Polymyxin B Peptide Hydrogel Coating: A Novel Approach to Prevent Ventilator-Associated Pneumonia.

Ventilator-associated pneumonia (VAP) remains one of the most common hospital-acquired infections (HAI). Considering the complicated diagnosis and the lack of effective treatment, prophylactic measures are suggested as the new standard to prevent the disease. Although VAP often manifests a polymicrobial nature, Pseudomonas aeruginosa remains one of the pathogens associated with the highest morbidity and mortality rates within these mechanically ventilated patients. In this paper, we report on the development of an antibacterial hydrogel coating using the polymyxin B (PMB) peptide to prevent bacterial adhesion to the polymeric substrate. We fully characterized the properties of the coating using atomic force microscopy (AFM), scanning electron microscopy (SEM), wettability analyses and Fourier-transform infrared (FTIR) and Raman spectroscopy. Furthermore, several biological assays confirmed the antibacterial and anti-biofilm effect of the tubing for at least 8 days against P. aeruginosa. On top of that, the produced coating is compliant with the requirements regarding cytocompatibility stated in the ISO (International Organization for Standardization) 10993 guidelines and an extended release of PMB over a period of at least 42 days was detected. In conclusion, this study serves as a foundation for peptide-releasing hydrogel formulas in the prevention of VAP.

In Vitro Effects of Silver Nanoparticles on Pathogenic Bacteria and on Metabolic Activity and Viability of Human Mesenchymal Stem Cells.

The rapid development of nanotechnology has led to the use of silver nanoparticles (Ag-NPs) in various biomedical fields. However, the effect of Ag-NPs on human mesenchymal stem cells (hMSCs) is not fully understood. Moreover, too frequent an exposure to products containing nanosilver in sublethal amounts raises widespread concerns that it will lead to the development of silver-resistant microorganisms. Therefore, this study aimed to evaluate the mechanism of action of Ag-NPs on hMSCs by analyzing the cellular uptake of Ag-NPs by the cells and its effect on their viability and to assess antimicrobial activity of Ag-NPs against emerging bacterial strains, including multidrug-resistant pathogens. For metabolic activity and viability evaluation, hMSCs were incubated with different concentrations of Ag-NPs (14 µg/mL, 7 µg/mL, and 3.5 µg/mL) for 10 min., 1 h and 24 h and subsequently analyzed for their viability by live-dead staining and metabolic activity by the MTS assay. The effect of Ag-NPs on bacterial pathogens was studied by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In conclusion, it was observed that exposure of hMSCs to Ag-NPs of size <10 nm has no cytotoxic effect on the metabolic activity of the cells at the concentration of 3.5 µg/mL, with minimal cytotoxic effect being observed at the concentration of 14 µg/mL after 24 h of incubation. Our findings also confirmed that Ag-NPs at the concentration of 4 µg/mL are effective broad-spectrum bactericidal agents, regardless of the antibiotic-resistance mechanism present in bacteria.

Indocyanine green and iohexol loaded hydroxyapatite in poly(L-lactide-co-caprolactone)-based composite for bimodal near-infrared fluorescence- and X-ray-based imaging.

This study aimed to develop material for multimodal imaging by means of X-ray and near-infrared containing FDA- and EMA-approved iohexol and indocyanine green (ICG). The mentioned contrast agents (CAs) are hydrophilic and amphiphilic, respectively, which creates difficulties in fabrication of functional polymeric composites for fiducial markers (FMs) with usage thereof. Therefore, this study exploited for the first time the possibility of enhancing the radiopacity and introduction of the NIR fluorescence of FMs by adsorption of the CAs on hydroxyapatite (HAp) nanoparticles. The particles were embedded in the poly(L-lactide-co-caprolactone) (P[LAcoCL]) matrix resulting in the composite material for bimodal near-infrared fluorescence- and X-ray-based imaging. The applied method of material preparation provided homogenous distribution of both CAs with high iohexol loading efficiency and improved fluorescence signal due to hindered ICG aggregation. The material possessed profound contrasting properties for both imaging modalities. Its stability was evaluated during in vitro experiments in phosphate-buffered saline (PBS) and foetal bovine serum (FBS) solutions. The addition of HAp nanoparticles had significant effect on the fluorescence signal. The X-ray radiopacity was stable within minimum 11 weeks, even though the addition of ICG contributed to a faster release of iohexol. The stiffness of the material was not affected by iohexol or ICG, but incorporation of HAp nanoparticles elevated the values of bending modulus by approximately 70%. Moreover, the performed cell study revealed that all tested materials were not cytotoxic. Thus, the developed material can be successfully used for fabrication of FMs.

3D-printed wound dressings containing a fosmidomycin-derivative prevent Acinetobacter baumannii biofilm formation.

Acinetobacter baumannii causes a wide range of infections, including wound infections. Multidrug-resistant A. baumannii is a major healthcare concern and the development of novel treatments against these infections is needed. Fosmidomycin is a repurposed antimalarial drug targeting the non-mevalonate pathway, and several derivatives show activity toward A. baumannii. We evaluated the antimicrobial activity of CC366, a fosmidomycin prodrug, against a collection of A. baumannii strains, using various in vitro and in vivo models; emphasis was placed on the evaluation of its anti-biofilm activity. We also developed a 3D-printed wound dressing containing CC366, using melt electrowriting technology. Minimal inhibitory concentrations of CC366 ranged from 1 to 64 µg/mL, and CC366 showed good biofilm inhibitory and moderate biofilm eradicating activity in vitro. CC366 successfully eluted from a 3D-printed dressing, the dressings prevented the formation of A. baumannnii wound biofilms in vitro and reduced A. baumannii infection in an in vivo mouse model.

Effect of adipose-derived stem cells seeding and surgical prefabrication on composite scaffold vascularization.

The study aimed to evaluate an angiogenic effect of adipose-derived stem cells (ASCs) seeding and surgical prefabrication (placing a vascular pedicle inside the scaffold) on developed composite scaffolds made of poly-ε-caprolactone (PCL), ß-tricalcium phosphate (ß-TCP), and poly (lactic-co-glycolic acid) (PLGA) (PCL+ß-TCP+PLGA). Moreover, we aimed to compare our data with previously tested PCL scaffolds to assess whether the new material has better angiogenic properties. The study included 18 inbred male WAG rats. There were three scaffold groups (six animals each): with non-seeded PCL+ß-TCP+PLGA scaffolds, with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and osteogenic-induced. Each rat was implanted with two scaffolds in the inguinal region (one prefabricated and one non-prefabricated). After 2 months from implantation, the scaffolds were explanted, and vessel density was determined by histopathological examination. Prefabricated ASC-seeded PCL+ß-TCP+PLGA scaffolds promoted greater vessel formation than non-seeded scaffolds (19.73 ± 5.46 vs 12.54 ± 0.81; p = .006) and those seeded with osteogenic-induced ASCs (19.73 ± 5.46 vs 11.87±2.21; p = .004). The developed composite scaffold promotes vessel formation more effectively than the previously described PCL scaffold.

Deep eutectic solvent-assisted fabrication of bioinspired 3D carbon-calcium phosphate scaffolds for bone tissue engineering.

Tissue engineering is a burgeoning field focused on repairing damaged tissues through the combination of bodily cells with highly porous scaffold biomaterials, which serve as templates for tissue regeneration, thus facilitating the growth of new tissue. Carbon materials, constituting an emerging class of superior materials, are currently experiencing remarkable scientific and technological advancements. Consequently, the development of novel 3D carbon-based composite materials has become significant for biomedicine. There is an urgent need for the development of hybrids that will combine the unique bioactivity of ceramics with the performance of carbonaceous materials. Considering these requirements, herein, we propose a straightforward method of producing a 3D carbon-based scaffold that resembles the structural features of spongin, even on the nanometric level of their hierarchical organization. The modification of spongin with calcium phosphate was achieved in a deep eutectic solvent (choline chloride : urea, 1 : 2). The holistic characterization of the scaffolds confirms their remarkable structural features (i.e., porosity, connectivity), along with the biocompatibility of α-tricalcium phosphate (α-TCP), rendering them a promising candidate for stem cell-based tissue-engineering. Culturing human bone marrow mesenchymal stem cells (hMSC) on the surface of the biomimetic scaffold further verifies its growth-facilitating properties, promoting the differentiation of these cells in the osteogenesis direction. ALP activity was significantly higher in osteogenic medium compared to proliferation, indicating the differentiation of hMSC towards osteoblasts. However, no significant difference between C and C-αTCP in the same medium type was observed.

Cytotoxic Properties of Polyurethane Foams for Biomedical Applications as a Function of Isocyanate Index.

Polyurethane foams are widely used in biomedical applications due to their desirable mechanical properties and biocompatibility. However, the cytotoxicity of its raw materials can limit their use in certain applications. In this study, a group of open-cell polyurethane foams were investigated for their cytotoxic properties as a function of the isocyanate index, a critical parameter in the synthesis of polyurethanes. The foams were synthesized using a variety of isocyanate indices and characterized for their chemical structure and cytotoxicity. This study indicates that the isocyanate index highly influences the chemical structure of polyurethane foams, also causing changes in cytotoxicity. These findings have important implications for designing and using polyurethane foams as composite matrices in biomedical applications, as careful consideration of the isocyanate index is necessary to ensure biocompatibility.

Toward osteomimetic formation of calcium phosphate coatings with carbonated hydroxyapatite.

Biomimetic production of coatings on various types of scaffolds is based mainly on simulated body fluid precipitation (SBF) of apatites, or, if the HCO3- is present, carbonated apatites. Recently, we proposed formation of calcium phosphates (CaP) precipitates by alkaline phosphatase (ALP) hydrolysing glycerophosphate in presence of calcium ions as an alternative to SBF. Since apatites synthesized in bone by the ALP activity contain carbonate anions, it was tempting to investigate whether the phosphatase method could be advanced into osteomimetic one. Therefore, taking example from the SBF studies, phosphatase incubation medium was enriched with carbonate ions at 4.2 and 27 mM concentration. X-ray diffraction of the precipitates disclosed peaks typical for hydroxyapatite (HAP). FTIR analysis showed that at both concentration of carbonate ions, apatites underwent both B and A substitution, more extensive at higher concentration. Thus, osteomimetic approach produced carbonated hydroxyapatites of the type encountered in bone tissue even at HCO3- concentration as low as 4.2 mM. Composite plates made of poly(ε-caprolactone) and mixture of ß-tricalcium phosphate and hydroxyapatite at mass ratio of 1:0.5:0.5, respectively, were covered by CaP coatings, i.e., CaP-0, CaP-4.2, CaP-27, by incubation in phosphatase medium containing 0, 4.2 or 27 mM of NaHCO3, respectively. Pristine or coated PCL50 plates were used to study release of calcium and adsorption/desorption of proteins, or seeded with human bone marrow mesenchymal stem cells (hMSC) for study of cell adhesion, spreading and osteogenic differentiation. Introduction of carbonate into the CaP coatings significantly increased release of Ca2+ in a carbonate concentration-dependent manner; the release was up to 4 times higher, when compared to CaP-0 coating, and reached 0.41 ± 0.01 mM for CaP-27 after first 24 h. Coating CaP-4.2 yielded significantly higher adsorption of bovine serum albumin and cytochrome C than CaP-0. All of the CaP coatings improved significantly hMSC adhesion, however, only CaP-4.2 provided 2 times higher cell number than PCL50 after 2 weeks of culture. Interestingly, ALP activity calculated per cell number was the highest on pristine plates, presumably because hMSC differentiate preferentially into osteoblasts at lower seeding densities. It appears, therefore, that the osteomimetic approach may be useful for production of carbonated hydroxyapatite coatings, but requires further studies and replacing intestinal phosphatase used in this work with one originating from bone.

Naturally prefabricated 3D chitinous skeletal scaffold of marine demosponge origin, biomineralized ex vivo as a functional biomaterial.

Solutions developed by nature for structural and functional optimization of three-dimensional (3D) skeletal structures provide unique windows not only into the evolutionary pathways of organisms, but also into bioinspired materials science and biomimetics. Great examples are naturally formed 3D chitinous scaffolds of marine sponge remain a focus of modern biomedicine and tissue engineering. Due to its properties like renewability, bioactivity, and biodegradability such constructs became very interesting players as components of organic-inorganic biocomposites. Herein, we developed chitin-based biocomposites by biomimetic ex vivo deposition of calcium carbonate particles using hemolymph from the cultivated mollusk Cornu aspersum and chitinous matrix from the marine demosponge Aplysina fistularis. The biological potential of the developed biofunctionalized scaffolds for bone tissue engineering was evaluated by investigating the spreading and viability of a human fetal osteoblast cell line has been determined for the first time. Performed analyses like dynamic mechanical analysis and atomic force microscopy shown that biofunctionalized scaffold possess about 4 times higher mechanical resistance. Moreover, several topographical changes have been observed, as e.g., surface roughness (Rq) increased from 31.75 ± 2.7 nm to 120.7 ± 0.3 nm. The results are indicating its potential for use in the modification of cell delivery systems in future biomedical applications.

The effect of diameter of fibre on formation of hydrogen bonds and mechanical properties of 3D-printed PCL.

Fused Deposition Modelling (FDM) technique has been widely utilized in fabrication of 3D porous scaffolds for tissue engineering (TE) applications. Surprisingly, although there are many publications devoted to the architectural features of the 3D scaffolds fabricated by the FDM, none of them give us evident information about the impact of the diameter of the fibres on material properties. Therefore, the aim of this study was to investigate, for the first time, the effect of the diameter of 3D-printed PCL fibres on variations in their microstructure and resulting mechanical behaviour. The fibres made of poly(ε-caprolactone) (PCL) were extruded through commonly used types of nozzles (inner diameter ranging from 0.18 mm to 1.07 mm) by means of FDM technique. Static tensile test and atomic force microscopy working in force spectroscopy mode revealed strong decrease in the Young's modulus and yield strength with increasing fibre diameter in the investigated range. To explain this phenomenon, we conducted differential scanning calorimetry, wide-angle X-ray-scattering, Fourier-transform infrared spectroscopy, infrared and polarized light microscopy imaging. The obtained results clearly showed that the most prominent effect on the obtained microstructures and mechanical properties had different cooling and shear rates during fabrication process causing changes in supramolecular interactions of PCL. The observed fibre size-dependent formation of hydrogen bonds affected the crystalline structure and its stability. Summarising, this study clearly demonstrates that the diameter of 3D-printed fibres has a strong effect on obtained microstructure and mechanical properties, therefore should be taken into consideration during design of the 3D TE scaffolds.

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

3D bioprinted hydrogel model incorporating ß-tricalcium phosphate for calcified cartilage tissue engineering.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

Formation of calcium phosphate coatings within polycaprolactone scaffolds by simple, alkaline phosphatase based method.

The paper presents a novel approach to the production of calcium phosphate coatings of scaffolds. Mineral deposits were formed during incubation of polycaprolactone (PCL) scaffolds with bovine intestinal alkaline phosphatase in sodium glycerophosphate and calcium chloride medium. To modify hydrophobic surface of scaffolds and intensify attachment of coating, scaffolds were incubated at 50 °C (thermal activation, TA) or at 37 °C after short exposition to lipase (lipase activation, LA). Micro-computed tomography observations demonstrated that both methods resulted in deposition of mineral on the surface of external and internal walls of the scaffolds. Precipitate formed after thermal and lipase activation contained particles with average size of 200-400 nm, and the shape of donuts. In thermal activated PCL coatings X-ray diffraction disclosed peaks typical for hydroxyapatite (HAp), while after lipase activation these peaks could be precisely defined only if left for 6 days in the incubation medium. The Fourier-transform infrared spectroscopy suggested crystalline structure of HAp both after thermal and lipase activation. The adherence of bone marrow mesenchymal stem cells was initially higher on coated than pristine PCL, but during 7 days of culture the cell number increased and was similar on all tested samples. Alkaline phosphatase activity, considered as a sign of osteogenic differentiation, measured on PCL samples after 7 days was 2-3 times lower on pristine PCL than on the coated samples, but after 2 weeks increased significantly and reached similar value as on the calcium phosphate substrates.

X-ray physics-based CT-to-composition conversion applied to a tissue engineering scaffold, enabling multiscale simulation of its elastic behavior.

Nowadays, the assessment of the mechanical competence of tissue engineering scaffolds based on computer simulations is a well-accepted technology. Typically, such simulations are performed by means of the Finite Element (FE) method, with the underlying structural model being created based on micro-computed tomography (microCT). Here, this analysis modality is applied to a new, ternary composite, consisting of PHBV, i.e. poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PLGA, i.e. poly(lactic-co-glycolide), as well as of TCP, i.e. tricalcium phosphate hydrate. The studied scaffold structure is made up by fibers of this new composite material, manufactured by means of the rapid prototyping method. The data collected from microCT is utilized for adequately defining the mechanical properties of the FE model. In particular, the three-dimensional field of grey values is interpreted in terms of the underlying field of attenuation coefficients, taking into account the photon energy employed in microCT imaging, eventually allowing for calculation of the three-dimensionally distributed, voxel-specific composition of the studied material. For the sake of keeping the FE simulations as efficient as possible, groups of voxels are combined into one finite element; the grey value of the latter is obtained by volume averaging. Employing a two-step micromechanical homogenization scheme, the experimentally accessible stiffness of the three constituents (PHBV, PLGA, and TCP) is then, finite element by finite element, upscaled to the composition-dependent stiffness of the composite material. The plausibility and adequacy of the FE model is demonstrated by simulating the effects of uniaxial compression on the scaffold structure, in terms of resulting stress and strain fields, highlighting the importance of the fiber junctions (as they are the mechanically most stressed regions), and that neglecting the material heterogeneity would lead to a potentially significant underestimation of stresses and strains. Finally, a comparison is made of the employed analysis modality of microCT data with a previously pursued, simplified analysis strategy, highlighting the conceptual superiority of the former, and pointing out the application limits of the latter.

The influence of chemical polishing of titanium scaffolds on their mechanical strength and in-vitro cell response.

Selective Laser Melting (SLM) is a powder-bed-based additive manufacturing method, using a laser beam, which can be used to produce metallic scaffolds for bone regeneration. However, this process also has a few disadvantages. One of its drawbacks is the necessity of post-processing in order to improve the surface finish. Another drawback lies in the removal of unmelted powder particles from the build. In this study, the influence of chemical polishing of SLM fabricated titanium scaffolds on their mechanical strength and in vitro cellular response was investigated. Scaffolds with bimodal pore size (200 µm core and 500 µm shell) were fabricated by SLM from commercially pure titanium powder and then chemically treated in HF/HNO3 solutions to remove unmelted powder particles. The cell viability and mechanical strength were compared between as-made and chemically-treated scaffolds. The chemical treatment was successful in the removal of unmelted powder particles from the titanium scaffold. The Young's modulus of the fabricated cellular structures was of 42.7 and 13.3 GPa for as-made and chemically-treated scaffolds respectively. These values are very similar to the Young's modulus of living human bone. Chemical treatment did not affect negatively cell proliferation and differentiation. Additionally, the chemically-treated scaffolds had a twofold increase in colonization of osteoblast cells migrating out of multicellular spheroids. Furthermore, X-ray computed microtomography confirmed that chemically-treated scaffolds met the dimensions originally set in the CAD models. Therefore, chemical-treatment can be used as a tool to cancel the discrepancies between the designed and fabricated objects, thus enabling fabrication of finer structures with regular struts and high resolution.

The Influence of Selective Laser Melting (SLM) Process Parameters on In-Vitro Cell Response.

The use of laser 3D printers is very perspective in the fabrication of solid and porous implants made of various polymers, metals, and its alloys. The Selective Laser Melting (SLM) process, in which consolidated powders are fully melted on each layer, gives the possibility of fabrication personalized implants based on the Computer Aid Design (CAD) model. During SLM fabrication on a 3D printer, depending on the system applied, there is a possibility for setting the amount of energy density (J/mm³) transferred to the consolidated powders, thus controlling its porosity, contact angle and roughness. In this study, we have controlled energy density in a range 8⁻45 J/mm³ delivered to titanium powder by setting various levels of laser power (25⁻45 W), exposure time (20⁻80 µs) and distance between exposure points (20⁻60 µm). The growing energy density within studied range increased from 63 to 90% and decreased from 31 to 13 µm samples density and Ra parameter, respectively. The surface energy 55⁻466 mN/m was achieved with contact angles in range 72⁻128° and 53⁻105° for water and formamide, respectively. The human mesenchymal stem cells (hMSCs) adhesion after 4 h decreased with increasing energy density delivered during processing within each parameter group. The differences in cells proliferation were clearly seen after a 7-day incubation. We have observed that proliferation was decreasing with increasing density of energy delivered to the samples. This phenomenon was explained by chemical composition of oxide layers affecting surface energy and internal stresses. We have noticed that TiO2, which is the main oxide of raw titanium powder, disintegrated during selective laser melting process and oxygen was transferred into metallic titanium. The typical for 3D printed parts post-processing methods such as chemical polishing in hydrofluoric (HF) or hydrofluoric/nitric (HF/HNO3) acid solutions and thermal treatments were used to restore surface chemistry of raw powders and improve surface.

3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage formation.

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>10(7) cells ml(-1)), high cell viability (85 ÷ 90%) and high printing resolution (≈100 µm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Influence of biodegradable polymer coatings on corrosion, cytocompatibility and cell functionality of Mg-2.0Zn-0.98Mn magnesium alloy.

Four kinds of biodegradable polymers were employed to prepare bioresorbable coatings on Mg-2.0Zn-0.98Mn (ZM21) alloy to understand the relationship between polymer characteristics, protective effects on substrate corrosion, cytocompatibility and cell functionality. Poly-l-lactide (PLLA), poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or poly(lactic-co-glycolic) acid (PLGA) was spin-coated on ZM21, obtaining a smooth, non-porous coating less than 0.5µm in thickness. Polymer coating characterization, a degradation study, and biocompatibility evaluations were performed. After 4 w of immersion into cell culture medium, degradation of PLGA and PLLA coatings were confirmed by ATR-FTIR observation. The coatings of PLLA, PHB and PHBV, which have lower water permeability and slower degradation than PLGA, provide better suppression of initial ZM21 degradation and faster promotion of human osteosarcoma cell growth and differentiation.

Post Processing and Biological Evaluation of the Titanium Scaffolds for Bone Tissue Engineering.

Nowadays, post-surgical or post-accidental bone loss can be substituted by custom-made scaffolds fabricated by additive manufacturing (AM) methods from metallic powders. However, the partially melted powder particles must be removed in a post-process chemical treatment. The aim of this study was to investigate the effect of the chemical polishing with various acid baths on novel scaffolds' morphology, porosity and mechanical properties. In the first stage, Magics software (Materialise NV, Leuven, Belgium) was used to design a porous scaffolds with pore size equal to (A) 200 µm, (B) 500 µm and (C) 200 + 500 µm, and diamond cell structure. The scaffolds were fabricated from commercially pure titanium powder (CP Ti) using a SLM50 3D printing machine (Realizer GmbH, Borchen, Germany). The selective laser melting (SLM) process was optimized and the laser beam energy density in range of 91-151 J/mm³ was applied to receive 3D structures with fully dense struts. To remove not fully melted titanium particles the scaffolds were chemically polished using various HF and HF-HNO3 acid solutions. Based on scaffolds mass loss and scanning electron (SEM) observations, baths which provided most uniform surface cleaning were proposed for each porosity. The pore and strut size after chemical treatments was calculated based on the micro-computed tomography (µ-CT) and SEM images. The mechanical tests showed that the treated scaffolds had Young's modulus close to that of compact bone. Additionally, the effect of pore size of chemically polished scaffolds on cell retention, proliferation and differentiation was studied using human mesenchymal stem cells. Small pores yielded higher cell retention within the scaffolds, which then affected their growth. This shows that in vitro cell performance can be controlled to certain extent by varying pore sizes.