Influence of titanium nanoscale surface roughness on fibrinogen and albumin protein adsorption kinetics and platelet responses.

Biomaterials with nanoscale topography have been increasingly investigated for medical device applications to improve tissue-material interactions. This study assessed the impact of nanoengineered titanium surface domain sizes on early biological responses that can significantly affect tissue interactions. Nanostructured titanium coatings with distinct nanoscale surface roughness were deposited on quartz crystal microbalance with dissipation (QCM-D) sensors by physical vapor deposition. Physico-chemical characterization was conducted to assess nanoscale surface roughness, nano-topographical morphology, wettability, and atomic composition. The results demonstrated increased projected surface area and hydrophilicity with increasing nanoscale surface roughness. The adsorption properties of albumin and fibrinogen, two major plasma proteins that readily encounter implanted surfaces, on the nanostructured surfaces were measured using QCM-D. Significant differences in the amounts and viscoelastic properties of adsorbed proteins were observed, dependent on the surface roughness, protein type, protein concentration, and protein binding affinity. The impact of protein adsorption on subsequent biological responses was also examined using qualitative and quantitative in vitro evaluation of human platelet adhesion, aggregation, and activation. Qualitative platelet morphology assessment indicated increased platelet activation/aggregation on titanium surfaces with increased roughness. These data suggest that nanoscale differences in titanium surface roughness influence biological responses that may affect implant integration.

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.

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.

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.

Nanostructured Medical Adhesives.

Suturing has been the gold standard approach to close wounds for many decades. However, suturing causes tissue damage, which is accompanied by foreign body reaction, entry of pathogens, complications, infection, or death. In addition, the procedure is usually time-consuming, requiring manual dexterity and free moving space. Other adhesive approaches have been proposed and demonstrated with great potential, including laser-assisted tissue closure with either photothermal or photochemical reactions, application of nanoparticles, glues, constructs based on extracellular matrix (ECM), microbarbs, bio-inspired structures, and tape. The quality of closure has been evaluated by histological methods, indexing, morphology, tensile testing, patency rate, leakage pressure, and burst pressure. All the novel tissue joining methods aim to provide an adhesive with appropriate strength, non-cytotoxicity, and minimal damage. The capability for rapid attachment and release may further reduce surgical procedure time. More research is needed to prove the feasibility of new tissue joining techniques based on the type of tissue, surface chemistry, and working environment.

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.

Current Advancements in Transdermal Biosensing and Targeted Drug Delivery.

In this manuscript, recent advancements in the area of minimally-invasive transdermal biosensing and drug delivery are reviewed. The administration of therapeutic entities through the skin is complicated by the stratum corneum layer, which serves as a barrier to entry and retards bioavailability. A variety of strategies have been adopted for the enhancement of transdermal permeation for drug delivery and biosensing of various substances. Physical techniques such as iontophoresis, reverse iontophoresis, electroporation, and microneedles offer (a) electrical amplification for transdermal sensing of biomolecules and (b) transport of amphiphilic drug molecules to the targeted site in a minimally invasive manner. Iontophoretic delivery involves the application of low currents to the skin as well as the migration of polarized and neutral molecules across it. Transdermal biosensing via microneedles has emerged as a novel approach to replace hypodermic needles. In addition, microneedles have facilitated minimally invasive detection of analytes in body fluids. This review considers recent innovations in the structure and performance of transdermal systems.

Biological responses to immobilized microscale and nanoscale surface topographies.

Cellular responses are highly influenced by biochemical and biomechanical interactions with the extracellular matrix (ECM). Due to the impact of ECM architecture on cellular responses, significant research has been dedicated towards developing biomaterials that mimic the physiological environment for design of improved medical devices and tissue engineering scaffolds. Surface topographies with microscale and nanoscale features have demonstrated an effect on numerous cellular responses, including cell adhesion, migration, proliferation, gene expression, protein production, and differentiation; however, relationships between biological responses and surface topographies are difficult to establish due to differences in cell types and biomaterial surface properties. Therefore, it is important to optimize implant surface feature characteristics to elicit desirable biological responses for specific applications. The goal of this work was to review studies investigating the effects of microstructured and nanostructured biomaterials on in vitro biological responses through fabrication of microscale and nanoscale surface topographies, physico-chemical characterization of material surface properties, investigation of protein adsorption dynamics, and evaluation of cellular responses in specific biomedical applications.

Effects of nanotopography on the in vitro hemocompatibility of nanocrystalline diamond coatings.

Nanocrystalline diamond (NCD) coatings have been investigated for improved wear resistance and enhanced hemocompatibility of cardiovascular devices. The goal of this study was to evaluate the effects of NCD surface nanotopography on in vitro hemocompatibility. NCD coatings with small (NCD-S) and large (NCD-L) grain sizes were deposited using microwave plasma chemical vapor deposition and characterized using scanning electron microscopy, atomic force microscopy, contact angle testing, and Raman spectroscopy. NCD-S coatings exhibited average grain sizes of 50-80 nm (RMS 5.8 nm), while NCD-L coatings exhibited average grain sizes of 200-280 nm (RMS 23.1 nm). In vitro hemocompatibility testing using human blood included protein adsorption, hemolysis, nonactivated partial thromboplastin time, platelet adhesion, and platelet activation. Both NCD coatings demonstrated low protein adsorption, a nonhemolytic response, and minimal activation of the plasma coagulation cascade. Furthermore, the NCD coatings exhibited low thrombogenicity with minimal platelet adhesion and aggregation, and similar morphological changes to surface-bound platelets (i.e., activation) in comparison to the HDPE negative control material. For all assays, there were no significant differences in the blood-material interactions of NCD-S versus NCD-L. The two tested NCD coatings, regardless of nanotopography, had similar hemocompatibility profiles compared to the negative control material (HDPE) and should be further evaluated for use in blood-contacting medical devices. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 253-264, 2017.

Biological evaluation of ultrananocrystalline and nanocrystalline diamond coatings.

Nanostructured biomaterials have been investigated for achieving desirable tissue-material interactions in medical implants. Ultrananocrystalline diamond (UNCD) and nanocrystalline diamond (NCD) coatings are the two most studied classes of synthetic diamond coatings; these materials are grown using chemical vapor deposition and are classified based on their nanostructure, grain size, and sp3 content. UNCD and NCD are mechanically robust, chemically inert, biocompatible, and wear resistant, making them ideal implant coatings. UNCD and NCD have been recently investigated for ophthalmic, cardiovascular, dental, and orthopaedic device applications. The aim of this study was (a) to evaluate the in vitro biocompatibility of UNCD and NCD coatings and (b) to determine if variations in surface topography and sp3 content affect cellular response. Diamond coatings with various nanoscale topographies (grain sizes 5-400 nm) were deposited on silicon substrates using microwave plasma chemical vapor deposition. Scanning electron microscopy and atomic force microscopy revealed uniform coatings with different scales of surface topography; Raman spectroscopy confirmed the presence of carbon bonding typical of diamond coatings. Cell viability, proliferation, and morphology responses of human bone marrow-derived mesenchymal stem cells (hBMSCs) to UNCD and NCD surfaces were evaluated. The hBMSCs on UNCD and NCD coatings exhibited similar cell viability, proliferation, and morphology as those on the control material, tissue culture polystyrene. No significant differences in cellular response were observed on UNCD and NCD coatings with different nanoscale topographies. Our data shows that both UNCD and NCD coatings demonstrate in vitro biocompatibility irrespective of surface topography.

Inkjet deposition of itraconazole onto poly(glycolic acid) microneedle arrays.

Poly(glycolic acid) microneedle arrays were fabricated using a drawing lithography process; these arrays were modified with a drug release agent and an antifungal agent by piezoelectric inkjet printing. Coatings containing poly(methyl vinyl ether-co-maleic anhydride), a water-soluble drug release layer, and itraconazole (an antifungal agent), were applied to the microneedles by piezoelectric inkjet printing. Microscopic evaluation of the microneedles indicated that the modified microneedles contained the piezoelectric inkjet printing-deposited agents and that the surface coatings were released in porcine skin. Energy dispersive x-ray spectrometry aided in confirmation that the piezoelectric inkjet printing-deposited agents were successfully applied to the desired target areas of the microneedle surface. Fourier transform infrared spectroscopy was used to confirm the presence of the component materials in the piezoelectric inkjet printing-deposited material. Itraconazole-modified microneedle arrays incubated with agar plates containing Candida albicans cultures showed zones of growth inhibition.

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.

Cytotoxic evaluation of nanostructured zinc oxide (ZnO) thin films and leachates.

Nanostructured ZnO films have potential use as coatings on medical devices and food packaging due to their antimicrobial and UV-protection properties. However, their influence on mammalian cells during clinical use is not fully understood. This study investigated the potential cytotoxicity of ZnO thin films in RAW 264.7 macrophages. ZnO thin films (∼96nm thick with a 50nm grain) were deposited onto silicon wafers using pulsed laser deposition. Cells grown directly on ZnO thin film coatings exhibited less toxicity than cells exposed to extracts of the coatings. Cells on ZnO thin films exhibited a 43% and 68% decrease in cell viability using the MTT and 7-AAD/Annexin V flow cytometry assays, respectively, after a 24-h exposure as compared to controls. Undiluted 100% 24- and 48-h extracts decreased viability by 89%, increased cell death by LDH release to 76% 24h after treatment, and increased ROS after 5-24h of exposure. In contrast, no cytotoxicity or ROS were observed for 25% and 50% extracts, indicating a tolerable concentration. Roughly 24 and 34µg/m(2) Zn leached off the surfaces after 24 and 48h of incubation, respectively. ZnO coatings may produce gradual ion release which becomes toxic after a certain level and should be evaluated using both direct exposure and extraction methods.

Stereolithography in tissue engineering.

Several recent research efforts have focused on use of computer-aided additive fabrication technologies, commonly referred to as additive manufacturing, rapid prototyping, solid freeform fabrication, or three-dimensional printing technologies, to create structures for tissue engineering. For example, scaffolds for tissue engineering may be processed using rapid prototyping technologies, which serve as matrices for cell ingrowth, vascularization, as well as transport of nutrients and waste. Stereolithography is a photopolymerization-based rapid prototyping technology that involves computer-driven and spatially controlled irradiation of liquid resin. This technology enables structures with precise microscale features to be prepared directly from a computer model. In this review, use of stereolithography for processing trimethylene carbonate, polycaprolactone, and poly(D,L-lactide) poly(propylene fumarate)-based materials is considered. In addition, incorporation of bioceramic fillers for fabrication of bioceramic scaffolds is reviewed. Use of stereolithography for processing of patient-specific implantable scaffolds is also discussed. In addition, use of photopolymerization-based rapid prototyping technology, known as two-photon polymerization, for production of tissue engineering scaffolds with smaller features than conventional stereolithography technology is considered.

Cytotoxicity of cultured macrophages exposed to antimicrobial zinc oxide (ZnO) coatings on nanoporous aluminum oxide membranes.

Zinc oxide (ZnO) is a widely used commercial material that is finding use in wound healing applications due to its antimicrobial properties. Our study demonstrates a novel approach for coating ZnO with precise thickness control onto 20 nm and 100 nm pore diameter anodized aluminum oxide using atomic layer deposition (ALD). ZnO was deposited throughout the nanoporous structure of the anodized aluminum oxide membranes. An 8 nm-thick coating of ZnO, previously noted to have antimicrobial properties, was cytotoxic to cultured macrophages. After 48 h, ZnO-coated 20 nm and 100 nm pore anodized aluminum oxide significantly decreased cell viability by ≈65% and 54%, respectively, compared with cells grown on uncoated anodized aluminum oxide membranes and cells grown on tissue culture plates. Pore diameter (20-200 nm) did not influence cell viability.

Hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin. Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements. This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH, which are important metabolic indicators of disease states (e.g., cancer proliferation) and exercise-induced acidosis.

Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.

The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure(®)) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-h period.