Reduced patency in left-sided arteriovenous grafts in a porcine model.

OBJECTIVE: The porcine arteriovenous graft model is commonly used to study hemodialysis vascular access failure, with most studies using a bilateral, paired-site approach in either the neck or femoral vessels. In humans, left- and right-sided central veins have different anatomy and diameters, and left-sided central vein catheters have worse outcomes. We assessed the effect of laterality on arteriovenous prosthetic graft patency and hypothesized that left-sided carotid-jugular arteriovenous prosthetic grafts have reduced patency in the porcine model. METHODS: Arteriovenous polytetrafluoroethylene grafts were placed ipsilaterally or bilaterally in 10 Yorkshire male pigs from the common carotid artery to the internal jugular vein. Ultrasound measurements of blood flow velocities and diameters were assessed before graft placement. Animals were sacrificed at 1 week, 2 weeks, or 3 weeks. Patency was determined clinically; grafts and perianastomotic vessels were excised and analyzed with histology and immunostaining. RESULTS: At baseline, left- and right-sided veins and arteries had similar blood flow velocities. Although internal jugular veins had similar diameters at baseline, left-sided carotid arteries had 11% smaller outer diameters (P = .0354). There were 10 left-sided and 8 right-sided polytetrafluoroethylene grafts placed; only 4 of 10 (40%) grafts were patent on the left compared with 7 of 8 (88%) grafts patent on the right (P = .04). Left-sided grafts had increased macrophages at the arterial anastomosis (P = .0007). Left-sided perianastomotic arteries had thicker walls (0.74 vs 0.60 mm; P = .0211) with increased intima-media area (1.14 vs 0.77 mm2; P = .0169) as well as a trend toward 38% smaller luminal diameter (1.6 vs 2.5 mm; P = .0668) and 20% smaller outer diameter (3.0 vs 3.7 mm; P = .0861). Left- and right-sided perianastomotic veins were similar histologically, but left-sided veins had decreased expression of phosphorylated endothelial nitric oxide synthase (P = .0032) and increased numbers of α-actin-positive smooth muscle cells (P = .0022). CONCLUSIONS: Left-sided arteriovenous grafts are associated with reduced short-term patency compared with right-sided grafts in the Yorkshire pig preclinical model of arteriovenous prosthetic grafts. Laterality must be considered in planning and interpreting surgical preclinical models.

Susceptibility of ePTFE vascular grafts and bioengineered human acellular vessels to infection.

BACKGROUND: Synthetic expanded polytetrafluorethylene (ePTFE) grafts are routinely used for vascular repair and reconstruction but prone to sustained bacterial infections. Investigational bioengineered human acellular vessels (HAVs) have shown clinical success and may confer lower susceptibility to infection. Here we directly compared the susceptibility of ePTFE grafts and HAV to bacterial contamination in a preclinical model of infection. MATERIALS AND METHODS: Sections (1 cm2) of ePTFE (n = 42) or HAV (n = 42) were inserted within bilateral subcutaneous pockets on the dorsum of rats and inoculated with Staphylococcus aureus (107 CFU/0.25 mL) or Escherichia coli (108 CFU/0.25 mL) before wound closure. Two weeks later, the implant sites were scored for abscess formation and explanted materials were halved for quantification of microbial recovery and histological analyses. RESULTS: The ePTFE implants had significantly higher abscess formation scores for both S. aureus and E. coli inoculations compared to that of HAV. In addition, significantly more bacteria were recovered from explanted ePTFE compared to HAV. Gram staining of explanted tissue sections revealed interstitial bacterial contamination within ePTFE, whereas no bacteria were identified in HAV tissue sections. Numerous CD45+ leukocytes, predominantly neutrophils, were found surrounding the ePTFE implants but minimal intact neutrophils were observed within the ePTFE matrix. The host cells surrounding and infiltrating the HAV explants were primarily nonleukocytes (CD45-). CONCLUSIONS: In an established animal model of infection, HAV was significantly less susceptible to bacterial colonization and abscess formation than ePTFE. The preclinical findings presented in this manuscript, combined with previously published clinical observations, suggest that bioengineered HAV may exhibit low rates of infection.

Bioengineered human acellular vessels for dialysis access in patients with end-stage renal disease: two phase 2 single-arm trials.

BACKGROUND: For patients with end-stage renal disease who are not candidates for fistula, dialysis access grafts are the best option for chronic haemodialysis. However, polytetrafluoroethylene arteriovenous grafts are prone to thrombosis, infection, and intimal hyperplasia at the venous anastomosis. We developed and tested a bioengineered human acellular vessel as a potential solution to these limitations in dialysis access. METHODS: We did two single-arm phase 2 trials at six centres in the USA and Poland. We enrolled adults with end-stage renal disease. A novel bioengineered human acellular vessel was implanted into the arms of patients for haemodialysis access. Primary endpoints were safety (freedom from immune response or infection, aneurysm, or mechanical failure, and incidence of adverse events), and efficacy as assessed by primary, primary assisted, and secondary patencies at 6 months. All patients were followed up for at least 1 year, or had a censoring event. These trials are registered with ClinicalTrials.gov, NCT01744418 and NCT01840956. FINDINGS: Human acellular vessels were implanted into 60 patients. Mean follow-up was 16 months (SD 7·6). One vessel became infected during 82 patient-years of follow-up. The vessels had no dilatation and rarely had post-cannulation bleeding. At 6 months, 63% (95% CI 47-72) of patients had primary patency, 73% (57-81) had primary assisted patency, and 97% (85-98) had secondary patency, with most loss of primary patency because of thrombosis. At 12 months, 28% (17-40) had primary patency, 38% (26-51) had primary assisted patency, and 89% (74-93) had secondary patency. INTERPRETATION: Bioengineered human acellular vessels seem to provide safe and functional haemodialysis access, and warrant further study in randomised controlled trials. FUNDING: Humacyte and US National Institutes of Health.

Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

Engineering of arteries in vitro.

This review will focus on two elements that are essential for functional arterial regeneration in vitro: the mechanical environment and the bioreactors used for tissue growth. The importance of the mechanical environment to embryological development, vascular functionality, and vascular graft regeneration will be discussed. Bioreactors generate mechanical stimuli to simulate biomechanical environment of arterial system. This system has been used to reconstruct arterial grafts with appropriate mechanical strength for implantation by controlling the chemical and mechanical environments in which the grafts are grown. Bioreactors are powerful tools to study the effect of mechanical stimuli on extracellular matrix architecture and mechanical properties of engineered vessels. Hence, biomimetic systems enable us to optimize chemo-biomechanical culture conditions to regenerate engineered vessels with physiological properties similar to those of native arteries. In addition, this article reviews various bioreactors designed especially to apply axial loading to engineered arteries. This review will also introduce and examine different approaches and techniques that have been used to engineer biologically based vascular grafts, including collagen-based grafts, fibrin-gel grafts, cell sheet engineering, biodegradable polymers, and decellularization of native vessels.

Evaluation of vascular repair by tissue-engineered human acellular vessels or expanded polytetrafluoroethylene grafts in a porcine model of limb ischemia and reperfusion.

BACKGROUND: This study evaluated performance of a tissue-engineered human acellular vessel (HAV) in a porcine model of acute vascular injury and ischemia. The HAV is an engineered blood vessel consisted of human vascular extracellular matrix proteins. Limb reperfusion and vascular outcomes of the HAV were compared with those from synthetic expanded polytetrafluoroethylene (ePTFE) grafts. METHODS: Thirty-six pigs were randomly assigned to four treatment groups, receiving either the HAV or a PTFE graft following a hind limb ischemia period of either 0 or 6 hours. All grafts were 3-cm-long interposition 6-mm diameter grafts implanted within the right iliac artery. Animals were not immunosuppressed and followed for up to 28 days after surgery. Assessments performed preoperatively and postoperatively included evaluation of graft patency, hind limb function, and biochemical markers of tissue ischemia or reperfusion injury. Histological analysis was performed on explants to assess host cell responses. RESULTS: Postoperative gait assessment and biochemical analysis confirmed that ischemia and reperfusion injury were caused by 6-hour ischemia, regardless of vascular graft type. Hind limb function and tissue damage biomarkers improved in all groups postoperatively. Final patency rates at postoperative day 28 were higher for HAV than for ePTFE graft in both the 0-hour (HAV, 85.7%; ePTFE, 66.7%) and 6-hour (HAV, 100%; ePTFE, 75%) ischemia groups, but these differences were not statistically significant. Histological analyses identified some intimal hyperplasia and host reactivity to the xenogeneic HAV and also to the synthetic ePTFE graft. Positive host integration and vascular cell infiltration were identified in HAV but not ePTFE explants. CONCLUSION: Based on the functional performance and the histologic profile of explanted HAVs, this study supports further investigation to evaluate long-term performance of the HAV when used to repair traumatic vascular injuries.

Patency of ePTFE Arteriovenous Graft Placements in Hemodialysis Patients: Systematic Literature Review and Meta-Analysis.

Arteriovenous grafts (AVGs) are an appropriate option for vascular access in certain hemodialysis patients. Expanded polytetrafluoroethylene (ePTFE) has become the dominant material for such grafts, due in part to innovations in graft design and surgical interventions to reduce complications and improve patency rates. Comprehensive evidence syntheses have not been conducted to update AVG performance in an era in which both access choice and ePTFE graft functioning may have changed. We conducted a systematic review and meta-analysis summarizing outcomes from recent studies of ePTFE AVGs in hemodialysis, following PRISMA standards. Literature searches were conducted in multiple databases to identify observational and interventional studies of AVG patency and infection risk. Primary, primary-assisted, and secondary patency rates were analyzed at 6, 12, 18, and 24 months postplacement. Kaplan-Meier graft survival plots were digitized to recreate individual patient-level data. Patency rates were pooled using a random effects model. We identified 32 studies meeting our selection criteria that were published from 2004 through 2019. A total of 38 study arms of ePTFE grafts were included, representing 3381 AVG accesses placed. The mean primary, primary-assisted, and secondary patency rates at 1 year were 41% (95% CI, 35% to 47%), 46% (95% CI, 41% to 51%), and 70% (95% CI, 64% to 75%), respectively. Mean 24-month patency rates were 28% (95% CI, 22% to 33%), 34% (95% CI, 27% to 41%), and 54% (95% CI, 47% to 61%), respectively. A high degree of heterogeneity across studies was observed. Overall risk of infection was not consistently reported, but among available studies the pooled estimate was 9% per patient-year (95% CI, 6% to 12%). This meta-analysis provides an up-to-date estimate of the performance of ePTFE AVGs, within the context of improved graft designs and improved interventional techniques.

Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes.

Single ventricle heart defects (SVDs) are congenital disorders that result in a variety of complications, including increased ventricular mechanical strain and mixing of oxygenated and deoxygenated blood, leading to heart failure without surgical intervention. Corrective surgery for SVDs are traditionally handled by the Fontan procedure, requiring a vascular conduit for completion. Although effective, current conduits are limited by their inability to aid in pumping blood into the pulmonary circulation. In this report, we propose an innovative and versatile design strategy for a tissue engineered pulsatile conduit (TEPC) to aid circulation through the pulmonary system by producing contractile force. Several design strategies were tested for production of a functional TEPC. Ultimately, we found that porcine extracellular matrix (ECM)-based engineered heart tissue (EHT) composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary cardiac fibroblasts (HCF) wrapped around decellularized human umbilical artery (HUA) made an efficacious basal TEPC. Importantly, the TEPCs showed effective electrical and mechanical function. Initial pressure readings from our TEPC in vitro (0.68 mmHg) displayed efficient electrical conductivity enabling them to follow electrical pacing up to a 2 Hz frequency. This work represents a proof of principle study for our current TEPC design strategy. Refinement and optimization of this promising TEPC design will lay the groundwork for testing the construct's therapeutic potential in the future. Together this work represents a progressive step toward developing an improved treatment for SVD patients. STATEMENT OF SIGNIFICANCE: Single Ventricle Cardiac defects (SVD) are a form of congenital disorder with a morbid prognosis without surgical intervention. These patients are treated through the Fontan procedure which requires vascular conduits to complete. Fontan conduits have been traditionally made from stable or biodegradable materials with no pumping activity. Here, we propose a tissue engineered pulsatile conduit (TEPC) for use in Fontan circulation to alleviate excess strain in SVD patients. In contrast to previous strategies for making a pulsatile Fontan conduit, we employ a modular design strategy that allows for the optimization of each component individually to make a standalone tissue. This work sets the foundation for an in vitro, trainable human induced pluripotent stem cell based TEPC.

Fas ligand and nitric oxide combination to control smooth muscle growth while sparing endothelium.

Metallic stents cause vascular wall damage with subsequent smooth muscle cell (SMC) proliferation, neointimal hyperplasia, and treatment failure. To combat in-stent restenosis, drug-eluting stents (DES) delivering mTOR inhibitors such as sirolimus or everolimus have become standard for coronary stenting. However, the relatively non-specific action of mTOR inhibitors prevents efficient endothelium recovery and mandates dual antiplatelet therapy to prevent thrombosis. Unfortunately, long-term dual antiplatelet therapy leads to increased risk of bleeding/stroke and, paradoxically, myocardial infarction. Here, we took advantage of the fact that nitric oxide (NO) increases Fas receptors on the SMC surface. Fas forms a death-inducing complex upon binding to Fas ligand (FasL), while endothelial cells (ECs) are relatively resistant to this pathway. Selected doses of FasL and NO donor synergistically increased SMC apoptosis and inhibited SMC growth more potently than did everolimus or sirolimus, while having no significant effect on EC viability and proliferation. This differential effect was corroborated in ex vivo pig coronaries, where the neointimal formation was inhibited by the drug combination, but endothelial viability was retained. We also deployed FasL-NO donor-releasing ethylene-vinyl acetate copolymer (EVAc)-coated stents into pig coronary arteries, and cultured them in perfusion bioreactors for one week. FasL and NO donor, released from the stent coating, killed SMCs close to the stent struts, even in the presence of flow rates mimicking those of native arteries. Thus, the FasL-NO donor-combination has a potential to prevent intimal hyperplasia and in-stent restenosis, without harming endothelial restoration, and hence may be a superior drug delivery strategy for DES.

Vascularization of Natural and Synthetic Bone Scaffolds.

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.

Engineered Microvasculature in PDMS Networks Using Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

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 3ß 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.

Vascular smooth muscle cells derived from inbred swine induced pluripotent stem cells for vascular tissue engineering.

Development of autologous tissue-engineered vascular constructs using vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (iPSCs) holds great potential in treating patients with vascular disease. However, preclinical, large animal iPSC-based cellular and tissue models are required to evaluate safety and efficacy prior to clinical application. Herein, swine iPSC (siPSC) lines were established by introducing doxycycline-inducible reprogramming factors into fetal fibroblasts from a line of inbred Massachusetts General Hospital miniature swine that accept tissue and organ transplants without immunosuppression within the line. Highly enriched, functional VSMCs were derived from siPSCs based on addition of ascorbic acid and inactivation of reprogramming factor via doxycycline withdrawal. Moreover, siPSC-VSMCs seeded onto biodegradable polyglycolic acid (PGA) scaffolds readily formed vascular tissues, which were implanted subcutaneously into immunodeficient mice and showed further maturation revealed by expression of the mature VSMC marker, smooth muscle myosin heavy chain. Finally, using a robust cellular self-assembly approach, we developed 3D scaffold-free tissue rings from siPSC-VSMCs that showed comparable mechanical properties and contractile function to those developed from swine primary VSMCs. These engineered vascular constructs, prepared from doxycycline-inducible inbred siPSCs, offer new opportunities for preclinical investigation of autologous human iPSC-based vascular tissues for patient treatment.

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

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.

Click-coated, heparinized, decellularized vascular grafts.

A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through "alkyne-azide" click chemistry, affording a tenfold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. X-ray photoelectron spectroscopy, nuclear magnetic resonance imaging, mass spectrometry and Fourier transform infrared FTIR spectroscopy were used to characterize the synthesis steps, building the final heparin layered coatings. The continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. The efficacy of heparin linkage was demonstrated with factor Xa anti-thrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels.

Requirements for growing tissue-engineered vascular grafts.

Small diameter (< 6 mm) vascular grafts are in large demand for coronary and peripheral bypass procedures. Although synthetic grafts have been developed, tissue-based vascular grafts that can better mimic native vessels will likely yield superior results. The success of a tissue-based graft depends on its ability to meet several requirements. First, a graft must possess a confluent, adherent and quiescent endothelium to resist thrombosis in vivo. The mechanical behavior of the graft must mimic the mechanical properties of a native vessel. Hence, a graft must have a highly organized collagen matrix to impart tissue strength. Finally, a graft must contain an elastin network to provide compliance and recoil.

Decellularized native and engineered arterial scaffolds for transplantation.

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.

Effects of polyglycolic acid on porcine smooth muscle cell growth and differentiation.

Polyglycolic acid (PGA) is commonly used as a scaffold for tissue engineering. Recent studies utilized PGA as a scaffold for vascular tissue engineering using bovine and porcine smooth muscle cells (SMCs). In engineered vessels, the SMCs displayed high rates of mitosis and dedifferentiation in areas where PGA fragments were present. We hypothesized that PGA breakdown products, sequestered within a SMC vessel at the conclusion of culture, led to increased proliferation and dedifferentiation of vascular SMCs. To test this hypothesis, the current study assessed possible means by which PGA breakdown products could lead to changes in SMC phenotype. SMCs grown in high concentrations of PGA breakdown products showed, by Western blotting, decreased expression of calponin, a marker for SMC differentiation. The same was true for SMCs grown in glycolic acid (GA), which also showed decreased expression of proliferating cell nuclear antigen (PCNA), a marker for SMC proliferation. In contrast, cells grown in varying amounts of NaCl or HCl showed little change in differentiation. We conclude that, independent of acidity or osmolality, plausible products of PGA degradation appear to induce dedifferentiation of porcine SMCs in vitro. Because of dedifferentiation and decreased mitosis, commercially available PGA may not represent an optimal scaffold for vascular tissue engineering.

Engineering porcine arteries: effects of scaffold modification.

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.

Engineering biological-based vascular grafts using a pulsatile bioreactor.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels. Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC). A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM). To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Development of novel biodegradable polymer scaffolds for vascular tissue engineering.

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.