Long-Term In Vitro Assessment of Biodegradable Radiopaque Composites for Fiducial Marker Fabrication.

Biodegradable polymer-based composite materials may be successfully utilised to fabricate fiducial markers (FMs), which are intended to precisely label tumour margins during image-guided surgery or radiotherapy. However, due to matrix degradability, the stability of the functional properties of FMs depends on the chosen polymer. Thus, this study aimed to investigate novel radiopaque composites which varied in the polymeric matrix-polycaprolactone (PCL), poly(L-lactide-co-caprolactone) (P[LAcoCL]) with two molar ratios (70:30 and 85:15), and poly(L-lactide-co-glycolide) (with molar ratio 82:18). The radiopaque component of the materials was a mixture of barium sulphate and hydroxyapatite. The changes in water contact angle, stiffness, and radiopacity occurring during the 24-week-long degradation experiment were examined for the first time. This study comprehensively analyses the microstructural causes of composites behaviour within degradation experiments using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), gel permitted chromatography (GPC), and scanning electron microscopy (SEM). The obtained results suggest that the utilized biodegradable matrix plays an essential role in radiopaque composite properties and stability thereof. This long-term in vitro assessment enabled a comparison of the materials and aided in choosing the most favourable composite for FMs' fabrication.

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.

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.

Osteogenic Potential of Sheep Mesenchymal Stem Cells Preconditioned with BMP-2 and FGF-2 and Seeded on an nHAP-Coated PCL/HAP/ß-TCP Scaffold.

Mesenchymal stem cells (MSCs) attract interest in regenerative medicine for their potential application in bone regeneration. However, direct transplantation of cells into damaged tissue is not efficient enough to regenerate large bone defects. This problem could be solved with a biocompatible scaffold. Consequently, bone tissue engineering constructs based on biomaterial scaffolds, MSCs, and osteogenic cytokines are promising tools for bone regeneration. The aim of this study was to evaluate the effect of FGF-2 and BMP-2 on the osteogenic potential of ovine bone marrow-derived MSCs seeded onto an nHAP-coated PCL/HAP/ß-TCP scaffold in vitro and its in vivo biocompatibility in a sheep model. In vitro analysis revealed that cells preconditioned with FGF-2 and BMP-2 showed a better capacity to adhere and proliferate on the scaffold than untreated cells. BM-MSCs cultured in an osteogenic medium supplemented with FGF-2 and BMP-2 had the highest osteogenic differentiation potential, as assessed based on Alizarin Red S staining and ALP activity. qRT-PCR analysis showed increased expression of osteogenic marker genes in FGF-2- and BMP-2-treated BM-MSCs. Our pilot in vivo research showed that the implantation of an nHAP-coated PCL/HAP/ß-TCP scaffold with BM-MSCs preconditioned with FGF-2 and BMP-2 did not have an adverse effect in the sheep mandibular region and induced bone regeneration. The biocompatibility of the implanted scaffold-BM-MSC construct with sheep tissues was confirmed by the expression of early (collagen type I) and late (osteocalcin) osteogenic proteins and a lack of an elevated level of proinflammatory cytokines. These findings suggest that FGF-2 and BMP-2 enhance the osteogenic differentiation potential of MSCs grown on a scaffold, and that such a tissue engineering construct may be used to regenerate large bone defects.

Biodegradable Fiducial Markers for Bimodal Near-Infrared Fluorescence- and X-ray-Based Imaging.

This study aimed to evaluate, for the first time, implantable, biodegradable fiducial markers (FMs), which were designed for bimodal, near-infrared fluorescence-based (NIRF) and X-ray-based imaging. The developed FMs had poly(l-lactide-co-caprolactone)-based core-shell structures made of radiopaque (core) and fluorescent (shell) composites with a poly(l-lactide-co-caprolactone) matrix. The approved for human use contrast agents were utilized as fillers. Indocyanine green was applied to the shell material, whereas in the core materials, iohexol and barium sulfate were compared. Moreover, the possibility of tailoring the stability of the properties of the core materials by the addition of hydroxyapatite (HAp) was examined. The performed in situ (porcine tissue) and in vivo experiment (rat model) confirmed that the developed FMs possessed pronounced contrasting properties in NIRF and X-ray imaging. The presence of HAp improved the radiopacity of FMs at the initial state. It was also proved that, in iohexol-containing FMs, the presence of HAp slightly decreased the stability of contrasting properties, while in BaSO4-containing ones, changes were less pronounced. A comprehensive material analysis explaining the differences in the stability of the contrasting properties was also presented. The tissue response around the FMs with composite cores was comparable to that of the FMs with a pristine polymeric core. The developed composite FMs did not cause serious adverse effects on the surrounding tissues even when irradiated in vivo. The developed FMs ensured good visibility for NIRF image-supported tumor surgery and the following X-ray image-guided radiotherapy. Moreover, this study replenishes a scanty report regarding similar biodegradable composite materials with a high potential for application.

Coupling Additive Manufacturing with Hot Melt Extrusion Technologies to Validate a Ventilator-Associated Pneumonia Mouse Model.

Additive manufacturing is widely used to produce highly complex structures. Moreover, this technology has proven its superiority in producing tools which can be used in different applications. We designed and produced an extrusion nozzle that allowed us to hot melt extrude drug-loaded tubes. The tubes were an essential part of a new mouse ventilator-associated pneumonia (VAP) model. Ciprofloxacin (CPX) was selected for its expected activity against the pathogen Staphylococcus aureus and ease of incorporation into thermoplastic polyurethane (TPU). TPU was selected as the carrier polymer for its biocompatibility and use in a variety of medical devices such as tubing and catheters. The effect of loading CPX within the TPU polymeric matrix and the physicochemical properties of the produced tubes were investigated. CPX showed good thermal stability and in vitro activity in preventing S. aureus biofilm formation after loading within the tube's polymeric matrix. Moreover, the produced tubes showed anti-infective efficacy in vivo. The produced tubes, which were extruded via our novel nozzle, were vital for the validation of our mouse VAP model. This model can be adopted to investigate other antibacterial and antibiofilm compounds incorporated in polymeric tubes using hot melt extrusion.

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-Printed Drug Delivery Systems: The Effects of Drug Incorporation Methods on Their Release and Antibacterial Efficiency.

Additive manufacturing technologies have been widely used in the medical field. More specifically, fused filament fabrication (FFF) 3D-printing technology has been thoroughly investigated to produce drug delivery systems. Recently, few researchers have explored the possibility of directly 3D printing such systems without the need for producing a filament which is usually the feedstock material for the printer. This was possible via direct feeding of a mixture consisting of the carrier polymer and the required drug. However, as this direct feeding approach shows limited homogenizing abilities, it is vital to investigate the effect of the pre-mixing step on the quality of the 3D printed products. Our study investigates the two commonly used mixing approaches-solvent casting and powder mixing. For this purpose, polycaprolactone (PCL) was used as the main polymer under investigation and gentamicin sulfate (GS) was selected as a reference. The produced systems' efficacy was investigated for bacterial and biofilm prevention. Our data show that the solvent casting approach offers improved drug distribution within the polymeric matrix, as was observed from micro-computed topography and scanning electron microscopy visualization. Moreover, this approach shows a higher drug release rate and thus improved antibacterial efficacy. However, there were no differences among the tested approaches in terms of thermal and mechanical properties.

Processing of (Co)Poly(2-oxazoline)s by Electrospinning and Extrusion from Melt and the Postprocessing Properties of the (Co)Polymers.

Poly(2-oxazoline) (POx) matrices in the form of non-woven fibrous mats and three-dimensional moulds were obtained by electrospinning and fused deposition modelling (FDM), respectively. To obtain these materials, poly(2-isopropyl-2-oxazoline) (PiPrOx) and gradient copolymers of 2-isopropyl- with 2-n-propyl-2-oxazoline (P(iPrOx-nPrOx)), with relatively low molar masses and low dispersity values, were processed. The conditions for the electrospinning of POx were optimised for both water and the organic solvent. Also, the FDM conditions for the fabrication of POx multi-layer moulds of cylindrical or cubical shape were optimised. The properties of the POx after electrospinning and extrusion from melt were determined. The molar mass of all (co)poly(2-oxazoline)s did not change after electrospinning. Also, FDM did not influence the molar masses of the (co)polymers; however, the long processing of the material caused degradation and an increase in molar mass dispersity. The thermal properties changed significantly after processing of POx what was monitored by increase in enthalpy of exo- and endothermic peaks in differential scanning calorimetry (DSC) curve. The influence of the processing conditions on the structure and properties of the final material were evaluated having in a mind their potential application as scaffolds.

Solventless Conducting Paste Based on Graphene Nanoplatelets for Printing of Flexible, Standalone Routes in Room Temperature.

Novel printable composites based on high aspect ratio graphene nanoplatelets (GNPs), fabricated without using solvents, and at room temperature, that can be employed for flexible, standalone conducting lines for wearable electronics are presented. The percolation threshold of examined composites was determined to be as low as 0.147 vol% content of GNPs. Obtained sheet resistance values were as low as 6.1 Ω/sq. Stretching and bending tests are presented, proving suitability of the composite for flexible applications as the composite retains its conductivity even after 180° folding and 13.5% elongation.

Spongelike Porous Silica Nanosheets: From "Soft" Molecular Trapping to DNA Delivery.

Spongelike porous silica nanosheets, with nanometer thicknesses and pores whose diameters are on the hundreds-of-nanometers scale, have been used as a novel carrier for molecular immobilization of different guests. Enhanced properties of encapsulation were shown for drug molecules of different dimensions due to "softness" caused by the specific nanometric features of the porous structure. The encapsulating effect of the structure results in sustained and stimuli-responsive release behavior of immobilized guest molecules. By studying the adsorption process of DNA molecules on spongelike porous nanosheets or on solid nanoparticles by use of a quartz crystal microbalance, we show that better elasticity of surfaces of the porous nanosheets over that of solid nanoparticles can improve the immobilization of guest molecules. The coating of porous silica nanosheets onto various substrates was also found to effectively mediate DNA delivery to mammalian cells.

Biodegradable fiducial markers for X-ray imaging - soft tissue integration and biocompatibility.

This study aims at the development of materials for biodegradable fiducial markers for X-ray based medical imaging and their anchorage in soft tissue. Towards this goal a degradable polymer matrix of poly(l-lactide-co-ε-caprolactone) (P[LAcoCL]) was combined with barium sulfate (BaSO4) and hydroxyapatite (HAp) as radio-opaque fillers. Low pressure plasma treatment was applied to the composite materials to improve cell adhesion and subsequent tissue integration. In particular, the effects of oxygen and ammonia plasmas were evaluated and compared using X-ray photoelectron spectroscopy, atomic force microscopy and dynamic water contact angle measurements as well as in vitro studies using the murine fibroblast cell line L929. To exclude the cytotoxic effects of degradation products of P[LAcoCL] and released BaSO4 or HAp cytotoxicity assays with the degradation products of the composite materials were conducted. The results obtained by this broad range of analytical techniques suggest the application of composites of P[LAcoCL] with BaSO4 and HAp as promising material systems for innovative fiducial markers for soft tissue in X-ray based medical imaging.