Effect of simulated body fluid formulation on orthopedic device apatite-forming ability assessment.

Integration of native bone into orthopedic devices is a key factor in long-term implant success. The material-tissue interface is generally accepted to consist of a hydroxyapatite layer so bioactive materials that can spontaneously generate this hydroxyapatite layer after implantation may improve patient outcomes. Per the ISO 22317:2014 standard, "Implants for surgery - In vitro evaluation for apatite-forming ability of implant materials," bioactivity performance statements can be assessed by soaking the material in simulated body fluid (SBF) and evaluating the surface for the formation of a hydroxyapatite layer; however, variations in test methods may alter hydroxyapatite formation and result in false-positive assessments. The goal of this study was to identify the effect of SBF formulation on bioactivity assessment. Bioglass® (45S5 and S53P4) and non-bioactive Ti-6Al-4V were exposed to SBF formulations varying in calcium ion and phosphate concentrations as well as supporting ion concentrations. Scanning electron microscopy and X-ray powder diffraction evaluation of the resulting hydroxyapatite layers revealed that SBF enriched with double or quadruple the calcium and phosphate ion concentrations increased hydroxyapatite crystal size and quantity compared to the standard formulation and can induce hydroxyapatite crystallization on surfaces traditionally considered non-bioactive. Altering concentrations of other ions, for example, bicarbonate, changed hydroxyapatite induction time, quantity, and morphology. For studies evaluating the apatite-forming ability of a material to support bioactivity performance statements, test method parameters must be adequately described and controlled. It is unclear if apatite formation after exposure to any of the SBF formulations is representative of an in vivo biological response. The ISO 23317 standard test method should be further developed to provide additional guidance on apatite characterization and interpretation of the results.

Micromolding of Amphotericin-B-Loaded Methoxyethylene-Maleic Anhydride Copolymer Microneedles.

Biocompatible and biodegradable materials have been used for fabricating polymeric microneedles to deliver therapeutic drug molecules through the skin. Microneedles have advantages over other drug delivery methods, such as low manufacturing cost, controlled drug release, and the reduction or absence of pain. The study examined the delivery of amphotericin B, an antifungal agent, using microneedles that were fabricated using a micromolding technique. The microneedle matrix was made from GantrezTM AN-119 BF, a benzene-free methyl vinyl ether/maleic anhydride copolymer. The GantrezTM AN-119 BF was mixed with water; after water evaporation, the polymer exhibited sufficient strength for microneedle fabrication. Molds cured at room temperature remained sharp and straight. SEM images showed straight and sharp needle tips; a confocal microscope was used to determine the height and tip diameter for the microneedles. Nanoindentation was used to obtain the hardness and Young's modulus values of the polymer. Load-displacement testing was used to assess the failure force of the needles under compressive loading. These two mechanical tests confirmed the mechanical properties of the needles. In vitro studies validated the presence of amphotericin B in the needles and the antifungal properties of the needles. Amphotericin B GantrezTM microneedles fabricated in this study showed appropriate characteristics for clinical translation in terms of mechanical properties, sharpness, and antifungal properties.

Impact of Surface Chemistry of Ultrasmall Superparamagnetic Iron Oxide Nanoparticles on Protein Corona Formation and Endothelial Cell Uptake, Toxicity, and Barrier Function.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) have been investigated for biomedical applications, including novel contrast agents, magnetic tracers for tumor imaging, targeted drug delivery vehicles, and magneto-mechanical actuators for hyperthermia and thrombolysis. Despite significant progress, recent clinical reports have raised concerns regarding USPION safety related to endothelial cell dysfunction; however, there is limited information on factors contributing to these clinical responses. The influence of USPION surface chemistry on nanoparticle interactions with proteins may impact endothelial cell function leading to adverse responses. Therefore, the goal of this study was to assess the effects of carboxyl-functionalized USPION (CU) or amine-functionalized USPION (AU) (approximately 30 nm diameter) on biological responses in human coronary artery endothelial cells. Increased protein adsorption was observed for AU compared with CU after exposure to serum proteins. Exposure to CU, but not AU, resulted in a concentration-dependent decrease in cell viability and perinuclear accumulation inside cytoplasmic vesicles. Internalization of CU was correlated with endothelial cell functional changes under non-cytotoxic conditions, as evidenced by a marked decreased expression of endothelial-specific adhesion proteins (eg, vascular endothelial-cadherin and platelet endothelial cell adhesion molecule-1) and increased endothelial permeability. Evaluation of downstream signaling indicated endothelial permeability is associated with actin cytoskeleton remodeling, possibly elicited by intracellular events involving reactive oxygen species, calcium ions, and the nanoparticle cellular uptake pathway. This study demonstrated that USPION surface chemistry significantly impacts protein adsorption and endothelial cell uptake, viability, and barrier function. This information will advance the current toxicological profile of USPION and improve development, safety assessment, and clinical outcomes of USPION-enabled medical products.

Matrix-Assisted Pulsed laser Evaporation-deposited Rapamycin Thin Films Maintain Antiproliferative Activity.

Matrix-assisted pulsed laser evaporation (MAPLE) has many benefits over conventional methods (e.g., dip-coating, spin coating, and Langmuir-Blodgett dip-coating) for manufacturing coatings containing pharmacologic agents on medical devices. In particular, the thickness of the coating that is applied to the surface of the medical device can be tightly controlled. In this study, MAPLE was used to deposit rapamycin-polyvinylpyrrolidone (rapamycin-PVP) thin films onto silicon and borosilicate optical glass substrates. Alamar Blue and PicoGreen studies were used to measure the metabolic health and DNA content of L929 mouse fibroblasts as measures of viability and proliferation, respectively. The cells on the MAPLE-deposited rapamycin-PVP surfaces exhibited 70.6% viability and 53.7% proliferation compared to a borosilicate glass control. These data indicate that the antiproliferative properties of rapamycin were maintained after MAPLE deposition.

Argon and oxygen plasma treatment increases hydrophilicity and reduces adhesion of silicon-incorporated diamond-like coatings.

In this study, the structure, adhesion, and cell viability characteristics of silicon-incorporated diamond-like carbon (Si-DLC) coatings on fused silica substrates were investigated. The effects of argon and oxygen postprocessing plasma treatments on the Si-DLC coatings were also studied. The contact angle results showed that the Si-DLC coatings were more hydrophilic than the uncoated surfaces, and postprocessing plasma treatment increased the hydrophilicity of the Si-DLC coatings. Atomic force microscopy and profilometry confirmed that postprocessing plasma treatment increased the thickness and roughness of the Si-DLC coatings. The results of microscratch testing indicated that the plasma treatments reduced the adhesion of the coatings. The x-ray photoelectron spectroscopy (XPS) showed the presence of carbon, oxygen, and silicon in the Si-DLC coatings before and after the plasma treatments. These results show that the postprocessing plasma treatment significantly reduced the atomic percentage of the carbon in the Si-DLC coatings. XPS also confirmed the presence of carbon in the form of sp3(C-C), sp2(C=C), C-O, and C=O bonds in the Si-DLC coatings; it showed that postprocessing treatments significantly increased the percentage of oxygen in the Si-DLC coatings. Fourier transform infrared spectroscopy (FTIR) analysis showed features associated with C-OH stretching, C-H bending, as well as Si-CH2 and C-H bending in the Si-DLC coating. The XPS and FTIR results confirmed that the plasma treatment caused dissociation of the sp2 and sp3 bonds and formation of C-OH bonds. The contact angle data indicated that postprocessing treatment increased the hydrophilicity of the Si-DLC coating. Similar to the uncoated substrates, L929 cells showed no change in cell viability when cultured on Si-DLC coatings. These results of the study indicate the suitability of Si-DLC coatings as inert coatings for medical and biotechnology applications.

The Photoinitiator Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate with Exposure to 405 nm Light Is Cytotoxic to Mammalian Cells but Not Mutagenic in Bacterial Reverse Mutation Assays.

Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) is a free radical photo-initiator used to initiate free radical chain polymerization upon light exposure, and is combined with gelatin methacryloyl (GelMA) to produce a photopolymer used in bioprinting. The free radicals produced under bioprinting conditions are potentially cytotoxic and mutagenic. Since these photo-generated free radicals are highly-reactive but short-lived, toxicity assessments should be conducted with light exposure. In this study, photorheology determined that 10 min exposure to 9.6 mW/cm2 405 nm light from an LED light source fully crosslinked 10 wt % GelMA with >3.4 mmol/L LAP, conditions that were used for subsequent cytotoxicity and mutagenicity assessments. These conditions were cytotoxic to M-1 mouse kidney collecting duct cells, a cell type susceptible to lithium toxicity. Exposure to ≤17 mmol/L (0.5 wt %) LAP without light was not cytotoxic; however, concurrent exposure to ≥3.4 mmol/L LAP and light was cytotoxic. No condition of LAP and/or light exposure evaluated was mutagenic in bacterial reverse mutation assays using S. typhimurium strains TA98, TA100 and E. coli WP2 uvrA. These data indicate that the combination of LAP and free radicals generated from photo-excited LAP is cytotoxic, but mutagenicity was not observed in bacteria under typical bioprinting conditions.

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) possess reactive surfaces, are metabolized and exhibit unique magnetic properties. These properties are desirable for designing novel theranostic biomedical products; however, toxicity mechanisms of USPION are not completely elucidated. The goal of this study was to investigate cell interactions (uptake and cytotoxicity) of USPION using human coronary artery endothelial cells as a vascular cell model. Polyvinylpirrolidone-coated USPION were characterized: average diameter 17 nm (transmission electron microscopy [TEM]), average hydrodynamic diameter 44 nm (dynamic light scattering) and zeta potential -38.75 mV. Cells were exposed to 0 (control), 25, 50, 100 or 200 µg/mL USPION. Concentration- and time-dependent cytotoxicity were observed after 3-6 hours through 24 hours of exposure using Alamar Blue and Real-Time Cell Electronic Sensing assays. Cell uptake was evaluated by imaging using live-dead confocal microscopy, actin and nuclear fluorescent staining, and TEM. Phase-contrast, confocal microscopy, and TEM imaging showed significant USPION internalization as early as 3 hours after exposure to 25 µg/mL. TEM imaging demonstrated particle internalization in secondary lysosomes with perinuclear localization. Three orthogonal assays were conducted to assess apoptosis. TUNEL staining demonstrated a marked increase in fragmented DNA, a response pathognomonic of apoptosis, after a 4-hour exposure. Cells subjected to agarose gel electrophoresis exhibited degraded DNA 3 hours after exposure. Caspase-3/7 activity increased after a 3-hour exposure. USPION uptake resulted in cytotoxicity involving apoptosis and these results contribute to further mechanistic understanding of the USPION toxicity in vitro in cardiovascular endothelial cells.

Toxicity and photosensitizing assessment of gelatin methacryloyl-based hydrogels photoinitiated with lithium phenyl-2,4,6-trimethylbenzoylphosphinate in human primary renal proximal tubule epithelial cells.

Gelatin methacryloyl (GelMA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator are commonly used in combination to produce a photosensitive polymer but there are concerns that must be addressed: the presence of unreacted monomer is well known to be cytotoxic, and lithium salts are known to cause acute kidney injury. In this study, acellular 10% GelMA hydrogels cross-linked with different LAP concentrations and cross-linking illumination times were evaluated for their cytotoxicity, photosensitizing potential, and elastic moduli. Alamar Blue and CyQuant Direct Cell viability assays were performed on human primary renal proximal tubule epithelial cells (hRPTECs) exposed to extracts of each formulation. UV exposure during cross-linking was not found to affect extract cytotoxicity in either assay. LAP concentration did not affect extract cytotoxicity as determined by the Alamar Blue assay but reduced hRPTEC viability in the CyQuant Direct cell assay. Photocatalytic activity of formulation extracts toward NADH oxidation was used as a screening method for photosensitizing potential; longer UV exposure durations yielded extracts with less photocatalytic activity. Finally, elastic moduli determined using nanoindentation was found to plateau to approximately 20-25 kPa after exposure to 342 mJ/cm2 at 2.87 mW of UV-A exposure regardless of LAP concentration. LAP at concentrations commonly used in bioprinting (<0.5% w/w) was not found to be cytotoxic although the differences in cytotoxicity evaluation determined from the two viability assays imply cell membrane damage and should be investigated further. Complete cross-linking of all formulations decreased photocatalytic activity while maintaining predictable final elastic moduli.

Correction to 'Ultrananocrystalline diamond-coated nanoporous membranes support SK-N-SH neuroblastoma endothelial cell attachment'.

[This corrects the article DOI: 10.1098/rsfs.2017.0063.].

Microneedle-Based Delivery of Amphotericin B for Treatment of Cutaneous Leishmaniasis.

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm2. By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 µM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.

Ultrananocrystalline diamond-coated nanoporous membranes support SK-N-SH neuroblastoma epithelial [corrected] cell attachment.

Ultrananocrystalline diamond (UNCD) has been demonstrated to have attractive features for biomedical applications and can be combined with nanoporous membranes for applications in drug delivery systems, biosensing, immunoisolation and single molecule analysis. In this study, free-standing nanoporous UNCD membranes with pore sizes of 100 or 400 nm were fabricated by directly depositing ultrathin UNCD films on nanoporous silicon nitride membranes and then etching away silicon nitride using reactive ion etching. Successful deposition of UNCD on the substrate with a novel process was confirmed with Raman spectroscopy, X-ray photoelectron spectroscopy, cross-section scanning electron microscopy (SEM) and transmission electron microscopy. Both sample types exhibited uniform geometry and maintained a clear hexagonal pore arrangement. Cellular attachment of SK-N-SH neuroblastoma endothelial cells was examined using confocal microscopy and SEM. Attachment of SK-N-SH cells onto UNCD membranes on both porous regions and solid surfaces was shown, indicating the potential use of UNCD membranes in biomedical applications such as biosensors and tissue engineering scaffolds.

Liquid-Phase Laser Induced Forward Transfer for Complex Organic Inks and Tissue Engineering.

Laser induced forward transfer (LIFT) acts as a novel alternative to incumbent plotting techniques such as inkjet printing due to its ability to precisely deposit and position picoliter-sized droplets while being gentle enough to preserve sensitive structures within the ink. Materials as simple as screen printing ink to complex eukaryotic cells have been printed with applications spanning from microelectronics to tissue engineering. Biotechnology can benefit from this technique due to the efficient use of low volumes of reagent and the compatibility with a wide range of rheological properties. In addition, LIFT can be performed in a simple lab environment, not requiring vacuum or other extreme conditions. Although the basic apparatus is simple, many strategies exist to optimize the performance considering the ink and the desired pattern. The basic mechanism is similar between studies so the large number of variants can be summarized into a couple of categories and reported on with respect to their specific applications. In particular, precise and gentle deposition of complex molecules and eukaryotic cells represent the unique abilities of this technology. LIFT has demonstrated not only marked improvements in the quality of sensors and related medical devices over those manufactured with incumbent technologies but also great applicability in tissue engineering due to the high viability of printed cells.

Two-photon polymerization of 3-D zirconium oxide hybrid scaffolds for long-term stem cell growth.

Two-photon polymerization is a technique that involves simultaneous absorption of two photons from a femtosecond laser for selective polymerization of a photosensitive material. In this study, two-photon polymerization was used for layer-by-layer fabrication of 3-D scaffolds composed of an inorganic-organic zirconium oxide hybrid material. Four types of scaffold microarchitectures were created, which exhibit layers of parallel line features at various orientations as well as pores between the line features. Long-term cell culture studies involving human bone marrow stromal cells were conducted using these 3-D scaffolds. Cellular adhesion and proliferation were demonstrated on all of the scaffold types; tissuelike structure was shown to span the pores. This study indicates that two-photon polymerization may be used to create microstructured scaffolds out of an inorganic-organic zirconium oxide hybrid material for use in 3-D tissue culture systems.

Two-photon polymerization of polyethylene glycol diacrylate scaffolds with riboflavin and triethanolamine used as a water-soluble photoinitiator.

AIM: In this study, the suitability of a mixture containing riboflavin (vitamin B2) and triethanolamine (TEOHA) as a novel biocompatible photoinitiator for two-photon polymerization (2PP) processing was investigated. MATERIALS & METHODS: Polyethylene glycol diacrylate was crosslinked using Irgacure(®) 369, Irgacure 2959 or a riboflavin-TEOHA mixture; biocompatibility of the photopolymer extract solutions was subsequently assessed via endothelial cell proliferation assay, endothelial cell viability assay and single-cell gel electrophoresis (comet) assay. Use of a riboflavin-TEOHA mixture as a photoinitiator for 2PP processing of a tissue engineering scaffold and subsequent seeding of this scaffold with GM-7373 bovine aortic endothelial cells was also demonstrated. RESULTS: The riboflavin-TEOHA mixture was found to produce much more biocompatible scaffolds than those produced with Irgacure 369 or Irgacure 2959. CONCLUSION: The results suggest that riboflavin is a promising component of photoinitiators for 2PP fabrication of tissue engineering scaffolds and other medically relevant structures (e.g., biomicroelectromechanical systems).