The maintenance of healthy bone tissue depends upon the ability of osteocytes to respond to mechanical cues on the cellular level. The combination of digital volume correlation and second harmonic generation microscopy offers the opportunity to investigate the mechanical microenvironment of intact bone on the scale of individual osteocytes. Adult human femurs were imaged under tensile loads of 5 and 15MPa and volumes of approximately 492×429×31μm(3) were analyzed, along with an image of a bone microcrack under the same loading conditions. Principal strains were significantly higher in three-dimensional digital volume correlation when compared to two-dimensional digital image correlation. The average maximum principal strain magnitude was 5.06-fold greater than the applied global strain, with peak strains of up to 23.14-fold over global strains measured at the borders of osteocyte lacunae. Finally, a microcrack that initiated at an osteocyte lacunae had its greatest tensile strain magnitudes at the crack expansion front in the direction of a second lacunae, but strain at the crack border was reduced to background strain magnitudes upon breaching the second lacunae. This serveed to demonstrate the role of lacunae in initiating, mediating and terminating microcrack growth.

Optical tomography has a wide range of biomedical applications. Accurate prediction of photon transport in media is critical, as it directly affects the accuracy of the reconstructions. The radiative transfer equation (RTE) is the most accurate deterministic forward model, yet it has not been widely employed in practice due to the challenges in robust and efficient numerical implementations in high dimensions. Herein, we propose a method that combines the discrete ordinate method (DOM) with a streamline diffusion modified continuous Galerkin method to numerically solve RTE. Additionally, a phase function normalization technique was employed to dramatically reduce the instability of the DOM with fewer discrete angular points. To illustrate the accuracy and robustness of our method, the computed solutions to RTE were compared with Monte Carlo (MC) simulations when two types of sources (ideal pencil beam and Gaussian beam) and multiple optical properties were tested. Results show that with standard optical properties of human tissue, photon densities obtained using RTE are, on average, around 5% of those predicted by MC simulations in the entire/deeper region. These results suggest that this implementation of the finite element method-RTE is an accurate forward model for optical tomography in human tissues.

Upconverting nanoparticles (UCNP) have the unique ability to emit both in the NIR and visible light upon NIR excitation. Herein we investigate experimentally the potential to use UCNPs in dental imaging ex vivo.

We determined inhomogeneity of strains around discontinuities as well as changes in orientation of collagen fibrils under applied load in skin. Second Harmonic Generation (SHG) images of collagen fibrils were obtained at different strain magnitudes. Changes in collagen orientation were analyzed using Fast Fourier Transforms (FFT) while strain inhomogeneity was determined at different distances from hair follicles using Digital Image Correlation (DIC). A parameter, defined as the Collagen Orientation Index (COI), is introduced that accounts for the increasingly ellipsoidal nature of the FFT amplitude images upon loading. We show that the COI demonstrates two distinct mechanical regimes, one at low strains (0%, 2.5%, 5% strain) in which randomly oriented collagen fibrils align in the direction of applied deformation. In the second regime, beginning at 5% strain, collagen fibrils elongate in response to applied deformation. Furthermore, the COI is also found to be linearly correlated with the applied stress indicating that collagen fibrils orient to take the applied load. DIC results indicated that major principal strains were found to increase with increased load at all locations. In contrast, minimum principal strain was dependent on distance from hair follicles. These findings are significant because global and local changes in collagen deformations are expected to be changed by disease, and could affect stem cell populations surrounding hair follicles, including mesenchymal stem cells within the outer root sheath.

Ultrasound induces mechanical vibration and heat, causing differentiation and proliferation in osteoblasts. All known in vitro evaluations of ultrasound are, however, performed with longitudinal ultrasound waves. We addressed a question: Do other forms of ultrasound waves, such as guided waves (longitudinal and guided flexural) transduced at a remote location, enhance differentiation of osteoblast cells? In this study, we employed guided Lamb waves that were induced in a borosilicate glass slide (cortical bone mimic). An average energy of 10–30 mW/cm2 for 20 min per day was applied to MC3T3 osteoblast-like cells, which were placed 30–75 mm distant from the transducer. The result revealed that guided waves significantly stimulated the differentiation and mineralization of MC3T3 cells. In particular, guided waves elevated mRNA expression levels of bone formation-related genes such as alkaline phosphatase, osteopontin, osteocalcin, osteoprotegerin, and bone sialoprotein on days 8 and 16. In addition, the amount of mineralization found via Alizarin red staining was increased by 157 % (p = 0.034). The amount of mineralization was found to be independent of distance from the transducer (p = 0.967). We demonstrate herein that ultrasound in a form of guided Lamb waves is capable of inducing osteoblast differentiation in vitro, and it may enable the stimulation of osteoblasts in vivo over a distance from the site of ultrasound application.

In this work, we investigated the effects of negative pressure, applied using a pump designed for Negative Pressure Wound Therapy (NPWT), on the process of wound healing in vitro via initiation of the Wnt signaling pathway. Results indicate that negative pressure enhanced Wnt signaling and migration into a simulated wound in vitro in NIH-3T3 murine fibroblast cells. Increasing doses of lithium (upto 15 mM) increased basal Wnt signaling and enhanced cell migration into the simulated wound site. A combination of negative pressure and increased doses of lithium synergistically increased Wnt signaling and demonstrated further enhanced cell migration into simulated wound sites, with maximal filling of the simulated wound observed at lithium concentrations of at least 10mM.

Polycationic nanocomplexes are a robust means for achieving nucleic acid condensation and efficient intracellular gene deliveries. To enhance delivery, a multilayered nanoparticle consisting of a core of electrostatically bound elements was used. These included a histone-mimetic peptides, poly-l-arginine and poly-d-glutamic acid was coated with silicate before surface functionalization with poly-l-arginine. Transfection efficiencies and duration of expression were similar when using green fluorescent protein (GFP) plasmid DNA (pDNA) or GFP mRNA. These nanoparticles demonstrated significantly higher (>100%) and significantly longer (15 vs. 4 days) transfection efficiencies in comparison to a commercial transfection agent (Lipofectamine 2000). Reprogramming of human foreskin fibroblasts using mRNA to the Sox2 transcription factor resulted in three-fold higher neurosphere formation in comparison to the commercial reagent. These results demonstrate the potential of these nanoparticles as ideal vectors for gene delivery.

The objective was to determine the effects of oral lithium chloride supplementation on bone strength and mass in broiler chickens. Ninety-six broilers were assigned to 1 of 2 treatment groups (lithium chloride or control; n=48/treatment). Beginning at 1 or 3 wk of age, chickens were administered lithium chloride (20 mg/kg body weight) or water daily by oral gavage. At 6 wk of age, chickens were euthanized and bone and muscle samples were collected. A 24 h lithium chloride (20 mg/kg body weight) challenge determined that serum lithium chloride increased within 2 h and cleared the system within 24 h, demonstrating the effective delivery of lithium chloride. Treatment did not influence body weight (P≥0.20) or feed intake (P≥0.81), demonstrating that lithium chloride did not negatively affect broiler growth. To determine bone strength, 3-point bending was performed on the femora and tibiae obtained from control and lithium chloride-treated birds in the 1 wk group. Lithium chloride-treated birds had a 22% reduction in stiffness compared with control in the femora (P=0.02) without a corresponding reduction in elastic modulus. No differences were observed in yield or ultimate load and in the corresponding calculations of stresses (P≥0.26). The toughness of tibiae was not altered in lithium chloride compared with control (P=0.11). Bone length and micro-CT imaging were performed on the tibiae of control and lithium chloride groups. No differences (P≥0.52) in bone length, cortical or trabecular bone volume, trabecular thickness, number, or spacing were observed. Lithium chloride treatment did not affect pectoralis muscle color or lipid oxidation (P>0.05). In conclusion, lithium chloride treatment in broilers did not negatively affect growth or meat quality. A reduction in bone stiffness of the femur with lithium chloride treatment was observed, however unlike the mouse model, the dosages of lithium chloride used in the current study did not result in anabolic effects on broiler long bones.

Centrifugal jet spinning (CJS) is a highly efficient, low-cost, and versatile method for fabricating polymer nanofiber assemblies, especially in comparison to electrospinning. The process uses centrifugal forces coupled with the viscoelastic properties and the mass transfer characteristics of spinning solutions to promote the controlled thinning of a polymer solution filament into nanofibers. In this study, three different spinning stages (jet initiation, jet extension, and fiber formation) were analyzed in terms of the roles of fluid viscoelasticity, centrifugal forces, and solvent mass transfer. Four different polymer solution systems were used, which enables a wide range of fluid viscoelasticity properties and solvent mass transfer properties, and polymer fibers were fabricated under different rotational speeds for these polymer solutions. The key dimensionless groups that determine the product morphology (beads, beads-on-fiber, and continuous fiber) and the radius of the fiber (when fibers are formed)...

A novel silicone-epoxy oligomer was synthesized and evaluated for its use as the polymer in photopolymerizable dental composites. This synthesized oligomer contained rigid and non-rigid groups with 1–8 epoxy functionalities as characterized using 1H NMR and 29Si NMR and MALDI-TOF analysis. In comparison to the traditional BisGMA/TEGDMA monomer system, the photo-polymerized silicone-epoxy demonstrated significantly improved material properties (148% greater elastic modulus, 12% greater ultimate strength, 48% greater fracture toughness), as well as 61% lower polymerization shrinkage and 58% lower polymerization stresses. Furthermore, the silicone-epoxy system demonstrated enhanced resistance to degradation of its material properties after accelerated (24hr) aging, i.e. exposure to severe hydrolytic (boiling in ethanol at 100 □C), oxidative (exposed to 5% H2O2), and low pH (0.05M acetic acid) stress. Under these conditions, the properties of conventional BisGMA/TEGDMA systems deteriorated by 22–47%, while the properties of the silicone-epoxy composites decreased by 2–10%. Bisphosphonate additives enhanced the precipitation of mineral in a dose-dependent manner, but inhibited polymerization due to interactions with epoxy groups. Bisphosphonate additives also dose-dependently demonstrated anti-bacterial efficacy as demonstrated using live-dead, MTT and crystral violet assays. The silicone-epoxy polymer was demonstrated to be biocompatible when compared to tissue culture plastic. When calcium fluoride was incorporated into this system, fluoride was found to be released quantities significant enough to engender anti-bacterial effects. In summary, the designed multifunctional dental resin exhibits higher stability as demonstrated by lower chemical, mechanical and enzymatic degradation.

A rapid and efficient method consisting of two simple steps, centrifugal jet spinning (CJS) and annealing, is introduced to fabricate multilevel structured silica micro-/nanofibers. Using this technique, which is 500 times faster than electrospinning, silica micro-/nanofibers with a hollow or porous internal structure are formed as a result of non-solvent evaporation induced phase separation in the spinning solution. Silica nanofibers with solid cross sections (364 nm and 781 nm), hollow cross sections (outer and internal diameters of 458 nm and 216 nm respectively), and encapsulated voids (outside diameter of 1.4 μm where bi-continuous nano-pores 118 nm are observed) are fabricated by tuning the amount of non-solvent in the spinning solutions. This technique can be readily extended to large-scale and efficient fabrication of various ceramic materials with multileveled fibrous structures.

Silica nanotubes are widely used in nanotechnology because of their unique properties. In this paper, silica nanotube fibers with wall thickness less than 100 nm and outer diameters ranging between 300 nm and 500 nm are fabricated using the centrifugal jet spinning (CJS) technique with two steps: spinning and annealing. Polyvinylpyrrolidone-silica (PVP-silica) composite fibers are initially spun with engineered spinning solutions. By controlling the degree of tetraethyl orthosilicate (TEOS) hydrolysis, unreacted excess TEOS is introduced into spinning solutions to form a dual-solvent system with ethanol. Because of the differential vapor pressures of TEOS and ethanol as well as their differential solubility of PVP and dispersion of silica, dual-solvent evaporation induces phase separation in the spinning solution. This promotes the formation of hollow structure in PVP-silica fibers during spinning. Silica nanotube fibers are obtained with subsequent thermal annealing of PVP-silica composite fibers. The diameter and wall thickness of the silica nanotubes can be controlled by tuning the content of TEOS in the initial spinning solutions. The mechanism of tubular structure formation is also explained and examined with the help of elemental analysis. The method used in this paper can provide a new direction for efficient and large-scale fabrication of nanomaterials with controlled morphologies.

Dental lesions located in the pulp are quite difficult to identify based on anatomical contrast, and, hence, to diagnose using traditional imaging methods such as dental CT. However, such lesions could lead to functional and/or molecular optical contrast. Herein, we report on the preliminary investigation of using Laminar Optical Tomography (LOT) to image the pulp and root canals in teeth. LOT is a non-contact, high resolution, molecular and functional mesoscopic optical imaging modality. To investigate the potential of LOT for dental imaging, we injected an optical dye into ex vivo teeth samples and imaged them using LOT and micro-CT simultaneously. A rigid image registration between the LOT and micro-CT reconstruction was obtained, validating the potential of LOT to image molecular optical contrast deep in the teeth with accuracy, non-invasively. We demonstrate that LOT can retrieve the 3D bio-distribution of molecular probes at depths up to 2mm with a resolution of several hundred microns in teeth.

Some dental lesions are difficult to detect with traditional anatomical imaging methods, such as, with visual observation, dental radiography and X-ray computed tomography (CT). Therefore, we investigated the viability of using an optical imaging technique, Mesoscopic Fluorescence Molecular Tomography (MFMT) to retrieve molecular contrast in dental samples. To establish feasibility of obtaining 3-D images in teeth using MFMT, molecular contrast was simulated using a dye-filled capillary that was placed in the lower half of human tooth ex vivo. The dye and excitation wavelength were chosen to be excited at 650–660 nm in order to simulate a carious lesion. The location of the capillary was varied by changing the depth from the surface at which the dye, at various concentrations, was introduced. MFMT reconstructions were benchmarked against micro-CT. Overall; MFMT exhibited a location accuracy of ~15% and a volume accuracy of ~15%, up to 2 mm depth with moderate dye concentrations. These results demonstrate the potential of MFMT to retrieve molecular contrast in 3-D in highly scattering tissues, such as teeth.

The structure and material properties of bones along with applied boundary conditions determine the region of peak stresses, where fracture is expected to occur. As the site of peak stresses is not influenced by the magnitude of applied load, the fracture site is not expected to change during fatigue loading of whole bone at different loads. However, in a highly curved bone such as the rat ulna, the magnitude of applied loads was found to influence the fracture site. Fatigue loading was conducted under load control on intact rat forearms and on excised ulnae. The distance to the site of failure from the proximal olecranon process of ulnae was determined. In intact forearms, the site of failure demonstrated a linear progression distally, towards sites with lower moment of inertia (or sites exhibiting lower section modulus). Intact rat forearms and excised ulnae loaded to failure at low loads fractured 2–3 mm distal to where they failed when applying high loads. This indicates a shift in the site of failure by approximately 10% of whole bone length just by varying the applied load magnitude. The site of failure in excised ulnae was similar when loading at 2 Hz or at 4 Hz, suggesting that this was frequency independent in this range and indicating that strain rate was not an important contributing factor. Creep loading of excised ulnae also demonstrated similar changes in the site of failure, indicating that magnitude of loads, and not type of loading were important in determining the site of failure. This has important implications with regards to the volume of bone that undergoes damage under physiological loading, before it fails.

PLLA fibrous tissue scaffolds with controlled fiber nanoscale surface roughness are fabricated with a novel centrifugal jet spinning process. The centrifugal jet spinning technique is a highly efficient synthesis method for micron- to nano-sized fibers with a production rate up to 0.5 g min(-1). During the centrifugal jet spinning process, a polymer solution jet is stretched by the centrifugal force of a rotating chamber. By engineering the rheological properties of the polymer solution, solvent evaporation rate and centrifugal force that are applied on the solution jet, polyvinylpyrrolidone (PVP) and poly(l-lactic acid) (PLLA) composite fibers with various diameters are fabricated. Viscosity measurements of polymer solutions allowed us to determine critical polymer chain entanglement limits that allow the generation of continuous fiber as opposed to beads or beaded fibers. Above a critical concentration at which polymer chains are partially or fully entangled, lower polymer concentrations and higher centrifugal forces resulted in thinner fibers. Etching of PVP from the PLLA-PVP composite fibers doped with increasing PVP concentrations yielded PLLA fibers with increasing nano-scale surface roughness and porosity, which increased the fiber hydrophilicity dramatically. Scanning electron micrographs of the etched composite fibers suggest that PVP and PLLA were co-contiguously phase separated within the composite fibers during spinning and nano-scale roughness features were created after the partial etching of PVP. To study the tissue regeneration efficacy of the engineered PLLA fiber matrix, human dermal fibroblasts are used to simulate partial skin graft. Fibers with increased PLLA surface roughness and porosity demonstrated a trend towards higher cell attachment and proliferation.

The use of hydrogels for bone regeneration has been limited due to their inherent low modulus to support cell adhesion and proliferation as well as their susceptibility to bacterial infections at the wound site. To overcome these limitations, we evaluated multifunctional polysaccharide hydrogels of varying stiffness to obtain the optimum stiffness at which the gels (1) induce proliferation of human dermal fibroblasts, human umbilical vascular endothelial cells (HUVECs), and murine preosteoblasts (MC3T3-E1), (2) induce osteoblast differentiation and mineralization, and (3) exhibit an antibacterial activity. Rheological studies demonstrated that the stiffness of hydrogels made of a polysaccharide blend of methylcellulose, chitosan, and agarose was increased by crosslinking the chitosan component to different extents with increasing amounts of genipin. The gelation time decreased (from 210 to 60 min) with increasing genipin concentrations. Proliferation of HUVECs decreased by 10.7 times with increasing gel stiffness, in contrast to fibroblasts and osteoblasts, where it increased with gel stiffness by 6.37 and 7.8 times, respectively. At day 14 up to day 24, osteoblast expression of differentiation markers-osteocalcin, osteopontin-and early mineralization marker-alkaline phosphatase, were significantly enhanced in the 0.5% (w/v) crosslinked gel, which also demonstrated enhanced mineralization by day 25. The antibacterial efficacy of the hydrogels decreased with the increasing degree of crosslinking as demonstrated by biofilm formation experiments, but gels crosslinked with 0.5% (w/v) genipin still demonstrated significant bacterial inhibition. Based on these results, gels crosslinked with 0.5% (w/v) genipin, where 33% of available groups on chitosan were crosslinked, exhibited a stiffness of 502±64.5 Pa and demonstrated the optimal characteristics to support bone regeneration.

The micromechanical environment of bone is crucial to understanding both bone fracture and mechanobiological responses of osteocytes, yet few techniques exist that are capable of measuring strains on the micrometer scale. A method for measuring micrometer level strains has been developed based on digital image correlation (DIC) of second harmonic generation microscopy (SHGM) images. Bovine tibias milled into thin sections were imaged using SHGM under loads of 0 and 15 MPa. Strains were measured using DIC and compared to applied strain values. First and second principal strains decreased in magnitude as the analysis region area increased from 1750 µm(2) to 60,920 µm(2), converging to 1.23 ± 0.74 and -0.745 ± 0.9816 times the applied strain respectively. A representative sample histogram revealed regions of pure tensile and compressive strain, and that strains were highly heterogeneous ranging from 8410 to -8840 microstrain for an applied 2870 microstrain. Comparison with applied strain measures suggested that analysis sizes of 1750 µm(2) and greater were measuring strains on the tissue scale, and higher resolution is required for collagen fibrillar strains. Regions of low SHGM intensity ("dark" regions) were seen which are believed to be lacunar and perilacunar regions of low collagen density. However, no significant differences in strain magnitude were present in dark regions versus regions of high signal intensity. The proposed technique is effective for strains on the size order of bone microarchitecture, and would be useful for studies into the mechanical microenvironment during loading. The technique also has potential for in vivo studies in small animal models.

The centrifugal jet spinning (CJS) method has been developed to enable large-scale synthesis of barium titanate nanofibers. Barium titanate nanofibers with fiber diameters down to 50 nm and grain sizes around 25 nm were prepared with CJS by spinning a sol-gel solution of barium titanate and poly(vinylpyrrolidone) with subsequent heat treatment at 850 °C. XRD and FTIR analysis demonstrated high purity and tetragonal perovskite structured barium titanate nanofibers. SEM and TEM images confirm the continuous high aspect ratio structure of barium titanate nanofibers after heat treatment. It is demonstrated that the CJS technique offers a highly efficient method for large-scale fabrication of ceramic nanofibers at production rates of up to 0.3 gram/minute.