Percentage of CD34(−) capillaries in human choriocapillaris.

<p>(A) Average values segregated by disease phenotype. Y = young (mean age  = 31.1); CTL = age-matched controls; ARM = early/dry AMD; GA = geographic atrophy; CNV = choroidal neovascularization. Error bars indicate the standard error of the mean. (B) All donors plotted as percent capillaries without CD34 versus age. Note the age related increase in CD34(−) capillaries (p<10⁻⁵).</p

Tandem triple labeling of CD34/UEA-I and endogenous alkaline phosphatase.

<p>Following epifluorescence photomicrography (A, B,C), coverslips were removed and sections reacted with NBT/BCIP. Note the good correspondence between alkaline phosphatase activity (D) and UEA-I binding (A) that includes capillaries that do not react with CD34 antibody (B). CC, choriocapillaris; scalebar = 50 µm.</p

Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels

The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that, after adequate surfactant removal, these materials are nontoxic, support the attachment of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration

Expression of CD34 in pathologic blood vessels in choroidal neovascular membranes (CNVMs) from 3 donors with neovascular AMD.

<p>Abnormal vessels within the CNVMs were invariably strongly positive (arrows). RPE, dystrophic retinal pigment epithelium; BlamD, layer of basal laminar deposit; CC, choriocapillaris. Scalebar in C, 50 µm.</p

Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications

Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose <i>in situ</i> has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly­(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and three-dimensional printing via stereolithography. PCL prepolymers with different molecular weights and functionalities were studied to understand the role of molecular structure in reaction kinetics, mechanical properties, and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High-molecular weight, low-functionality PCLDA prepolymers exhibited a lower modulus and a higher strain at break, while low-molecular weight, high-functionality PCLTA prepolymers exhibited a lower strain at break and a higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than those of more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both <i>in vitro</i> and <i>in vivo</i>, causing no observed detrimental effects on seeded murine-induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create three-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low-molecular weight, high-functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 μm. This work lays the foundation for future work in fabricating microscale PCL structures for a wide range of tissue regeneration applications

OCT abnormalities in <i>rd16;Nrl^−/−</i> mice.

<p>(A) Upper panels: Representative OCT scans vertically across ∼2 mm of retina (centered at the ONH, optic nerve head) in a WT mouse and in two <i>rd16;Nrl^−/−</i> mice of different ages. Lower panels: Magnified parts of the superior region of the retinal sections with overlaid longitudinal reflectivity profiles (LRPs) to demonstrate the reflective abnormalities in the outer retinal region in <i>rd16;Nrl^−/−</i> mice (b and c) compared with C57BL6 WT (a). (B) Upper two panels: Vertical OCT sections quantified for ONL+ thickness in two age groups of <i>rd16;Nrl^−/−</i> mice. Regions of outer retina with pseudorosettes were excluded in the measurement. ONL+ profiles in the older (P83–89, n = 12 eyes) age group were thinner than those in younger (P31–41, n = 35 eyes) mice; gray bands in the P83–89 plot represent mean±2 SD for ONL+ thickness of the P31–41 mice. For reference, insets at lower right of the upper two plots show original raw data before suppression of pseudorosette regions. Third panel from top: Means of ONL+ data across the vertical meridian in two age groups (error bar: ± SD; P31–41, open circles; P83–89, filled triangles). Lowest panel: Histograms showing average ONL+ fraction across vertical meridian of two age groups (*represents <i>p</i><0.001). (C) Histological sections of <i>rd16;Nrl^−/−</i> retina at 4 different ages from P21 to P80, compared with a WT retinal section. Histograms show ONL fraction (based on the earlier age group) in <i>rd16;Nrl^−/−</i> mice from peripheral retina (n = 6 eyes in each of the two age groups, *represents <i>p</i> = 0.01).</p

Structure and function in the <i>rd16;Nrl^−/−</i> mouse retina.

<p>(A) ERG b-wave amplitudes of responses to UV- and M-cone stimuli as a function of age in <i>rd16;Nrl^−/−</i> mice from P34 to P83 (n = 95) with comparisons to data from previously recorded signals in <i>Nrl^−/−</i> (squares <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cheng1" target="_blank">[11]</a>; square with cross <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Mears1" target="_blank">[19]</a>), and <i>rd16</i> (crosses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cideciyan3" target="_blank">[7]</a>) mice. Upper: Cone b-wave responses to ultraviolet (UV, 360 nm peak) stimuli in the <i>rd16;Nrl^−/−</i> mice are severely reduced compared with those of <i>Nrl^−/−</i> mice at comparable ages. Amplitudes in <i>rd16</i> mice are low compared to the other mice. It is also notable that ERGs of the <i>Nrl^−/−</i> mice remain relatively stable throughout this age range, while ERGs of the <i>rd16; Nrl^−/−</i> and <i>rd16</i> mice decline in amplitude with age. Lower: Responses to green (510 nm) stimuli are substantially lower in amplitude than those from UV-cone stimuli. Again, <i>Nrl^−/−</i> mice have the largest amplitudes and do not decline with increasing age within this time period. The <i>rd16;Nrl^−/−</i> waveforms are lower in amplitude and there is a reduction with age. Only limited data were available for <i>rd16</i> mice and these fell within the range of <i>rd16;Nrl^−/−</i> amplitudes. Waveforms for representative <i>rd16;Nrl^−/−</i> mice at various ages (grey-filled circles) are illustrated in the panels at right. Grey lines: linear regression fit to log-converted data (dashes) and 95% prediction intervals (solid). Squares with cross at earliest age in graphs: <i>Nrl^−/−</i> data from Mears et al., 2001 (B) Photoreceptor structure (ONL+) as a function of the combined UV- and M-cone ERG b-wave amplitudes. ONL+ remains similar to the value at P31 (youngest age <i>rd16;Nrl^−/−</i> we studied) across various degrees of ERG amplitude reduction. Horizontal dashed line is the reference level for the lower limit of retinal structure thickness at P31 (−2SD from the mean at this age); photoreceptor structure above this lower limit indicates no difference compared to the data of P31 (error bars, +2SD from mean).</p

Spatial and temporal distribution of pseudorosettes in <i>rd16;Nrl^−/−</i> retina.

<p>(A) Histological sections from peripheral retina of two <i>rd16;Nrl^−/−</i> mice at different ages demonstrating the presence of pseudorosettes (arrows). Calibration = 50 μm. (B) Schematic drawing of the mouse retina indicating the coverage of the central OCT raster scans (red circle). ONH is centered in the drawing. Integrated <i>en face</i> image of the central region of a P41 <i>rd16;Nrl^−/−</i> mouse showing how pseudorosettes appear as white dots (B, right panel, red circle). (C) Pseudorosette distribution within the central retinal region in a young (P31) and an older (P83) <i>rd16;Nrl^−/−</i> eyes. Insets (up and right) show average pseudorosettes as density in different sectors of the central retinal region sampled (n = 8 eyes for both age groups). (D) Upper: Histograms comparing number of pseudorosettes in the central retina by OCT at two different ages (P31, n = 10 eyes; P83, n = 8 eyes). Lower: Pseudorosette counts from histological sections of peripheral retina of two different age groups (P21–40, n = 6 eyes; P60–80, n = 6 eyes). Both data sets in <i>rd16;Nrl^−/−</i> mice indicate that the number of rosettes decreases with age (*represents <i>p</i><0.001 and <i>p = </i>0.01 for the upper and lower graphs, respectively). Error bars, ± SD from the mean.</p

Expression of photoreceptor proteins is reduced over time in <i>rd16;Nrl^−/−</i> mice.

<p>Representative cross sections (20X) from central and peripheral retina of P21, P40, P60 and P80 <i>rd16;Nrl^−/−</i> mice were immunostained for the presence of cone transducin alpha (GNAT2) (A) or S- cone opsin (B) and PNA (A,B). Despite maintenance of cone outer segment sheaths (PNA), both GNAT2 and S-opsin expression are markedly reduced by P60 in both central and peripheral retina. INL- inner nuclear layer, ONL- outer nuclear layer, IS/OS- inner segments/outer segments. Calibration = 35 μm.</p

Structure and function in the central retina of <i>CEP290</i>-LCA patients.

<p>(A) Cross-sectional OCT scans along the horizontal meridian through the fovea in a normal subject, a <i>CEP290</i>-LCA patient, and an RP patient. ONL is highlighted in blue. Inset shows location of scan. (B) Relationship of foveal ONL thickness and visual acuity in <i>CEP290</i>-LCA patients. Bar graph represents the average ±1SD foveal ONL thickness of eyes in the different visual acuity ranges (n = 3, for 0.1–1 LogMAR; n = 5, for 1–2 LogMAR; and n = 11, for 2–NLP). Dashed line is lower limit of normal and emphasizes that despite low acuities, foveal ONL is within normal limits. Inset, data from a series of RP patients plotted similarly to show the more expected relationship between structure and visual acuity in retinal degenerations (n = 3, for 0–0.2 LogMAR; n = 20, for 0.2–1 LogMAR; and n = 3 for 1–2 LogMAR). Dashed line is also lower limit of normal. (C) Relationship in <i>CEP290</i>-LCA patients of width of the ONL in the central retina and patient age at time of examination. ONL width was unable to be defined in a 32-year-old <i>CEP290</i>-LCA patient with maculopathy. Solid line is linear regression. Inset, traced central ONL peaks in representative patients of different ages.</p