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

Abstract

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.

Figure 1. Dobutamine Echocardiography

Dobutamine echocardiography results after chronic implantation of human and baboon bioengineered and control valves. Source species and process preparation for each group noted in figure. Porcine (light blue) were glutaraldehyde crosslinked stentless valves (Medtronic Freestyle®). Transvalvular gradients were uniformly lower for all bioengineered valves (red) at rest and during dobutamine challenge as compared to the clinical reference valves (green) (Panel A). The human bioengineered valves (orange) functionally had low gradients and normal indexed EOA at maximum dobutamine stimulation (Panel B). *p≤0.005. BEV=Bioengineered Valves.

Figure 2. Examples of PRA Time Curves

Panel A: Animal #3 received a cryopreserved allograft and developed both Class I&II PRA's by week 3, sustained through 10 weeks. Panel B: Animal #9 received a bioengineered allograft and did not elevate Class II, and was the only bioengineered allograft recipient in which Class I rose at all. Titer returned to zero by 10 weeks. Panel C: Animal #13 received a human bioengineered valve which provoked Class II antibodies at week 7, but returned to baseline by weeks 26 and 27; no Class I antibodies were detected. These serial measurements demonstrate the potential difficulties in interpretation of random isolated PRA titer measurements after tissue transplants.

Figure 3. Implanted Valve Sizes

Nomogram of BSA normalized human pediatric pulmonary valve annulus diameters derived from 14,128 normal pediatric transthoracic echocardiograms archived in the Children's Mercy Cardiac Database and on which the 14 implanted valve annulus diameters measured by transesophageal echocardiography at baseline after implant surgery (day 0) are graphed (green dots). Note that all but 3 were above the 50% median; the smallest valve for the recipient size was a baboon bioengineered (#8). An inadvertent “downsizing” bias would not explain the greater functional deterioration of the cryopreserved valves. Numbers identify recipient animals for each valve as identified in Table 2.

eFigure 1. Relative Primate Homology

Time domain is estimated with fossil or mtDNA evidence ; Ma = million years ago. Primates first appeared as fossils dated to c. 95 Ma. The depicted divergences represent generalized combinations of physical anthropological findings and computational algorithms based on genetic sequencing data. Of the Old World monkeys, the baboon has the most genomic homology to humans. Data from multiple sources including: Raaum RL, Sterner KN, Noviello CM, Stewart C-B, Disotell TR: Catarrhine primate divergence dates estimated from complete mitochondrial genomes: Concordance with fossil and nuclear DNA evidence. Journal of Human Evolution. 2005;48:237-257.

eFigure 2. C-reactive Protein titers

C-reactive protein serum titers for each animal consistently peaked 1 week postoperatively indicating classic proinflammatory effects of surgery and cardiopulmonary bypass. Pre = preoperatively; Post = immediately after surgery.

eFigure 3. Histopathology

Histologic and immunohistochemical stains of pulmonary valves. A, Histologic section of cryopreserved human pulmonary valve explanted at 10 weeks (H&E, magnification 2.5x) exemplifying a microscopic field that would rate a mild inflammation score. Elsewhere in the specimen, areas of marked inflammation were observed, thus the overall score for this valve explant was “marked” (Animal #5, Table 2). B, Immunohistochemical stain for B-cells (red, alkaline phosphatase, magnification 10x) within the conduit wall inflammatory infiltrate shown in Panel A. C, Histologic section of cryopreserved papio pulmonary valve explanted at 10 weeks (H&E, magnification 2.5x) exemplifying a score of “marked” inflammation (Animal #4). D, Immunohistochemical stain for B-cells (red, alkaline phosphatase, magnification 10x) within the inflammatory infiltrate in the conduit of PRA⁺ recipient in Panel C.