Tuna Cornea as Biomaterial for Cardiac Applications

Abstract

Among available biomaterials, cornea is almost completely devoid of cells and is composed only of collagen fibers oriented in an orderly pattern, which contributes to low antigenicity. Thunnus thynnus, the Atlantic bluefin tuna, is a fish with large eyes that can withstand pressures of approximately 10 MPa. We evaluated the potential of this tuna cornea in cardiac bioimplantation.

Eyes from freshly caught Atlantic bluefin tuna were harvested and preserved in a fixative solution. Sterilized samples of corneal stroma were embedded in paraffin and stained with hematoxylin and eosin, and the histologic features were studied. Physical and mechanical resistance tests were performed in comparison with bovine pericardial strips and porcine mitral valves. Corneal material was implanted subcutaneously in 7 rats, to evaluate in vivo calcification rates. Mitral valves made from tuna corneal leaflets were implanted in 9 sheep.

We found that the corneal tissue consisted only of parallel collagen fibers without evidence of vascular or neural structures. In tensile strength, the tuna corneal specimens were substantially similar to bovine pericardium. After 23 days, the rat-implanted samples showed no calcium or calcium salt deposition. Hydrodynamic and fatigue testing of valve prototypes yielded acceptable functional and long-term behavioral results. In the sheep, valvular performance was stable during the 180-day follow-up period, with no instrumental sign of calcification at the end of observation.

We conclude that low antigenicity and favorable physical properties qualify tuna cornea as a potential material for durable bioimplantation. Further study is warranted.

Animal xenografts are widely and effectively used in human surgery.¹ However, factors that limit their application include antigenicity² and tissue degeneration that lead to implantation-related pathologic conditions. Both of these factors are mainly dependent on tissue cellular presence. Bovine pericardium, for example, is a versatile material because of its fibrous nature and scarcity of animal cells; however, it requires eventual replacement because of calcium deposition³ that alters the mechanical characteristics of collagen fibers.

Among biological tissues, cornea is known to be almost completely devoid of cells. It is composed only of collagen fibers oriented in an orderly pattern. However, its typically small size greatly limits its medical uses, because even large mammals rarely have a corneal surface area that would be adequate for medical xenografts.

Thunnus thynnus, the Atlantic bluefin tuna, can exceed 150 kg in weight. Its large eyes, unprotected by eyelids, can withstand pressures of approximately 10 MPa. The size, composition, and mechanical properties of these corneas appear to qualify them for use as medical xenografts. We evaluated the potential of tuna cornea as a material for medical bioimplantation.

Materials and Methods

From January through June 2003, experiments and measurements were performed by Italian investigators at the University of São Paulo, Brazil. Data analysis was performed at the University of Modena, Italy. An investigator at the University of Genoa, Italy, provided the necessary expertise in marine biology. All animal experiments were conducted in accordance with Brazilian institutional regulations.

Specimen Collection and Histologic Examination. The eyes from freshly caught Atlantic bluefin tuna were removed and preserved in 0.3% glutaraldehyde solution⁴ until skilled personnel with blunt surgical scissors collected corneal stroma. After lavage with normal saline solution, the specimens were stored on a rigid surface and immersed in 0.2 M acetate buffer (pH, 4.6) in 0.3% glutaraldehyde solution for 15 days. The corneal material was then treated for 24 hours with the same acetate buffer in 4% formaldehyde solution, for sterilization and to remove residual fixative traces. Afterwards, the specimens were embedded in paraffin and stained with hematoxylin and eosin (H & E).

Physical Testing. Shrinkage temperature was determined for the tuna corneal strips. In compliance with standards from the American Society for Testing and Materials, we measured the tensile strength of 5 equally shaped strips of bovine pericardium, porcine mitral valve, and tuna cornea (average length, 50.8 mm; average width, 6 mm) to determine elongation and peak load.⁵

In vivo Calcification. To evaluate the in vivo calcification rate of the tuna cornea, a pledget-shaped, 10 × 0.5-mm corneal specimen was implanted subcutaneously in the dorsal interscapular region of 7 male CD® SPF rats (age, 3 wk; weight, 75 kg) (Charles River Laboratories International, Inc.; Wilmington, Mass). The rats were humanely killed 23 days later, and the specimens were explanted in order to evaluate local host reaction to the xenograft. Histologic examination for calcium deposition was conducted on the specimens after H & E and Von Kossa staining.

Cardiovascular Application. Tuna corneal stroma was used to manufacture mitral valves. A 40-mm biomaterial disc was shaped into 3 triangular portions in accordance with the orientation of the collagen fibers. Each leaflet was sutured onto an 0.6-mm acetyl resin Delrin® 500 support ring (DuPont Engineering Polymers; Wilmington, Del), which was then covered with bovine pericardium to prevent abrasion phenomena and contact with blood during surgical placement. The external annular margin of each prosthesis was covered with 2 layers of Dacron, to enable suturing to myocardial tissue. Figure 1 shows a prototype of the corneal mitral valve. Two slightly different valve designs were produced as part of the prototype definition process.

Fig. 1 Photograph shows a prototype of a mitral valve made from Thunnus thynnus cornea.

A Celcon® M270™ holder (Ticona Engineering Polymers, a business of Celanese; Florence, Ky) was chosen to facilitate implantation. The prosthesis was packaged in a polycarbonate container in a preservative solution of 4% formaldehyde. Random bioprosthetic samples were tested for bacterial and fungal contamination.

Hydrodynamic Testing. Ten 23-mm-diameter mitral valve prostheses were tested⁶ at different simulated heart rates (approximately 50, 70, and 100 beats/min) with use of a Shelhigh Pulse Duplicator System v. 4.0 (Shelhigh Inc.; Union, NJ). This was done to determine closing volume, leakage volume, regurgitant fraction, effective orifice area, mean flow, mean gradient, performance index, and efficiency index.

Durability Testing. Fatigue tests were performed to measure potential duration and failure modes.⁷ Five mitral valve prostheses were inserted into a closed circuit at room temperature and subjected to a 90- to 95-mmHg closing pressure. A Shelhigh Fatigue Test System 300™ (Shelhigh) was used to attain a simulated heart rate of 1,500 beats/min for an uninterrupted period of 90 days, up to a total of 70 million cycles.

Mitral Valve Replacement. Nine sheep (age range, 3–4 yr; weight, 35–50 kg) underwent beating-heart mitral valve replacement. Anterolateral thoracotomy enabled access to the mediastinum. The right ventricle was cannulated through the pulmonary artery for the venous line, and through the descending aorta for the arterial line. After beating-heart posterolateral left atrial chamber dissection, the native valve was removed, and the prosthesis was implanted with use of separated stitches reinforced with pledgets.

After the operation and during a 180-day follow-up period, the sheep underwent regular echocardiographic evaluation of valve behavior and cardiovascular function. At the end of observation, the sheep were humanely killed and the valves were explanted. X-ray and atomic absorption spectrometry were used to detect possible calcium deposits on the valves.

Statistical Analysis

For statistical analysis, we used Stata® version 10.0 software (StataCorp LP; College Station, Texas). Data are expressed as mean ± SD and as number and percentage. Because of the novelty of the biomaterial under study and the limited preliminary data of this initial investigation, only the results of descriptive analysis are reported here.

Results

Results of H & E staining showed that the tuna corneal tissue was composed only of parallel collagen fibers,⁸ oriented according to force lines along the samples, without evidence of vascular or neural structures.

In regard to shrinkage, the 5 corneal samples showed 20% length contraction at 88 °C, which can be considered a satisfactory outcome after aldehyde fixation. Table I shows the comparative tensile strengths of the 5 bovine pericardial, porcine mitral valve, and tuna corneal strips; the tuna specimens were substantially similar to the bovine pericardial samples.

TABLE I. Comparison of Tensile Strength between Equal-Sized Strips of Tuna Cornea, Porcine Mitral Valve, and Bovine Pericardium

The corneal samples explanted from the rats were macroscopically unaltered after contact with murine cells. No calcium deposition was visible, and a thin vascular network had developed on the corneal surface as part of the wound-healing process. Staining with H & E revealed an intense proliferation of giant multinucleate cells along the xenografts' perimeters. Macrophages and lymphocytes were present within the collagen fibers, but no calcium salt precipitates were documented. Initial vascular neogenesis was observed in several specimens (Fig. 2). Von Kossa staining, performed to reveal calcium or calcium salt deposits, was positive in an analyzed control sample and negative in all 7 explanted specimens (Fig. 3). Morphologic diagnosis was compatible with wound-healing reactions. Foreign-body granulomatous lesions were concentrated along the sutures.

Fig. 2 Photomicrograph shows initial vascular neogenesis and little inflammatory response in a tuna corneal xenograft, 23 days after subcutaneous implantation in a rat (H & E, orig. ×3.2).

Fig. 3 Photomicrograph shows no calcium salt deposition in a tuna corneal xenograft, 23 days after subcutaneous implantation in a rat (Von Kossa stain, orig. ×10).

Table II is a summary of the hydrodynamic performance of the ten 23-mm mitral valves. The difference in effective orifice area among mitral valves was because of the slightly different valve designs. After 70 million cycles at a simulated heart rate of 1,500 beats/min, the 5 analyzed mitral valves showed no relevant morphologic or functional alterations.

TABLE II. Performance of Ten 23-mm Tuna-Cornea Mitral Valve Prostheses at Different Simulated Heart Rates

During the 180-day observation period, the 9 sheep that underwent mitral valve replacement had no complications or hemodynamic alterations, and no valvular functional abnormalities were detected on echocardiography. Examination of the explanted prostheses by x-ray and atomic absorption spectrometry revealed no mineralization (Fig. 4).

Fig. 4 Atomic absorption spectrometry shows no mineralization on a tuna-cornea mitral valve after 180 days of implantation in a sheep.

Discussion

For bioapplications, tuna cornea has several interesting characteristics: its strength, size, ready availability, and histologic composition with a low antigenic profile. By virtue of a highly pressurized marine environment, Atlantic bluefin tuna have developed strong corneal stroma—enough to withstand pressures of about 10 MPa, without protection from eyelids. Tuna corneal stroma is widely available at a relatively low cost, because tuna heads are normally a waste material. With little necessary expertise, tuna corneas could be harvested and immediately preserved in fixative solution at the fishing site. To our knowledge, no differences exist as far as tuna species is concerned; there appear to be no data on the topic. The size of the available material is the only limiting factor in the cornea-selection process for a specific application.

There are several pathologic conditions that can affect tuna eyes, most of which are due to bacterial or parasitic infection that can easily be detected by simple visual inspection.¹¹ In our study, every harvested specimen was buffered, sterilized, and embedded in paraffin. No signs of stromal alteration were noted in the samples studied.

The process after harvesting is relatively simple and yields a roughly circular, uniform tissue portion with a 40-mm diameter. This peculiar morphology—crucial to potential bioapplication—qualifies Atlantic bluefin tuna cornea as a suitable material from which to make complex 3-dimensional devices such as heart valves. Another implantation application might be as a carrier structure for stem cells, to sustain them and afford mechanical protection until new tissue is generated.

The results of our physical tests indicated that tuna corneal stroma has characteristics similar to those of bovine pericardium already in use. The porcine specimens seemed to have a more favorable profile in terms of peak load and maximum elongation. Further research is needed to confirm these preliminary data.

As suggested by our results in the rats, one of the most notable features of tuna corneal stroma is a low calcium-deposition rate.¹² This characteristic lengthens the life of xenografts and preserves their physical and mechanical properties. In contrast, implanted bovine and porcine bioprostheses are vulnerable to inevitable calcification, which alters their functionality.

Calcium deposition is directly proportional to tissue antigenicity, which determines a subsequent inflammatory activation with calcium salt deposition, as a consequence of the immune process. Tuna cornea is almost antigen-free and has very mild reaction profiles, as shown in the histologic specimens that we analyzed.

The cardiac valves that we made from Thunnus thynnus cornea behaved similarly to porcine or bovine pericardial valves. When this functional result is combined with low antigenicity and calcium deposition, tuna corneas are potentially a realistic substitute for long-term implantation. Furthermore, there are no religious contraindications to the medical use of tuna, whereas attempts to use bovine or porcine materials can evoke religious resistance in several cultures.

Although our prototypes were partially made of bovine pericardium to facilitate the production process, the objective is to manufacture a valve made solely of tuna cornea. Our initial supply of corneal samples ready for medical use was limited, because of acquisition and production processes that still needed to be implemented. As a result, we could produce valves only in small quantities. This determined the size of our sample and our reliance on components not specifically engineered for this application, such as the support rings covered with bovine pericardium.

Tuna corneal bioimplants have a potentially longer working life than do other biomaterials. Tuna cornea is suitable for long-term application, as suggested by our fatigue testing that revealed no marked defects even after 70 million cycles.

Conclusion

Tuna cornea is a widely available biomaterial with medically useful physical and mechanical properties: size, strength, and homogeneous composition. Its composition of pure collagen fiber and its low antigenicity make it very suitable for durable bioimplantation, because of low calcium deposition and negligible immune response. Further research is warranted in order to extend our comprehension of this biomaterial and to explore further medical uses.