Novel digital image analysis using fractal dimension for assessment of skin radiance.

BACKGROUND: Despite a strong desire to quantify skin radiance in the field of cosmetics, there does not exist a robust method to characterize it. Classical shine that quantifies the specular reflection from skin has been commonly used as the metric to characterize radiance. However, it does not always correlate with the perceived radiance as there are many other parameters that inform radiance perception including spatial distribution of shine and color homogeneity. MATERIALS AND METHODS: In this work, we propose a novel method using fractal analysis to better characterize radiance by considering the spatial heterogeneity of pixel intensities as well as color evenness. A simulated image library (nine images) from very dull to very bright was created using bare face images of 20 panelists. Product images taken post-product usage were ranked along this library by finding the image in the library that most resembles the product image by our algorithm as well as experts. Additionally, classical shine and color measurements were made as benchmarks. RESULTS: Our results confirm a strong correlation (R2  = 0.99) between the expert radiance rankings and the rankings by fractal dimension algorithm. The new algorithm offers an improved product differentiation compared with classical shine or color measurements. CONCLUSION: Fractal dimension calculation offers higher sensitivity and resolution compared with other descriptors such as classical shine or color heterogeneity. In cases where the image rank is dominated by pixel intensities rather than color evenness, the image ranks resulting from calculating the fractal dimension is comparable with use of classical shine as the ranking parameter.

Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes.

Live imaging has become the favorite method in recent years to study the protein transport, localization and dynamics in live cells. Protein transport is extremely essential for proper function of photoreceptors. Aberration in the proper transport of proteins gives rise to the loss of photoreceptor and blindness. On the other hand, the ease of generation of transgenic Xenopus laevis tadpoles and the advantage of high resolution live confocal imaging provide new insight into understanding protein dynamics in photoreceptors. There are several steps for quantifying and visualizing fluorescently tagged proteins in photoreceptors starting with assembly of plasmids, generation of transgenic tadpoles, preparation of retinal tissues, imaging the transgenic photoreceptors and finally analyzing the recorded data. The focus of this manuscript is to describe how to prepare retinal tissues suited for live cell imaging and provide our readers with a tutorial video. We also give a summary of steps leading to a successful experiment that might be designed for imaging the ultrastructures of photoreceptors, the expression of two or more different fluorescently tagged proteins, their localization, distribution, or protein dynamics within photoreceptors. •Retinal tissue live imaging demonstrates the ultrastructures of photoreceptors.•High resolution live confocal imaging provides new insight into understanding the pathophysiology of photoreceptors.

Enhanced differentiation of dental pulp cells cultured on microtubular polymer scaffolds in vitro.

Dental caries (tooth decay) is the most common chronic disease. Dental tissue engineering is a promising alternative approach to alleviate the shortcomings of the currently available restorative materials. Mimicking the natural extracellular matrix (ECM) could enhance the performance of tissue engineering scaffolds. In this study, we developed microtubular (~20 µm diameter) polymethyl methacrylate (PMMA) scaffolds resembling the tubular (~2.5 µm diameter) structure of dentin, the collagen-based mineralized tissue that forms the major portion of teeth, to study the effect of scaffold architecture on differentiation of mouse dental pulp cells in vitro. Flat (control), plasma-treated solid and microtubular PMMA scaffolds with densities of 240±15, 459±51 and 480±116 tubules/mm2 were first characterized using scanning electron microscopy and contact angle measurements. Dental pulp cells were cultured on the surface of the scaffolds for up to 21 days and examined using various assays. Cell proliferation and mineralization were examined using Alamar Blue and Xylenol Orange (XO) staining assays, respectively. The differentiation of pulp cells into odontoblasts was examined by immunostaining for Nestin and by quantitative PCR analysis for dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and osteocalcin (Ocn). Our results showed that the highest tubular density scaffolds significantly (p<0.05) enhanced differentiation of pulp cells into odontoblasts as compared to control flat scaffolds, as evidenced by increased expression of Nestin (5.4x). However, mineralization was suppressed on all surfaces, possibly due to low cell density. These results suggest that the microtubular architecture may be a desirable feature of scaffolds developed for clinical applications. LAY SUMMARY: Regenerative engineering of diseased or traumatized tooth structure could avoid the deficiencies of traditional dental restorative (filling) materials. Cells in the dental pulp have the potential to differentiate to dentin-producing odontoblast cells. Furthermore, cell-supporting scaffolds that mimic a natural extracellular matrix (ECM) are known to influence behavior of progenitor cells. Accordingly, we hypothesized that a dentin-like microtubular scaffold would enhance differentiation of dental pulp cells. The hypothesis was proven true and differentiation to odontoblasts increased with increasing density of the microtubules. However, mineralization was suppressed, possibly due to a low density of cells. The results demonstrate the potential benefits of a microtubular scaffold design to promote odontoblast cells for regeneration of dentin.

Study of cellular dynamics on polarized CoCrMo alloy using time-lapse live-cell imaging.

The physico-chemical processes and phenomena occurring at the interface of metallic biomedical implants and the body dictate their successful integration in vivo. Changes in the surface potential and the associated redox reactions at metallic implants can significantly influence several aspects of biomaterial/cell interactions such as cell adhesion and survival in vitro. Accordingly, there is a voltage viability range (voltages which do not compromise cellular viability of the cells cultured on the polarized metal) for metallic implants. We report on cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesion complexes in transiently transfected MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy surfaces polarized at the cathodic and anodic edges of its voltage viability range (-400 and +500 mV (Ag/AgCl), respectively). Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) were also studied on the polarized metal at -1000, -400 and +500 mV (Ag/AgCl). Our results show that at -400 mV, where reduction reactions dominate, a gradual loss of adhesion occurs over 24 h while cells shrink in size during this time. At +500 mV, where oxidation reactions dominate (i.e. metal ions form, including Cr6+), cells become non-viable after 5h without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at -1000 mV decreased sharply within 15 min after polarization, which rendered the cells completely non-viable. No significant amount of ROS release by cells was detected on the polarized CoCrMo at any of these voltages.

Voltage-controlled cellular viability of preosteoblasts on polarized cpTi with varying surface oxide thickness.

Cathodic voltage shifts of metallic biomaterials were recently shown to induce cell apoptosis in-vitro. The details of the reduction-based physico-chemical phenomena have not yet been fully elucidated. This study shows how surface oxide thickness of commercially pure titanium affects the voltage viability range, and whether anodic oxidation can extend this range. Cell viability, cytoskeletal organization, and cellular adhesion on bare and anodized Ti, at -500, -400 mV(Ag/AgCl) and open circuit potential were assessed. Surfaces were characterized using contact angle measurement and atomic force microscopy, and the observed cellular response was related to the changes in electrochemical currents, and impedance of the samples. Results show that anodization at 9 V in phosphate buffer saline generates a compact surface oxide with comparable surface roughness and energy to the starting bare surface. The anodized surface extends the viability range at 24h from -400 mV(Ag/AgCl) by about -100 mV, which corresponds to an increase in impedance of the surface from 58 kΩ cm(2) to 29 MΩ cm(2) at -400 mV(Ag/AgCl) and results in low average current densities below 0.1 µA cm(-2). The results demonstrate that the voltage range for cell viability under cathodic polarization is expanded due to anodization of the surface oxide and lowering of cathodic currents.

Electrochemical control of cell death by reduction-induced intrinsic apoptosis and oxidation-induced necrosis on CoCrMo alloy in vitro.

Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. These excursions may compromise the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. The in vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 < V < +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 µA cm(-2) and anodic current densities of 10 µA cm(-2). Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p < 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo.