Tear cytokine profiling in patients with superior limbic keratoconjunctivitis who underwent medical treatment or in conjunction with surgical management.

PURPOSE: To determine the correlation between superior limbic keratoconjunctivitis (SLK) and selected tear cytokines and to evaluate the efficacy of these cytokines in monitoring the response of patients with SLK to either medical treatment alone or in combination with conjunctival resection. DESIGN: A cohort study. METHODS: Twenty-five eyes of 13 patients with SLK were assigned to either medically responsive or surgical treatment groups depending on their responses 1 month after initial medical treatment. Treatment efficacy was assessed by improvements in clinical grading and decreases in the levels of tear cytokines. RESULTS: Fourteen eyes were improved by medical treatment alone, whereas satisfactory outcomes were achieved for the remaining 11 eyes after surgical management. The overall grading prior to medical treatment was lower in medically responsive group compared with surgical treatment group (p=0.0139). Among the examined tear cytokines, monocyte chemoattractant protein (MCP)-1 was positively associated with the severity of clinical grading (p=0.0251). While both treatments significantly decreased the levels of MCP-1 and interleukin-6, surgical treatment also decreased the levels of interferon-γ and tumour necrosis factor-α. Notably, overall cytokine levels after surgical treatment were lower than those after medical treatment alone. CONCLUSIONS: Since an association of tear MCP-1 level with the clinical grading and treatment response of SLK was observed in this study, tear MCP-1 may be a potential indicator of SLK disease severity. According to the degree to which the tear cytokine levels were decreased, surgical treatment appears to be an effective treatment modality for patients with SLK who are refractory to medical treatment alone.

Human antigen R protein modulates vascular endothelial growth factor expression in human corneal epithelial cells under hypoxia.

PURPOSE: Corneal avascularity is critical for corneal transparency; therefore, a tailored process has been presumed to minimize corneal neovascularization (NV). In most cell types, the transcription of vascular endothelial growth factor (VEGF) is up-regulated, and the stability of VEGF mRNA is sustained by human antigen R (HuR) during hypoxia; however, whether such response applies to corneal epithelial cells is unclear. METHODS: Human corneal epithelial cells (HCECs) and MCF-7 cells that serves as the control were incubated under 0.5% oxygen, and the levels of VEGF and HuR were examined time-dependently. The alteration of HuR was also examined in vivo using the closed-eye contact lens-induced corneal neovascularization rabbit model and immunohistochemistry. Additionally, the expression of HuR was modulated by transfection of plasmids encoding HuR or siRNA targeting HuR to validate the role of HuR in VEGF expression. RESULTS: We found that, unlike in control cells, the level of VEGF was not up-regulated, and the HuR expression was declined in HCECs following hypoxia. The HuR immunostaining intensities were decreased in corneal epithelial cells of rabbits wearing contact lenses. In addition, HuR overexpression restored the ability of HCECs to up-regulate VEGF under hypoxia; however, knockdown of HuR suppressed hypoxia-induced VEGF in control cells but did not further decrease VEGF in HCECs. These findings suggest that HCECs may modulate HuR to suppress hypoxia-mediated up-regulation of VEGF. CONCLUSION: Our study revealed a distinct regulation of VEGF via HuR in HCECs following hypoxia, which likely contributes to minimizing corneal NV and/or maintenance of corneal avascularity.

Monocarboxylate Transporters Mediate Fluorescein Uptake in Corneal Epithelial Cells.

Purpose: To determine the presence of monocarboxylate transporter (MCT) in human and rabbit corneal epithelium and its role in transcellular fluorescein transportation in the cornea. Methods: The presence of MCTs in human and rabbit corneal epithelium was determined by RT-PCR and immunohistochemistry. Intracellular fluorescein uptake experiment was performed using cultured human corneal epithelial cells (HCECs). The involvement of MCT in fluorescein uptake was determined by addition of MCT inhibitors to HCECs and acute dry eye model on New Zealand albino rabbits by spectrophotometry, corneal impression cytology, and external eye photographs. Results: MCT-1 and MCT-4 were identified in both human and rabbit corneal epithelia. A longer treatment period and a lower pH value in culture medium increased fluorescein uptake in HCECs. Fluorescein uptake in HCECs was decreased following addition of MCT inhibitors in a concentration-dependent manner. Impression cytology under fluorescent microscopy showed intracellular fluorescein staining in the rabbit cornea with acute dry eye treatment that was decreased following topical treatment of MCT inhibitors. Conclusions: Fluorescein ingress in corneal epithelial cells is mediated by the MCT family. Further study of MCT-mediated transport on HCECs may potentially benefit differential diagnosis and contribute better understandings of ocular surface disorders.

Stem Cell Factor and Thymic Stromal Lymphopoietin Overexpression With Correlation to Mast Cells in Superior Limbic Keratoconjunctivitis.

PURPOSE: To investigate the differential expression of stem cell factor (SCF) and thymic stromal lymphopoietin (TSLP) and their correlation to mast cells, between patients diagnosed with superior limbic keratoconjunctivitis (SLK) and normal subjects. METHODS: A laboratory investigation included 22 surgical specimens of the superior bulbar conjunctiva from 17 patients with medically refractory SLK and 5 control subjects who underwent cataract or retinal surgery. Protein expression of tryptase, SCF, and TSLP in conjunctival specimens was detected by immunohistochemistry. The number of mast cells was correlated with immunohistochemistry intensity of SCF and TSLP. RESULTS: In patients with SLK, higher immunostaining intensity of SCF and TSLP was found in the conjunctival epithelium than that in the conjunctival subepithelial stroma. SCF and TSLP staining in the conjunctival epithelium was significantly more intense in patients with SLK than in normal subjects. In addition, there was a significant correlation between the number of tryptase (+) mast cells in conjunctival subepithelial stroma and TSLP immunointensity in the conjunctival epithelium and subepithelial stroma. CONCLUSIONS: Overexpression of SCF and TSLP was found in the conjunctival epithelium of patients with SLK. Significant correlation between TSLP grading and the number of mast cells was also found. SCF and TSLP may be involved in promoting mast cell migration and activation contributing to the pathogenesis of SLK.

Bone marrow mesenchymal stem cells, platelet-rich plasma and nanohydroxyapatite-type I collagen beads were integral parts of biomimetic bone substitutes for bone regeneration.

Platelet rich plasma (PRP), which includes many growth factors, can activate osteoid production, collagen synthesis and cell proliferation. Nanohydroxyapatite-type I collagen beads (CIB), which mimetic natural bone components, are not only flexible fillers for bone defect but also encourage osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) are often used as an abundant cell source for tissue engineering. We used a rabbit model to combine PRP, CIB and BMSCs (CIB+PRP+BMSC) into a bone-like substitute to study its impact on bone regeneration, when compared to defect alone, PRP, CIB+PRP, and PRP+BMSC. CIB+PRP upregulated more alkaline phosphatase (ALP) activity in BMSCs than PRP alone at 4 weeks postoperation. CIB+PRP+BMSC and PRP+BMSC did not differ significantly in DNA content, total collagen content, and ALP activity at 8 weeks. In histological assay, both CIB+PRP+BMSC and PRP+BMSC showed more bone regeneration at 4 and 8 weeks. Higher trabecular bone volume in tissue volume (BV/TV) (31.15±2.67% and 36.93±1.01%), fractal dimension (FD) (2.30±0.18 and 2.65±0.02) and lower trabecular separation (Tb.Sp) (2.30±0.18 and 1.35±0.16) of CIB+PRP+BMSC than of other groups at 4 and 8 weeks, and approach to of bone tissue (BV/TV=24.35±2.13%; FD=2.65±0.06; Tb.Sp=4.19±0.95). CIB+PRP+BMSC significantly enhanced new bone formation at 4 week. Therefore, nanohydroxyapatite-type I collagen beads combined with PRP and BMSCs produced a bone substitute with efficiently improved bone regeneration that shows promise to repair bone defects.

MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix.

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.

Preparation and characterization of microspheres comprised of collagen, chondroitin sulfate, and apatite as carriers for the osteoblast-like cell MG63.

Numerous studies about bone matrix fabrication focus on how the species and concentrations of components affect the cellular response. However, there are few studies that investigate how the related spatial arrangement of the components influences cellular activity. The aim of this work was to develop a novel method to biomimetically manufacture a three-dimensional mineral bone matrix and study the effect of apatite-collagen-chondroitin sulfate (CS) microspheres on the adhesion rate and activity of osteoblast-like cells. Although previous studies used a crosslinking agent or lyophilized methods to fabricated three-dimensional collagen microspheres, we produced beads composed of collagen and CS under mild reaction conditions. This process not only maintains collagen self-assembly into fibrils with a D-periodic pattern ability but also simultaneously introduces two major native bone matrix elements, collagen and CS, into the beads. Furthermore, we mimic the native in vivo bone matrix formation process by the direct nucleation and growth of apatite crystals on collagen fibrils. The apatite crystals are similar in composition to human bone mineral via X-ray diffraction and energy-dispersive X-ray spectrometric analysis. The cellular attachment rate of MG63 osteoblast-like cells is significantly higher for collagen-CS-apatite gel beads than for collagen-CS gel beads. In addition, with regard to the osteoblast bioactivity, we observed that alkaline phosphatase activity of MG63 cells on the collagen-CS-apatite gel beads higher than on the collagen-CS gel beads on day 14.

Electrospun hyaluronate-collagen nanofibrous matrix and the effects of varying the concentration of hyaluronate on the characteristics of foreskin fibroblast cells.

In this study we propose a novel electrospinning fabrication process for the production of a nanofibrous matrix composed of collagen and hyaluronate. This procedure utilized 1,1,1,3,3,3-hexafluoro-2-propanol and formic acid as a mixed solvent. Fluorescence microscopy photographs revealed that the resulting electrospun nanofibers contained both collagen and hyaluronate. The mean diameter of the composite nanofibrous matrix (as observed using scanning electron micrographs) was approximately 200nm; this dimension is similar to that of native fibrous protein within the extracellular matrix. The expression of proteinases (e.g. matrix metalloproteinases, MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in epidermal repair during wound healing. Moreover, the characteristics of scarless wounds are known to be related to a decreased ratio of TIMP to MMP expression. In the present study the ratio of expression of TIMP1 to MMP1 was lower in foreskin fibroblast cells that were cultured on a hyaluronate-collagen composite nanofibrous matrix than in those cultured on an exclusively collagen nanofibrous matrix. This indicates that the hyaluronate-collagen composite nanofibrous matrix could potentially be used as a wound dressing for the regeneration of scarless skin.