A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types.

Surfactin, a negatively charged amphiphilic lipopeptide biosurfactant, is synthesized by the bacterium Bacillus subtilis. It consists of a cyclic heptapeptide and an 11-15C ß-hydroxy fatty acid. To probe how the modification of the molecular skeleton of surfactin influences its selectivity and activity against breast cancer, six synthetic surfactins were generated. Modifications were accomplished by conjugating amine-functionalized molecules to the Glu and Asp carboxyl moieties of the heptapeptide. The resulting synthetic surfactins provided a diverse series of molecules with differences in charge, size, and hydrophilicity. After purification and structural analysis, insights into biological activity and specificity were generated for each compound. Dose-dependent growth inhibition was determined for four tumorigenic breast cancer cell lines in monolayer and spheroid morphologies, as well as nontumorigenic fibroblasts and sheep erythrocytes, which were utilized to determine selectivity indices. Results indicated that two compounds, which have amplified anionic charge, had increased activity on breast cancer, with reduced activity on nontumorigenic fibroblasts and erythrocytes. Cationic derivative surf-ethylenediamine has increased activity on all cell lines tested. Novel correlations between dose-response activities and physicochemical properties of all compounds determined that there is a significant correlation between the critical micelle concentration and activity against multiple cell lines.

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models.

Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

Can biomechanics turn youth sports into a venue for informal STEM engagement?

A common pitfall of existing Science, Technology, Engineering, and Math (STEM) outreach programs is that they preferentially engage youth with a preexisting interest in STEM. Biomechanics has the unique potential to broaden access to STEM enrichment due to its direct applicability to sports and human performance. In this study we examine whether biomechanics within youth sports can be used as a venue for STEM outreach, and whether recruiting participants through youth sports programs could broaden access to the STEM pipeline. We created a four-hour sports science clinic that was performed as part of National Biomechanics Day and invited two groups of student participants: youth recruited through local high school sports programs ("Sports Cohort", N = 80) and youth recruited through existing STEM enrichment programs ("STEM Cohort", N = 31). We evaluated interest in STEM, Sports Science, and Sports using a pre-post survey. Somewhat expectedly, youth recruited through sports programs (Sports Cohort) had a lower baseline interest in STEM and a higher baseline interest in sports, compared to those recruited through STEM programs (STEM Cohort). The Sports Cohort exhibited a statistically significant increase in STEM interest following participation in the clinic, while youth in the STEM Cohort maintained their high baseline of STEM interest. These findings provide evidence that youth sports programs can serve as an attractive partner for biomechanists engaged in STEM outreach, and that situating STEM within sports through biomechanical analysis has potential to introduce STEM interest to a wider audience and to broaden access to the STEM fields among diverse youth.

Tendon cell and tissue culture: Perspectives and recommendations.

The wide variety of cell and tissue culture systems used to study and engineer tendons can make it difficult to choose the best approach and "optimal" culture conditions to test a given hypothesis. Therefore, a breakout session was organized at the 2022 ORS Tendon Section Meeting that focused on establishing a set of guidelines for conducting cell and tissue culture studies of tendon. This paper summarizes the outcomes of that discussion and presents recommendations for future studies. In the case of studying tendon cell behavior, cell and tissue culture systems are reductionist models in which the culture conditions should be strictly defined to approximate the in vivo condition as closely as possible. In contrast, for tissue engineering tendon replacements, the culture conditions do not need to replicate native tendon, but the outcome measures for success should be narrowly defined for the specific clinical application. Common recommendations for both applications are that researchers should perform a baseline phenotypic characterization of the cells that are ultimately used for experimentation. For models of tendon cell behavior, culture conditions should be well justified by existing literature and meticulously reported, tissue explant viability should be assessed, and comparisons to in vivo conditions should be made to determine baseline physiological relevance. For tissue engineering applications, the functional/structural/compositional outcome targets should be defined by the specific tendons they seek to replace, with key biologic and material properties prioritized for construct assessment. Lastly, when engineering tendon replacements, researchers should utilize clinically approved cGMP materials to facilitate clinical translation.

Viscoelastic Properties of Bioprinted Alginate Microbeads Compared to Their Bulk Hydrogel Analogs.

Hydrogel microbeads are engineered spherical microgels widely used for biomedical applications in cell cultures, tissue engineering, and drug delivery. Their mechanical and physical properties (i.e., modulus, porosity, diffusion) heavily influence their utility by affecting encapsulated cellular behavior, biopayload elution kinetics, and stability for longer term cultures. There is a need to quantify these properties to guide microbead design for effective application. However, there are few techniques with the µN-level resolution required to evaluate these relatively small, compliant constructs. To circumvent mechanically testing individual microbeads, researchers often approximate microbead properties by characterizing larger bulk gel analogs of the same material formulation. This approach provides some insight into the hydrogel properties. However, bulk gels possess key structural and mechanical differences compared to their microbead equivalents, which may limit their accuracy and utility as analogs for estimating microbead properties. Herein, we explore how microbead properties are influenced by hydrogel formulation (i.e., alginate concentration, divalent cation crosslinker, and crosslinker concentration), and whether these trends are accurately reflected in bulk gel analogs. To accomplish this, we utilize laser direct-write bioprinting to create 12 × 12 arrays of alginate microbeads and characterize all 144 microbeads in parallel using a commercially available microcompression system. In this way, the compressive load is distributed across a large number of beads, thus amplifying sample signal. Comparing microbead properties to those of their bulk gel analogs, we found that their trends in modulus, porosity, and diffusion with hydrogel formulation are consistent, yet bulk gels exhibit significant discrepancies in their measured values.

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 µm zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 µm of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

Sophorolipids: Anti-cancer activities and mechanisms.

Sophorolipids (SLs) are biosurfactants synthesized as secondary metabolites by non-pathogenic yeasts and other microorganisms. They are members of glycolipid microbial surfactant family that consists of a sophorose polar head group and, most often, an ω-1 hydroxylated fatty acid glycosidically linked to the sophorose moiety. Since the fermentative production of SLs is high (>200 g/L), SLs have the potential to provide low-cost therapeutics. Natural and modified SLs possess anti-cancer activity against a wide range of cancer cell lines such as those derived from breast, cervical, colon, liver, brain, and the pancreas. Corresponding data on their cytotoxicity against noncancerous cell lines including human embryo kidney, umbilical vein, and mouse fibroblasts is also discussed. These results are compiled to elucidate trends in SL-structures that lead to higher efficacy against cancer cell lines and lower cytotoxicity for normal cell lines. While extrapolation of these results provides some insights into the design of SLs with optimal therapeutic indices, we also provide a critical assessment of gaps and inconsistencies in the literature as well as the lack of data connecting structure-to-anticancer and cytotoxicity on normal cells. Furthermore, SL-mechanism of action against cancer cell lines, that includes proliferation inhibition, induction of apoptosis, membrane disruption and mitochondria mediated pathways are discussed. Perspectives on future research to develop SL anticancer therapeutics is discussed.

Lipase-Catalyzed Poly(glycerol-1,8-octanediol-sebacate): Biomaterial Engineering by Combining Compositional and Crosslinking Variables.

This study examined poly(glycerol-1,8-octanediol-sebacate) (PGOS) copolymers with low-level substitution of O (1,8-octanediol) for G (glycerol) units (G/O ratios 0.5:0.5, 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09) prepared in bulk by immobilized Candida antarctica Lipase B (N435) catalysis. The central question explored was the extent that exchanging less than half of poly(glycerol sebacate) (PGS) glycerol units with 1,8-octanediol can be used as a strategy to fine-tune biomaterial properties. Synthesized copolymers having G/O ratios of 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09 have similar molecular weights, where Mw varied from 52,800 to 63,800 g/mol, Mn varied from 5100 to 6450 g/mol, and DM (molecular mass dispersity, Mw/Mn) values were also similar (8.4-11.4). All of the copolymers were branched, and dendritic glycerol units reached 11% for PGOS-0.91:0.09:1.0. Analysis of DSC second heating scans revealed that copolymers with higher 1,8-octanediol contents have relatively higher Tm and ΔHf values. Over the copolymer compositional range studied herein, Tm and ΔHf values varied from 5.3 to 21.1 °C and 8.0 to 23.1 J/g, respectively. Stress-strain curves of PGOS copolymers cured at 140 °C for 48 h exhibited either a unimodal, bimodal, or trimodal response to tensile loading. Varying G/O from 10:1 to 2:1 resulted in significant increases in the peak stress (0.26-4.01 MPa), preyield modulus (0.65-62.59 MPa), failure to strain (64-110%), and failure toughness (0.1-0.56 MPa). This demonstrates that altering the G/O ratio over a narrow compositional range provides biomaterials with widely different yet tunable mechanical properties. Further investigation of PGOS-0.75:0.25:1.0 films revealed that varying the cure conditions from 120 to 160 °C for periods of 24-72 h provides access to biomaterials with a failure strain range from 15 to 224% and Young's modulus from 1.17 to 10.85 MPa. Hence, using the dual variables of compositional variation and changes in cure conditions provides an exciting platform for PGS analogues to optimize material-tissue interactions. Increased contents of 1,8-octanediol slowed in vitro degradation. Slowed degradation of PGOS relative to PGS will be valuable for use in slower healing wounds.

A Bioreactor for Controlled Electrical and Mechanical Stimulation of Developing Scaffold-Free Constructs.

Bioreactors are commonly used to apply biophysically relevant stimulations to tissue-engineered constructs in order to explore how these stimuli influence tissue development, healing, and homeostasis, and they offer great flexibility because key features of the stimuli (e.g., duty cycle, frequency, amplitude, and duration) can be controlled to elicit a desired cellular response. However, most bioreactors that apply mechanical and electrical stimulations do so to a scaffold after the construct has developed, preventing study of the influence these stimuli have on early construct development. To enable such exploration, there is a need for a bioreactor that allows the direct application of mechanical and electrical stimulation to constructs as they develop. Herein, we develop and calibrate a bioreactor, based on our previously established modified Flexcell system, to deliver precise mechanical and electrical stimulation, either independently or in combination, to developing scaffold-free tissue constructs. Linear calibration curves were established, then used to apply precise dynamic mechanical and electrical stimulations, over a range of physiologically relevant strains (0.50%, 0.70%, 0.75%, 1.0%, and 1.5%) and voltages (1.5 and 3.5 V), respectively. Following calibration, applied mechanical and electrical stimulations were not statistically different from their desired target values and were consistent whether delivered independently or in combination. Concurrent delivery of mechanical and electrical stimulation resulted in a negligible change in mechanical (<2%) and electrical (<1%) values, compared to their independently delivered values. With this calibrated bioreactor, we can apply precise, controlled, reproducible mechanical and electrical stimulations, alone or in combination, to scaffold-free, tissue-engineered constructs as they develop.

Mechanobiology in Tendon, Ligament, and Skeletal Muscle Tissue Engineering.

Tendon, ligament, and skeletal muscle are highly organized tissues that largely rely on a hierarchical collagenous matrix to withstand high tensile loads experienced in activities of daily life. This critical biomechanical role predisposes these tissues to injury, and current treatments fail to recapitulate the biomechanical function of native tissue. This has prompted researchers to pursue engineering functional tissue replacements, or dysfunction/disease/development models, by emulating in vivo stimuli within in vitro tissue engineering platforms-specifically mechanical stimulation, as well as active contraction in skeletal muscle. Mechanical loading is critical for matrix production and organization in the development, maturation, and maintenance of native tendon, ligament, and skeletal muscle, as well as their interfaces. Tissue engineers seek to harness these mechanobiological benefits using bioreactors to apply both static and dynamic mechanical stimulation to tissue constructs, and induce active contraction in engineered skeletal muscle. The vast majority of engineering approaches in these tissues are scaffold-based, providing interim structure and support to engineered constructs, and sufficient integrity to withstand mechanical loading. Alternatively, some recent studies have employed developmentally inspired scaffold-free techniques, relying on cellular self-assembly and matrix production to form tissue constructs. Whether utilizing a scaffold or not, incorporation of mechanobiological stimuli has been shown to improve the composition, structure, and biomechanical function of engineered tendon, ligament, and skeletal muscle. Together, these findings highlight the importance of mechanobiology and suggest how it can be leveraged to engineer these tissues and their interfaces, and to create functional multitissue constructs.

Non-Destructive Tumor Aggregate Morphology and Viability Quantification at Cellular Resolution, During Development and in Response to Drug.

Three-dimensional (3D) tissue-engineered in vitro models, particularly multicellular spheroids and organoids, have become important tools to explore disease progression and guide the development of novel therapeutic strategies. These avascular constructs are particularly powerful in oncological research due to their ability to mimic several key aspects of in vivo tumors, such as 3D structure and pathophysiologic gradients. Advancement of spheroid models requires characterization of critical features (i.e., size, shape, cellular density, and viability) during model development, and in response to treatment. However, evaluation of these characteristics longitudinally, quantitatively and non-invasively remains a challenge. Herein, Optical Coherence Tomography (OCT) is used as a label-free tool to assess 3D morphologies and cellular densities of tumor spheroids generated via the liquid overlay technique. We utilize this quantitative tool to assess Matrigel's influence on spheroid morphologic development, finding that the absence of Matrigel produces flattened, disk-like aggregates rather than 3D spheroids with physiologically-relevant features. Furthermore, this technology is adapted to quantify cell number within tumor spheroids, and to discern between live and dead cells, to non-destructively provide valuable information on tissue/construct viability, as well as a proof-of-concept for longitudinal drug efficacy studies. Together, these findings demonstrate OCT as a promising noninvasive, quantitative, label-free, longitudinal and cell-based method that can assess development and drug response in 3D cellular aggregates at a mesoscopic scale.

Enzymatic Polymerization of Poly(glycerol-1,8-octanediol-sebacate): Versatile Poly(glycerol sebacate) Analogues that Form Monocomponent Biodegradable Fiber Scaffolds.

A family of poly(glycerol sebacate) (PGS) analogues were synthesized by Candida antarctica lipase B (CALB) catalysis to tailor biomaterial properties. Different fractions of glycerol (G) units in PGS were replaced by 1,8-octanediol (O) units. Poly(glycerol-1,8-octanediol-sebacate), PGOS, synthesized by CALB catalysis with a 1:3 molar ratio of G to O units has Mn and Mw values of 9500 and 92,000, respectively. PGS undergoes fiber fusion during electrospinning, and cross-linked PGS rapidly resorbs when implanted. By decreasing the molar ratio of glycerol-to-octanediol from 1:1 to 1:4, the peak melting temperature (Tm) increased from 27 to 47 °C. PGOS with 1:3 G to O units was electrospun into nanofibers without the need for a second component. The copolymer is semicrystalline and, when cross-linked, undergoes slow in vitro mass loss (3.5 ± 1.0% in 31 days) at pH 7.4 and 37 °C. Furthermore, PGOS cross-linked films have an elastic modulus of 106.1 ± 18.6 MPa, which is more than 100 times that of cross-linked PGS. New PGOS polymers showed tunable molecular weights, better thermal properties, and excellent electrospinnability. This work expanded PGS analogues' function, making these suitable biodegradable polymers for various biomedical applications.

Characterization of Spatially Graded Biomechanical Scaffolds.

Advances in fabrication have allowed tissue engineers to better mimic complex structures and tissue interfaces by designing nanofibrous scaffolds with spatially graded material properties. However, the nonuniform properties that grant the desired biomechanical function also make these constructs difficult to characterize. In light of this, we developed a novel procedure to create graded nanofibrous scaffolds and determine the spatial distribution of their material properties. Multilayered nanofiber constructs were synthesized, controlling spatial gradation of the stiffness to mimic the soft tissue gradients found in tendon or ligament tissue. Constructs were characterized using uniaxial tension testing with digital image correlation (DIC) to measure the displacements throughout the sample, in a noncontacting fashion, as it deformed. Noise was removed from the displacement data using principal component analysis (PCA), and the final denoised field served as the input to an inverse elasticity problem whose solution determines the spatial distribution of the Young's modulus throughout the material, up to a multiplicative factor. Our approach was able to construct, characterize, and determine the spatially varying moduli, in four electrospun scaffolds, highlighting its great promise for analyzing tissues and engineered constructs with spatial gradations in modulus, such as those at the interfaces between two disparate tissues (e.g., myotendinous junction, tendon- and ligament-to-bone entheses).

Multi-modal characterization of polymeric gels to determine the influence of testing method on observed elastic modulus.

Demand for materials that mechanically replicate native tissue has driven development and characterization of various new biomaterials. However, a consequence of materials and characterization technique diversity is a lack of consensus within the field, with no clear way to compare values measured via different modalities. This likely contributes to the difficulty in replicating findings across the research community; recent evidence suggests that different modalities do not yield the same mechanical measurements within a material, and direct comparisons cannot be made across different testing platforms. Herein, we examine whether "material properties" are characterization modality-specific by analyzing the elastic moduli determined by five typical biomaterial mechanical characterization techniques: unconfined-compression, tensiometry, rheometry, and micro-indentation at the macroscopic level, and microscopically using nanoindentation. These analyses were performed in two different polymeric gels frequently used for biological applications, polydimethylsiloxane (PDMS) and agarose. Each was fabricated to span a range of moduli, from physiologic to supraphysiologic values. All five techniques identified the same overall trend within each material group, supporting their ability to appreciate relative moduli differences. However, significant differences were found across modalities, illustrating a difference in absolute moduli values, and thereby precluding direct comparison of measurements from different characterization modalities. These observed differences may depend on material compliance, viscoelasticity, and microstructure. While determining the underlying mechanism(s) of these differences was beyond the scope of this work, these results demonstrate how each modality affects the measured moduli of the same material, and the sensitivity of each modality to changes in sample material composition.

Laser-based 3D bioprinting for spatial and size control of tumor spheroids and embryoid bodies.

3D multicellular aggregates, and more advanced organotypic systems, have become central tools in recent years to study a wide variety of complex biological processes. Most notably, these model systems have become mainstream within oncology (multicellular tumor spheroids) and regenerative medicine (embryoid bodies) research. However, the biological behavior of these in vitro tissue surrogates is extremely sensitive to their aggregate size and geometry. Indeed, both of these geometrical parameters are key in producing pathophysiological gradients responsible for cellular and structural heterogeneity, replicating in vivo observations. Moreover, the fabrication techniques most widely used for producing these models lack the ability to accurately control cellular spatial location, an essential component for regulating homotypic and heterotypic cell signaling. Herein, we report on a 3D bioprinting technique, laser direct-write (LDW), that enables precise control of both spatial patterning and size of cell-encapsulating microbeads. The generated cell-laden beads are further processed into core-shelled structures, allowing for the growth and formation of self-contained, self-aggregating cells (e.g., breast cancer cells, embryonic stem cells). Within these structures we demonstrate our ability to produce multicellular tumor spheroids (MCTSs) and embryoid bodies (EBs) with well-controlled overall size and shape, that can be designed on demand. Furthermore, we investigated the impact of aggregate size on the uptake of a commonly employed ligand for receptor-mediated drug delivery, Transferrin, indicating that larger tumor spheroids exhibit greater spatial heterogeneity in ligand uptake. Taken together, these findings establish LDW as a versatile biomanufacturing platform for bioprinting and patterning core-shelled structures to generate size-controlled 3D multicellular aggregates. STATEMENT OF SIGNIFICANCE: Multicellular 3D aggregates are powerful in vitro models used to study a wide variety of complex biological processes, particularly within oncology and regenerative medicine. These tissue surrogates are fabricated using environments that encourage cellular self-assembly. However, specific applications require control of aggregate size and position to recapitulate key in vivo parameters (e.g., pathophysiological gradients and homotypic/heterotypic cell signaling). Herein, we demonstrate the ability to create and spatially pattern size-controlled embryoid bodies and tumor spheroids, using laser-based 3D bioprinting. Furthermore, we investigated the effect of tumor spheroid size on internalization of Transferrin, a common ligand for targeted therapy, finding greater spatial heterogeneity in our large aggregates. Overall, this technique offers incredible promise and flexibility for fabricating idealized 3D in vitro models.

On-Demand Radial Electrodeposition of Alginate Tubular Structures.

We present an electrodeposition technique for fabricating tubular alginate structures. In this technique, two electrodes (anode and cathode) are suspended in a solution of alginate and insoluble calcium carbonate particles, and the application of an electrical potential produces a localized pH change at the anode surface causing suspended divalent cations to become soluble and cross-link the alginate. We robustly characterize how the fabrication parameters influence the rate of radial deposition on the anode, including deposition time, applied voltage, alginate concentration, type of divalent cation and concentration, and anode diameter. Furthermore, we produce gels with a range of tailorable features, including mechanical properties, dimensions (thick-ness and lumen size), customizable tubular geometries, and radial compositional heterogeneity.

Cyclic Uniaxial Tensile Strain Enhances the Mechanical Properties of Engineered, Scaffold-Free Tendon Fibers.

The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.

Solvent retention in electrospun fibers affects scaffold mechanical properties.

Electrospinning is a robust material fabrication method allowing for fine control of mechanical, chemical, and functional properties in scaffold manufacturing. Electrospun fiber scaffolds have gained prominence for their potential in a variety of applications such as tissue engineering and textile manufacturing, yet none have assessed the impact of solvent retention in fibers on the scaffold's mechanical properties. In this study, we hypothesized that retained electrospinning solvent acts as a plasticizer, and gradual solvent evaporation, by storing fibers in ambient air, will cause significant increases in electrospun fiber scaffold brittleness and stiffness, and a significant decrease in scaffold toughness. Thermogravimetric analysis indicated solvent retention in PGA, PLCL, and PET fibers, and not in PU and PCL fibers. Differential scanning calorimetry revealed that polymers that were electrospun below their glass transition temperature (T g ) retained solvent and polymers electrospun above T g did not. Young's moduli increased and yield strain decreased for solventretaining PGA, PLCL, and PET fiber scaffolds as solvent evaporated from the scaffolds over a period of 14 days. Toughness and failure strain decreased for PGA and PET scaffolds as solvent evaporated. No significant differences were observed in the mechanical properties of PU and PCL scaffolds that did not retain solvent. These observations highlight the need to consider solvent retention following electrospinning and its potential effects on scaffold mechanical properties.