APL Bioengineering最新文献

In tissues and organs, the extracellular matrix (ECM) helps maintain inter- and intracellular architectures that sustain the structure-function relationships defining physiological homeostasis. Combining fiber scaffolds and cells to form engineered tissues is a means of replicating these relationships. Engineered tissues' fiber scaffolds are designed to mimic the topology and chemical composition of the ECM network. Here, we asked how cells found in the heart compare in their propensity to align their cytoskeleton and self-organize in response to topological cues in fibrous scaffolds. We studied cardiomyocytes, valvular interstitial cells, and vascular endothelial cells as they adapted their inter- and intracellular architectures to the extracellular space. We used focused rotary jet spinning to manufacture aligned fibrous scaffolds to mimic the length scale and three-dimensional (3D) nature of the native ECM in the muscular, valvular, and vascular tissues of the heart. The representative cardiovascular cell types were seeded onto fiber scaffolds and infiltrated the fibrous network. We measured different cell types' propensity for cytoskeletal alignment in response to fiber scaffolds with differing levels of anisotropy. The results indicated that valvular interstitial cells on moderately anisotropic substrates have a higher propensity for cytoskeletal alignment than cardiomyocytes and vascular endothelial cells. However, all cell types displayed similar levels of alignment on more extreme (isotropic and highly anisotropic) fiber scaffold organizations. These data suggest that in the hierarchy of signals that dictate the spatiotemporal organization of a tissue, geometric cues within the ECM and cellular networks may homogenize behaviors across cell populations and demographics.

There is critical need for a predictive model of human cardiac physiology in drug development to assess compound effects on human tissues. In vitro two-dimensional monolayer cultures of cardiomyocytes provide biochemical and cellular readouts, and in vivo animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to their incomplete recapitulation of adult human cardiovascular physiology. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in three-dimensional tissue structure. Here, we advance this paradigm by implementing FRESH™ 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its drug response. We combined hiPSC-CMs, endothelial cells, and fibroblasts in a cellular bioink and FRESH™ 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. We show that cardiac tissues could be fabricated directly in a 24-well plate format were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL and, within 14 days, demonstrated reproducible calcium transients and a fast conduction velocity of ∼16 cm/s. Interrogation of these cardiac tissues with the β-adrenergic receptor agonist isoproterenol showed responses consistent with positive chronotropy and inotropy. Treatment with calcium channel blocker verapamil demonstrated responses expected of hiPSC-CM derived cardiac tissues. These results confirm that FRESH™ 3D bioprinted cardiac tissues represent an in vitro platform that provides data on human physiological response.

[This corrects the article DOI: 10.1063/5.0163264.].

Fractional-pixel CO₂ laser therapy shows promise for treating the genitourinary syndrome of menopause (GSM). Nevertheless, it remains controversial in the field of female pelvic medicine. This is due to the inherent difficulties in obtaining noninvasive biopsies to evaluate the treatment's efficacy and safety objectively. To address this challenge, we developed a noninvasive intravaginal optical coherence tomography (OCT)/OCT angiography (OCTA) endoscopic system, whose probe features a shape identical to the laser treatment probe. This system can provide high-resolution OCT images to identify the microstructure of vaginal tissue and visualize the vasculature network in vivo. We conducted clinical research on 25 post-menopausal patients with GSM. OCT/OCTA scans were acquired at four different locations of the vagina (distal anterior, distal posterior, proximal anterior, and proximal posterior) during the whole laser treatment session. A U-Net deep learning model was applied to segment the vaginal epithelium for assessing vaginal epithelial thickness (VET). Blood vessel density and VET were quantified to monitor the efficacy of fractional-pixel CO₂ laser therapy. Statistical correlation analyses between these metrics and other clinical scores were conducted, validating the utility of our system. This OCT/OCTA endoscopic system has great potential to serve as a noninvasive biopsy tool in gynecological studies to screen, evaluate, and guide laser treatment for GSM.

Real-time closed-loop control of neuromodulation devices requires long-term monitoring of neural activity in the peripheral nervous system. Although many signal extraction methods exist, few are both clinically viable and designed for extracting small signals from fragile peripheral visceral nerves. Here, we report that our minimally invasive recording and analysis technology extracts low to negative signal to noise ratio (SNR) neural activity from a visceral nerve with a high degree of specificity for fiber type and class. Complex activity was recorded from the rat pelvic nerve that was physiologically evoked during controlled bladder filling and voiding, in an extensively characterized in vivo model that provided an excellent test bed to validate our technology. Urethane-anesthetized male rats (n = 12) were implanted with a four-electrode planar array and the bladder instrumented for continuous-flow cystometry, which measures urodynamic function by recording bladder pressure changes during constant infusion of saline. We demonstrated that differential bipolar recordings and cross-correlation analyses extracts afferent and efferent activity, and discriminated between subpopulations of fibers based on conduction velocity. Integrated Aδ afferent fiber activity correlated with bladder pressure during voiding (r²: 0.66 ± 0.06) and was not affected by activating nociceptive afferents with intravesical capsaicin (r²: 0.59 ± 0.14, P = 0.54, and n = 3). Collectively, these results demonstrate our minimally invasive recording and analysis technology is selective in extracting mixed neural activity with low/negative SNR. Furthermore, integrated afferent activity reliably correlates with bladder pressure and is a promising first step in developing closed-loop technology for bladder control.

Spinal cord injury (SCI) arises from damage to the spinal cord, often caused by trauma or disease. The resulting sensorimotor dysfunction is variable and dependent on the extent of the injury. Despite years of research, curative options for SCI remain limited. However, recent advancements in electric field stimulated axonal regrowth have shown promise for neuronal regeneration. One roadblock in the development of therapeutic treatments based on this is a lack of understanding of the exogenous electric field distribution in the injured tissue, and in particular, how this is influenced by electrode geometry and placement. To better understand this electric field, and provide a means by which it can be optimized, we have developed a finite element model of such spinal cord treatment. We investigate the impact of variations in electrode geometry, spinal cord size, and applied current magnitude as well as looking at several injury models in relation to clinically observed outcomes. Through this, we show that electrode shape has little effect on the induced electric field, that the placement of these electrodes has a noticeable influence on the field distribution, and that the magnitude of this field is governed by both the applied current and the spinal cord morphology. We also show that the injury modality influences the induced field distribution and that a stronger understanding of the injury will help decide treatment parameters. This work provides guidance in the design of electrodes for future clinical application in direct current electric field stimulation for axonal regeneration.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.