Evaluation of In Vivo Biocompatibility in Preclinical Studies of a Finger Implant Medical Device Correlated with Mechanical Properties and Microstructure.

The aim of the experiment was to evaluate the biocompatibility of four 3D-printed biomaterials planned for use in the surgical treatment of finger amputees: Ti-6Al-4 V (Ti64), ZrO2-Al2O3 ceramic material (ATZ20), and osteoconductive (anodized Ti64) and antibacterial (Hydroxyapatite, HAp) coatings that adhere well to materials dedicated to finger bone implants. The work concerns the correlation of mechanical, microstructural, and biological properties of dedicated materials. Biological tests consisted of determining the overall cytotoxicity of the organism on the basis of in vivo tests carried out in accordance with the ISO 10993-6 and ISO 10993-11 standards. Clinical observations followed by diagnostic examinations, histopathological evaluation, and biochemical characterization showed no significant differences between control and tested groups of animals. The wound healed without complication, and no pathological effects were found. The wear test showed the fragility of the hydroxyapatite thin layer and the mechanical stability of the zirconia-based ceramic substrate. Electron microscopy observations revealed the layered structure of tested substrates and coatings.

Characterization of biomaterials with reference to biocompatibility dedicated for patient-specific finger implants.

PURPOSE: The research was focused on determining basic mechanical properties, surface, and phase structure taking into consideration basic cytotoxicity analysis towards human cells. METHODS: Biological tests were performed on human C-12302 fibroblasts cells using 3D-printed Ti6Al4V alloy (Ti64), produced by laser-based powder bed fusion (LB-PBF) and Alumina Toughened Zirconia 20 (ATZ20), produced by lithography-based ceramic manufacturing (LCM). Surface modifications included electropolishing and hydroxyapatite or hydroxyapatite/zinc coating. Structure analysis was carried out using a variety of techniques such as X-Ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM), followed by mechanical properties evaluation using nanoindentation testing. RESULTS: Samples subjected to surface modifications showed diversity among surface and phase structure and mechanical properties. However, the cytotoxicity towards tested cells was not significantly higher than the control. Though, a trend was noted among the materials analysed, indicating that HAp/Zn coating on Ti64 and ATZ20 resulted in the best biological performance increasing cell survivability by more than 10%. CONCLUSIONS: Hydroxyapatite coating on Ti64 and ATZ20 resulted in the best biological properties. Tested materials are suitable for in vivo toxicity testin.

Antimicrobial materials with improved efficacy dedicated to large craniofacial bone defects after tumor resection.

The research was focused on alternative treatment techniques, separating immediate and long-term reconstruction stages. The work involved development of ceramic materials dedicated to reconstruction of the temporomandibular joint area. They were based on alumina (aluminum oxide) and characterized by varying porosities. A broad spectrum of studies was conducted to test the proposed material and determine its suitability for mandibular reconstruction. They compared the effects of substrate properties of ceramic materials in terms of biocompatibility, microbiology and systemic toxicity in in vivo studies. Finally it was concluded that Alumina LithaLox 350D is best suited for jawbone implants.

Hemocompatibile Thin Films Assessed under Blood Flow Shear Forces.

The aim of this study was to minimize the risk of life-threatening thromboembolism in the ventricle through the use of a new biomimetic heart valve based on metal-polymer composites. Finite volume element simulations of blood adhesion to the material were carried out, encompassing radial flow and the cone and plane test together with determination of the effect of boundary conditions. Both tilt-disc and bicuspid valves do not have optimized blood flow due to their design based on rigid valve materials (leaflet made of pyrolytic carbon). The main objective was the development of materials with specific properties dedicated to contact with blood. Materials were evaluated by dynamic tests using blood, concentrates, and whole human blood. Hemostability tests under hydrodynamic conditions were related to the mechanical properties of thin-film materials obtained from tribological tests. The quality of the coatings was high enough to avoid damage to the coating even as they were exposed up to maximum loading. Analysis towards blood concentrates of the hydrogenated carbon sample and the nitrogen-doped hydrogenated carbon sample revealed that the interaction of the coating with erythrocytes was the strongest. Hemocompatibility evaluation under hydrodynamic conditions confirmed very good properties of the developed coatings.

Interdisciplinary Methods for Zoonotic Tissue Acellularization for Natural Heart Valve Substitute of Biomimetic Materials.

The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.

Experimental and numerical analysis of blood flow in roughness impact-R test.

PURPOSE: The present paper covers simulation of blood flow in a roughness impact-R test model to anticipate the hemodynamic conditions of adhesion of blood elements to the modified surface. It was performed using numerical modelling of this process. The aim of these simulations was to create a surface morphology that stimulates the adhesion of blood elements to the surface of base plate of impact-R test. METHODS: The morphology of base plate of impact-R test was developed using a vacuum powder sintering of commercial purity titanium powder (CP-Ti) on Ti6Al7Nb substrate. The finite volume method (FVM) and disperse particle method (DPM) were applied to develop the target model of a roughness impact-R test. The morphology of modified surfaces was documented with digital microscope and SEM (scanning electron microscopy). RESULTS: The impact-R test developed using the two-phase blood model performed on regularly structured base plate resulted in shear stress values higher than the analogous for the model lacking such modification. The most significant reduction in maximum values of shear stress occurred in case of the DPM model and especially in the model with regular structures. CONCLUSIONS: The proposed models are very effective in modeling of the analysis of blood flow in roughness impact-R test.

Discrete phase model of blood flow in a roughness microchannel simulating the formation of pseudointima.

PURPOSE: The goal of the present study was the development of discrete phase model to simulate the phenomenon of backfilling a morphologically complex surface by red blood cells (RBCs) in a flow microchannel and to anticipate the conditions of forming a pseudointima. The objective of the experimental studies that inspired the development of the simulation was to create a surface that stimulates the formation of the pseudointima layer. METHODS: The finite volume method (FVM) and discrete particle method (DPM) were applied to develop the target model. In addition, a mixture model and a roughness model of bottom layer were tested in the present study to show their influence on simulation the phenomenon of backfilling a morphologically complex surface by RBCs in a flow microchannel. RESULTS: Numerical models were developed including: a) FVM models to compare the effect of applying boundary conditions with/without roughness and cubes, as well as the analysis of their influence on blood velocity and shear stress; b) mixture models to compare the effect of applying different boundary conditions and cubes on computed results; c) DPM models to compare the effect of applying and not applying roughness as a boundary condition; d) DPM models with a morphologically complex surface and RBCs collisions to present RBCs concentration, velocity and time distributions during flow in a channel. CONCLUSIONS: The analysis carried out for the developed numerical models indicates that DPM model with cubes computes the best results. It also shows the backfilling of a morphologically complex surface of the bottom microchannel with RBCs.

Antibacterial Optimization of Highly Deformed Titanium Alloys for Spinal Implants.

The goal of the work was to develop materials dedicated to spine surgery that minimized the potential for infection originating from the transfer of bacteria during long surgeries. The bacteria form biofilms, causing implant loosening, pain and finally, a risk of paralysis for patients. Our strategy focused both on improvement of antibacterial properties against bacteria adhesion and on wear and corrosion resistance of tools for spine surgery. Further, a ~35% decrease in implant and tool dimensions was expected by introducing ultrahigh-strength titanium alloys for less-invasive surgeries. The tested materials, in the form of thin, multi-layered coatings, showed nanocrystalline microstructures. Performed direct-cytotoxicity studies (including lactate dehydrogenase activity measurement) showed that there was a low probability of adverse effects on surrounding SAOS-2 (Homo sapiens bone osteosarcoma) cells. The microbiological studies (e.g., ISO 22196 contact tests) showed that implanting Ag nanoparticles into Ti/TixN coatings inhibited the growth of E. coli and S. aureus cells and reduced their adhesion to the material surface. These findings suggest that Ag-nanoparticles present in implant coatings may potentially minimize infection risk and lower inherent stress.

Surface modification of polyurethane with eptifibatide-loaded degradable nanoparticles reducing risk of blood coagulation.

The main purpose of the work was to develop a drug releasing coatings on the surface of medical devices exposed to blood flow, what should enable effective inhibition of blood coagulation process. As a part of the work, the process of encapsulating the anticoagulant drug eptifibatide (EPT) in poly(DL-lactic-co-glycolic acid) (PLGA) nanoparticles was developed. EPT encapsulation efficiency was 29.1 ± 2.1%, while the EPT loading percentage in the nanoparticles was 4.2 ± 0.3%. The PLGA nanoparticles were suspended in a polyanion solution (hyaluronic acid (HA)) and deposited on the surface-treated thermoplastic polyurethane (TPU) by a layer-by-layer method. As a polycation poly-L-lysine (PLL) was used. The influence of released EPT on the activation of the coagulation system was analyzed using dynamic blood tester. Performed experiments show an effective delivery of the drug to the bloodstream and low risk of platelets (membrane receptor) activation. The dynamic blood test process, including its physical phenomenon, was described using numerical methods, i.e. a finite volume cone-and-plate test model as well as non-Newtonian blood models. The values of shear stress and blood flow velocity under the fast-rotating cone were computed applying boundary conditions of cylinder wall imitating blood-nanomaterial interaction. Implementing boundary conditions as initial shear stress values of bottom cylinder wall resulted in the increase of shear stress in blood under rotating cone. The developed system combining drug eluting polymeric nanoparticles with the polyelectrolyte "layer-by-layer" coating can be easily introduced to medical implants of various shape, with the advantages of resorbable drug carriers allowing for local and controllable delivery of anti-thrombogenic drugs.

In vitro haemocompatibility assessment of acrylic acid deposited on solid, polyurethane substrate.

The main purpose of the work was to assess the haemocompatible properties of polyurethane discs with a modified surface dedicated to cardiovascular system regeneration. They were coated with acrylic acid-based material to inhibit the activation of the blood coagulation cascade. This coating improved the wettability of the material, leading to the prevention of protein adsorption on the surface. The blood-material interaction was analyzed in dynamic conditions with a specially designed tester, which helps to control blood-material interaction under high shear stress conditions. The corresponding numerical model of the tester was also developed by finite volume method (FVM). The 3D FVM model allows the determination of shear stresses applying different flow and boundary conditions representing blood-material interactions. The haemocompatibility analyses were performed through in vitro tests using a blood flow simulator. They revealed a low probability of activation of blood coagulation and low leukocyte activation. The original mechanical set-up to test the blood-material interaction helped to prove that acrylic acid-based coatings expressed good haemocompatible properties.

The interaction of laser radiation with tissue in the aspect of generating the process of decellularization in the preparation of animal origin autologous tissue.

PURPOSE: The aim of the work was to create an appropriate substrate for organ transplantation using bioactive tissue-based scaffold populated by cells of the graft recipient. The purpose of the modeling was to investigate the mechanical effects of wave loading of aortic and pulmonary tissue material. METHODS: The biological properties of tissues of aortic and pulmonary valves were modified by the process of decellularization. The host cells were removed by various physical methods with focus on minimal degradation of the extracellular matrix. Thus, the decellularization process was controlled by histological methods. The tissue decellularization process was simulated by finite element modelling. RESULTS: The mechanical results represented by a displacement at the center of the sample were coherent and the heterogeneity of the distribution of the caves on the surface of the samples was confirmed, both by experiment and in the simulation by the alternate occurrence of local minima and maxima. The latter results from the uneven removal of cells from the effect of the wave causing decellularization were also predicted by the numerical model. Laser radiation had a destructive effect on the components of the extracellular matrix (e.g., collagen and elastic fibers), mainly depending on the fluence and number of pulses in a single exposure. CONCLUSIONS: The differences between the valve tissue materials were shown, and the impact of the process of decellularization on the properties of the tissues was analyzed. It should be emphasized that due to low absorption and high scattering, laser radiation can deeply penetrate the tissue, which allows for effective decellularization process in the entire volume of irradiated tissue.

Development of multi-phase models of blood flow for medium-sized vessels with stenosis.

PURPOSE: The purpose of the work was to develop two-phase non-Newtonian blood models for medium-sized vessels with stenosis using power law and Herschel-Bulkley models. METHODS: The blood flow was simulated in 3D models of blood vessels with 60% stenosis. The Ansys Fluent software was applied to implement the two-phase non-Newtonian blood models. In the present paper, the mixture model was selected to model the two phases of blood: plasma and red blood cells. RESULTS: Simulations were carried out for four blood models: a) single-phase non-Newtonian, b) two-phase non-Newtonian, c) two-phase Herschel-Bulkley with yield stress 0 mPa, and d) two-phase Herschel-Bulkley with yield stress 10 mPa for blood plasma, while flow took place in vessel with stenosis 60%. Presentation of results in this paper shows that stenosis can substantially affect blood flow in the artery, causing variations of velocity and wall shear stress. Thus, the results in the present paper are maximum values of blood velocity and wall shear stress, profiles and distributions of blood velocity and wall shear stress computed for single- and two-phase blood models for medium-sized vessels with stenosis. CONCLUSIONS: For the two-phase blood models the influence of initial velocity on blood flow in the stenosis zone is not observed, the velocity profiles are symmetric and parabolic. Contrary, for the single phase non-Newtonian blood model, the velocity profile is flat in the stenosis zone and distribution of velocity is disturbed just behind the stenosis zone. The shapes of wall shear stress profiles for two-phase blood models are similar and symmetric in the center of stenosis. The biggest differences in maximum values of velocities and wall shear stress are observed between single phase non-Newtonian power law and Herschel-Bulkley blood models. The comparison of the obtained results with the literature indicates that the two-phase Herschel-Bulkley model is the most suitable for describing flow in medium-sized vessels with stenosis.

Qualitative and quantitative interpretation of SEM image using digital image processing.

The aim of the this study is improvement of qualitative and quantitative analysis of scanning electron microscope micrographs by development of computer program, which enables automatic crack analysis of scanning electron microscopy (SEM) micrographs. Micromechanical tests of pneumatic ventricular assist devices result in a large number of micrographs. Therefore, the analysis must be automatic. Tests for athrombogenic titanium nitride/gold coatings deposited on polymeric substrates (Bionate II) are performed. These tests include microshear, microtension and fatigue analysis. Anisotropic surface defects observed in the SEM micrographs require support for qualitative and quantitative interpretation. Improvement of qualitative analysis of scanning electron microscope images was achieved by a set of computational tools that includes binarization, simplified expanding, expanding, simple image statistic thresholding, the filters Laplacian 1, and Laplacian 2, Otsu and reverse binarization. Several modifications of the known image processing techniques and combinations of the selected image processing techniques were applied. The introduced quantitative analysis of digital scanning electron microscope images enables computation of stereological parameters such as area, crack angle, crack length, and total crack length per unit area. This study also compares the functionality of the developed computer program of digital image processing with existing applications. The described pre- and postprocessing may be helpful in scanning electron microscopy and transmission electron microscopy surface investigations.

Digital image correlation of coated and uncoated Religa Heart_Ext ventricular assist device.

The digital image correlation is used to estimate influence of deposited heamocompatible coatings (gold and titanium nitride) on mechanical response of ventricular assist device Religa Heart_Ext made of Bionate II (thermoplastic polycarbonate urethane) under working conditions by comparison of the coated Religa Heart_Ext with uncoated Religa Heart_Ext. The DIC is applied for experimental investigation of the strains and displacements distribution on external surface of the blood chamber of ventricular assist device during loading. The experiment was conducted in a hydraulic system with water at operating temperatures of 25 and 37 °C, as well as under static pressures: 80, 120, 180, 220 and 280 mmHg, and static underpressures: -25, -45, -75 mmHg. The subsequent images were taken after stabilization of pressure on a set level. The applied research method shows that the nano-coating of 30 nm in thickness significantly affects deformation of the blood chamber of Religa Heart_Ext in macro scale. The proposed composition of coatings increases strain on external surface of the ventricular assist device.

Shape optimisation of a ventricular assist device using a VADFEM computer program.

The Polish ventricular assist device, POLVAD_EXT, is made of a polymer designed to be covered with a nanocoating of titanium nitride to improve haemocompatibility. A loss of cohesion can occur between the coating and the substrate. An analysis of stress and strain states in a multi-scale model of the blood chamber was performed in the finite element computer program, VADFEM. The multiscale model is composed of a macro model of the blood chamber and a micro model of the titanium nitride (TiN) deposited on the polymer. The finite element method and the goal function, based on the triaxiality factor, are used to solve the problems formulated. The theories of non-linear elasticity and elasto-plasticity are applied. The goal of the paper is to optimise the construction of the POLVAD_EXT with respect to shape parameters.

Numerical modelling and verification of Polish ventricular assist device.

The developed multiscale model of blood chamber of POLVAD (Polish ventricular assist device) was introduced. The tension test for polymer and digital image correlation (DIC) were performed for verification of the strains and displacements obtained in the numerical model of POLVAD_EXT. The numerical simulations were carried out in conditions given in the experiment to compare the results obtained on external surfaces of blood chamber of the POLVAD_EXT. The examined polymer applied in the POLVADs is sensitive to changes of temperature and this observation is considered in all prepared numerical models. The comparison of experimental and numerical results shows acceptable coincidence. There are some heterogeneous distributions of strains in experiment with respect to analysis of computed parameters. The comparison of two versions of blood chambers (POLVAD and POLVAD_EXT) in numerical analysis shows that POLVAD_EXT construction is better with respect to analysis of strain and stress. The maximum values of computed parameters are located in the regions between connectors on the internal surfaces of blood chambers of POLVAD.

Comparative analysis of ventricular assist devices (POLVAD and POLVAD_EXT) based on multiscale FEM model.

The prosthesis - pulsatory ventricular assist device (VAD) - is made of polyurethane (PU) and biocompatible TiN deposited by pulsed laser deposition (PLD) method. The paper discusses the numerical modelling and computer-aided design of such an artificial organ. Two types of VADs: POLVAD and POLVAD_EXT are investigated. The main tasks and assumptions of the computer program developed are presented. The multiscale model of VAD based on finite element method (FEM) is introduced and the analysis of the stress-strain state in macroscale for the blood chamber in both versions of VAD is shown, as well as the verification of the results calculated by applying ABAQUS, a commercial FEM code. The FEM code developed is based on a new approach to the simulation of multilayer materials obtained by using PLD method. The model in microscale includes two components, i.e., model of initial stresses (residual stress) caused by the deposition process and simulation of active loadings observed in the blood chamber of POLVAD and POLVAD_EXT. The computed distributions of stresses and strains in macro- and microscales are helpful in defining precisely the regions of blood chamber, which can be defined as the failure-source areas.

Microscale analysis of strain-stress state for TiN nanocoating of POLVAD and POLVAD_EXT.

The main purpose of the research was to develop the micromodel of biocompatible titanium nitride nanocoating deposited on polymer by pulsed laser deposition method in blood chambers of Polish ventricular assist devices: POLVAD and POLVAD_EXT. The analysis of the parameters of micromodel crucial for the phenomenon of loss of cohesion occurring between coating and substrate was carried out as well. The micromodel takes into account residual stress, material model of nanocoating, stress resulting from blood pressure in chamber, the thickness of coating and wave parameters of nanocoating (wavelength and antinode). The investigation shows that thickness and residual stress are the most influential parameters. The phenomenon of the loss of cohesion will be observed more frequently for thicker coatings with higher residual stresses.

Multiscale FEM model of artificial heart chamber composed of nanocoatings.

The purpose of the present work was to construct the multiscale FEM model of artificial heart chamber composed of nanocoatings. This goal is reached by the development of the FEM macromodel of artificial blood chamber using the commercial code and by the creation of the micromodel based on our own FEM code. The latter uses strain state obtained after loading in macromodel. The loci of failure initiation in the material of chamber were precisely determined and these results are used as an input data for the new micromodel.