Acellular Porcine Corneal Stroma Research Articles

Small-diameter acellular porcine corneal stroma for peripheral corneal ulceration treatment.

To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma (SAPS) for the treatment of peripheral corneal ulceration (PCU). This retrospective clinical study included 18 patients (18 eyes) with PCU between April 2018 and December 2020. All patients had PCU and underwent lamellar keratoplasty with SAPS. Observation indicators included preoperative and postoperative best-corrected visual acuity (BCVA) and transparency of SAPS. The infection control rate in the surgical eye-lesion area was also calculated. Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU. None of the patients experienced rejection after 6mo (18/18) and 12mo (16/16) of follow-up. The BCVA (0.47±0.30) at the 6mo follow-up after operation was significantly improved compared with the baseline (0.99±0.80), and the difference was statistically significant (Z=-3.415, P<0.05). The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo (Z=0, P=1). With time, the SAPS graft gradually became transparent. At the 6mo (18/18) and 12mo (16/16) follow-up, none of the patients had recurrent corneal infection. SAPS is clinically effective in the treatment of PCU, improving the patient's BCVA and reducing the incidence of rejection after keratoplasty.

Reconstructing auto tissue engineering lamellar cornea with aspartic acid modified acellular porcine corneal stroma and preconditioned limbal stem cell for corneal regeneration

Tissue engineering cornea has shown great clinical potential for cornea reconstruction, but efficient recovery of natural structure and physiological function remains great challenges. In this study, the acellular porcine corneal stroma (APCS) was prepared by a phospholipase A2 decellularization method and further crosslinked with aspartic acid (Asp). The modified APCS-Asp scaffold showed significant increase of hydration degree, ultrastructure regularity, corneal viscoelasticity and anti-degradation ability compared to APCS. Autologous rabbit limbal tissue was pre-treated by tumor necrosis factor-alpha (TNF-α) and collagenase IV, and the pretreated primary limbal stem cells (LSCs) were cultured with embryonic stem cells conditioned medium (ESCM), and LSCs showed 3D cell sphere structure and improved stem cell properties compared to the control group. The auto-tissue engineering lamellar cornea (ATELC) was quickly reconstructed by using peptide hydrogel with a dynamic culture system. With intact and functional epithelial cell layer, the reconstructed ATELC quickly recovered natural optical characteristics 1 week post transplantation in the rabbit lamellar keratoplasty model and satisfying neural regrowth as well as favorable stromal repopulation were observed in the transplanted eyes in the 6 months following up post-surgery. In summary, this study provides a comprehensive optimized reconstruction strategy for ATELC, which maybe similar medical application to natural cornea.

Tissue-Engineered Corneal Endothelial Sheets Using Ultrathin Acellular Porcine Corneal Stroma Substrates for Endothelial Keratoplasty.

Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 μm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 μm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm2 and expressed functional biomarkers Na+/K+-ATPase and zonula occludens. After 14 days, RCECs formed an early stage of Descemet's membrane-like structure by synthesizing collagen IV and laminin. Human corneal endothelial cells were also used to further validate the supportive effect of ultrathin APCS on cells. The resulting constructs were flexible and tough enough to implant into rabbits' anterior chambers through small incisions. TECES adhered to the posterior corneal stroma, and the thickness of cornea gradually reduced to normal after grafting. These results indicate that the ultrathin APCS can serve as a tissue engineering carrier and might be a suitable alternative for endothelial cells expansion in endothelial keratoplasty.

Rejection of Acellular Porcine Corneal Stroma Transplantation During Coronavirus Disease 2019 Pandemic.

:To report 2 successfully managed cases of graft rejection with acellular porcine corneal stroma (APCS) transplantation in patients with fungal corneal ulcer. Two patients were diagnosed with fungal corneal ulcer and received APCS transplantation. Graft rejection developed due to the lost follow-up during the period of coronavirus disease 2019 outbreak. Amniotic membranes transplantation and cauterization of neovascularization was performed, respectively. The graft failure resolved successfully after the procedure. To the best of our knowledge, amniotic membranes transplantation and cauterization of new vessels are the firstly reported in treating APCS graft failure. Amniotic membranes transplantation or cauterization of neovascularization appear to be a safe and costeffective method for treating graft failure.

Hydrogels derived from acellular porcine corneal stroma enhance corneal wound healing

Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. Statement of significanceExtracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Efficacy of Lamellar Keratoplasty with Acellular Porcine Corneal Stroma in Treatment for Infectious Central and Peripheral Corneal Ulcers.

BackgroundThe aim of this study was to investigate the efficacy of acellular porcine corneal stroma (APCS) transplantation in the treatment of infectious central and peripheral corneal ulcers.MethodsA total of 45 patients with infectious corneal ulcers who had undergone lamellar keratoplasty using APCS grafts were included. Among these, 24 had lesions located near the pupil (infectious central corneal ulcer group) and 21 had lesions located in the limbus or around the cornea (infectious peripheral corneal ulcer group). Efficacy was assessed in terms of best-corrected visual acuity, graft transparency, corneal neovascularization, corneal reepithelialization, survival rate, and postoperative complications.ResultsBaseline characteristics showed that poor visual acuity and larger-diameter APCS graft in the infectious central corneal ulcer group were comparable with the infectious peripheral corneal ulcer grouper group (P<0.05). After lamellar keratoplasty using APCS grafts, no obvious differences were observed in aspects of graft transparency, corneal neovascularization, or survival rate (P>0.05). Postoperative complications, ie, delayed corneal epithelial healing, rejection episode, recurrence of infection, and graft melting, were not significantly different between the two groups (P>0.05). Visual acuity in bothgroups had improved significantly at 3 months and 6 months postoperation, respectively.ConclusionAPCS transplantation is safe and efficacious for treating infectious central and peripheral corneal ulcers. Despite its good efficacy, APCS-graft size, implant position, patient indications, and postoperative management should be kept in mind in treatment for infectious corneal ulcers in different locations.

Invited commentary on "Deep Anterior lamellar keratoplasty with Cross-linked Acellular Porcine Corneal Stroma to Manage Fungal Keratitis".

Invited commentary on "Deep Anterior lamellar keratoplasty with Cross-linked Acellular Porcine Corneal Stroma to Manage Fungal Keratitis".

Deep anterior lamellar keratoplasty with cross-linked acellular porcine corneal stroma to manage fungal keratitis.

To evaluate the effects of deep anterior lamellar keratoplasty (DALK) with cross-linked acellular porcine corneal stroma (APCS) and post-operative topical tacrolimus treatment in patients with fungal keratitis. This multicenter prospective study involved 25 cases of fungal keratitis that were treated by DALK with cross-linked APCSs and post-operative topical tacrolimus from December 2013 to November 2014 at the Wenzhou Eye Hospital and the Henan provincial Eye Hospital. Signs of post-operative inflammation, corneal reepithelialization, corneal neovascularization, and graft rejection were assessed, and best corrected visual acuity (BCVA), intraocular pressure (IOP), and APCS graft transparency were monitored for the 12-month follow-up period. All 25 patients underwent DALK without Descemet's membrane perforation. Corneal epithelium recovered completely in 17 patients in the first week, and APCS grafts maintained transparency in 18 patients at 1-year follow-up. The mean BCVA significantly improved from 2.16±0.32 (LogMAR) at baseline to 1.56±0.70 at 1-week (P<.001), 0.95±0.57 at 1-month (P<.001), and 0.70±0.51 at 3-month follow-ups (P<.001). The BCVA kept stable at 6-month and 12-month follow-ups. Post-operative topical tacrolimus alleviated the ciliary injection, except in one case which acute stromal rejection occurred. One patient developed fungal reinfection and underwent penetrating keratoplasty. Graft rejection occurred in three patients. No case was noted with graft splitting, elevated IOP or tacrolimus intolerance. DALK using cross-linked APCS combining topical tacrolimus treatment is safe and effective in managing fungal keratitis. It may ameliorate the shortage of corneal donation globally.

Natural cross-linker-stabilized acellular porcine corneal stroma for lamellar keratoplasty

Acellular porcine corneal stroma (APCS) is a promising alternative to human donor cornea for lamellar keratoplasty (LKP). However, the detergents, enzymes and physical forces used during decellularization unavoidably alter the cornea's extracellular matrix composition and disrupt its ultrastructure, making it less mechanically stable and liable to degradation both in vitro and in vivo. Herein, we systematically analyzed the low biomechanics and easy degradability of APCS in terms of structure and protein composition. Then, we introduced natural cross-linkers, namely proanthocyanidin (PA), epigallocatechin-3-gallate and genipin, to stabilize the APCS that exhibited color variations during crosslinking. Then, we developed a protective crosslinking system by combining cross-linkers with bovine serum albumin (BSA) to reduce color change, maintain transparency and improve the mechanical property of APCS. PA/BSA-crosslinked APCS (PA/BSA-APCS) shows favorable corneal transparency and swelling property; the improved overall and surface corneal biomechanics were comparable to those of human cornea, revealing strong resistance to enzymatic degradation and good biocompatibility. Results from LKP in the rabbit model showed complete re-epithelialization without graft melting, the stitches were scarcely loosened after the operation and more host keratocytes had migrated in PA/BSA-APCS at six months post-operation. Therefore, PA/BSA-APCS could be useful as a corneal substitute for tissue regeneration and the protective crosslinking system could be applicable in other bioengineering fields.

Natural Cross-Linkers Stabilizedacellular Porcine Corneal Stroma for Lamellar Transplantation

Acellular porcine corneal stroma (APCS) is a promising alternative to human donor cornea for lamellar keratoplasty (LKP). However, the detergents, enzymes and physical forces during decellularization unavoidably alter cornea extracellular matrix composition and disrupt its ultrastructure, making it less mechanically stable and liable to degradation in vitro and in vivo. Herein, we systematically analyzed the low biomechanics and easy degradability of APCS from structure and protein composition. Then, we introduced natural cross-linkers to stabilize the APCS that exhibited color variations during cross-linking. Herein, we developed a protective cross-linking system by combining cross-linkers with bovine serum albumin (BSA) to reduce color change, maintain transparency, and improve the mechanical properties of APCS. Proanthocyanidin (PA)/BSA-cross-linked APCS showed favorable corneal transparency and swelling property; the improved overall and surface corneal biomechanics were comparable to those of human donor cornea, revealing good cytocompatibility and strong resistance to enzymatic degradation. Results from corneal micropocket implantation indicated that the PA/BSA-cross-linked APCS gets more corneal stroma cells migrated and LKP in the rabbit model showed complete re-epithelialization without graft melt, and the stitches had scarcely loosened one month after the operation. The proposed method may provide more stable cornea substitutes and be applicable in other bioengineering fields.

Cover page

XenotransplantationVolume 27, Issue 2 ISSUE INFORMATIONFree Access Cover page First published: 15 April 2020 https://doi.org/10.1111/xen.12593AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract The cover image is based on the Original Article Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty by Saiqun Li et al., https://doi.org/10.1111/xen.12566. Volume27, Issue2March 2020 RelatedInformation

Risk factors influencing survival of acellular porcine corneal stroma in infectious keratitis: a prospective clinical study

BackgroundA worldwide lack of donor corneas demands the bioengineered corneas be developed as an alternative. The primary objective of the current study was to evaluate the efficacy of acellular porcine corneal stroma (APCS) transplantation in various types of infectious keratitis and identify risk factors that may increase APCS graft failure.MethodsIn this prospective interventional study, 39 patients with progressive infectious keratitis underwent therapeutic lamellar keratoplasty using APCS and were followed up for 12 months. Data collected for analysis included preoperative characteristics, visual acuity, graft survival and complications. Graft survival was evaluated by the Kaplan–Meier method and compared with the log-rank test.ResultsThe percentage of eyes that had a visual acuity of 20/40 or better increased from 10.3% preoperatively to 51.2% at 12 months postoperatively. Twelve patients (30.8%) experienced graft failure within the follow-up period. The primary reasons given for graft failure was noninfectious graft melting (n = 5), and the other causes included recurrence of primary infection (n = 4) and extensive graft neovascularization (n = 3). No graft rejection was observed during the follow-up period. A higher relative risk (RR) of graft failure was associated with herpetic keratitis (RR = 8.0, P = 0.046) and graft size larger than 8 mm (RR = 6.5, P < 0.001).ConclusionsAPCS transplantation is an alternative treatment option for eyes with medically unresponsive infectious keratitis. Despite the efficacy of therapeutic lamellar keratoplasty with APCS, to achieve a good prognosis, restriction of surgical indications, careful selection of patients and postoperative management must be emphasized.Trial registration Prospective Study of Deep Anterior Lamellar Keratoplasty Using Acellular Porcine Cornea, NCT03105466. Registered 31 August 2016, ClinicalTrails.gov

Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty.

Acellular porcine corneal stroma (APCS) has proven to be a promising alternative to traditional corneal grafts. This prospective case series was conducted to further investigate the healing characteristics of APCS following keratoplasty. Twenty-seven patients undergoing APCS implantation to treat infectious keratitis were included. The patients were followed up for 12months after surgery. The main outcome measures included visual acuity, corneal transparency, graft thickness, and cellular and nerve regeneration. In the operated eyes, the best-corrected visual acuity (BCVA, in logarithm of the minimal angle of resolution [logMAR] units) increased from 1.23±0.95 logMAR before surgery to 0.23±0.18 logMAR at 12months after surgery (P<.001). The contrast sensitivity was still evidently reduced, especially at higher spatial frequencies. Gradual transparency improvement was observed in APCS grafts post-operatively. After implantation, the APCS graft thickness initially increased (day 1=592.41±52.69µm) but then continuously decreased until 3months after surgery (1month=449.26±50.38µm; 3months=359.63±34.14µm, P<.001). Graft reepithelialization was completed within 1week. In the in vivo confocal microscopy scans, host keratocytes began to repopulate the APCS grafts between 3 and 6months post-operatively; subbasal nerve regeneration was only noted in 18.52% (5/27) of the eyes by 12months after surgery. Acellular porcine corneal stroma functions as an effective alternative to human corneal tissue in lamellar keratoplasty. However, APCS is somewhat different from fresh human cornea in term of the post-operative healing process, which warrants the attention of both clinicians and patients.

The lived experiences of patients undergoing acellular porcine corneal stroma transplantation

To explore the lived experiences of patients undergoing acellular porcine corneal stroma (APCS) transplantation, a descriptive, qualitative design was performed. A purposive sample of 13 patients who underwent APCS transplantation to treat progressive infectious keratitis were enrolled in the semi-structured, open-ended interviews. The taped and transcribed interviews were analyzed using a thematic analysis approach. Alterations in the transparency of APCS grafts were accompanied by a gradual improved visual acuity (before surgery: 1.38± 0.91 logMAR; 3mo postoperatively: 0.40±0.24 logMAR, respectively). Accordingly, in terms of lived experiences, the patients generally reported "negative" experiences before the operation and during the early postoperative period, but this was greatly improved 3mo after surgery. Four main themes were derived: anxiety and fear, stigma, lifestyle change, and gratitude and insights. Conclusively, health care professionals should provide holistic care for patients, proactively promoting patients' physical and mental health.

Preparation and Biomechanical Properties of an Acellular Porcine Corneal Stroma.

To construct an acellular porcine corneal stroma (aPCS) as a human corneal stroma alternative and to further explore its biomechanical properties. A combination of DNA-RNA enzymes and ultrasound technology was used to strip the native porcine corneal cells. The microstructure of aPCS was observed by H&E staining, DAPI staining, and α-Gal tests. The mechanical properties were detected by a tension machine. Cytotoxicity of aPCS was measured by the MTT assay. The subcutaneous embedding experiment in rats was also used to detect immunity and degradation. The aPCS was transplanted into the rabbit cornea by lamellar keratoplasty, general observations were made at 3 days, 1 week, 1 month, and 3 months after implantation, respectively. The microstructure and mechanical properties of aPCS were not damaged during the decellularization process. The aPCS extracts had no significant cytotoxicity on human corneal stroma cells. Moreover, the subcutaneous embedding experiment in rats demonstrated that aPCS could not be degraded and induced no immune reaction in and around the transplanted discs. More important is that the aPCS reconstructed normal corneal stroma and maintained corneal transparency and thickness, with almost no neovascularization and inflammation at 3 months after surgery. The aPCS prepared in this study had good biocompatibility, safety, and low antigenicity, which has great potential for corneal disease treatment.

Characterization of Ex Vivo Expanded Oral Mucosal Epithelium Cells on Acellular Porcine Corneal Stroma for Ocular Surface Reconstruction.

Purpose To ex vivo expand oral mucosal epithelium cells (OMECs) on acellular porcine corneal stroma (APCS) without using feeder cells and serum and to compare the morphologic and phenotypic characteristics of cultured oral cells on APCS to those of cells on deluded human amniotic membrane (HAM). Methods SD rat oral mucosal biopsies were cultured on APCS and HAM. Reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to analyze the characterization of stem cells and epithelial differentiation of the outgrowth products. Results Stratified and optimal transplantable OMECs were obtained after being cultured three to four weeks. Both RT-PCR and immunohistochemistry showed that cultured OMECs expressed markers of epithelial differentiation cytokeratin K3 and epithelial stem cell markers of p63 and ABCG2. Conclusions OMECs can be successfully cultured on APCS without using xenobiotic feeder cells and serum. Characterization showed that these sheets retain the morphologic and phenotypic characteristics of OMECs within differentiated cells and stem cells. The optimal transplantable sheets can prove to be particularly beneficial to both bilateral limbal stem cell deficiency and deep corneal lesions.

Formation of cell sheet on acellular porcine corneal stroma with human corneal endothelial cells cocultured by mouse embryonic stem cell conditioned medium

Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro, and acellular porcine corneal stroma (APCS) was a good saffold material.However, whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied. Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet. Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured, and the cultured medium was centrifuged and mixed with 75% human corneal endothelium medium (CEM) at a proportion of 1∶3 to prepare the 25% ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane, and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution, and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1) and Na+ -K+ -ATPase in the cell sheet were detected by immunofluorescence staining. Results The second generation of HCECs cultured with 25% ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent, and no corneal cells were seen, the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+ -K+ -ATPase were positively expressed on the HCECs sheet. Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation. Key words: Corneal stroma/transplantation; Endothelial cells, corneal/cytology; Humans; Tissue engineering/methods; Tissue scaffolds; Culture media, conditioned; Embryonic stem cells, mouse; Acellular corneal stroma, porcine

Corneal Stroma Regeneration with Acellular Corneal Stroma Sheets and Keratocytes in a Rabbit Model.

Acellular corneal stroma matrix has been used for corneal stroma engineering. However, because of its compact tissue structure, regrowth of keratocytes into the scaffold is difficult. Previously, we developed a sandwich model for cartilage engineering using acellular cartilage sheets. In the present study, we tested this model for corneal stroma regeneration using acellular porcine corneal stroma (APCS) sheets and keratocytes. Porcine corneas were decellularized by NaCl treatment, and the APCS was cut into 20-μm-thick sheets. A rabbit corneal stroma defect model was created by lamellar keratoplasty and repaired by transplantation of five pieces of APCS sheets with keratocytes. Six months after transplantation, transparent corneas were present in the experimental group, which were confirmed by anterior segment optical coherence tomography examination and transmittance examination. The biomechanical properties in the experimental group were similar to those of normal cornea. Histological analyses showed an even distribution of keratocytes and well-oriented matrix in the stroma layer in the experimental group. Together, these results demonstrated that the sandwich model using acellular corneal stroma sheets and keratocytes could be potentially useful for corneal stroma regeneration.

Lamellar Keratoplasty Treatment of Fungal Corneal Ulcers With Acellular Porcine Corneal Stroma

The fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To explore an alternative to donor corneas, we conducted a clinical study in which implanted acellular porcine corneal stromas (APCSs) replaced the pathologic anterior corneas in 47 patients who had experienced fungal corneal infections. Subsequently, we demonstrated the safety and efficacy of APCSs in human keratoplasty for a minimum follow-up period of 6 months, during which time no recurrence of infection was observed. All corneal ulcers healed with the return of neovascularization. In addition, our results indicated that epithelialization occurred in all APCS grafts except four grafts; for these four, the grafts dissolved to varying degrees. Furthermore, most porcine grafts (n = 41) gradually became transparent without rejection, and an improvement of more than two lines in best corrected visual acuity (BCVA) was achieved in 34 eyes (∼72%). Finally, no patients showed any severe adverse reaction or any significant change in postoperative systemic safety indicators. Thus, we concluded that APCS grafts are safe and efficacious during lamellar keratoplasty in treating corneal fungal ulcers and potentially for other clinical diseases.

Reconstruction of Highly Proliferative Auto-Tissue-Engineered Lamellar Cornea Enhanced by Embryonic Stem Cell.

To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0 × 10(6) corneal epithelial cells (CECs) were combined with 3 × 10(5) mouse embryonic stem cells (ESCs) pretransfected with the HSV-tk gene (CECs + ESCs-TK group), and 3.3×10(6) corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (four to five layers) formed in the CECs + ESCs-TK group compared with that observed in the CECs group (two to three layers). More stem/progenitor cell (K3 -, p63+, ABCG2+, and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs + ESCs-TK group compared with the CECs group using immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs + ESCs-TK group (16.18% ± 3.98%, 28.45 ± 2.03 h) and CECs group (11.96% ± 2.60%, 36.3 ± 1.15 h). In a rabbit lamellar transplantation model, the CECs + ESCs-TK group had better epithelial barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering.