BACKGROUNDFrozen or dried corneal grafts are commonly used for stromal transplantation such as lamellar keratoplasty (full or partial thickness), keratophakia, epikeratophakia. Structural properties are important for the final optical results of these surgeries but the effects of freezing/thawing and drying/rehydration on the properties of the stroma are known little compared with the corneal endothelium, mainly because of lack of non-invasive technique to evaluate the stromal structure. This study aimed to investigate the swelling and structural properties of the bovine corneal stroma following freezing or drying by X-ray diffraction which was a non-invasive technique and could give ultra-structural information in hydrated tissues.

METHODSBovine corneas were either frozen at -40 degrees C or dried to constant weight in a dessicator over silica gel. Swelling was carried out by placing the corneas into dialysis tubing and equilibrating them against various concentrations of polyethylene glycol (PEG) to obtain a range of tissue hydrations. This method minimises the loss of soluble tissue components during the swelling process. Synchrotron X-ray diffraction was used to measure the average intermolecular spacing, the interfibrillar spacing and the fibril diameter as a function of hydration. Changes in light scattering were detected using a microdensitometer.

RESULTSFreezing and thawing of the cornea caused an increase in light scattering by 63.9% at tissue hydration (H) = 3.4, and by 50.0% at H = 4.9. Repeated freezing and thawing causes further increased by 38.9% at the second time and another 36.0% at the third time (P < 0.05). There was a tendency for both the frozen and the dried corneas to lose some swelling ability, achieving hydrations respectively of 10% and 18% below those of fresh corneas at 0 PEG. There were no changes in the fibril diameters, interfibrillar or intermolecular spacings as measured by X-ray diffraction in the equilibrated fresh, pre-frozen and pre-dried corneas.

CONCLUSIONSThe increase in light scattering and the loss of swelling ability after freezing and thawing probably results from structural changes following the close association of the collagen molecules and fibrils whilst the tissue is in the dry or frozen state. Some unknown changes in the extracellular matrix between the collagen fibrils may also play a role in the light scattering. The equilibration technique may improve the quality of rehydrated corneal graft or lenticules used for corneal surgeries.

Animals ; Cattle ; Cornea ; chemistry ; Desiccation ; Freezing ; X-Ray Diffraction

OBJECTIVETo study the corneal penetration of PAMAM dendrimers-coated puerarin liposomes in rabbits.

METHODEvaluated PAMAM (G2, G3) dendrimers-coated puerarin liposomes were prepared and the in vitro transcorneal penetration were compared to puerarin drop solution and uncoated liposomes. The effect of different proportion of PAMAM to phospholipids in formulation on corneal penetration and the penetration parameters were investigated.

RESULTThe steady state fluxes and permeability coefficients of puerarin by PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated puerarin liposomes were greater than that by puerarin drop solution and uncoated liposomess (P < 0.01), meanwhile the PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated liposomes were better than other ratios of coated liposomes for improvement of corneal penetration (P < 0.01).

CONCLUSIONThe PAMAM coated liposomes is able to enhance the corneal penetration of puerarin and promising as an ocular drug carriers.

Animals ; Cornea ; metabolism ; Dendrimers ; chemistry ; metabolism ; In Vitro Techniques ; Isoflavones ; chemistry ; Liposomes ; chemistry ; metabolism ; Rabbits

It is important to design a long-period transparent bioactive material for corneal repair in the process of corneal tissue renovation. This article discusses the silk fibroin and formamide blend membranes as a corneal stroma repair material. Silk fibroin solution was mixed with formamide in different proportions to obtain insoluble transparent silk fibroin film by casting method. The blending membranes had excellent mechanical properties, cell compatibility and long-term transparent properties. Rabbit corneal stromal cells were seeded on the sterilized composite films. The rate of cell surface adhesion was over 90% after cells were placed on it for 5 hours. When cells were seeded on blend membranes from one day to seven days, Alma Blue was added to complete medium. Compared with the cell culture plate, there was no significant difference in cell proliferation on formamide/silk films. The results indicated that formamide/silk films might be used as a corneal stroma repair material and worth of further investigatinn
Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; Cell Proliferation ; Cornea ; cytology ; Fibroins ; chemistry ; Rabbits ; Regeneration

A novel cornea tissue scaffold material was prepared with N-vinly pyrrolidome (NVP) and a biodegradable crosslinking agent by radical polymerization, using azoisobutyronitrile (AIBN) as initiator. Water absorption test and contact angle measure were conducted, and the degradation process of material was investigated. The biocompatibility evaluation was carried out by implantation of material in the rabbits, and by cell culture. The water absorption was over 104%, the contact angle was lower than 41degrees, and the degradation speed in vitro kept steady. The results of implantation in the rabbits showed that the material was almost degraded 3 months later and lots of collagen and cornea stroma cells appeared in it,but there was no inflammation around it. The result of epithelial cells culture showed that the cells conglutinated on the material, but no remarkable cytotoxicity was noted.
Animals ; Biocompatible Materials ; chemistry ; Cornea ; cytology ; Epithelium, Corneal ; cytology ; Materials Testing ; Nitriles ; chemistry ; Prostheses and Implants ; Pyrrolidinones ; chemistry ; Rabbits ; Tissue Engineering

OBJECTIVETo prepare sparfloxcain lactate (SPLX) loaded liposomes and study its corneal penetration and bacterial inhibitory in vitro.

METHODSLiposomal SPLX (mass ratio of phospholipids/ cholesterol/drug at 18:6:1) was prepared by pH-gradients. The transcorneal penetration experiments of liposomal SPLX were performed in modified Franz's cells with the rabbit's corneal. The concentration of SPLX was determined by high-performance liquid chromatography. The penetration parameters were calculated. The in vitro antibacterial activities on S. aureus, P. aeruginusa, E. coli, and B. subtilis were determined by two fold dilutions.

RESULTSThe entrapment efficiency of SPLX in the liposomes by pH-gradients was (81.61 +/- 1.98)%, which was significantly higher than that by film dispersion method (11.48 +/- 0.86)% and reverse evaporating method (18.64 +/- 1.05)% (both P < 0.01). The permeability coefficient and corneal deposition quantity of SPLX liposomes were 1.65- and 4.98-folds higher as compared with those of free drug solutions. The minimal inhibitory concentrations (MICs) of liposomal SPLX against S. aureus, P. aeruginosa, E. coli, and B. subtilis were 1/4, 1/2, 1/1, 1/17 times lower than those of free drug, respectively, and the minimal bactericide concentrations (MBCs) were 1/4, 1/2, 1/1, 1/4 times lower than those. In addition, the time-kill values of liposomal SPLX were better than those of free.

CONCLUSIONThe pH gradient technique is suitable for preparing SPLX liposomes, which can improve the transcorneal penetration and antibacterial activity of SPLX in vitro.

Animals ; Anti-Bacterial Agents ; chemistry ; pharmacology ; Bacteria ; drug effects ; Cornea ; chemistry ; drug effects ; Female ; Liposomes ; chemistry ; pharmacology ; Male ; Permeability ; Rabbits

OBJECTIVETo investigate the dynamic changes of matrix metalloproteinase-9 (MMP-9) level in tear fluid within 12 months after laser in situ keratomileusis (LASIK).

METHODSTwenty-two myopic patients undergoing uneventful LASIK were enrolled in this study. Tear fluid samples were collected from the patients for measurements of MMP-9 level using Western blotting preoperatively, at 7 and 14 days, and at 1, 2, 3, 6, and 12 months after the surgery.

RESULTSMMP-9 concentrations in the tear fluid of post-LASIK patients showed a time-dependent variation pattern. MMP-9 reached its peak level in the tear fluid at 14 days postoperatively, which was 2.70 times the preoperative level; it gradually decreased thereafter but was still 1.38 times the preoperative level at 12 months after the surgery.

CONCLUSIONSMMP-9 concentrations in the tear fluid of post-LASIK patients show a time-dependent variation pattern and remains higher than the preoperative level even at 12 months after the surgery, suggesting that corneal wound healing after LASIK lasts for more than 12 months.

Cornea ; Humans ; Keratomileusis, Laser In Situ ; Matrix Metalloproteinase 9 ; chemistry ; Myopia ; surgery ; Postoperative Period ; Prospective Studies ; Tears ; chemistry ; Wound Healing

To study corneal lymphangiogenesis after corneal transplantation, corneal allogenic transplantation models were established in rats. 8 female Wister rats were used as donors, and 16 Sprague Dawley (SD) rats were used as recipients and 2 SD served as controls. Corneal lymphangiogenesis and hemangiogenesis was examined by electron microscopy 1 and 2 weeks after corneal penetrating transplantation, and the expression of lymphatic vessel endothelial receptor (LYVE-1) was examined 1, 3, 7, 14 days after the transplantation respectively. In addition, 19 allograft failed human corneas were examined by 5'-nase-alkaline phosphatase (5'-NA-ALP) double-enzyme-histochemistry staining to detect corneal lymphangiogenesis and hemangiogenesis. By immunohistochemistry for LYVE-1, it was found that blown lymphatics were localized in the stroma 3 days after the corneal transplantation. With electron microscopy, new lymphatic vessels and blood vessels were found 1 and 2 weeks after the corneal transplantation. By 5'-NA-ALP enzyme-histochemistry, corneal hemangiogenesis was found in all allograft failed human corneas and 5 of 19 (26.3 %) cases had developed corneal lymphangiogenesis. It is concluded that corneal lymphangiogenesis is present after corneal transplantation, which may play an important role in allograft rejection.
Cornea/*blood supply ; Cornea/chemistry ; Cornea/ultrastructure ; Corneal Neovascularization/etiology ; Corneal Neovascularization/metabolism ; Corneal Transplantation/adverse effects ; Corneal Transplantation/*methods ; Immunohistochemistry ; Lymphangiogenesis ; Microscopy, Electron ; Rats, Sprague-Dawley ; Rats, Wistar ; Vesicular Transport Proteins/biosynthesis

OBJECTIVETo investigate the effects of labrasol, solutol HS 15 and transcutol P on the corneal permeability of mangiferin in vitro.

METHODThe effects of three penetration enhancers on the corneal permeability of mangiferin were investigated in vitro by using isolated rabbit corneas.

RESULTThe apparent Papp enhancements were increased 1.80, 3.27, 3.41 and 4.76-folds with Lab at 1.0%, 1.5%, 2.0% and 3.0% (P < 0.01), respectively. The apparent Papp increased 1.98 and 3.07-folds with Sol at 0.2% and 0.4% (P < 0.01), respectively, but reduced with 0.010%-0.03% Trans.

CONCLUSIONThe Papp value of mangiferin is significantly enhanced by 1.0%-3.0% Lab, 0.2% and 0.4% Sol, however the Papp value of mangiferin is reduced by 0.01%-0.03% Trans.

Animals ; Cornea ; drug effects ; metabolism ; Drug Carriers ; chemistry ; Ethylene Glycols ; chemistry ; Glycerides ; In Vitro Techniques ; Organic Chemicals ; chemistry ; Permeability ; Plant Extracts ; pharmacokinetics ; Polyethylene Glycols ; chemistry ; Rabbits ; Stearic Acids ; chemistry ; Xanthones ; pharmacokinetics

Some different membranes were prepared by Chitosan with the degree of deacetylation (DD) of 63.7%, 73.7%, 83% and 97% respectively. To study the biocompatibility of Chitosan membrane toward corneal stromal cells, the rabbit cells were cultured on the surface of different DD chitosan membranes. The morphological characteristics, the cell-adhesion, the cell proliferation and the activity of LDH in the medium were investigated. The results of experiment shows that the DD of Chitosan has very significant effect on the biocompatibility of Chitosan membrane toward corneal stromal cells. The more DD of Chitosan, the less injury was made to corneal stromal cells by the chitosan membrane, which is favor of the growing and adhesion of corneal stromal cells. The biocompatibility of the membrane made with low DD Chitosan with corneal stromal cells became worse.
Acetylation ; Animals ; Biocompatible Materials ; chemistry ; pharmacology ; Cell Adhesion ; drug effects ; Cell Division ; drug effects ; Chitosan ; chemistry ; pharmacology ; Cornea ; cytology ; Materials Testing ; Membranes, Artificial ; Rabbits ; Stromal Cells ; drug effects

The study is to design chitosan-coated pilocarpine nitrate submicro emulsion (CS-PN/SE) for the development of a novel mucoadhesive submicro emulsion, aiming to prolong the precorneal retention time and improve the ocular absorption. CS-PN/SE was fabricated in two steps: firstly, pilocarpine nitrate submicro emulsion (PN/SE) was prepared by high-speed shear with medium chain triglycerides (MCT) as oil phase and Tween 80 as the main emulsifier, and then incubated with chitosan (CS) acetic solution. The preparation process was optimized by central composite design-response surface methodology. Besides the particle size, zeta potential, entrapment efficiency and micromorphology were investigated, CS-PN/SE's precorneal residence properties and miotic effect were especially studied using New Zealand rabbits as the animal model. When CS-PN/SE was administered topically to rabbit eyes, the ocular clearance and the mean resident time (MRT) of pilocarpine nitrate were found to be dramatically improved (P < 0.05) compared with PN/SE and pilocarpine nitrate solution (PNs), since the K(CS-PN/SE) was declined to 0.006 4 +/- 0.000 3 min(-1) while MRT was prolonged up to 155.4 min. Pharmacodynamics results showed that the maximum miosis of CS-PN/SE was as high as 46.3%, while the miotic response lasted 480 min which is 255 min and 105 min longer than that of PNs and PN/SE, respectively. A larger area under the miotic percentage vs time curve (AUC) of CS-PN/SE was exhibited which is 1.6 folds and 1.2 folds as much as that of PNs and PN/SE, respectively (P < 0.05). Therefore, CS-PN/SE could enhance the duration of action and ocular bioavailability by improving the precorneal residence and ocular absorption significantly.
Absorption ; Animals ; Area Under Curve ; Biological Availability ; Chitosan ; chemistry ; Cornea ; metabolism ; Emulsions ; Microscopy, Electron, Transmission ; Miotics ; administration & dosage ; chemistry ; pharmacokinetics ; Ophthalmic Solutions ; Particle Size ; Pilocarpine ; administration & dosage ; chemistry ; pharmacokinetics ; Rabbits ; Random Allocation ; Solubility ; Tears ; metabolism