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Ann Thorac Surg 1996;62:169-174
© 1996 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Calcification of Bovine Pericardium: Glutaraldehyde Versus No-React Biomodification

Departments of Cardiothoracic Surgery and Preventive Medicine and Community Health, New Jersey Medical School; and the Department of Applied Chemistry, New Jersey Institute of Technology, Newark, New Jersey

Accepted for publication March 7, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Calcific degeneration is the most frequent cause of clinical dysfunction of glutaraldehyde (GA)-pretreated bioprosthetic heart valves. The No-React (NR) process has been shown to be a promising anticalcification treatment. In this comparative study, our objective was to delineate the advantages of the NR treatment over GA.

Methods. Bovine pericardial strips pretreated with GA and NR were individually incubated in calcium phosphate solution for 21 days at 37°C. The pretreated bovine pericardium then was implanted subcutaneously in rats and retrieved at 14, 21, and 35 days after-implantation. Mineral and morphologic analyses were performed on each specimen.

Results. The NR-treated pericardium revealed significantly reduced in vitro calcification compared with the GA-treated tissue (mean tissue calcium content 1.3 ± 0.2 versus 5.9 ± 0.7 µg/mg; p < 0.001). Mineral analysis showed progressive calcification of the GA-pretreated pericardium over the period of implantation (calcium content increasing from 49.6 ± 9.6 µg/mg after 2 weeks to 134.3 ± 9.1 µg/mg at 5 weeks after-implantation). The NR-treated implants had calcified significantly less (p < 0.05) at each corresponding interval. Moreover, morphologic examinations demonstrated a protracted inflammatory response in the form of giant cell and mononuclear cell infiltration associated with intrinsic collagen disruption in the GA-treated tissue; the NR-treated pericardium maintained morphologic integrity with a mild inflammatory response.

Conclusions. The NR biochemical process appears not only to attenuate pericardial calcification, but also to abort the host's destructive inflammatory response to the xenograft.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Fabrication of a durable bioprosthetic cardiac valve from chemically treated xenograft tissues continues to be a challenging prospect. Despite the inherent advantages of biologic valve prostheses, such as pseudoanatomic central flow with superior hemodynamic indices and low thrombogenicity, negating the need for chronic anticoagulation therapy [1], clinical bioprosthetic valve implantation has been plagued by progressive tissue degeneration, resulting in valve dysfunction and ultimate replacement. Reoperation is an eventual outcome in approximately 20% to 30% of bioprosthetic valve recipients by the tenth postoperative year; most failures are attributed to primary tissue failure in the form of calcific degeneration [1].

The pathophysiology of calcific mineralization of bioprosthetic valves has been examined by numerous investigators over the past 2 decades, yet the exact mechanism and contributing factors have not been defined clearly. During the early 1980s, the potential role of the host's immune response in xenograft calcific degeneration was refuted by the description of equivalent chemical and morphologic characteristics in the calcification of porcine bioprosthetic aortic valve cusps in athymic mice [2]. In addition, Levy and associates [3] assigned negligible importance to the host's inflammatory response based on their millipore diffusion chamber subcutaneous implant model, simulating the intrinsic calcification that was observed in other experimental models despite the absence of attached host cells. Furthermore, although the in vitro pulse accelerator (fatigue tester) models have demonstrated stress-induced collagen deterioration and its potential role in accelerating the calcification process [4–6], the biopathologic mechanism of bioprosthetic mineralization cannot be explained solely on the basis of dynamic stress.

For the past several years, the pendulum of investigations in the area of bioprosthetic calcification has swung toward exploring the seemingly fundamental role of the xenograft biochemical preparation method in inducing tissue calcification. Glutaraldehyde (GA) is currently the standard reagent for preservation and biochemical fixation of fresh bioprosthetic leaflet materials of either bovine pericardium or porcine aortic valve cusp origin. It imparts intrinsic tissue stability (biodegradation resistance) and reduces the antigenicity of the material [7]. However, multiple recent reports have suggested a detrimental role of aldehyde-induced intra- and intermolecular collagen cross-linkages in initiating tissue mineralization [8–11]. In addition, GA has been implicated in devitalization of the intrinsic connective tissue cells of the bioprosthesis, thus resulting in breakdown of transmembrane calcium regulation and hence contributing to cell-associated calcific deposits [12].

A battery of alternative modification regimens have been proposed, with variable success. Among the most notable anticalcification reagents are 2-aminooleic acid, sodium dodecyl sulfate, and diphosphonates [13–15]. However, GA remains the final sterilization agent in all of these alternative treatment cocktails, with its inherent injurious effects on both cellular and collagenous components of the bioprosthetic tissue. Replacing GA with an alternative preservative would have profound clinical implications if the proposed biochemical modification process retained the advantages of aldehyde fixation while inhibiting or minimizing implant calcification.

In a series of pilot studies conducted previously in our laboratory, we have tested bovine pericardial and porcine aortic valve cusp tissue fragments treated with a novel anticalcification process known as No-React (NR), introduced by Biocor (Belo Horizonte, Brazil). These preliminary studies have shown great promise of a potentially viable alternative for chemical preparation of cardiac valve bioprostheses, with comparable stress tolerance and superior cytocompatibility [16]. Although the intricacies of the process have not been disclosed fully because of commercial incentives, the treatment involves the following: (1) aldehydes cross-linkage to achieve high resistance to biodegradation, (2) a detoxification process in solutions of natural endogenous substances with multiple physical variables, and (3) incubation with a surfactant that presumably supports in vivo nonreactivity. The objectives of the present study were to characterize the beneficial biologic properties of the NR treatment and to verify its merit as an anticalcification process. We also examined the role of local foreign body reaction and the host's inflammatory response in implant degeneration.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Static In Vitro Model
Static in vitro calcification testing was conducted as described previously by Bernacca and colleagues [17]. Bovine pericardial tissue strips of 1 x 5 cm, pretreated with either conventional GA or NR, were placed in individual vials containing a solution of 135 mmol/L NaCl, 2.2 mmol/L CaCl₂•2H₂O, and 1.2 mmol/L KH₂ PO₄ in 0.05 mmol/L 3-(N-morpholino) propane-sulfonic acid, buffered to pH 7.40. In a parallel control group, tissue samples were placed in a solution containing 135 mmol/L NaCl in 0.05 mmol/L 3-(N-morpholino) propane-sulfonic acid, with pH adjusted to 7.40. Solutions were filter sterilized under laminar-flow conditions. The vials then were kept in a 37°C incubator for 21 days; the calcifying solution was changed weekly under sterile conditions in half of the vials. The samples and the vials were inspected regularly for evidence of gross extrinsic calcific deposits or bacterial overgrowth. On completion of the incubation period, the tissue samples were retrieved from the vials, washed in several changes of sterile 0.9% NaCl, dried at 90°C overnight, and analyzed for calcium content by atomic absorption spectrophotometry.

Subcutaneous Implantation in Rats
Weanling Sprague-Dawley rats (SD Strain; Taconic Laboratories, German Town, NY) were used at age 4 weeks (60 to 80 g). The animals were injected intraperitoneally with two distinct anesthetic cocktails of equal efficacy; either pentobarbital (40 to 60 mg/kg) or ketamine/xylazine (ketamine 40 to 87 mg/kg; xylazine 5 to 13 mg/kg). Each animal received two strips of tissue, one each of GA- and NR-pretreated bovine pericardium, in separate subcutaneous pouches in the anterior abdominal wall. The wounds were closed with 5.0 Vicryl suture material. The rats were fed Lab Rodent Diet (Purina Meals Inc). They received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985). At 14, 21, and 35 days after-implantation, the animals were sacrificed with a lethal intraperitoneal dose of thiopental (300 mg/kg), and the pericardial specimens were retrieved. A small portion of each specimen was fixed immediately in 10% neutral buffered formalin for light microscopy examination. The remainder of each sample was used for mineral analysis.

Mineral Analyses
Pericardial tissue was washed in sterile saline and dried to constant weight in a 90°C desiccator oven. The tissue concentration of calcium was determined using previously described techniques [18] by flame atomic absorption spectrophotometry (Perkin-Elmer Model 603; Perkin-Elmer, Norwalk, CT) after digestion with a 3:1 mixture of double-distilled 70% nitric and perchloric acids (GFS Chemicals, Columbus, OH). National Institutes of Standards and Technology bovine liver (SRM 1577a, Gaithersburg, MD) was used as a quality control sample for all analyses. Concentrations were expressed as µg/mg dry tissue weight (mean ± standard error of the mean). Statistical significance was determined by a two-tailed independent t test.

Morphologic Analyses
Sample fragments removed for histologic evaluation were fixed immediately in 10% neutral buffered formalin, dehydrated in graded concentrations of ethanol, cleared in xylene, and embedded in paraffin according to standard methods. Sections 5 µm thick were stained with hematoxylin and eosin and Von-Kossa stain for demonstration of calcium deposits.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Mineral Analyses
STATIC IN VITRO TESTING.
Figure 1 summarizes the differential in vitro calcification of GA- versus NR-treated pericardial tissues with either weekly changes or no changes of the calcifying solution. With weekly changes of solution, the mean calcium content of GA-treated pericardium after 21 days of incubation (5.9 ± 0.7 µg/mg) was significantly higher than the calcium content of NR-treated pericardium (1.3 ± 0.2 µg/mg) (p < 0.001). With no changes of solution, the absolute calcium content was less in either tissue, but was still significantly higher in GA-treated pericardium (p < 0.001).

View larger version (34K): [in this window] [in a new window]   Fig 1. . In vitro calcification of bovine pericardium, pretreated with either conventional glutaraldehyde or No-React, after 21 days of incubation at 37°C in a physiologic calcium solution (mean tissue calcium content expressed in µg/mg ± standard error of the mean; n = 5). Tissue incubation in saline solution over the same time period was used as a control.

SUBCUTANEOUS IMPLANTATION.
Figure 2 summarizes the results for in vivo calcification of the GA- versus NR-treated bovine pericardium at 2, 3, and 5 weeks after-implantation. The mean calcium content of GA-treated pericardium after 2, 3, and 5 weeks of subcutaneous implantation was 49.6 ± 9.6, 82.5 ± 10.4, and 134.3 ± 9.1 µg/mg, respectively. Comparatively, the mean calcium content of the NR-treated pericardium was significantly lower (p < 0.05) at each corresponding interval (19.6 ± 6.0, 32.3 ± 12.2, and 21.4 ± 5.2 µg/mg, respectively). Furthermore, there was no evidence of a trend of progression of calcification in the NR-treated pericardium over the 5 weeks of observation.

View larger version (40K): [in this window] [in a new window]   Fig 2. . Comparison of trends of calcification of No-React- versus glutaraldehyde-pretreated bovine pericardium implanted subcutaneously in rats (mean tissue calcium content expressed in µg/mg ± standard error of the mean; n = 7).

View larger version (54K): [in this window] [in a new window]   Fig 3. . Light photomicrographs of No-React (NR)- and glutaraldehyde (GA)-pretreated bovine pericardial explants after 3 weeks. (A) NR-pretreated implant showing well-preserved collagen fascicular bundles with no foreign body reaction (hematoxylin and eosin). (B) NR-pretreated implant stained with the von Kossa technique; no calcific deposits are seen. (C) GA-preserved explant, illustrating intense inflammatory cellular infiltration (InF-R) of the collagen structure and giant cell formation (Gc) (hematoxylin and eosin). (D) van Kossa stain of GA-pretreated tissue demonstrating destructive calcium (CA++) deposits. (A, x100; B, x100; C, x200; D, x100.)

View larger version (54K): [in this window] [in a new window]   Fig 4. . Light photomicrographs of No-React (NR)- and glutaraldehyde (GA)-pretreated bovine pericardium harvested 5 weeks after subcutaneous implantation in rats. (A) NR-treated pericardium maintained intrinsic collagen architecture (hematoxylin and eosin). (B) GA-pretreated explant demonstrating persistent cellular permeation of the pericardial fibrosa accompanied by intrinsic disruption (FBR) (hematoxylin and eosin). (C) Higher magnification of slide in 4B illustrating multinuclear giant cells with associated destruction of collagen bundles (CoL-B) (hematoxylin and eosin). (D) GA-pretreated explant showing calcific deposits (CA++), detectable even with the hematoxylin and eosin technique. (InF-R = inflammatory cellular infiltration.) (A, x100; B, x100; C, x400; D, x100.)

View larger version (38K): [in this window] [in a new window]   Fig 5. . Light photomicrographs of 5-week No-React– and glutaraldehyde-pretreated subcutaneous implants stained with the von Kossa technique. (A) No-React–treated pericardial tissue showing a few small punctuated calcium crystals with remarkably preserved collagenous morphology. (B) Severe intrinsic disruption and surface ulcerations of glutaraldehyde-pretreated implant, with confluent sheets of calcific deposits (CA++) replacing normal architecture. (x100.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

In conclusion, the NR anticalcification treatment is a superior alternative to conventional GA treatment as a biologic modifier of xenograft tissue; it appears not only to arrest the degenerative mineralization of tissue implants, but also to abolish the host's inflammatory response to the xenograft. Long-term clinical in vivo circulatory trials are needed before recommending this modality as the preferred chemical modification treatment for cardiac valve bioprostheses.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We express our gratitude for the assistance of Mr Francis W. Kemp and Dr Shenggao Han with the atomic absorption spectroscopy analyses. We also thank Mr James A. Jetko for his superb technical assistance with histologic preparations.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Gabbay, Department of Cardiothoracic Surgery, UMDNJ-New Jersey Medical School, 185 South Orange Ave, Room G-502, Newark, NJ 07103.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

1. Schoen FJ, Kujovich JL, Levy RJ, Sutton MSJ. Bioprosthetic valve failure. Cardiovasc Clin 1987;18:289–97.
2. Levy RJ, Schoen FJ, Howard SL. Mechanism of calcification of porcine bioprosthetic aortic valve cusps: role of T lymphocytes. Am J Cardiol 1983;52:629–31.[Medline]
3. Levy RJ, Schoen FJ, Levy JT, Nelson AC, Howard SL, Oshry LJ. Biologic determinants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J Pathol 1983;113:143–54.[Abstract]
4. Gabbay S, Kresh J. Bioengineering of mechanical and biologic heart valve substitutes. In: Morse D, Steiner RM, Fernandez J, eds. Guide to prosthetic cardiac valves. New York: Springer-Verlag, 1985:239.
5. Gabbay S, Bortolotti U, Iosif M. Mechanical factors influencing the durability of heart valve pericardial bioprostheses. Trans Am Soc Artif Intern Organs 1985;32:282–7.
6. Thurbikar MJ, Deck JD, Aovad J, et al. Role of mechanical stress in calcification of aortic bioprosthetic valves. J Thorac Cardiovasc Surg 1983;86:115–25.[Abstract]
7. Carpentier A, Lemaigre G, Robert L, et al. Biological factors affecting long-term results of valvular heterografts. J Thorac Cardiovasc Surg 1969;58:467–83.
8. Angell WW, Angell JD, Kosek JC. Twelve-year experience with glutaraldehyde-preserved porcine xenografts. J Thorac Cardiovasc Surg 1982;83:493–502.[Abstract]
9. Schoen FJ, Collins JJ, Cohn LH. Long term failure rate and morphologic correlations in porcine bioprosthetic heart valve. Am J Cardiol 1983;51:957–64.[Medline]
10. Inamura E, Sawotani O, Koyanagi H, et al. Epoxy compounds as new cross linking agent for porcine aortic leaflets. Subcutaneous implant studies in rats. J Cardiovasc Surg 1989;4:50–7.
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12. Schoen FJ, Tsao JW, Levy RJ. Calcification of bovine pericardium used in cardiac valve bioprostheses: implications for the mechanism of bioprosthetic tissue mineralization. Am J Pathol 1986;123:134–45.[Abstract]
13. Chen W, Schoen FJ, Levy RJ. Mechanism of efficacy of 2-amino oleic acid for inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic heart valves. Circulation 1994;90:323–9.[Abstract/Free Full Text]
14. Levy RJ, Levy JT, Schoen FJ, et al. Prevention of bioprosthetic heart valve calcification. Circulation 1983;68(Suppl 3):395.
15. Levy RJ, Hawley MA, Schoen FJ, et al. Inhibition by diphosphonate compounds of calcification of porcine bioprosthetic heart valve cusps implanted subcutaneously in rats. Circulation 1985;71:349–56.[Abstract/Free Full Text]
16. Gabbay S, Chuback JA, Khavarian C, Donahoo J, Oyarzun JR, Scoma R. In vitro and animal evaluation with the Biocor No-React^TM anticalcification treatment for bioprostheses (new concept in anticalcification treatment). In: Gabbay S, Frater RW, eds. New horizons and the future of heart valve bioprostheses. Austin, Texas: Silent Partners, Inc, 1994:73–91.
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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Amir Abolhoda Shlomo Gabbay Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abolhoda, A. Articles by Gabbay, S. Search for Related Content PubMed PubMed Citation Articles by Abolhoda, A. Articles by Gabbay, S.