Decellularized Porcine Cornea Research Articles

Preclinical evaluation of the safety and effectiveness of a new bioartificial cornea.

Cross-linking agents are frequently used to restore corneal properties after decellularization, and it is especially important to select an appropriate method to avoid excessive cross-linking. In addition, how to promote wound healing and how to improve scar formation require further investigation. To ensure the safety and efficacy of animal-derived products, we designed bioartificial corneas (BACs) according to the criteria for Class III medical devices. Our BACs do not require cross-linking agents and increase mechanical strength via self-cross-linking of aldehyde-modified hyaluronic acid (AHA) and carboxymethyl chitosan (CMC) on the surface of decellularized porcine corneas (DPCs). The results showed that the BACs had good biocompatibility and transparency, and the modification enhanced their antibacterial and anti-inflammatory properties in vitro. Preclinical animal studies showed that the BACs can rapidly regenerate the epithelium and restore vision within a month. After 3 months, the BACs were gradually filled with epithelial, stromal, and neuronal cells, and after 6 months, their transparency and histology were almost normal. In addition, side effects such as corneal neovascularization, conjunctival hyperemia, and ciliary body hyperemia rarely occur in vivo. Therefore, these BACs show promise for clinical application for the treatment of infectious corneal ulcers and as a temporary covering for corneal perforations to achieve the more time.

A Light-Curable and Tunable Extracellular Matrix Hydrogel for In Situ Suture-Free Corneal Repair.

Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative bio-integrating materials for in-situ repair and replacement of corneal tissue. Here, we introduce Light-curable COrnea Matrix (LC-COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un-denatured collagen and sulfated glycosaminoglycans. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross-linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. Cross-linked LC-COMatrix strongly adheres to human corneas ex vivo and effectively closes full-thickness corneal perforations with tissue loss. Likewise, in vivo, LC-COMatrix seals large corneal perforations, replaces partial-corneal stromal defects and bio-integrates into the tissue in rabbit models. LC-COMatrix is a natural ready-to-apply bio-integrating adhesive that is representative of native corneal matrix with potential applications in corneal and ocular surgeries.

Comparative evaluation of decellularized porcine cornea and small intestine submucosa for repairing of corneal defect in rabbit model

A study was conducted from December 2013 to March 2014 to compare the healing potential of decellularized porcine cornea (DPC) and small intestinal submucosa (DPSIS) for repairing of partial thickness corneal defect in rabbit model. Eighteen clinically healthy adult New Zealand White rabbits of either sex were divided into two groups. The porcine corneas and small intestinal submucosa were collected from abattoir and made acellular by treating with 1% sodium dodecyl sulphate. Under general anesthesia, a 5 mm diameter lamellar keratectomy wound was created over the peripheral cornea of rabbits under operating microscope in both the groups. The defect was repaired with DPC in Gr A and with DPSIS in Gr B. Corneal clarity was better in Gr A with less stromal scarring and incited less immune response than those of Gr B. Histopathological findings confirmed increased corneal neovascularization and fibroblast proliferation in Gr B than Gr A. On the basis of clinical, pathological and scanning electron microscopic examinations, DPC showed better corneal healing than DPSIS for repairing of corneal defect in rabbits.

The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

In-situ porcine corneal matrix hydrogel as ocular surface bandage

PurposeBioactive substrates can be used therapeutically to enhance wound healing. Here, we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects. MethodsCOMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1β, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem. ResultsIn vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P < 0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P < 0.01). There was significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control (P < 0.05). ConclusionsCOMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing.

Fabrication, Rheological, and Compositional Characterization of Thermoresponsive Hydrogel from Cornea.

Fabricating thermoresponsive hydrogels from decellularized tissues is a trending and promising approach to develop novel biomaterials for tissue engineering and therapeutic purposes. There are differences in the characteristics of the produced hydrogels related to the source tissue as well as the decellularization and solubilization protocols used. Detailed characterization of the hydrogels will support the efforts to optimize their application as biomaterials for tissue engineering and therapeutics. Here, we describe an optimized method for fabricating an in situ thermoresponsive hydrogel from decellularized porcine cornea extracellular matrix (COMatrix), and provide a detailed characterization of its structure, thermoresponsive rheological behavior (heat-induced sol-gel transition), as well as exploring its protein composition using proteomics. COMatrix forms a transparent gel (10-min time to gelation) after in situ curing with heat, characterized by alteration in light absorbance and rheological indexes. The rheological characterization of heat-formed COMatrix gel shows similar behavior to common biomaterials utilized in tissue engineering. The fibrillar structure of COMatrix gel was observed by scanning electron microscopy showing that the density of fibers attenuates in lower concentrations. Mass spectrometry-based proteomic analysis revealed that COMatrix hydrogel is rich in proteins with known regenerative properties such as lumican, keratocan, and laminins in addition to structural collagen proteins (Data is available via ProteomeXchange with identifier PXD020606). COMatrix hydrogel is a naturally driven biomaterial with favorable biomechanical properties and protein content with potential application as a therapeutic biomaterial in ocular regeneration and tissue engineering. Impact statement Fabrication and application of decellularized porcine corneal extracellular matrix is an emerging approach for corneal tissue engineering and regeneration. There are several protocols for decellularization of porcine cornea with various efficiencies. Here, we are presenting an optimized protocol for decellularization of porcine cornea followed by fabrication of a thermoresponsive hydrogel from the decellularized cornea matrix. Moreover, the fabricated hydrogel was rheologically and compositionally characterized as crucial features to be employed for further application of this hydrogel in corneal tissue engineering and regeneration.

Clinical Characteristics and Outcomes of Short-term Repeat Corneal Transplantation

ABSTRACT Purpose To explore the primary indications, direct causes and graft survival of short-term repeat keratoplasty. Methods An analytical cross-sectional study. Results The primary indications for keratoplasty were infectious keratitis (67.9%) and immune keratopathy (12.8%). The direct causes of graft failure were infectious ulcer (43.6%) and recurrence (30.8%). Pre-operative hypopyon (P = 0.017, HR = 1.757) and use of decellularized porcine corneas as grafts (P = 0.005, HR = 2.690) were independent risk factors for graft failure. The Kaplan–Meier analysis revealed that the 1-year survival rate of 88 repeat grafts was 85.2%, and the 5-year survival rate was 79.2%. The survival rates of the repeat grafts were significantly higher than those of the first grafts (P < 0.001). Conclusion Infectious keratitis is the most common indication of short-term graft failure and is often failed by infectious ulcer and keratitis recurrence. While the decellularized porcine cornea is a promising application in developing countries, certain difficulties, such as graft melting, remain unresolved.

Hybrid natural hydrogels integrated with voriconazole-loaded microspheres for ocular antifungal applications.

Fungal keratitis is a major threat to ocular morbidity and blindness. The therapeutic efficacy of eye drops against fungal keratitis is limited by their poor bioavailability, especially in patients with corneal stromal ulcers that lead to tissue defects. Therefore, intervention measures that can control fungal infection while promoting tissue regeneration are desirable. Herein, we designed and fabricated natural antifungal hydrogels that comprise a decellularized porcine cornea (DPC), gelatin and microspheres (MCs) containing voriconazole (Vor) for the management of fungal keratitis with focal corneal stromal defects. The size and structure of the Vor-loaded MCs were characterized by scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The antifungal drug Vor showed that continuous release from the hybrid hydrogel system for up to 7 days and that MCs loading did not affect the mechanical properties, in vitro release profile or cytocompatibility of the hybrid hydrogel. Moreover, the Vor-loaded hydrogels exhibited excellent antifungal properties against F. solani and A. fumigatus. The efficiency of the natural antifungal hydrogel was evaluated by an ex vivo infectious rabbit corneal defect model. After 24 h, the number of fungal colony-forming units (CFU) significantly decreased in antifungal hydrogel-treated corneal tissue compared with that in non-treated corneas and corneas treated with hydrogels without Vor. The above results demonstrated that this natural hydrogel-based drug delivery system holds great promise for preventing fungal keratitis infection while promoting focal corneal stromal regeneration. Additionally, natural hybrid hydrogels might be a candidate material for use with various drugs in the effective treatment of many other ocular diseases.

PREPARATION OF ULTRA-THIN DESCEMET’S MEMBRANE GRAFT USING DECELLULARIZED PORCINE CORNEA

A corneal endothelial layer which is the bottom layer of cornea perform a role to maintain a clarity of cornea. When the number of corneal endothelial cell decrease due to aging or hereditary condition, it is hard to restore because its mitosis capacity is limited. As fundamental treatment, a corneal transplantation is conducted widely. However, there are lots of transplantation waiting lists through a shortage of donor cornea. In this study, we optimize a protocol to prepare ultra-thin descemet’s membrane (DM) graft using decellularized porcine cornea. Using fresh porcine eyeball, a cornea is excised including sclera and conjunctiva. With biopsy punch, endothelium, DM including stroma are partially incised. Next, the cornea was decellularized in 0.1% SDS for 5 hours. Then, a graft is detached DM including stroma. Again, the graft is decelluarized in 0.1% SDS for 10hour at room temperature. The graft was dried in dry oven at 40 celsius overnight. The induced pluripotent stem cell derived corneal endothelial cells was reseeded. Finally, a thickness of dried acellular DM graft was 57.6±7.92um and recelluarized wet graft was 99.2±5.98um. The clarity of recellularized DM graft was 91.8±2.38%. Although the future in-vivo trials would be warranted, tissue engineering DM graft using porcine decellularized corneas might be efficient tools to resolve refractory corneal disease. This work was supported by the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017K1A4A3014959) and NRF-2018R1D1A1B07050014. This work was also supported by the research grant of the Chungbuk National University in 2017.

Decellularized porcine cornea-derived hydrogels for the regeneration of epithelium and stroma in focal corneal defects

PurposeHydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable and transparent hydrogel for the regeneration of epithelium and stroma in focal corneal defects. MethodsThe DPC-derived hydrogel was prepared with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate/N-hydroxysuccinimide (CMC/NHS) as cross-linkers. The characteristics of the hydrogel were analyzed and its cytocompatibility was assessed by Live/Dead and Cell Counting Kit (CCK)-8 assays. Immunofluorescence staining, quantitative PCR and Western blot analyses were performed to assess the relative protein and gene expression in corneal fibroblasts on hydrogel. The safety and efficiency of the hydrogel for repairing focal corneal defects in rabbit were measured by slit-lamp, anterior segment optical coherence tomography (AS-OCT), confocal microscopy and histological analyses. ResultsThe DPC-derived hydrogel cross-linked with CMC/NHS assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected onto rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface on the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of the corneal epithelium and stroma was restored at 12 weeks after surgery without significant inflammation or scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium. ConclusionsThe DPC-derived hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.

Corneal xenotransplantation: Where are we standing?

The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine.

Decellularized Porcine Cornea-Based Hydrogels for Regeneration of Corneal Epithelium and Stroma

Hydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable hydrogel, characterized the biocompatibility and transparency, and evaluated the use to repair corneal stromal defect in rabbit. Upon cross-linking with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate (CMC), the DPC-based hydrogel assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected on rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface upon the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of corneal epithelium and stroma was restored at 12 weeks postoperatively without significant inflammation and scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium. Considering to the advantages of low cost and ease of production and use, the DPC-based hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.

Decellularization methods for developing porcine corneal xenografts and future perspectives.

Corneal transplantation is the only option to cure corneal opacities. However, there is an imbalance between supply and demand of corneal tissues in the world. To solve the problem of corneal shortage, corneal xenotransplantation studies have been implemented in the past years using porcine corneas. The corneal xenografts could come from (a) wild-type pigs, (b) genetically engineered pigs, (c) decellularized porcine corneas, and (d) decellularized porcine corneas that are recellularized with human corneal cells, eventually with patients' own cells, as in all type of xenografts. All approaches except, the former would reduce or mitigate recipient immune responses. Although several techniques in decellularization have been reported, there is still no standardized protocol for the complete decellularization of corneal tissue. Herein, we reviewed different decellularization methods for porcine corneas based on the mechanism of action, decellularization efficacy, biocompatibility, and the undesirable effects on corneal ultrastructure. We compared 9 decellularization methods including: (a) sodium dodecyl sulfate, (b) triton x-100, (c) hypertonic saline, (d) human serum with electrophoresis, (e) high hydrostatic pressure, (f) freeze-thaw, (h) nitrogen gas, (h) phospholipase A2 , and (i) glycerol with chemical crosslinking methods. It appears that combined methods could be more useful to perform efficient corneal decellularization.

Protectively Decellularized Porcine Cornea versus Human Donor Cornea for Lamellar Transplantation

AbstractDecellularization can reduce the immune barrier of xenotransplantation, but tissue swelling‐caused structural and functional damage remains unsolved, including corneal transparency loss after decellularization. Here, a protective decellularization strategy is developed for the preparation of decellularized porcine cornea (DPC), in which corneas are treated by detergent and endonuclease in the protective medium with 50 mmHg colloid osmotic pressure. A nonrandomized open‐label trial is conducted to evaluate the clinical outcome of lamellar transplantation with DPC versus human donor cornea (HDC) as grafts. Through the protective corneal decellularization, major xenoantigen DNA and α‐gal are efficiently removed, while corneal original structural and transparency characteristics are preserved. Among the 23 patients with DPC transplantation for 12 months, 22 grafts survive without ulcer recurrence or immune rejection, 1 graft demonstrate melting. Compared with HDC grafts, DPC grafts showed early suture loosing, but no complication is observed with timely removal. The epithelial regeneration rate, graft transparency restoration, best‐corrected visual acuity improvement, and mechanical properties achieve equivalent levels compared with that of HDC grafts. Collectively, the results suggest that the porcine cornea through protective decellularization may provide an effective “off‐the‐shelf” substitute of globally‐shortened human donor tissue for lamellar transplantation.

Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts

To address the shortcomings associated with corneal transplants, substantial efforts have been focused on developing new modalities such as xenotransplantion. Xenogeneic corneas are anatomically and biomechanically similar to the human cornea, yet their applications require prior decellularization to remove the antigenic components to avoid rejection. In the context of bringing decellularized corneas into clinical use, sterilization is a crucial step that determines the success of the transplantation. Well-standardized sterilization methods, such as gamma irradiation (GI), have been applied to decellularized porcine corneas (DPC) to avoid graft-associated infections in human recipients. However, little is known about the effect of GI on decellularized corneal xenografts. Here, we evaluated the radiation effect on the ultrastructure, optical, mechanical and biological properties of DPC. Transmission electron microscopy revealed that gamma irradiated decellularized porcine cornea (G-DPC) preserved its structural integrity. Moreover, the radiation did not reduce the optical properties of the tissue. Neither DPC nor G-DPC led to further activation of complement system compared to native porcine cornea when exposed to plasma. Although, DPC were mechanically comparable to the native tissue, GI increased the mechanical strength, tissue hydrophobicity and resistance to enzymatic degradation. Despite these changes, human corneal epithelial, stromal, endothelial and hybrid neuroblastoma cells grew and differentiated on DPC and G-DPC. Thus, GI may achieve effective tissue sterilization without affecting critical properties that are essential for corneal transplant survival.

Rapid porcine corneal decellularization through the use of sodium N-lauroyl glutamate and supernuclease

Corneal decellularization represents a promising alternative source of human donor with global shortage. Multiple methods have been developed for the preparation of decellularized porcine corneal stroma. However, most strategies relied on long-time treatment to facilitate the entry of detergents or nucleases, which may cause irreversible ultrastructural damage. Here, we developed a rapid decellularization method for porcine corneal stroma through the combined mild detergent sodium N-lauroyl glutamate (SLG) and supernuclease. Compared with traditional methods, the novel decellularization method allowed the efficient removal of xenoantigen DNA within 3 h, while retaining the ultrastructure, transparency, and mechanical properties of porcine corneas. When transplanted in rabbit model for 1 month, the decellularized porcine corneal grafts presented favorable transparency and biocompatibility without immune rejection. Therefore, the combined use of detergent SLG and supernuclease may serve as a promising method for the clinical use of decellularized porcine cornea.

Decellularized porcine cornea alters the extent of complement activation and cytokine production when in contact with human blood

Decellularized porcine cornea alters the extent of complement activation and cytokine production when in contact with human blood

Evaluation of bio-engineered corneal scaffold for the repair of corneal defect in rabbit model

Clinically healthy adult New Zealand white rabbits (27) of either sex, were randomly divided into three groups (A, B and C) having 9 animals each. Porcine cornea was made acellular by treating it with 1% sodium dodecyl sulphate (SDS). Rabbit bone marrow derived mesenchymal stem cells were seeded over this acellular matrix. A 5mm diameter lamellar keratectomy wound was created over the peripheral cornea of rabbits in all the 3 groups. Ingroup A, the corneal defect was managed by simple tarsorrhaphy without any graft and is treated as control. In group B, defect was repaired with decellularized porcine cornea and in group C, corneal defect was repaired with r-MSC seeded decellularized cornea. On the basis of clinical, pathological and scanning electron microscopic examinations, mesenchymal stem cell seeded corneal scaffold showed better healing and vision when compared tononseeded scaffolds. Cell seeded corneal matrix was found to be an alternative to conventional means of surgical management of corneal ulcer.

Preparation of acellular scaffold for corneal tissue engineering by supercritical carbon dioxide extraction technology.

We decellularized the porcine cornea using SCCO2 extraction technology and investigated the characteristics, mechanical properties, and biocompatibility of the decellularized porcine cornea by lamellar keratoplasty in rabbits. To the best of our knowledge, this is the first report describing the use of SCCO2 extraction technology for preparation of acellular corneal scaffold. We proved that the cellular components of porcine corneas had been efficiently removed, and the biomechanical properties of the scaffold were well preserved by SCCO2 extraction technology. SCCO2-treated corneas maintained optical transparency and exhibited appropriate strength to withstand surgical procedures. In vivo, the transplanted corneas showed no evidence of immunological reactions and exhibited good biocompatibility and long-term stability. Our results suggested that the APCs developed by SCCO2 extraction technology could be an ideal and useful scaffold for corneal replacement and corneal tissue engineering.

Исследование эффективности интраламеллярной кератопластики эквивалента стромы роговицы на основе децеллюляризированной роговицы свиньи в эксперименте

Исследование эффективности интраламеллярной кератопластики эквивалента стромы роговицы на основе децеллюляризированной роговицы свиньи в эксперименте