Type of animal
In the past, a wide variety of animal species have been used in cardiovascular research, the most common being dogs, goats, pigs, calves and sheep. No universally accepted animal model exist, but 3 species (sheep 78%, pig 9.5% and calf 3.8%) account for 95% of the animals used[3]. The physical size and the rapid growth rate of calves present problems for postoperative husbandry and non-invasive valvular functional assessment[3, 4]. Goats have been found to be a good model for valve testing, but their availability is often limited[5]. The relative susceptibility of dogs to sepsis and thrombosis has marked these animals as less than optimal as well as the moral objections raised by anti-vivisectionist movements[6, 7].
Sheep are the most utilized subjects for preclinical valve studies. Almost 80% of the animals used for valve testing are sheep[8]. Sheep have several advantages that make them ideal candidates. Sheep are widely available from venders at adequate size for valve replacement at a reasonable price. Sheep are docile and easy to handle. The somatic growth is acceptable and adult weight is around 45-100kg for female sheep, well within the husbandry limits even for the adult animals. Their docile nature allows extensive perioperative care and follow-up investigations with minimal or no sedation[7, 9]. Sheep anatomy is comparable to human with similar annulus size, systemic and pulmonary pressures[10].
While the sheep appeared to form a perfect model for valve testing, serious concern was raised when unexpectedly high rate of valve thrombosis was observed in the Medtronic Parallel valve, despite good preclinical results in sheep[2]. Several authors reported reduced platelet activity in sheep when compared to humans, rendering sheep less suited for thrombogenicity testing[11–13]. This led to the withdrawal of this valve from clinical practice and initiated the search for alternatives to sheep.
Pigs are a promising alternative, due to similar coagulation cascades, fibrinolytic mechanisms and platelet activity. Swine studies evaluating coronary artery stents and vascular prostheses are common [13–15]. Grehan et al. described their model of mitral valve replacement in pigs[16]. The model was limited by high incidence of haemorrhagic complications leading to a very high mortality, marked fibrous sheath formation and associated thrombosis and increased incidence of perivalvular defects due to somatic growth. This discouraged elaborating the swine model for future research.
Somatic growth can be substantial when compared to sheep. Pig or swine can gain up to 1-2kg body mass a day, especially in the absence of dietary restrictions[16, 17]. This somatic growth leads to prosthesis-to-animal mismatch and eventually to perivalvular leakage[16]. To bypass the somatic growth, some authors suggested the use of mini-pigs[18, 19]. Their limited availability and high obtaining cost impede their use in most centres.
Porcine vs. ovine coagulation system
There is ample literature comparing the blood-material interaction of different species[20–23]. These studies, however, do not compare how platelets from different animal models react to and form thrombi on materials. Goodman et al. compared in a well-established in vitro platelet spreading assay the reaction of human, pig and sheep platelets to four materials known for their clinical applicability and ability to induce a platelet response: isotropic PYC leaflets, polyethylene (PE), silicone rubber (SIL) and Formvar (FVR)[12]. He concluded that sheep platelets do not spread nor attach to nearly to same extent as human platelets. The material of our interest is PYC, as it is now the most commonly used material in mechanical heart valves. Sheep platelets did not reach fully spread forms on PYC (or any other material), which is in bare contrast with human platelets who spread extensively on PYC as a base for the formation of large thrombi[24, 25]. Contrarily, pig platelets spread more than human platelets on PYC and showed greater cohesion on PYC.
Mizuno et al. compared different clotting parameters (clotting time (CT), clot formation time (CFT), kinetics of fibrin formation (α-angle), maximum clot firmness (MCF), time until MCF (MCF-t)) of humans, calves, goats and pigs[26]. The clotting values of humans and minipigs were most closely related, with the exception of MCF and MCF-t. The shortened MCF-t in pigs indicates a relative hypercoagulability of pigs compared to humans, as confirmed by other authors[27, 28]. These findings strengthen our belief that the pig is the better alternative to sheep for mechanical valve testing.
Ortho- vs heterotopic
There is a choice to be made between ortho- vs heterotopic positioning of the new prosthetic valve. Orthopic position of the valve means it is situated in a native valve position. The orthotopic position has the advantage of physiological flow, normal valve movement, normal washout of the valve leaflets and hinge regions. The downside of orthotopic position is higher cost due to the indispensable need for cardiopulmonary bypass (CPB) and the higher associated mortality due to the inherent risks of cannulation and bleeding due to heparinization.
This led to the development of a heterotopic model by McKellar et al., where an extra-anatomical, modified valve conduit bypassing the native descending aorta is created[19]. The new valve is incorporated in this bypass circuit. After partially clamping the descending the aorta and establishing both anastomoses, the native descending aorta is ligated. This model has several advantages, the most important one being its cost reduction due to the absence of CPB requirement. It is easier to perform, minimizing the risk of technical errors and has fewer complications such as perioperative arrhythmias and haemorrhages. Due to no CPB, there is less systemic inflammatory response. When McKellar first published his model in 2007 and compared ortho- vs heterotopic implantation, the survival in the heterotopic cohort was 100% (7/7) at 30 days, compared to only 43% (6/14) in orthotopic position. Another main advantage is no risk of periprosthetic leakage due to somatic growth, a well-known liability of orthotopic models limiting the longevity of the follow-up period[18]. Lastly, the high frequency of infection seen in standard mitral valve replacement is reduced in the heterotopic models[19]. His findings instigated several other research groups to adopt this model, testing various anticoagulation strategies[29–33].
The main downside of the heterotopic model is the absence of normal physiological, pulsatile flow over the valve in the bypass circuit. This impedes normal valve movement due to an absent backflow, leading to valve leaflets being in a fixed ‘open’ position[19]. The important wash out of the hinge regions and struts is thus absent[34]. This tripled the weight of the thrombus when compared to the orthotopic position. While this model allows for comparing thrombus weight for different anticoagulation and antiplatelet therapies, it cannot properly assess valve function when compared to an orthotopically placed valve.
In conclusion, the heterotopic model is most useful for testing new non-warfarin alternatives, a rapidly emerging field requiring robust animal models. For testing of new valve designs and materials, normal valve movement and wash-out remains paramount which is only possible in a truly orthotopic model.
Valve position
Several possibilities are to be discussed when considering a truly orthotopic approach. In humans, most commonly replaced valve is the aortic valve followed by the mitral valve. Ideally, the researched valve should be positioned accordingly. However, some concerns are to be addressed.
Aortic position in porcine models is avoided due to the high mortality associated with aortic surgery in pigs. The aortic wall is short and fragile compared to other species, with a high risk of shearing and rupture of the aortic wall despite proper suturing techniques.
The mitral position is equally related to a relative high mortality. Grehan et al. concluded, due to a 50% (11/22) mortality, that the porcine model was unsuited for mechanical valve implantation[16]. This discouraged further experiments in this direction. However, Demin et al. performed mitral valve replacement with a mechanical valve and noted a mortality of 2/16 at 1 month, increasing to 8/16 at 3 months follow-up[35]. Umashanker et al. had only one early death in a series of 12 miniature pigs after mitral valve replacement[36]. However, in the non-anticoagulated control group, animals suffered from pulmonary oedema and hydrothorax due to mitral valve thrombosis, leading to early euthanization of 3 animals and only 2 control animals completing the designated 20 week follow-up period.
Smerup et al. had significantly lower mortality after implantation of a mechanical valve in mitral position in 16 pigs, with 14 of these completing the follow-up period[18]. McKellar et al. developed and compared the heterotopic model to the orthotopic mitral valve replacement model. All animals in the heterotopic model completed the follow-up period, but only 6/14 in the orthotopic model completed the follow-up period[19].
These findings conclude that while the mitral position is deemed second best compared to aorta, it is not always as feasible and robust. A lot of survival variability within different research groups exists, and the animals may suffer from a mitral valve thrombosis leading to hydrothorax and pulmonary oedema, impeding completion of follow up. This especially seems the case in the often non-anticoagulated control groups.
The model we aim to design needs to be more robust and reliable. It is known that pulmonary valve replacement with a mechanical valve causes thrombotic changes in these valves[37]. In a previous study at our centre, 9 Yucatan pigs where implanted with On-X valves in pulmonary position[38]. All animals completed the follow-up period. It appeared that the pulmonary position was highly thrombotic but that normal valve movement was preserved. Another benefit of the pulmonary position is that is can be done without the need of cardioplegia, minimizing weaning and rhythm difficulties which are notorious in pigs. This minimizes operative risk and improves survivability. Besides, the consequences of a thrombosed pulmonary valve are generally well tolerated, compared to the dire effects of a thrombosed mitral valve[19, 36].