Systematic Review of the Association Between Physical Fitness and Musculoskeletal Injury Risk: Part 3-Flexibility, Power, Speed, Balance, and Agility.

de la Motte, SJ, Lisman, P, Gribbin, TC, Murphy, K, and Deuster, PA. Systematic review of the association between physical fitness and musculoskeletal injury risk: part 3-flexibility, power, speed, balance, and agility. J Strength Cond Res 33(6): 1723-1735, 2019-We performed a systematic review and evaluation of the existing scientific literature on the association between flexibility, power, speed, balance, and agility, and musculoskeletal injury (MSK-I) risk in military and civilian populations. MEDLINE, EBSCO, EMBASE, and the Defense Technical Information Center were searched for original studies published from 1970 to 2015 that examined associations between these physical fitness measures (flexibility, power, speed, balance, and agility) and MSK-I. Methodological quality and strength of the evidence were determined after criteria adapted from previously published systematic reviews. Twenty-seven of 4,229 citations met our inclusion criteria. Primary findings indicate that there is (a) moderate evidence that hamstring flexibility, as measured by performance on a sit-and-reach test or active straight leg raise test assessed with goniometry, and ankle flexibility, assessed with goniometry, are associated with MSK-I risk; (b) moderate evidence that lower body power, as measured by performance on a standing broad jump or vertical jump with no countermovement, is associated with MSK-I risk; (c) moderate evidence that slow sprint speed is associated with MSK-I risk; (d) moderate evidence that poor performance on a single-leg balance test is associated with increased risk for ankle sprain; and (e) insufficient evidence that agility is associated with MSK-I risk. Several measures of flexibility, power, speed, and balance are risk factors for training-related MSK-I in military and civilian athletic populations. Importantly, these findings can be useful for military, first responder, and athletic communities who are seeking evidence-based metrics for assessing or stratifying populations for risk of MSK-I.

Restoring vasculogenic potential of endothelial cells from diabetic patients through spheroid formation.

INTRODUCTION: Diabetes is an emerging epidemic in the developing world and represents a major risk factor for cardiovascular disease. Among other issues, patients with diabetes suffer from diminished endothelial cell (EC) function, which contributes to impaired vasculogenesis and recovery from ischemic insult. The formation of cells into three-dimensional spheroids promotes cell survival and activates key signaling pathways through the upregulation of cell-cell contacts, providing an opportunity to overcome shortcomings associated with individual autologous cells. METHODS: We hypothesized that forming human microvascular endothelial cells (HMVECs) from diabetic patients into spheroids would restore their vasculogenic potential following upregulation of these cell-cell interactions. HMVEC spheroids were formed and suspended in fibrin gels to quantify vasculogenic potential. RESULTS: Individual HMVECs from diabetic patients exhibited similar proliferative and chemotactic potential to cells from healthy donors but reduced tubulogenesis. HMVEC spheroids formed from diabetic donors formed more sprouts than spheroids from healthy donors, and more sprouts than individual cells from either population. CONCLUSIONS: Compared to cells from healthy donors, sprout formation was more efficiently abrogated in HMVECs from diabetic patients by blocking matrix metalloproteinase activity. This study demonstrates a promising approach for restoring the diminished vasculogenic potential of endothelial cells in diabetic patients.

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids.

Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu.

Systematic Review of the Association Between Physical Fitness and Musculoskeletal Injury Risk: Part 2-Muscular Endurance and Muscular Strength.

de la Motte, SJ, Gribbin, TC, Lisman, P, Murphy, K, and Deuster, PA. A systematic review of the association between physical fitness and musculoskeletal injury risk: part 2-muscular endurance and muscular strength. J Strength Cond Res 31(11): 3218-3234, 2017-This is a systematic review and evaluation of the current evidence on the association between both muscular endurance (ME) and muscular strength (MS) and musculoskeletal injury (MSK-I) risk in military and civilian populations. MEDLINE, EBSCO, EMBASE, and the Defense Technical Information Center were searched for original studies published from 1970 through 2015 which examined associations between physical fitness (ME and MS) and MSK-I in military or civilian populations. Methodological quality and strength of the evidence were determined following criteria adapted from previously published systematic reviews. Forty-five of 4,229 citations met our inclusion criteria. Although results for some tests did vary by sex, taken together, our primary findings indicate there is (a) a strong evidence that poor performance in a push-up test is associated with MSK-I risk; (b) moderate evidence that poor performance in sit-up test is associated with MSK-I risk; (c) moderate evidence that isokinetic ankle and knee flexion strength, and isometric strength assessments at the back, elbow, or knee are associated with MSK-I risk; and (d) limited evidence that poor performance in a pull-up test and isotonic assessments of muscular strength are associated with MSK-I. Several measures of ME/MS are moderately or strongly associated with risk of MSK-I, but additional research is needed to identify and recommend specific assessments of ME/MS that predict MSK-I in both men and women. Future studies should also consider measures of ME and MS as a function of upper body, lower body, and core strength, and their potential association with specific, rather than general, MSK-I.

A Systematic Review of the Association Between Physical Fitness and Musculoskeletal Injury Risk: Part 1-Cardiorespiratory Endurance.

Musculoskeletal injuries (MSK-Is) are a significant health problem for both military and athletic populations. Research indicates that MSK-I is associated with physical fitness; however, the association between specific components of physical fitness and MSK-I in military and athletic populations has not been systematically examined. Our goal was to systematically review the literature to provide a best evidence synthesis on the relationship between components of physical fitness and MSK-I risk in military and civilian athletic populations. This first of 3 manuscripts focuses on cardiorespiratory endurance (CRE). MEDLINE, EBSCO, EMBASE, and the Defense Technical Information Center were searched for original studies published from 1970 through 2015 that examined associations between physical fitness and MSK-I. Forty-nine of 4,229 citations met our inclusion criteria. Primary findings indicate that there is (a) strong evidence that poor performance on a set distance run for time is a predictor for MSK-I risk in both genders; (b) strong evidence that poor performance on timed shuttle runs is a predictor for MSK-I risk in males; (c) moderate evidence in males and limited evidence in females that poor performance on a timed step test is a predictor of MSK-I risk; and (d) limited or insufficient evidence that poor performance on the Cooper run test, maximal and submaximal aerobic graded exercise tests, and the Conconi test are predictors of MSK-I risk in males or females or both. Several measures of CRE are risk factors for training-related MSK-I in military and civilian athletic populations, indicating that CRE may be an important measure for MSK-I risk stratification.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2 (PGE2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504.

Measurement of oxygen tension within mesenchymal stem cell spheroids.

Spheroids formed of mesenchymal stem cells (MSCs) exhibit increased cell survival and trophic factor secretion compared with dissociated MSCs, making them therapeutically advantageous for cell therapy. Presently, there is no consensus for the mechanism of action. Many hypothesize that spheroid formation potentiates cell function by generating a hypoxic core within spheroids of sufficiently large diameters. The purpose of this study was to experimentally determine whether a hypoxic core is generated in MSC spheroids by measuring oxygen tension in aggregates of increasing diameter and correlating oxygen tension values with cell function. MSC spheroids were formed with 15 000, 30 000 or 60 000 cells per spheroid, resulting in radii of 176 ± 8 µm, 251 ± 12 µm and 353 ± 18 µm, respectively. Oxygen tension values coupled with mathematical modelling revealed a gradient that varied less than 10% from the outer diameter within the largest spheroids. Despite the modest radial variance in oxygen tension, cellular metabolism from spheroids significantly decreased as the number of cells and resultant spheroid size increased. This may be due to adaptive reductions in matrix deposition and packing density with increases in spheroid diameter, enabling spheroids to avoid the formation of a hypoxic core. Overall, these data provide evidence that the enhanced function of MSC spheroids is not oxygen mediated.

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2ß1 Signaling.

UNLABELLED: : The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2ß1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. SIGNIFICANCE: Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair.

Fabrication of petal-shaped masks for suppression of the on-axis Poisson spot in telescope systems.

The presence of a bright (Poisson) spot in the geometrical shadow of circular/spherical shapes has been known for the past two centuries. A broad class of telescopes that involve simultaneous transmit and receive require suppression of the reflected light from the secondary mirror on the detector. For instance, the on-axis design of optical telescope for the evolved Laser Interferometric Space Antenna (eLISA), a re-scoped version of the baseline LISA mission concept, requires suppression of reflected laser light from the secondary mirror on the detector. In the past few years, the hypergaussian functions with petal-shaped realization have been shown to significantly suppress intensity along the optical axis. This work reports on fabrication of a series of petal-shaped masks using a variety of techniques such as 3D printing, photolithography, and wire Electro Discharge Machining. These masks are designed and fabricated to operate in the range of Fresnel numbers between 4 and 120. This paper discusses the challenges, successes, and failures of each fabrication technique and the optical performance of typical masks with suggestions for potential follow up work.

Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels.

UNLABELLED: Mesenchymal stem cell (MSC)-based therapies are under broad investigation for applications in tissue repair but suffer from poor cell persistence and engraftment upon transplantation. MSC spheroids exhibit improved survival, anti-inflammatory, and angiogenic potential in vitro, while also promoting vascularization when implanted in vivo. However, these benefits are lost once cells engage the tissue extracellular matrix and migrate from the aggregate. The efficacy of cell therapy is consistently improved when using engineered materials, motivating the need to investigate the role of biomaterials to instruct spheroid function. In order to assess the contribution of adhesivity on spheroid activity in engineered materials and promote the bone-forming potential of MSCs, we compared the function of MSC spheroids when entrapped in Arg-Gly-Asp (RGD)-modified alginate hydrogels to nonfouling unmodified alginate. Regardless of material, MSC spheroids exhibited reduced caspase activity and greater vascular endothelial growth factor (VEGF) secretion compared with equal numbers of dissociated cells. MSC spheroids in RGD-modified hydrogels demonstrated significantly greater cell survival than spheroids in unmodified alginate. After 5 days in culture, spheroids in RGD-modified gels had similar levels of apoptosis, but more than a twofold increase in VEGF secretion compared with spheroids in unmodified gels. All gels contained mineralized tissue 8 weeks after subcutaneous implantation, and cells entrapped in RGD-modified alginate exhibited greater mineralization versus cells in unmodified gels. Immunohistochemistry confirmed more diffuse osteocalcin staining in gels containing spheroids compared with dissociated controls. This study demonstrates the promise of cell-instructive biomaterials to direct survival and function of MSC spheroids for bone tissue engineering applications. SIGNIFICANCE: Mesenchymal stem cell (MSC) spheroids exhibit improved therapeutic potential in vitro compared with dissociated MSCs, yet spheroids are directly injected into tissues, ceding control of cell function to the extracellular matrix and potentially limiting the duration of improvement. Cell delivery using adhesive biomaterials promotes cell retention and function. These studies explored the role of adhesion to the surrounding matrix on spheroid function. When entrapped in an adhesive biomaterial, MSC spheroids exhibited improved survival and proangiogenic growth factor secretion in vitro and bone formation in vivo compared with cells in nonadhesive hydrogels. These findings demonstrate the value of deploying MSC spheroids in instructive biomaterials to improve cell function.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

Engineered Fibrin Gels for Parallel Stimulation of Mesenchymal Stem Cell Proangiogenic and Osteogenic Potential.

Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo.

The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.

Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing.

Mesenchymal stem cells (MSCs) have great therapeutic potential for the repair of nonhealing bone defects, because of their proliferative capacity, multilineage potential, trophic factor secretion and lack of immunogenicity. However, a major challenge to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesize that forming MSCs into more physiologic three-dimensional spheroids, rather than employing dissociated cells from two-dimensional monolayer culture, will enhance their survival when exposed to a harsh microenvironment but maintain their osteogenic potential. MSC spheroids were formed by using the hanging drop method with increasing cell numbers. Compared with larger spheroids, the smallest spheroids, which contained 15,000 cells, exhibited increased metabolic activity, reduced apoptosis and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free medium and 1 % oxygen. Compared with identical numbers of dissociated MSCs in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more vascular endothelial growth factor. Moreover, fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. Thus, MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential while maintaining similar osteogenic potential to dissociated MSCs entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair.

Ultrastructure and growth factor content of equine platelet-rich fibrin gels.

OBJECTIVE: To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). SAMPLE: PRFGs and conventional FGs prepared from the blood of 10 healthy horses. PROCEDURES: Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/µL, 250 × 10³ platelets/µL, 500 × 10³ platelets/µL, and 1,000 × 10³ platelets/µL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-ß1 concentrations were quantified with ELISAs. RESULTS: Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-ß1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/µL. CONCLUSIONS AND CLINICAL RELEVANCE: The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

Nutrition as a component of the performance triad: how healthy eating behaviors contribute to soldier performance and military readiness.

OBJECTIVE: Nutrition is a critical element of Soldier health and performance. Food choices, meal timing, and dietary intake behaviors contribute to nutritional fitness. The objectives of this study were to describe Soldier dietary behaviors and quantify the association between healthy eating behaviors and demographic, lifestyle, and psychosocial factors. METHODS: The Comprehensive Soldier and Family Fitness Global Assessment Tool (GAT) assesses emotional, social, family, and spiritual fitness. In 2012, 57 pilot questions were added to the GAT to create a physical dimension that included nutrition assessments. Participants included 13,858 Active Duty, Reserve, and National Guard Soldiers: 83% male; 85% enlisted; a mean age of 28±9 years. A Healthy Eating Score (HES-5) was calculated from 5 questions assessing frequency of fruit, vegetable, whole grain, dairy, and fish intake (Cronbach α=0.81). Associations between HES-5 and other dietary habits, physical activity patterns, and GAT psychosocial dimension scores were examined. RESULTS: Soldiers who ate breakfast regularly (6 times/week or more), drank 7 servings or more of water/day, and met weekly exercise recommendations were more likely to be in the highest HES-5 quartile than those who did not. Those who passed their Army Physical Fitness Test (APFT) in the top quartile were also more likely to report high HES-5 scores than those who failed (P<.001). Soldiers with healthy anthropometric measures and the highest emotional, social, family, and spiritual fitness scores were also more likely to be in the top HES-5 quartile than those with unhealthy measures and with the lowest fitness scores (P<.001). CONCLUSION: The HES-5 may be a useful index for characterizing dietary intake behaviors. Healthy dietary intake behaviors are associated with all dimensions of health, physical fitness, and psychosocial status.

Sleep as a component of the performance triad: the importance of sleep in a military population.

OBJECTIVE: Sleep habits among military populations are problematic. Poor sleep hygiene occurs in parallel with the global increase in obesity and metabolic syndrome and contributes to a decrease in performance. The extent of sleep issues needs to be quantified to provide feedback for optimizing warfighter performance and readiness. This study assessed various health behaviors and habits of US Army Soldiers and their relationship with poor sleep quality by introducing a set of new questions into the Comprehensive Soldier and Family Fitness (CSF2) Global Assessment Tool (GAT). METHODS: Subjects included 14,148 US Army Active, Reserve, and National Guard members (83.4% male) who completed the GAT, a self-report questionnaire that measures 4 fitness dimensions: social, family, emotional, and spiritual. Approximately 60 new questions, including ones on sleep quality, within the fifth CSF2 dimension (physical) were also answered. A sleep score was calculated from 2 questions validated in the Pittsburgh Insomnia Rating Scale (0 to 6). RESULTS: Poor sleepers (5-6) were significantly (P<.001) more likely than good sleepers (0-1) to consider themselves in fair or poor health, be overweight or obese, and score in the lowest quartile of the emotional, social, family, and spiritual fitness dimensions. Additionally, poor sleepers were significantly (P<.001) less likely to have a healthy body mass index and waist circumference, eat breakfast 6 or more times a week, meet aerobic exercise and resistance training recommendations, and pass their Army Physical Fitness Test in the top quartile. CONCLUSION: This study examined sleep quality in a group of military personnel and indicated significant associations between quality of sleep and physical performance, nutritional habits, measures of obesity, lifestyle behaviors and measures of psychosocial status. Targeted educational interventions and resources are needed to improve sleep patterns based on behaviors that can be most easily modified.

A reproducible, high throughput method for fabricating fibrin gels.

BACKGROUND: Fibrin gels are a promising biomaterial for tissue engineering. However, current fabrication methods are time intensive with inherent variation. There is a pressing need to develop new and consistent approaches for producing fibrin-based hydrogels for examination. FINDINGS: We developed a high throughput method for creating fibrin gels using molds fabricated from polydimethylsiloxane (PDMS). Fibrin gels were produced by adding solutions of fibrinogen and thrombin to cylindrical defects in a PDMS sheet. Undisturbed gels were collected by removing the sheet, and fibrin gels were characterized. The characteristics of resulting gels were compared to published data by measuring compressive stiffness and osteogenic response of entrapped human mesenchymal stem cells (MSCs). Gels exhibited compressive moduli nearly identical to our previously reported fabrication method. Trends in alkaline phosphatase activity, an early marker of osteogenic differentiation in MSCs, were also consistent with previous data. CONCLUSIONS: These findings demonstrate a streamlined approach to fibrin gel production that drastically reduces the time required to make fibrin gels, while also reducing variability between gel batches. This fabrication technique provides a valuable tool for generating large numbers of gels in a cost-effective manner.

Functionalization of reactive polymer multilayers with RGD and an antifouling motif: RGD density provides control over human corneal epithelial cell-substrate interactions.

Our study demonstrates that substrates fabricated using a "reactive" layer-by-layer approach promote well-defined cell-substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating "reactive" deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell-substrate interactions enables the use of these films in a wide range of biological applications.

Ceramic identity contributes to mechanical properties and osteoblast behavior on macroporous composite scaffolds.

Implants formed of metals, bioceramics, or polymers may provide an alternative to autografts for treating large bone defects. However, limitations to each material motivate the examination of composites to capitalize on the beneficial aspects of individual components and to address the need for conferring bioactive behavior to the polymer matrix. We hypothesized that the inclusion of different bioceramics in a ceramic-polymer composite would alter the physical properties of the implant and the cellular osteogenic response. To test this, composite scaffolds formed from poly(lactide-co-glycolide) (PLG) and either hydroxyapatite (HA), ß-tricalcium phosphate (TCP), or bioactive glass (Bioglass 45S®, BG) were fabricated, and the physical properties of each scaffold were examined. We quantified cell proliferation by DNA content, osteogenic response of human osteoblasts (NHOsts) to composite scaffolds by alkaline phosphatase (ALP) activity, and changes in gene expression by qPCR. Compared to BG-PLG scaffolds, HA-PLG and TCP-PLG composite scaffolds possessed greater compressive moduli. NHOsts on BG-PLG substrates exhibited higher ALP activity than those on control, HA-, or TCP-PLG scaffolds after 21 days, and cells on composites exhibited a 3-fold increase in ALP activity between 7 and 21 days versus a minimal increase on control scaffolds. Compared to cells on PLG controls, RUNX2 expression in NHOsts on composite scaffolds was lower at both 7 and 21 days, while expression of genes encoding for bone matrix proteins (COL1A1 and SPARC) was higher on BG-PLG scaffolds at both time points. These data demonstrate the importance of selecting a ceramic when fabricating composites applied for bone healing.