Re-epithelialization and remodeling of decellularized corneal matrix in a rabbit corneal epithelial wound model.

This study investigated the in vivo correlation between re-epithelialization and remodeling of a decellularized corneal matrix prepared by a high-hydrostatic pressure (HHP) method in rabbits. Decellularized corneal matrices were transplanted in a 6-mm-diameter recipient corneal interlamellar pocket with a 2 mm epithelial defect. The time course of graft status in rabbits was examined daily for 6 months by biomicroscopy and scored for clarity and re-epithelialization, after which the rabbits were sacrificed for histological analysis. Fluorescein staining revealed that the corneal epithelial cells had migrated onto the decellularized corneal matrix. Histological analysis revealed that the implanted decellularized corneal matrix was completely integrated with the recipient rabbit cornea and the stratified corneal epithelia consisting of multiple layers were regenerated, similar to that in the normal cornea. The recipient keratocytes infiltrated into the decellularized corneal matrix at 6 months after the operation and the decellularized corneal matrix was gradually remodeled into the recipient tissue. Transmission electron microscopy revealed that the ultrastructure of the decellularized corneal matrix was rearranged, similar to the normal cornea. These findings suggest that the decellularized corneal matrix serves as a template for remodeling. The decellularized corneal matrix obtained through HHP is a useful graft for corneal tissue regeneration.

Ultrastructural analysis of the decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty in a rabbit model.

The decellularized cornea has received considerable attention for use as an artificial cornea. The decellularized cornea is free from cellular components and other immunogens, but maintains the integrity of the extracellular matrix. However, the ultrastructure of the decellularized cornea has yet to be demonstrated in detail. We investigated the influence of high hydrostatic pressure (HHP) on the decellularization of the corneal ultrastructure and its involvement in transparency, and assessed the in vivo behaviour of the decellularized cornea using two animal transplantation models, in relation to remodelling of collagen fibrils. Decellularized corneas were prepared by the HHP method. The decellularized corneas were executed by haematoxylin and eosin and Masson's trichrome staining to demonstrate the complete removal of corneal cells. Transmission electron microscopy revealed that the ultrastructure of the decellularized cornea prepared by the HHP method was better maintained than that of the decellularized cornea prepared by the detergent method. The decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty using a rabbit model was stable and remained transparent without ultrastructural alterations. We conclude that the superior properties of the decellularized cornea prepared by the HHP method were attributed to the preservation of the corneal ultrastructure.

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix.

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 µm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

Embossed-carving processing of cytoskeletons of cultured cells by using focused ion beam technology.

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga(+) onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga(+) sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi-3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells.

Origin and possible role of males in hermaphroditic androgenetic Corbicula clams.

Hermaphroditic Corbicula leana clams reproduce by androgenesis and have been regarded as simultaneous hermaphrodites. To date, there has been no report on the occurrence of male clams in hermaphroditic Corbicula. In an irrigation ditch in Shiga Prefecture, we found that 78.2% of C. leana specimens were males and 21.8% were hermaphrodites. Microfluorometric analysis revealed that males were diploids and hermaphrodites were triploids. All males produced nonreductional and biflagellate spermatozoa. The sequence analysis of mitochondrial DNA (cytochrome b, 621 bp) for 31 specimens of C. leana showed that four male and nine hermaphrodites shared the same H2 mtDNA haplotype; H1 was detected from 17 males and H3 was detected from one hermaphrodite. Coexisting C. fluminea clams also have haplotypes H1 and H2. Phylogenetic tree by a neighborjoining method based on the partial sequence of cytochrome b revealed that the haplotypes (H1- 3) of C. leana were evidently different from those of dioecious C. sandai (S1 and S2) and C. japonica (J1 and J2). These results suggest that males may be derived from hermaphrodite C. leana clams. The role of males in hermaphroditic populations is unknown. However, if the spermatozoon from a male is able to fertilize an egg from a hermaphrodite and the nuclear genome of the egg is expelled as polar bodies, the sperm nucleus could form a zygote nucleus. This mode of reproduction would allow the replacement of the nuclear genome.

Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering.

To prepare acellular corneal scaffold, we used high-hydrostatic pressurization (HHP) to decellularize porcine cornea. The HHP method disrupts cells by hydrostatic pressurization, and then the disrupted cells' components are removed by washing with a cell culture medium. Porcine corneas were hydrostatically pressed at 980 MPa at 10 or 30 degrees C for 10 min to make them opaque. There was no change in the thickness of the corneas immediately after the pressurization, but they swelled during the washing process. The cornea swelling caused by HHP was suppressed when medium containing 3.5% w/v dextran was used. For H-E staining of the cornea decellularized with the HHP method, the complete removal of corneal cells was confirmed. Furthermore, when the corneas were immersed in glycerol for 1 hour, their optical properties were restored to those of native corneas. In an animal study, when acellular porcine corneas were implanted into rabbit cornea, no immune reaction occurred and the turbid corneas became clear. The decellularized corneas obtained through HHP could be useful as a corneal scaffold for tissue regeneration.

In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas.

PURPOSE: To evaluate the stability and biocompatibility of artificial corneal stroma that was prepared by using ultrahigh hydrostatic pressurization treatment to decellularize corneas. METHODS: The porcine cornea was decellularized by two methods, a detergent method and an ultrahigh hydrostatic pressure (UHP) method. Either 1% w/v Triton X-100 or sodium dodecyl sulfate (SDS) was used for the detergent method, and 10,000 atmospheres (atm; 7.6x10(6) mmHg) was applied to the cornea for 10 min at 10 degrees C by a high-pressure machine for the UHP method. Hematoxylin-eosin staining was performed to confirm the removal of the corneal cells, and then decellularized porcine corneal stroma was implanted into rabbit corneal pockets. After eight weeks, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. RESULTS: Complete decellularization was confirmed only in corneas treated by the UHP method, and little inflammation was seen when they were implanted into the rabbit corneal pockets. CONCLUSIONS: Porcine corneal stroma completely decellularized by the UHP method has extremely high biocompatibility and is a possible corneal scaffold for an artificial cornea.