Off-Axis Loading Fixture for Spine Biomechanics: Combined Compression and Bending.

The spine is a multi-tissue musculoskeletal system that supports large multi-axial loads and motions during physiological activities. The healthy and pathological biomechanical function of the spine and its subtissues are generally studied using cadaveric specimens that often require multi-axis biomechanical test systems to mimic the complex loading environment of the spine. Unfortunately, an off-the-shelf device can easily exceed 200,000 USD, while a custom device requires extensive time and experience in mechatronics. Our goal was to develop a cost-appropriate compression and bending (flexion-extension and lateral bending) spine testing system that requires little time and minimal technical knowledge. Our solution was an off-axis loading fixture (OLaF) that mounts to an existing uni-axial test frame and requires no additional actuators. OLaF requires little machining, with most components purchased off-the-shelf, and costs less than 10,000 USD. The only external transducer required is a six-axis load cell. Furthermore, OLaF is controlled using the existing uni-axial test frame's software, while the load data is collected using the software included with the six-axis load cell. Here we provide the design rationale for how OLaF develops primary motions and loads and minimizes off-axis secondary constraints, verify the primary kinematics using motion capture, and demonstrate that the system is capable of applying physiologically relevant, noninjurious, axial compression and bending. While OLaF is limited to compression and bending studies it produces repeatable physiologically relevant biomechanics, with high quality data, and minimal startup costs.

Detrimental effects of long sedentary bouts on the biomechanical response of cartilage to sliding.

Purpose/Aim: Epidemiological evidence suggests, contrary to popular mythos, that increased exercise/joint activity does not place articular cartilage at increased risk of disease, but instead promotes joint health. One explanation for this might be activity-induced cartilage rehydration; where joint articulation drives restoration of tissue hydration, thickness, and dependent tribomechanical outcomes (e.g., load support, stiffness, and lubricity) lost to joint loading. However, there have been no studies investigating how patterning of intermittent articulation influences the hydration and biomechanical functions of cartilage.Materials and Methods: Here we leveraged the convergent stationary contact area (cSCA) testing configuration and its unique ability to drive tribological rehydration, to elucidate how intermittency of activity affects the biomechanical functions of bovine stifle cartilage under well-controlled sliding conditions that have been designed to model a typical "day" of human joint activity.Results: For a fixed volume of "daily" activity (30 min) and sedentary time (60 min), breaking up intermittent activity into longer and less-frequent bouts (corresponding to longer continuous sedentary periods) resulted in the exposure of articular cartilage to markedly greater strains, losses of interstitial pressure, and friction coefficients.Conclusions: These results demonstrated that the regularity of ex vivo activity regimens, specifically the duration of sedentary bouts, had a dominant effect on the biomechanical functions of articular cartilage. In more practical terms, the results suggest that brief but regular movement patterns (e.g., every hour) may be biomechanically preferred to long and infrequent movement patterns (e.g., a long walk after a sedentary day) when controlling for daily activity volume (e.g., 30 min).

Can axial loading restore in vivo disc geometry, opening pressure, and T2 relaxation time?

Background: Cadaveric intervertebral discs are often studied for a variety of research questions, and outcomes are interpreted in the in vivo context. Unfortunately, the cadaveric disc does not inherently represent the LIVE condition, such that the disc structure (geometry), composition (T2 relaxation time), and mechanical function (opening pressure, OP) measured in the cadaver do not necessarily represent the in vivo disc. Methods: We conducted serial evaluations in the Yucatan minipig of disc geometry, T2 relaxation time, and OP to quantify the changes that occur with progressive dissection and used axial loading to restore the in vivo condition. Results: We found no difference in any parameter from LIVE to TORSO; thus, within 2 h of sacrifice, the TORSO disc can represent the LIVE condition. With serial dissection and sample preparation the disc height increased (SEGMENT height 18% higher than TORSO), OP decreased (POTTED was 67% lower than TORSO), and T2 time was unchanged. With axial loading, an imposed stress of 0.20-0.33 MPa returned the disc to in vivo, LIVE disc geometry and OP, although T2 time was decreased. There was a linear correlation between applied stress and OP, and this was conserved across multiple studies and species. Conclusion: To restore the LIVE disc state in human studies or other animal models, we recommend measuring the OP/stress relationship and using this relationship to select the applied stress necessary to recover the in vivo condition.

Minimum design requirements for a poroelastic mimic of articular cartilage.

The exceptional functional performance of articular cartilage (load-bearing and lubrication) is attributed to its poroelastic structure and resulting interstitial fluid pressure. Despite this, there remains no engineered cartilage repair material capable of achieving physiologically relevant poroelasticity. In this work we develop in silico models to guide the design approach for poroelastic mimics of articular cartilage. We implement the constitutive models in FEBio, a PDE solver for multiphasic mechanics problems in biological and soft materials. We investigate the influence of strain rate, boundary conditions at the contact interface, and fiber modulus on the reaction force and load sharing between the solid and fluid phases. The results agree with the existing literature that when fibers are incorporated the fraction of load supported by fluid pressure is greatly amplified and increases with the fiber modulus. This result demonstrates that a stiff fibrous phase is a primary design requirement for poroelastic mimics of articular cartilage. The poroelastic model is fit to experimental stress-relaxation data from bovine and porcine cartilage to determine if sufficient design constraints have been identified. In addition, we fit experimental data from FiHy™, an engineered material which is claimed to be poroelastic. The fiber-reinforced poroelastic model was able to capture the primary physics of these materials and demonstrates that FiHy™ is beginning to approach a cartilage-like poroelastic response. We also develop a fiber-reinforced poroelastic model with a bonded interface (rigid contact) to fit stress relaxation data from an osteochondral explant and FiHy™ + bone substitute. The model fit quality is similar for both the chondral and osteochondral configurations and clearly captures the first order physics. Based on this, we propose that physiological poroelastic mimics of articular cartilage should be developed under a fiber-reinforced poroelastic framework.

Microfibrous Scaffolds Guide Stem Cell Lumenogenesis and Brain Organoid Engineering.

3D organoids are widely used as tractable in vitro models capable of elucidating aspects of human development and disease. However, the manual and low-throughput culture methods, coupled with a low reproducibility and geometric heterogeneity, restrict the scope and application of organoid research. Combining expertise from stem cell biology and bioengineering offers a promising approach to address some of these limitations. Here, melt electrospinning writing is used to generate tuneable grid scaffolds that can guide the self-organization of pluripotent stem cells into patterned arrays of embryoid bodies. Grid geometry is shown to be a key determinant of stem cell self-organization, guiding the position and size of emerging lumens via curvature-controlled tissue growth. Two distinct methods for culturing scaffold-grown embryoid bodies into either interconnected or spatially discrete cerebral organoids are reported. These scaffolds provide a high-throughput method to generate, culture, and analyze large numbers of organoids, substantially reducing the time investment and manual labor involved in conventional methods of organoid culture. It is anticipated that this methodological development will open up new opportunities for guiding pluripotent stem cell culture, studying lumenogenesis, and generating large numbers of uniform organoids for high-throughput screening.

Translating musculoskeletal bioengineering into tissue regeneration therapies.

Musculoskeletal injuries and disorders are the leading cause of physical disability worldwide and a considerable socioeconomic burden. The lack of effective therapies has driven the development of novel bioengineering approaches that have recently started to gain clinical approvals. In this review, we first discuss the self-repair capacity of the musculoskeletal tissues and describe causes of musculoskeletal dysfunction. We then review the development of novel biomaterial, immunomodulatory, cellular, and gene therapies to treat musculoskeletal disorders. Last, we consider the recent regulatory changes and future areas of technological progress that can accelerate translation of these therapies to clinical practice.

In vitro and in vivo investigation of a zonal microstructured scaffold for osteochondral defect repair.

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [ϵ-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen™, and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

A Novel Ventilator Design for COVID-19 and Resource-Limited Settings.

There has existed a severe ventilator deficit in much of the world for many years, due in part to the high cost and complexity of traditional ICU ventilators. This was highlighted and exacerbated by the emergence of the COVID-19 pandemic, during which the increase in ventilator production rapidly overran the global supply chains for components. In response, we propose a new approach to ventilator design that meets the performance requirements for COVID-19 patients, while using components that minimise interference with the existing ventilator supply chains. The majority of current ventilator designs use proportional valves and flow sensors, which remain in short supply over a year into the pandemic. In the proposed design, the core components are on-off valves. Unlike proportional valves, on-off valves are widely available, but accurate control of ventilation using on-off valves is not straightforward. Our proposed solution combines four on-off valves, a two-litre reservoir, an oxygen sensor and two pressure sensors. Benchtop testing of a prototype was performed with a commercially available flow analyser and test lungs. We investigated the accuracy and precision of the prototype using both compressed gas supplies and a portable oxygen concentrator, and demonstrated the long-term durability over 15 days. The precision and accuracy of ventilation parameters were within the ranges specified in international guidelines in all tests. A numerical model of the system was developed and validated against experimental data. The model was used to determine usable ranges of valve flow coefficients to increase supply chain flexibility. This new design provides the performance necessary for the majority of patients that require ventilation. Applications include COVID-19 as well as pneumonia, influenza, and tuberculosis, which remain major causes of mortality in low and middle income countries. The robustness, energy efficiency, ease of maintenance, price and availability of on-off valves are all advantageous over proportional valves. As a result, the proposed ventilator design will cost significantly less to manufacture and maintain than current market designs and has the potential to increase global ventilator availability.

Size-Tunable Nanoneedle Arrays for Influencing Stem Cell Morphology, Gene Expression, and Nuclear Membrane Curvature.

High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols were used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that could be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used changes in the nanoneedle tip diameter to control the morphology, nuclear size, and F-actin alignment of interfaced hMSCs and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes, and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays, and the degree of nuclear membrane impingement, with the latter clearly visualized using focused ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring.

Buoyancy-Driven Gradients for Biomaterial Fabrication and Tissue Engineering.

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment and cannot be generally applied to the wide range of systems used for tissue engineering. Here, the fundamental physical principle of buoyancy is exploited as a generalized approach for generating materials bearing well-defined compositional, mechanical, or biochemical gradients. Gradient formation is demonstrated across a range of different materials (e.g., polymers and hydrogels) and cargos (e.g., liposomes, nanoparticles, extracellular vesicles, macromolecules, and small molecules). As well as providing versatility, this buoyancy-driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique is readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, is used to locally stimulate osteogenesis and mineralization in order to produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.

Spatial Patterning of Molecular Cues and Vascular Cells in Fully Integrated Hydrogel Channels via Interfacial Bioorthogonal Cross-Linking.

Fully integrated hydrogel channels were fabricated via interfacial bioorthogonal cross-linking, a diffusion-controlled method for the creation and patterning of synthetic matrices based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) dienophiles. Injecting an aqueous solution of a bisTCO cross-linker into a reservoir of tetrazine-modified hyaluronic acid (HA-Tz), while simultaneously drawing the syringe needle through the reservoir, yielded a cross-linked hydrogel channel that was mechanically robust. Fluorescent tags and biochemical signals were spatially patterned into the channel wall through time-dependent perfusion of TCO-conjugated molecules into the lumen of the channel. Different cell populations were spatially encapsulated in the channel wall via temporal alteration of cells in the HA-Tz reservoir. The interfacial approach enabled the spatial patterning of vascular cells, including human abdominal aorta endothelial cells, aortic vascular smooth muscle cells, and aortic adventitial fibroblasts, into the hydrogel channels with high viability and proper morphology in the anatomical order found in human arteries. The bioorthogonal platform does not rely on external triggers and represents the first step toward the engineering of functional and implantable arteries.

Mapping the spatiotemporal evolution of solute transport in articular cartilage explants reveals how cartilage recovers fluid within the contact area during sliding.

The interstitial fluid within articular cartilage shields the matrix from mechanical stresses, reduces friction and wear, enables biochemical processes, and transports solutes into and out of the avascular extracellular matrix. The balanced competition between fluid exudation and recovery under load is thus critical to the mechanical and biological functions of the tissue. We recently discovered that sliding alone can induce rapid solute transport into buried cartilage contact areas via a phenomenon termed tribological rehydration. In this study, we use in situ confocal microscopy measurements to track the spatiotemporal propagation of a small neutral solute into the buried contact area to clarify the fluid mechanics underlying the tribological rehydration phenomenon. Sliding experiments were interrupted by periodic static loading to enable scanning of the entire contact area. Spatiotemporal patterns of solute transport combined with tribological data suggested pressure driven flow through the extracellular matrix from the contact periphery rather than into the surface via a fluid film. Interestingly, these testing interruptions also revealed dynamic, repeatable and history-independent fluid loss and recovery processes consistent with those observed in vivo. Unlike the migrating contact area, which preserves hydration by moving faster than interstitial fluid can flow, our results demonstrate that the stationary contact area can maintain and actively recover hydration through a dynamic competition between load-induced exudation and sliding-induced recovery. The results demonstrate that sliding contributes to the recovery of fluid and solutes by cartilage within the contact area while clarifying the means by which it occurs.

Cellular interactions with hydrogel microfibers synthesized via interfacial tetrazine ligation.

Fibrous proteins found in the natural extracellular matrix (ECM) function as host substrates for migration and growth of endogenous cells during wound healing and tissue repair processes. Although various fibrous scaffolds have been developed to recapitulate the microstructures of the native ECM, facile synthesis of hydrogel microfibers that are mechanically robust and biologically active have been elusive. Described herein is the use of interfacial bioorthogonal polymerization to create hydrogel-based microfibrous scaffolds via tetrazine ligation. Combination of a trifunctional strained trans-cyclooctene monomer and a difunctional s-tetrazine monomer at the oil-water interface led to the formation of microfibers that were stable under cell culture conditions. The bioorthogonal nature of the synthesis allows for direct incorporation of tetrazine-conjugated peptides or proteins with site-selectively, genetically encoded tetrazines. The microfibers provide physical guidance and biochemical signals to promote the attachment, division and migration of fibroblasts. Mechanistic investigations revealed that fiber-guided cell migration was both F-actin and microtubule-dependent, confirming contact guidance by the microfibers. Prolonged culture of fibroblasts in the presence of an isolated microfiber resulted in the formation of a multilayered cell sheet wrapping around the fiber core. A fibrous mesh provided a 3D template to promote cell infiltration and tissue-like growth. Overall, the bioorthogonal approach led to the straightforward synthesis of crosslinked hydrogel microfibers that can potentially be used as instructive materials for tissue repair and regeneration.

Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering.

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.

Unexpected Tribological Synergy in Polymer Blend Coatings: Leveraging Phase Separation to Isolate Domain Size Effects and Reduce Friction.

We employed a systematic processing approach to control phase separation in polymer blend thin films and significantly reduce dynamic friction coefficients (µ)s. We leveraged this modulation of phase separation to generate composite surfaces with dynamic friction coefficients that were substantially lower than expected on the basis of simple mixing rules, and in several cases, these friction coefficients were lower than those of both pure components. Using a model polyisoprene [PI]/polystyrene [PS] composite system, a minimum µ was found in films with PS mass fractions between 0.60 and 0.80 (µblend = 0.11 ± 0.03); that value was significantly lower than the friction coefficient of PS (µPS = 0.52 ± 0.01) or PI (µPI = 1.3 ± 0.09) homopolymers and was comparable to the friction coefficient of poly(tetrafluoroethylene) [PTFE] (µPTFE = 0.09 ± 0.01) measured under similar conditions. Additionally, through experiments in which the domain size was systematically varied at constant composition (through an annealing process), we demonstrated that µ decreased with decreasing characteristic domain size. Thus, the tribological synergy between PS and PI domains (discrete size, physical domain isolation, and overall film composition) was shown to play an integral role in the friction and wear of these PS/PI composites. Overall, our results suggest that even high friction polymers can be used to create low friction polymer blends by following appropriate design rules and demonstrate that engineering microstructure is critical for controlling the friction and adhesion properties of composite films for tribologically relevant coatings.