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.

Biocompatibility testing of composite biomaterial designed for a new petal valve construction for pulsatile ventricular assist device.

This paper presents the results of biocompatibility testing performed on several biomaterial variants for manufacturing a newly designed petal valve intended for use in a pulsatile ventricular assist device or blood pump. Both physical vapor deposition (PVD) and plasma-enhanced chemical vapor deposition (PECVD) were used to coat titanium-based substrates with hydrogenated tetrahedral amorphous carbon (ta-C:H) or amorphous hydrogenated carbon (a-C:H and a-C:H, N). Experiments were carried out using whole human blood under arterial shear stress conditions in a cone-plate analyzer (ap. 1800 1/s). In most cases, tested coatings showed good or very good haemocompatibility. Type a-C:H, N coating proved to be superior in terms of activation, risk of aggregation, and the effects of generating microparticles of apoptotic origin, and also demonstrated excellent mechanical properties. Therefore, a-C:H, N coatings were selected for further in vivo studies. In vivo animal studies were carried out according to the ISO 10993 standard. Intradermal reactivity was assessed in three rabbits and sub-acute toxicity and local effects after implantation were examined in 12 rabbits. Based on postmortem examination, no organ failure or wound tissue damage occurred during the required period of observation. In summary, our investigations demonstrated high biocompatibility of the biomaterials in relation to thrombogenicity, toxicity, and wound healing. Prototypes of the petal valves were manufactured and mounted on the pulsatile ventricular assist device. Hydrodynamic features and impact on red blood cells (hemolysis) as well as coagulation (systemic thrombogenicity) were assessed in whole blood.

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.

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.

Design, Manufacturing Technology and In-Vitro Evaluation of Original, Polyurethane, Petal Valves for Application in Pulsating Ventricular Assist Devices.

Minimizing of the life-threatening thrombo-emboli formation in pulsatile heart assist devices by a new biomimetic heart valve design is presently one of the most important problems in medicine. As part of this work, an original valve structure was proposed intended for pneumatic, extracorporeal ventricular assist devices. The valve design allows for direct integration with other parts of the pulsating blood pump. Strengthening in the form of the titanium or steel frame has been introduced into the polyurethane lagging, which allows for maintaining material continuity and eliminating the risk of blood clotting. The prototype of the valve was made by the injection molding method assisted by numerical simulation of this process. The prototype was introduced into a modified pulsating, extracorporeal heart assist pump ReligaHeart EXT (developed for tilting disc valves) and examined in-vitro using the "artificial patient" model in order to determine hydrodynamic properties of the valve in the environment similar to physiological conditions. Fundamental blood tests, like hemolysis and thrombogenicity have been carried out. Very low backflow through the closed valve was observed despite their slight distortion due to pressure. On the basis of immunofluorescence tests, only slight activation of platelets was found on the inlet valve and slight increased risk of clotting of the outlet valve commissures as a result of poor valve leaflets assembling in the prototype device. No blood hemolysis was observed. Few of the clots formed only in places where the valve surfaces were not smooth enough.

Patient specific implants for jawbone reconstruction after tumor resection.

In case of benign and malignant tumours affecting the maxillofacial region, the resection of jawbone reflects the standard therapy in more than 5.000 cases per year within the European Union. The resulting large bone defects lead to scarred, mangled facial appearance, loss of mastication and probably speech, requiring aesthetic and functional surgery as a basis for physical and physiological rehabilitation. Although autologous vascularized bone autografts reflect the current golden standard, the portion of bone available for the procedure is limited and subsequent high-dose anti-cancer chemo-/radiotherapy can lead to local tissue necrosis. Autologous vascularized bone from fibular or iliac-crest autografts is current golden standard in jawbone resection post-treatment, however, the portion of transplantable bone is limited and subsequent high-dose anti-cancer chemo-/radiotherapy often results in tissue necrosis Our research focuses on alternative treatment techniques: tissue reconstruction via novel patient-specifically manufactured maxillofacial implant that stimulates bone tissue growth. The planned neoformation of vascularized bone in such implants within the patient's own body as "bioreactor" is the safest approach in tissue engineering. The works described herein included the design of the metallic substrate of the implant with the use of computed tomography basing on real patients scans and then 3D-printing the substrates from the Ti6Al7Nb powder. The metal core was then evaluated in terms of structural characteristic, cytotoxicity and gene expression through the in vitro tests. Further experiments were focused on fabrication of the biocompatible coating for outer surface of the bone implant that would enhance the healing process and accelerate the tissue growth. Functional polymeric granulate dedicated for osteoconductive, osteoinductive and osteogenesis properties were elaborated. Another approach including the coating for the implant surface with two-phase biocompatible layer including polymeric microspheres and hydrogel carrier, which would provide long-time release of bone and cartilage growth factors around the implant were also done. The polymeric granulate containing ßTCP improved bone cells growth, but it some modification has to be done in order to improve structural pores to ensure for better osteoconductivity. The biocompatible coating including PVP hydrogel and polymeric microspheres is still in the development process.

Rolling or Two-Stage Aggregation of Platelets on the Surface of Thin Ceramic Coatings under in Vitro Simulated Blood Flow Conditions.

The process of modern cardiovascular device fabrication should always be associated with an investigation of how surface properties modulate its hemocompatibility through plasma protein adsorption as well as blood morphotic element activation and adhesion. In this work, a package of novel assays was used to correlate the physicochemical properties of thin ceramic coatings with hemocompatibility under dynamic conditions. Different variants of carbon-based films were prepared on polymer substrates using the magnetron sputtering method. The microstructural, mechanical, and surface physicochemical tests were performed to characterize the coatings, followed by investigation of whole human blood quality changes under blood flow conditions using the "Impact R" test, tubes' tester, and radial flow chamber assay. The applied methodology allowed us to determine that aggregate formation on hydrophobic and hydrophilic carbon-based coatings may follow one of the two different mechanisms dependent on the type and conformational changes of adsorbed blood plasma proteins.

Surface modification of metallic materials designed for a new generation of artificial heart valves.

PURPOSE:: The main goal of this work was to develop haemocompatibile thin film materials dedicated to novel flexible mechanical heart valves intended for pulsatile ventricle assist devices. METHODS:: The studies performed have led to the selection of a material for the surface modification of the metallic scaffold. Haemocompatible, biofunctional, ultra-elastic, thin carbon-based coatings were proposed. The surface was designed to eliminate thrombogenic and microbial construction by a reduction in turbulence and sufficient washing of the biofunctional-adapted surfaces, thus allowing for extended use for temporary heart support. The article presents the influence of the mechanical properties of coatings and their influence on the haemocompatibility. In this study, we investigated a simplified model of the whole blood shear stress based on a cone and plate rotational viscometer. Several indices of platelet activation were analysed, including platelet and granulocyte-platelet aggregates, platelet activation markers and platelet-derived microparticles. RESULTS:: The shear stress induced a platelet aggregate count in the range from 2% to 30% of the CD61 positive cells. For polyurethane (PU), the average value of platelet aggregates was on the level of 7%. The analyses have demonstrated that the cytometric methods of the direct determination of platelet-derived microparticles in plasma are reproducible and reliable. Considering the generation of microparticles on the tested coatings under hydrodynamic conditions, the best properties were observed for the coating a-C:H,N. CONCLUSION:: The results indicate that a-C:H-based coatings with the thickness of 110 nm do not induce an immune response and do not influence the origin of platelet microparticle formation; thus, these type of coatings are the most promising for the parts which are planned to withstand blood contact under the high value of shear stress.

Biomimetics in thin film design: Niche-like wrinkles designed for i-cell progenitor cell differentiation.

The future and development of science are in interdisciplinary areas, such as biomedical engineering. Self-assembled structures, similar to stem cell niches, inhibit rapid cellular division processes and enable the capture of stem cells from blood flow. By modifying the surface topography and stiffness properties, progenitor cells were differentiated towards the formation of endothelial cell monolayers to effectively inhibit the coagulation cascade. Wrinkled material layers in the form of thin polymeric coatings were prepared. An optimized surface topography led to proper cell differentiation and influenced the appropriate formation of endothelial cell monolayers. Blood activation was decelerated by the formed endothelium.

Effects of the surface modification of polyurethane substrates on genotoxicity and blood activation processes.

The aim of this study was to determine the mutagenic and thrombogenic potential of a material composed of a thin coating deposited on a polymeric substrate. In this work, a surface was modified in a manner that would mimic the function of cellular niches. Finally, the surfaces should actively capture and differentiate progenitor cells from the blood stream. Thin films with 10 to 500nm thicknesses were deposited by unbalanced, pulsed DC magnetron sputtering on smooth polyurethane. Such high energy conditions led to a stiffening of the polymer surface layers by pseudodiffusion during the initial stages of film growth. Both the high intrinsic film stress due to high energy film growth and the huge difference in the elastic properties of the films and polymer substrates resulted in hierarchical and self-adapting nanowrinkling. Surface modifications of synthetic materials for future use in regeneration of the circulatory system must be tested in terms of their thrombogenicity and mutagenicity. Point mutations in many cases can lead to many serious haematologic complications. Genotoxicity was determined by testing for reverse histidine mutations in selected strains of Salmonella typhimurium. The analysis was performed in the presence and absence of metabolic activation system S9 containing liver microsomal fraction of rats. Based on these results, no mutagenicity of the tested material was observed. The interaction of blood and the material under dynamic conditions was described. Blood from above the analysed surface was collected after the test, and the quality of the blood was assessed along with the type of cellular response to the surface. In the obtained results of the coagulation processes, it was found that the tested material reduced the process of platelet activation under hydrodynamic conditions in comparison to the control material, polyurethane.

Diamond-like carbon coatings with zirconium-containing interlayers for orthopedic implants.

Six types of diamond-like carbon (DLC) coatings with zirconium (Zr)-containing interlayers on titanium alloy (Ti-6Al-4V) were investigated for improving the biotribological performance of orthopedic implants. The coatings consist of three layers: above the substrate a layer stack of 32 alternating Zr and ZrN sublayers (Zr:ZrN), followed by a layer comprised of Zr and DLC (Zr:DLC), and finally a N-doped DLC layer. The Zr:ZrN layer is designed for increasing load carrying capacity and corrosion resistance; the Zr:DLC layer is for gradual transition of stress, thus enhancing layer adhesion; and the N-doped DLC layer is for decreasing friction, squeaking noises and wear. Biotribological experiments were performed in simulated body fluid employing a ball-on-disc contact with a Si3N4 ball and a rotational oscillating motion to mimic hip motion in terms of gait angle, dynamic contact pressures, speed and body temperature. The results showed that the Zr:DLC layer has a substantial influence on eliminating delamination of the DLC from the substrates. The DLC/Si3N4 pairs significantly reduced friction coefficient, squeaking noise and wear of both the Si3N4 balls and the discs compared to those of the Ti-6Al-4V/Si3N4 pair after testing for a duration that is equivalent to one year of hip motion in vivo.

In vitro hemocompatibility on thin ceramic and hydrogel films deposited on polymer substrate performed in arterial flow conditions.

Hydrogel coatings were stabilized by titanium carbonitride a-C:H:Ti:N buffer layers deposited directly onto the polyurethane (PU) substrate beneath a final hydrogel coating. Coatings of a-C:H:Ti:N were deposited using a hybrid method of pulsed laser deposition (PLD) and magnetron sputtering (MS) under high vacuum conditions. The influence of the buffer a-C:H:Ti:N layer on the hydrogel coating was analysed by means of a multi-scale microstructure study. Mechanical tests were performed at an indentation load of 5 mN using Berkovich indenter geometry. Haemocompatible analyses were performed in vitro using a blood flow simulator. The blood-material interaction was analysed under dynamic conditions. The coating fabrication procedure improved the coating stability due to the deposition of the amorphous titanium carbonitride buffer layer.

Thrombogenicity and biocompatibility studies of reduced graphene oxide modified acellular pulmonary valve tissue.

Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.

Tribology of bio-inspired nanowrinkled films on ultrasoft substrates.

Biomimetic design of new materials uses nature as antetype, learning from billions of years of evolution. This work emphasizes the mechanical and tribological properties of skin, combining both hardness and wear resistance of its surface (the stratum corneum) with high elasticity of the bulk (epidermis, dermis, hypodermis). The key for combination of such opposite properties is wrinkling, being consequence of intrinsic stresses in the bulk (soft tissue): Tribological contact to counterparts below the stress threshold for tissue trauma occurs on the thick hard stratum corneum layer pads, while tensile loads smooth out wrinkles in between these pads. Similar mechanism offers high tribological resistance to hard films on soft, flexible polymers, which is shown for diamond-like carbon (DLC) and titanium nitride thin films on ultrasoft polyurethane and harder polycarbonate substrates. The choice of these two compared substrate materials will show that ultra-soft substrate materials are decisive for the distinct tribological material. Hierarchical wrinkled structures of films on these substrates are due to high intrinsic compressive stress, which evolves during high energetic film growth. Incremental relaxation of these stresses occurs by compound deformation of film and elastic substrate surface, appearing in hierarchical nano-wrinkles. Nano-wrinkled topographies enable high elastic deformability of thin hard films, while overstressing results in zigzag film fracture along larger hierarchical wrinkle structures. Tribologically, these fracture mechanisms are highly important for ploughing and sliding of sharp and flat counterparts on hard-coated ultra-soft substrates like polyurethane. Concentration of polyurethane deformation under the applied normal loads occurs below these zigzag cracks. Unloading closes these cracks again. Even cyclic testing do not lead to film delamination and retain low friction behavior, if the adhesion to the substrate is high and the initial friction coefficient of the film against the sliding counterpart low, e.g. found for DLC.

Hemocompatibility of Inorganic Physical Vapor Deposition (PVD) Coatings on Thermoplastic Polyurethane Polymers.

Biocompatibility improvements for blood contacting materials are of increasing interest for implanted devices and interventional tools. The current study focuses on inorganic (titanium, titanium nitride, titanium oxide) as well as diamond-like carbon (DLC) coating materials on polymer surfaces (thermoplastic polyurethane), deposited by magnetron sputtering und pulsed laser deposition at room temperature. DLC was used pure (a-C:H) as well as doped with silicon, titanium, and nitrogen + titanium (a-C:H:Si, a-C:H:Ti, a-C:H:N:Ti). In-vitro testing of the hemocompatibility requires mandatory dynamic test conditions to simulate in-vivo conditions, e.g., realized by a cone-and-plate analyzer. In such tests, titanium- and nitrogen-doped DLC and titanium nitride were found to be optimally anti-thrombotic and better than state-of-the-art polyurethane polymers. This is mainly due to the low tendency to platelet microparticle formation, a high content of remaining platelets in the whole blood after testing and low concentration of platelet activation and aggregation markers. Comparing this result to shear-flow induced cell motility tests with e.g., Dictostelium discoideum cell model organism reveals similar tendencies for the investigated materials.

Hemocompatible, pulsed laser deposited coatings on polymers.

State-of-the-art non-thrombogenic blood contacting surfaces are based on heparin and struggle with the problem of bleeding. However, appropriate blood flow characteristics are essential for clinical application. Thus, there is increasing demand to develop new coating materials for improved human body acceptance. Materials deposited by vacuum coating techniques would be an excellent alternative if the coating temperatures can be kept low because of the applied substrate materials of low temperature resistance (polymers). Most of the recently used plasma-based deposition techniques cannot fulfill this demand. However, adequate film structure and high adhesion can be reached by the pulsed laser deposition at room temperature, which was developed to an industrial-scaled process at Laser Center Leoben. Here, this process is described in detail and the resulting structural film properties are shown for titanium, titanium nitride, titanium carbonitride, and diamond-like carbon on polyurethane, titanium and silicon substrates. Additionally, we present the biological response of blood cells and the kinetic mechanism of eukaryote cell attachment. In conclusion, high biological acceptance and distinct differences for the critical delamination shear stress were found for the coatings, indicating higher adhesion at higher carbon contents.