Bovine Pericardial Tissue in Heart Valves

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Substitute heart valves

Substitute heart valves used since the early 1990s have resulted in a change of prospects for people with cardiac valvular disease. Bioprosthetic heart valves have been found to be superior to mechanical prostheses (Hwang, 1982). Anticoagulant therapy is unnecessary with the bioprosthetic valves. However major problems are still present with them. Mechanical failure due to calcification of leaflet regions which have flexural stresses during valve opening, high tensile stresses during valve closure and tearing of the cusps even without calcification are common problems (Krucinski, 1993). Apart from calcification other problems noted are the inability to be durable on a long term basis and problems of functional and haemodynamic performance (Sung et al, 1993, p. M532).

Chemical treatments of prostheses have brought about some changes but problems have not abated. The problems of the prostheses are related to the cross linking reagents of glutaraldehyde or formaldehyde which are used to fix the bioprostheses.

Ideal heart valve

The modeling of an active and non-antigenic bioprosthesis free from calcification and fibroblast proliferation induced by both glutaraldehyde treatment and cell remnants is essential (Colomb et al, 1987). The most ideal bioprosthesis must be in addition be free from thrombogenic property, durable, have the potential to grow and have no immunogenic properties that cause rejection (Ikada, 2006, p. 159).

Prosthetic heart valves

These are not at all ideal. Long term benefits are hard to be elicited from them. The foreign materials used are thromobogenic. Bioprostheses and homografts available commercially have advantages and limitations (Ikada, 2006, p.158). Porcine valves have low resistance to flow, larger diameter and lack of anticoagulation requirement but have a higher rate of inflammatory deterioration with valve failure where tearing occurs even without calcification. Tissue engineered heart valves (TEHV) are made by seeding autologous cells on anatomically shaped porous scaffolds (Ikada, 2006, p. 158).

Bioprosthetic heart valves have superior fluid dynamic properties and do not need anti-coagulant therapy. Reduction in the mineralization rate can be achieved with new chemical treatments but mechanical failure still results (Sacks, 1998, p. 899).

Bovine Tissue

Allogeneic and xenogeneic type I collagen of bovine tissue is a useful scaffold with low antigenecity (Ikada, 2006, p.7). Bovine tissue is used for studies due to its abundance. Extraction of the Type I collagen is through alkaline or enzymatic processes.

The telopeptide portions with the antigenic properties are eliminated during extraction. This collagen has the disadvantages of low mechanical stiffness and speedy biodegradation if left untreated. The biodegradation can be prevented by two methods of cross linking. The first process is physically done by photooxidation, uv irradiation or dehydrothermal treatment (DHT). The chemical process could be by using glutaraldehyde or water soluble carbodiimide or other chemicals (Ikada, 2006, p.7). Glutaraldehyde is the best and has the advantages of water solubility, high cross-linking efficiency and low cost. It has however the disadvantage of cytotoxicity.

Bovine pericardial tissue has been found to be anisotropic with important directional variation. A study found it to be more extensible in the circumferential direction than in the root apex direction and had a greater mean strength too in the circumferential direction. It had been found to be optically anisotropic also and there was a relationship between the two anisotropies (Zioupos et al., 1992).

The sites for taking the specimens of bovine tissue have been specified as the left ventricle and the superior aspect of the right ventricle (Sacks, 1998, p. 899).

Photo-oxidation or glutaraldehyde fixation for cross-LINKING?

Photo-oxidation is a physical catalytic process causing modification and cross-link formation within existing matrix components. Glutaraldehyde fixation is associated with extensive cross-linking and polymer formation which together contribute to matrix complexity and thermal stability. Alterations in the architecture of the fibers determine how chemicals can influence the mechanical properties (Sacks, 1998, p.900). A pre-stretched specimen was similar to a glutaraldehyde-fixed specimen in stiffness but the cross-fiber was unchanged. Even without chemical treatment a wide range of mechanical properties is possible. A bovine pericardial tissue with a high degree of mechanical anisotropy could be used by reducing excessive deformed leaflets along the circumferential direction and maintaining a good leaflet coaptation in the radial direction (Sacks, 1998, p. 900). Sack’s study showed that chemical treatment affected the tissue’s mechanical properties in the low stress region.

The accepted prosthesis

The bovine pericardium prosthesis is now commonly accepted for heart valve replacement (Tattini, 2007, p. 1). Glutaraldehyde has been used as the cross-linking agent. The actions of glutaraldehyde include the stabilization of the collagen structure, prevention of tissue digestion by enzymes or bacteria and reduction of the antigenecity of the bovine pericardium (Nimni, 1987).

Basic structure of collagen

Collagen is a triple helical structure with a long protein chain to which three coiled subunits of abundant amino acids, glycine, proline and hydroxyproline are attached. The various arrangements of the helical and non helical parts form the fibrils and sheets and the cross-linking of different collagen types. Water maintains the conformation of the collagen molecules and the properties of the fibrils. Three forms of water that are found are the structural, bound and free or bulk (Tattini, 2007, p. 1). The triple helix is stabilized by participating in the H bond backbone through the structural water. The hydrogen-bonded water that lies between the triple helices and the microfibrils is the bound form. The free water lies between the fibrils and microfibrils. Intermolecular cross-links between the collagen molecules in the fibres help to bear load and prevent slippage under it (Avery and Bailey, 2008, p.81).

The alignment of the cross-linked fibers decides the strength in different directions. Cross-linking of parallel fibers produce strength in the longitudinal direction, random organization allows compliance, laminated fibers flexibility and concentric layers produce strength. The cross-link profile varies with the tissue. The profiles would be different in bones, cartilage, muscle and tendons. Major cross-links are dependent on the ‘oxidative deamination of the ε-amino groups of specific lysine residues by the enzyme lysyl oxidase to form lysine-aldehydes’ (Avery, 2008, p.83). They act on the telopeptide lysines and hydroxylysines. The aldehydes formed would then have a reaction in the triple helical region allowing the formation of a Schiff base intermolecular cross-link. Stabilization of a collagen fiber to acquire optimal functionality is possible through the enzyme lysyl oxidase and the formation of trivalent cross-links by the spontaneous conversion of the divalent aldimine and ketamine (Avery, 2008, p. 91). Inhibition of the cross links reduces the mechanical strength of the collagen tissue. During maturation, the initial divalent links react with the adjacent fibers to form trivalent cross-links further increasing its strength. This system of cross-linking exists throughout the animal kingdom. Unidentified and unusual cross-links are present in some tissues. Different tissues exhibit different kinds of cross-linking. In other words, cross-linking is tissue-specific and not species specific (Avery, 2008, p.81). The extent of hydroxylation of the telopeptide and triple helical lysines involved with the cross-link and the collagen metabolic rate decide the type of cross-links and the tissue.

The cross-linking and close packing of the collagen molecules in the fibers make them inextensible. However some elasticity still remains. The strength of collagen tissue increases with age. An accumulation of glucose oxidation products that form intermolecular cross-links cause the fiber to become stiff and unable to perform normal function and result in the low turnover of collagen (Avery, 2008, p. 81). This is seen in aging and diabetes mellitus. Specific glycation cross-link breakers can decrease the ill effect.

Significance in Industries

Industry has taken advantage of the high mechanical strength of collagen, resistance to heat and degradation of fibers (Avery, 2008, p.81). Collagen biomaterials are widely used. A ‘specific increase in mechanical strength, denaturation temperature, enzyme resistance and biocompatibility can be achieved by a correct choice from a wide range of available cross-linking agents’ (Avery, 2008, p. 104).

Shrinking of the tissue

An increase in the denaturation temperature has been related to the decreased water content of collagen caused by the cross-linking agent which produces tight binding of the molecules. The effect is not totally understood (Avery, 2008, p.105).

Heat induced denaturation of collagen is one main topic of study in the hunt for a fit bioprosthesis. The resulting tissue shortening is related to the hydrothermal shrinkage temperature (Tattini, 2006, p.1). The temperature at which the denaturation and thereby shrinkage begins under constant load is the shrinkage temperature. A tensometer and a water bath may be used for the study. This hydrothermal method is limited in that it measures only on a macroscopic scale, has a maximum workable temperature of 100 °C and the load may hinder the contraction and give a wrong reading for the shrinkage temperature (Tattini, 2006, p.1).

Differential scanning calorimetry (DSC) is another method of recording shrinkage temperature. The absorption of heat produces an endothermic peak over the shrinkage temperature range of the tissue. This technique employs the comparison of the temperatures of the tissue bathed in a solution in a pan on one side and a pan with a solution only on the other. It allows a measurement of the transitions.

Freeze-drying microscopy is a method which determines the melting point. This process can use a microscope or a camera and imaging system with a computer (Tattini, 2006, p.1). The DSC and the freeze-drying methods appear to give similar results by a study by Tattini in 2006. Both methods showed excellent sensitivity and reproducibility. Both also provided information on the thermal shrinkage transition through the thermodynamical parameters.

Renaturation is a process by which a heated pericardial tissue is returned to prior basal temperature after the heating. This partial renaturation is due partly to the ‘absorption of hydrogen bonds or water bridges’ inside the molecule (Chen, 1998, p. 1236). Stress-strain tests done before the heating and after the recovery indicate that heat- induced changes depend on the extent of thermal damage and not the reason for the damage which could be thermo-mechanical.

Thermal reasons for causing the denaturation produces changes at the molecular level (Wall, 1999, p.343). This is a phase transition which changes the collagen molecule into an ‘amorphous, contracted, random, coil formation’ which becomes rubber like (Wall, 1998, p. 343). This conformational change which occurs mainly in the hydrogen bonds takes place after disrupting the stabilizing forces. The shrinkage is a rate-dependent process over a range of temperatures.

Instantaneous shrinkage (at a high rate) and incipient shrinkage (at a low rate) have been recognized (Pankhurst, 1947). Several factors can influence the result. The collagen source or age and architecture are significant. The density and packing orientation decides the amount of shrinkage possible. The next factor is the fibril orientation. Shrinkage measurements must be oriented to the long axis of the maximum fibrils. Temperature and the time when this temperature lasted must be recorded. The ‘shrinkage process is sensitive to temperature’ (Wall, 1998, p.343). Weir reported that a rise of temperature of 2 degrees caused the shrinkage to double. The mechanical properties of the tissue reduced with increasing shrinkage. The goal would be not to overshoot the shrinkage. Incipient shrinkage would have lesser risk of overshooting it.

Calcification

This problem is noticed in bovine pericardium bioprostheses. This has been attributed to the standard glutaraldehyde fixation causing a limitation to durability (Sucu, 2004, p.89). It allows formation of complexes with calcium inducing devitalization of the cells. This leads to disruption of cellular calcium regulation. The effect of ethylenediaminetetraacetic acid (EDTA) on calcific degeneration was investigated in a study on bovine pericardial tissue treated with glutaraldehyde first (Sucu, 2004, p. 89). Calcium levels were found to be lower than in the controls without the EDTA (Sucu, 2004, p.91). The EDTA bound the calcium in the pericardial tissue and prevented the formation of calcium phosphate.

Cross-linking by glutaraldehyde

The bioprostheses become immunologically acceptable to the human being through the cross-linking by glutaraldehyde (Carpentier, 1969). This preservation agent, glutaraldehyde, also increases the mechanical resistance and reduces the thrombogenecity of the biomaterials (Schoen, 1987). However the later structural deterioration limits the use of bioprosthetic valves, more commonly due to calcification and degeneration (Schoen, 1983).

References

Avery, N.C. and Bailey, A.J. (2008). “Restraining Cross-Links Responsible for the Mechanical Properties of Collagen Fibers: Natural and Artificial”. in Chapter 4 “Collagen: Structure and mechanics” by Fratzel P., Springer Science+Business Media, LLC 2008
Carpentier A, Lemaigre G, Carpentier S, Dubost C (1969) Biological factors affecting long-term results in valvular heterografts. J Thorac Cardivasc Surg 58:467–483
Chen, S.S. et al. (1998). “Phenomenological Evolution Equations for Heat-Induced Shrinkage of a Collagenous Tissue”. IEEE Transactions on Biomedical Engineering, Vol.45, No.10, 1998
Colomb, G. et al, (1987). “The role of glutaraldehyde-induced cross links in calcification of bovine pericardium used in cardiac valve prostheses”. American Journal of Pathology, Vol. 127, p.122-130.
Freeman, W.H. (2000). “Integrating cells into tissues’. Chapter 22 in “Molecular cell Biology”, 2000, 4^th Ed., New York: W.H.Freeman Company
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Ikada, Yoshito. (2006). “Tissue Engineering: Fundamentals and applications”. Academic Press, 2006
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Pankhurst K. (1947). “Incipient shrinkage of collagen and gelatin”..Nature1 947; 159538.
Sacks, M.S. and Chuong, C.J. (1998). “Orthotropic Mechanical Properties of Chemically Treated Bovine Pericardium”. Annals of Biomedical Engineering, Vol. 26, pp. 892–902, 1998: Biomedical Engineering Society.
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Sucu, N. et al. (2004). “The effect of ethylenediaminetetraacetic acid on calcific degeneration in bovine pericardium”. Heart vessels, Vol. 19, Pgs. 89-93.
Sung, H.W. et al. (1993). “Comparison of the cross-liking characteristics of porcine heart valves fixed with glutaraldehyde or epoxy compounds”. ASAIO Journal, 1993, Vol 39, M532-536
Tattini Jr, V. et al. (2007). “Evaluation of Shrinkage Temperature of Bovine Pericardium Tissue for Bioprosthetic Heart Valve Application by Differential Scanning Calorimetry and Freeze-drying Microscopy”. Materials Research, Vol. 10, No.1, p.1-4, 2007
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