| Περίληψη: | Cardiovascular diseases are the most recurrent cause of death in the developed world. Calcification is the condition with the highest incidence among heart diseases, accounting for almost 34 %. Calcification also poses as the leading cause for long term dysfunction of biological heart valve prostheses. Up to the present, no medical therapy to prevent the progression of the disease is available. The only treatment relies on constant clinical monitoring to indicate when a valvular replacement is required. Biological heart valve prostheses used to replace a diseased heart valve suffer from constant mechanical fatigue and calcification, leading to tearing, leaflet stenosis, or regurgitation. Techniques used to enhance the biocompatibility and structural integrity of the implant prevent the natural remodeling of the valve, potentially becoming a potential site of calcification. The deposition of calcium phosphate crystals during calcification alters the biomaterial structure and natural mechanical properties. Novel tissue-engineered prostheses try to reduce the immune response to the implant as well as preventing or retarding the progress of calcification.
In recent years, the blooming of tissue-engineered concepts presented a need for a system capable of evaluating the performance and life span of the new prostheses in a fast, accurate, and economical way. In vivo models are accurate yet time consuming and expensive ways to validate early-stage valvular developments. An in vitro model that could replicate the in vivo conditions could be a tool to pre-screen biomaterials used in tissue engineering. Such a model should consider the chemical and mechanical cues present in the human body, besides being fast, reliable, and reproducible. This work describes several models and tools to recreate the physicochemical and mechanical environment that the bioprostheses undergo in vivo.
The in vitro methods developed in the present work closely simulated the chemical environment present in vivo, where the supersaturation levels with respect to calcium phosphate mineral phases remain constant. The initial model was an improvement from the previously developed constant supersaturation reactor (CSR) by our group. This test rig maintained the supersaturation levels constant at values close to those found in vivo. pH and temperature levels were also maintained constant at the respective physiological values, providing a suitable environment for the calcium-phosphate crystals to grow in a controlled, mineral specific, highly reproducible, and reliable manner. Kinetics of the calcification process were measured to provide the means for comparison between the test samples and the control of the prevalent mineralization mechanism.
The system consisted of a temperature-controlled reactor attached to a pH-driven syringe pump that replenished the consumed ions during crystallization of the calcific deposits. The improved system included a real-time data processing screen, semi-automatic calibration screens for the pH electrode and syringe pump, and a monitoring algorithm that ensured the correct titrant volumes. The validation of the system and software was done by studying the calcification kinetics of hydroxyapatite (HAP, n = 14) seeds. Biological tissues were also assessed with respect to their calcification potential. Frozen untreated bovine and porcine pericardium (FBP, n = 4; FPP n = 3) were studied as untreated control groups, while glutaraldehyde-fixed bovine pericardium (BPG, n = 12) was studied at different supersaturation levels and compared with the data previously gathered by our group. Furthermore, the fibrosa (n = 4) and serosa (n = 4) layers of the BPG were also investigated for their calcification potential.
The system was later modified and integrated into a circulatory loop that replicated the mechanical load and flow conditions at the aortic valve location. This dynamic system consisted of a set of stations driven by a metallic bellow that provided the required ejection profile and volume for the experiments. Temperature, pH, flow, pressures, and supersaturation levels were also monitored to accurately simulate in vivo conditions. The overall calcification kinetics was monitored by the system, providing an estimate of the calcification process based on the rates of titrants added in time. The reference experiments were done using mechanical, non-reactive prosthetic valves (MHV, n = 5). Glutaraldehyde-fixed porcine aortic valves (GAV, n = 5) were later compared with decellularized specimens (DCV, n = 6) for their calcification potential. The concentration of species in solutions was regularly checked by atomic absorption spectroscopy (AAS) for calcium and UV-Visible spectrophotometry for phosphate to confirm constant solution composition. The solid samples were later assessed by scanning electron microscopy (SEM) and energy dispersive microanalysis (EDS) to identify mineral deposits.
CSR studies showed that the device developed was capable of maintaining the physicochemical parameters during mineral formation. Calcification rates for experiments with HAP were in good agreement with literature reports. Frozen tissues (FPP, FBP) did not calcify compared to tissues pre-treated with a fixating agent (BPG). The latter yielded calcification rates proportional to the supersaturation levels. The serosa surface of the BPG pericardium mineralized at lower rates than the fibrosa side at the same conditions. Dynamic in vitro experiments at mild flow conditions with GAVs showed higher mineralization rates than MHV, used as a reference. DCVs showed a higher, statistically non-significant, mineralization rate than GAV. SEM-EDS observations corroborated the results, suggesting a prevalence of deposits on the fibrosa side of the heart valve leaflets compared to the opposite ventricularis side. The location and extent of the deposits in GAV was assessed by digital mammographic X-ray imaging.
The in vitro models presented allowed for the measurements of the calcification process kinetics for different biomaterials. The developed models increased the reproducibility and accuracy of the kinetic measurements of calcification. In the current study, only the inorganic components of the blood plasma were considered, not considering the effect of the biological factors. Although in vitro models cannot fully simulate the in vivo conditions, the models presented may serve as a pre-screening method to assess the performance and structural stability of new biomaterials. The specimens investigated showed that different treatments often used to control the in vivo structural stability and immunomodulation produce a deterioration of the collagen-rich tissue structures, affecting mineralization rates. The dynamic model developed in this work simulated valvular blood flow conditions and mechanical stresses in vivo. The formation of calcific deposits suggested that the location and extent were related to the mechanical load and stress concentration areas. Supersaturation levels of blood serum with respect to calcium phosphate were related to the extent and rate of the respective mineral phase deposited in all cases. The tissue surface's structure and composition affected the calcification rates, confirming that calcification is also a surface-structure related process.
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