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The development of a tracheal graft to replace long-segment defects has thwarted clinicians and engineers alike for over 100 years. To better understand the challenges facing this field today, we have consolidated all published reports of engineered tracheal grafts used to repair long-segment circumferential defects in humans, from the first in 1898 to the most recent in 2018, totaling 290 clinical cases. Distinct trends emerge in the types of grafts used over time, including repair using autologous fascia, rigid tubes of various inert materials, and pretreated cadaveric allografts. Our analysis of maximum clinical follow-up, as a proxy for graft performance, revealed that the Leuven protocol has a significantly longer clinical follow-up time than all other methods of airway reconstruction. This method involves transplanting a cadaveric tracheal allograft that is first prevascularized heterotopically in the recipient. We further quantified graft-related causes of mortality, revealing failure modes that have been resolved, and those that remain a hurdle, such as graft mechanics. Finally, we briefly summarize recent preclinical work in tracheal graft development. In conclusion, we synthesized top clinical care priorities and design criteria to inform and inspire collaboration between engineers and clinicians toward the development of a functional tracheal replacement graft. Impact statement The field of tracheal engineering has floundered in recent years due to multiple article retractions. However, with recent advances in biofabrication and tissue analysis techniques, the field remains ripe for advancement through collaboration between engineers and clinicians. With a long history of clinical application of tracheal replacements, engineered tracheas are arguably the regenerative technology with the greatest potential for translation. This work describes the many phases of engineered tracheal replacements that have been applied in human patients over the past 100 years with the goal of carrying forward critical lessons into development of the next generation of engineered tracheal graft.

Effective vascularization is vital for survival and functionality of complex tissue-engineered organs. The formation of the microvasculature, composed of endothelial cells (ECs) alone, has been mostly used to restore the vascular networks in organs. However, recent heterocellular studies demonstrate that co-culturing is a more effective approach in revascularization of engineered organs. This review presents key considerations for manufacturing of artificial vascularized composite tissues. We summarize the importance of co-cultures and the multicellular interactions with ECs, as well as design and use of bioreactors, as critical considerations for tissue vascularization. In addition, as an emerging scaffolding technique, this review also highlights the current caveats and hurdles associated with three-dimensional bioprinting and discusses recent developments in bioprinting strategies such as four-dimensional bioprinting and its future outlook for manufacturing of vascularized tissue constructs. Finally, the review concludes with addressing the critical challenges in the regulatory pathway and clinical translation of artificial composite tissue grafts. Impact statement Regeneration of composite tissues is critical as biophysical and biochemical characteristics differ between various types of tissues. Engineering a vascularized composite tissue has remained unresolved and requires additional evaluations along with optimization of methodologies and standard operating procedures. To this end, the main hurdle is creating a viable vascular endothelium that remains functional for a longer duration postimplantation, and can be manufactured using clinically appropriate source of cell lines that are scalable in vitro for the fabrication of human-scale organs. This review presents key considerations for regeneration and manufacturing of vascularized composite tissues as the field advances.

The pancreatic islet is a highly vascularized micro-organ, and rapid revascularization postislet transplantation is important for islet survival and function. However, the various mechanisms involved in islet revascularization are not fully understood, and we currently lack good in vitro platforms to explore this. Our aim for this study was to generate perfusable microvascular networks in a microfluidic chip device, in which islets could be easily integrated, to establish an in vitro platform for investigations on islet-microvasculature interactions. We compared the ability of mesenchymal stem cells (MSCs) and fibroblasts to support microvascular network formation by human umbilical vein endothelial cells (HUVECs) and human induced pluripotent stem cell-derived endothelial colony-forming cell in two-dimensional and three-dimensional models of angiogenesis, and tested the effect of different culture media on microvessel formation. HUVECs that were supported by MSCs formed patent and perfusable networks in a fibrin gel, whereas networks supported by fibroblasts rapidly regressed. Network morphology could be controlled by adjusting relative cell numbers and densities. Incorporation of isolated rat islets demonstrated that islets recruit local microvasculature in vitro , but that the microvessels did not invade islets, at least during the course of these studies. This in vitro microvascularization platform can provide a useful tool to study how various parameters affect islet integration with microvascular networks and could also be utilized for studies of vascularization of other organ systems. Impact statement To improve pancreatic islet graft survival and function posttransplantation, rapid and adequate revascularization is critical. Efforts to improve islet revascularization are demanding due to an insufficient understanding of the mechanisms involved in the process. We have applied a microfluidics platform to generate microvascular networks, and by incorporating pancreatic islets, we were able to study microvasculature-islet interactions in real time. This platform can provide a useful tool to study islet integration with microvascular networks, and could be utilized for studies of vascularization of other organ systems. Moreover, this work may be adapted toward developing a prevascularized islet construct for transplantation.

Impact Statement: Recent advances in tissue engineering using decellularized organ scaffolds have expanded the possibilities for organ replacement therapy. However, detergent-based decellularization itself damages the extracellular matrix (ECM), which results in failure associated with the transplanted bioengineered organ. This study determined that potassium laurate (PL), a natural detergent, significantly reduces lung ECM damage during the decellularization process compared with protocols using sodium dodecyl sulfate. PL-decellularized lungs showed better microarchitecture preservation and low biological reactions after subcutaneous implantation. PL-decellularized scaffolds supported rat lung endothelial cell attachment/proliferation and the bioengineered lungs significantly reduced lung congestion after transplantation.

Pluripotent stem cell-derived endothelial cells (ECs) have great potential to be used in vascular therapy or tissue engineering. It is also much desired to obtain arterial or venous ECs for specific applications. Factors that are critical for the proper arterial or venous differentiation from pluripotent stem cells still need to be understood. Here, we aim at investigating this problem deeper by examining neuropilin-1 (Nrp1), an early arterial marker that may be critical for arterial cell fate commitment. Using murine embryonic stem cells as the model system, this study investigates the neuropilin-1 (Nrp1) expression during the differentiation of pluripotent stem cells toward a vascular progenitor population. We hypothesize that Nrp1, an early arterial marker present in a developing embryo, may be more responsive when further induced in vitro toward an arterial fate. We developed a two-step differentiation approach that yielded a large percentage of Nrp1 + vascular progenitor cells (VPCs) and investigated their potential to become arterial ECs. We have defined the culture parameters that contribute greatly to the emergence of Nrp1 + VPCs: certain soluble factors, especially Wnt and BMP4, early cell-cell contact, and hypoxia. Subsequent isolation of this population demonstrated a highly proliferative and network-forming behavior. The Nrp1 + VPCs exhibited increased gene expression of several Notch pathway-related arterial markers compared with Nrp1 - VPCs. Most importantly, Nrp1 + VPCs demonstrated a dramatically greater response to hemodynamic stimuli by upregulating many arterial markers whereas Nrp1 - VPCs have very little response. Surprisingly, these differences between Nrp1 + and Nrp1 - VPCs are not evident with vascular endothelial growth factor (VEGF) treatment. Our data suggest that Nrp1 + VPCs may serve as the arterial progenitor by enhanced response to hemodynamic flow but not to VEGF, whereas Nrp1 - VPCs lack the plasticity to become arterial ECs. The findings of this research indicate that Nrp1 + VPCs in the murine model act as an important step in the arterial differentiation process.

Here we report the creation of a novel tracheal construct in the form of an engineered, acellular tissue-stent biocomposite trachea (TSBT). Allogeneic or xenogeneic smooth muscle cells are cultured on polyglycolic acid polymer-metal stent scaffold leading to the formation of a tissue comprising cells, their deposited collagenous matrix, and the stent material. Thorough decellularization then produces a final acellular tubular construct. Engineered TSBTs were tested as end-to-end tracheal replacements in 11 rats and 3 nonhuman primates. Over a period of 8 weeks, no instances of airway perforation, infection, stent migration, or erosion were observed. Histological analyses reveal that the patent implants remodel adaptively with native host cells, including formation of connective tissue in the tracheal wall and formation of a confluent, columnar epithelium in the graft lumen, although some instances of airway stenosis were observed. Overall, TSBTs resisted collapse and compression that often limit the function of other decellularized tracheal replacements, and additionally do not require any cells from the intended recipient. Such engineered TSBTs represent a model for future efforts in tracheal regeneration.

Tissue-engineered blood vessels (TEVs) are typically produced using the pulsatile, uniaxial circumferential stretch to mechanically condition and strengthen the arterial grafts. Despite improvements in the mechanical integrity of TEVs after uniaxial conditioning, these tissues fail to achieve critical properties of native arteries such as matrix content, collagen fiber orientation, and mechanical strength. As a result, uniaxially loaded TEVs can result in mechanical failure, thrombus, or stenosis on implantation. In planar tissue equivalents such as artificial skin, biaxial loading has been shown to improve matrix production and mechanical properties. To date however, multiaxial loading has not been examined as a means to improve mechanical and biochemical properties of TEVs during culture. Therefore, we developed a novel bioreactor that utilizes both circumferential and axial stretch that more closely simulates loading conditions in native arteries, and we examined the suture strength, matrix production, fiber orientation, and cell proliferation. After 3 months of biaxial loading, TEVs developed a formation of mature elastic fibers that consisted of elastin cores and microfibril sheaths. Furthermore, the distinctive features of collagen undulation and crimp in the biaxial TEVs were absent in both uniaxial and static TEVs. Relative to the uniaxially loaded TEVs, tissues that underwent biaxial loading remodeled and realigned collagen fibers toward a more physiologic, native-like organization. The biaxial TEVs also showed increased mechanical strength (suture retention load of 303 ± 14.53 g, with a wall thickness of 0.76 ± 0.028 mm) and increased compliance. The increase in compliance was due to combinatorial effects of mature elastic fibers, undulated collagen fibers, and collagen matrix orientation. In conclusion, biaxial stretching is a potential means to regenerate TEVs with improved matrix production, collagen organization, and mechanical properties.

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO2) that can achieve a sterility assurance level 10(-6) in decellularized lung matrix. The effects of ScCO2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current study's results indicate that ScCO2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes.

Kidney transplantation is often the most effective therapy for end-stage renal disease, but there are not enough donor organs to meet the rising demand. Tissue engineering of kidneys is a potential solution to this organ shortage. Achieving microvascular perfusion has been a major barrier to engineering tissues beyond thin muscularized sheets such as the bladder wall. Our laboratory has previously reported that human umbilical vein endothelial cells (ECs) transduced with the antiapoptotic protein Bcl-2 will spontaneously organize into perfused microvessels within type I collagen gels when implanted in immunodeficient mice. To test if this system can be used to perfuse more complex structures, we combined Bcl-2-transduced ECs (Bcl-2-ECs) with renal glomeruli, the specialized vascular filtration units of the kidney. Microdissected green fluorescent protein-expressing rat glomeruli suspended in type I collagen gels were implanted within immunodeficient mice with or without the inclusion of Bcl-2-ECs. Survival of rat glomeruli was enhanced by coimplantation with Bcl-2-ECs. Intravital rhodamine dextran injections demonstrated that surviving glomeruli were perfused through Bcl-2-EC-derived microvessels. Perfused glomeruli maintained podocin staining, but transmission electron microscopy revealed endothelial swelling and podocyte foot process effacement. Anastomosis of microvessels derived from Bcl-2-ECs with glomerular capillaries provides proof of concept that self-assembled microvessels can perfuse specialized organ structures such as glomeruli, but that perfusion alone may be insufficient to maintain normal structure.

Conventional bioreactors are used to enhance extracellular matrix (ECM) production and mechanical strength of tissue-engineered vessels (TEVs) by applying circumferential strain, which is uniaxial stretching. However, the resulting TEVs still suffer from inadequate mechanical properties, where rupture strengths and compliance values are still very different from native arteries. The biomechanical milieu of native arteries consists of both circumferential and axial loading. Therefore, to better simulate the physiological stresses acting on native arteries, we built a novel bioreactor system to enable biaxial stretching of engineered arteries during culture. This new bioreactor system allows for independent control of circumferential and axial stretching parameters, such as displacement and beat rate. The assembly and setup processes for this biaxial bioreactor system are reliable with a success rate greater than 75% for completion of long-term sterile culture. This bioreactor also supports side-by-side assessments of TEVs that are cultured under three types of mechanical conditions (static, uniaxial, and biaxial), all within the same biochemical environment. Using this bioreactor, we examined the impact of biaxial stretching on arterial wall remodeling of TEVs. Biaxial TEVs developed the greatest wall thickness compared with static and uniaxial TEVs. Unlike uniaxial loading, biaxial loading led to undulated collagen fibers that are commonly found in native arteries. More importantly, the biaxial TEVs developed the most mature elastin in the ECM, both qualitatively and quantitatively. The presence of mature extracellular elastin along with the undulated collagen fibers may contribute to the observed vascular compliance in the biaxial TEVs. The current work shows that biaxial stretching is a novel and promising means to improve TEV generation. Furthermore, this novel system allows us to optimize biomechanical conditioning by unraveling the interrelationships among the applied mechanical stress, the resulting ECM properties, and the mechanics of TEVs.

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-βR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-βR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes.

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7-21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells.

The creation of decellularized organs for use in regenerative medicine requires the preservation of the organ extracellular matrix (ECM) as a means to provide critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. The purpose of this study was to assess the influence of varying pH levels on the preservation of key ECM components during the decellularization of rat lungs. Herein, we show that the pH of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-based decellularization solution influences ECM retention, cell removal, and also the potential for host response upon implantation of acellular lung tissue. The preservation of ECM components, including elastin, fibronectin, and laminin, were better retained in the lower pH conditions that were tested (pH ranges tested: 8, 10, 12); glycosaminoglycans were preserved to a higher extent in the lower pH groups as well. The DNA content following decellularization of the rat lung was inversely correlated with the pH of the decellularization solution. Despite detectible levels of cyotoskeletal proteins and significant residual DNA, tissues decellularized at pH 8 demonstrated the greatest tissue architecture maintenance and the least induction of host response of all acellular conditions. These results highlight the effect of pH on the results obtained by organ decellularization and suggest that altering the pH of the solutions used for decellularization may influence the ability of cells to properly differentiate and home to appropriate locations within the scaffold, based on the preservation of key ECM components and implantation results.

Recent advances in three-dimensional (3D) tissue engineering have concomitantly generated a need for new methods to visualize and assess the tissue. In particular, methods for imaging intact volumes of whole tissue, rather than a single plane, are required. Herein, we describe the use of multiphoton microscopy, combined with optical clearing, to noninvasively probe decellularized lung extracellular matrix scaffolds and decellularized, tissue-engineered blood vessels. We also evaluate recellularized lung tissue scaffolds. In addition to nondestructive imaging of tissue volumes greater than 4 mm(3), the lung tissue can be visualized using three distinct signals, combined or singly, that allow for simple separation of cells and different components of the extracellular matrix. Because the 3D volumes are not reconstructions, they do not require registration algorithms to generate digital volumes, and maintenance of isotropic resolution is not required when acquiring stacks of images. Once a virtual volume of tissue is generated, structures that have innate 3D features, such as the lumens of vessels and airways, are easily animated and explored in all dimensions. In blood vessels, individual collagen fibers can be visualized at the micron scale and their alignment assessed at various depths through the tissue, potentially providing some nondestructive measure of vessel integrity and mechanics. Finally, both the lungs and vessels assayed here were optically cleared, imaged, and visualized in a matter of hours, such that the added benefits of these techniques can be achieved with little more hassle or processing time than that associated with traditional histological methods.

Identification of appropriate donor cell types is important for lung cell therapy and for lung regeneration. Previous studies have indicated that mesenchymal stromal cells derived from human bone marrow (hBM-MSCs) and from human adipose tissue (hAT-MSCs) may have the ability to trans-differentiate into lung epithelial cells. However, these data remain controversial. Herein, the ability of hBM-MSCs and hAT-MSCs to repopulate acellular rodent lung tissue was evaluated. hBM-MSCs and hAT-MSCs were isolated from bone marrow aspirate and lipoaspirate, respectively. Rat lungs were decellularized with CHAPS detergent, followed by seeding the matrix with hBM-MSCs and hAT-MSCs. Under appropriate culture conditions, both human MSC populations attached to and proliferated within the lung tissue scaffold. In addition, cells were capable of type 2 pneumocyte differentiation, as assessed by marker expression of surfactant protein C (pro-SPC) at the protein and the RNA level, and by the presence of lamellar bodies by transmission electron microscopy. Additionally, hAT-MSCs contributed to Clara-like cells that lined the airways in the lung scaffolds, whereas the hBM-MSCs did not. We also tested the differentiation potential of MSCs on different extracellular matrix components in vitro, and found that protein substrate influences MSC epithelial differentiation. Together our data show the capacity for human MSCs to differentiate toward lung epithelial phenotypes, and the possibility of using these cells for lung cell therapies and tissue engineering.

Tissue-engineered small-diameter vascular grafts have been developed as a promising alternative to native veins or arteries for replacement therapy. However, there is still a crucial need to improve the current approaches to render the tissue-engineered blood vessels more favorable for clinical applications. A completely biological blood vessel (3-mm inner diameter) was constructed by culturing a 50:50 mixture of bovine smooth muscle cells (SMCs) with neonatal human dermal fibroblasts in fibrin gels. After 30 days of culture under pulsatile stretching, the engineered blood vessels demonstrated an average burst pressure of 913.3±150.1 mmHg (n=6), a suture retention (53.3±15.4 g) that is suitable for implantation, and a compliance (3.1%±2.5% per 100 mmHg) that is comparable to native vessels. These engineered grafts contained circumferentially aligned collagen fibers, microfibrils and elastic fibers, and differentiated SMCs, mimicking a native artery. These promising mechanical and biochemical properties were achieved in a very short culture time of 30 days, suggesting the potential of co-culturing SMCs with fibroblasts in fibrin gels to generate functional small-diameter vascular grafts for vascular reconstruction surgery.

Functional connective tissues have been developed using tissue engineering approach by seeding cells on biodegradable scaffolds such as polyglycolic acid (PGA). However, a major drawback of tissue engineering approaches that utilize synthetic polymers is the persistence of polymer remnants in engineered tissues at the end of culture. Such polymer fragments may significantly degrade tissue mechanics and stimulate local inflammatory responses in vivo. In this study, several polymeric materials with a range of degradation profiles were developed and evaluated for their potential applications in construction of collagen matrix-rich tissues, particularly tissue-engineered blood vessels. The degradation characteristics of these polymers were compared as were their characteristics vis-à-vis cell adhesion and proliferation, collagen synthesis, and ability to support growth of engineered vessels. Under aqueous conditions at 37°C, Polymer I (comprising 84% glycolide and 16% trimethylene carbonate [TMC]) had a similar degradation profile to PGA, Polymer II (comprising 84% glycolide, 14% TMC, and 2% polyethylene succinate) degradedly more slowly, but Polymer III (comprising 87% glycolide, 7% TMC, and 6% polyethylene glycol) had a more extensive degradation as compared to PGA. All polymers supported cell proliferation, but Polymer III improved collagen production and engineered vessel mechanics as compared with PGA. In addition, more slowly degrading polymers were associated with poorer final vessel mechanics. These results suggest that polymers that degrade more quickly during tissue culture actually result in improved engineered tissue mechanics, by virtue of decreased disruption of collagenous extracellular matrix.

Although the importance of fluid flow for proper vascular development and function in vivo is well recognized, microvascular formation in response to flow has not been well evaluated in a three-dimensional (3D) environment in vitro. In this study, we developed a novel 3D in vitro perfusion system that allows direct investigation of the effects of shear stress on the development of microvasculature in vitro. This system utilizes a 3D collagen gel for suspension of vascular cells and mesenchymal stem cells, through which flow is directly perfused. We characterized the flow conditions and demonstrate the impact of flow on the development of microvasculature using a coculture of endothelial cells and mesenchymal stem cells. With the unique ability to apply bulk flow through the collagen gels, and to estimate shear stress within the constructs, this perfusion system provides a flexible platform for developing a controllable biomimetic environment that can be adapted for a variety of investigations of microvascularization.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Decellularization of native tissues is a promising technique with numerous applications in tissue engineering and regenerative medicine. However, there are various limitations of currently available decellularization methods, such as alteration of extracellular matrix mechanics and restricted use on certain tissues. This study was conducted to explore the effect of serum on the decellularization of various types of tissues. Fetal bovine serum-containing cell culture medium endothelial growth media-2 removed DNA but not cellular beta-actin from human umbilical artery after detergent treatment, without compromising the tissue mechanical strength assessed by burst pressure. In addition, the effect of serum-containing endothelial growth media-2 on DNA removal was replicated in other types of tissues such as tissue-engineered vessels and myocardium. Other types of serum, including human serum, were also shown to remove DNA from detergent-pretreated tissues. In conclusion, we describe a novel utilization of serum that may have broad applications in tissue decellularization.

Objective: Developing a tissue-engineered small-diameter (<6mm) vascular graft for reconstructive surgery has remained a challenge for the past several decades. This study was conducted to develop a decellularized umbilical artery and to evaluate its composition, endothelial cell compatibility, mechanical properties, and in vivo stability for potential use as a small-diameter vascular graft., Methods and Results: Human umbilical arteries were isolated and decellularized by incubation in CHAPS and sodium dodecyl sulfate buffers followed by incubation in endothelial growth media-2. Decellularized umbilical arteries were completely devoid of cellular and nuclear material while retaining the integrity of extracellular collagenous matrix. The mechanical strength of the decellularized umbilical artery as assessed by its burst pressure in vitro showed no significant change from its native form. Decellularized umbilical arteries supported endothelial adherence as indicated by the re-endotheliazation with a monolayer of human umbilical vein endothelial cells. Furthermore, decellularized vessels that were implanted into nude rats as abdominal aorta interposition grafts remained mechanically intact and patent for up to 8 weeks., Conclusion: Decellularized human umbilical arteries preserved the extracellular matrix, supported endothelialization, and retained function in vivo for up to 8 weeks. These properties suggest the potential use of decellularized umbilical arteries as small-diameter vascular grafts.

Human bone marrow-derived mesenchymal stem cells (hMSCs) represent an appealing source of smooth muscle cells (SMCs) for engineering small-diameter vascular grafts due to the limited availability and replicative capacity of somatic SMCs. However, lack of standardization of hMSC culture conditions has limited some progress in hMSC research. Because, at the moment, a chemically defined, serum-free medium without growth factors is not capable of amplifying hMSCs in vitro, the usage of serum (either human serum or fetal bovine serum [FBS]) continues in hMSC research. The emergence of commercial hMSCs and hMSC media opened a series of questions regarding the compatibility of commercial and homemade hMSCs and hMSC media. In this study, two types of commonly used FBS-containing hMSC media-MSCGM (containing 10% FBS) and MesenPro (containing 2% FBS), along with our homemade medium (low-glucose Dulbecco's modified Eagle's medium plus 10% selected lot FBS)-were compared in their ability to support SMC differentiation from hMSCs. The effects of FBS level, medium supplements (ascorbic acid, copper, etc.), and growth factors (transforming growth factor beta1) were also examined for their impact on SMC differentiation. It was discovered that MesenPro and transforming growth factor beta1 are the strongest SMC inducers from hMSCs. In contrast, hMSCs grown in homemade (10% Dulbecco's modified Eagle's medium) and commercial MSCGM media remained undifferentiated. FBS concentration did not affect SMC differentiation when 10% FBS was compared with 2%. Finally, the mechanism underlying SMC differentiation from hMSCs grown in FBS-containing medium was explored by following the expression changes of serum response factor during the establishment of hMSC culture.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Cardiovascular disease is the leading cause of morbidity and mortality in Western society. More than 1 million arterial bypass procedures are performed annually in the United States, where either autologous veins or synthetic grafts are used to replace arteries in the coronary or peripheral circulation. Tissue engineering of blood vessels from autologous cells has the potential to produce biological grafts for use in bypass surgery. Ex vivo development of vascular grafts also provides an ideal target of site-specific gene therapy to optimize the physiology of the developing conduit, and for the possible delivery of other therapeutic genes to a vascular bed of interest. In this article, we demonstrate that by using a novel retroviral gene delivery system, a target gene of interest can be specifically delivered to the endothelial cells of a developing engineered vessel. Further, we demonstrate that this technique results in stable incorporation of the delivered gene into the target endothelial cells for more than 30 days. These data demonstrate the utility of the retroviral gene delivery approach for optimizing the biologic phenotype of engineered vessels. This also provides the framework for testing an array of genes that may improve the function of engineered blood vessels after surgical implantation.