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Ann Thorac Surg 2006;81:918-926
© 2006 The Society of Thoracic Surgeons

Original article: Cardiovascular

Impact of Cryopreservation on Extracellular Matrix Structures of Heart Valve Leaflets

Katja Schenke-Layland, PhD a , b , * , Navid Madershahian, MD a , Iris Riemann, PhD c , Barry Starcher, PhD d , Karl-Jürgen Halbhuber, MD e , Karsten König, PhD c , Ulrich A. Stock, MD a , f

b Cardiovascular Research Laboratory, University of California Los Angeles (UCLA), Los Angeles, California
a Department of Cardiothoracic and Vascular Surgery, Friedrich-Schiller-University, Jena, Germany
c Fraunhofer Institute of Biomedical Technology, St. Ingbert, Germany
d University of Texas Health Center at Tyler, Tyler, Texas
e Institute of Anatomy II, Friedrich-Schiller-University, Jena, Germany
f Department of Medical Physics and Biophysics, Humboldt University, University Hospital Charité, and Department of Cardiac Surgery, Heart Center Brandenburg, Bernau, Berlin, Germany

Accepted for publication September 9, 2005.

* Address correspondence to Dr Schenke-Layland, University of California Los Angeles, Cardiovascular Research Laboratory, 675 Charles E. Young Dr S, MRL 3-579, Los Angeles, CA 90095-1760 (Email: kschenkelayland{at}mednet.ucla.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Transplantation of cryopreserved allografts represents a well-established valve replacement option. Despite their clinical use for more than 40 years, the integrity of the extracellular matrix (ECM) of these valves after thawing has not been determined. The purpose of this study was to investigate and compare ECM structures of fresh and cryopreserved porcine heart valve leaflets with special emphasis on the condition of collagenous and elastic fibers.

METHODS: Pulmonary valves were excised from unprocessed porcine hearts under sterile conditions. After treatment with antibiotics, the valves were incubated in a cryoprotective solution, cryopreserved stepwise, and stored at –196°C for 1 week. Two groups of heart valves (fresh untreated and thawed cryopreserved [each, n = 8]) were analyzed using biochemical (collagen, elastin, desmosine), histologic (hematoxylin-eosin, Movat-pentachrome, resorcin-fuchsin), and immunohistochemical (antibodies against collagen I, III, IV, and elastin) methods. Near-infrared femtosecond multiphoton laser scanning microscopy and second harmonic generation were used for high-resolution three-dimensional imaging of ECM structures.

RESULTS: Biochemical testing demonstrated similar amounts of collagen and desmosine, but a minor loss of elastin in the cryopreserved specimens. Conventional histology revealed almost comparable cell and ECM formations in fresh and cryopreserved valve leaflets. In contrast, laser-induced autofluorescence imaging showed substantial ultrastructural deterioration and disintegration of most collagenous structures. Second harmonic generation was not inducible.

CONCLUSIONS: Conventional cryopreservation of heart valves is accompanied by serious alterations and destruction of leaflet ECM structures, specifically demonstrated by multiphoton imaging. Further in-depth studies to clarify the impact of alternative cryopreservation techniques proposed for clinical use, such as vitrification, are crucial.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Despite advantages such as superior hemodynamic properties, resistance to infections, and low incidence of thromboembolic complications, the long-term durability of cryopreserved allogeneic homografts remains limited, especially in children, young adults, and patients with terminal renal failure. In the majority of cases, tissue deterioration, manifested as structural and calcific degeneration of the valves, leads to graft dysfunction, and eventually reoperations are required [1, 2].

The identification and understanding of the possibly multifactorial mechanisms of cryopreserved homograft failure has been the subject of studies for several decades. These investigations were concentrated on the relevance of cellular viability [3–5], the role of immune responses [6], and biochemical aspects of extracellular matrix (ECM) elements such as proteoglycans [7], elastin [8], or collagen [4, 9–11]. Others focused on the impact of damage caused by ice formation after cryopreservation [12] of human, porcine, ovine, and rat aortic or pulmonary heart valves. Neither the relative contributions of cellular viability, immune responses, and ECM durability nor the immediate effects of cryopreservation on structural properties of the amorphous and fibrillar valve matrix have yet been clearly identified.

The valvular ECM contains a variety of structures, underlies and surrounds the interstitial cells, and performs many essential functions, including mechanical support and physical strength [13–15]. Moreover, the ECM exerts profound influences on cell adherence, migration, and differentiation as well as the pattern of gene expression of the cells in contact with it [16, 17]. Since the quality of the structural matrix at implantation may predetermine durability or failure of a cryopreserved heart valve, our goal was to carefully examine state and quality of the major ECM elements (collagenous bundles and elastin-containing fibers) based on different techniques before implantation. Besides biochemical assays and conventional matrix visualization methods such as histology and immunohistochemical staining, near-infrared (NIR) femtosecond multiphoton laser scanning imaging and second harmonic generation (SHG) microscopy have been applied in this study as novel non-contact optical technology for three-dimensional resolved ECM component imaging and heart valve tissue state diagnosis [18, 19].


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Tissue Preparation and Cryopreservation
Hearts of 9- to 12-week-old pigs (weight, 25 to 35 kg) were obtained from a local slaughterhouse and immediately transferred to the laboratory. Pulmonary valves were excised under sterile conditions and gently rinsed free of blood in sterile phosphate-buffered saline (PBS [1x; Invitrogen, Carlsbad, California]). Eight specimens of the untreated, fresh valves were directly processed for histology, immunohistochemistry, NIR multiphoton imaging, and biochemistry. The remaining valves (n = 8) were incubated each in 100 mL of a solution containing a combination of antibiotics (1.2 mg amikacin; 3 mg flucytosin; 1.2 mg vancomycin; 0.3 mg ciprofloxacin; 1.2 mg metronidazol in 1 mL aqua ad injection) and medium 199 (M199; Invitrogen) at 4°C for 24 hours. After this sterilization, each valve was placed separately in cryopreservation bags containing 100 mL of a cryoprotective solution (M199; 10% dimethyl sulfoxide [DMSO (Me2SO)]; Sigma, St. Louis, Missouri), and immediately controlled-rate frozen at –1°C per minute for 60 minutes, down to –40°C, and at 5°C per minute until a temperature of –196°C was attained (Ice Cube 1810CD; SY-LAB GmbH, Purkersdorf, Austria). The valves were stored in the vapor phase of liquid nitrogen at approximately –196°C (Cryo 200; Thermo Forma, Marietta, Ohio). After 1 week of storage, the valves were fast thawed using a 37°C water bath (total thawing time approximately 10 minutes).

Histology, Immunohistochemistry, and Transmission Confocal Laser Scanning Microscopy
Samples of each heart valve (each, n = 8) were processed as described before [19, 20]. All specimens were treated and sectioned similarly. Each leaflet considered for histologic or immunohistochemical analysis was processed in serial sections. To determine general cellular and tissue morphology, representative sections of the paraffin-embedded samples were stained with hematoxylin-eosin (HE). A modified Movat-pentachrome stain [21] was used to demonstrate ECM components such as collagen, elastin, and proteoglycans/glycosaminoglycans (GAGs). For an improved histologic imaging of the elastic fiber system, tissue slides were prepared and stained with resorcin-fuchsin [22]. After staining, all sections were dehydrated in ethanol (Mallinckrodt Baker, Deventer, Netherlands), cleared in xylene (Merck KGaA, Darmstadt, Germany), mounted using Entellan (Merck), analyzed, and documented using routine bright-field light microscopy (Axiovert S 100 System; Carl Zeiss, Jena, Germany).

Immunohistochemical analysis was carried out using cryostat sections and standard indirect immunoperoxidase (strept)avidin-biotin techniques. The primary antibodies used in this study were as follows: a monoclonal mouse antibody to elastin (1:50; Acris Antibodies GmbH, Hiddenhausen, Germany), a polyclonal rabbit antibody to collagen type I (1:50; Acris), a polyclonal rabbit antibody to collagen type III (1:50; Acris), and a polyclonal rabbit antibody to collagen type IV (1:50; Acris). The (strept)avidin-biotin-complex technique was performed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, California). Secondary antibodies were a biotin-labeled anti-mouse antibody (elastin) and a biotin-labeled anti-rabbit antibody (collagens [each 1:200; DakoCytomation GmbH, Hamburg, Germany]). The immunobound peroxidase activity was also visualized using the chromogenic substrate Jenchrom px blue (MoBiTech GmbH, Göttingen, Germany) according to the manufacturer's instructions. Negative controls were performed without primary antibodies. All sections were analyzed and documented using a routine bright-field microscope (Axiovert S 100 System; Zeiss) or a confocal laser scanning microscope system (LSM 310; Zeiss). Jenchrom px blue-stained sections were additionally examined by the use of the transmission mode of the LSM 310, equipped with a helium-neon gas laser (excitation wavelength 543 nm) and an argon ion laser (excitation wavelength 488 nm). Further processing of the stored transmission images was performed using digital image analysis based on features provided by the Carl Zeiss LSM 310 software. After subtraction of the background based on electronic noise, room light, or dust on the optical system, the image underwent gray value inversion [23], which enhanced the ability to assess the images by eye.

Multiphoton Imaging
Shortly after dissection, fresh and thawed cryopreserved leaflet specimens (each, n = 8) were examined using a NIR femtosecond laser scanning microscope system, as described previously [24]. Extracellular-matrix-dependent autofluorescence and SHG were induced using wavelengths of 760 nm and 840 nm. Non-invasive serial optical horizontal sections from both the inflow (ventricularis) and the outflow side (arterialis-fibrosa) of the different leaflet specimens were taken in z-steps of 5 µm and 10 µm. Induction of SHG radiation, which can be detected at half of the incident laser wavelength, was demonstrated with a filter FB420-10 (central wavelength, 420 nm; full width half maximum, 10 nm [Thorlabs, Newton, New Jersey]) in front of the detector. A 700 nm short pass filter (E700SP; Chroma Technology, Brattleboro, Vermont) was used to block ultraviolet radiation (transmission range, 390 nm to 700 nm) and to prevent the scattered laser radiation from reaching the detector.

Biochemical Assays
Biochemical assays were used for the quantitative analysis of ECM components of a representative number of fresh and cryopreserved valve leaflet tissues (each, n = 8). Crude protein extracts were prepared by manual disruption of the leaflets using liquid nitrogen, a pistil and mortar. All samples were normalized to the dry weight. Total collagen and elastin contents were quantified using SIRCOL and FASTIN assays (Biocolor, Belfast, Northern Ireland), respectively, as described before [25].

Radioimmunoassay for Desmosine
The elastin quantity in fresh and cryopreserved heart valve tissues (each, n = 8) was also determined by quantifying desmosine and protein after hydrolysis, as described in detail elsewhere [26]. Desmosine represents a rare amino acid cross-link specific to elastin, formed by condensation of four molecules of lysine into a pyridinium ring.

Statistics
All results are presented as mean values ± SD. Significant differences between the heart valve tissues were assessed by the non-parametric Mann-Whitney test, using the commercially available software package SPSS for Windows, version 11.0 (SPSS GmbH Software, München, Germany). Any p values of 0.05 or less were defined as statistically significant differences.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Macroscopic Appearance
Macroscopic analysis showed no structural differences between fresh unprocessed, antibiotic-treated, and thawed cryopreserved valves (Fig 1). The gross dimensions and thickness appeared comparable between all specimens.


Figure 1
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  		Fig 1. Macroscopical appearance of representative specimens of (A) fresh, (B) with antibiotics treated and (C) thawed cryopreserved heart valves. No macroscopical cracks or other apparent damages were noticed after thawing.

 
Routine Histology and Immunohistochemical Analysis
Routine histology revealed almost comparable morphologic features of the ECM of fresh and cryopreserved leaflets. A layered structure, including the elastic fiber-containing ventricularis, the spongiosa layer with the majority of GAGs, and a collagen-rich fibrosa with the adjoining arterialis as the outflow side, was visible (Fig 2). However, we observed two differences between the fresh and cryopreserved samples. Only a few collagen-containing structures were detectable within the ventricularis layer of all thawed cryopreserved heart valve leaflets. The second finding was an overall more fragmented appearance of the histoarchitecture of thawed cryopreserved heart valve leaflets, especially between the spongiosa and fibrosa layers (Fig 2B).


Figure 2
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  		Fig 2. Images of representative parts of fresh (A) and cryopreserved (B) leaflets (longitudinal sections). (A, B) Both specimens show a normal interstitial cellularity (cell nuclei: dark magenta) and the expected layered structure of the leaflet with the ventricularis (v) as inflow side, spongiosa (s) as inner layer, followed by fibrosa (f) and arterialis (a) as the outflow side. (B) Only a few collagen-containing structures (yellow) are detectable within the ventricularis layer of the thawed cryopreserved leaflets. Furthermore, a more loose structure with a slight degree of partial detachment of the fibrosa from the spongiosa layer is detectable within these specimens. (Movat-pentachrome stain for visualization of the major matrix proteins: collagen, yellow; glycosaminoglycans, green-blue; elastin, black.) Magnification is x10. Scale bar equals 100 µm.

 
Immunohistochemical staining of fresh and cryopreserved leaflet tissues with an elastin-specific antibody revealed a normal pattern of elastic fibers, particularly within the ventricularis layer (Fig 3). Staining with antibodies against collagen type I, III, and IV showed almost similar results within the fibrosa of fresh and cryopreserved specimens (Fig 4A through F). The majority of the fibers were positive for collagen types I and III. Collagen IV showed moderate staining. However, differences appeared in the geometric properties of collagenous structures of cryopreserved tissues. The birefringent nature of collagen enables the visualization of crimp pattern [27]. Compared with fresh leaflet sections (Fig 4A, B), the cross-sections of cryopreserved tissues stained with collagen type I and type III antibodies did not show these crimped, interweaving pattern (Fig 4D, E). The ventricularis layer of fresh heart valve leaflets principally contains of collagen types I and III. The inflow side of cryopreserved leaflets showed a diffuse moderate staining for collagen type III (Fig 4H). Collagen types I and IV were barely detectable (Fig 4I). No interweaving pattern could be discerned.


Figure 3
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  		Fig 3. Immunohistochemical staining of representative cross-sections (ventricularis; z = 25 µm to 30 µm) of fresh (A, B) and cryopreserved specimens (C, D) with an antibody against elastin at two different magnifications. Magnification of A, C is x40; magnification of B, D is x63. Scale bars equal 100 µm.

 

Figure 4
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  		Fig 4. Transmission confocal laser scanning microscopy images, converted by gray value inversion, show cross-sections of the fibrosa of fresh leaflets (A, B, C), and the fibrosa (D, E, F) and ventricularis (G, H, I) of cryopreserved leaflets. Extracellular matrix structures stained positive for collagen type I (A, D, G), type III (B, E, H), and type IV (C, F, I) appear yellow (pseudofluorescence). The images are representative for all fresh and cryopreserved specimens. Magnification is x63. Scale bar equals 100 µm.

 
Biochemical Data
Quantitative tissue analysis data are summarized in Table 1. The amounts of collagen (fresh, 46 ± 6 µg/mg; cryopreserved, 45 ± 4 µg/mg) and desmosine (fresh, 743 ± 156 pmol/mg; cryopreserved, 741 ± 130 pmol/mg) were comparable in fresh and cryopreserved leaflet tissues. The difference found in the elastin content between fresh (33 ± 6 µg/mg) and cryopreserved (29 ± 8 µg/mg) heart valve leaflets was not statistically significant.


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  		Table 1. Collagen, Desmosine, and Elastin Contents of Fresh and Cryopreserved Heart Valve Tissues (each n = 8)
 
Multiphoton Imaging
State and quality of collagenous bundles and elastin-containing fibers of fresh and cryopreserved heart valve tissues were assessed using NIR multiphoton excitation at wavelengths of 760 nm (cells and single branched elastic fibers) and 840 nm (collagenous structures; Figs 5, 6). Go


Figure 5
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  		Fig 5. Autofluorescence images of the elastin-rich ventricular side of fresh and cryopreserved heart valve leaflets induced with 760 nm laser pulses. Single cells with fluorescent mitochondria based on two-photon excited reduced coenzyme NAD(P)H, and nonfluorescent nuclei are visible especially at the surface of fresh tissues (z = 0 µm). Leaflet cells of the cryopreserved specimens appear to be damaged or non-viable, indicated by the total strong-fluorescent cellular structures, including cell nuclei. Arrows indicate cell debris within the cryopreserved tissue. Compared with the distinct elastin network of the fresh tissue, just a few elastic fibers are detectable at depths of 10 to 15 µm of the cryopreserved leaflets. The images shown in this figure are representative for all monitored leaflet tissues. Scale bar equals 30 µm.

 

Figure 6
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  		Fig 6. Multiphoton imaging of the outflow side of representative fresh and cryopreserved leaflets induced with 840 nm near-infrared femtosecond laser pulses show substantial ultrastructural deterioration and disintegration of most collagenous structures in cryopreserved tissues. Second harmonic generation was only inducible in fresh tissue structures. Scale bar equals 30 µm.

 
Exposure of fresh tissue material to laser pulses at 760 nm revealed living cells mainly at the surface, and autofluorescent elastic fibers within the tissue in depths of 5 to 40 µm (Fig 5, fresh). In contrast, cells at the surface and within the whole tissue of the cryopreserved specimens appeared to be damaged, indicated by a strong aberrant autofluorescence, including cell nuclei [28]. Only a few delicate elastic fibers were detectable at depths of 10 to 15 µm of the cryopreserved leaflets (Fig 5, cryopreserved).

When imaging intratissue regions, in particular the outflow sides of the fresh specimens at a higher wavelength of 840 nm, wavy autofluorescent structures became visible—the collagenous fibers. Within these, SHG signals could be induced as demonstrated with filter FB420-10 (Fig 6, fresh). In contrast, in cryopreserved valvular samples was a dramatic loss of autofluorescence and SHG throughout the entire leaflet, indicating substantial structural deterioration especially of the collagenous fibers (Fig 6, cryopreserved).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The earliest method of sterilization of homografts was to expose them to a solution of various antibiotics. Repeated cultures of this solution were performed to confirm the absence of any infection before using the valves for replacement in humans [29]. Because this method cannot be used for long-term storage, cryopreservation techniques were developed that enabled valves to be stored for prolonged periods [30]. The discovery of cryoprotective properties of different agents such as glycerol or DMSO spurred the expansion of the field of cryoprotection and represented a tremendous step forward in providing valve availability for replacement efforts [31, 32]. The most common cryopreservation protocol for heart valves involves freezing in 10% DMSO and storage in liquid nitrogen. However, a significant controversy exists regarding the actual preservation of cellular and extracellular valve elements. Despite a body of literature that documents the different effects on cell viability [33–36], it remains unclear whether the process of cryopreservation itself is preservative or detrimental to valvular ECM structures. As destruction of the amorphous and particularly of the fibrillar matrix structures may predispose cryopreserved valve grafts to structural failure, we thoroughly investigated and compared in the present study the ECM of fresh and cryopreserved porcine heart valve leaflets, with special emphasis on the condition of collagenous and elastic fibers.

For quantitative and qualitative matrix evaluations, biochemistry, immunohistochemical screening, and routine histology were performed. Near-infrared femtosecond multiphoton laser scanning microscopy and SHG detection have been used as novel technologies for tissue state diagnosis of unprocessed fresh and cryopreserved leaflets.

The principal finding of our experiments is that conventional cryopreservation of heart valves causes alteration and significant deterioration of crucial leaflet matrix structures. Collagenous and elastic fibers, well known as critical elements for the biomechanical profile of heart valves, were impaired at different degrees. Interestingly, biochemical testing demonstrated no significant differences between the amounts of collagen, desmosine, and elastin of fresh and cryopreserved specimens. Conventional histologic staining using resorcin-fuchsin and Movat-pentachrome revealed almost comparable cell and ECM formations, although two conspicuous differences were observed. In contrast to fresh leaflet tissues, only a few collagen-containing structures were detectable within the ventricularis layer of thawed cryopreserved heart valve leaflets. Staining with saffron solution exclusively and picrosirius red confirmed this result (data not shown). The second finding was an overall more disintegrated and detrimental appearance of the histoarchitecture of thawed cryopreserved heart valve leaflets, especially between the spongiosa and the fibrosa layers. This damage is most likely due to ice formation within the highly hydrated proteoglycan- and glycosaminoglycan-containing, gel-like matrix of the spongiosa.

Immunohistochemical staining using an antibody against elastin revealed no differences between fresh and cryopreserved tissues, which was similar to the results obtained by routine histology. The collagen expression pattern of fresh and cryopreserved leaflet tissues showed almost similar results within the fibrosa. Within the ventricularis of cryopreserved leaflets, only collagen type III was sufficiently detectable. However, the most apparent difference appeared in the geometric properties of collagenous structures. No crimped, interlacing collagenous fiber pattern could be discerned within the cryopreserved tissues.

Laser-induced autofluorescence imaging confirmed the substantial ultrastructural deterioration and disintegration of most collagenous structures. In contrast to fresh leaflet tissues, no wavy autofluorescent structures became visible by exposure of the cryopreserved tissues to laser pulses at 840 nm. Second harmonic generation was not inducible. Moreover, multiphoton imaging revealed an alteration of the elastic fiber system within cryopreserved specimens, which was not detectable using routine histology or immunohistochemistry. Only a few delicate fibers could be discerned. The cells at the surface and within the whole tissue of the cryopreserved specimens appeared to be damaged and nonviable.

In conclusion, conventional approaches to cryopreservation of heart valves have a detrimental and destructive effect on crucial leaflet matrix elements. Our results demonstrate serious alteration and significant deterioration of collagenous and elastic fiber structures, accompanied by a general damage of the leaflet histoarchitecture caused by extracellular ice formation. If these structural changes might have an unfavorable impact on the biomechanical performance of the heart valve leaflets needs to be tested in more detail. Further in-depth studies to improve the conventional cryopreservation process or to clarify the impact of alternative preservation techniques proposed for clinical use, such as vitrification [37, 38], D-hydro-, or glycerol-treatment [39, 40], are crucial.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
We are grateful to Sabine Hitschke and Ilka Degenkolbe for their excellent technical assistance. We would like to thank Penny Thomas (University of Southern California) for her helpful comments on the manuscript. This study was financially supported by the Interdisciplinary Center of Clinical Research Jena (IZKF 01ZZ0105 [Drs König and Stock]) and the German Science Foundation (DFG Sto359/2-3, Sto359/4-1 [Dr Stock]; and Sche701/2-1, Sche701/3-1 [Dr Schenke-Layland]).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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