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Ann Thorac Surg 2001;71:S428-S432
© 2001 The Society of Thoracic Surgeons

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Basic research

Decellularized human valve allografts

^a University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, USA
^b CryoLife, Inc, Kennesaw, Georgia, USA

Address reprint requests to Ms Dawson, CryoLife, Inc, 1655 Roberts Blvd, NW, Kennesaw, GA 30144
e-mail: dawson.patti{at}cryolife.com

Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.

	Abstract

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Variable performance of allograft tissues in children and some adults may be linked to an immune response and could be mitigated by reducing implant antigenicity.

Methods. As endothelial and fibroblast cells are the likely source of valve antigenicity, human (CryoValve SG) and sheep pulmonary valves were decellularized using the SynerGraft treatment process. Treated valves were evaluated in vitro using histochemical, biomechanical, and hydrodynamic methods, and compared with standard cryopreserved valves. Four SynerGraft-treated and two cryopreserved sheep pulmonary valves were implanted as root replacements in the right ventricular outflow tract of growing sheep and monitored echocardiographically and histologically at 3 and 6 months. CryoValve SG human pulmonary valves were implanted in 36 patients.

Results. SynerGraft treatment reduced tissue antigen expression but did not alter human valve biomechanics or strength. Decellularized sheep allograft valves were functional during the implantation period, and, they became progressively recellularized with recipient cells. In humans, CryoValve SG pulmonary valves did not provoke a panel reactive antibody response.

Conclusions. SynerGraft decellularization leaves the physical properties of valves unaltered and substantially diminishes antigen content. Reduction in implant cellularity enables host recellularization of the matrix, which should favorably impact long-term graft durability.

	Introduction

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Allograft heart valves are used for replacement of diseased or damaged heart valves in adults and children and have shown clinical durability [1], except in children. Although functionally superior to other bioprosthetic options, the variable durability has resulted in unpredictable valve longevity, specifically in children [2, 3]. Host antigen recognition and antibody development may be linked to early onset of tissue calcification and structural valve deterioration [4].

Allograft tissues not matched for human leukocyte antigens result in elevated human leukocyte antigen antibodies after implantation. Smith and associates [5] suggested a relationship between patient sensitization as assessed by panel reactive antibody (PRA) evaluation and increased structural valve deterioration. Although elevated PRA levels have been reported in pediatric and adult recipients, data correlating this response to long-term valve function have not been demonstrated [6, 7]. It is likely that the component cells are the antigenic element of the valve as they express class I and class II human leukocyte antigens [8]. We have reported that removal of cells and reduction of major histocompatibility complexes I and II and other porcine-specific epitopes in the connective tissue matrix yields a nonantigenic material, allowing cross-species implantation of these tissues [9]. Porcine decellularized heart valve tissues have been successfully implanted into sheep and humans, as aortic and pulmonary valve replacements.

This study investigated the application of antigen reduction through decellularization of allograft heart valves. Human pulmonary allograft valves were decellularized by the SynerGraft process and cryopreserved (CryoValve SG). Treated valves were examined microscopically for reduction in tissue cellularity and antigen expression. The acute safety of CryoValve SG was assessed, demonstrating the preservation of tissue strength, biomechanics, and valvar hydrodynamic function relative to cryopreserved tissues. Chronic in vivo stability was assessed in pulmonary valve replacements in a weanling sheep model. Clinical utility of CryoValve SG was evaluated by PRA assessments in both patients requiring primary valve replacement and those requiring replacement of dysfunctional allografts.

	Material and methods

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Sheep and human pulmonary valves were treated using the SynerGraft process [9] to reduce cellularity and maintain an intact extracellular matrix. After cell lysis and washout, the processed valves were cryopreserved using a patented controlled rate freeze protocol [10]. Unprocessed human and sheep pulmonary valves were also cryopreserved, and served as study controls.

In vitro testing
Biomechanics
Samples of CryoValve SG human pulmonary artery and valve leaflets were evaluated under uniaxial tension using an Instron model 5565 universal load frame (Instron Corp, Canton MA) and compared with cryopreserved human pulmonary valve tissues. Samples of conduit and myocardium were also evaluated for suture retention ability.

Valve hydrodynamics
Cryopreserved controls and CryoValve SG human pulmonary valves were mounted as intact roots into a modified left heart pulse duplicator (ViVitro Systems, Victoria, BC, Canada). System variables were adjusted to obtain standard cardiac flow conditions (70 cycles/min, 30% to 40% systolic fraction). The mean arterial pressure (averaged for the entire cardiac cycle) was adjusted to between 25 and 180 mm Hg to simulate normal pulmonary artery through hypertensive aortic pressure conditions. Stroke volume was adjusted to achieve pulsatile flow rates from 2 to 7 L/min. Three of each test and control valves were studied under all flow conditions.

Calorimetry
Tissue denaturation temperature for each tissue type and processing technique was determined from the endothermal peak obtained from differential scanning calorimetry data obtained between 20°C and 100°C.

Histology and immunohistology
Samples of processed leaflet and conduit or explanted tissue specimens were studied with hematoxylin and eosin–stained paraffin sections or immunostained frozen sections. Antibodies for immunohistochemistry included monoclonal antibody to class I and class II major histocompatibility antigens (VMRD Inc, Pullman, WA).

In vivo study
Sheep implants
SynerGraft-treated sheep cryopreserved pulmonary valves (n = 4) and untreated cryopreserved pulmonary allografts (n = 2), 17 to 19 mm diameter, were implanted into the right ventricular outflow tract of 3- to 6-month-old sheep. Echocardiographic assessment was obtained immediately after implantation and at 1, 3, and 6 months.

Two test valves were explanted at 3 months, and two test valves and one control were explanted at 6 months, with hemodynamic measurements taken before sacrifice. All sheep involved in this study received humane care according to the principles in the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985).

Clinical performance
CryoValve SG pulmonary valves were implanted in 32 patients as an adjunct to Ross aortic valve replacements and in 4 patients with a dysfunctional pulmonary allograft. Patients ranged in age from 0.27 to 51.2 years. Antibody response to the implant was monitored with a cytotoxicity-based assessment of PRAs both before and after implant (1 and 3 months). Postimplant valve function was assessed by echocardiography 7 to 10 days after operation, and at 3 and 6 months postoperatively.

	Results

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
In vitro testing
Tissue mechanics and valvular hydrodynamics
The biomechanical properties of CryoValve SG and cryopreserved human pulmonary leaflet and conduit tissues were compared for specific tissue mechanical properties after treatment. As a nominal value of one indicates no change in the property, we could detect no alterations in the properties indicated (Fig 1).

View larger version (17K): [in this window] [in a new window]   Fig 1. Comparison of uniaxial tensile biomechanics and suture pull strength of cryopreserved and CryoValve SG human pulmonary valves. Midpoint line indicates the ratios of each characteristic as determined in the CryoValve SG tissue versus its cryopreserved counterpart (full bar shows experimental range). A ratio of one indicates equivalency of the biomechanical feature; greater than one indicates CryoValve SG valve is greater than that of CryoValve, less than one indicates the converse.

CryoValve SG and cryopreserved valves showed equivalent hydrodynamic performance with mean systolic pressure gradients less than 5 mm Hg at all flow conditions. At normal pulmonary pressures all valves demonstrated small closure volumes and no significant diastolic regurgitation. Elevation of the mean arterial pressure from 25 mm Hg through 180 mm Hg caused a corresponding dilation in the valve root diameters with a marginal increase in the closure volume, remaining less than 5% of forward flow.

Antigen reduction by the decellularization process
Removal of cells and tissue antigens was demonstrated by histologic and immunohistochemical analysis. Figure 2 compares representative sections of cryopreserved conduit and CryoValve SG leaflets and conduit of human pulmonary valve. The reduction in hematoxylin and eosin staining of endothelial and interstitial cellular elements was at least 99%. Similarly, staining of the CryoValve SG tissues for class I and class II major histocompatability antigens was markedly reduced by the decellularization process. Although cryopreserved human pulmonary conduit possesses significant expression of both types of tissue antigens (Figs 3A, 3C), little major histocompatibility complex class I (Fig 3B) or class II (Fig 3D) staining was found in CryoValve SG.

View larger version (75K): [in this window] [in a new window]   Fig 2. Histologic preparations of cryopreserved and CryoValve SG human pulmonary valve tissues. Shown are hematoxylin and eosin–stained sections (6 to 8 µm) of cryopreserved human pulmonary valve leaflet (A) and CryoValve SG human pulmonary valve leaflet (B) and conduit (C). All specimens are shown at an original magnification of x40.

View larger version (147K): [in this window] [in a new window]   Fig 3. Immunohistochemistry for tissue antigens in cryopreserved and CryoValve SG human pulmonary valve conduit. Cryosections of either cryopreserved human pulmonary valve conduit (A, B) or CryoValve SG conduit (C, D) are shown after reaction with major histocompatibility complex anti-class I (A, C) or anti-class II (B, D) antisera. Sectioning and magnification are as in Figure 2.

Histologic analysis of heart valves explanted from sheep at 3 months demonstrated substantial recellularization of the full thickness of the conduit, and initial recellularization of the ventricular surface of the leaflets. By the sixth month of implantation, leaflet recellularization had progressed into the leaflet matrix and extended as much as 75% of the leaflet length (Fig 4).

View larger version (133K): [in this window] [in a new window]   Fig 4. Histology of explants of SynerGraft-treated sheep pulmonary valve tissues after various times of engraftment in the right ventricular outflow tract of sheep. Shown are representative sections of conduit explanted at 3 (A) or 6 months (B), and of leaflet base (C) or midsection (D) explanted after 6 months. Leaflet orientation is identified by V on the ventricularis surface and F on the fibrosal surface.

The 4 patients requiring allograft pulmonary valve replacements had elevated preoperative PRA (29% to 88%), and at 3 months postoperatively their levels remained stable at 48% to 88%.

Postoperative echocardiographic evaluations at 7 to 10 days in all patients, and at 3 months postoperatively in 8 patients, have shown normal allograft valve function.

	Comment

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Although there is no established causal relationship between allograft antigenicity and valve durability, there is growing concern over donor-specific cellular responses and elevated levels of tissue-specific antibodies found in allograft valve recipients. The present investigation provided an opportunity to evaluate the systematic removal of antigens (by removing the cellular component) on the integrity of the tissue matrix.

The in vitro results demonstrate that tissue decellularization in CryoValve SG is highly effective in antigen reduction, although causing no significant impact on tissue strength or biomechanics. Immunohistochemical staining shows the positive link between cellularity and antigen and shows that antigen reduction can be achieved through decellularization.

The preclinical animal studies verified acceptability of the SynerGraft treatment process as predicted by the biomechanical and hydrodynamic tests, and demonstrated the suitability of the tissues in a biologic environment. The SynerGraft-treated pulmonary allografts in the sheep showed progressive recellularization of the leaflet and conduit matrices, providing the potential for tissue maintenance and repair similar to SynerGraft xenogeneic valve implants [9].

Clinically, the absence of measurable PRA after implantation of the CryoValve SG pulmonary valves in the previously PRA-negative patient cohort demonstrates that cell reduction effectively eliminates presentable tissue antigen, and thus the opportunity to develop an antibody response.

Allograft valve durability may be enhanced through application of this antigen reduction technology. In addition, as certain valve recipient’s medical status progresses to requiring heart transplantation, limiting the risk for PRA development enhances the likelihood of matching that individual with a suitable donor heart.

	Acknowledgments

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We thank BioSurg, Inc, for oversight of the sheep study and Carribeth Bair, Kathleen Kite-Powell, Amy Cameron, and Kimberly Greene for their excellent technical assistance.

	Footnotes

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Dr Elkins is a member of the Board of Directors and a paid consultant for CryoLife, Inc. Patti Dawson, Steven Goldstein, Steven Walsh, and Kirby Black are all paid, full-time employees of CryoLife, Inc.

	References

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 

1. O’Brien M.F., Stafford E.G., Gardner M.A.H., et al. Allograft aortic valve replacement: long-term follow-up. Ann Thorac Surg 1995;60:S65-S70.
2. Clarke D.R., Campbell D.N., Hayward A.R., Bishop D.A. Degeneration of aortic valve allografts in young recipients. J Thoracic Cardiovasc Surg 1993;105:934-942.[Abstract]
3. Niwaya K., Knott-Craig C.J., Lane M.M., Chandrasekaren K., Overholt E.D., Elkins R.C. Cryopreserved homograft valves in the pulmonary position: risk analysis for intermediate-term failure. J Thoracic Cardiovasc Surg 1999;117:141-146.[Abstract/Free Full Text]
4. Yankah C.A., Alex-Meskhishvili V., Weng Y., Schorn K., Lange P., Hetzer R. Accelerated degeneration of allografts in the first two years of life. Ann Thorac Surg 1995;60:S71-S77.
5. Smith J.D., Ogino H., Hunt D., Laylor R.M., Rose M.L., Yacoub M.H. Humoral immune response to human aortic valve homografts. Ann Thorac Surg 1995;60:S127-S130.
6. Hoekstra F.M.E., Witvliet M., Knoop C.Y., et al. Immunogenic human leukocyte antigen class II antigens on human cardiac valves induce specific alloantibodies. Ann Thorac Surg 1998;66:2022-2026.[Abstract/Free Full Text]
7. Hawkins J.A., Breinholt J.P., Lambert L.M., et al. Class I and class II anti-HLA antibodies after implantation of cryopreserved allograft material in pediatric patients. J Thorac Cardiovasc Surg 2000;119:324-330.[Abstract/Free Full Text]
8. Yankah A.C., Wottge H.-U., Muller-Ruchholtz W. Antigenicity and fate of cellular components of heart valve allografts. In: Yankah A.C., Hetzer R., Miller D.C., Ross D., Somerville J., Yacoub M.H., eds. Cardiac valve allografts 1962–1987. Darmstadt: Steinkopff Verlag, 1988:77-87.
9. O’Brien M.F., Goldstein S., Walsh S., Black K.S., Elkins R., Clarke D. The SynerGraft valve: a new acellular (nonglutaraldehyde-fixed) tissue heart valve for autologous recellularization first experimental studies before clinical implantation. Semin Thorac Cardiovasc Surg 1999;11:194-200.[Medline]
10. McNally RT, Heacox A, Brockbank KGM, Bank HL. US Patent No. 4,890,457. 1990.

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