Cardiac Valve Bioreactor for Physiological Conditioning and Hydrodynamic Performance Assessment.

PURPOSE: Tissue engineered heart valves (TEHV) are being investigated to address the limitations of currently available valve prostheses. In order to advance a wide variety of TEHV approaches, the goal of this study was to develop a cardiac valve bioreactor system capable of conditioning living valves with a range of hydrodynamic conditions as well as capable of assessing hydrodynamic performance to ISO 5840 standards. METHODS: A bioreactor system was designed based on the Windkessel approach. Novel features including a purpose-built valve chamber and pressure feedback control were incorporated to maintain asepsis while achieving a range of hydrodynamic conditions. The system was validated by testing hydrodynamic conditions with a bioprosthesis and by operating with cell culture medium for 4 weeks and living cells for 2 weeks. RESULTS: The bioreactor system was able to produce a range of pressure and flow conditions from static to resting adult left ventricular outflow tract to pathological including hypertension. The system operated aseptically for 4 weeks and cell viability was maintained for 2 weeks. The system was also able to record the pressure and flow data needed to calculate effective orifice area and regurgitant fraction. CONCLUSIONS: We have developed a single bioreactor system that allows for step-wise conditioning protocols to be developed for each unique TEHV design as well as allows for hydrodynamic performance assessment.

Biomaterial characterization of off-the-shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications.

Fixed pericardial tissue is commonly used for commercially available xenograft valve implants, and has proven durability, but lacks the capability to remodel and grow. Decellularized porcine pericardial tissue has the promise to outperform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile properties was performed on decellularized, decellularized-sterilized, fixed, and native porcine pericardial tissue versus native valve leaflet cusps. The results of non-parametric analysis showed statistically significant differences (p < .05) between the stiffness of decellularized versus native pericardium and native cusps as well as fixed tissue, respectively; however, decellularized tissue showed large increases in elastic properties. Porosity testing of the tissues showed no statistical difference between decellularized and decell-sterilized tissue compared with native cusps (p > .05). Scanning electron microscopy confirmed that valvular endothelial and interstitial cells colonized the decellularized pericardial surface when seeded and grown for 30 days in static culture. Collagen assays and transmission electron microscopy analysis showed limited reductions in collagen with processing; yet glycosaminoglycan assays showed great reductions in the processed pericardium relative to native cusps. Decellularized pericardium had comparatively low mechanical properties among the groups studied; yet the stiffness was comparatively similar to the native cusps and demonstrated a lack of cytotoxicity. Suture retention, accelerated wear, and hydrodynamic testing of prototype decellularized and decell-sterilized valves showed positive functionality. Sterilized tissue could mimic valvular mechanical environment in vitro, therefore making it a viable potential candidate for off-the-shelf tissue-engineered valvular applications.

Recellularization of a novel off-the-shelf valve following xenogenic implantation into the right ventricular outflow tract.

Current research on valvular heart repair has focused on tissue-engineered heart valves (TEHV) because of its potential to grow similarly to native heart valves. Decellularized xenografts are a promising solution; however, host recellularization remains challenging. In this study, decellularized porcine aortic valves were implanted into the right ventricular outflow tract (RVOT) of sheep to investigate recellularization potential. Porcine aortic valves, decellularized with sodium dodecyl sulfate (SDS), were sterilized by supercritical carbon dioxide (scCO2) and implanted into the RVOT of five juvenile polypay sheep for 5 months (n = 5). During implantation, functionality of the valves was assessed by serial echocardiography, blood tests, and right heart pulmonary artery catheterization measurements. The explanted valves were characterized through gross examination, mechanical characterization, and immunohistochemical analysis including cell viability, phenotype, proliferation, and extracellular matrix generation. Gross examination of the valve cusps demonstrated the absence of thrombosis. Bacterial and fungal stains were negative for pathogenic microbes. Immunohistochemical analysis showed the presence of myofibroblast-like cell infiltration with formation of new collagen fibrils and the existence of an endothelial layer at the surface of the explant. Analysis of cell phenotype and morphology showed no lymphoplasmacytic infiltration. Tensile mechanical testing of valve cusps revealed an increase in stiffness while strength was maintained during implantation. The increased tensile stiffness confirms the recellularization of the cusps by collagen synthesizing cells. The current study demonstrated the feasibility of the trans-species implantation of a non-fixed decellularized porcine aortic valve into the RVOT of sheep. The implantation resulted in recellularization of the valve with sufficient hemodynamic function for the 5-month study. Thus, the study supports a potential role for use of a TEHV for the treatment of valve disease in humans.

Xenoantigenicity of porcine decellularized valves.

BACKGROUND: The xenoantigenicity of porcine bioprosthetic valves is implicated as an etiology leading to calcification and subsequent valve failure. Decellularization of porcine valves theoretically could erase the antigenicity of the tissue leading to more durable prosthetic valves, but the effectiveness of decellularization protocols in regard to completely removing antigens has yet to be verified. Our hypothesis was that decellularization would remove the more abundant α-gal antigens but not remove all the non α-gal antigens, which could mount a response. METHODS: Porcine aortic valves were decellularized with 1% sodium dodecyl sulfate for 4 days. Decellularized cusps were evaluated for α-gal epitopes by ELISA. To test for non α-gal antigens, valves were implanted into sheep. Serum was obtained from the sheep preoperatively and 1 week, 1 month, and 2 months postoperatively. This serum was utilized for anti-porcine antibody staining and for quantification of anti-pig IgM and IgG antibodies and complement. RESULTS: Decellularized porcine cusps had 2.8 ± 2.0% relative α-gal epitope as compared to fresh porcine aortic valve cusps and was not statistically significantly different (p = 0.4) from the human aortic valve cusp which had a 2.0 ± 0.4% relative concentration. Anti-pig IgM and IgG increased postoperatively from baseline levels. Preoperatively anti-pig IgM was 27.7 ± 1.7 µg/mL and it increased to 71.9 ± 12.1 µg/mL average of all time points postoperatively (p = 0.04). Preoperatively anti-pig IgG in sheep serum was 44.9 ± 1.5 µg/mL and it increased to 72.6 ± 6.0 µg/mL average of all time points postoperatively (p = 0.01). There was a statistically significant difference (p = 0.00007) in the serum C1q concentration before valve implantation (2.5 ± 0.2 IU/mL) and at averaged time points after valve implantation (5.3 ± 0.3 IU/mL). CONCLUSIONS: Decellularization with 1% sodium dodecyl sulfate does not fully eliminate non α-gal antigens; however, significant reduction in α-gal presence on decellularized cusps was observed. Clinical implications of the non α-gal antigenic response are yet to be determined. As such, evaluation of any novel decellularized xenografts must include rigorous antigen testing prior to human trials.

Supercritical carbon dioxide-based sterilization of decellularized heart valves.

OBJECTIVE: The goal of this research project encompasses finding the most efficient and effective method of decellularized tissue sterilization. BACKGROUND: Aortic tissue grafts have been utilized to repair damaged or diseased valves. Although, the tissues for grafting are collected aseptically, it does not eradicate the risk of contamination nor disease transfer. Thus, sterilization of grafts is mandatory. Several techniques have been applied to sterilize grafts; however, each technique shows drawbacks. In this study, we compared several sterilization techniques: supercritical carbon dioxide, electrolyzed water, gamma radiation, ethanol-peracetic acid, and hydrogen peroxide for impact on the sterility and mechanical integrity of porcine decellularized aortic valves. METHODS: Valve sterility was characterized by histology, microbe culture, and electron microscopy. Uniaxial tensile testing was conducted on the valve cusps along their circumferential orientation to study these sterilization techniques on their integrity. RESULTS: Ethanol-peracetic acid and supercritical carbon dioxide treated valves were found to be sterile. The tensile strength of supercritical carbon dioxide treated valves (4.28 ± 0.22 MPa) was higher to those valves treated with electrolyzed water, gamma radiation, ethanol-peracetic acid and hydrogen peroxide (1.02 ± 0.15, 1.25 ± 0.25, 3.53 ± 0.41 and 0.37 ± 0.04 MPa, respectively). CONCLUSIONS: Superior sterility and integrity were found in the decellularized porcine aortic valves with supercritical carbon dioxide sterilization. This sterilization technique may hold promise for other decellularized soft tissues. SUMMARY: Sterilization of grafts is essential. Supercritical carbon dioxide, electrolyzed water, gamma radiation, ethanol-peracetic acid, and hydrogen peroxide techniques were compared for impact on sterility and mechanical integrity of porcine decellularized aortic valves. Ethanol-peracetic acid and supercritical carbon dioxide treated valves were found to be sterile using histology, microbe culture and electron microscopy assays. The cusp tensile properties of supercritical carbon dioxide treated valves were higher compared to valves treated with other techniques. Superior sterility and integrity was found in the decellularized valves treated with supercritical carbon dioxide sterilization. This sterilization technique may hold promise for other decellularized soft tissues.

Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo.

BACKGROUND: Decellularized heart valves are emerging as a potential alternative to current bioprostheses for valve replacement. Whereas techniques of decellularization have been thoroughly examined, terminal sterilization techniques have not received the same scrutiny. METHODS: This study evaluated low-dose gamma irradiation as a sterilization method for decellularized heart valves. Incubation of valves and transmission electron microscopy evaluation after different doses of gamma irradiation were used to determine the optimal dose of gamma irradiation. Quantitative evaluation of mechanical properties was done by tensile mechanical testing of isolated cusps. Sterilized decellularized heart valves were tested in a sheep model (n = 3 [1 at 1,500 Gy and 2 at 3,000 Gy]) of pulmonary valve replacement. RESULTS: Valves sterilized with gamma radiation between 1,000 Gy and 3,000 Gy were found to be optimal with in vitro testing. However, in vivo testing showed deteriorating valve function within 2 months. On explant, the valve with 1,500 Gy gamma irradiation showed signs of endocarditis with neutrophils on hematoxylin and eosin staining, and positive gram stain resembling streptococcus infection. The 3,000 Gy valves had no evidence of infection, but the hematoxylin and eosin staining showed evidence of wound remodeling with macrophages and fibroblasts. Tensile strength testing showed decreased strength (0 Gy: 2.53 ± 0.98 MPa, 1,500 Gy: 2.03 ± 1.23 MPa, and 3,000 Gy: 1.26 ± 0.90 MPa) with increasing levels of irradiation. CONCLUSIONS: Low-dose gamma irradiation does not maintain the mechanical integrity of valves, and the balance between sterilization and damage may not be able to be achieved with gamma irradiation. Other methods of terminal sterilization must be pursued and evaluated.