The Effects of Simulated and Real Microgravity on Vascular Smooth Muscle Cells.

As considerations are being made for the limitations and safety of long-term human spaceflight, the vasculature is important given its connection to and impact on numerous organ systems. As a major constituent of blood vessels, vascular smooth muscle cells are of interest due to their influence over vascular tone and function. Additionally, vascular smooth muscle cells are responsive to pressure and flow changes. Therefore, alterations in these parameters under conditions of microgravity can be functionally disruptive. As such, here we review and discuss the existing literature that assesses the effects of microgravity, both actual and simulated, on smooth muscle cells. This includes the various methods for achieving or simulating microgravity, the animal models or cells used, and the various durations of microgravity assessed. We also discuss the various reported findings in the field, which include changes to cell proliferation, gene expression and phenotypic shifts, and renin-angiotensin-aldosterone system (RAAS), nitric oxide synthase (NOS), and Ca2+ signaling. Additionally, we briefly summarize the literature on smooth muscle tissue engineering in microgravity as well as considerations of radiation as another key component of spaceflight to contextualize spaceflight experiments, which by their nature include radiation exposure. Finally, we provide general recommendations based on the existing literature's focus and limitations.

Spaceflight effects on human vascular smooth muscle cell phenotype and function.

The cardiovascular system is strongly impacted by the hazards of spaceflight. Astronauts spending steadily increasing lengths of time in microgravity are subject to cardiovascular deconditioning resulting in loss of vascular tone, reduced total blood volume, and diminished cardiac output. Appreciating the mechanisms by which the cells of the vasculature are altered during spaceflight will be integral to understanding and combating these deleterious effects as the human presence in space advances. In this study, we performed RNA-Seq analysis coupled with review by QIAGEN Ingenuity Pathway Analysis software on human aortic smooth muscle cells (HASMCs) cultured for 3 days in microgravity and aboard the International Space Station to assess the transcriptomic changes that occur during spaceflight. The results of our RNA-Seq analysis show that SMCs undergo a wide range of transcriptional alteration while in space, significantly affecting 4422 genes. SMCs largely down-regulate markers of the contractile, synthetic, and osteogenic phenotypes including smooth muscle alpha actin (αSMA), matrix metalloproteinases (MMPs), and bone morphogenic proteins (BMPs). Additionally, components of several cellular signaling pathways were strongly impacted including the STAT3, NFκB, PI3K/AKT, HIF1α, and Endothelin pathways. This study highlights the significant changes in transcriptional behavior SMCs exhibit during spaceflight and puts these changes in context to better understand vascular function in space.

Sex as a Biological Variable in Tissue Engineering and Regenerative Medicine.

Although sex differences have been noted in cellular function and behavior, therapy efficacy, and disease incidence and outcomes, the adoption of sex as a biological variable in tissue engineering and regenerative medicine remains limited. Furthering the development of personalized, precision medicine requires considering biological sex at the bench and in the clinic. This review provides the basis for considering biological sex when designing tissue-engineered constructs and regenerative therapies by contextualizing sex as a biological variable within the tissue engineering triad of cells, matrices, and signals. To achieve equity in biological sex within medicine requires a cultural shift in science and engineering research, with active engagement by researchers, clinicians, companies, policymakers, and funding agencies.

Ancestry of cells must be considered in bioengineering.

Bioengineered platforms, intended to be used in the investigation of human health and disease, often incorporate cells of unknown ancestry or that lack diversity. To develop tools and platforms that benefit the entire human population, we must consider the ancestry of cells and intentionally diversify the cells we use in our designs.

Ancestral Background Is Underreported in Regenerative Engineering.

Purpose: The ancestral background of human cells may play a role in cells' behavior and response to therapeutic interventions in vitro. We investigate the prevalence of ancestry reporting in current biological research and suggest that increased reporting would be beneficial to the field. Methods: Articles published over a six-month period in ten different journals were reviewed for their use of human primary cells and immortalized cell lines, and were analyzed based on whether or not the ancestral or ethnic information of cell donors was ascertainable. Results: The vast majority of literature published in the journals and timeframe we investigated did not report on the ancestral or ethnic origins of the human cells used. Conclusion: There is currently a substantial lack of reporting on the ancestral background of human cells used for research. We suggest that increased ancestral reporting should be implemented in order to improve the development of precision medicine. Lay Summary: Many diseases affect patients of different ancestral backgrounds in a variety of ways. In this perspective article, we raise the concern that, since many scientists do not consider ancestry when designing their studies, their results may not apply to all patients. We use data to show that very few scientists report on the ancestry of the donors who contribute cells and tissues to their research. We suggest that broader reporting on donor ancestry would improve biomedical research and would help doctors to personalize treatments for their patients.Future work includes further increasing awareness of the importance of including ancestry as a variable in experimental design, as well as promoting increased reporting on ancestry in the research community. Supplementary Information: The online version contains supplementary material available at 10.1007/s40883-021-00237-8.

Engineering Vascular Grafts with Multiphase Structures.

Blood vessels in the body are multiphasic organs with microenvironmental niches specific to the cells that inhabit each section. Electrospinning is a fabrication technique used to produce nano- to microfibrous architectures capable of mimicking native extracellular matrix structure. Likewise, polycitrate elastomers are favorable luminal materials for vascular applications because of their hemocompatibility and mechanical properties. Here we describe the procedure for fabricating a biphasic polycitrate elastomer, collagen, and elastin electrospun composite to spatially tailor both composition and architecture for recapitulating the intimal and medial layers of the blood vessel in a vascular graft.

Hemocompatibility of all-trans retinoic acid-loaded citrate polymer coatings for vascular stents.

Purpose: Current strategies implementing drug-eluting polymer stent coatings fail to fully address the lasting effects of endothelial suppression which ultimately result in delayed reendothelialization and thrombogenic complications. The present study investigates the in vitro hemocompatibility of all-trans retinoic acid loaded poly (1,8-octanediol-co-citrate) coatings (AtRA-POC coatings) for advanced intravascular stent technology. The ability of these materials in supporting endothelial restoration via migration and proliferation while inhibiting smooth muscle cell growth is also explored. Methods: Using in vitro models, the hemocompatibility of AtRA-loaded POC-coated cobalt chromium (CoCr) vascular stents was evaluated in terms of platelet and inflammatory activity. Platelet activity was quantified by platelet adhesion and platelet activation, further supported by SEM visualization. Inflammatory activity was quantified by the production of proinflammatory cytokines by THP1 monocytes. Lastly, in vitro wound healing and an 5-Ethynyl-2'deoxyuridine (EdU) and pico green DNA assays were used in quantitating endothelial and smooth muscle cell migration and proliferation. Results: Experimental examinations of platelet adhesion and activation demonstrate significant reductions in the platelet response to POC coated AtRA loaded stents when compared to bare CoCr stents. Such findings reveal AtRA-POC coatings to have significantly improved hemocompatibility compared to that of bare metal stents and at least as good as POC alone. Similarly, in reference to LPS-stimulated controls, Human monocyte-like THP1 cells in culture with AtRA-POC-CoCr stents for 24 hours showed reduced detection of proinflammatory cytokines, comparable to that of bare CoCr and untreated controls. This result supports AtRA-POC coatings as possessing limited immunological potential. Observations from in vitro endothelial and smooth muscle cell investigations demonstrate the ability of the drug AtRA to allow cell processes involved in restoration of the endothelium while inhibiting smooth muscle cell processes. Conclusion: This study demonstrates AtRA loaded POC coatings are hemocompatible, noninflammatory, and provide a promising strategy in enhancing vascular stent techniques and clinical integration. Possessing hemocompatibility and immunological compatibility that is at least as good as bare metal stents as clinical standards support the use of AtRA-POC coatings for vascular applications. Additionally, selectively reducing smooth muscle cell proliferation while supporting endothelial cell proliferation and migration further demonstrates the potential of these materials in significantly improving the state of vascular stent technology in the area of stent thrombosis and neointimal hyperplasia.

The Effects of Simulated Microgravity on Macrophage Phenotype.

The effects of spaceflight, including prolonged exposure to microgravity, can have significant effects on the immune system and human health. Altered immune cell function can lead to adverse health events, though precisely how and to what extent a microgravity environment impacts these cells remains uncertain. Macrophages, a key immune cell, effect the inflammatory response as well as tissue remodeling and repair. Specifically, macrophage function can be dictated by phenotype that can exist between spectrums of M0 macrophage: the classically activated, pro-inflammatory M1, and the alternatively activated, pro-healing M2 phenotypes. This work assesses the effects of simulated microgravity via clinorotation on M0, M1, and M2 macrophage phenotypes. We focus on phenotypic, inflammatory, and angiogenic gene and protein expression. Our results show that across all three phenotypes, microgravity results in a decrease in TNF-α expression and an increase in IL-12 and VEGF expression. IL-10 was also significantly increased in M1 and M2, but not M0 macrophages. The phenotypic cytokine expression profiles observed may be related to specific gravisensitive signal transduction pathways previously implicated in microgravity regulation of macrophage gene and protein expression. Our results highlight the far-reaching effects that simulated microgravity has on macrophage function and provides insight into macrophage phenotypic function in microgravity.

Sex-Specific Response to Combinations of Shear Stress and Substrate Stiffness by Endothelial Cells In Vitro.

By using a full factorial design of experiment, the combinatorial effects of biological sex, shear stress, and substrate stiffness on human umbilical vein endothelial cell (HUVEC) spreading and Yes-associated protein 1 (YAP1) activity are able to be efficiently evaluated. Within the range of shear stress (0.5-1.5 Pa) and substrate stiffness (10-100 kPa), male HUVECs are smaller than female HUVECs. Only with sufficient mechanical stimulation do they spread to a similar size. More importantly, YAP1 nuclear localization in female HUVECs is invariant to mechanical stimulation within the range of tested conditions whereas for male HUVECs it increases nonlinearly with increasing shear stress and substrate stiffness. The sex-specific response of HUVECs to combinations of shear stress and substrate stiffness reinforces the need to include sex as a biological variable and multiple mechanical stimuli in experiments, informs the design of precision biomaterials, and offers insight for understanding cardiovascular disease sexual dimorphisms. Moreover, here it is illustrated that different complex mechanical microenvironments can lead to sex-specific phenotypes and sex invariant phenotypes in cultured endothelial cells.

Macrophages in microgravity: the impact of space on immune cells.

The effects of a microgravity environment on the myriad types of immune cells present within the human body have been assessed both by bench-scale simulation and suborbital methods, as well as in true spaceflight. Macrophages have garnered increased research interest in this context in recent years. Their functionality in both immune response and tissue remodeling makes them a unique cell to investigate in regards to gravisensitive effects as well as parameters of interest that could impact astronaut health. Here, we review and summarize the literature investigating the effects of microgravity on macrophages and monocytes regarding the microgravity environment simulation/generation methods, cell sources, experiment durations, and parameters of interest utilized within the field. We discuss reported findings on the impacts of microgravity on macrophage/monocyte structure, adhesion and migration, proliferation, genetic expression, cytokine secretion, and reactive oxygen species production, as well as polarization. Based on this body of data, we make recommendations to the field for careful consideration of experimental design to complement existing reports, as the multitude of disparate study methods previously published can make drawing direct comparisons difficult. However, the breadth of different testing methodologies can also lend itself to attempting to identify the most robust and consistent responses to microgravity across various testing conditions.

Self-assembled VEGF-R2 targeting DNA aptamer-collagen fibers stimulate an angiogenic-like endothelial cell phenotype.

Vascularization of engineered tissue is one of the hallmark challenges of tissue engineering. Leveraging self-assembled nucleic acid-collagen complexes (NACCs), we mixed a VEGF-R2 targeting aptamer or its receptor agonist divalent assembly with type I collagen to assemble NACC microfibers. Human umbilical vein endothelial cells (HUVECs) quickly remodeled these fibers into tubulogenic-like structures over 48 h. Moreover, NACCs made with the receptor agonist divalent aptamer assembly promoted enhanced expression of von Willebrand factor (vWF), angiopoietin-2 (ANGPT-2), and matrix metalloproteinase-2 (MMP-2) by HUVECs as measured by either immunocytochemistry or ELISA. The findings suggest, endothelial cell phenotype was directed by both biochemical cues afforded by the agonist behavior of the divalent aptamer assembly as well as by the biophysical cues afforded by the fibrous topography. Collectively, these results support the development of an angiogenic endothelial cell phenotype stimulated by the VEGF-R2 agonist NACC fibers. Thus, the combination of engineered DNA aptamer nanotechnology and DNA-collagen complexation phenomena is a promising biofunctional natural scaffold material system for tissue engineering and regenerative medicine applications.

Fund Black scientists.

Our nationwide network of BME women faculty collectively argue that racial funding disparity by the National Institutes of Health (NIH) remains the most insidious barrier to success of Black faculty in our profession. We thus refocus attention on this critical barrier and suggest solutions on how it can be dismantled.

Let's Talk About Sex-Biological Sex Is Underreported in Biomaterial Studies.

Precision medicine aims to better individualize healthcare. It requires that biomaterials be designed for the physiological characteristics of a specific patient. To make this a reality, biomaterials research and development must address differences of biological sex. More specifically, biomaterials should be designed with properties optimized and appropriate for male and female patients. In analyzing research articles from seven prominent biomaterials journals, sex as a biological variable is missing from an overwhelming majority of in vitro biomaterial studies. From the survey, the reporting of the sex of primary cell cultures happened only 10.3% of the time. Contributing to this trend is that commercial vendors bias cell lines toward one sex or another by not disclosing information of cell line sex at the time of purchase; researchers do not communicate this pertinent information in published studies; and many journal policies have little to no requirements for reporting cell line characteristics. Omitting this valuable information leads to a gap in the understanding of sex-specific cell-biomaterial interactions and it creates a bias in research findings towards one sex or another. To curb this concerning trend and make precision biomaterials a reality will require the biomaterials field to "talk about sex" by reporting cell sex more broadly.

Anti-VEGF-R2 Aptamer and RGD Peptide Synergize in a Bifunctional Hydrogel for Enhanced Angiogenic Potential.

Hydrogels have gained interest for use in tissue regeneration and wound healing because of their absorbing and swelling properties as well as their ability to mimic the natural extracellular matrix. Their use in wound healing specifically may be in the form of a patch or wound dressing or they may be administered within the wound bed as a filler, gel in situ, to promote healing. Thiolated hyaluronic acid-polyethylene diacrylate (tHA-PEGDA) hydrogels are ideal for this purpose due to their short gelation times at physiological temperature and pH. But these hydrogels alone are not enough and require added components to gain bioactivity. In this work, RGD adhesion peptides and an antivascular endothelial growth factor receptor-2 (VEGF-R2) DNA aptamer are incorporated into a tHA-PEGDA hydrogel to make a bifunctional hyaluronic acid hydrogel. RGD peptides promote attachment and growth of cells while the anti-VEGF-R2 DNA aptamer seems to improve cell viability, induce cell migration, and spur the onset of angiogenesis by tube formation by endothelial cells. This bifunctional hydrogel supports cell culture and has improved biological properties. The data suggest that these hydrogels can be used for advanced tissue regeneration applications such as in wound healing.

Mineralized DNA-collagen complex-based biomaterials for bone tissue engineering.

DNA is a highly polyanionic biomolecule that complexes with both collagen and hydroxyapatite. By combining these complexes, we synthesized nucleic-acid collagen complexes (NACC) mineralized with hydroxyapatite. The composite complexes were made using a short, monodisperse single-stranded DNA, type I collagen, and mineralizing medium. They rapidly self-assembled into both mineralized NACC microfibers and 3D NACC gels. At the nanoscale, these complexes are hierarchical, interwoven, curly nanofibrils resembling native extracellular matrix, which mineralized an interpenetrating nanocrystalline hydroxyapatite phase. Mineralization was able to be done either before or after NACC formation enabling temporal control of the process. In response to the NACC material, primary human osteoblasts took on an osteocyte-like morphology. Moreover, the cells agglomerated and remodeled the NACC gels into densified, tissue-like structures within 3 days. NACC fibers and gels have promise not only as osteoconductive coatings and scaffolds, but as coatings and scaffolds for any tissue using this new form of naturally-derived biomaterials.

Palm readings: Manicaria saccifera palm fibers are biocompatible textiles with low immunogenicity.

Plant-based fibers are a potential alternative to synthetic polymer fibers that can yield enhanced biocompatibility and mechanical properties matching those properties of tissue. Given the unique morphology of the bract of the Manicaria saccifera palm, being an interwoven meshwork of fibers, we believe that these fibers with this built-in structure could prove useful as a tissue engineering scaffold material. Thus, we first investigated the fiber's in vitro biocompatibility and immunogenicity. We cultured NIH/3T3 mouse fibroblasts, human aortic smooth muscle cells, and human adipose-derived mesenchymal stem cells on the fiber mats, which all readily attached and over 21 days grew to engulf the fibers. Importantly, this was achieved without treating the plant tissue with extracellular matrix proteins or any adhesion ligands. In addition, we measured the gene expression and protein secretion of three target inflammatory cytokines (IL-1ß, IL-8, and TNFα) from THP-1 human leukemia monocytes cultured in the presence of the biotextile as an in vitro immunological model. After 24 h of culture, gene expression and protein secretion were largely the same as the control, demonstrating the low immunogenicity of Manicaria saccifera fibers. We also measured the tensile mechanical properties of the fibers. Individual fibers after processing had a Young's modulus of 9.51 ± 4.38 GPa and a tensile strength of 68.62 ± 27.93 MPa. We investigated the tensile mechanical properties of the fiber mats perpendicular to the fiber axis (transverse loading), which displayed upwards of 100% strain, but with a concession in strength compared to longitudinal loading. Collectively, our in vitro assessments point toward Manicaria saccifera as a highly biocompatible biotextile, with a range of potential clinical and engineering applications.

Oligomer Length Defines the Self-Assembly of Single-Stranded DNA-Collagen Complex Fibers.

Collagen and single-stranded DNA (ssDNA) complex to self-assemble into fibers depending on the length of the ssDNA and the relative amounts of collagen and ssDNA in solution. We report for the first time that when monodisperse, random sequences of ssDNA in the range of 15-90 nucleotides and type I collagen were mixed together at room temperature, fibers several tens of micrometers in length and as large as 10 µm in diameter were formed. Fiber formation was rapid and spontaneous, requiring no further treatment after mixing. Most notably, more ssDNA oligomers were incorporated into the fibers formed using shorter ssDNA oligomers. Endothelial cells formed angiogenic-like structures using the fibers with elevated expression of von Willebrand factor for cells in direct contact with the fibers. These fibers open the door to future applications in the administration and functionality of ssDNA and collagen.

In vitro and in vivo biocompatibility assessment of free radical scavenging nanocomposite scaffolds for bone tissue regeneration.

Bone is the second most transplanted tissue in the world, resulting in increased demand for bone grafts leading to the fabrication of synthetic scaffold grafting alternatives. Fracture sites are under increased oxidative stress after injuries, affecting osteoblast function and hindering fracture healing and remodeling. To counter oxidative stress, free radical scavenging agents, such as cerium oxide nanoparticles, have gained traction in tissue engineering. Toward the goal of developing a functional synthetic system for bone tissue engineering, we characterized the biocompatibility of a porous, bioactive, free radical scavenging nanocomposite scaffold composed of poly(1,8 octanediol-co-citrate), beta-tricalcium phosphate, and cerium oxide nanoparticles. We studied cellular and tissue compatibility utilizing in vitro and in vivo models to assess nanocomposite interactions with both human osteoblast cells and rat subcutaneous tissue. We found the scaffolds were biocompatible in both models and supported cell attachment, proliferation, mineralization, and infiltration. Using hydrogen peroxide, we simulated oxidative stress to study the protective properties of the nanocomposite scaffolds via a reduction in cytotoxicity and recovered mineralization of osteoblast cells in vitro. We also found after implantation in vivo the scaffolds exhibited biocompatible properties essential for successful scaffolds for bone tissue engineering. Cells were able to infiltrate through the scaffolds, the surrounding tissues elicited a minimal immune response, and there were signs of scaffold degradation after 30 days of implantation. After the array of biological characterization, we had confirmed the development of a nanocomposite scaffold system capable of supporting bone-remodeling processes while providing a protective free radical scavenging effect.

Multi-layer approaches to scaffold-based small diameter vessel engineering: A review.

Cardiovascular disease is one of the leading causes of death in the world. A characteristic symptom of cardiovascular disease is occlusion of vessels. Once vascular occlusion occurs there is a critical need to re-establish flow to prevent ischemia in the downstream tissues. In the most advanced cases, flow is re-established by creating a secondary flow path around the blockage, bypass grafting. For large diameter applications, synthetic conduits are successfully implanted, however in small diameter applications re-occlusion occurs and there is a critical need for new vascular grafts. There are many strategies and approaches that are being employed to design an effective and successful vascular graft. However, to date, there are no clinically available small diameter vascular grafts that are consistently successful in vivo long term (>7 years). As an effort to develop a successful graft there are several tissue engineering approaches: cell sheets, synthetic and natural biomaterial platforms, and decellularized extracellular matrices that are being investigated. While each area has its advantages, scaffold-based approaches are among the most widely studied. Scaffold based approaches are extensively studied due to tailorability and the availability of synthetic and natural polymers. Within the area of scaffold-based approaches, biomimicry has become an increasingly studied area, and structural biomimicry is one of the many approaches. The focus of this review paper is to analyze scaffold-based approaches. Particularly the advantages and disadvantages of using multi-layer scaffold-based approaches to engineer conduits for small diameter applications.

Fabrication of a bilayer scaffold for small diameter vascular applications.

One of the greatest challenges plaguing cardiovascular tissue engineering has been the development of a compliant vascular graft. In this work, we report the development of a synthetic vascular graft with compliance similar to native arteries at physiological pressures. A bilayer scaffold was fabricated from a solid polymeric lumen made from poly(1,8 octanediol-co-citrate) (POC) and a microfibrous medial layer composed of type I collagen, elastin, and POC. Mechanical analysis revealed dynamic compliance, ~6.9% within 1% of native vessels, 5.9%. The burst pressure was an order of magnitude lower than native vessels (~400 mmHg vs. ~3000 mmHg) but was above physiological pressure ranges. Biocompatibility studies indicated the scaffold posed no acute cytotoxic risk to relevant cell types and supported the proliferation of vascular smooth muscle cells. In addition, upon exposure of the scaffold to whole blood, there was no statistically significant hemolysis, <2%. Overall this is a promising material system and scaffold to develop a biodegradable tissue-engineered vascular graft. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2850-2862, 2018.