DurImplant – Analysis of implant behavior after bioreactor maturation in vitro
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Background
A reduced durability of implants due to pathological calcification tendency is particularly pronounced in the field of avital bioprosthetic heart valves, which primarily affects the aortic position. In principle, two different implant groups are available for replacing diseased or defective heart valves: mechanical valves made of metal (titanium) or pyrolytic carbon and biological heart valves from human donors (homografts) or from animal (avital xenogeneic) tissue (porcine valves, porcine or bovine pericardial valves). Although mechanical valve prostheses generally have a life-time durability, show no calcification, but due to the coagulation-activating foreign material surface, they are more prone to thrombus formation than biological valves. This necessitates lifelong anticoagulation of the patients. After avital bioprosthetic valve replacement only a maximal three-month anticoagulation is required, but here the progressive aging up to the failure of the valve, due to calcification-related stiffening of the leaflets after about 10 - 12 years, is a serious problem. As cause of this phenomenon, both mechanical, chemical and biological processes are accountable.
Decades of research in this field has led to various explanations regarding the process and approaches to valve conditioning, but so far no standardized and sustainably effective prevention or therapeutic measures have been produced. The explanatory approaches regarding influencing factors are still discussed controversially, such as: the question of whether the initial crystallization process is a purely physicochemical precipitation of metastable solution (blood) with possibly subsequent cell and protein involvement, or whether the nucleation itself is already controlled by the cells. In the healthy organism, a balance of calcification inducers and inhibitors is assumed to prevent mineralization at the undesirable site. As cause for the pathological occurrence of calcifications a disorder of the inhibition mechanism is held responsible. It is likely that the interaction of a number of effects is responsible for pathological crystallization, from purely physicochemical processes over biologically induced nucleation to cell-controlled calcium phosphate deposition.
Just at this point, the overall project PAK 961, which is concerned with the generation of a maturation model for biohybrid implants, formulates the hypothesis that biohybrid heart valve prostheses with a vital, functionally active endothelium constitute a natural barrier to calcification. This is supported by the assumption that just the glutaraldehyde fixation of avital bioprosthetic standard valves prevents their permanent re-endothelialization and thereby promotes calcification. Other relevant factors of both mechanical and biological nature are the mechanical stress, the surface stress and structure of the valve material, as well as the endogenous, often anamnestic inflammation processes.
Objective
The aim of project DurImplant is the development of an in vitro methodology for the investigation of the durability of biohybrid implants with main focus on the propensity to calcification as a decisive limiting factor of the implant lifetime and function. For this purpose a biomimetic test environment as close to reality as possible should be created. To test avital bioprosthetic heart valves, the institute already has a dynamic durability test system, which has hitherto been based on a purely mechanical and physicochemical basis, but does not offer a suitable test environment for cellularized material either from a physiological or cytotoxicological point of view. This aspect will be addressed by the development of a biohybrid-compatible test system. Important parameters with regard to the test environment are the cell compatibility with simultaneous calcification potential of the fluid, physiological pH, temperature and flow conditions as well as the online detection of the formation and progression of calcifications. In addition to the test environment, individual factors, whose role in calcification have not yet been fully clarified, have to be taken into account. These include blood, inflammation, material and surface parameters, calcification inducers and inhibitors, and not least cell involvement.
Methods
The aforementioned factors are to be investigated first in a miniaturized flow chamber system on material patches. The advantages of a miniaturized system for research purposes consist i) in a better flexibility in the analysis of variable fluids and fluid-substrate combinations, so that individual calcification parameters can be investigated separately, ii) a constant composition of the fluid in the test chamber through an open flow system (single-way passape) and iii) the possibility of parallel operation of multiple flow chambers with microscopic online tracking of the calcification process. Comparably small amounts of substrate (sizes) allow the testing of different materials with and without living cells. For this purpose, a corresponding flow chamber is specially developed and built at CVE. For testing and detection the Fetuin-A-based imaging, inflammatory reporter cell assays and (patho)-biomimetic fluids with high-molecular-weight mineral precursors developed at the Biointerface Laboratory at Helmholtz-Institute for Biomedical Engineering (ZMG) are used. The influence of surface structures and anomalies on the calcification behavior of the biohybrid constructs will be investigated using optical coherence tomography of the starting materials and correlation with the calcification patterns occurring after testing.
| Funding | German research foundation Project number 403041552 |
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| Sub-project P5 of PAK 961: DFG project "Towards a model based control of biohybrid implant maturation" |
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