Three-Dimensional Insights into Interfacial Segregation at the Atomic Scale in a Nanocrystalline Glass-Ceramic.

The distribution of dopant atoms plays a key role in the effectiveness of doping, thereby requiring delicate characterizations. In this study, we found that energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS) techniques in scanning transmission electron microscopy (STEM) were not adequate to reveal the distribution of yttrium and the chemical composition of the ZrO2/SiO2 heterophase interface in an yttrium-doped ZrO2-SiO2 nanocrystalline glass-ceramic. Atom probe tomography (APT) is rarely utilized to characterize ceramics due to some inherent difficulties. However, we successfully revealed the three-dimensional distribution of ZrO2 nanocrystallites and SiO2 matrix at the atomic scale with APT under optimized and well-controlled conditions. We also found that the ZrO2 nanocrystallites had a special core-shell structure, with a thin Zr/Si interfacial layer as a shell and a ZrO2 solid solution as a core. Yttrium dopants showed interfacial segregation at both ZrO2 grain boundaries and the ZrO2/SiO2 heterophase interfaces.

Simultaneous Visualization of Wet Cells and Nanostructured Biomaterials in SEM using Ionic Liquids.

This work presents a successful methodology to image mammalian cells adhered to nanostructured titanium by using scanning electron microscopy (SEM) operating in low-vacuum mode following ionic liquid treatment. Human osteoblast-like Saos-2 cells were treated with a room-temperature ionic liquid, 1-ethyl-3-methylimidazolium tetrafluoroborate, and subsequently imaged on titanium by SEM. Titanium substrates were modified to create laser-induced periodic surface structures (LIPSS) for visualization at the submicron scale. By using a combination of fluorescence-based cell metabolism along with light microscopy and SEM image analysis, the shape and location of irradiated cells were confirmed to be unchanged after multiple irradiation sessions; the viability of minimally irradiated cells was also unaltered. The wet imaging conditions combined with a rapid facile protocol using ionic liquid allows this technique to fulfill a niche in examining cellular behavior on biomaterials with submicron surface features. The demonstrated method to track observed cell adhesion to submicron surface features by SEM has great implications for understanding cell migration on nanostructured surfaces as well as the exploration of simpler SEM preparation methods for cellular imaging.

Ti-5Al-5Mo-5V-3Cr bone implants with dual-scale topography: a promising alternative to Ti-6Al-4V.

Modifications to the compositional, topographical and morphological aspects of bone implants can lead to improved osseointegration, thus increasing the success of bone implant procedures. This study investigates the creation of dual-scale topography on Ti-5Al-5Mo-5V-3Cr (Ti5553), an alloy not presently used in the biomedical field, and compares it to Ti-6Al-4V (Ti64), the most used Ti alloy for bone implants. Dual-scale surface topography was obtained by combining selective laser melting (SLM) and electrochemical anodization, which resulted in micro- and nanoscale surface features, respectively. Ti5553 and Ti64 samples were manufactured by SLM and showed comparable surface topography. Subsequent electrochemical anodization succeeded in forming titania nanotubes (TNTs) on both alloys, with larger nanotubes obtained with Ti5553 at all investigated anodization voltages. At an anodization voltage of 40 V, a minimum time of 20 min was necessary to have nanotube formation on the surface of either alloy, while only nanopores were evident for shorter times. Seeded Saos-2 cells showed ideal interactions with surface-modified structures, with filopodia extending to both surface microparticles characteristic of SLM and to the interior of TNTs. Attractiveness of Ti5553 lies in its lower elastic modulus (E = 72 GPa) compared to Ti64, which should mitigate stress-shielding phenomena in vivo. This, combined with the analogous results obtained in terms of dual-scale surface topography and cell-substrate interaction, could indicate Ti5553 as a promising alternative to the widely-employed Ti64 for bone implant device manufacturing.

Fabrication of polycaprolactone electrospun nanofibers doped with silver nanoparticles formed by air plasma treatment.

Implanted devices are prone to bacterial infections, which can result in implant loosening and device failure. Mitigating these infections is important to both implant stability and patient health. The development of antibacterial implant coatings can decrease the presence of bacterial colonies, reducing the risk for bacterial-dependent implant failure. Here, we show that electrospun polycaprolactone (PCL) fibers doped with silver nanoparticles (NPs) from a silver nitrate precursor have the potential to decrease the prevalence of Streptococcus pneumoniae while supporting osteoblast attachment and proliferation. An air plasma reduction method of PCL electrospun fibers was used to prepare fibers doped with silver NPs. Fibers were characterized using scanning electron microscopy and transmission electron microscopy for qualitative evaluation of NP distribution and quantitative analysis of fiber diameters. Antibacterial testing against S. pneumoniae was performed with successful inhibition observed after 24 h of exposure. In vitro testing was completed using Saos-2 cells and suggests that the negative surface charge has the potential to increase mammalian cell viability even in the presence of fibers containing NPs. In conclusion, this study describes a novel method to produce bioresorbable implant coatings with the ability to reduce bacterial infections surrounding the implant surface while remaining biocompatible to the host.

Micrometer-Sized Magnesium Whitlockite Crystals in Micropetrosis of Bisphosphonate-Exposed Human Alveolar Bone.

Osteocytes are contained within spaces called lacunae and play a central role in bone remodelling. Administered frequently to prevent osteoporotic fractures, antiresorptive agents such as bisphosphonates suppress osteocyte apoptosis and may be localized within osteocyte lacunae. Bisphosphonates also reduce osteoclast viability and thereby hinder the repair of damaged tissue. Osteocyte lacunae contribute to toughening mechanisms. Following osteocyte apoptosis, the lacunar space undergoes mineralization, termed "micropetrosis". Hypermineralized lacunae are believed to increase bone fragility. Using nanoanalytical electron microscopy with complementary spectroscopic and crystallographic experiments, postapoptotic mineralization of osteocyte lacunae in bisphosphonate-exposed human bone was investigated. We report an unprecedented presence of ∼80 nm to ∼3 µm wide, distinctly faceted, magnesium whitlockite [Ca18Mg2(HPO4)2(PO4)12] crystals and consequently altered local nanomechanical properties. These findings have broad implications on the role of therapeutic agents in driving biomineralization and shed new insights into a possible relationship between bisphosphonate exposure, availability of intracellular magnesium, and pathological calcification inside lacunae.

Visualization of Collagen-Mineral Arrangement Using Atom Probe Tomography.

Bone is a functional material comprised of mainly two phases: an organic collagenous phase and an inorganic mineral phase. Collagen-mineral arrangement has implications for bone function, aging, and disease. However, theories on collagen-mineral arrangement have been confined to studies with low spatial and/or compositional resolution resulting in an extensive debate over the location of mineral with respect to collagen. Herein, a strategy is developed to extract a single mineralized collagen fibril from bone and analyze its composition and structure atom-by-atom with 3D sub-nanometer accuracy and compositional clarity using atom probe tomography (APT). It is shown for the first time a method to probe fibril-level mineralization and collagen-mineral arrangement from an in vivo system with both the spatial and compositional precision required to comment on nanoscale collagen-mineral arrangement. APT of leporine bone shows distinct and helical collagen fibrils with mineralized deposits both encapsulating and incorporated into the collagenous structures. This study demonstrates a novel fibril-level detection method that can be used to probe the composition of bone and contribute new insights to the structure and organization of mineralized materials such as bones and teeth.

Response of Saos-2 osteoblast-like cells to kilohertz-resonance excitation in porous metallic scaffolds.

Post-operative therapy for joint replacement is often performed to optimize bone volume and bone-implant contact. Methods, such as pulsed therapeutic ultrasound, have been shown to be a valuable addition to regular physiotherapy to increase bone regeneration. To evaluate the efficacy of kilohertz-frequency (kHz) resonant stimuli to additively manufactured implant analogues, Saos-2 cells were seeded onto porous stainless steel scaffolds and flat substrates. Resonant frequency modes were mapped in the low kHz range, and cells were subjected to daily stimulus for 10 min at a frequency of 1.278 kHz. kHz-frequency excitation was found to increase normalized alkaline phosphatase production by almost twofold on metallic substrates relative to non-vibrated control scaffolds, while peak velocity influenced alkaline phosphatase production on porous scaffolds but not flat substrates. Total cell proliferation was downregulated by excitation, and all excited samples displayed larger variability. This work indicates that vibration within the range of 0.16-0.48 mm/s may reduce cell proliferation, but favour osteogenic gene expression. This study highlights the potential of using kHz-resonance therapy to mitigate early-onset pore occlusion to achieve uniform osseointegration through porous metallic scaffolds.

Identification of collagen fibrils in cross sections of bone by electron energy loss spectroscopy (EELS).

Transmission electron microscopic (TEM) images of ion-milled bovid cortical bone cut approximately normal to the axes of fibrils show that mineral occurs in the form of plates surrounding and laying between circular or elliptical features about 50 nm in diameter. The classification of these features as either pores or collagen fibrils is highly debated. Electron energy loss spectroscopy (EELS) mapping of these features in ion milled sections shows that they are lacking significant amounts of mineral or collagen, although their appearance suggests that they are cross sections of collagen fibrils. However, analogous sections prepared using an ultramicrotome show that, while these circular features show reduced concentrations of calcium and phosphorus, some of them contain quantities of carbon and nitrogen in bonding states comparable to the composition of collagen. This work demonstrates that the observed circular features are sections of collagen fibrils, but that bombardment by argon ions during broad beam ion milling destroys the collagen and associated gap-zone mineral.

Niobium pentoxide and hydroxyapatite particle loaded electrospun polycaprolactone/gelatin membranes for bone tissue engineering.

Effective methods of accelerating the bone regeneration healing process are in demand for a number of bone-related diseases and trauma. This work developed scaffolds with improved properties for bone tissue engineering by electrospinning composite polycaprolactone-gelatin-hydroxyapatite-niobium pentoxide (PGHANb) membranes. Composite membranes, with average fiber diameters ranging from 123 to 156 nm, were produced by adding hydroxyapatite (HA) and varying concentrations of niobium pentoxide (Nb2O5) particles (0, 3, 7, and 10 wt%) to a polycaprolactone (PCL) and gelatin (GL) matrix prior to electrospinning. The morphology, mechanical, chemical and biological properties of resultant membranes were evaluated. Bioactivity was assessed using simulated body fluid (SBF) and it confirmed that the presence of particles induced the formation of hydroxyapatite crystals on the surface of the membranes. Samples were hydrophilic and cell metabolism results showed that the niobium-containing membranes were non-toxic while improving cell proliferation and differentiation compared to controls. This study demonstrated that electrospun membranes containing HA and Nb2O5 particles have potential to promote cell adhesion and proliferation while exhibiting bioactive properties. PGHANb membranes are promising candidates for bone tissue engineering applications.

A Bioprinted In Vitro Model for Osteoblast to Osteocyte Transformation by Changing Mechanical Properties of the ECM.

Osteocytes are key contributors to bone remodeling. During the remodeling process, trapped osteoblasts undergo a phenotypic change to become osteocytes. The specific mechanisms by which osteocytes work are still debatable and models that exist to study them are sparse. This work presents an in vitro, bioprinted model based on the previously developed technique, ExCeL, in which a cell-embedded hydrogel is printed and immediately crosslinked using paper as a crosslinker-storing substrate. This process mimics the phenotypical change of osteoblast to osteocyte by altering the mechanical properties of the hydrogel. By printing Saos-2, osteosarcoma cells, embedded in the alginate hydrogel with differing mechanical properties, their morphology, protein, and gene expression can be changed from osteoblast-like to osteocyte-like. The stiffer gel is 30 times stiffer and results in significantly smaller cells with reduced alkaline phosphatase activity and expression of osteoblast-marker genes such as MMP9 and TIMP2. There is no change in viability between cells despite encapsulation in gels with different mechanical properties. The results show that the phenomenon of osteoblasts becoming encapsulated during the bone remodeling process can be replicated using the ExCeL bioprinting technique. This model has potential for studying how osteocytes can interact with external mechanical stimuli or drugs.

Cross-linked cellulose nanocrystal aerogels as viable bone tissue scaffolds.

Chemically cross-linked cellulose nanocrystal (CNC) aerogels possess many properties beneficial for bone tissue scaffolding applications. CNCs were extracted using sulfuric acid or phosphoric acid, to produce CNCs with sulfate and phosphate half-ester surface groups, respectively. Hydrazone cross-linked aerogels fabricated from the two types of CNCs were investigated using scanning electron microscopy, X-ray micro-computed tomography, X-ray photoelectron spectroscopy, nitrogen sorption isotherms, and compression testing. CNC aerogels were evaluatedin vitrowith osteoblast-like Saos-2 cells and showed an increase in cell metabolism up to 7 days while alkaline phosphatase assays revealed that cells maintained their phenotype. All aerogels demonstrated hydroxyapatite growth over 14 days while submerged in simulated body fluid solution with a 0.1 M CaCl2 pre-treatment. Sulfated CNC aerogels slightly outperformed phosphated CNC aerogels in terms of compressive strength and long-term stability in liquid environments, and were implanted into the calvarian bone of adult male Long Evans rats. Compared to controls at 3 and 12 week time points, sulfated CNC aerogels showed increased bone volume fraction of 33% and 50%, respectively, compared to controls, and evidence of osteoconductivity. These results demonstrate that cross-linked CNC aerogels are flexible, porous and effectively facilitate bone growth after they are implanted in bone defects. STATEMENT OF SIGNIFICANCE: Due to the potential complications associated with autografts, there is a need for synthetic bone tissue scaffolds. Here, we report a new naturally-based aerogel material for bone regeneration made solely from chemically cross-linked cellulose nanocrystals (CNC). These highly porous CNC aerogels were shown to promote the proliferation of bone-like cells and support the growth of hydroxyapatite on their surface in vitro. The first in vivo study on these materials was conducted in rats and showed their osteconductive properties and an increase in bone volume up to 50% compared to sham sites. This study demonstrates the potential of using functionalized cellulose nanocrystals as the basis for aerogel scaffolds for bone tissue engineering.

Osseointegration of a 3D Printed Stemmed Titanium Dental Implant: A Pilot Study.

In this pilot study, a 3D printed Grade V titanium dental implant with a novel dual-stemmed design was investigated for its biocompatibility in vivo. Both dual-stemmed (n = 12) and conventional stainless steel conical (n = 4) implants were inserted into the tibial metaphysis of New Zealand white rabbits for 3 and 12 weeks and then retrieved with the surrounding bone, fixed, dehydrated, and embedded into epoxy resin. The implants were analyzed using correlative histology, microcomputed tomography, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The histological presence of multinucleated osteoclasts and cuboidal osteoblasts revealed active bone remodeling in the stemmed implant starting at 3 weeks and by 12 weeks in the conventional implant. Bone-implant contact values indicated that the stemmed implants supported bone growth along the implant from the coronal crest at both 3- and 12-week time periods and showed bone growth into microporosities of the 3D printed surface after 12 weeks. In some cases, new bone formation was noted in between the stems of the device. Conventional implants showed mechanical interlocking but did have indications of stress cracking and bone debris. This study demonstrates the comparable biocompatibility of these 3D printed stemmed implants in rabbits up to 12 weeks.