Tomáš Suchý

Support for the initial attachment, growth and differentiation of MG-63 cells: a comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites

Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0–N25 or M0–M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%–15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%–15% (v/v) nano-size or micro-size HA seems to be optimum.

Generating standardized image data for testing and calibrating quantification of volumes, surfaces, lengths, and object counts in fibrous and porous materials using X-ray microtomography

Quantification of the structure and composition of biomaterials using micro‐CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three‐dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro‐CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro‐CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro‐CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro‐CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open‐source software for calibrating quantitative micro‐CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three‐dimensional fibrillar and porous structures in micro‐CT scans.

Effects of thermal ageing on the static and cyclic mechanical properties of carbon fibres/PDMS composites for use in medicine.

composites for use in medicine T. Suchý, R. Sedláček*, Z. Sucharda, K. Balı́k and T. Bouda Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical Engineering, Czech Technical University in Prague, Technická 4, 166 07 Prague 6, Czech Republic; Department of Composite and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, v.v.i, V Holešovickách 41, Prague 8, Czech Republic

Dry versus hydrated collagen scaffolds: are dry states representative of hydrated states?

AbstractCollagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

The release kinetics, antimicrobial activity and cytocompatibility of differently prepared collagen/hydroxyapatite/vancomycin layers: Microstructure vs. nanostructure

&NA; The aim of this study was to develop an osteo‐inductive resorbable layer allowing the controlled elution of antibiotics to be used as a bone/implant bioactive interface particularly in the case of prosthetic joint infections, or as a preventative procedure with respect to primary joint replacement at a potentially infected site. An evaluation was performed of the vancomycin release kinetics, antimicrobial efficiency and cytocompatibility of collagen/hydroxyapatite layers containing vancomycin prepared employing different hydroxyapatite concentrations. Collagen layers with various levels of porosity and structure were prepared using three different methods: by means of the lyophilisation and electrospinning of dispersions with 0, 5 and 15 wt% of hydroxyapatite and 10 wt% of vancomycin, and by means of the electrospinning of dispersions with 0, 5 and 15 wt% of hydroxyapatite followed by impregnation with 10 wt% of vancomycin. The maximum concentration of the released active form of vancomycin characterised by means of HPLC was achieved via the vancomycin impregnation of the electrospun layers, whereas the lowest concentration was determined for those layers electrospun directly from a collagen solution containing vancomycin. Agar diffusion testing revealed that the electrospun impregnated layers exhibited the highest level of activity. It was determined that modification using hydroxyapatite exerts no strong effect on vancomycin evolution. All the tested samples exhibited sufficient cytocompatibility with no indication of cytotoxic effects using human osteoblastic cells in direct contact with the layers or in 24‐hour infusions thereof. The results herein suggest that nano‐structured collagen‐hydroxyapatite layers impregnated with vancomycin following cross‐linking provide suitable candidates for use as local drug delivery carriers. Graphical abstract Figure. No caption available.

Optimizing and evaluating the biocompatibility of fiber composites with calcium phosphate additives.

SummaryComposite materials based on a polyamide fabric (aramid) and a polydymethylsiloxane (PDMS) matrix were designed for application in bone surgery. In order to increase the bioactivity, 2, 5, 10, 15, 20, and 25 vol.% of nano/micro hydroxyapatite (HA) and tricalcium phosphate (TCP) were added. We studied the effect of the additives on the biocompatibility of the composite. It appears that nano additives have a more favorable effect on mechanical properties than microparticles. 15 vol.% of nano hydroxyapatite additive is an optimum amount for final application of the composites as substitutes for bone tissue: in this case both the mechanical properties and the biological properties are optimized without distinct changes in the inner structure of the composite.

Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the epiphysis of the porcine femur for morphometric evaluation

The aim of this study was to compare data on the volume fraction of bone and the thickness of the cortical compact bone acquired during microcomputed tomography (micro-CT) analysis with data acquired from identical samples using stereological analysis of either decalcified paraffin sections or ground sections. Additionally, we aimed to compare adjacent tissue samples taken from the major trochanter of the porcine femur to map the basic biological variability of trabecular bone. Fifteen pairs of adjacent tissue blocks were removed from the major trochanter of the proximal epiphyses of porcine femurs (female pigs aged 24-39 months, weight=59.16±8.15kg). In each sample, the volume of the cortical compact bone, the volume of the trabecular bone, and the thickness of the cortical compact bone was assessed using micro-CT. Afterwards, half of the samples were decalcified and processed using paraffin histological sections. Another half was processed into ground sections. The volume and thickness of bone was assessed in histological sections using stereological techniques. There were no significant differences in the bone volumes and thicknesses measured by micro-CT and the corresponding values quantified in decalcified sections. Similarly, there were no differences between the results from micro-CT and the analysis of the corresponding ground sections. Histomorphometric studies based on relatively low numbers of undecalcified ground sections or demineralized paraffin sections of bone yield data on bone volume and the thickness of cortical compact bone that is comparable with three-dimensional micro-CT examination. The pilot data on the variability of cortical compact bone and trabecular bone volumes in the porcine major trochanter provided in this study aim for planning experiments in the field of bone healing and implantology.

The influence of sterilisation processes on the micromechanical properties of polyamide fibre-reinforced PDMS composites for orthopaedic applications

fibre-reinforced PDMS composites for orthopaedic applications R. Sedláček*, T. Suchý, Z. Sucharda, K. Balı́k, M. Sochor, J. Šepitka and J. Lukeš Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical Engineering, Czech Technical University in Prague, Technická 4, 166 07 Prague 6, Czech Republic; Department of Composite and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, v.v.i, V Holešovickách 41, Prague 8, Czech Republic

Nanoindentation characterisation of poly(dl-lactide)/collagen nanocomposites

T. Suchý*, Š. Rýglová, Z. Sucharda, K. Balı́k, J. Šepitka and J. Lukeš Department of Composite and CarbonMaterials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, v.v.i, V Holešovickách 41, Prague 8, Czech Republic; Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical Engineering, Czech Technical University in Prague, Technická 4, 166 07 Prague 6, Czech Republic