Keratocyte Proliferation Research Articles

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at −10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m2/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.

Manual Suction versus Femtosecond Laser Trephination for Penetrating Keratoplasty: Intraocular Pressure, Endothelial Cell Damage, Incision Geometry, and Wound Healing Responses

To measure real-time intraocular pressure (IOP) during trephination with a manual suction trephine (MST) and the femtosecond laser (FSL), and to assess endothelial cell damage, incision geometry, and wound healing response with these procedures. IOP was monitored with an intracameral sensor. Eight rabbits underwent manual suction trephination. Eight rabbits had FSL trephination (FSL-T). Slit lamp photography, confocal microscopy, and anterior segment optical coherence tomography (AS-OCT) were performed at baseline and postoperatively. Animals were sacrificed at 4 hours and 3 days. Tissue was examined with scanning electron microscopy (SEM) and immunohistochemistry for an array of wound-healing markers. Separately, 6 human corneas had MST (3) and FSL-T (3). Incision geometry was imaged with high resolution Optovue AS-OCT. The average IOP during MST and FSL-T was similar (37 mm Hg). There was wider IOP fluctuation during the MST cutting phase (60 mm Hg maximum). There were 1-2 rows of endothelial loss on either side of the incision for FSL-T and 2-5 rows deep for MST. Immune cell responses at 4 hours (CD11b) were comparable, greater apoptosis with FSL-T (TUNEL) occurred at 4 hours, and there was increased keratocyte proliferation at 3 days (Ki67) with FSL-T. There was significantly greater undercutting of the cornea with MST (46.86 degrees versus 16.72 degrees). There is more IOP variation during MST. Average IOP is 37 mm Hg for both techniques. More endothelial damage and undercutting of the cornea occurs with MST. The wound healing response to FSL-T appears greater at 3 days.

Mitomycin C: Biological Effects and Use in Refractive Surgery

To provide an overview of the safety and efficacy of mitomycin C (MMC) as adjuvant therapy after refractive surgery procedures. Literature review. Over the past 10 years, MMC has been used by refractive surgeons to prophylactically decrease haze after surface ablation procedures and therapeutically in the treatment of preexisting haze. Development of MMC treatments has had a significant role in the revival of surface ablation techniques. We reviewed the literature regarding mechanism of action of MMC, its role in modulating wound healing after refractive surgery, and its safety and efficacy as adjuvant therapy applied after primary photorefractive keratectomy surgery or after photorefractive keratectomy re-treatment after laser in situ keratomileusis and other corneal surgeries and disorders. The drug is a potent mitotic inhibitor that effectively blocks keratocyte activation, proliferation, and myofibroblast differentiation. Many studies have suggested that MMC is safe and effective in doses used by anterior surface surgeons, although there continue to be concerns regarding long-term safety. After initial depletion of anterior keratocytes, keratocyte density seems to return to normal 6 to 12 months after the use of MMC when corneas are examined with the confocal microscope. Most clinical studies found no difference between preoperative and postoperative corneal endothelial cell densities when MMC 0.02% was applied during refractive surgery, with exposure time of 2 minutes or less. After more than 10 years of use, MMC has been found to be effective when used for prevention and treatment of corneal haze. Questions remain regarding optimal treatment parameters and long-term safety.

Hydroxyapatite for Keratoprosthesis Biointegration

Integration of keratoprosthesis with the surrounding cornea is very important in preventing bacterial invasion, which may cause ocular injury. Here the authors investigated whether hydroxyapatite (HAp) coating can improve keratoprosthesis (KPro) biointegration, using polymethyl methacrylate (PMMA)--the principal component of the Boston KPro--as a model polymer. HAp coatings were induced on PMMA discs after treatment with concentrated NaOH and coating with poly-dopamine (PDA) or polydopamine and then with 11-mercaptoundecanoic acid (11-MUA). Coatings were characterized chemically (Fourier transform infrared spectroscopy [FTIR], energy dispersive X-ray spectroscopy [EDX]) and morphologically (SEM) and were used as substrates for keratocyte growth in vitro. Cylinders of coated PMMA were implanted in porcine corneas ex vivo for 2 weeks, and the force required to pull them out was measured. The inflammatory reaction to coated discs was assessed in the rabbit cornea in vivo. FTIR of the coatings showed absorption bands characteristic of phosphate groups, and EDX showed that the Ca/P ratios were close to those of HAp. By SEM, each method resulted in morphologically distinct HAp films; the 11-MUA group had the most uniform coating. The hydroxyapatite coatings caused comparable enhancement of keratocyte proliferation compared with unmodified PMMA surfaces. HAp coating significantly increased the force and work required to pull PMMA cylinders out of porcine corneas ex vivo. HAp coating of implants reduced the inflammatory response around the PMMA implants in vivo. These results are encouraging for the potential of HAp-coated surfaces for use in keratoprostheses.

Plasma Rich in Growth Factors (PRGF-Endoret) Stimulates Proliferation and Migration of Primary Keratocytes and Conjunctival Fibroblasts and Inhibits and Reverts TGF-β1–Induced Myodifferentiation

Plasma rich in growth factors (PRGF-Endoret) technology is an autologous platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, and growth and differentiation. Because ocular surface wound healing is mediated by different growth factors, we decided to explore the potential of PRGF-Endoret technology in stimulating the biological processes related with fibroblast-induced tissue repair. Furthermore, the anti-fibrotic properties of this technology were also studied. Blood from healthy donors was collected, centrifuged and, whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were drawn off, avoiding the buffy coat. Primary human cells including keratocytes and conjunctival fibroblasts were used to perform the "in vitro" investigations. The potential of PRGF-Endoret in promoting wound healing was evaluated by means of a proliferation and migration assays. Fibroblast cells were induced to myofibroblast differentiation after the treatment with 2.5 ng/mL of TGF-β1. The capability of WP and F3 to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results show that this autologous approach significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, plasma rich in growth factors prevents and inhibits TGF-β1-induced myofibroblast differentiation. No differences were found between WP and F3 plasma fractions. These results suggest that PRGF-Endoret could reduce scarring while stimulating wound healing in ocular surface. F3 or whole plasma column show similar biological effects in keratocytes and conjunctival fibroblast cells.

Inhibitory effect of curcumin on corneal keratocytes fibrosis

Background The injury or surgery of cornea cause the proliferation of corneal stromal cells and scar formation.Recent research showed that cureumin can obviously reduce the degree of fibrosis of tissue.But if curcumm play inhibitory effect on corneal keratocytes fibrosis is rarely reported.Objecttve This studv was to investigate the effect of curcumin on the transformation of corneal keratocytes into fibroblasts in vitro and further explore the antifibrotic effect of curcumin on corneal keratocytes.Methods The murine corneal keratocytes from 150 BALB/c mice were isolated and primary culture in DMEM culture medium containing 10% fetal bovine serum and then divided into blank control group(inducer group,CG),low-dose group(CG+7.5 mg/L curcumin),mediumdose group(CG+10.0 mg/L curcumin),high-dose group(CG+12.5 mg/L curcumin),non-inducer group.Seven days following intervention,the expression of cell markers such as keratocan,aldehyde dehydrogenase(ALDH),decorin and fibronectin-1 in keratocytes were analyzed by RT-PCR.The effect of curcumin on cultured murine corneal keratocytes proliferation was evaluated by MTS technique.The expression of fibronectin-1 in murine cornea was investigated by immunofluorescence assay.Results The primarily cultured keratocytes showed tlIe fusiform-like shape with the abundant cytoplasm and big nuclei.In the presence of curcumin,the mRNA levels of keratocan and ALDH were down-regulated and those of CD90 and decorin were up-regulated,showing the significantly differences with the increase of dose(P<0.05),but the expression pf fibronectin-i was not obviously changed with the alteration of dose of curcumin. MTS showed that the inhibitory rates of curcumin on keratocytes in 10.0 mg/L and 2. 5 mg/L groups were enhanced in comparison with 7.5 mg/L group, showing statistically significant difference among three groups( F = 956.00, P<0.05). The expression of fibronectin-1 was found in the corneal keratocytes with the red fluorescence in stroma. Conclusion Curcumin can inhibit the fibrosis of corneal keratoeytes in a dose-dependent manner. These results offer a preliminary theoretical basis for the application of curcumin in controlling corneal scar formation during wound healing. Key words: Curcumin; Keratocyte; Fibroblast; Fibrosis

Comparison of Candidate Materials for a Synthetic Osteo-Odonto Keratoprosthesis Device

Osteo-odonto keratoprosthesis is one of the most successful forms of keratoprosthesis surgery for end-stage corneal and ocular surface disease. There is a lack of detailed comparison studies on the biocompatibilities of different materials used in keratoprosthesis. The aim of this investigation was to compare synthetic bioinert materials used for keratoprosthesis surgery with hydroxyapatite (HA) as a reference. Test materials were sintered titanium oxide (TiO(2)), aluminum oxide (Al(2)O(3)), and yttria-stabilized zirconia (YSZ) with density >95%. Bacterial adhesion on the substrates was evaluated using scanning electron microscopy and the spread plate method. Surface properties of the implant discs were scanned using optical microscopy. Human keratocyte attachment and proliferation rates were assessed by cell counting and MTT assay at different time points. Morphologic analysis and immunoblotting were used to evaluate focal adhesion formation, whereas cell adhesion force was measured with a multimode atomic force microscope. The authors found that bacterial adhesion on the TiO(2), Al(2)O(3), and YSZ surfaces were lower than that on HA substrates. TiO(2) significantly promoted keratocyte proliferation and viability compared with HA, Al(2)O(3,) and YSZ. Immunofluorescent imaging analyses, immunoblotting, and atomic force microscope measurement revealed that TiO(2) surfaces enhanced cell spreading and cell adhesion compared with HA and Al(2)O(3). TiO(2) is the most suitable replacement candidate for use as skirt material because it enhanced cell functions and reduced bacterial adhesion. This would, in turn, enhance tissue integration and reduce device failure rates during keratoprosthesis surgery.

Effect of Mitomycin C, Dexamethasone, and Cyclosporine A 0.05% on the Proliferation of Human Corneal Keratocytes

Effect of Mitomycin C, Dexamethasone, and Cyclosporine A 0.05% on the Proliferation of Human Corneal Keratocytes

Polydopamine coatings enhance biointegration of a model polymeric implant

The biointegration of implants affects their function, stability and safety. Although most research on this topic has focused on bone and other hard tissues, biointegration with soft tissues is important in numerous applications, such as in prosthetic corneas. Here, we have adapted polydopamine-based adhesive surface chemistry to enhance the biointegration with soft tissue of a model polymer—poly(methyl methacrylate) (PMMA), commonly used in prosthetic corneas. Polydopamine coating (PDA) and subsequent modification with the cell-adhesive peptide RGD (PDA-PEG-RGD) significantly enhanced cellular proliferation of corneal epithelial cells and keratocytes without causing excessive secretion of pro-inflammatory cytokines (e.g.IL-6) by either cell type. PDA adhered tightly to collagen gels, while PDA-PEG-RGD and uncoated PMMA did not. PDA's adhesion to collagen was greatly reduced by preincubation in serum. Tissue reaction to both polydopamine-coated surfaces was benign after 45 days of subcutaneous implantation. However, in contrast to the findings with collagen gels, PDA-PEG-RGD bound much more tightly to tissue than did PDA—although both bound better than unmodified PMMA. Polydopamine-based surface chemistries are potentially useful in enhancing tissue integration of implants with soft tissues.

Inhibition of human keratocyte proliferation following the transfection of cyclinD1 antisense gene

目的 探讨反义cyclinD1基因转染后人角膜基质细胞增生的改变.方法 人角膜组织来源于北京大学第一医院眼库体外培养人角膜基质细胞,采用第2代或第3代细胞用于实验.利用阳离子脂质体lipofectamine介导反义cyclinD1基因转染入体外培养的人角膜基质细胞.同时设非转染组(以PBS代替cyclinD1质粒及lipofectamine)及lipofectamine转染组(以PBS代替反义cyclinD1质粒)作为对照.应用免疫细胞化学法观察lipofectamine介导反义cyclinD1基因转染对人角膜基质细胞中cyclinD1蛋白表达的影响,应用四甲基偶氮唑盐(MTT)法观察其对人角膜基质细胞增生的影响,并用质量分数0.5%锥虫蓝染色观察活细胞比率.结果 非转染组和lipofectamine转染组均有大量人角膜基质细胞的细胞核cyclinD1染色呈棕褐色,反义cyclinD1转染组只有少量人角膜基质细胞细胞核cyclinD1染色呈棕褐色.与非转染组和lipofectamine转染组比较,反义cyclinD1转染组在转染后2、6、10d cyclinD1蛋白在培养的人角膜基质细胞中的表达明显降低,差异均有统计学意义(t=6.050、t=5.180、t=5.040,P＜0.01;t=6.190、t=5.670、t=5.010,P＜0.01),而非转染组与lipofectamine转染组间比较,差异均无统计学意义(t=1.120、t=1.310、t=1.010,P＞0.05).转染后2、6、10d,反义cyclinD1转染组人角膜基质细胞的A570值分别低于非转染组和lipofectamine转染组,差异均有统计学意义(t=5.830、t=5.210、t=4.920,P＜0.01;t=5.130、t=5.010、t=4.850,P＜0.01),说明抑制了基质细胞的增生,而非转染组与lipofectamine转染组间比较,差异均无统计学意义(t=1.110、t=1.230、t=1.120,P＞0.05).0.5%锥虫蓝染色显示各组活细胞比率均在90%以上,未见明显的细胞毒性.结论 阳离子脂质体介导的反义cyclinD1基因转染在转染后一定时间内可降低人角膜基质细胞中cyclinD1蛋白的表达,抑制人角膜基质细胞的增生,未见明显细胞毒性,为角膜基质过度愈合反应的基因治疗奠定了基础。

Cell proliferation studies of novel biomaterials to be used as synthetic keratoprosthesis skirts

Abstract Purpose In vitro studies of human cornea stroma keratocyte proliferation and cytokine secretion on the various kerato‐prosthesis skirt material. In the development of an artificial cornea, an appropriate skirt material which encourages keratocytes proliferation is important to help wound healing and integration. Epidermal growth factor (EGF), insulin growth factor (IGF‐1) and hepatocyte growth factor (HGF) stimulate corneal keratocytes proliferation. IGF‐1 and EGF have an anti‐apoptotic effect of keratocytes. Conclusion: Methods Three materials were studied: Hydroxyappatite (HA), titanium substate (TiO2) and aluminium oxide substrate (Al2O3). Corneal stromal were seeded on top of these materials for EDU image assay and real time PCR analysing. Conditioned cell culture medium was collected for cytokine array. Results Cytokine array showed the levels of EGF, IGF‐1 and HGF were no statistical significance among the HA, TiO2 and Al2O3. RT‐PCR, showed that the expression level of EGF, IGF‐1 and HGF were highest with TiO2 substrates and lowest with HA substrates with statistical significance. Edu assay showed a comparable number of proliferating cells on the HA and TiO2 with significantly more cells on the Al2O3 dics. Conclusion TiO2 is a comparable material to HA at promoting corneal keratocytes proliferation, hence is a possible alternative material to generate synthetic keratoprosthetic skirts.

Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C

To study the effects of laser-assisted subepithelial keratectomy (LASEK) with mitomycin C (MMC) on the keratocyte population. Prospective, nonrandomized, interventional, comparative case series. Fifty-six eyes treated at Vissum Santa Hortensia, Madrid, Spain, were included in the study. We compared 28 eyes treated with LASEK with intraoperative 0.02% MMC versus 28 non-treated eyes. Keratocyte density was measured 3 months after the surgery in the anterior, mid, and posterior stroma and was compared with the corresponding layers in the control eyes. The anterior layer in the LASEK group was compared with 2 layers in the control group: the most anterior stromal layer and the 80 μm-deep layer, because that was the mean ablation depth performed in eyes that underwent LASEK. We found a statistically significantly lower keratocyte population in the most anterior stromal layer after LASEK with MMC compared with both the most anterior stromal layer and the 80 μm-deep layer in controls. On the contrary, the treated group showed a significantly higher keratocyte density in both the mid stroma and the deep stroma. The comparison between the average densities through the entire cornea showed a significantly higher keratocyte population in the LASEK with MMC group. LASEK with MMC seems to cause a decrease in the anterior stromal cells 3 months after the surgery compared with nonoperated corneas. There seems to be a compensating proliferation of keratocytes in the deeper corneal layers, suggesting that the ability of keratocytes to repopulate the cornea is maintained after the surgical procedure.

Effects of antifungal drugs and delivery vehicles on morphology and proliferation of equine corneal keratocytes in vitro

To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxymethylcellulose and a combination vehicle composed of polyethylene glycol, methylparaben, and propylparaben were also evaluated. Morphological changes and cellular proliferation were assessed 24, 48, and 72 hours after application. At the highest concentrations tested, all antifungals caused marked cellular morphological changes and inhibited proliferation. At low concentrations, natamycin and miconazole induced rounding, shrinking, and detaching of the cells with inhibition of cellular proliferation. Natamycin caused the most severe cellular changes. Itraconazole, at the low concentrations, caused minimal morphological changes and had a minimal effect on proliferation. All vehicles tested had significantly less effects on cellular morphology and proliferation when compared with the antifungals, except for the combination vehicle, which caused severe morphological changes and inhibited proliferation, even at low concentrations. The DMSO had minimal effects on cellular morphology and proliferation, even at high concentrations. Itraconazole had significantly less cytotoxic effects on equine keratocytes in culture than did natamycin or miconazole. Natamycin had severe cytotoxic effects in vitro.

Role of transforming growth factor Beta in corneal function, biology and pathology.

Transforming growth factor-beta (TGFbeta) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFbeta are known in mammals and the human cornea expresses all of them. TGFbeta1 has been shown to play a central role in scar formation in adult corneas whereas TGFbeta2 and TGFbeta3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFbeta in the cornea have been shown to follow Smad dependent as well as Smad-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFbeta expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFbeta is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFbeta is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFbeta-mediated corneal scarring are under investigation. These include blocking of TGFbeta, TGFbeta receptor, TGFbeta function and/or TGFbeta maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFbeta has also been demonstrated recently.

This article has been retracted: A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblasts

Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis.The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). RT-PCR and Western Analysis were used to investigate the presence of NFAT-3 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-3 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-3 localisation in these biopsies using a well characterized anti-NFAT-3 antibody. The NFAT-3 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. The expression of NFAT-3 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. As with NFAT 1, NFAT 2 and recently NFAT 5, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-3 in cultured keratocytes but with different kinetics. These data provide the first evidence of that NFAT-3 is expressed in normal skin, psoriasis and that NFAT-3 functionally active in human keratocytes and that nuclear translocation of NFAT-3 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-3 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis.

Twist2: Role in Corneal Stromal Keratocyte Proliferation and Corneal Thickness

Twist2 is a member of a family of bHLH transcription factors critical for normal mesenchymal proliferation and differentiation. In this study, the authors analyzed the role of Twist2 in the eye and cornea through examination of a Twist2 loss-of-function mouse mutant. Twist2 expression during eye development in the mouse was investigated using RT-PCR and mRNA slide in situ hybridization. Lineage tracing was performed using Cre reporter mice. Morphometric analyses were performed, and cell proliferation and cell death were investigated by immunohistochemistry using Ki67 and cleaved caspase 3 antibodies, respectively. In the mouse, Twist2 is expressed first in the periocular mesenchyme and subsequently in the corneal stroma and endothelium of the developing eye. Loss of Twist2 function leads to corneal thinning and a reduced population of stromal keratocytes. The reduction in the stromal cell population can be traced back to embryonic stages during which the proliferation of stromal progenitor cells is impaired and to the reduced number of proliferating cells in the corneal limbus postnatally. Adult Twist2-null mice display enophthalmia and blepharophimosis. Corneal thinning in mutant mice is not accompanied by glaucoma, an association reported in human patients. Twist2 is required for normal corneal keratocyte proliferation and eyelid morphogenesis in the mouse. Loss of Twist2 function leads to corneal thinning because of the reduction in stromal keratocyte proliferation.

Live or Let Die: Epithelial Flap Vitality and Keratocyte Proliferation Following LASEK and Epi-LASIK in Human Donor and Porcine Eyes

To determine the relationship between epithelial flap vitality and stromal keratocyte proliferation following two epithelial refractive techniques: epi-LASIK and laser epithelial keratomileusis (LASEK). Human corneas were maintained in organ culture and underwent standard -6.00-diopter ablation. Rates of stromal keratocyte proliferation were detected 1 week postoperative using a Ki67 antibody specific to proliferating cells. Images were captured with a laser scanning confocal microscope and analyzed by a masked observer. Epithelial flap vitality was determined with propidium iodide using fresh porcine corneas. Epithelial flaps were created with Gebauer Epikeratome epi-LASIK or alcohol-assisted LASEK method. Flaps treated with 100% alcohol and uninjured corneas were used as controls. The number of proliferating keratocytes was greatest at 1 week in the epi-LASIK corneas (P<.001). Cell vitality was greatest in the epi-LASIK flaps and declined in the LASEK and 100% alcohol flaps (P<.001). In this in vitro setting, epi-LASIK results in an epithelial flap with significantly more live cells. There is also a greater number of proliferating stromal cells following epi-LASIK at 1 week. Based on these in vitro observations, epi-LASIK may result in greater levels of haze compared to LASEK.

Induction of Controlled Wound Healing with PMMA Segments in the Deep Stroma in Corneas of Hens

Polymethylmethacrylate (PMMA) segments are normally used in additive surgery to treat both corneal ectasia post laser-assisted in situ keratomileusis and keratoconus. The aim of this work was to develop an experimental animal model to induce wound healing in the deep stroma in corneas of hens. PMMA segments were implanted in the right eyes of 40 adult hens without suture in the wound incision. Animals were randomized for 5 time points to histopathology study (1, 3, 15, 30, and 90 days) being clinically evaluated during the experiment. Thirty-four eyes (85%) had a successful clinical outcome and intraoperative mistakes occurred in 6 eyes (15%), including anterior chamber perforation resulting in abscess (1), excess of lamellar dissection with segment migration (3), and peripheral incisions evolving with neovascularization (2). At 24 hours, all the epithelial injuries were completed in integrity, but a persistent stromal incision, with a fish mouth form, was observed until day 15. Corneal edema disappeared at the fifth day. Haze, keratocyte cell death, keratocyte proliferation, myofibroblast differentiation, and new matrix production were observed in length around the segment. Wound healing was induced in the deep corneal stroma by means of PMMA segment implantation in a new animal model developed in hens.

Effects of riboflavin/UVA corneal cross‐linking on keratocytes and collagen fibres in human cornea

To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in human corneas. Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross-linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter. Normal corneas exhibited no TUNEL-positive keratocytes and keratoconic and cross-linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP-ribose) polymerase assessed using Western blot. The Ki-67 staining showed a significant increase in the keratocyte proliferation in cross-linked corneas compared with normal and keratoconus. In cross-linked corneas CD34-positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross-linked corneas compared with control and keratoconus. Corneal cross-linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34-positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.

Effects of Gel Type Artificial Tears on Human Corneal Keratocytes and Conjunctival Epithelial Cells

Purpose: To evaluate the biological effects and cytotoxicity of gel-type artificial tears on human corneal keratocytes and conjunctival cells in vitro. Methods: Human corneal keratocytes and conjunctival epithelial cells were exposed to Soothe and Systane at variable concentrations. Evaluations were conducted through an MTT-based calorimetric assay to measure the metabolic activity and through a lactate dehydrogenase (LDH) assay to assess cellular damage. Apoptotic response was examined using fluorescent microscopy and flow cytometric analysis, and cellular morphologic results were evaluated with a transmission electron microscope. Results: The inhibitory effects of corneal keratocyte and conjunctival cell proliferations increased at higher concentrations and longer exposure times to Soothe and Systane. The LDH titers increased after Soothe exposure, but showed no significant difference after Systane exposure. Soothe and Systane treatments both produced fluorescence, representing apoptotic cells. In flow cytometry, the maximal apoptotic response was observed for both types of artificial tears, although Systane showed less edema, as well as reduced cytoplasmic and nuclear cell degeneration compared to those of Soothe. Conclusions: The apoptotic responses of Soothe and Systane are associated with inhibitory effects of human corneal keratocyte and conjunctival epithelial cell proliferations. To inhibit the cellular proliferation of human corneal keratocytes and conjunctival epithelial cells, Systane may be less severe than Soothe at higher concentrations and longer exposure times.