Overcoming big bottlenecks in vascular regeneration.

Bioengineering and regenerative medicine strategies are promising for the treatment of vascular diseases. However, current limitations inhibit the ability of these approaches to be translated to clinical practice. Here we summarize some of the big bottlenecks that inhibit vascular regeneration in the disease applications of aortic aneurysms, stroke, and peripheral artery disease. We also describe the bottlenecks preventing three-dimensional bioprinting of vascular networks for tissue engineering applications. Finally, we describe emerging technologies and opportunities to overcome these challenges to advance vascular regeneration.

Skeletal muscle-on-a-chip in microgravity as a platform for regeneration modeling and drug screening.

Microgravity has been shown to lead to both muscle atrophy and impaired muscle regeneration. The purpose was to study the efficacy of microgravity to model impaired muscle regeneration in an engineered muscle platform and then to demonstrate the feasibility of performing drug screening in this model. Engineered human muscle was launched to the International Space Station National Laboratory, where the effect of microgravity exposure for 7 days was examined by transcriptomics and proteomics approaches. Gene set enrichment analysis of engineered muscle cultured in microgravity, compared to normal gravity conditions, highlighted a metabolic shift toward lipid and fatty acid metabolism, along with increased apoptotic gene expression. The addition of pro-regenerative drugs, insulin-like growth factor-1 (IGF-1) and a 15-hydroxyprostaglandin dehydrogenase inhibitor (15-PGDH-i), partially inhibited the effects of microgravity. In summary, microgravity mimics aspects of impaired myogenesis, and the addition of these drugs could partially inhibit the effects induced by microgravity.

Institutional Support for the Career Advancement of Women Faculty in Science and Academic Medicine: Successes, Challenges, and Future Directions.

Institutional support is crucial for the successful career advancement of all faculty but in particular those who are women. Evolving from the past, in which gender disparities were prevalent in many institutions, recent decades have witnessed significant progress in supporting the career advancement of women faculty in science and academic medicine. However, continued advancement is necessary as previously unrecognized needs and new opportunities for improvement emerge. To identify the needs, opportunities, and potential challenges encountered by women faculty, the Women's Leadership Committee of the Arteriosclerosis, Thrombosis, and Vascular Biology Council developed an initiative termed GROWTH (Generating Resources and Opportunities for Women in Technology and Health). The committee designed a survey questionnaire and interviewed 19 leaders with roles and responsibilities in faculty development from a total of 12 institutions across various regions of the United States. The results were compiled, analyzed, and discussed. Based on our interviews and analyses, we present the current status of these representative institutions in supporting faculty development, highlighting efforts specific to women faculty. Through the experiences, insights, and vision of these leaders, we identified success stories, challenges, and future priorities. Our article provides a primer and a snapshot of institutional efforts to support the advancement of women faculty. Importantly, this article can serve as a reference and resource for academic entities seeking ideas to gauge their commitment level to women faculty and to implement new initiatives. Additionally, this article can provide guidance and strategies for women faculty as they seek support and resources from their current or prospective institutions when pursuing new career opportunities.

Bioengineering Cell Therapy for Treatment of Peripheral Artery Disease.

Peripheral artery disease is an atherosclerotic disease associated with limb ischemia that necessitates limb amputation in severe cases. Cell therapies comprised of adult mononuclear or stromal cells have been clinically tested and show moderate benefits. Bioengineering strategies can be applied to modify cell behavior and function in a controllable fashion. Using mechanically tunable or spatially controllable biomaterials, we highlight examples in which biomaterials can increase the survival and function of the transplanted cells to improve their revascularization efficacy in preclinical models. Biomaterials can be used in conjunction with soluble factors or genetic approaches to further modulate the behavior of transplanted cells and the locally implanted tissue environment in vivo. We critically assess the advances in bioengineering strategies such as 3-dimensional bioprinting and immunomodulatory biomaterials that can be applied to the treatment of peripheral artery disease and then discuss the current challenges and future directions in the implementation of bioengineering strategies.

Cardiovascular human organ-on-a-chip platform for disease modeling, drug development, and personalized therapy.

Cardiovascular organ-on-a-chip (OoC) devices are composed of engineered or native functional tissues that are cultured under controlled microenvironments inside microchips. These systems employ microfabrication and tissue engineering techniques to recapitulate human physiology. This review focuses on human OoC systems to model cardiovascular diseases, to perform drug screening, and to advance personalized medicine. We also address the challenges in the generation of organ chips that can revolutionize the large-scale application of these systems for drug development and personalized therapy.

Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

Development of a rat model of lymphedema and the implantation of a collagen-based medical device for therapeutic intervention.

Secondary lymphedema is a common condition among cancer survivors, and treatment strategies to prevent or treat lymphedema are in high demand. The development of novel strategies to diagnose or treat lymphedema would benefit from a robust experimental animal model of secondary lymphedema. The purpose of this methods paper is to describe and summarize our experience in developing and characterizing a rat hindlimb model of lymphedema. Here we describe a protocol to induce secondary lymphedema that takes advantage of micro computed tomography imaging for limb volume measurements and visualization of lymph drainage with near infrared imaging. To demonstrate the utility of this preclinical model for studying the therapeutic benefit of novel devices, we apply this animal model to test the efficacy of a biomaterials-based implantable medical device.

Chronic nicotine impairs the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells in a murine model of peripheral arterial disease.

Objective: Lifestyle choices such as tobacco and e-cigarette use are a risk factor for peripheral arterial disease (PAD) and may influence therapeutic outcomes. The effect of chronic nicotine exposure on the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) was assessed in a murine model of PAD. Methods: Mice were exposed to nicotine or phosphate-buffered saline (PBS) for 28 days, followed by induction of limb ischemia and iPSC-EC transplantation. Cells were injected into the ischemic limb immediately after induction of hindlimb ischemia and again 7 days later. Limb perfusion was assessed by laser Doppler spectroscopy, and transplant cell survival was monitored for 14 days afterward using bioluminescence imaging, followed by histological analysis of angiogenesis. Results: Transplant cell retention progressively decreased over time after implantation based on bioluminescence imaging, and there were no significant differences in cell survival between mice with chronic exposure to nicotine or PBS. However, compared with mice without nicotine exposure, mice with prior nicotine exposure had had an impaired therapeutic response to iPSC-EC therapy based on decreased vascular perfusion recovery. Mice with nicotine exposure, followed by cell transplantation, had significantly lower mean perfusion ratio after 14 days (0.47 ± 0.07) compared with mice undergoing cell transplantation without prior nicotine exposure (0.79 ± 0.11). This finding was further supported by histological analysis of capillary density, in which animals with prior nicotine exposure had a lower capillary density (45.9 ± 4.7 per mm2) compared with mice without nicotine exposure (66.5 ± 8.1 per mm2). Importantly, the ischemic limbs mice exposed to nicotine without cell therapy also showed significant impairment in perfusion recovery after 14 days, compared with mice that received PBS + iPSC-EC treatment. This result suggested that mice without chronic nicotine exposure could respond to iPSC-EC implantation into the ischemic limb by inducing perfusion recovery, whereas mice with chronic nicotine exposure did not respond to iPSC-EC therapy. Conclusions: Together, these findings show that chronic nicotine exposure adversely affects the ability of iPSC-EC therapy to promote vascular perfusion recovery and angiogenesis in a murine PAD model.

Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Biomedical Applications of Collagen.

Extracellular matrix proteins (ECMs) provide structural support and dynamic signaling cues that regulate cell behavior and tissue morphogenesis [...].

Engineering Spatiotemporal Control in Vascularized Tissues.

A major challenge in engineering scalable three-dimensional tissues is the generation of a functional and developed microvascular network for adequate perfusion of oxygen and growth factors. Current biological approaches to creating vascularized tissues include the use of vascular cells, soluble factors, and instructive biomaterials. Angiogenesis and the subsequent generation of a functional vascular bed within engineered tissues has gained attention and is actively being studied through combinations of physical and chemical signals, specifically through the presentation of topographical growth factor signals. The spatiotemporal control of angiogenic signals can generate vascular networks in large and dense engineered tissues. This review highlights the developments and studies in the spatiotemporal control of these biological approaches through the coordinated orchestration of angiogenic factors, differentiation of vascular cells, and microfabrication of complex vascular networks. Fabrication strategies to achieve spatiotemporal control of vascularization involves the incorporation or encapsulation of growth factors, topographical engineering approaches, and 3D bioprinting techniques. In this article, we highlight the vascularization of engineered tissues, with a focus on vascularized cardiac patches that are clinically scalable for myocardial repair. Finally, we discuss the present challenges for successful clinical translation of engineered tissues and biomaterials.

Scalable macroporous hydrogels enhance stem cell treatment of volumetric muscle loss.

Volumetric muscle loss (VML), characterized by an irreversible loss of skeletal muscle due to trauma or surgery, is accompanied by severe functional impairment and long-term disability. Tissue engineering strategies combining stem cells and biomaterials hold great promise for skeletal muscle regeneration. However, scaffolds, including decellularized extracellular matrix (dECM), hydrogels, and electrospun fibers, used for VML applications generally lack macroporosity. As a result, the scaffolds used typically delay host cell infiltration, transplanted cell proliferation, and new tissue formation. To overcome these limitations, we engineered a macroporous dECM-methacrylate (dECM-MA) hydrogel, which we will refer to as a dECM-MA sponge, and investigated its therapeutic potential in vivo. Our results demonstrate that dECM-MA sponges promoted early cellularization, endothelialization, and establishment of a pro-regenerative immune microenvironment in a mouse VML model. In addition, dECM-MA sponges enhanced the proliferation of transplanted primary muscle stem cells, muscle tissue regeneration, and functional recovery four weeks after implantation. Finally, we investigated the scale-up potential of our scaffolds using a rat VML model and found that dECM-MA sponges significantly improved transplanted cell proliferation and muscle regeneration compared to conventional dECM scaffolds. Together, these results validate macroporous hydrogels as novel scaffolds for VML treatment and skeletal muscle regeneration.

Advances in three-dimensional bioprinted stem cell-based tissue engineering for cardiovascular regeneration.

Three-dimensional (3D) bioprinting of cellular or biological components are an emerging field to develop tissue structures that mimic the spatial, mechanochemical and temporal characteristics of cardiovascular tissues. 3D multi-cellular and multi-domain organotypic biological constructs can better recapitulate in vivo physiology and can be utilized in a variety of applications. Such applications include in vitro cellular studies, high-throughput drug screening, disease modeling, biocompatibility analysis, drug testing and regenerative medicine. A major challenge of 3D bioprinting strategies is the inability of matrix molecules to reconstitute the complexity of the extracellular matrix and the intrinsic cellular morphologies and functions. An important factor is the inclusion of a vascular network to facilitate oxygen and nutrient perfusion in scalable and patterned 3D bioprinted tissues to promote cell viability and functionality. In this review, we summarize the new generation of 3D bioprinting techniques, the kinds of bioinks and printing materials employed for 3D bioprinting, along with the current state-of-the-art in engineered cardiovascular tissue models. We also highlight the translational applications of 3D bioprinting in engineering the myocardium cardiac valves, and vascular grafts. Finally, we discuss current challenges and perspectives of designing effective 3D bioprinted constructs with native vasculature, architecture and functionality for clinical translation and cardiovascular regeneration.

Comparative Effects of Basic Fibroblast Growth Factor Delivery or Voluntary Exercise on Muscle Regeneration after Volumetric Muscle Loss.

Volumetric muscle loss (VML) is associated with irreversibly impaired muscle function due to traumatic injury. Experimental approaches to treat VML include the delivery of basic fibroblast growth factor (bFGF) or rehabilitative exercise. The objective of this study was to compare the effects of spatially nanopatterned collagen scaffold implants with either bFGF delivery or in conjunction with voluntary exercise. Aligned nanofibrillar collagen scaffold bundles were adsorbed with bFGF, and the bioactivity of bFGF-laden scaffolds was examined by skeletal myoblast or endothelial cell proliferation. The therapeutic efficacy of scaffold implants with either bFGF release or exercise was examined in a murine VML model. Our results show an initial burst release of bFGF from the scaffolds, followed by a slower release over 21 days. The released bFGF induced myoblast and endothelial cell proliferation in vitro. After 3 weeks of implantation in a mouse VML model, twitch force generation was significantly higher in mice treated with bFGF-laden scaffolds compared to bFGF-laden scaffolds with exercise. However, myofiber density was not significantly improved with bFGF scaffolds or voluntary exercise. In contrast, the scaffold implant with exercise induced more re-innervation than all other groups. These results highlight the differential effects of bFGF and exercise on muscle regeneration.

Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis.

Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

Extracellular Matrix-Based Biomaterials for Cardiovascular Tissue Engineering.

Regenerative medicine and tissue engineering strategies have made remarkable progress in remodeling, replacing, and regenerating damaged cardiovascular tissues. The design of three-dimensional (3D) scaffolds with appropriate biochemical and mechanical characteristics is critical for engineering tissue-engineered replacements. The extracellular matrix (ECM) is a dynamic scaffolding structure characterized by tissue-specific biochemical, biophysical, and mechanical properties that modulates cellular behavior and activates highly regulated signaling pathways. In light of technological advancements, biomaterial-based scaffolds have been developed that better mimic physiological ECM properties, provide signaling cues that modulate cellular behavior, and form functional tissues and organs. In this review, we summarize the in vitro, pre-clinical, and clinical research models that have been employed in the design of ECM-based biomaterials for cardiovascular regenerative medicine. We highlight the research advancements in the incorporation of ECM components into biomaterial-based scaffolds, the engineering of increasingly complex structures using biofabrication and spatial patterning techniques, the regulation of ECMs on vascular differentiation and function, and the translation of ECM-based scaffolds for vascular graft applications. Finally, we discuss the challenges, future perspectives, and directions in the design of next-generation ECM-based biomaterials for cardiovascular tissue engineering and clinical translation.