Ann Thorac Surg 2008;86:491-495. doi:10.1016/j.athoracsur.2008.03.061
© 2008 The Society of Thoracic Surgeons
Original Articles: Adult Cardiac
Mechanical Stress: An Independent Determinant of Early Bioprosthetic Calcification in Humans
Kenneth K. Liao, MDa,*,
Xiaohuan Li, MDa,
Ranjit John, MDa,
Devesh M. Amatya, PhDa,
Lyle D. Joyce, MD, PhDa,
Soon J. Park, MDb,
Richard Bianco, MSa,
R. Morton Bolman, III, MDc
a Division of Cardiothoracic Surgery, University of Minnesota, Minneapolis
b Division of Cardiac Surgery, Mayo Clinic, Rochester, Minnesota
c Division of Cardiac Surgery, Brigham and Women's Hospital, Boston, Massachusetts
Accepted for publication March 25, 2008.
* Address correspondence to Dr Liao, Division of Cardiothoracic Surgery, University of Minnesota, MMC 207, 420 Delaware St SE, Minneapolis, MN 55455 (Email: liaox014{at}umn.edu).
Presented at the Poster Session of the Forty-fourth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007.
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Abstract
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Background: Mechanical stress is one of the contributing factors for bioprosthetic calcification. A HeartMate XVE (Thoratec, CA) left ventricular assist device (LVAD) has two identical porcine valves, one for inflow and the other for outflow. The inflow valve endures a higher closing pressure than the outflow valve; thus, an implanted LVAD offers an ideal human model to study the independent effect of stress on calcification.
Methods: Sixty-four pairs of LVAD inflow and outflow valves underwent gross examination, histologic study, and x-ray imaging. X-ray films were converted to digital images, and the calcification area was calculated. The distribution of calcium deposits was documented. The frequency and degree of calcification in both valves were analyzed (paired t test). Calcification of both valves in relationship to the duration of LVAD implantation and to the patient's age was also analyzed (linear regression).
Results: The mean age of patients supported with LVAD was 55 ± 12 years (range, 17 to 77 years). The mean duration of LVAD implantation was 265 ± 151 days (range, 3 to 630 days). Calcification developed more commonly in inflow valves. The calcification area (CA) was larger in the inflow valves (21.6 ± 30.7 mm2) than in the outflow valves (15.1 ± 26.2 mm2, p < 0.05). There was a positive relationship between CA and days of implantation for both valves (inflow CA = 4.96 ± 0.063 days; outflow, CA = 2.39 ± 0.047 days, linear regression; p < 0.05 for both).
Conclusions: Mechanical stress is an independent determinant of early bioprosthetic calcification in humans.
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Introduction
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More than 250,000 artificial heart valves are implanted worldwide each year, and nearly half are bioprostheses [1, 2]. Unfortunately, a significant number of bioprostheses cannot achieve long-term durability after implantation. The most common mode of failure is structural deterioration caused by progressive calcification.
Bioprosthetic calcification is a multifactorial process. Mechanical stress, chemical factors, host age, hormonal status, and duration of implantation all contribute to the calcification. In human studies, separating the role of stress from other biologic factors in the calcification process is difficult. Several such studies tried to investigate the relationship between mechanical stress and calcification, but key questions about that relationship remain unanswered [3, 4].
In our current study, we focused on patients who underwent implantation of a HeartMate XVE (Thoratec, CA) left ventricular assist device (LVAD). The LVAD is a volume-displacement pump that generates a pulse pressure that mimics the human heart. It has 2 identical 25-mm Hancock I porcine valves (Medtronic Inc, Minneapolis, MN). One of the valves is used as the inflow valve (ventricular valve), which is analogous in function to the mitral valve. The other is used as the outflow valve (aortic valve), which is analogous in function to the aortic valve. The ventricular valve encounters greater mechanical stress because of higher closing pressure (> 275 mm Hg) compared with the aortic valve. An LVAD pump generates a much higher first derivative of left ventricular pressure over time (dP/dt) during each cardiac cycle compared with a human heart. Other host facts being the same, an implanted LVAD offers an ideal human model to study the independent effect of mechanical stress on bioprosthetic calcification. We hypothesized that the mechanical stress is an independent determinant of bioprosthetic calcification.
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Material and Methods
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This study was approved by the University of Minnesota Institutional Review Board. The need for patient consent was waived because the patients in the study were not identified.
We removed and examined 64 pairs of inflow and outflow valves from explanted LVADs after the patients underwent either a heart transplant or LVAD exchange. We documented the calcification by gross examination, histologic study, and x-ray photography.
Gross examination of the valves was recorded by a Cybershot 4.1 Mega Pixel digital camera (Sony Corp, Tokyo, Japan). Before removing the valves from their metal housing, we took photographs of both sides of the valves. After the valves were completely isolated, we took another set of photographs.
We performed histologic study on the valve leaflets to examine the calcification. Sections of the leaflets were cut from the central part of the biggest leaflet of each valve, perpendicular to the base. The specimens were fixed in 10% formalin, dehydrated, cleared, and infiltrated with paraffin using an automated tissue processor (Tissue-Tek 3000, Miles Scientific, Naperville, IL). Tissues were embedded in paraffin and sectioned at 5 µm. The slides were stained with Harris Hematoxylin (Newcomer Supply, Middleton WI) and counterstained with Alcoholic Eosin (Newcomer Supply). Finally, the slides were coverslipped using a synthetic mounting media. The slides were studied under the microscope.
We placed each pair of valves in the Faxitron X-ray machine (Field Emission Corp, McMinnville, OR) in order to detect various degrees of calcification. A Polaroid film placed beneath the valves was exposed to 40 kV of x-rays for 2.5 minutes. The film was processed using sodium sulfite solution (220 g/L) and then distilled water. We scanned the x-ray photographs and converted them to digital images using an HP LaserJet 3330 (Hewlett-Packard Co, Palo Alto, CA). To manually trace and calculate the area of calcification, we used Image J software (National Institutes of Health, Bethesda, MD). The calcification appeared as dark spots in the films compared with the gray color of normal tissue.
We documented the distribution of calcium in the valves. The paired t test was used to compare the frequency and degree of calcification between the inflow and outflow valves. A value of p 
0.05 was statistically significant. To analyze calcification of both valves in relationship to the duration of LVAD implantation and to the patient's age, we used a linear regression. A value of p 0.05 was statistically significant.
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Results
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Patient Characteristics
The 64 patients (51 men, 13 women) who underwent LVAD implantation had a mean age of 55 ± 12 years (range, 17 to 77 years). The mean duration of LVAD implantation was 265 ± 151 days (range, 3 to 630 days). Patient characteristics, including diagnosis, outcome, and serum creatinine level, are reported in Table 1. The average serum creatinine level was not abnormally high in these patients because chronic renal failure was considered one of the contraindications for LVAD implantation.
Gross Inspection of Calcification
At gross examination, we noted that 22 inflow valves and 1 outflow valve showed clear signs of gross calcification (Fig 1). The remaining valves showed no gross calcification. We also observed other changes, such as valve conduit graft displacement and valve cusp migration or prolapse, which were more common in inflow valves than in outflow valves (Fig 2). Obvious tears or perforations were found in 9 inflow valves and 2 outflow valves. Endocarditis had developed in 2 inflow valves and 1 outflow valve. The severity and area of gross calcification were verified by the histologic examination.
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Fig 2. The black arrow points to gross calcification at the inflow valve commissure. The white arrow points to graft displacement of the valve housing unit.
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Incidence of Calcification and Distribution
In analyzing our x-ray photos of 128 valves, we noted various degrees of calcification in 45 (70%) of the 64 inflow valves and in 37 (58%) of the 64 outflow valves (Fig 3). The commissure and the base of the leaflets had more calcium deposits: 28 inflow and 28 outflow valves had calcium deposits at the commissure and bases simultaneously, 14 inflow and 8 outflow valves had calcium deposits at the commissure only, and 3 inflow valves and 1 outflow valve had calcium deposits at the bases only (Fig 4).
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Fig 3. The incidence of calcification (black bars) and absence of calcification (white bars) in the inflow and outflow valves was significantly different, with a calcification rate of 70% for the inflow values and 58% for the outflow valves.
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Fig 4. An analysis of the distribution of calcification in inflow (white bars) and outflow valves (black bars) showed that commissure and the base of valve leaflet were the most common sites of calcium deposit.
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Degree of Calcification
The degree of calcification was reflected by the calcification area. The calcification area was larger in the inflow valves than in the outflow valves (inflow calcification: 21.6 ± 30.7 mm2 vs outflow calcification: 15.1 ± 26.2 mm2, p < 0.05; Fig 5,
Fig 6).
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Fig 5. Degree of calcification area of the inflow valves (black bar) is significantly larger than that of the outflow valves (white bar). Inflow valve calcification: 21.6 ± 30.7 mm2 vs outflow valve calcification: 15.1 ± 26.2 mm2 (p < 0.05, paired t test).
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Fig 6. X-ray photograph of the inflow (A) and outflow (B) valves from the same left ventricular assist device shows significant calcification in the inflow valve (black arrow) vs no calcification in the outflow valve.
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Relationship of Calcification to the Duration of LVAD Implantation and Patient Age
The LVAD implantation duration in our patients ranged from 3 to 630 days. We found for both type of valves a positive relationship between a larger calcification area and a higher number of days of implantation (inflow calcification, 4.96 ± 0.063 days; outflow calcification, 2.39 ± 0.047 days; linear regression, p < 0.05 for both; Fig 7A,
7B). The two out of range calcification areas in Figure 7 were caused by calcified vegetations in the valves with endocarditis, which were verified by microscopic study.
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Fig 7. (A) Relationship of inflow valve calcification area to the duration of left ventricular assist device implantation. Inflow valve calcification (Y) = 4.96 ± 0.063 days (X); R2 = 0.096, p < 0.05, linear regression. (B) Relationship of outflow calcification area to the duration of left ventricular assist device implantation. Outflow valve calcification (Y) = 2.39 ± 0.047 days (X); R2 = 0.077, p < 0.05, linear regression.
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Our patients were 17 to 77 years old. We found no relationship between age and size of the calcification area for either the inflow valves or outflow valves.
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Comment
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Mechanical Stress as an Independent Factor in Bioprosthetic Calcification
Only a few studies in the literature have investigated stress-related factors in bioprosthetic degeneration by comparing bioprostheses at different anatomic locations [3, 4]. Pansini and colleagues [3] compared 25 pairs of porcine bioprostheses in the mitral and aortic position in patients and found that calcification occurred more extensively in the mitral position. Leprince and colleagues [4] reported more extensive degeneration in the mitral position compared with the tricuspid position in 7 patients and suggested that the high stress encountered in the mitral position contributed to more severe calcification, which was the major cause of bioprosthetic deterioration.
Those two studies were limited, however, by small patient samples. Furthermore, they only used morphologic analysis for the late stage of valve calcification, and their findings did not reflect the early or intermediate stages of calcification. Another drawback of both studies is that little is mentioned about the early calcification progress of bioprostheses in human patients.
In our study, we compared 64 pairs of bioprostheses taken from LVAD patients—a much larger patient sample size. The average duration of implantation was less than a year. Under the same human biologic conditions, the inflow valves clearly developed more calcification than the outflow valves. The inflow valves were exposed to a much higher mechanical pressure than the outflow valves. We attribute the calcification difference to the greater closing pressure and the greater pressure gradient experienced by the inflow valves compared with the outflow valves. A similar observation was made by Pool and colleagues [5] in an animal model.
The calcification of our patients' LVAD bioprostheses tended to develop much earlier compared with the bioprostheses used in valve replacement, in which calcification typically takes years to develop. The mean valve implantation duration was 95 ± 26 months in the Pansini and colleagues' study [3] and 7.5 ± 3.3 years in the Leprince and colleagues' [4] study. In our study, we noted bioprosthetic calcification as early as 66 days after LVAD implantation (Fig 8). In the inflow valve position, the LVAD pump is known to generate a high systolic pressure of between 275 450 mm Hg in every cardiac cycle. Such high pressure can accelerate the process of calcification. Unlike the Pansini [3] and Leprince and colleagues' studies [4], which only observed the late change of gross calcification, we were able to observe the early process of bioprosthetic calcification—clearly an advantage over other studies.
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Fig 8. X-ray photograph shows calcification area (arrows) in the (A) inflow and (B) outflow valves 66 days after implantation. The calcification area is significantly larger in the inflow valve than in the outflow valve.
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Patterns of Calcification Distribution
We found calcium deposits were more common in the commissure than in the base of the leaflets (Fig 4). This observation is similar to other studies, such as one by Bernacca and colleagues [6] of bovine bioprostheses that also reported the highest concentrations of calcium in the commissure. They believed that the commissure was subjected to high tensile stress concentrated at the retaining stitches, particularly when the valve closed under high back pressure. The high stresses in the leaflets caused them to degenerate, resulting in calcification of the leaflets. In a study of bovine pericardium bioprostheses, Black and colleagues [7] reported high stress areas at the commissure near the stent post. Chandran and colleagues [8] reported a high stress area at the commissure for a polyurethane valve. Krucinski and colleagues [9] described a similar observation for a pericardial valve.
In our study we also noted that calcification tended to develop in larger leaflets that were stretched or elongated. Such stretching or elongation was frequent in LVAD inflow valves when the Dacron grafts (DuPont, Wilmington, DE) were displaced (Fig 2). According to the Laplace's law, larger leaflets encounter more stress than smaller ones.
Methods of Analyzing Calcification Area
Several different methods have been used to analyze the calcification of bioprostheses, including x-ray photography [10, 11], holographic interferometry [12, 13], and atomic absorption spectrophotometry [8, 14]. These calcium measurement methods fail to show the area involved by calcification. We used x-ray photography and histologic study to detect calcification. To measure the calcification area of the leaflets, we used a new technique—Image J software—with which we can calculate the size of the calcification area and also measure the distances and angles. Such precise quantitative measurement seems to be more accurate than the other previously used methods.
Duration of Implantation and Degree of Calcification
In our study as in others, a longer duration of implantation was positively related to a larger calcification area for both inflow and outflow valves. However, we did not notice any relationship between patient age and the size of the calcification area; this discrepancy can be explained by the more important role of mechanical stress during a relatively short period of implantation time compared with a biologic factor like age.
Summary
Our results support other previous observations that mechanical stress plays an important role in bioprosthetic calcification. More important, we demonstrated that mechanical stress is an independent determinant of early bioprosthetic calcification, probably even more critical than biologic factors such as patient age.
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References
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Abstract
Introduction
