Performance of allogeneic bioengineered replacement pulmonary valves in rapidly growing young lambs.

BACKGROUND: Cardiac allometric organ growth after pediatric valve replacement can lead to patient-prosthesis size mismatch and valve re-replacement, which could be mitigated with allogeneic decellularized pulmonary valves treated with collagen conditioning solutions to enhance biological and mechanical performance, termed "bioengineered valves." In this study, we evaluated functional, dimensional, and biological responses of these bioengineered valves compared with traditional cryopreserved valves implanted in lambs during rapid somatic growth. METHODS: From a consanguineous flock of 13 lambs, the pulmonary valves of 10 lambs (mean weight, 19.6 ± 1.4 kg) were replaced with 7 bioengineered valves or 3 classically cryopreserved valves. After 6 months, the 10 lambs with implanted valves and 3 untreated flock mates were compared by echocardiography, cardiac catheterization, and explant pathology. RESULTS: Increases in body mass, valve geometric dimensions, and effective orifice areas were similar in the 2 groups of lambs. The bioengineered valves had higher median cusp-to-cusp coaptation areas (34.6%; interquartile range, 21.00%-35.13%) and were more similar to native valves (43.4%; interquartile range, 42.59%-44.01%) compared with cryopreserved valves (13.2%; interquartile range, 7.07%-13.91%) (P = .043). Cryopreserved valves cusps, but not bioengineered valve cusps, were thicker than native valve cusps (P = .01). Histologically, cryopreserved valves demonstrated less than native cellularity, whereas bioengineered valves that were acellular at the time of surgery gained surface endothelium and subsurface myofibroblast interstitial cells in pulmonary artery, sinus wall, and cusp base regions. CONCLUSIONS: Biological valve conduits can enlarge via passive dilatation without matrix synthesis, but this would result in decreased cusp coaptational areas. Bioengineered valves demonstrated similar annulus enlargement as cryopreserved valves but usually retained larger areas of cuspal coaptation. Heat-shock protein 47-positive (collagen-synthesizing) cells were present in previously acellular bioengineered sinus walls and cusp bases, but rarely in more distal cusp matrices.

Pulse Duplicator Hydrodynamic Testing of Bioengineered Biological Heart Valves.

There are many heart valve replacements currently available on the market; however, these devices are not ideal for pediatric patients with congenital heart valve defects. Decellularized valve substitutes offer potential for improved clinical outcomes and require pre-clinical testing guidelines and testing systems suitable for non-crosslinked, biological heart valves. The objective of this study was to assess the hydrodynamic performance of intact, bioengineered pulmonary valves using a custom pulse duplicator capable of testing intact biological valved conduits. The mechanical behavior of valve associated sinus and arterial tissue was also evaluated under biaxial loading. Cryopreserved, decellularized, extracellular matrix (ECM) conditioned and glutaraldehyde fixed valves showed reduced pressure gradients and increased effective orifice area for decellularized and ECM conditioned valves. ECM conditioning resulted in increased elastic modulus but decreased stretch in circumferential and longitudinal directions under biaxial loading. Overall, decellularization and ECM conditioning did not compromise the scaffolds, which exhibited satisfactory bench top performance.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Bioengineered human and allogeneic pulmonary valve conduits chronically implanted orthotopically in baboons: hemodynamic performance and immunologic consequences.

OBJECTIVE: This study assesses in a baboon model the hemodynamics and human leukocyte antigen immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds. METHODS: Fourteen baboons underwent pulmonary valve replacement, 8 with decellularized and conditioned (bioengineered) pulmonary valves derived from allogeneic (N = 3) or xenogeneic (human) (N = 5) hearts; for comparison, 6 baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel-reactive serum antibodies (human leukocyte antigen class I and II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon rank-sum tests. RESULTS: All animals survived and thrived, exhibiting excellent immediate implanted valve function by transesophageal echocardiograms. Over time, reference valves developed a smaller effective orifice area index (median, 0.84 cm(2)/m(2); range, 1.22 cm(2)/m(2)), whereas all bioengineered valves remained normal (effective orifice area index median, 2.45 cm(2)/m(2); range, 1.35 cm(2)/m(2); P = .005). None of the bioengineered valves developed elevated peak transvalvular gradients: 5.5 (6.0) mm Hg versus 12.5 (23.0) mm Hg (P = .003). Cryopreserved valves provoked the most intense antibody responses. Two of 5 human bioengineered and 2 of 3 baboon bioengineered valves did not provoke any class I antibodies. Bioengineered human (but not baboon) scaffolds provoked class II antibodies. C1q(+) antibodies developed in 4 recipients. CONCLUSIONS: Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.

Effects of cryopreservation, decellularization and novel extracellular matrix conditioning on the quasi-static and time-dependent properties of the pulmonary valve leaflet.

Decellularized allografts offer potential as heart valve substitutes and scaffolds for cell seeding. The effects of decellularization on the quasi-static and time-dependent mechanical behavior of the pulmonary valve leaflet under biaxial loading conditions have not previously been reported in the literature. In the current study, the stress-strain, relaxation and creep behaviors of the ovine pulmonary valve leaflet were investigated under planar-biaxial loading conditions to determine the effects of decellularization and a novel post-decellularization extracellular matrix (ECM) conditioning process. As expected, decellularization resulted in increased stretch along the loading axes. A reduction in relaxation was observed following decellularization. This was accompanied by a reduction in glycosaminoglycan (GAG) content. Based on previous implant studies, these changes may be of little functional consequence in the short term; however, the long term effects of decreased relaxation and GAG content remain unknown. Some restoration of relaxation was observed following ECM conditioning, especially in the circumferential specimen direction, which may help mitigate any detrimental effects due to decellularization. Regardless of processing, creep under biaxial loading was negligible.

Determining cell seeding dosages for tissue engineering human pulmonary valves.

BACKGROUND: This study examines in vitro seeding of decellularized human pulmonary valves (hPVs) with human valve interstitial cells (hVICs) isolated from unrelated donor aortic valve leaflets. An assay was developed to assess seeding using precut uniform sized biopsies from whole hPVs for sequential evaluation of seeding efficiency, proliferation, and migration. MATERIALS AND METHODS: Scaffolds for seeding were created from decellularized hPVs using a reciprocating osmolality, double detergent, enzyme, multiple solvent protocol. hVICs seeded decellularized leaflet and sinus wall scaffolds were incubated in either static or cyclic pressure bioreactors. Low, medium, and high initial cell seeding "dosing" densities were assayed at subsequent three time points, using eight replicates each (n = 576 biopsies including manufactured scaffold controls). Metabolically viable seeded cells were quantified by MTT assay. Histology defined cell locations and morphology. RESULTS: After 24 h of static seeding with 2.5 × 10(5) cells (medium dose), 100 ± 13 cells/mm(2) (2.5%) attached to leaflets, compared with 193 ± 21 cells/mm(2) (8%) for sinuses. Subsequent 4 d in static culture yielded 894 ± 84 and 838 ± 50 cells/mm(2)versus pulsatile culture yielding 80 ± 12 and 79 ± 12 cells/mm(2) for leaflet and sinus, respectively. However, 76.0% ± 12.2% of cells in leaflets in the pulsatile bioreactor were subsurface as compared to 21.4% ± 3.9% in statically cultured leaflets (P < 0.001). CONCLUSION: Different seeding modes suggest a tradeoff between surface proliferation resulting in higher absolute cell numbers for static seeding versus fewer cells in a cyclic pressure bioreactor but with a greater percentage having migrated into the matrix. The medium seeding dose determined to be optimal is actually feasible for tissue engineering heart valves, and can be achieved by fairly traditional cell amplification methods.

Performance and morphology of decellularized pulmonary valves implanted in juvenile sheep.

BACKGROUND: Because of cryopreserved heart valve-mediated immune responses, decellularized allograft valves are an attractive option in children and young adults. The objective of this study was to investigate the performance and morphologic features of decellularized pulmonary valves implanted in the right ventricular outflow tract of juvenile sheep. METHODS: Right ventricular outflow tract reconstructions in juvenile sheep (160±9 days) using cryopreserved pulmonary allografts (n=6), porcine aortic root bioprostheses (n=4), or detergent/enzyme-decellularized pulmonary allografts (n=8) were performed. Valve performance (echocardiography) and morphologic features (gross, radiographic, and histologic examination) were evaluated 20 weeks after implantation. RESULTS: Decellularization reduced DNA in valve cusps by 99.3%. Bioprosthetic valves had the largest peak and mean gradients versus decellularized valves (p=0.03; p<0.001) and cryopreserved valves (p=0.01; p=0.001), which were similar (p=0.45; p=0.40). Regurgitation was minimal and similar for all groups (p=0.16). No cusp calcification was observed in any valve type. Arterial wall calcification was present in cryopreserved and bioprosthetic grafts but not in decellularized valves. No autologous recellularization or inflammation occurred in bioprostheses, whereas cellularity progressively decreased in cryopreserved grafts. Autologous recellularization was present in decellularized arterial walls and variably extending into the cusps. CONCLUSIONS: Cryopreserved and decellularized graft hemodynamic performance was comparable. Autologous recellularization of the decellularized pulmonary arterial wall was consistently observed, with variable cusp recellularization. As demonstrated in this study, decellularized allograft valves have the potential for autologous recellularization.

Pediatric cardiopulmonary bypass adaptations for long-term survival of baboons undergoing pulmonary artery replacement.

Cardiopulmonary bypass (CPB) protocols of the baboon (Papio cynocephalus anubis) are limited to obtaining experimental data without concern for long-term survival. In the evaluation of pulmonary artery tissue engineered heart valves (TEHVs), pediatric CPB methods are adapted to accommodate the animals' unique physiology enabling survival up to 6 months until elective sacrifice. Aortic access was by a 14F arterial cannula and atrial access by a single 24F venous cannula.The CPB circuit includes a 3.3 L/min flow rated oxygenator, 1/4" x %" arterial-venous loop, 3/8" raceway, and bubble trap. The prime contains 700 mL Plasma-Lyte, 700 units heparin, 5 mL of 50% dextrose, and 20 mg amiodarone. Heparinization (200 u/kg) targets an activated clotting time of 350 seconds. Normothermic CPB was initiated at a 2.5 L/m2/min cardiac index with a mean arterial pressure of 55-80 mmHg. Weaning was monitored with transesophageal echocardiogram. Post-CPB circuit blood was re-infused. Chest tubes were removed with cessation of bleeding. Extubation was performed upon spontaneous breathing. The animals were conscious and upright 3 hours post-CPB. Bioprosthetic valves or TEHVs were implanted as pulmonary replacements in 20 baboons: weight = 27.5 +/- 5.6 kg, height = 73 +/- 7 cm, body surface area = 0.77 m2 +/- 0.08, mean blood flow = 1.973 +/- .254 L/min, core temperature = 37.1 +/- .1 degree C, and CPB time = 60 +/- 40 minutes. No acidosis accompanied CPB. Sixteen animals survived, four expired. Three died of right ventricular failure and one of an anaphylactoid reaction. Surviving animals had normally functioning replacement valves and ventricles. Baboon CPB requires modifications to include high systemic blood pressure for adequate perfusion into small coronary arteries, careful CPB weaning to prevent ventricular distention, and drug and fluid interventions to abate variable venous return related to a muscularized spleno-splanchnic venous capacity.

Heart valve collagens: cross-species comparison using immunohistological methods.

BACKGROUND AND AIM OF THE STUDY: Tissue engineering is an emerging strategy for the development of replacement heart valves where the properties of native tissues are to be replicated. The complexity of the distribution of various collagens in the aortic, mitral, and pulmonary valve leaflets of porcine, bovine, and ovine origin, has been examined. METHODS: Immunohistological and transmission electron microscopy analyses using monoclonal antibodies to types I, III, IV, V and VI collagens were performed. RESULTS: The results indicated that each collagen type has its own distinct distribution, with minimal variation between heart valve anatomic sites and species. Of particular interest was type VI collagen, which had an asymmetric distribution that was principally localized along the outflow surface of the valve. CONCLUSION: Successful tissue engineering constructs of heart valves may need to replicate the complex distribution of different collagens found in heart valve tissues.

Prototype anionic detergent technique used to decellularize allograft valve conduits evaluated in the right ventricular outflow tract in sheep.

BACKGROUND AND AIM OF THE STUDY: Biodegradable polymeric materials or extracellular matrix scaffolds are used in tissue-engineered heart valve designs, with the expectation of replicating the anatomic, histological and biomechanical characteristics of semi-lunar valves. The study aim was to evaluate the extent of in-vivo recellularization and the explant pathology findings of a prototype anionic, non-denaturing detergent and endonuclease technique used to decellularize allograft (homograft) valve conduits implanted in the right ventricular outflow tract (RVOT) of sheep, and to identify possible risks associated with tissue-engineered heart valve conduits based on decellularized allograft semilunar valve scaffolds. METHODS: Valve conduits were decellularized using a solution of N-lauroylsarcosinate and endonucleases, rinsed in lactated Ringers solution, and stored in an antibiotic solution at 4 degrees C until implanted. Explanted valves and unimplanted controls were examined macroscopically, radiographically (for calcification) and histologically using immunohistochemistry (IHC), routine and special histological stains, transmission electron microscopy (TEM) and polarized light microscopy (evaluation of collagen crimp). RESULTS: Cells and cellular remnants were uniformly absent in the decellularized cusps, but occasional focal sites of arterial wall smooth muscle cells and to a greater extent subvalvular cardiac myocytes were variably retained. The trilaminar histological structure of the cusp was preserved. Valve conduit-related pathology consisted of intracuspal hematoma formation, collagen fraying, thinning of the conduit wall, and inflammatory cells associated with cardiac myocyte remnants. Cuspal calcification was not seen, but elastic fibers in the conduit wall and retained subvalvular cardiac myocyte remnants were liable to calcification. Fibrous sheath formation was present on the luminal surface of the conduit and extended over the cuspal surfaces to a variable extent. Myofibroblast-like cells repopulated the conduit wall and the basal region of the cusp. Re-endothelialization was variably present on the cuspal surfaces. CONCLUSION: Explant pathology findings showed that in-vivo recellularization occurred, but was focally limited to regions of the arterial wall and cusp base. Safety concerns related to detergent and endonuclease use were identified. Methods to eliminate the potential for structural deterioration and enhance the rate and extent of recellularization of valve conduit tissue are required. Pathology findings showed implantation of valve conduits in the RVOT of juvenile sheep for 20 weeks to be a reliable animal model for the initial in-vivo assessment of decellularized valves. A 20-week period may be insufficient however to evaluate the long-term safety and effectiveness of a tissue-engineered valve conduit, as these depend on effective and phenotypically appropriate recellularization accompanied by sustained cell viability and function.

Effects of constant static pressure on the biological properties of porcine aortic valve leaflets.

An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. In this study, the effect of constant ambient pressure on the biological properties of heart valve leaflets was evaluated using a custom-designed pressure system. Native porcine aortic valve leaflets were exposed to static pressures of 100, 140, or 170 mmHg for 48 h. Collagen synthesis, DNA synthesis, sulfated glycoaminoglycan (sGAG) synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were examined. Results showed that elevated pressure caused an increase in collagen synthesis. This increase was not statistically significant at 100 mmHg, but at 140 mmHg and 170 mmHg collagen synthesis increased by 37.5 and 90%, respectively. No significant difference in DNA or sGAG synthesis was observed at elevated pressures, with the exception that DNA synthesis at 100 mmHg decreased. A notable decline in alpha-SMC actin was observed over the course of the experiments although no significant difference was observed between the pressure and control groups. It was concluded that elevated pressure caused a proportional increase in collagen synthesis of porcine aortic valve leaflets, but was unable to preserve alpha-SMC actin immunoreactive cells.

Explant pathology study of decellularized carotid artery vascular grafts.

The purpose of this study was to evaluate the morphologic findings in small-diameter freeze-dried decellularized carotid artery grafts implanted in goats as carotid artery interposition grafts for 6-7 months. Unimplanted decellularized carotid artery grafts did not contain intact cells; however, remnants of smooth muscle cells were present in the media. The extracellular matrix was well preserved. All decellularized grafts were patent at explant, without significant dimensional changes or aneurysm formation. Their luminal surfaces were lined by a thin neointima, consisting of myofibroblasts, collagen, and a discontinuous layer of endothelial cells. Histologic evidence of calcification within the explants was not observed; however, electron microscopy showed calcification of minute remnants of cell membranes. Inflammatory cells were not present in the graft wall. Host cell migration was greatest in the adventitia along the length of the graft. Migration of host cells into the media was more apparent close to the anastomoses, forming cellular nests rich in extracellular proteoglycans, whereas cell migration into areas subjacent to the lumen was minimal. Ingrowth of host blood vessels was not observed. These results demonstrate satisfactory structural and morphologic features of a decellularized carotid artery small-diameter graft implanted for up to 7 months.

Cyclic pressure affects the biological properties of porcine aortic valve leaflets in a magnitude and frequency dependent manner.

An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. Previous studies by this group have shown that exposure to constant static pressure leads to enhanced collagen synthesis in porcine aortic valve leaflets. In this study, the effect of cyclic pressure was evaluated using a custom-designed pressure system. Different pressure magnitudes (100, 140, and 170 mmHg) as well as pulse frequencies (0.5, 1.167, and 2 Hz) were studied. Collagen synthesis, cell proliferation, sGAG synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were chosen as markers for valvular biological responses. Results showed that aortic valve leaflets responded to cyclic pressure in a magnitude and frequency-dependent manner. Increases in pressure magnitude (with the frequency fixed at 1.167 Hz) resulted in significant increases in both collagen and sGAG synthesis, while DNA synthesis remained unchanged. Responses to pulse frequency (with the magnitude fixed at 100 mmHg) were more complex. Collagen and sGAG synthesis were increased by 25 and 14% respectively at 0.5 Hz; but were not affected at 1.167 and 2 Hz. In contrast, DNA synthesis increased by 72% at 2 Hz, but not at 0.5 and 1.167 Hz. Under extreme pressure conditions (170 mmHg, 2 Hz), collagen and sGAG synthesis were increased but to a lesser degree than at 170 mmHg, and 1.167 Hz. Cell proliferation was not affected. A notable decline in a-SMC actin was observed over the course of the experiments, although no significant difference was observed between the cyclic pressure and control groups. It was concluded that cyclic pressure affected biosynthetic activity of aortic valve leaflets in a magnitude and frequency dependent manner. Collagen and sGAG synthesis were positively correlated and more responsive to pressure magnitude than pulse frequency. DNA synthesis was more responsive to pulse frequency than pressure magnitude. However, when combined, pressure magnitude and pulse frequency appeared to have an attenuating effect on each other. The number of alpha-SMC actin positive cells did not vary with cyclic pressure, regardless of pulse frequency and pressure magnitude.