependent on Vimentin Intermediate Filaments.

Mesenchymal stem cells (MSCs) are being studied extensively due to their potential as a therapeutic cell source for many load-bearing tissues. Compression of tissues and the subsequent deformation of cells are just one type physical strain MSCs will need to withstand in vivo. Mechanotransduction by MSCs and their mechanical properties are partially controlled by the cytoskeleton, including vimentin intermediate filaments (IFs). Vimentin IF deficiency has been tied to changes in mechanosensing and mechanical properties of cells in some cell types. However, how vimentin IFs contribute to MSC deformability has not been comprehensively studied. Investigating the role of vimentin IFs in MSC mechanosensing and mechanical properties will assist in functional understanding and development of MSC therapies. In this study, we examined vimentin IFs' contribution to MSCs' ability to deform under external deformation using RNA interference. Our results indicate that a deficient vimentin IF network decreases the deformability of MSCs, and that this may be caused by the remaining cytoskeletal network compensating for the vimentin IF network alteration. Our observations introduce another piece of information regarding how vimentin IFs are involved in the complex role the cytoskeleton plays in the mechanical properties of cells.

When Should We Change Drill Bits? A Mechanical Comparison of New, Reprocessed, and Damaged Bits.

OBJECTIVES: We assessed how reprocessed and damaged drill bits perform relative-to-new drill bits in terms of drilling force required, heat generated at near and far cortices, and number of usable passes. METHODS: Nine pairs of nonosteoporotic human cadaveric femora were tested using 3 types of 3.2-mm drill bits (new, reprocessed, and damaged) in 3 investigations (force, temperature, and multiple usable passes). Operating room conditions were simulated. Force and temperature data were collected for each type. The multiple pass investigation measured only force. RESULTS: New and reprocessed drill bits performed similarly regarding force required and heat generated; both outperformed damaged bits. New and reprocessed bits had a similar number of usable passes in ideal conditions. Damaged bits required nearly 2.6 times as much force to maintain drilling rate. CONCLUSIONS: Reprocessed drill bits seem to be a viable alternative to new drill bits for fracture treatment surgery in terms of force required, heat generated, and number of usable passes. Drill bits that are damaged intraoperatively should be replaced. In ideal conditions, new and reprocessed drill bits can be used for multiple consecutive cases. CLINICAL RELEVANCE: Reprocessed drill bits may be as effective as new drill bits, representing potential cost savings for institutions. Both types can be considered for reuse.

Biomechanical Comparison of Cadaveric and Commercially Available Synthetic Osteoporotic Bone Analogues in a Locked Plate Fracture Model Under Torsional Loading.

OBJECTIVES: Biomechanical studies of osteoporotic bone have used synthetic models rather than cadaveric samples because of decreased variability, increased availability, and overall ease of the use of synthetic models. We compared the torsional mechanical properties of cadaveric osteoporotic bone with those of currently available synthetic osteoporotic bone analogues. METHODS: We tested 12 osteoporotic cadaveric humeri and 6 specimens each of 6 types of synthetic analogues. A 5-mm fracture gap model and posterior plating technique with 4.5-mm narrow 10-hole locking compression plate were used. Torque was applied to a peak of ±10 N·m for 1000 cycles at 0.3 Hz. Data were continuously collected during cyclical and ramped loading with a servohydraulic materials testing system. RESULTS: Cadaveric bone had a 17% failure rate before completing 1000 cycles. Three osteoporotic bone models had 100% failure (P < 0.05), 2 had 17% failure, and 1 had 0% failure before 1000 cycles. Significant differences in the stiffness of the 3 types of synthetic bone models that survived cyclic loading were noted compared with the cadaveric bone model (P < 0.05). Osteoporotic bone analogues had torsional mechanical properties different from those of osteoporotic cadaveric specimens. CONCLUSIONS: The differences between osteoporotic cadaveric humeri and synthetic osteoporotic bone analogues ranged from profound with complete catastrophic failure after a few cycles to subtler differences in stiffness and strain hardening. These findings suggest that different bone analogue models vary substantially in their torsional mechanical properties and might not be appropriate substitutes for cadaveric bone in biomechanical studies of osteoporotic bone.

Biomechanical and Cost Comparisons of Near-Far and Pin-Bar Constructs.

Orthopedic dogma states that external fixator stiffness is improved by placing 1 pin close to the fracture and 1 as distant as possible ("near-far"). This fixator construct is thought to be less expensive than placing pins a shorter distance apart and using "pin-bar" clamps that attach pins to outriggers. The authors therefore hypothesized that the near-far construct is stiffer and less expensive. They compared mechanical stiffness and costs of near-far and pin-bar constructs commonly used for temporary external fixation of femoral shaft fractures. Their testing model simulated femoral shaft fractures in damage control situations. Fourth-generation synthetic femora (n=18) were used. The near-far construct had 2 pins that were 106 mm apart, placed 25 mm from the gap on each side of the fracture. The pin-bar construct pins were 55 mm apart, placed 40 mm from the gap. Mechanical testing was performed on a material test system machine. Stiffness was determined in the linear portion of the load-displacement curve for both constructs in 4 modes: axial compression, torsional loading, frontal plane 3-point bending, and sagittal plane 3-point bending. Costs were determined from a 2012 price guide. Compared with the near-far construct, the pin-bar construct had stiffness increased by 58% in axial compression (P<.05) and by 52% in torsional loading (P<.05). The pin-bar construct increased cost by 11%. In contrast to the authors' hypothesis and existing orthopedic dogma, the near-far construct was less stiff than the pin-bar construct and was similarly priced. Use of the pin-bar construct is mechanically and economically reasonable. [Orthopedics. 2017; 40(2):e238-e241.].

A Biomechanical Comparison of Allograft Tendons for Ligament Reconstruction.

BACKGROUND: Allograft tendons are frequently used for ligament reconstruction about the knee, but they entail availability and cost challenges. The identification of other tissues that demonstrate equivalent performance to preferred tendons would improve limitations. Hypothesis/Purpose: We compared the biomechanical properties of 4 soft tissue allograft tendons: tibialis anterior (TA), tibialis posterior (TP), peroneus longus (PL), and semitendinosus (ST). We hypothesized that allograft properties would be similar when standardized by the looped diameter. STUDY DESIGN: Controlled laboratory study. METHODS: This study consisted of 2 arms evaluating large and small looped-diameter grafts: experiment A consisted of TA, TP, and PL tendons (n = 47 each) with larger looped diameters of 9.0 to 9.5 mm, and experiment B consisted of TA, TP, PL, and ST tendons (n = 53 each) with smaller looped diameters of 7.0 to 7.5 mm. Each specimen underwent mechanical testing to measure the modulus of elasticity (E), ultimate tensile force (UTF), maximal elongation at failure, ultimate tensile stress (UTS), and ultimate tensile strain (UTε). RESULTS: Experiment A: No significant differences were noted among tendons for UTF, maximal elongation at failure, and UTϵ. UTS was significantly higher for the PL (54 MPa) compared with the TA (44 MPa) and TP (43 MPa) tendons. E was significantly higher for the PL (501 MPa) compared with the TP (416 MPa) tendons. Equivalence testing showed that the TP and PL tendon properties were equivalent or superior to those of the TA tendons for all outcomes. Experiment B: All groups exhibited a similar E. UTF was again highest in the PL tendons (2294 N) but was significantly different from only the ST tendons (1915 N). UTϵ was significantly higher for the ST (0.22) compared with the TA (0.19) and TP (0.19) tendons. Equivalence testing showed that the TA, TP, and PL tendon properties were equivalent or superior to those of the ST tendons. CONCLUSION: Compared with TA tendons, TP and PL tendons of a given looped diameter exhibited noninferior initial biomechanical strength and stiffness characteristics. ST tendons were mostly similar to TA tendons but exhibited a significantly higher elongation/UTϵ and smaller cross-sectional area. For smaller looped-diameter grafts, all tissues were noninferior to ST tendons. In contrast to previous findings, PL tendons proved to be equally strong. CLINICAL RELEVANCE: The results of this study should encourage surgeons to use these soft tissue allografts interchangeably, which is important as the number of ligament reconstructions performed with allografts continues to rise.

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Ultrasonic Structural Health Monitoring to Assess the Integrity of Spinal Growing Rods In Vitro.

BACKGROUND: Rod fracture is a common complication of growing rods and can result in loss of correction, patient discomfort, and unplanned revision surgery. The ability to quantitate rod integrity at each lengthening would be advantageous to avoid this complication. We investigate the feasibility of applying structural health monitoring to evaluate the integrity of growing rods in vitro. METHODS: Single-rod titanium 4.5-mm growing rod constructs (n = 9), one screw proximally and one distally connected by in-line connectors, were assembled with pedicle screws fixed in polyethylene blocks. Proximal and distal ends were loaded and constructs subjected to cyclic axial compression (0-100 N at 1 Hz), with incrementally increasing maximum compressive loads of 10 N every 9k cycles until failure. Four piezoceramic transducers (PZTs) were mounted along the length the constructs to interrogate the integrity of the rods with an ultrasonic, guided lamb wave approach. Every 9k cycles, an 80 V excitatory voltage was applied to a PZT to generate high-frequency vibrations, which, after propagating through the construct, was detected by the remaining PZTs. Amplitude differences between pre- and postload waveform signals were calculated until rod failure. RESULTS: Average construct lifetime was 88,991 ± 13,398 cycles. All constructs failed due to rod fracture within 21 mm (mean = 15 ± 4.5 mm) of a screw or connector. Amplitude differences between pre- and postload increased in a stepwise fashion as constructs were cycled. Compared to baseline, we found a 1.8 ± 0.6-fold increase in amplitude 18k cycles before failure, a 2.2 ± 1.0-fold increase in amplitude 9k cycles before failure, and a 2.75 ± 1.5-fold increase in amplitude immediately before rod fracture. CONCLUSION: We describe a potential method for assessing the structural integrity of growing rods using ultrasonic structural health monitoring. These preliminary data demonstrate the ability of periodic rod assessment to detect structural changes in cycled growing rods, which appear to correspond to subclinical rod fatigue before rod fracture.

Optical Coherence Tomographic Elastography Reveals Mesoscale Shear Strain Inhomogeneities in the Annulus Fibrosus.

STUDY DESIGN: Basic science study using in vitro tissue testing and imaging to characterize local strains in annulus fibrosus (AF) tissue. OBJECTIVE: To characterize mesoscale strain inhomogeneities between lamellar and inter-/translamellar (ITL) matrix compartments during tissue shear loading. SUMMARY OF BACKGROUND DATA: The intervertebral disc is characterized by significant heterogeneities in tissue structure and plays a critical role in load distribution and force transmission in the spine. In particular, the AF possesses a lamellar architecture interdigitated by a complex network of extracellular matrix components that form a distinct ITL compartment. Currently, there is not a firm understanding of how the lamellar and ITL matrix coordinately support tissue loading. METHODS: AF tissue samples were prepared from frozen porcine lumbar spines and mounted onto custom fixtures of a materials testing system that incorporates optical coherence tomography (OCT) imaging to perform tissue elastography. Tissues were subjected to 20 and 40% nominal shear strain, and OCT images were captured and segmented to identify regions of interest corresponding to lamellar and ITL compartments. Images were analyzed using an optical flow algorithm to quantify local shear strains within each compartment. RESULTS: Using histology and OCT, we first verified our ability to visualize and discriminate the ITL matrix from the lamellar matrix in porcine AF tissues. Local AF strains in the ITL compartment (22.0 ±â€Š13.8, 31.1 ±â€Š16.9 at 20% and 40% applied shear, respectively) were significantly higher than corresponding strains in the surrounding lamellar compartment (12.1 ±â€Š5.6, 15.3 ±â€Š5.2) for all tissue samples (P < 0.05). CONCLUSION: Results from this study demonstrate that the lamellar and ITL compartments of the AF distribute strain unevenly during tissue loading. Specifically, shear strain is significantly higher in the ITL matrix, suggesting that these regions may be more susceptible to tissue damage and more mechanobiologically active. LEVEL OF EVIDENCE: N/A.

Effect of Dynamic Culture and Periodic Compression on Human Mesenchymal Stem Cell Proliferation and Chondrogenesis.

We have recently developed a bioreactor that can apply both shear and compressive forces to engineered tissues in dynamic culture. In our system, alginate hydrogel beads with encapsulated human mesenchymal stem cells (hMSCs) were cultured under different dynamic conditions while subjected to periodic, compressive force. A customized pressure sensor was developed to track the pressure fluctuations when shear forces and compressive forces were applied. Compared to static culture, dynamic culture can maintain a higher cell population throughout the study. With the application of only shear stress, qRT-PCR and immunohistochemistry revealed that hMSCs experienced less chondrogenic differentiation than the static group. The second study showed that chondrogenic differentiation was enhanced by additional mechanical compression. After 14 days, alcian blue staining showed more extracellular matrix formed in the compression group. The upregulation of the positive chondrogenic markers such as Sox 9, aggrecan, and type II collagen were demonstrated by qPCR. Our bioreactor provides a novel approach to apply mechanical forces to engineered cartilage. Results suggest that a combination of dynamic culture with proper mechanical stimulation may promote efficient progenitor cell expansion in vitro, thereby allowing the culture of clinically relevant articular chondrocytes for the treatment of articular cartilage defects.

Effects of angular frequency during clinorotation on mesenchymal stem cell morphology and migration.

AIMS: To determine the short-term effects of simulated microgravity on mesenchymal stem cell behaviors-as a function of clinorotation speed-using time-lapse microscopy. BACKGROUND: Ground-based microgravity simulation can reproduce the apparent effects of weightlessness in spaceflight using clinostats that continuously reorient the gravity vector on a specimen, creating a time-averaged nullification of gravity. In this work, we investigated the effects of clinorotation speed on the morphology, cytoarchitecture, and migration behavior of human mesenchymal stem cells (hMSCs). METHODS: We compared cell responses at clinorotation speeds of 0, 30, 60, and 75 rpm over 8 h in a recently developed lab-on-chip-based clinostat system. Time-lapse light microscopy was used to visualize changes in cell morphology during and after cessation of clinorotation. Cytoarchitecture was assessed by actin and vinculin staining, and chemotaxis was examined using time-lapse light microscopy of cells in NGF (100 ng/ml) gradients. RESULTS: Among clinorotated groups, cell area distributions indicated a greater inhibition of cell spreading with higher angular frequency (P<0.005), though average cell area at 30 rpm after 8 h became statistically similar to control (P=0.794). Cells at 75 rpm clinorotation remained viable and were able to re-spread after clinorotation. In chemotaxis chambers, clinorotation did not alter migration patterns in elongated cells, but most clinorotated cells exhibited cell retraction, which strongly compromised motility. CONCLUSIONS: These results indicate that hMSCs respond to clinorotation by adopting more rounded, less-spread morphologies. The angular frequency-dependence suggests that a cell's ability to sense the changing gravity vector is governed by the rate of perturbation. For migration studies, cells cultured in clinorotated chemotaxis chambers were generally less motile and exhibited retraction instead of migration.

Transient solid-fluid interactions in rat brain tissue under combined translational shear and fixed compression.

An external mechanical insult to the brain may create internal deformation waves, which have shear and longitudinal components that induce combined shear and compression of the brain tissue. To isolate such interactions and to investigate the role of the extracellular fluid (ECF) in the transient mechanical response, translational shear stretch up to 1.25 under either 0 or 33% fixed normal compression is applied without preconditioning to heterogeneous sagittal slices which are nearly the full length of the rat brain cerebrum. The normal stress contribution is estimated by separate unconfined compression stress-stretch curves at 0.0667/s and 1/s engineering strain rates to 33% strain. Unconfined compression deformation causes lateral dimension expansion less than that predicted for an incompressible material under large deformation and often a visible loss of internal fluid from the specimen so that the bulk brain tissue is not incompressible in vitro, as sometimes assumed for mathematical modeling. The response to both slow 0.001/s and moderate 1/s shear translational stretch rates is deformation rate dependent and hardening under no compression but under 33% compression is nearly linear perhaps because of increased solid-solid friction. Both shear and normal stress relaxation are faster after the fast rate deformation possibly because higher deformation rates produce higher ECF hydrostatic pressure that primarily drives stress relaxation. The experimental results on ECF behavior guide the form of our nonlinear viscoelastic mathematical model. Our data are closely fit by non-equilibrium evolution equations that involve at most three specimen-specific empirical parameters and that are based on the idea that stretch of axons and glial processes resists load-induced ECF pressure.