Donut-shaped Corneal Allogeneic Intrastromal Segment as an Alternative to Deep Anterior Lamellar Keratoplasty in Advanced Keratoconus.

PURPOSE: The aim of this study was to describe a new technique of selective corneal stromal transplantation for keratoconus treatment, donut-shaped CAIRS (corneal allogeneic intrastromal ring segment). METHODS: A donut-shaped corneal graft is obtained using a double-bladed trephine. Descemet membrane, endothelium, and epithelium are all removed from the graft. Only stromal tissue is transplanted. A wide 360-degree intrastromal tunnel is created using the femtosecond laser, with a 30-degree angulation. The diameter is from 5.4 mm to 8 mm optical zone. After dehydration, the corneal graft is inserted into the tunnel. We report the clinical and tomographic outcomes after the procedure in 3 patients. RESULTS: The mean follow-up time after donut-shaped CAIRS was 6.01 ± 1.02 months. In case 1, best spectacle-corrected visual acuity improved from 20/150 to 20/40. In case 2, it improved from 20/400 to 20/40, and in case 3 from 20/200 to 20/40. The mean preoperative K was 57.3 ± 4.5 D and reduced to 44.2 ± 2.5 D after donut-shaped CAIRS. The mean spherical equivalent decreased from -9.8 ± 3.2 preoperatively to -3.2 ± 2.2 postoperatively. No intraoperative or postoperative complications were observed. Anterior segment OCT showed a mid-stroma implant, fusiform in shape, equidistant from the epithelium and endothelium. CONCLUSIONS: Donut-shaped CAIRS is a variation of the traditional CAIRS technique and showed to be an alternative option for keratoconus treatment, especially in moderate to advanced cases with a central clear cornea without scars. The technique is minimally invasive, and the visual axis remains untouched.

Electron beam-irradiated donor cornea for on-demand lenticule implantation to treat corneal diseases and refractive error.

The cornea is the major contributor to the refractive power of the eye, and corneal diseases are a leading cause of reversible blindness. The main treatment for advanced corneal disease is keratoplasty: allograft transplantation of the cornea. Examples include lenticule implantation to treat corneal disorders (e.g. keratoconus) or correct refractive errors. These procedures are limited by the shelf-life of the corneal tissue, which must be discarded within 2-4 weeks. Electron-beam irradiation is an emerging sterilisation technique, which extends this shelf life to 2 years. Here, we produced lenticules from fresh and electron-beam (E-beam) irradiated corneas to establish a new source of tissue for lenticule implantation. In vitro, in vivo, and ex vivo experiments were conducted to compare fresh and E-beam-irradiated lenticules. Results were similar in terms of cutting accuracy, ultrastructure, optical transparency, ease of extraction and transplantation, resilience to mechanical handling, biocompatibility, and post-transplant wound healing process. Two main differences were noted. First, ∼59% reduction of glycosaminoglycans resulted in greater compression of E-beam-irradiated lenticules post-transplant, likely due to reduced corneal hydration-this appeared to affect keratometry after implantation. Cutting a thicker lenticule would be required to ameliorate the difference in refraction. Second, E-beam-sterilised lenticules exhibited lower Young's modulus which may indicate greater care with handling, although no damage or perforation was caused in our procedures. In summary, E-beam-irradiated corneas are a viable source of tissue for stromal lenticules, and may facilitate on-demand lenticule implantation to treat a wide range of corneal diseases. Our study suggested that its applications in human patients are warranted. STATEMENT OF SIGNIFICANCE: Corneal blindness affects over six million patients worldwide. For patients requiring corneal transplantation, current cadaver-based procedures are limited by the short shelf-life of donor tissue. Electron-beam (E-beam) sterilisation extends this shelf-life from weeks to years but there are few published studies of its use. We demonstrated that E-beam-irradiated corneas are a viable source of lenticules for implantation. We conducted in vitro, in vivo, and ex vivo comparisons of E-beam and fresh corneal lenticules. The only differences exhibited by E-beam-treated lenticules were reduced expression of glycosaminoglycans, resulting in greater tissue compression and lower refraction suggesting that a thicker cut is required to achieve the same optical and refractive outcome; and lower Young's modulus indicating extra care with handling.

SMILE lenticule versus amniotic membrane graft (AMG) augmented with platelet-rich plasma (PRP) for the treatment of perforated corneal ulcer.

PURPOSE: To evaluate the safety and efficacy of stromal lenticule obtained from small-incision lenticule extraction (SMILE) surgery versus amniotic membrane graft (AMG) augmented with platelet-rich plasma (PRP) for the treatment of perforated corneal ulcers and compare the results between the two groups. PATIENTS AND METHODS: This is a comparative retrospective study that included 40 eyes with medium-sized corneal perforations, which were classified into two equal groups of 20 eyes each; group (A) was treated with SMILE lenticule graft and group (B) was treated with AMG augmented with PRP. Pre- and postoperative evaluations were carried out using both slit-lamp (SL) examination and anterior segment optical coherence tomography (AS-OCT), including closure of perforation, complete healing, and best corrected visual acuity (BCVA). RESULTS: Complete closure of the perforation was achieved in both groups. However, healing was faster in the SMILE lenticule group than in the AMG with PRP group (P < 0.05). Complete healing was achieved in both groups: 100% in SMILE lenticule group and 95% in AMG with PRP group (P > 0.05). Both groups had few insignificant complications (30% in each), which were managed. CONCLUSION: Both methods achieved adequate healing of corneal perforations within few weeks without significant complications. However, the stromal lenticule obtained from small-incision lenticule extraction (SMILE) surgery tended to be safer with faster healing than AMG with PRP.

Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept.

PURPOSE: To demonstrate the suitability of using decellularized SMILE (Small-incision Lenticule Extraction) lenticules for culturing and transplanting the corneal endothelium (CE). METHODS: The SMILE lenticules, obtained during refractive surgery, were decellularized by incubating in CE culture medium and fetal bovine serum. Decellularization was confirmed by hematoxylin and eosin staining, DAPI staining, and gel electrophoresis. The amount of DNA per milligram of dry tissue weight was calculated to quantify the residual nuclear content. The transparency of the decellularized lenticules was determined by calculating the modulation transfer function. Immunostaining for stromal collagens and glycosaminoglycan was performed using specific antibodies. Engineered tissue was constructed by culturing the CE cells on lenticules and staining for ZO-1, Na/K ATPase, and N-cadherin. The functionality of the engineered tissues was assessed by transplanting them onto edematous human donor corneas and perfusing for 10 days ex-vivo. RESULTS: The residual DNA per milligram of dry tissue weight was found to be significantly reduced (p < 0.0001) in serum (0.255 µg/mg) and Opti-MEM (0.140 µg/mg) when compared to fresh lenticules (3.9 µg/mg). Decellularization did not alter the arrangement of the collagen fibers or the transparency of the lenticules. CE cells attached and matured to express ZO-1, Na/K ATPase, and N-cadherin at two weeks after seeding. The engineered tissue upon transplantation significantly reduced the corneal edema (p < 0.05) and the transplanted cells remained intact on the SMILE lenticule post-transplantation. CONCLUSION: This study demonstrates the suitability of using SMILE lenticules decellularized using a simple, chemical-free method for engineering the corneal endothelium for transplantation.

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

ABSTRACT: 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.

Effect of corneal stromal lenticule customization on neurite distribution and excitatory property.

Introduction: Refractive stromal lenticules from Small Incision Lenticule Extraction (SMILE), though usually discarded, hold a potential for various ophthalmic applications, including refractive correction, stromal volume expansion, and biomechanical strengthening of the cornea. Objectives: To investigate the effect of lenticule customization on lenticule neurite length profile and the excitatory response (calcium signaling) and the potential of reinnervation. Methods: Human and porcine stromal lenticules were treated by (1) excimer laser reshaping, (2) ultraviolet A-riboflavin crosslinking (CXL), and (3) decellularization by sodium dodecyl sulfate (SDS), respectively. The overall neurite scaffold immuno-positive to TuJ1 (neuron-specific class III ß-tubulin) expression and population of active neurite fragments with calcium response revealed by L-glutamate-induced Fluo-4-acetoxymethyl ester reaction were captured by wide-field laser-scanning confocal microscopy, followed by z-stack image construction. The NeuronJ plugin was used to measure neurite lengths for TuJ1 (NL-TuJ1) and calcium signal (NL-Ca). Reinnervation of lenticules was examined by the ex vivo grafting of chick dorsal root ganglia (DRG) to the decellularized human lenticules. Differences between groups and controls were analyzed with ANOVA and Mann-Whitney U test. Results: The customization methods significantly eliminated neurites inside the lenticules. NL-TuJ1 was significantly reduced by 84% after excimer laser reshaping, 54% after CXL, and 96% after decellularization. The neurite remnants from reshaping and CXL exhibited calcium signaling, indicative of residual excitatory response. Re-innervation occurred in the decellularized lenticules upon stimulation of the grafted chick embryo DRG with nerve growth factor (NGF 2.5S). Conclusion: All of the lenticule customization procedures reduced lenticule neurites, but the residual neurites still showed excitatory potential. Even though these neurite remnants seemed minimal, they could be advantageous to reinnervation with axon growth and guidance after lenticule reimplantation for refractive and volume restoration of the 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.

Ten-Year Follow-up of Lamellar Keratoplasty Treatment With Acellular Porcine Corneal Stroma: A Case Report.

PURPOSE: To report a 10-year follow-up case of the first lamellar keratoplasty treatment with acellular porcine corneal stroma (APCS). METHODS: A 62-year-old woman was diagnosed with a fungal corneal ulcer and received lamellar keratoplasty treatment with APCS in 2010. The 10-year follow-up results were evaluated by slit lamp biomicroscopy, anterior segment optical coherence tomography, in vivo confocal microscopy, and corneal biomechanics analysis. RESULTS: The APCS graft maintained good biocompatibility and physical properties in transparency, stromal regeneration, elasticity, and deformation resistance. However, some disadvantages were observed, including a protracted course to eventual clearing, a decreased thickness, corneal depositions, sparsely distributed neural fibers, and low stiffness. CONCLUSIONS: This case indicated that APCS remains stable over a 10-year follow-up period. APCS can serve as a functional stromal surrogate where donor human corneal tissue is unavailable.

Corneal calcification of acellular porcine corneal stroma following lamellar keratoplasty.

PURPOSE: To describe the corneal calcification of acellular porcine corneal stroma (APCS) following lamellar keratoplasty (LKP) and identify risk factors. METHODS: Two cases of APCS calcification were evaluated by slit-lamp photography and anterior segment optical coherence tomography (AS-OCT). von Kossa staining and scanning electron microscope/energy-dispersive spectrometry (SEM/EDS) were performed on pathologic tissue. Associated graft and postoperative risk factors were analysed. Acellular porcine corneal stroma (APCS) cleanliness and element content after rinsing with sterilized water were observed by SEM/EDS and inductively coupled plasma mass spectrometry. Calcium metabolism-related proteins were analysed by protein mass spectrometry. Corneal epithelial defects and postoperative medications were reviewed. RESULTS: Two cases of APCS calcification occurred at 23 and 22 days postoperatively. Anterior segment optical coherence tomography (AS-OCT) and von Kossa staining demonstrated calcium deposition in the superficial stroma composed of calcium, phosphorus and oxygen conforming to the Ca/P ratio of hydroxyapatite. Phosphate crystals were present on the APCS surface and decreased with number of rinsing times. The phosphorus content of APCS was minimal after rinsing 10 times and avoiding excessive corneal swelling. Calcium metabolism-related proteins were downregulated in APCS. Patients with corneal calcification had 1-week postoperative corneal epithelial defects and were treated with three types of phosphorous eyedrops. CONCLUSIONS: Acellular porcine corneal stroma (APCS) calcification occurs in the superficial corneal stroma about 1 month after LKP. The application of AS-OCT, von Kossa staining and SEM/EDS provides a basis for the clinical and pathological diagnosis of corneal calcification. The associated risk factors were mainly high phosphorus content and downregulated calcium metabolism-related proteins in APCS. Postoperative epithelial defects, inflammation and use of phosphorous eyedrops may promote corneal calcification.

The use of acellular porcine corneal stroma xenograft (BioCorneaVet™ ) for the treatment of deep stromal and full thickness corneal defects: A retrospective study of 40 cases (2019-2021).

OBJECTIVE: To document the effectiveness and outcome of corneal grafting using acellular porcine corneal stroma (APCS) for veterinary use (BioCorneaVet™ ) to restore corneal integrity in dogs. METHODS: A review of medical records of patients that underwent keratoplasty with APCS graft to repair deep corneal defects, descemetoceles, and perforations between 2019 and 2021 was carried out. Only animals with intact dazzle reflex, consensual PLR before the surgery and a minimum follow-up of four weeks were considered for the study, with forty dogs (1 eye each) meeting the inclusion criteria. RESULTS: Brachycephalic breeds were the most frequently represented, and 20 right eyes and 20 left eyes were affected with 25 perforations, 8 descemetoceles, and 9 deep stromal defects (1 eye had both perforation and descemetocele). Most of the patients had concurrent ocular diseases or had undergone previous surgery on the other eye. Two different thickness of xenograft was used (300 or 450 µm), and the diameter ranged from 3 to 10 mm. Postoperative complications included mild to severe corneal vascularization, partial dehiscence, melting, and glaucoma. Follow-up time ranged from 28 to 797 days (mean: 233 days). Ocular integrity was maintained in 37/40 cases (92.5%), and vision was preserved in 36 cases (90%). CONCLUSION: The use of APCS (BioCorneaVet™ ) is an effective surgical treatment for deep stromal defects, descemetocele, and perforations in dogs, providing a good tectonic support and preserving anatomical integrity and vision. The cosmetic appearance was considered good in all the cases and continued to improve with time.

Penetrating Keratoplasty in Dogs using Acellular Porcine Corneal Stroma (BioCorneaVet™): A prospective pilot study of five cases.

OBJECTIVE: This prospective pilot study was conducted to evaluate the outcome of a commercially available corneal stroma substitute, Acellular Porcine Corneal Stroma (APCS), in dogs undergoing penetrating keratoplasty (PK) to restore corneal integrity after having deep ulcers. METHOD: Five dogs (1 eye in each dog) underwent a PK using APCS (BioCorneaVet™) as a graft. The surgical procedure and peri- and postoperative treatment were standardized. All cases required a minimum 6 months follow-up. Ease of keratoprosthetic tissue handling, graft survival, anterior chamber stability, corneal opacity, neovascularization and re-epithelialization were noted. Presence of secondary uveitis was investigated. RESULTS: BioCorneaVet™ was easy to handle and, at all-time points, provided adequate tectonic support. Graft survival was achieved in all 5 cases. A minimum follow-up period of 10 months was available for the five eyes (22 months maximum). Degree and area of corneal graft opacity progressively improved resulting in minimal to moderate loss of transparency in all cases but one, where it was severe. Neovascularization degree was most severe 0.5-1 month after surgery and fully resolved 4-6 months post-surgery. Re-epithelialization was complete in the majority of grafts in 1 month. Secondary uveitis was not detected at any time in 4 of 5 dogs. CONCLUSION: BioCorneaVet™ seems to be an effective graft for PK in the dog. In this case series, APCS was convenient to handle during surgery and provided excellent tectonic support. The material showed good tissue biocompatibility and resulted in the majority of cases in minimal to moderate graft opacity, that ameliorates with time.

Sterile Excimer Laser Shaped Allograft Corneal Inlay for Hyperopia: One-year Clinical Results in 28 Eyes.

Purpose: This study aimed to evaluate the one-year clinical results of an allograft corneal inlay (ACI) implantation in a case series of 28 hyperopic eyes of 16 patients.Methods: Patients with manifest refraction spherical equivalent (MRSE) between +1.00 and +6.00 D and having a cylindrical refraction of less than 1 D were included in this prospective study. The refractive powers of excimer laser-shaped ACIs were determined based on the refractive error of the individual subject's eyes. After the creation of a femtosecond flap, the inlays were centered on the pupillary axis. Visual acuities, refractive results, and other clinical findings were reported for the 6- and 12-month follow-up exams.Results: The mean age of the patients included in the study was 36.2 ± 12.4 years (range 22-65 years). The mean pre-operative MSRE of 3.6 ± 1.51 D decreased to 0.21 ± 0.56 D (P < .001). The uncorrected distance and near visual acuity increased from 0.33 ± 0.22 and 0.17 ± 0.13 to 0.75 ± 0.22 (P < .001) and 0.72 ± 0.19 (P < .001), respectively. The corrected distance visual acuity remained unchanged (pre-OP: 0.79 ± 0.22; post-OP: 0.80 ± 0.21; P = .916), and the corrected near visual acuity increased from 0.78 ± 0.22 to 0.84 ± 0.20 (P = .003). The mean K-value and central corneal thickness increased from 42.57 ± 0.81 D and 557.5 ± 43.0 µm to 44.8 ± 1.4 D (P < .001) and 597.1 ± 58.1 µm (P < .001), respectively. No significant postoperative complications such as diffuse lamellar keratitis, epithelial ingrowth, or decentralization were observed.Conclusion: Excimer laser-shaped ACI offers an alternative treatment modality for patients with hyperopia. Acceptable visual results and similar regression rates were observed with ACI implantation compared with other laser refractive procedures.

Intrastromal Allogeneic Inclusions for the Management of Keratoconus: A Review of Current Literature.

ABSTRACT: A new therapeutic alternative has been developed in the past 6 years to treat severe keratoconus in young patients. Those patients had only corneal transplantation as an option, but now a variety of surgical alternatives in the form of allogeneic corneal inclusions have bloomed and are becoming more popular. Although Bowman layer transplantation is the most studied technique, recent studies have described different options with very promising preliminary results. Mostly all the techniques described improve corneal curvature, visual acuity, pachymetry, contact lens tolerance, and foremost, manage to avoid or postpone corneal transplantation. Very few complications have been described so far, which makes these techniques not only feasible but also safe. Herein, we focus on reviewing recently published studies describing these techniques and their first results.

A decellularized human corneal scaffold for anterior corneal surface reconstruction.

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Acellular Porcine Corneal Stroma May Not Be Optimal for Peripheral Keratoplasty: Reports of 2 Cases.

PURPOSE: Although acellular porcine corneal stroma (APCS) is a promising alternative to the human donor cornea for lamellar keratoplasty, here, we report 2 patients who exhibited persistent epithelial defects and sterile keratolysis after APCS transplantation to treat peripheral corneal diseases. METHODS: Two patients with different peripheral corneal diseases underwent lamellar keratoplasty by using D-shaped lamellar APCS as graft materials. Standard keratoplasty postoperative treatments, including topical antibiotic-corticosteroid, tacrolimus, and artificial tears, were applied. RESULTS: Patient 1 was a 7-year-old boy with limbal dermoid, and patient 2 was a 50-year-old man suffered from simultaneous Mooren ulcer with pterygium. Both patients developed persistent graft epithelial defects postoperatively, which were refractory to conventional nonsurgical therapies. The APCS grafts were noted to start sterile keratolysis at approximately 1 month after implantation and were completely dissolved within 3 months, leaving vascularized scars in the previously grafted area. CONCLUSIONS: These 2 cases demonstrated that given the high risk of postoperative persistent epithelial defect and sterile keratolysis, the application of APCS in peripheral keratoplasty may need further evaluation.

In Vivo Confocal Microscopy of Stromal Lenticule Addition Keratoplasty for Advanced Keratoconus.

PURPOSE: To investigate the in vivo corneal microscopic changes after femtosecond laser-assisted stromal lenticule addition keratoplasty in keratoconus by means of in vivo confocal microscopy. METHODS: Patients affected by advanced keratoconus were included in the study. Negative meniscus-shaped stromal lenticules, produced with a femtosecond laser (VisuMax; Carl Zeiss Meditec) from eye bank corneas were transplanted into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. In vivo confocal microscopy was performed during the 12-month follow-up to investigate changes of the corneal and lenticule structure. RESULTS: Ten patients were enrolled in the study. No changes of the dendritic cell population were documented during the follow-up period. Mild edema and stromal keratocyte activation gradually decreased during the first month. Subbasal nerve density returned to preoperative values after 6 months. Donor-recipient interfaces appeared hyperreflective but gradually improved over time with significantly reduced reflectivity after 3 months. No evidence of stromal inflammatory cell migration or matrix opacification was observed. Endothelial and keratocyte density remained stable over time. A variable degree of stromal radially distributed folds, not visible on biomicroscopy, was observed in the lenticule and in the posterior recipient stroma. CONCLUSIONS: Stromal lenticule addition keratoplasty produces transitory nerve plexus density reduction and minor inflammatory reaction that rapidly decreases during the first month. Donor-recipient interface reflectivity is comparable to a femtosecond laser refractive procedure with no sign of stromal opacification or stromal rejection in 1 year of follow-up. [J Refract Surg. 2020;36(8):544-550.].

Femtosecond Laser-Assisted Surgery for Implantation of Bioengineered Corneal Stroma to Promote Corneal Regeneration.

The femtosecond laser has achieved widespread use in ophthalmology owing to its ability to deliver focused high energy that is rapidly dissipated and thereby does not damage surrounding tissue outside the precise focal region. Extremely accurate and smooth cuts can be made by the laser, enabling a range of applications in anterior segment surgery. Minimally invasive corneal surgical procedures can be performed using the femtosecond laser, and here we describe the application of such procedures to improve implantation of bioengineered materials into the cornea. Bioengineered corneal tissue, including the collagenous corneal stroma, promises to provide a virtually unlimited supply of biocompatible tissue for treating multiple causes of corneal blindness globally, thereby circumventing problems of donor tissue shortages and access to tissue banking infrastructure. Optimal implantation of bioengineered materials, however, is required, in order to facilitate postoperative wound healing for the maintenance of corneal transparency and avoidance of postoperative complications such as scarring, inflammation, and neovascularization. Moreover, the avoidance of a detrimental physiological physiological wound healing response is critical for facilitating the corneal stromal regeneration enabled by the bioengineered stroma. Without proper implantation, the tissue response will favor inflammation and pathologic processes instead of quiescent keratocyte migration and new collagen production. Here we describe several procedures for optimized biomaterial implantation into the corneal stroma, that facilitate rapid wound healing and regenerative restoration of corneal transparency without the use of human donor tissue. A step-by-step methodology is provided for the use of the femtosecond laser and associated techniques, to enable seamless integration of bioengineered materials into the corneal stroma.

Corneal Stroma Cell Density Evolution in Keratoconus Corneas Following the Implantation of Adipose Mesenchymal Stem Cells and Corneal Laminas: An In Vivo Confocal Microscopy Study.

Purpose: To report the corneal stroma cell density evolution identified by in vivo corneal confocal microscopy in humans using injected autologous adipose-derived adult stem cells (ADASCs) and corneal decellularized laminas in corneas with advanced keratoconus. Methods: Interventional prospective, consecutive, randomized, comparative series of cases. A total of 14 keratoconic patients were randomly distributed into three groups for three types of surgical interventions: group 1 (G-1), autologous ADASC implantation (n = 5); group 2 (G-2), decellularized human corneal stroma (n = 5); and group 3 (G-3), autologous ADASCs + decellularized human corneal stroma (n = 4). Results: A gradual and significant increase (P < 0.001) was observed in the cellularity in the anterior and posterior stroma of patients in G-1, G-2, and G-3 a year after the surgery in comparison with the preoperative density level. The same result was observed at the mid-corneal stroma in G-1 and at the anterior and posterior surfaces and within the laminas in G-2 and G-3. The cell density of patients receiving ADASC recellularized laminas (G-3) was statistically significantly higher (P = 0.011) at the anterior surface and within the lamina (P = 0.029) and at the posterior surface than in those implanted only with decellularized laminas (G-2). Conclusions: A significant increase in cell density occurred up to 1 postoperative year at the corneal stroma following the implantation of ADASCs alone, as well as in those cases implanted with decellularized and recellularized laminas at the different levels of the analysis. However, this increase was significantly higher in the ADASC recellularized laminas.

Two-year observation of morphologic and histopathologic changes in the monkey cornea following small incision allogenic lenticule implantation.

PURPOSE: To observe the morphologic and histopathologic changes of femtosecond laser assisted small incision allogenic intrastromal lenticule implantation (AILI) in monkey corneas. METHODS: 6 healthy adult monkeys were included. One eye of two monkeys and both eyes of one monkey received femtosecond lenticule extraction with a -4.0 diopter (D) correction. Each extracted refractive donor lenticule was immediately allogeneically transplanted into a corneal stromal pocket created by a femtosecond laser in another monkey's eye. A postoperative two-year follow-up was performed with slit lamp microscopy, corneal topography, anterior segment optical coherence tomography and in vivo confocal microscopy. All eyes were enucleated for Hematoxylin-Eosin staining and transmission electron microscopy (TEM) observation. RESULTS: No complications were observed in the follow-up period. At postoperative 2 years, the corneas remained clear and the lenticules were integrated with the surrounding tissue under slit lamp microscopy. Nerve fiber regeneration was detected in the lenticule layer as observed through confocal microscopy. Corneal power was increased by 1.83 ± 1.36 D after 2 years, which was less than at 6 months (3.27 ± 1.2 D). Disordered fibers and decreased keratocytes in the implanted lenticules could be detected under light microscopy and TEM, with a clear boundary between the lenticules and the surrounding tissue. CONCLUSIONS: Small incision AILI is feasible and safe for reshaping the cornea. Corneal healing remained stable while refraction showed a moderate regression within postoperative 2 years.

Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty.

BACKGROUND: 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. METHODS: Twenty-seven patients undergoing APCS implantation to treat infectious keratitis were included. The patients were followed up for 12 months after surgery. The main outcome measures included visual acuity, corneal transparency, graft thickness, and cellular and nerve regeneration. RESULTS: 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 12 months 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 ± 52.69 µm) but then continuously decreased until 3 months after surgery (1 month = 449.26 ± 50.38 µm; 3 months = 359.63 ± 34.14 µm, P < .001). Graft reepithelialization was completed within 1 week. In the in vivo confocal microscopy scans, host keratocytes began to repopulate the APCS grafts between 3 and 6 months post-operatively; subbasal nerve regeneration was only noted in 18.52% (5/27) of the eyes by 12 months after surgery. CONCLUSIONS: 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.