Science for humanity.

This year marks the 175th anniversary of the American Association for the Advancement of Science (AAAS, the publisher of Science). In striving to advance its mission, the organization's theme for its annual meeting (2 to 5 March in Washington, DC), "Science for Humanity," reiterates its commitment to explore and make sense of the world through inquiry, evidence seeking, and discovery. The words of archbishop and Nobel laureate Desmond Tutu are a reminder that this pursuit must be shared by everyone if it is to serve all of society: "My humanity is bound up in yours, for we can only be human together." These words aptly capture the essence of the ties that bind us, which include our shared DNA, our need to bond with others, and our ability to collaborate. Science is intertwined with the human condition and stories of the human experience and is hence the great connector. The more we embrace our common humanity-and science as a unifier-the better we will understand what it means to be human and what it will take to sustain ourselves and our planet.

The Biggest Challenges Resulting from the COVID-19 Pandemic on Gender-Related Work from Home in Biomedical Fields-World-Wide Qualitative Survey Analysis.

(1) Background: This paper aims to present and discuss the most significant challenges encountered by STEM professionals associated with remote working during the COVID-19 lockdowns. (2) Methods: We performed a qualitative analysis of 921 responses from professionals from 76 countries to the open-ended question: "What has been most challenging during the lockdown for you, and/or your family?" (3) Findings: Participants reported challenges within the immediate family to include responsibilities for school, childcare, and children's wellbeing; and the loss of social interactions with family and friends. Participants reported increased domestic duties, blurred lines between home and work, and long workdays. Finding adequate workspace was a problem, and adaptations were necessary, especially when adults shared the same setting for working and childcare. Connectivity issues and concentration difficulties emerged. While some participants reported employers' expectations did not change, others revealed concerns about efficiency. Mental health issues were expressed as anxiety and depression symptoms, exhaustion and burnout, and no outlets for stress. Fear of becoming infected with COVID-19 and uncertainties about the future also emerged. Pressure points related to gender, relationship status, and ethnicities were also evaluated. Public policies differed substantially across countries, raising concerns about the adherence to unnecessary restrictions, and similarly, restrictions being not tight enough. Beyond challenges, some benefits emerged, such as increased productivity and less time spent getting ready for work and commuting. Confinement resulted in more quality time and stronger relationships with family. (4) Interpretation: Viewpoints on positive and negative aspects of remote working differed by gender. Females were more affected professionally, socially, and personally than males. Mental stress and the feeling of inadequate work efficiency in women were caused by employers' expectations and lack of flexibility. Working from home turned out to be challenging, primarily due to a lack of preparedness, limited access to a dedicated home-office, and lack of previous experience in multi-layer/multi-scale environments.

Engineering Solutions to COVID-19 and Racial and Ethnic Health Disparities.

The role of engineers in response to the COVID-19 pandemic and in the elimination of health disparities, while not always visible, has important implications for the attainment of impactful solutions. The design skills, systems approach, and innovative mindset that engineers bring all have the potential to combat crises in novel and impactful ways. When a disparities lens is applied, a lens that views gaps in access, resources, and care, the engineering solutions are bound to be more robust and equitable. The disproportionate impact of COVID-19 on the Black community and other communities of color is linked to inequities in health rooted in a centuries long structural racism. Engineers working collaboratively with physicians and healthcare providers are poised to close equity gaps and strengthen the collective response to COVID-19 and future pandemics.

In Vitro Evaluation of the Influence of Substrate Mechanics on Matrix-Assisted Human Chondrocyte Transplantation.

Matrix-assisted chondrocyte transplantation (MACT) is of great interest for the treatment of patients with cartilage lesions. However, the roles of the matrix properties in modulating cartilage tissue integration during MACT recovery have not been fully understood. The objective of this study was to uncover the effects of substrate mechanics on the integration of implanted chondrocyte-laden hydrogels with native cartilage tissues. To this end, agarose hydrogels with Young's moduli ranging from 0.49 kPa (0.5%, w/v) to 23.08 kPa (10%) were prepared and incorporated into an in vitro human cartilage explant model. The hydrogel-cartilage composites were cultivated for up to 12 weeks and harvested for evaluation via scanning electron microscopy, histology, and a push-through test. Our results demonstrated that integration strength at the hydrogel-cartilage interface in the 1.0% (0.93 kPa) and 2.5% (3.30 kPa) agarose groups significantly increased over time, whereas hydrogels with higher stiffness (>8.78 kPa) led to poor integration with articular cartilage. Extensive sprouting of extracellular matrix in the interfacial regions was only observed in the 0.5% to 2.5% agarose groups. Collectively, our findings suggest that while neocartilage development and its integration with native cartilage are modulated by substrate elasticity, an optimal Young's modulus (3.30 kPa) possessed by agarose hydrogels is identified such that superior quality of tissue integration is achieved without compromising tissue properties of implanted constructs.

Adipogenic and Osteogenic Differentiation of In Vitro Aged Human Mesenchymal Stem Cells.

Multipotent mesenchymal stem cells (MSCs) are an attractive candidate for regeneration of damaged cells, tissues, and organs. Due to limited availabilities, MSC populations must be rapidly expanded to satisfy clinical needs. However, senescence attributed to extensive in vitro expansion compromises the regenerative and therapeutic potential of MSCs. In this chapter, we describe a step-by-step protocol that aims to induce adipogenic and osteogenic differentiation of in vitro aged human MSCs and highlight noteworthy issues that may arise during the process.

Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro.

BACKGROUND: Adult mesenchymal stem cells (MSCs) hold great promise for regenerative medicine because of their self-renewal, multipotency, and trophic and immunosuppressive effects. Due to the rareness and high heterogeneity of freshly isolated MSCs, extensive in-vitro passage is required to expand their populations prior to clinical use; however, senescence usually accompanies and can potentially affect MSC characteristics and functionality. Therefore, a thorough characterization of the variations in phenotype and differentiation potential of in-vitro aging MSCs must be sought. METHODS: Human bone marrow-derived MSCs were passaged in vitro and cultivated with either DMEM-based or αMEM-based expansion media. Cells were prepared for subculture every 10 days up to passage 8 and were analyzed for cell morphology, proliferative capacity, and surface marker expression at the end of each passage. The gene expression profile and adipogenic and osteogenic differentiation capability of MSCs at early (passage 4) and late (passage 8) passages were also evaluated. RESULTS: In-vitro aging MSCs gradually lost the typical fibroblast-like spindle shape, leading to elevated morphological abnormality and inhomogeneity. While the DMEM-based expansion medium better facilitated MSC proliferation in the early passages, the cell population doubling rate reduced over time in both DMEM and αMEM groups. CD146 expression decreased with increasing passage number only when MSCs were cultured under the DMEM-based condition. Senescence also resulted in MSCs with genetic instability, which was further regulated by the medium recipe. Regardless of the expansion condition, MSCs at both passages 4 and 8 could differentiate into adipocyte-like cells whereas osteogenesis of aged MSCs was significantly compromised. For osteogenic induction, use of the αMEM-based expansion medium yielded longer osteogenesis and better quality. CONCLUSIONS: Human MSCs subjected to extensive in-vitro passage can undergo morphological, phenotypic, and genetic changes. These properties are also modulated by the medium composition employed to expand the cell populations. In addition, adipogenic potential may be better preserved over osteogenesis in aged MSCs, suggesting that MSCs at early passages must be used for osteogenic differentiation. The current study presents valuable information for future basic science research and clinical applications leading to the development of novel MSC-based therapeutic strategies for different diseases.

Internal Viscosity-Dependent Margination of Red Blood Cells in Microfluidic Channels.

Cytoplasmic viscosity-dependent margination of red blood cells (RBC) for flow inside microchannels was studied using numerical simulations, and the results were verified with microfluidic experiments. Wide range of suspension volume fractions or hematocrits was considered in this study. Lattice Boltzmann method for fluid-phase coupled with spectrin-link method for RBC membrane deformation was used for accurate analysis of cell margination. RBC margination behavior shows strong dependence on the internal viscosity of the RBCs. At equilibrium, RBCs with higher internal viscosity marginate closer to the channel wall and the RBCs with normal internal viscosity migrate to the central core of the channel. Same margination pattern has been verified through experiments conducted with straight channel microfluidic devices. Segregation between RBCs of different internal viscosity is enhanced as the shear rate and the hematocrit increases. Stronger separation between normal RBCs and RBCs with high internal viscosity is obtained as the width of a high aspect ratio channel is reduced. Overall, the margination behavior of RBCs with different internal viscosities resembles with the margination behavior of RBCs with different levels of deformability. Observations from this work will be useful in designing microfluidic devices for separating the subpopulations of RBCs with different levels of deformability that appear in many hematologic diseases such as sickle cell disease (SCD), malaria, or cancer.

Cultivation of agarose-based microfluidic hydrogel promotes the development of large, full-thickness, tissue-engineered articular cartilage constructs.

The fabrication of tissue-engineered constructs of clinically relevant sizes continues to be plagued by poor nutrient transport to the interior of the construct. Consequences of poor mass transfer to the construct core include large gradients in cell viability and matrix deposition, as well as inadequate mechanical functionality. Prior literature has shown that embedded microfluidic channels offer the potential to control the spatial and temporal presentation of hydrodynamic and chemical cues within the developing tissue construct toward improved mass transfer. The current state of the art in microfluidic constructs, however, has fallen short of achieving sufficient thickness and robustness of constructs for further development towards translation. Towards this goal, we designed a microfluidic tissue construct and established bioprocessing conditions to meet nutrient transport requirements of a large, full-thickness, articular cartilage construct over a 2 week culture period. Our microfluidic constructs of 2.5 and 5 mm thicknesses showed enhanced cell proliferation relative to statically cultured constructs. These constructs, which are both thick and robust to culture periods of sufficient length to support extracellular matrix development, represent an important improvement over previously reported constructs which were thinner and lacking in extracellular matrix (most likely attributable to too-short culture periods). Copyright © 2014 John Wiley & Sons, Ltd.

Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products.

Hydrodynamic loading in concomitance with exogenous cytokine stimulation modulates differentiation of bovine mesenchymal stem cells towards osteochondral lineages.

BACKGROUND: Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors. METHODS: Expanded bovine MSCs were suspended in 2.5 % agarose, cast in a custom mold, and placed into either static or one of two dynamic culture environments consisting of "high" and "low" magnitude shear conditions. Constructs were supplemented with varying concentrations (0, 1, 10, 100 ng/mL) of either TGF-ß3 or BMP-2 throughout cultivation with tissue samples being collected following each week of culture. RESULTS: In the absence of exogenous supplementation, hydrodynamic loading had little effect on cell phenotype at either magnitude of stimulation. When cultures were supplemented with BMP-2 and TGF-ß3, MSCs gene expression progressed towards the osteogenic and chondrogenic pathways, respectively. This progression was enhanced by the presence of hydrodynamic loading, particularly under high shear conditions, but may point the chondrogenic cultures down a hypertrophic path toward osteogenesis reminiscent of endochondral ossification if TGF-ß3 supplementation is insufficient. CONCLUSIONS: Moving forward, these results suggest bioprocessing conditions which minimize exposure of chondrogenic cultures to fluid shear stress to avoid undesirable differentiation of the MSC population.

Type I collagen-based fibrous capsule enhances integration of tissue-engineered cartilage with native articular cartilage.

Successful integration of engineered constructs with host tissues is crucial for cartilage repair, yet achieving it remains challenging. A collagen I-based fibrous capsule characterized by increased cell density and decreased glycosaminoglycan deposition usually forms at the periphery of tissue-engineered cartilage. The current study aimed to evaluate the effects of a solid fibrous capsule on construct integration with native articular cartilage. To this end, capsule-containing (CC) and capsule-free (CF) constructs were grown by culturing chondrocyte-seeded scaffolds with insulin-like growth factor-1 and transforming growth factor-ß1, respectively, in a wavy-walled bioreactor that imparts hydrodynamic forces for 4 weeks. The ability of harvested constructs to integrate with native cartilage was determined using a cartilage explant model. Our results revealed that adhesive stress between native cartilage and the CC constructs was 57% higher than that in the CF group, potentially due to the absence of glycosaminoglycans and increased cell density in the capsule region and deposition of denser and thicker collagen fibrils at the integration site. The present work demonstrates that the fibrous capsule can effectively enhance early integration of engineered and native cartilage tissues and thus suggests the need to include the capsule as a variable in the development of cartilage tissue engineering strategies.

Differential morphology and homogeneity of tissue-engineered cartilage in hydrodynamic cultivation with transient exposure to insulin-like growth factor-1 and transforming growth factor-ß1.

Successful tissue-engineering strategies for cartilage repair must maximize the efficacy of chondrocytes within their limited life span. To that end, the combination of exogenous growth factors with mechanical stimuli holds promise for development of clinically relevant cartilage tissue substitutes. The current study aimed to determine whether incorporation of transient exposure to growth factors into a hydrodynamic bioreactor system can improve the functional maturation of tissue-engineered cartilage. Chondrocyte-seeded polyglycolic acid scaffolds were cultivated within a wavy-walled bioreactor that imparts fluid flow-induced shear stress for 4 weeks. Constructs were nourished with 100 ng/mL insulin-like growth factor-1 (IGF-1) or 10 ng/mL transforming growth factor-ß1 (TGF-ß1) either for the first 15 days of the culture (transient) or throughout the entire cultivation (continuous). Transiently treated constructs were found to exhibit better functional properties than continuously nourished constructs. The limited development of engineered tissues continuously stimulated by IGF-1 or TGF-ß1 was related to massive growth factor leftovers in the environments that downregulated the expression of the associated receptors. Treatment with TGF-ß1 eliminated the formation of a fibrous capsule at the construct periphery possibly through suppression of Smad3 phosphorylation, yielding constructs with greater homogeneity. Furthermore, TGF-ß1 reversely regulated Smad2 and Smad3 pathways in articular chondrocytes under hydrodynamic stimuli partially via Smad7. Collectively, transient exposure to growth factors is likely to maintain chondrocyte homeostasis, and thus promotes their anabolic activities under hydrodynamic stimuli. The present work suggests that robust hydrodynamically engineered neocartilage with a reduced fibrotic response and enhanced tissue homogeneity can be achieved through optimization of growth factor supplementation protocols and potentially through manipulation of intracellular signals such as Smad.

Coculture-driven mesenchymal stem cell-differentiated articular chondrocyte-like cells support neocartilage development.

Controlled differentiation of mesenchymal stem cells (MSCs) into the chondrogenic lineage is crucial for in vitro generation of neocartilage, yet achieving it remains challenging. Traditional protocols for MSC differentiation using exogenous inductive molecules, such as transforming growth factor-ß, fall short in meeting the needs of clinical applications because they yield differentiated cells that exhibit hypertrophic characteristics and subsequently facilitate endochondral bone formation. The objective of the current study was to deliver endogenous inductive factors from juvenile articular chondrocytes to bone marrow-derived MSCs to drive MSC chondrogenic differentiation through cocultivation of the two cell types in the absence of direct physical contact and exogenous stimulators. An initial chondrocyte/MSC ratio of 63:1 was identified as the appropriate proportion of the two cell populations to ensure that coculture-driven MSC-differentiated (CDMD) cells replicated the cellular morphology, behavior, and phenotype of articular chondrocytes. In a three-dimensional agarose system, CDMD cells were further shown to develop into robust neocartilage structurally and mechanically stronger than chondrocyte-laden constructs and with reduced hypertrophic potential. Although MSCs tended to lose the ability to express CD44, an important regulator in cartilage biology, during the coculture induction, CDMD cells regained this function in the three-dimensional tissue cultivation. The present work establishes a chondrocyte/MSC coculture model that serves as a template to better understand chondrocyte-driven MSC differentiation and provides insights for improved strategies to develop clinically relevant cartilage tissue replacements.

Modeling of bioreactor hydrodynamic environment and its effects on tissue growth.

The design of optimal bioreactor systems for tissue engineering applications requires a sophisticated understanding of the complexities of the bioreactor environment and the role that it plays in the formation of engineered tissues. To this end, a tissue growth model is developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in a wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: (1) a computational fluid dynamics (CFD) model to characterize the complex hydrodynamic environment in the bioreactor, (2) a kinetic growth model to characterize the cell growth and extracellular matrix production dynamics, (3) an artificial neural network (ANN) that empirically correlates hydrodynamic parameters with kinetic constants, and (4) a second ANN that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict optimal bioprocessing conditions required to achieve desired tissue properties.

Requirement for serum in medium supplemented with insulin-transferrin-selenium for hydrodynamic cultivation of engineered cartilage.

Achievement of viable engineered tissues through in vitro cultivation in bioreactor systems requires a thorough understanding of the complex interplay between hydrodynamic forces and biochemical cues such as serum. To this end, chondrocyte-seeded constructs were cultured under continuous fluid-induced shear forces with reduced serum content (0%-2%, v/v), which was partially or completely replaced by a potential substitute, insulin-transferrin-selenium, to minimize deleterious effects associated with the use of culture media containing high levels of serum (10%-20%). Low-serum cultures yielded constructs with similar biochemical properties to those cultivated with high-serum supplements, whereas the serum-free constructs exhibited poor cell proliferation, insufficient extracellular matrix production, and rapid degradation of and/or shear-induced damage to polyglycolic acid scaffolds. A fibrous outer capsule typically observed in hydrodynamic cultures and characterized by increased cell density and decreased (virtually none) glycosaminoglycan deposition was eliminated when serum concentration was equal to or <0.2% in the presence of hydrodynamic stimuli. Our findings suggest that serum is a requirement in insulin-transferrin-selenium-supplemented cultures in order for constructs to exhibit improved properties in response to hydrodynamic forces, and that mechanical and biochemical stimuli may synergistically modulate tissue properties and morphology through shear-responsive signals.

Synergic effects of crypt-like topography and ECM proteins on intestinal cell behavior in collagen based membranes.

The basement membrane of small intestinal epithelium possesses complex topography at multiple scales ranging from the mesoscale to nanoscale. Specifically, intestinal crypt-villus units are comprised of hundred-micron-scale well-like invaginations and finger-like projections; intestinal cell phenotype is related to location on this crypt-villus unit. A biomimetic intestinal cell culture system composed of type I collagen based permeable cell culture membranes incorporating both micron-scale intestinal crypt-like topography and nanometer scale topography was fabricated. Membranes were pre-incubated with either laminin (Ln) or fibronectin (Fn), inoculated with intestinal epithelial Caco-2 cells and cultured for 1-21 days to study the relative significance of influence of crypt-like topography and biomimetic substrate chemistry on cell phenotype. Crypt-like topography inhibited Caco-2 differentiation during early culture, as evidenced by slower cell spreading and lower brush border enzyme activity. For example, alanine aminopeptidase activity was lower on Ln-coated patterned collagen ( approximately 3.4+/-0.24mU/mg) compared to flat collagen (10.84+/-0.55mU/mg) at day 7. Caco-2 cultured on Fn-coated collagen started to spread earlier (1 day vs 3 days) and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation, as the maximum activity of a cell differentiation marker (alkaline phosphatase) was 2-3 times higher than on Fn-coated collagen, and maintained differentiated phenotype in long term (up to 21 days) culture. In general, compared to substrate topography, coating with ECM protein had more prominent and longer effect on cell behavior. Crypt-like topography affected Caco-2 spreading and differentiation during early culture, however the effect diminished as culture progressed. This information will benefit intestinal tissue engineering scaffold design, and modification of in vitro intestinal cell models.