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Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells. TPMD-induced calcium increases (Ca++i) were measured in Ca++-containing and Ca++-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-α-glycyrrhetinic acid (18α-GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca++ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca++ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.

Purpose In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. Methods We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. Results AQP3 was present in human corneas in situ, and AQP3 and PLD2 were co-immunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3- and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). Conclusions These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)(2)D3 and 24R,25(OH)(2)D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)(2)D3 and 24R,25(OH)(2)D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR (−/−)) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)(2)D3 or 24R,25(OH)(2)D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca(++) (Ca(i)(++)) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)(2)D3 and 24R,25(OH)(2)D3. Cx30 and Cx43 protein levels were also significantly increased in VDR (−/−) MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)(2)D3 and 1,25(OH)(2)D3. Ca(i)(++) was not affected by 1,25(OH)(2)D3 or 24R,25(OH)(2)D3 in HCEC or MPCEC. We conclude that both 1,25(OH)(2)D3 and 24R,25(OH)(2)D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca(i)(++).

This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure.Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy.25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3.There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

Objectives. SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl - current (ICI LPA ) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls. Methods. Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure ICI LPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. Results. Fibroblasts cultured from SSc skin show significantly increased ICI LPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. Conclusions. Elevated ICI LPA activity is a hallmark of SSc skin fibroblasts. Blocking ICI LPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.

Vitamin D deficiency may have implications for cardiovascular health. The purpose of this study was to determine the relationship of 25-hydroxyvitamin D (25[OH]D) to cholesterol and lipoprotein particles and to determine whether increasing 25(OH)D through ultraviolet (UV) irradiation impacted on these parameters in healthy young men and women. This was a randomized trial of 51 adults exposed to suberythemal doses of whole-body irradiation using UV lamps that emitted UV-A and UV-B radiation, compared with a control group, twice weekly for 12 weeks. 25-Hydroxyvitamin D, cholesterol, and lipoprotein subfractions were measured at baseline and after 12 weeks. There was a significant (P < .03) positive association between 25(OH)D and apolipoprotein A-I (Apo A-I) and lipoprotein A-I (Lp A-I). The ratio of low-density lipoprotein to high-density lipoprotein was significantly (P < or = .044) negatively correlated with 25(OH)D levels. The levels of 25(OH)D increased significantly in the treated compared with control group (P < .05). Overall, there were no significant differences between the treated and control groups in any lipoproteins or apolipoproteins after administration of UV irradiation. Subgroup analysis for Apo A-II confined to those with 25(OH)D insufficiency (25[OH]D

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.

Summary A single infusion of pamidronate was given to patients with systemic sclerosis (scleroderma, SSc) to assess effects on cytokine production by peripheral blood mononuclear cells (PBMC) and lymphocyte subsets. Eighteen patients with SSc received a single intravenous dose of 60 mg of pamidronate and were followed for 6 months. Assessment of cytokine production [interferon (IFN)-γ, interleukin (IL)-10, transforming growth factor (TGF)-β1, tumour necrosis factor (TNF)-α and IL-4] by PBMC and lymphocyte subsets by flow cytometry was carried out before and after the pamidronate infusion. Unstimulated PBMC produced increased amounts of IFN-γ and TNF-α and reduced levels of TGF-β1 for up to 24 weeks after the infusion. γδ T cells from patients with SSc were activated in vitro and produced increased IFN-γ. The effects of pamidronate on modulation of cytokine profiles in patients with SSc may merit future study.

Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl− current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1–5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2–5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.

Gap junctional communication in the epithelium and endothelium of human corneas was studied. The influence of corneal storage on endothelial gap junctions was also examined.Donor human corneal cells were injected with carboxyfluorescein while surrounding cells were monitored for traces of fluorescence. Dye-spread coefficients were measured in corneal endothelial cells. Western blot and immunohistochemical analysis of the endothelium and epithelium was employed to determine if connexin 43 was present.Dye coupling occurs in both the epithelium and endothelium of the human cornea. Epithelial dye coupling was extensive in the basal layers but less apparent in the superficial layers. Endothelial dye coupling was similar to that reported for rabbit corneas. Western blot and immunohistochemical analyses demonstrated the presence of connexin 43 in both cell types.Gap junctional communication occurs in the endothelium and epithelium of human corneas, and both cell types express connexin 43. These results are similar to previous rabbit studies, thereby strengthening the use of the rabbit cornea as a gap junction model.

We investigated the effects of demographic, lifestyle (self-reported smoking status and physical activity levels), cancer-related treatment factors (radiation and chemotherapy), and diet (calcium and vitamin D intake) on bone turnover and the relationship of bone turnover to lumbar spine bone mineral density (BMD) Z-scores (LS-BMD Z-scores) determined by quantitative computed tomography (QCT) in 418 ≥5-year survivors of childhood acute lymphoblastic leukemia (ALL).Bone turnover was assessed by biomarkers including serum bone-specific alkaline phosphatase (BALP), osteocalcin (OC), and urinary N-telopeptide of type I collagen indexed to creatinine (NTX/Cr). The 215 males ranged in age from 9 to 36 years (median age 17 years).Age and tanner score were inversely associated with all biomarkers (BALP, OC, NTX/Cr) (P 0.001). Males had higher BALP and OC than females (P 0.001). Body mass index (BMI) was inversely associated with OC and NTX/Cr (P 0.001). There was no significant association of biomarkers with lifestyle related factors, ALL treatment-related factors, dietary calcium, vitamin D, or LS-BMD Z-score.In this population of long-term survivors of ALL, bone turnover was significantly associated with age, gender, tanner stage, and BMI. ALL-related treatments did not influence bone turnover and bone turnover was not predictive of volumetric LS-BMD Z-score.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

Myofibroblasts are a differentiated cell type essential for wound healing, participating in tissue remodeling following insult. Myofibroblasts are typically activated fibroblasts, although they can also be derived from other cell types, including epithelial cells, endothelial cells, and mononuclear cells. In most organ systems, cell signals initiated following tissue-specific insult or during the metastatic process lead to differentiation of fibroblasts or other precursor cells to the myofibroblast phenotype. In addition to their beneficial and necessary role in wound healing, myofibroblasts also contribute to a number of pathologies, primarily fibrotic processes and tumor invasiveness. This review explores both traditional and nontraditional concepts of myofibroblast differentiation in the cornea, skin, heart, and other tissues, as well as some of the pathologies associated with myofibroblast activities.

Systemic sclerosis (SSc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA), a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD). Sphingosine 1-phosphate (S1P), a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC). The major results were that serum levels of arachidonoyl (20:4)-LPA and S1P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S1P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

Institutionalized adults with severe developmental disabilities have a high rate of minimal trauma and appendicular fracture. There is little information about osteoporosis treatment in this population. In this efficacy and safety study, men and women with severe developmental disabilities and osteoporosis received 20 mcg teriparatide subcutaneously daily for 18-24 months. Markers of bone formation [procollagen type 1 intact N-terminal propeptide (P1NP)] and resorption [C-telopeptide (CTx)] were measured at three-month intervals. Serum calcium was measured at two-week intervals for 12 weeks and thereafter at three-month intervals. Twenty-seven individuals received at least one injection. The incidence of hypercalcemia was 11.1% but was persistent and led to medication discontinuation in only one participant. Biomarkers of bone formation increased rapidly, doubling by three months. At 12 months, P1NP and CTx remained elevated from baseline; P1NP had risen from 66.95 +/- 83.71 microg/l (mean +/- SD) to 142.42 +/- 113.85 microg/l (P = 0.05), and CTx had increased from 0.377 +/- 0.253 to 1.016 +/- 1.048 ng/ml (P = 0.01). The majority of participants had an increase in P1NP of over 10 microg/l. In conclusion, teriparatide is safe and effective in developmentally disabled institutionalized adults. Serial calcium measurements are warranted, particularly during the first three months of therapy.

A biointeractive collagen-phospholipid corneal Substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed ail overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and, regeneration (despite MPCs known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea.In addition, the glucose and albumin permeability were comparable to those Of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be Substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal Source materials.

PURPOSE. To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS. Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS. Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS. Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl−currents ( ICl,LPAand ICl,VRAC, respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure ICl,LPAand ICl,VRACcurrents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased ICl,LPAand ICl,VRACactivity in the presence of transforming growth factor-β1(TGF-β1) compared with controls, whereas ClC-3 overexpression resulted in increased ICl,LPAactivity in the absence of TGF-β1. ClC-3 knockdown also resulted in a reduction of α-smooth muscle actin (α-SMA) protein levels in the presence of TGF-β1, whereas ClC-3 overexpression increased α-SMA protein expression in the absence of TGF-β1. In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the ICl,LPAcurrent activity, and participates in the fibroblast-to-myofibroblast transition.

To determine bone mineral content (BMC), bone mineral density (BMD), Z scores, and markers of bone turnover in African American children with juvenile rheumatoid arthritis (JRA).Eight children with JRA with no prior exposure to corticosteroids were evaluated. Lumbar spine (L1-L4) and total body and total hip BMC and BMD were determined using dual x-ray absorptiometry (DXA), and Z scores (BMD) were calculated. Serum samples of markers of bone turnover including pyridinoline (PYR), N-terminal propeptide of type I procollagen (P1NP), osteocalcin (OC), and bone-specific alkaline phosphatase (BSAP) were measured.The mean Z score (BMD) at the lumbar spine (L1-L4) in patients with JRA was -1.2+/-0.8. Z scores for total body and total hip were within 1 standard deviation of normal compared with healthy historical controls matched for age, sex, and race.BMD was normal for chronological age (defined as Z scoreor= 2.0) in African American children with JRA who had not previously been treated with corticosteroids. Further studies are needed on the effects of JRA on skeletal health in African American children.

PURPOSE. To develop a simple corneal substitute from crosslinked collagen. METHODS. Porcine type I collagen (10%; pH 5), was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and Nhydroxysuccinimide (NHS). The final homogenous solution was molded to corneal dimensions, cured, and then implanted into rabbits and minipigs by lamellar keratoplasty. The implants were followed for up to 6 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, topography and esthesiometry for nerve function. Histopathologic examinations were also performed on rabbit corneas harvested after 6 months. RESULTS. Cross-linked collagen (refractive index, 1.35) had optical clarity superior to human corneas. Implanted into rabbit and porcine corneas, only 1 of 24 of the surgical corneas showed a slight haze at 6 months after surgery. All other implants showed no adverse reactions and remained optically clear. Topography showed a smooth surface and a profile similar to that of the contralateral nonsurgical eye. The implanted matrices promoted regeneration of corneal cells, tear film, and nerves. Touch sensitivity was restored, indicating some restoration of function. The corneas with implants showed no significant loss of thickness and demonstrated stable host‐graft integration. CONCLUSIONS. Collagen can be adequately stabilized, using water soluble carbodiimides as protein cross-linking reagents, in the fabrication of corneal matrix substitutes for implantation. The simple cross-linking methodology would allow for easy fabrication of matrices for transplantation in centers where there is a shortage of corneas, or where there is need for temporary patches to repair perforations in emergency situations. (Invest Ophthalmol Vis Sci. 2006;47:1869‐1875)

Osteoporosis is a serious public health problem, and dietary interventions may potentially be helpful in preventing this disorder. The purpose of this study was to determine the effects of a low sodium diet on bone metabolism in postmenopausal women. This was a longitudinal study to determine the effects of a low sodium (2-g/day) diet on bone. Forty postmenopausal African-American and Caucasian women were enrolled in a 2-g/day sodium diet for 6 months. Sodium and calcium excretion, bone turnover, and calcitropic hormones (intact parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D) were measured before and 6 months after the intervention. In women who had baseline sodium excretions equal to or greater than the average sodium intake in the United States (or =3.4 g/day), the low sodium diet resulted in significant decreases in sodium excretion (P = 0.01), in calcium excretion (P = 0.01), and in a biomarker of bone turnover, aminoterminal propeptide of type I collagen (P = 0.04). However, there were no significant changes in calcitropic hormones, including intact PTH (P = 0.97) or 1,25 dihydroxyvitamin D (P = 0.49) with the low sodium diet. These findings suggest that in postmenopausal women with sodium intakesor =3.4 g/day, a low sodium diet may have benefits for skeletal health.

It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl−channel current ( ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPAon fibroblast-to-myofibroblast differentiation. We recorded ICl-LPAusing the amphotericin perforated-patch technique. We activated ICl-LPAusing LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPAcurrent density compared with control fibroblasts. ICl-LPAactivation was blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of ICl-LPAby LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPAactivity in fibroblasts. This Cl−channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

A sensory nerve supply is crucial for optimal tissue function. However, the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood. Engineered tissue substitutes can provide controllable environments in which to study tissue innervation. We have therefore engineered human corneal substitutes that promote nerve in-growth in a pattern similar to in vivo re-innervation. We demonstrate that these nerves (a) are morphologically equivalent to natural corneal nerves; (b) make appropriate contact with target cells; (c) can generate action potentials; (d) respond to chemical and physical stimuli; and (e) play an important role in the overall functioning of the bioengineered tissue. This model can be used for studying the more general topics of nerve ingrowth or regeneration and the interaction between nerves and their target cells and, more specifically, the role of nerves in corneal function. This model could also be used as an in vitro alternative to animals for safety and efficacy testing of chemicals and drugs.

Purpose. To review the development of artificial corneas (prostheses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods. Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results. Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions. Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

There are no published clinical or basic science studies examining the presence, metabolism, or physiological role of native vitamin D (Vit D) in the anterior segment of the eye. Given that >50% of the population may be suffering from Vit D insufficiency,1–3 these are critically important topics for investigation. Low circulating Vit D levels could affect the levels of Vit D and its active metabolite, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), in the vitreous humor, aqueous humor, and cornea. Anterior segment hypovitaminosis D could be particularly important after corneal injury, where Vit D may play a role in cell redifferentiation of the different cell layers during the wound healing process. The classical systemic route for activation of Vit D is initial 25-hydroxylation to 25-dihydroxyvitamin D3 (25(OH)D3) in the liver via cytochrome P-450 containing enzymes, followed by renal conversion to the active 1,25(OH)2D by 1α-hydroxylase. 1,25(OH)2D can be subsequently degraded to 24R,25-dihydroxyvitamin D by 25-hydroxyvitamin D-24-hydroxylase, which is found in target cells throughout the body and is strongly induced by 1,25(OH)2D.4 More recently, 1α-hydroxylase activity has been found in such diverse tissue as the colon,5 vascular smooth muscle,6 and breast tissue.7 Since the discovery that 1-α-hydroxylase is active in tissues outside of the kidney, it has become clear that Vit D acts as much more than a regulator of intestinal Ca2+ absorption. While the most thoroughly studied actions of Vit D are related to its management of calcium concentration in the plasma, 1,25(OH)2D has been found to induce keratinocyte differentiation and proliferation in the skin.8 Studies in Vit D receptor (VDR) knockout mice determined that VDR is essential for normal keratinocyte stem cell function.9 Vit D has also been found to affect extracellular matrix turnover and regulate growth factor release, in part through regulation of metalloproteinases.10 In addition, the VDR signaling partner, retinoid X receptor (RXR), has been shown to play a significant role in the differentiation and maintenance of epithelial cells, as well as in the mediation of tight junction proteins and barrier function.11,12 This barrier function mediation may be the result of the role Vit D plays in cell differentiation. Here we show that both 25(OH)D3 and 1,25(OH)2D3 are present in the eye, as are the VDR and 1α-hydroxylase, and we demonstrate that 25(OH)D3 and 1,25(OH)2D3 can both enhance the barrier function of corneal epithelial cells.

We present experimental procedures describing the creation of perforated patches by use of amphotericin B. In 13 different cellular preparations, access resistances below 10 M omega were achieved and with blunt electrode tips, access resistances of 3-4 M omega were possible. In addition to using the techniques to measure whole cell currents, we have used them to measure single channel currents in a new "outside-out patch" preparation and we have utilized them to measure the resting voltage of epithelial monolayers. We conclude that these new approaches can provide a substantial increase in versatility and quality for many kinds of electrophysiological measurements.

Endothelial permeability was examined in rabbit and human corneas using an in vitro perfusion system with 5(6)-carboxyfluorescein as the permeability tracer. Following endothelial removal, the permeability of de-epithelialized rabbit corneas increased from 3.19 x 10(-4) cm min-1 to 31.21 x 10(-4) cm min-1, and de-epithelialized human donor corneal permeability values increased from 2.26 to 12.85. In human corneas, no correlation was seen between endothelial permeability and donor age, moist chamber storage time, endothelial cell density, coefficient of variation of cell area, or percent hexagonal cells. A positive correlation was found between permeability and time between donor death and enucleation. Donor eyes from three separate populations, having undergone cataract surgery prior to death (aphakic, posterior and anterior chamber implants), all showed a significant increase in permeability. Neither Type I nor Type II diabetes had an effect on the endothelial permeability of human donor corneas.

This comparative study shows the surface area ratio of conjunctiva to cornea to be two times larger in humans than in rabbits. This large heretofore unrecognized interspecies difference may affect the applicability of drug pharmacokinetic data obtained using rabbit models and should be taken into consideration in topical drug development and future comparative drug penetration studies between rabbit and man.