| Background and ObjectiveIt is generally accepted that irreversible opacification of the cornea from various diseases is a major cause of vision loss, second only to cataracts as the leading cause of blindness. And corneal transplantation, penetrating keratoplasty (PKP), has been used as the standard procedure for treating cornea-induced blindness and millions of people have enjoyed restoration of useful sight. However, there are still many hurdles to be overcome in PKP, such as the disadvantages of corneal transplantation, primarily immune rejection and the shortage of donor corneas. The high-risk patients with severe diseases causing total destruction of the limbal tissue are considered contraindications for PKP. Therefore, it is clearly beneficial to seek new paradigm shifts, corneal replacements.Corneal replacements can be divided into two categories, keratoprostheses and tissue-engineered corneal equivalents (TECE). TECE is an entirely cell-based tissue engineered corneal replacement that mimics the structure of the native cornea, incorporating the collagenous structural component and the main cell types present in the natural cornea and can perform many of its functions. In order to create a tissue-engineered biologic equivalent, an appropriate three dimensions (3D) matrix for culture of the cells is necessary. Natural biopolymer hydrogels, including those based on collagen, have been shown to support the 3D growth of cells and have been widely used in tissue engineering applications. However, few of the TECE were used as clinical applications, possibly due to poor mechanical properties of the currently available collagen scaffolds.Decellularization processes of xenogeneic or allogeneic tissues and organs are to lower inflammation and immunological responses, to minimize the disruption of extracellular material (ECM), and thus retain native mechanical properties and biologic properties of the resultant. As we know, decellularized biological scaffolds have been successfully used in both pre-clinical animal studies and in human clinical applications including heart valves, blood vessels, skin, nerves, skeletal muscle, tendons, ligaments, small intestinal submucosa (SIS), urinary bladder and liver for tissue engineering and regenerative medicine applications. But little information is available on the xenogeneic corneal acellularized matrix in tissue-engineered cornea.The aim of this study was to develop a new decellularization method or protocol depended upon the natural corneal properties and to harvest an ideal scaffold with good biocompatibilities and mechanical properties. We hope to reconstruct TECE ex vivo and use plain corneal acellularized matrix (CAM) or TECE in PKP animal models and evaluated the possibility of recellularization for plain CAM in vivo and verified the inhibition of TECE in angiogenesis. Materials and Methods1. Preparation of CAM100 fresh porcine eyeballs were obtained from the local abattoir immediately after sacrifice and sectioned at the corneal limbal plane. After osmotic shock with a hypotonic solution (deionized water), each swollen corneal button was split into two equal parts, the endothelial sides and the epithelial sides, and marked with interrupted sutures of black monofilament 10-0 nylon. After freezing and thawing, enzymatic techniques (including the use of trypsin digestion and nucleases) and mechanical agitation were chosen to remove the resultant cellular remnants from the corneal tissues. Finally standard histological staining with HE were used to determine if nuclear structures could be observed. One air-dry sample was mounted and sputter-coated with gold for viewing by scanning electron microscopy.2. Evaluation of the biocompatibility of CAM22 adult male New Zealand white rabbits with a body weight of 2-2.5 kg and 2 neonatal rabbits were used in this study. Evaluation of the biocompatibility of CAM was performed in vitro by cell seeding on CAM and in vivo by CAM direct implantation within the mid-depth stromal pockets or the anterior chamber.3. Corneal ulcer repaired with plain CAM in rabbit LKP models or PKP models12 rabbit corneal ulcer models were prepared a week before operation and all received PKP or LKP with plain CAM. The rabbit with postoperative complications, such as secondary glaucoma, hyphema, was excluded (n=1). The left were analyzed by slit lamp, ultrasound sound microscopy, transmission electron microscopy and light microscopy.4. TECE reconstruction ex vivo and the inhibition of angiogenesis of TECE in SD rat PKP models in vivo20 SD rats with a body weight of 200-250g were divided into two groups randomly and each received PKP with a plain CAM graft or a TECE graft (CAM seeded with primary corneal cells). TECE reconstructed with red-emitting PKH26 labeled corneal epithelial cells and keratocytes seeded on CAM. The neovascularization, opacity and autologous repopulation of plain CAM or TECE were observed. Corneal buttons were excised at 2, 4, 8, 16, 20 weeks after surgery and HE staining analysis or transmission electron microscopy (TEM) evaluation was performed. Results1. Morphology of CAMCAM was noted as transparent, smooth and strong enough to withstand suturing. However, CAM showed abnormal morphology without natural corneal curvance. The decellularization procedure resulted in a complete loss of cells in the corneal stroma and the collagen lamellae were well preserved.2. The biocompatibility of CAMBy day 7, HE staining showed a monolayer of the adhension of keratocytes on the CAM surface. One month postoperatively, the rabbits were sacrificed and histopathologic sections of the materials in anterior chamber revealed that they were entrapped with fibroblasts. The scaffolds implanted within the corneal stroma pockets did not invoke an adverse immune response by the host and no keratocytes were observed in the pores of the CAM.3. Autologous repopulation in plain CAM.Autologous cell-reseeding was found in each case. The reconstructed ocular surface showed uneven and irregular fluorescein staining absent of epithelial defects during 1-2 weeks after surgery, and the defects were gradually reepithelialized from the surrounding epithelium. HE showed epithelial cells were confluent and stratified 4 weeks after surgery. TEM revealed keratocytes migrated into pores of the CAM along the collagen layers and secreted collagen fibrils. CNV occurred on days 4-7 and pannus grew across the anastomoses in day 14-20 and minimized till the 32nd week. The cornea grafts became nearly transparent with normal curvance.4. TECE in vivo and ex vivoCorneal epithelial cells without red pseudocolor were stratified on CAM the same as those normal counterpart and keratocytes transformed into fibroblasts and infiltrated along the lamella of CAM and secreted collagen fibril in TECE group. TECE grafts showed apparently inhibition to neovascularization compared to plain CAM grafts. Cornea nerves were observed in the TECE grafts at 24 weeks. Conclusions1. The decellularization protocol is effective.The protocol including the combination of Osmotic shock with deionized water, agitation, freezing and thawing, protease digestion, and sodium hydroxide (NaOH)-treating showed complete removal of cells from a porcine corneal split stroma and the desirable components of the ECM were retained at the same time. After air drying, the resultant CAM was transparent with ideal mechanical properties though loss of the normal appearance of a native cornea. Basement membrane and DM was observed on the CAM with highly ordered collagen layers.2. CAM showed very good biocompatibility in vivo and ex vivo. There are no toxic residual chemicals in CAM. Biodegradation rates of the CAM in rabbit capsules were low and the expected lifetime of a transplant was long enough for the matrices regeneration by recruited cells.3. Plain CAM grafts healed rabbits of corneal ulcer The CAM used in LKP or PKP of rabbit corneal ulcer models promoted normal epithelial overgrowth and stratification, keratocytes infiltration and collagen fibrils secretion which led to integration with the host tissue. Finally, the CAM grafts became nearly transparent with normal curvature in LKP group.4. TECE restrained the cornea from angiogenesis. Preimplantation repopulation of the CAM with autologous cells will be protective in wound healing and be inhibitive in corneal angiogenesis. The TECE allowed nerve fibers growing into the implant at the epithelial-stromal interface either.Above all, CAM with excellent performance in vitro (keratocyte seeding) and in vivo (nearly transparence, normal curvance, neurite penetration through the grafts) may be one of the promising scaffolds for tissue-engineered cornea. It supports host cells growth in 3D and integrates with the host tissue. And the plain CAM can be used in the treatment of corneal ulcer. Air-dry CAM for long-term sample storage and then rehydration just before use may be advantageous for production, shipping, and storage.
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