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Abstract The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.

Abstract Engineered whole lungs may one day expand therapeutic options for patients with end-stage lung disease. However, the feasibility of ex vivo lung regeneration remains limited by the inability to recapitulate mature, functional alveolar epithelium. Here, we modulate multimodal components of the alveolar epithelial type 2 cell (AEC2) niche in decellularized lung scaffolds in order to guide AEC2 behavior for epithelial regeneration. First, endothelial cells coordinate with fibroblasts, in the presence of soluble growth and maturation factors, to promote alveolar scaffold population with surfactant-secreting AEC2s. Subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-magnitude mechanical strain to induce the differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an epithelium of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial alveolar-like units, and highlight the critical interplay amongst cellular, biochemical, and mechanical niche cues within the reconstituting alveolus.

Summary: Despite much concerted effort to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feedforward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.

Tracheal replacement using tissue engineering technologies offers great potential to improve previously intractable clinical interventions, and interest in this area has increased in recent years. Many engineered airway constructs currently rely on decellularized native tracheas to serve as the scaffold for tissue repair. Yet, mechanical failure leading to airway narrowing and collapse remains a major cause of morbidity and mortality following clinical implantation of decellularized tracheal grafts. To understand better the factors contributing to mechanical failure in vivo, we characterized the histo-mechanical properties of tracheas following two different decellularization protocols, including one that has been used clinically. All decellularized tracheas deviated from native mechanical behavior, which may provide insights into observed in vivo graft failures. We further analyzed protein content by western blot and analyzed microstructure by histological staining and found that the specific method of decellularization resulted in significant differences in the depletion of proteoglycans and degradation of collagens I, II, III, and elastin. Taken together, this work demonstrates that the heterogeneous architecture and mechanical behavior of the trachea is severely compromised by decellularization. Such structural deterioration may contribute to graft failure clinically and limit the potential of decellularized native tracheas as viable long-term orthotopic airway replacements.

Abstract Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell–cell and ligand–receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell–cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand–receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell–cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

The study of human microglia function in health and disease is limited by the availability of sound models. Here, the authors develop a method to generate functional microglia in human cortical organoids and investigate the role of human microglia during amyloid beta1-42- induced inflammation.

SARS-CoV-2 infection can lead to progressive pathology in patients with COVID-19, but information for this disease progression is sparse. Here the authors use multi-omics approach to profile the immune responses of patients, assessing immune repertoire and effects of tocilizumab treatments, to find a dyssynchrony between innate and adaptive immunity in progressive COVID-19.

Chronic obstructive pulmonary disease is a leading cause of death worldwide, while our understanding of cell-specific mechanisms underlying its pathobiology remains incomplete. Here the authors perform scRNA-seq of human lung tissue to identify transcriptional changes in alveolar niche cells associated with the disease.

Objective: Patients with end stage renal failure who require haemodialysis suffer morbidity and mortality due to vascular access. Bioengineered human acellular vessels (HAVs) may provide a haemodialysis access option with fewer complications than other grafts. In a prospective phase II trial from 2012 to 2014 (NCT01744418), HAVs were implanted into 40 haemodialysis patients at three sites in Poland. The trial protocol for this “first in man” use of the HAV contemplated only two years of follow up, and the trial results were initially reported in 2016. In light of the retained HAV function seen in many of the patients at the two year time point, follow up for patients who were still alive was extended to a total of 10 years. This interim follow up report, at the long term time point of five years, assessed patient and conduit status in those who continued routine dialysis with the HAV. Methods: HAVs are bioengineered by culturing human vascular smooth muscle cells on a biodegradable polymer matrix. In this study, patients with patent HAV implants at 24 months were followed every three months, starting at month 27 through to month 60, or at least five years post-implantation. This report contains the follow up functional and histological data on 29 of the original 40 patients who demonstrated HAV function at the 24 month time point. Results: Eleven patients completed at month 60. One patient maintained primary patency, and 10 maintained secondary patency. Secondary patency was estimated at 58.2% (95% confidence interval 39.2–73.1) at five years, after censoring for deaths (n = 8) and withdrawals (n = 1). No HAV conduit infections were reported during the follow up period. Conclusion: This phase II long term follow up shows that the human acellular vessel (HAV) may provide durable and functional haemodialysis access for patients with end stage renal disease.

Objective: Synthetic expanded polytetrafluoroethylene (ePTFE) grafts are known to be susceptible to bacterial infection. Results from preclinical and clinical studies of bioengineered human acellular vessels (HAVs) have shown relatively low rates of infection. This study evaluates the interactions of human neutrophils and bacteria with ePTFE and HAV vascular conduits to determine whether there is a correlation between neutrophil-conduit interactions and observed differences of their infectivity in vivo. Methods: A phase III comparative clinical study between investigational HAVs (n = 177) and commercial ePTFE grafts (n = 178) used for hemodialysis access (ClinicalTrials.gov Identifier: NCT02644941) was evaluated for conduit infection rates followed by histological analyses of HAV and ePTFE tissue explants. The clinical histopathology of HAV and ePTFE conduits reported to be infected was compared with immunohistochemistry of explanted materials from a preclinical model of bacterial contamination. Mechanistic in vitro studies were then conducted using isolated human neutrophils seeded directly onto HAV and ePTFE materials to analyze neutrophil viability, morphology, and function. Results: Clinical trial results showed that the HAV had a significantly lower (0.93%; P = .0413) infection rate than that of ePTFE (4.54%). Histological analysis of sections from infected grafts explanted approximately 1 year after implantation revealed gram-positive bacteria near cannulation sites. Immunohistochemistry of HAV and ePTFE implanted in a well-controlled rodent infection model suggested that the ePTFE matrix permitted bacterial infiltration and colonization but may be inaccessible to neutrophils. In the same model, the HAV showed host recellularization and lacked detectable bacteria at the 2-week explant. In vitro results demonstrated that the viability of human neutrophils decreased significantly upon exposure to ePTFE, which was associated with neutrophil elastase release in the absence of bacteria. In contrast, neutrophils exposed to the HAV material retained high viability and native morphology. Cocultures of neutrophils and Staphylococcus aureus on the conduit materials demonstrated that neutrophils were more effective at ensnaring and degrading bacteria on the HAV than on ePTFE. Conclusions: The HAV material seems to demonstrate a resistance to bacterial infection. This infection resistance is likely due to the HAV's native-like material composition, which may be more biocompatible with host neutrophils than synthetic vascular graft material. : Clinical Relevance: Clinical trial results to date have shown that human acellular vessels may be a promising alternative to synthetic conduits in certain indications based on mechanical function, host remodeling, and low infection rates. Resistance to infection may be inherent to the human acellular vessel based on its biological composition, which supports the migration and normal function of a patient's own cells after implantation.

Objective: The human acellular vessel (HAV) was evaluated for surgical bypass in a phase II study. The primary results at 24 months after implantation have been reported, and the patients will be evaluated for ≤10 years. Methods: In the present report, we have described the 6-year results of a prospective, open-label, single-treatment arm, multicenter study. Patients with advanced peripheral artery disease (PAD) requiring above-the-knee femoropopliteal bypass surgery without available autologous graft options had undergone implantation with the HAV, a bioengineered human tissue replacement blood vessel. The patients who completed the 24-month primary portion of the study will be evaluated for ≤10 years after implantation. The present mid-term analysis was performed at the 6-year milestone (72 months) for patients followed up for 24 to 72 months. Results: HAVs were implanted in 20 patients at three sites in Poland. Seven patients had discontinued the study before completing the 2-year portion of the study: four after graft occlusion had occurred and three who had died of causes deemed unrelated to the conduit, with the HAV reported as functional at their last visit. The primary results at 24 months showed primary, primary assisted, and secondary patency rates of 58%, 58%, and 74%, respectively. One vessel had developed a pseudoaneurysm deemed possibly iatrogenic; no other signs of structural failure were reported. No rejections or infections of the HAV occurred, and no patient had required amputation of the implanted limb. Of the 20 patients, 13 had completed the primary portion of the study; however, 1 patient had died shortly after 24 months. Of the remaining 12 patients, 3 died of causes unrelated to the HAV. One patient had required thrombectomy twice, with secondary patency achieved. No other interventions were recorded between 24 and 72 months. At 72 months, five patients had a patent HAV, including four patients with primary patency. For the entire study population from day 1 to month 72, the overall primary, primary assisted, and secondary patency rate estimated using Kaplan-Meier analysis was 44%, 45%, and 60% respectively, with censoring for death. No patient had experienced rejection or infection of the HAV, and no patient had required amputation of the implanted limb. Conclusions: The infection-resistant, off-the-shelf HAV could provide a durable alternative conduit in the arterial circuit setting to restore the lower extremity blood supply in patients with PAD, with remodeling into the recipient’s own vessel over time. The HAV is currently being evaluated in seven clinical trials to treat PAD, vascular trauma, and as a hemodialysis access conduit. : Clinical Relevance: Patients with peripheral artery disease who require surgical revascularization need options when autologous grafts are not available. The human acellular vessel (HAV) has been demonstrated to have characteristics similar to those of autologous vessels in terms of resistance to infection, mechanics, and a very low risk of rejection. Safety and performance were evaluated for ≤6 years after implantation of an HAV in a femoropopliteal position. Overall, the secondary patency rate estimated using the Kaplan-Meier method was 60% at 72 months, with 45% primary patency. No infection or rejection episodes had occurred with the HAV conduits. These data have demonstrated the durability of the HAV and suggest the occurrence of cellular remodeling by the host.

Abstract A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back—at least partially—toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Summary: Cell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, here, we isolate and culture a population of pharmacologically expanded basal cells (peBCs) from rat tracheas. At peak basal marker expression, we simultaneously split peBCs into four in vitro platforms: organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples are evaluated using single-cell RNA sequencing (scRNA-seq) and computational pipelines are developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq as a control for engineered epithelium. Overall, this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications. : Greaney et al. compare pulmonary epithelial regeneration across multiple modalities in vitro, finding that decellularized scaffolds achieved the most physiologic differentiation over more artificial platforms. scRNA-seq enables high-resolution comparison between engineered and native cell populations, thereby better gauging progress toward the generation of a tissue that may function on implantation. Keywords: Pulmonary Cell Biology, Regenerative Medicine, Tissue Engineering, Primary Epithelial Cells

Abstract Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Abstract Vascular tissue engineering has significant potential to make a major impact on a wide array of clinical problems. Continued progress in understanding basic vascular biology will be invaluable in making further advancements. Past and current achievements in tissue engineering of microvasculature to perfuse organ specific constructs, small vessels for dialysis grafts, and modified synthetic and pediatric large caliber-vessel grafts will be discussed. An emphasis will be placed on clinical trial results with small and large-caliber vessel grafts. Challenges to achieving engineered constructs that satisfy the physiologic, immunologic, and manufacturing demands of engineered vasculature will be explored.

Objective: Invasive coronary interventions can fail due to intimal hyperplasia and restenosis. Endothelial cell (EC) seeding to the vessel lumen, accelerating re-endothelialization, or local release of mTOR pathway inhibitors have helped reduce intimal hyperplasia after vessel injury. While animal models are powerful tools, they are complex and expensive, and not always reflective of human physiology. Therefore, we developed an in vitro 3D vascular model validating previous in vivo animal models and utilizing isolated human arteries to study vascular remodeling after injury. Approach: We utilized a bioreactor that enables the control of intramural pressure and shear stress in vessel conduits to investigate the vascular response in both rat and human arteries to intraluminal injury. Results: Culturing rat aorta segments in vitro , we show that vigorous removal of luminal ECs results in vessel injury, causing medial proliferation by Day-4 and neointima formation, with the observation of SCA1 + cells (stem cell antigen-1) in the intima by Day-7, in the absence of flow. Conversely, when endothelial-denuded rat aortae and human umbilical arteries were subjected to arterial shear stress, pre-seeding with human umbilical ECs decreased the number and proliferation of smooth muscle cell (SMC) significantly in the media of both rat and human vessels. Conclusion: Our bioreactor system provides a novel platform for correlating ex vivo findings with vascular outcomes in vivo . The present in vitro human arterial injury model can be helpful in the study of EC-SMC interactions and vascular remodeling, by allowing for the separation of mechanical, cellular, and soluble factors.

Vascularization of engineered bone tissue is critical for ensuring its survival after implantation. In vitro pre-vascularization of bone grafts with endothelial cells is a promising strategy to improve implant survival. In this study, we pre-cultured human smooth muscle cells (hSMCs) on bone scaffolds for 3 weeks followed by seeding of human umbilical vein endothelial cells (HUVECs), which produced a desirable environment for microvasculature formation. The sequential cell-seeding protocol was successfully applied to both natural (decellularized native bone, or DB) and synthetic (3D-printed Hyperelastic “Bone” scaffolds, or HB) scaffolds, demonstrating a comprehensive platform for developing natural and synthetic-based in vitro vascularized bone grafts. Using this sequential cell-seeding process, the HUVECs formed lumen structures throughout the DB scaffolds as well as vascular tissue bridging 3D-printed fibers within the HB. The pre-cultured hSMCs were essential for endothelial cell (EC) lumen formation within DB scaffolds, as well as for upregulating EC-specific gene expression of HUVECs grown on HB scaffolds. We further applied this co-culture protocol to DB scaffolds using a perfusion bioreactor, to overcome the limitations of diffusive mass transport into the interiors of the scaffolds. Compared with static culture, panoramic histological sections of DB scaffolds cultured in bioreactors showed improved cellular density, as well as a nominal increase in the number of lumen structures formed by ECs in the interior regions of the scaffolds. In conclusion, we have demonstrated that the sequential seeding of hSMCs and HUVECs can serve to generate early microvascular networks that could further support the in vitro tissue engineering of naturally or synthetically derived bone grafts and in both random (DB) and ordered (HB) pore networks. Combined with the preliminary bioreactor study, this process also shows potential to generate clinically sized, vascularized bone scaffolds for tissue and regenerative engineering.

In this study, we used a polydimethylsiloxane (PDMS)-based platform for the generation of intact, perfusion-competent microvascular networks in vitro. COMSOL Multiphysics, a finite-element analysis and simulation software package, was used to obtain simulated velocity, pressure, and shear stress profiles. Transgene-free human induced pluripotent stem cells (hiPSCs) were differentiated into partially arterialized endothelial cells (hiPSC-ECs) in 5 d under completely chemically defined conditions, using the small molecule glycogen synthase kinase 3β inhibitor CHIR99021 and were thoroughly characterized for functionality and arterial-like marker expression. These cells, along with primary human umbilical vein endothelial cells (HUVECs), were seeded in the PDMS system to generate microvascular networks that were subjected to shear stress. Engineered microvessels had patent lumens and expressed VE-cadherin along their periphery. Shear stress caused by flowing medium increased the secretion of nitric oxide and caused endothelial cells s to align and to redistribute actin filaments parallel to the direction of the laminar flow. Shear stress also caused significant increases in gene expression for arterial markers Notch1 and EphrinB2 as well as antithrombotic markers Kruppel-like factor 2 (KLF-2)/4. These changes in response to shear stress in the microvascular platform were observed in hiPSC-EC microvessels but not in microvessels that were derived from HUVECs, which indicated that hiPSC-ECs may be more plastic in modulating their phenotype under flow than are HUVECs. Taken together, we demonstrate the feasibly of generating intact, engineered microvessels in vitro, which replicate some of the key biological features of native microvessels.

In this article we describe the design and validation of a bioreactor for the in vitro culture of whole rodent lung tissue. Many current systems only enable large segments of lung tissue to be studied ex vivo for up to a few hours in the laboratory. This limitation restricts the study of pulmonary biology in controlled laboratory settings, and also impacts the ability to reliably culture engineered lung tissues in the laboratory. Therefore, we designed, built, and validated a bioreactor intended to provide sufficient nutrient supply and mechanical stimulation to support cell survival and differentiation in cultured lung tissue. We also studied the effects of perfusion and ventilation on pulmonary cell survival and maintenance of cell differentiation state. The final bioreactor design described herein is capable of supporting the culture of whole native lung tissue for up to 1 week in the laboratory, and offers promise in the study of pulmonary biology and the development of engineered lung tissues in the laboratory.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1–TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1–TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

It has been shown that mechanical stimulation affects the physical properties of multiple types of engineered tissues. However, the optimum regimen for applying cyclic radial stretch to engineered arteries is not well understood. To this end, the effect of mechanical stretch on the development of engineered blood vessels was analyzed in constructs grown from porcine vascular smooth muscle cells. Cyclic radial distension was applied during vessel culture at three rates: 0 beats per minute (bpm), 90 bpm, and 165 bpm. At the end of the 7-week culture period, harvested vessels were analyzed with respect to physical characteristics. Importantly, mechanical stretch at 165 bpm resulted in a significant increase in rupture strength in engineered constructs over nonstretched controls. Stress–strain data and maximal elastic moduli from vessels grown at the three stretch rates indicate enhanced physical properties with increasing pulse rate. In order to investigate the role of collagen cross-linking in the improved mechanical characteristics, collagen cross-link density was quantified by HPLC. Vessels grown with mechanical stretch had somewhat more collagen and higher burst pressures than nonpulsed control vessels. Pulsation did not increase collagen cross-link density. Thus, increased wall thickness and somewhat elevated collagen concentrations, but not collagen cross-link density, appeared to be responsible for increased burst strength.

Engineering solid tissues, including cardiac muscle, requires the inclusion of a microvasculature. Prevascularization in vitro will likely be dependent upon coculturing parenchymal cells with vascular cells, on a matrix that is sufficiently porous to allow microvessel formation. In this study, we examined the behavior and function of endothelial cells on a highly porous elastomeric 3D poly(glycerol sebacate) (PGS) scaffold, to provide a flexible and biocompatible endothelial cell delivery system for developing cardiac engineered tissues with neovascularization potential. Both static and perfusion cell seeding methods were used, and the effects of surface treatment of the scaffold with various extracellular matrix components were examined. Endothelial cell adhesion and phenotype on the PGS scaffold under various flow conditions were also determined. Surface coating with laminin markedly improved the endothelial cell adhesion, survival, and proliferation. The anticoagulant phenotype of adhered endothelial cells was further regulated by the application of flow through regulation of nitric oxide expression. By providing a highly porous scaffolding that contains endothelium with anticoagulant properties, the endothelial cell-seeded PGS scaffold could provide a new basis for subsequent coculture studies with various cell types to develop complex engineered tissue constructs with vascularization capacity.

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50–70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-β1 (TGF-β1) differentiated into a homogeneous population expressing α-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use. In contrast, decellularized tissue-engineered arteries could serve as grafts for immediate implantation, as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. The goal of this study was to quantify the effects of decellularization on vascular matrix and mechanical properties. Specifically, we compared cellular elimination, extracellular matrix retention, and mechanical characteristics of porcine carotid arteries before and after treatment with three decellularization methods. In addition, for the first time, tissue-engineered arteries were decellularized. Decellularized native arteries were also used as a scaffold onto which vascular cells were seeded. These studies identified a decellularization method for native and engineered arteries that maximized cellular elimination, without greatly compromising mechanical integrity. We showed that engineered tissues could be decellularized, and demonstrated the feasibility of reseeding decellularized vessels with vascular cells.