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Eur J Cardiothorac Surg 2008;34:418-422. doi:10.1016/j.ejcts.2008.05.012
Copyright © 2008, European Association for Cardio-thoracic Surgery. Published by Elsevier. All rights reserved.
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Octanediol treatment of glutaraldehyde fixed bovine pericardium: evidence of anticalcification efficacy in the subcutaneous rat model

Elena Pettenazzo, Marialuisa Valente, Gaetano Thiene*

Department of Medical Diagnostic Sciences, University of Padua Medical School, Via A. Gabelli, 61, 35121 Padua, Italy

Received 2 November 2007; received in revised form 5 May 2008; accepted 6 May 2008.

* Corresponding author. Tel.: +39 049 8272283; fax: +39 049 8272284. (Email: gaetano.thiene{at}unipd.it).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Anticalcification strategies of glutaraldehyde-fixed xenograft tissue aim to extract lipids or to neutralize toxic aldehyde residuals. The purpose of this study was to evaluate the efficacy of octanediol compared to standard treatments of glutaraldehyde-fixed bovine pericardium in the subdermal rat model. Octanediol treatment is an ethanolic solution (40%) containing a long chain aliphatic alcohol (5% 1,2-octanediol) that removes lipids without diminishing the stability of collagen. Methods: Octanediol and standard glutaraldehyde fixed bovine pericardium were both implanted in 24 Sprague-Dawley rats, explanted after 30–75 days (12 animals each) and submitted to X-ray (score 0–4), histology, electron microscopy and elemental analysis by spectroscopy (Ca and P content). Unimplanted octanediol and standard glutaraldehyde fixed pericardium served as control. Results: At 30 days octanediol-treated pericardium showed calcium content of 0.20 ± 0.1 vs 20.07 ± 36.79 mg/g dry weight for standard pericardium. The difference was also evident at 75 days: calcium content of 2.36 ± 7.38 mg/g dry weight for octanediol vs 165.61 ± 23.35 mg/g dry weight for standard (p < 0.0001). Differences were also detected at X-ray (mean score 0.7 ± 0.6 octanediol vs 3.8 ± 0.4 standard at 75 days). Equally, mean P content was 11.69 ± 21.33 mg/g dry weight for standard vs 0.60 ± 1.45 mg/g dry weight for octanediol samples at 30 days, and 90.90 ± 12.61 mg/g dry weight for standard vs 1.42 ± 4.34 mg/g dry weight for octanediol at 75 days (p < 0.0001). At electron microscopy collagen appeared well preserved regardless of the type of treatment; in octanediol treated pericardium cell membranes almost disappeared and only few profiles of endoplasmic reticulum and rare mitochondria were visible. Conclusions: Treatment with octanediol strongly prevents calcification of glutaraldehyde fixed bovine pericardium in rat subdermal model, even in the long-term. Evidence of octanediol efficacy may entail important implications for new generation bioprosthetic valves.

Key Words: Bovine pericardium • Rat subcutaneous model • Anticalcification treatment


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The primary problem of bioprostheses, whether porcine or pericardial, is structural valve deterioration at long-term, mainly due to dystrophic calcification [1,2]. However, many improvements have been implemented in order to prolong long-term durability [3,4].

Cross-linkage of xenograft tissue with glutaraldehyde (GA) was revealed to be successful in suppressing host immunological reactivity and to obtain stabilization of collagen. However, GA fixation promotes dystrophic calcification as a consequence of xenograft cells devitalization and a toxic effect of unstable cross-linking [5–7]. The mechanism of mineralization of GA-fixed bioprostheses consists of attraction and precipitation of calcium upon lipid-based cell debris, which are full of phospholipids [8].

Considerable efforts over many years through basic research have been directed toward developing a tissue treatment process to prevent calcification in GA-fixed xenograft tissue. The main anticalcification strategies aim to extract lipids [9,10] or to neutralize toxic aldehyde residuals [11–13].

The aim of this study was to evaluate the efficacy of octanediol (ON) anticalcification treatment of GA-fixed bovine pericardium (BP), compared to standard (ST) GA-fixed BP, in the subcutaneous rat model. ON treatment is an ethanolic solution containing a long chain aliphatic alcohol (5% 1,2-octanediol), which removes lipids without diminishing the stability of GA-fixed collagen. The major purpose of the investigation was to test a new post-fixation treatment effective enough to justify subsequent investigation in the large animal circulatory implant.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Tissue preparation and ON treatment
BP, obtained from the abattoir, was pre-fixed with 0.2% GA and then assembled into 1 cm squares. Each sample was fixed by immersion in 0.5% GA. Post-fixation treatment was accomplished in a sterile hood with a solution of 5% 1,2-octanediol in 40% ethanol for 68 h at 37 °C.

2.2 Subcutaneous implants
BP square samples (1 cm x 1 cm), both ST and ON treated, were implanted subcutaneously in 24 Sprague-Dawley rats (3 weeks old) and retrieved after 30 or 75 days (12 animals each). Handling of the animals complied with care of animal according to Italian and European regulations. The explants were fixed in formalin.

2.3 Morphological evaluation
Each BP samples were submitted to mammography X-ray and the presence of calcium deposits was quantified on the basis of a 0–4 score as follows (1): 0 = absent; 1 = focal, pinpoint, <1 mm of diameter; 2 = focal, >1 mm of diameter or pinpoint multiple; 3 = multiple >1 mm of diameter; 4 = massive deposition. Each specimen was cut in half: one half was used for microscopic examinations, whereas the remaining half was used for elemental analysis (calcium and phosphorous quantification) as described below.

2.4 Microscopic examinations
2.4.1 Histology
The samples, dehydrated in crescent ethanol series, were paraffin embedded and 5–7 µm thick sections stained with hematoxylin-eosin, Weigert Van Gieson, Heidenhain trichrome and Von Kossa.

The following parameters were evaluated:

(a) Presence, amount and type of mineralization (nodular and laminar, whether calcium deposits occurred on cells, collagen fibers or both) on Von Kossa stained sections.
(b) Presence, amount and type of inflammatory reaction (whether granulomatous, lymphocytic or neutrophilic) on hematoxylin-eosin stained sections.

A semiquantitative method was applied on histologic sections to evaluate the amount of mineralization and inflammatory infiltrate: (0) absent; (1) minimal; (2) mild; (3) moderate; and (4) severe.

2.4.2 Electron microscopy
Small pieces (1 mm), complementary to histology samples, were harvested from each square. Post-fixation was accomplished in 1% osmium tetroxide in buffered sodium phosphate 0.1 M. Samples were then dehydrated in crescent ethanol series and embedded in epoxy resin. Semithin sections were stained with toluidine blue and observed at the light microscope. Ultrathin sections of selected fields were stained with uranyl acetate in 50% ethanol and Reynolds lead citrate and examined with a Hitachi H7000 microscope.

2.5 Elemental analysis by spectroscopy
For assessment of calcium content by atomic absorption spectroscopy, samples were dried to a constant weight in a desiccator oven and hydrolyzed with HNO3 (0.75 mol/l) at 68 °C for 15 h. After centrifugation at 2500 x g, the fluid was removed, diluted, and the calcium content was determined with the atomic absorption spectrometer UNICAM Solaar 989. A calibration curve was plotted using a set of crescent concentrations of a calcium standard solution and distilled water blank. Calcium content, expressed as mg/g tissue dry weight, represents the mean of at least three different spectrometric determinations.

Phosphorous (P) content (expressed as mg/g tissue dry weight) was assessed by inductively coupled plasma (ICP) analysis.

We evaluated if there was a significant difference between Ca and P content in ST and ON treatment with Tukey–Kramer test for multiple comparison.

2.6 Unimplanted pericardium
Two unimplanted bovine pericardium samples, ST and ON treated, served as controls and were submitted to the same study protocol.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Unimplanted pericardium
At electron microscopy, both ST and ON treated BP showed optimal preservation of collagen fibers that had multidirectional orientation and regular periodicity. Rare elastic fibers were scattered within the interstitium and appeared intact.

Different to ST treated BP, where the cells appeared almost intact (Fig. 1A), ON treated BP cellular ghosts were observed with empty cytoplasm, fragmented cells and nuclear membranes as well as cytoplasmic debris (Fig. 1B).


Figure 1
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  		Fig. 1. Electron microscopy of unimplanted glutaraldehyde treated bovine pericardium (BP) samples. (A) ST-treated BP with integrity of cell component. Original magnification, x5000. (B) ON-treated BP shows cellular ghost with fragmentation of cellular membrane and cytoplasmic debris (arrows). Original magnification, x3000.

 
3.2 Post-implant features
3.2.1 30 Days explants: X-ray and histology
One ON treated sample showed focal neutrophilic inflammatory infiltrate, necrosis and calcification in keeping with pocket infection and thus was excluded from analysis and data elaboration, since mineralization was considered secondary to infection.

At 30 days a sharp difference was noted in terms of mineralization between ST and ON treated BP. Mean score at X-ray were 3.6 ± 1.2 for ST vs 0.3 ± 0.6 for ON samples. Semiquantitative histologic analysis of calcium content showed similar findings: mean score 3.2 ± 1.1 for ST samples vs no calcification of ON samples.

Mineralization of ST samples was laminar and involved both cells and collagen fibers. Mild to moderate granulomatous inflammatory reaction was found in both ST and ON samples with an equal mean score (2.2 ± 0.9 vs 2.6 ± 0.5, respectively).

3.2.2 75 Days explants: X-ray and histology
The difference in calcification noted in the mid-term was confirmed at 75 days (Fig. 2 ). At X-ray, ON-treated BP showed negligible mineralization with a mean score of 0.7 ± 0.6, whereas calcification of ST samples was massive (mean score 3.8 ± 0.4). At histology mean calcium score was 3.6 ± 0.7 for ST vs 0.3 ± 1.1 for ON samples.


Figure 2
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  		Fig. 2. X-ray and histology of a 75-days explant. (A) X-ray of ST-treated BP with score 4 of calcification. (B) Severe histological calcification extended to full thickness of ST-treated BP sample. Von Kossa stain, original magnification, x3. (C) X-ray of ON-treated BP with score 0 of calcification. (D) Absent calcification in the histology of ON-treated BP sample. Von Kossa stain, original magnification, x3.

 
Mineralization was laminar and involved both cells and collagen fibers.

Mild inflammatory infiltrate was still present with no difference between ON and ST treatment (mean score 2 ± 0.6 vs 2.1 ± 0.9, respectively).

3.3 Electron microscopy findings
At both 30 and 75 days collagen appeared intact and well preserved in all the cases, regardless of the treatment (Fig. 3A and B). Mineralization of ST samples involved both cells and collagen (Fig. 3C).


Figure 3
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  		Fig. 3. Electron microscopy of ST and ON-treated samples. Collagen fibers appear well preserved with a normally banded structure either in ST-treated (A) or in ON-treated (B) samples. Original magnification, x 20,000 (A); x30,000 (B). (C) Calcification involves collagen and elastic fibers (arrows) in a ST-treated BP explanted after 30 days. Calcospherulae (head arrows), resulting from calcium deposition upon cells, are also present. Original magnification, x4000. (D) ON-treated samples appeared mostly decellularized with no calcification. Mitochondria (arrows) and debris of endoplasmic reticulum (head arrow) are visible. Original magnification, x6000.

 
In ON treated pericardium cell membranes almost disappeared and only few small profiles of cell membranes, endoplasmic reticulum and rare mitochondria were visible (Fig. 3D).

3.4 Elemental analysis
Calcium content was quantified in unimplanted glutaraldehyde fixed BP with a value of 0.22 ± 0.01 mg/g dry weight and 0.20 ± 0.02 mg/g dry weight for ON and ST samples, respectively (Fig. 4A).


Figure 4
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  		Fig. 4. Histogram of calcium and phosphorous content expressed as mg/g tissue dry weight. (A) Mean calcium content was: 20.07 ± 36.79 mg/g for ST vs 0.20 ± 0.1 mg/g for ON samples at 30 days; 165.61 ± 23.35 mg/g for ST vs 2.36 ± 7.38 mg/g for ON samples (p < 0.0001) at 75 days. (B) Mean phosphorous content was: 11.69 ± 21.33 mg/g for ST vs 0.60 ± 1.45 mg/g for ON samples at 30 days; 90.90 ± 12.61 mg/g for ST vs 1.42 ± 4.34 mg/g for ON samples (p < 0.0001) at 75 days.

 
At 30 days mean calcium content was 0.20 ± 0.1 mg/g dry weight for ON vs 20.07 ± 36.79 mg/g dry weight for ST samples (Fig. 4A).

At 75 days the difference in calcium content between the two groups was also clear-cut (Fig. 4A): 2.36 ± 7.38 mg/g dry weight for ON vs 165.61 ± 23.35 mg/g dry weight for ST (p < 0.0001).

Mean P content was 11.69 ± 21.33 mg/g dry weight for ST vs 0.60 ± 1.45 mg/g dry weight for ON samples at 30 days, and 90.90 ± 12.61 mg/g dry weight for ST vs 1.42 ± 4.34 mg/g dry weight for ON at 75 days (p < 0.0001) (Fig. 4B).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Prosthetic valve replacement still represents the treatment of choice for severe cardiac valve disease and GA-fixed porcine aortic or bovine pericardial valves, either stented or unstented, are widely used. Despite numerous improvements that have been accomplished in terms of design and tissue pretreatment, the long-term durability of bioprostheses is still not optimal. Structural valve deterioration, mainly due to dystrophic calcification, represents the major cause of failure within 10 years post-implantation [1,2].

Xenograft tissue structure and its modifications following fixation play an important role in inducing dystrophic calcification. GA fixation devitalizes but does not remove connective tissue cells that are prone to be the initial site of calcium deposition. In fact, viable cells have an approximately 10,000-fold gradient of calcium from outside to inside (10–3 M vs 10–7 M) and the low intracellular calcium concentrations is maintained by energy-dependent pumps at the intact cell membranes. In cells rendered non-viable by GA fixation, calcium pumps are impaired and calcium passively diffuses into the cytoplasm and reacts with phospholipids of cells and organelles membranes [8,14,15]. Calcification of bioprostheses at first occurs upon cell membranes and then involves collagen fibers and interfibrillar spaces [3,8,16].

Prevention of bioprosthetic dystrophic calcification has been directed into two different pathways: removal of phospholipids from the xenograft tissue [9,10] or neutralization of the toxic residual aldehyde groups [11–13].

In this study, we tested the efficacy of the ON anticalcification treatment, a long chain aliphatic alcohol, on GA-fixed BP in a subcutaneous rat model.

Previous reports have been published about the use of substances that extract lipids as anticalcification strategies. In 1994 Jorge-Herrero et al. published a study about the effect of the extraction of lipids from the porcine valve tissue using chloroform–methanol in a subcutaneous rat model: glutaraldehyde treated aortic and pulmonary porcine valves, post-treated with chloroform-methanol, calcify much less than those treated only with glutaraldehyde [17]. More recently Vyavahare et al. demonstrated that ethanol post-fixation treatment effectively inhibits calcification of glutaraldehyde-pretreated porcine aortic leaflets, without affecting tissue morphology and biocompatibility, in both subdermal rat and large animals circulatory implants [10,18].

Surfactants, such as polysorbate 80, Triton X-100, and N-lauryl sarcosine, have been investigated as anticalcification substances. Jones et al, in a study published in 1988 [19], found that surfactants substantially reduce calcification in bioprostheses implanted in intracardiac position, but Triton X-100 and N-lauryl sarcosine also induce tissue alteration that decrease the durability of the valves. Sodium dodecyl sulphate, a detergent, has been employed as anticalcification treatment with satisfactory results, both in the growing sheep model [20] and in humans [21,22]. Recently, a multicenter study about the clinical experience with the Hancock II valve (a porcine bioprosthesis treated with sodium dodecyl sulphate) showed that optimal 15-year durability can be expected for aortic valve replacement in patients 60 years and older [23]. This is an important demonstration that the application of an anticalcification technology not only prolongs long-term durability, but also extends the age limits for the use of bioprosthetic valve xenografts.

We chose the alcohol ON because is a long chain aliphatic alcohol structurally similar to phospholipids, with an hydrophobic tail allowing solubility in lipids and an hydrophilic head providing solubility in water. We used a buffered ethanolic solution in order to improve ON solubility in water and its penetration in the tissue. ON should act both removing lipids from the tissue and neutralizing toxic GA residuals.

In our experiments, a significant difference in terms of mineralization was observed at 30 days, with 0.20 ± 0.1 mg/g dry weight in ON samples vs 20.07 ± 36.79 mg/g dry weight in ST treated BP. A sharp difference was also evident at 75 days: mean calcium content of 2.36 ± 7.38 mg/g dry weight for ON and 165.61 ± 23.35 mg/g dry weight for ST treated BP.

Histological score of calcification confirmed this clear-cut difference (mean score 0.3 ± 1.1 ON vs 3.6 ± 0.7 ST at 75 days).

The pioneer work of Vyavahare et al., about the use of 80% ethanol as anticalcification treatment of GA-fixed porcine aortic valves, reported a mean calcium content of 1.87 ± 12.0 µg/mg vs 236 ± 6.1 µg/mg in controls after 60 days rat subcutaneous implantation [10].

In 2001 Cunanan et al. [24], published a quite interesting investigation of a subcutaneous rat model in which they compared the calcium levels of eight different commercial types of GA-fixed bioprosthetic valves, both porcine and bovine, with or without anticalcification treatment. After 90 days of subcutaneous implantation both untreated porcine (St Jude Medical Toronto SPV) and bovine (Mitroflow Model 12) showed a remarkable calcification (244.43 ± 55.69 µg/mg and 214.60 ± 11.44 µg/mg, respectively). As far as those treated with anticalcification, Carpentier-Edwards Duraflex and Supra annular valve, both porcine and treated with XenoLogiX, containing ethanol and surfactant Tween-80, showed a negligible mineralization (2.13 ± 5.99 µg/mg vs 0.76 ± 0.91 µg/mg, respectively), and Carpentier-Edwards Perimount, consisting of bovine pericardium treated with XenoLogiX, had a mean calcium content of 3.3 ± 8.4 µg/mg. The Medtronic porcine valves, with anticalcification treatment consisting of sodium dodecyl sulphate (Hancock II) and amino-oleic acid (Mosaic and Freestyle) presented a mean calcium content of 8.24 ± 28.75 µg/mg, 25.37 ± 57.68 µg/mg and 9.54 ± 36.07 µg/mg, respectively.

Our experiment with ON-treated bovine pericardium in the subcutaneous rat model, showed similar findings as Perimount (2.36 ± 7.38 µg/mg) at 75 days.

It is noticeable that in a previous study, Pathak et al. [25] demonstrated that treatment with 5% ON in 40% ethanol solution sharply reduce phospholipids content in unimplanted BP tissue compared to fresh and GA-fixed BP tissue (0.21 ± 0.05 µg/mg vs 6.7 ± 3.0 µg/mg vs 3.9 ± 0.48 µg/mg). In contrast, at the same ethanol concentration (40%) without ON, the investigation of Vyavahare et al. [10] showed only a negligible decrease of phospholipids content in ethanol treated, unimplanted porcine tissue when compared to GA-fixed porcine tissue (16.5 ± 1.5 nmol/mg dry weight vs 17.2 ± 0.8 nmol/mg dry weight). However, it should be emphasized that in the Vyavahare experiment the incubation time and the temperature were much less (25 h and 25 °C, respectively).

In our experiment, quantification of phosphorous revealed a significant difference between ST and ON-treated samples, with a mean content of 90.90 ± 12.61 mg/g dry weight vs 1.42 ± 4.34 mg/g dry weight, respectively at 75 days (p < 0.0001).

The demonstration of a sharp decrease of P content as well as of cell remnants removal at electron microscopy, following octanediol treatment in 40% ethanol, supports the notion of a prevention of calcification through the mechanism of phospholipid extraction.

A limitation of our study is a lack of investigation on mechanical properties like elasticity and strength as well as resistance to enzymatic and cellular digestion of the ON-treated BP compared to untreated. However, at electron microscopy, elastic and collagen fibers were intact with perfect periodicity of collagen fibrils in both types of treatments.

In conclusion, treatment with ON strongly prevents BP calcification in rat subdermal model even in the long-term. Evidence of ON efficacy may entail important implications in the development of new generation bioprosthetic valves. In this regard, circulatory implant of pericardial valve bioprostheses, both ON and ST-treated, in mitral position of juvenile sheep is mandatory.


   Footnotes
 
{star} This research was supported by an unrestrictive grant of SORIN GROUP, Saluggia (VC), by MIUR, Rome and by the University of Padua, Italy.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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