Autologous Limbal Transplantation Research Articles

Cultivated Autologous Limbal Epithelial Cell Transplantation: New Frontier in the Treatment of Limbal Stem Cell Deficiency

Taking into consideration prior human experience with treating limbal stem cell deficiency (LSCD) with cultivated limbal epithelial cells (CLEC) from other countries, we have set a goal to optimize and standardize the techniques of CLEC preparation (called CALEC by our group) for the clinical trial in the United States. We performed an extensive literature review of all human trials, case series, and reports involving autologous cultivated limbal epithelial cell transplantation. Allogeneic cultivated limbal epithelial cell transplantations were reported only when combined with autologous studies. We also searched prior animal data aiding in detailing regulatory toxicology requirements. Between 1997 and 2020, the analysis of human trials revealed 21 studies on autologous grafts, and 13 studies analyzing both autologous grafts and allogeneic grafts. Of a total of 34 studies, 6 studies used good manufacturing process (GMP) facilities, and 11 studies had no animal-derived products or murine feeder layers, whereas only 1 study had both. Overall, the treatment with autologous CLEC grafts was 68.9% successful. In total there were 6 preclinical studies using rabbits, serving as surrogate studies to assess the safety and toxicity of cultivated limbal epithelial cells for human trials. Based on prior human experience, we further optimized the manufacturing conditions with GMP-grade and serum and animal-free reagents, and developed cell characterization assays for the CALEC product release. These data were used to develop a novel and consistent manufacturing process using only qualified and validated reagents for performing the first clinical trial on CALEC transplantation to treat LSCD in the United States.

Indications for intraoperative anterior segment optical coherence tomography in corneal surgery

PurposeRecently, intraoperative optical coherence tomography (iOCT) has evolved in the field of ophthalmic surgery. So far, the use of iOCT was mainly focused to lamellar keratoplasty, especially deep anterior lamellar keratoplasty (DALK) and Descemet membrane endothelial keratoplasty (DMEK). The aim of this study was to report our experiences with iOCT to introduce new possibilities of this application.MethodsWe used iOCT in 18 patients who underwent the following surgeries: DALK, DMEK, penetrating keratoplasty, autologous limbal transplantation, transscleral suture fixation of a posterior chamber lens, pannus removal on corneal surface and newborn investigation in Peters’ anomaly. We obtained qualitative video data for all procedures.ResultsWith the iOCT, the cannula placement during DALK preparation of the recipient cornea and bubble formation could be visualized to improve the success rate of the big bubble injection. In DMEK, the iOCT enables the visualization of Descemet’s membrane removal in the recipient and graft orientation as well as better control of graft attachment. The iOCT enables intraoperative visualization of the graft–host interface during penetrating keratoplasty. During autologous limbal transplantation, transscleral suture fixation of a posterior chamber lens and removal of corneal surface pannus the iOCT is capable of showing the thickness of lamellar preparations to avoid penetrations and to save healthy recipient’s tissue.ConclusionThe iOCT is a helpful device for intraoperative anterior segment imaging not only for DALK and DMEK. It is also beneficial in penetrating keratoplasty and every other form of lamellar preparation during corneoscleral surgery.

Outcomes of Limbal Stem Cell Transplant

Limbal stem cell transplant (LSCT) can be categorized as direct autologous limbal transplant (AULT), direct allogenic limbal transplant (ALLT), cultivated autologous limbal stem cells transplant (cAULT), and cultivated allogenic limbal stem cells transplant (cALLT). To our knowledge, there is no study directly comparing the outcomes and complications of these procedures. To evaluate the outcomes of different LSCT procedures. We searched PubMed, EMBASE, Web of Science, and Cochrane without language filter for peer-reviewed articles about LSCT. The latest search was performed on June 30, 2019. Clinical studies with the outcome of at least 20 eyes after LSCT were included. Animal studies and studies of other surgical interventions were excluded. Two reviewers independently abstracted the data from each study. Heterogeneity was evaluated with the I2 statistic, and a meta-analysis was performed using the random-effects model. Outcome measures included the improvement of ocular surface, visual acuity (VA), and adverse events of recipient eyes and donor eyes. Forty studies (2202 eyes) with a mean (SD) follow-up of 31.3 (20.9) months met the inclusion criteria. The mean (SD) age of study participants was 38.4 (13.1) years, and men accounted for 74%. The number of eyes that underwent AULT, ALLT, cAULT, and cALLT were 505, 742, 771, and 184, respectively. Improvement of the ocular surface was achieved in 74.5% of all eyes, 85.7% of eyes after AULT (95% CI, 79.5%-90.3%), 84.7% after cAULT (95% CI, 77.2%-90.0%), 57.8% after ALLT (95% CI, 49.0%-66.1%), and 63.2% after cALLT (95% CI, 49.3%-75.2%). Autologous limbal transplantation resulted in a greater VA improvement rate (76%) than did the other 3 procedures (cAULT: 56.4%; ALLT: 52.3%; cALLT: 43.3%; all P < .001). The most common adverse events in all recipient eyes were recurrent/persistent epithelial erosion (10.5%; 95% CI, 7.2%-23.3%) and elevated intraocular pressure (intraocular pressure, 1.7%; 95% CI, 0.5%-7.8%). Patients who underwent ALLT had the highest rate of recurrent epithelial erosion (27.8%; 95% CI, 17.1%-41.9%) and intraocular pressure elevation (6.3%; 95% CI, 1.8%-19.4%). These findings suggest LSCT can improve or stabilize the corneal surface with a low rate of severe ocular complications and that autologous LSCT may have a higher success rate and fewer complications than allogenic LSCT.

OUTCOME OF AUTOLOGOUS LIMBAL CONJUNCTIVAL TRANSPLANTATION IN TERMS OF RECURRENCE RATE, AS THE MAIN OUTCOME MEASURE AND COMPLICATIONS AS THE SECONDARY OUTCOME

Purpose: To find the outcome of autologous limbal conjunctival transplantation in terms of recurrence rate, as the main outcome measure and complications as the secondary outcome.&#x0D; Methods: The present study retrospective study conducted from a tertiary eye care hospital. Pterygium excision with limbal conjunctival autograft from the affected eye was performed for primary pterygium. Secondary pterygia resulting from inflammation, trauma and other diseases were excluded. Patients were followed up for 18 months for recurrence and other complications.&#x0D; Results: A total of 36 eyes of 36 patients with primary pterygia from November 2016 and October 2017 were included in the study. The study recruited 19 (52.7%) males and 17 (47.2%) females with mean age of 38.2 ±10.42 years. Pterygium was diagnosed more commonly in left eye(55.5%) as compare to right eye(44.4%). Out of 36 cases 32 (88.8%) cases of nasal and 4 (11.1%) cases of temporal pterygium noted. Out of 36 cases Graft edema in 1 (2.7%) case, graft retraction in 2 (5.5%) cases and 1 (2.7%) case of granuloma were noted. Any kind of pterygium recurrence and graft necrosis was not noted in present study.&#x0D; Conclusions: Pterygium excision with autologous limbal conjunctival autograft is safe and effective method to treat primary pterygium.

Preserving Basement Membranes during Detachment of Cultivated Oral Mucosal Epithelial Cell Sheets for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

Total bilateral limbal stem cell deficiency leading to loss of corneal clarity, potential vision loss, pain, photophobia, and keratoplasty failure cannot be treated by autologous limbal transplantation, and allogeneic limbal transplantation requires subsequent immunosuppressive treatment. Cultured autologous oral mucosal epithelial cells have been shown to be safe and effective alternatives. These cells can be transplanted on supports or without support after detachment from the culture dishes. Dispase, known for epidermal sheet detachment, is reported as not usable for oral mucosa. The objective was to find an optimized detachment method providing a sufficiently resistant and adhesive cultured oral mucosal epithelium (COME), which can be grafted without sutures. Enzymatic treatments (dispase or collagenase at different concentrations) were compared to enzyme-free mechanical detachment. Histological immunofluorescence (IF) and Western blotting (WB) were used to examine the impact on adhesion markers (laminin-332, β1-integrin, and type VII collagen) and junctional markers (E-cadherin, P-cadherin). Finally, the COME ability to adhere to the cornea and produce a differentiated epithelium 15 d after grafting onto an ex vivo porcine stroma model were investigated by histology, IF, and transmission electron microscopy. Collagenase at 0.5 mg/mL and dispase at 5 mg/mL were selected for comparative study on adhesive expression marker by IF and WB showed that levels of basement membrane proteins and cell–cell and cell–matrix junction proteins were not significantly different between the 3 detachment methods. Collagenase 0.5 mg/mL was selected for the next step validation because of the better reproducibility, 100% success (vs. 33% with dispase 5 mg/mL). Grafted onto porcine de-epithelialized corneal stroma, collagenase 0.5 mg/mL detached COME were found to adhere, stratify, and continue to ensure renewal of the epithelium. For COME, collagenase 0.5 mg/mL enzymatic detachment was selected and validated on its resistance and adhesive marker expression as well as their anchorage onto our new ex vivo de-epithelialized stroma model.

Conjunctival-limbal autograft in total unilateral limbal stem cell deficiency

Background : Corneal epithelium is renewed by stem cells (SC) that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD), characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC) from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU) that we combined with amniotic membrane (AM) use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury. Methods : Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently. Results : CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new epithelium from the limbal grafts was noted on day 3–6 after CLAU. The cornea was completely epithelized within 2 weeks in 2 patients and after 35 days in one patient. In two patients the corneal epithelium remained clear, smooth and stable during the follow up of 3.5 years and 4 months, respectively. In one patient, uneven epithelium probably representing a mosaic of corneal and conjunctival cells was noted in the central corneal region, where a small corneal ulcer developed 5 months after CLAU. In donor eyes no postoperative complications were noted, the donor sites epithelized within few days. Conclusions : Autologous limbal transplantation according to CLAU surgical technique combined with the use of AM is a successful and safe therapy for restoring corneal surface in total unilateral LSCD after chemical injury. It enables further surgical procedures for restoring the vision such as corneal transplantation and cataract surgery.

Avtologni veznično-limbalni presadek pri enostranskem popolnem pomanjkanju limbalnih epitelnih matičnih celic

Background: Corneal epithelium is renewed by stem cells (SC) that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD), characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC) from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU) that we combined with amniotic membrane (AM) use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury.Methods: Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently.Results: CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new epithelium from the limbal grafts was noted on day 3–6 after CLAU. The cornea was completely epithelized within 2 weeks in 2 patients and after 35 days in one patient. In two patients the corneal epithelium remained clear, smooth and stable during the follow up of 3.5 years and 4 months, respectively. In one patient, uneven epithelium probably representing a mosaic of corneal and conjunctival cells was noted in the central corneal region, where a small corneal ulcer developed 5 months after CLAU. In donor eyes no postoperative complications were noted, the donor sites epithelized within few days.Conclusions: Autologous limbal transplantation according to CLAU surgical technique combined with the use of AM is a successful and safe therapy for restoring corneal surface in total unilateral LSCD after chemical injury. It enables further surgical procedures for restoring the vision such as corneal transplantation and cataract surgery.

Variations in the technique for autologous limbal transplantation

Abstract Objective To present the results on the use of a single block limbal autograft, combined with amniotic membrane transplantation and sectoral sequential postoperative epitheliectomy of the conjunctiva in 2 patients with unilateral total limbal stem cell deficiency. Conclusions A single block limbal autograft combined with amniotic membrane transplantation may be sufficient to restore a stable corneal surface, but sometimes sequential sectoral conjunctival epitheliectomy may be required to treat anomalous epithelial remnants.

Keratolimbal autograft transplantation as a possible new treatment of Lisch epithelial corneal dystrophy

Case reportThe case concerns 64-year-old woman with visual acuity of 20/40 in the right eye. Slit-lamp examination revealed a grey, feathery corneal opacification with intraepithelial microcysts compatible with Lisch epithelial corneal dystrophy (LECD). It was treated with epithelial debridements, contact lenses and mitomycin C, but the opacification recurred within months. The removal of limbus sector and autologous limbal transplantation (KLAT) were used successfully without recurrence. ConclusionsAfter removal of damaged limbus, KLAT should be considered as a treatment option for asymmetric LECD when other treatments have failed.

Trasplante autólogo de limbo como nuevo tratamiento de la distrofia corneal epitelial de Lisch

Case reportThe case concerns 64-year-old woman with visual acuity of 20/40 in the right eye. Slit-lamp examination revealed a grey, feathery corneal opacification with intraepithelial microcysts compatible with Lisch epithelial corneal dystrophy (LECD). It was treated with epithelial debridements, contact lenses and mitomycin C, but the opacification recurred within months. The removal of limbus sector and autologous limbal transplantation (KLAT) were used successfully without recurrence. ConclusionsAfter removal of damaged limbus, KLAT should be considered as a treatment option for asymmetric LECD when other treatments have failed.

The treatment of corneal perforation with defect of tissue

Objective To evaluate the feasibility and clinical efficacy of scleral graft or combined with autologous limbal transplantation for the treatment of peripheral corneal perforation with defect of tissue. Methods Twelve eyes of 12 inpatients with peripheral corneal perforating wound in our hospital from August 1992 to February 2013 were retrospectively analyzed.Seven patients received scleral graft transplantation only, and 5 patients received preserved scleral graft combined with autologous limbal transplantation.The follow-up period was at least 12 months after the transplantation.Postoperative visual acuity and complications were studied in the follow-up period. Results All perforations(12 eyes) were closed, no significant complication occurred during follow-up period, and visual functions were improved. Conclusion Either scleral graft or combined with autologous limbal transplantation for the treatment of peripheral corneal perforation may be a new effective method for the treatment of emergent peripheral corneal perforation and defect. Key words: Wound, perforating, peripheral cornea; Defect, tissue; Transplantation, scleral graft, limbus, autologous

Safety profile and complications of autologous limbal conjunctival transplantation for primary pterygium

PurposePrimary pterygium is a fibrovascular proliferation over the nasal cornea, probably resulting from the limbal stem cell deficiency. Intraoperative mitomycin-C application seems to associate with reduced recurrences, however produced ocular surface problems and vision threatening complications. The present clinical study investigated the safety profile of autologous limbal conjunctival transplantation in terms of recurrence rate, as the main outcome measure and complications as the secondary outcome. MethodsThe present study was randomised, interventional and prospective clinical study conducted from a tertiary Hospital. Pterygium excision was performed with limbal conjunctival autograft availed from the affected eye. Secondary pterygia resulting from inflammation, trauma and other diseases were excluded. Patients were followed up for 18months for recurrence and other complications. Microsoft Office Excel 2007 was used for statistical analysis. ResultsA total of 71 eyes of sixty-eight patients with primary pterygia included between November 2007 and October 2010. The study recruited 35 (51%) males and 33 (49%) females with mean age of 36.9 with ±12.82years standard deviation (mean, SD) ranging from 19 to 75years. Age grouped by range intervals categorised into six groups. Pterygium was diagnosed in 32 (45%) right eyes and 39 (55%) left eyes. There were 65 (91.55%) nasal and 4 (5.63%) temporal pterygium and no case of double head pterygia found. Average horizontal extension of the pterygium measured was 1.67mm (±4.23) from the apex to the corneal limbus. Graft oedema in 1 (0.71%) patient, graft bleed in 2 (1.42%) cases and 1 (0.72%) case of granuloma observed. No recurrences encountered during 18months follow up. ConclusionsPterygium occurred predominantly in the younger population group 36.9mm (±12.82) probably due to the increased outdoor activity with high exposure to sunlight and dusty atmosphere. Absence of recurrences was probably attributable to the smaller pterygium size of 1.67mm (±4.23), use of the autologous limbal conjunctival graft and treatable intra and post operative complications successfully.

Tissue-Regenerating, Vision-Restoring Corneal Epithelial Stem Cells

The cornea, the most anterior segment of the eye, provides us with exquisite vision. Unlike other vital tissues, it is poorly protected from the environment and is thus reliant on a self-renewal program to preserve integrity. This function is reserved for corneal epithelial stem cells located in the basal layer of the limbus, a narrow transition zone that segregates the peripheral cornea from the adjacent conjunctiva. Under physiological conditions, these cells replenish the corneal epithelium when mature or traumatized cells are lost. However, when the limbus is extensively damaged, stem cell activity is compromised, resulting in a condition known as limbal stem cell deficiency (LSCD). This disease is characterized by corneal neovascularization and persistent epithelial defects which impair vision. Over the past 20 years a myriad of treatment options have been developed for LSCD, most of which incorporate stem cell transplantation. Due to the disadvantages associated with the use of allogeneic and xenogeneic material, researchers are currently focusing on refining techniques involving autologous limbal tissue transplantation and are delving into the possibility that stem cells found in other organs can provide an alternative source of corneal epithelium. Determining where donor stem cells reside on the recipient's ocular surface and how long they remain viable will provide further insights into improving current therapeutic options for patients with LSCD.

Ocular Surface Reconstruction

The most difficult part in ocular surface reconstruction for total limbal stem cell deficiency is restoring a healthy and stable corneal epithelium. Recently, there has been a shift toward a cellular-based transplantation that eliminates the need for harvesting large amounts of limbal tissues from a living donor. The autologous oral epithelium can be used as a source for ocular surface reconstruction instead of the limbal epithelial cells. This paper describes the method of harvesting autologous oral epithelial cells, culturing the cells on the amniotic membrane, and transplanting the amniotic membranewith the cultured oral epithelial cells on the ocular surface after removal of the fibrovascular scar tissue. Once the corneal epithelium is stable, penetrating keratoplasty may be performed at a later stage to restore the corneal clarity. (Tech Ophthalmology 2009;7: 118Y123) HISTORICAL REVIEW Ocular surface diseases with partial or total stem cell deficiency can usually be distinguished into 2 types: (a) diseases with a conjunctivalized cornea, where there is a viable tear film and the surface is wet, and (b) a completely dry and keratinized ocular surface. The former may be subject to a series of procedures collectively termed ocular surface reconstruction, whereas in the latter, only keratoprosthesis can be performed. This paper will discuss the latest advances in ocular surface reconstruction and will focus on the shift from tissue transplantation, where autologous or allogenic limbal tissue is transplanted, to cellbased therapy, where epithelial cells are isolated and transplanted to the ocular surface using different carriers. The treatment strategy in ocular surface reconstruction involves a multistep approach (Table 1). Improving the ocular surface health is the first step, which includes managing tear film problems, controlling blepharitis, suppressing inflammation, and treating recurrent corneal epithelial erosions and chronic ulcers. The second step includes the major surgical procedures, which start with reconstruction of the fornices and symblepharon repair and lid surgery to repair entropion. The next surgical step, which is the most challenging part in ocular surface reconstruction is aimed at creating a stable corneal epithelium. This includes autologous or allogenic transplantation of limbal tissue or transplantation of cultured epithelial cells that were expanded ex vivo on a carrier. These procedures will be further discussed in the following paragraphs. If a stable ocular surface was established and the corneal stroma is opaque, the next surgical step is corneal transplantation. The third step is maintaining the ocular surface defense mechanisms by continuous attention to the tear film, lid problems, and epithelial integrity (Table 1). The mainstay of ocular surface reconstruction involves the creation of a stable surface epithelium. Twomajor approaches are available today to establish this goal (Table 2). The first approach involves tissue transplantation, whereas the second is based on cell therapy that involves ex vivo cultured epithelial cells. Tissue transplantation involves transplantation of the limbal region, which may be harvested either from the contralateral healthy eye (autologous limbal transplantation), a living related donor, or a cadaveric source (allogenic limbal transplantation). Autologous limbal transplantation was first described by Kenyon and Tseng in 1989 (Table 3) and is probably the most successful surgical procedure in ocular surface reconstruction. However, this procedure is not suitable in cases of bilateral limbal deficiency andmay be avoided in patients who are afraid of operating on their only eye. The safety and favorable results of this procedure have been demonstrated in many studies. Allogenic limbal transplantation is performed in cases of bilateral limbal deficiency. Its disadvantages include the need for a long-term systemic immune suppression, a high incidence of graft rejection, and gradual loss of the donor cells with time. TABLE 1. Treatment Strategies in Ocular Surface Diseases With Limbal Deficiency: A Multi-Step Approach

Utilização de adesivo de fibrina em cirurgias oftalmológicas

To evaluate the use of fibrin glue and its complications in different ophthalmic surgeries. A retrospective and non-comparative case series study. 161 eyes were evaluated. The mean age was 51.8 years. Fibrin glue (Beriplast--Aventis Behring, Germany) was used in all patients for tissue adherence or incisional closure. Main preoperatives diseases were pterygium (76 patients), superior limbic keratoconjunctivitis (4 patients), limbal tumor (4 patients), conjunctivochalasis (21 patients), bullous keratopathy (31 patients), limbal stem cell deficiency (7 patients), alkali injury (6 patients), Stevens-Johnson syndrome (2 patients), cataract (5 patients) and glaucoma (5 patients). Complications occurred in 28 patients (17.4%). Detachment of the fixed tissue and granulomatous foreign body reaction were the main complications. Detachment occurred in 10 cases (5.6%) (7 in amniotic membrane transplantation in bullous keratopathy, 2 in autologous conjunctival grafting for pterygium surgery, 1 in autologous limbal transplantation in a Stevens-Johnson syndrome). Granulomatous foreign body reaction was described in 9 patients (5.6%) (5 in amniotic membrane transplantation due to acute alkali injury, 1 in autologous limbal graft due to chronic burn injury, and 3 in autologous conjunctival transplant due to pterygium surgery). All the complications described occurred in the first week after surgery. Despite the limitations related to the retrospective and non-comparative setting, this study suggests that the use of fibrin glue seems to be safe and effective for tissue fixation or incisional closure presenting low rates of complications in ocular surface and anterior segment surgeries.

Persistence of Transplanted Oral Mucosal Epithelial Cells in Human Cornea

To determine the expression of differentiation and progenitor cell markers in corneal tissues that previously underwent autologous cultivated oral mucosal epithelial transplantation (COMET). Four eyes from three alkaline-injured patients and one thermally injured patient underwent COMET to promote re-epithelialization or corneal reconstruction. Between 10 and 22 months (mean, 14.2 +/- 5.5 months [SD]) after COMET, the corneal tissues were obtained after penetrating keratoplasty (n = 1) or autologous limbal transplantation (n = 3). Immunoconfocal microscopy for keratin (K)3, -12, -4, -13, and -8; connexin (Cx)43; MUC5AC; laminin-5; pan-p63; ABCG2; and p75 was performed in those specimens as well as in the oral mucosa and cultivated oral mucosal epithelial cells (OMECs). All four specimens were unanimously positive for K3, -4, and -13 but negative for K8 and MUC5AC, suggesting that the keratinocytes were oral mucosa-derived. However, peripheral K12 staining was positive only in patient 2, suggesting a mixed oral and corneal epithelium in that case. Cx43 staining in the basal epithelium was negative in patients 1, 2, and 3, but was positive in patient 4. Small, compact keratinocytes in the basal epithelium preferentially expressed pan-p63, ABCG2, and p75. Although the staining of pan-p63 and ABCG2 tended to be more than one layer, signal for p75 was consistently localized only to the basal layer. The study demonstrated the persistence of transplanted OMECs in human corneas. In addition, small, compact cells in the basal epithelium preferentially expressed the keratinocyte stem/progenitor cell markers, which may be indicative of the engraftment of the progenitor cells after transplantation.

Combined autologous and allograft limbal cell transplantation with penetrating keratoplasty in a case of chemical corneal burn patient

A patient with chemical corneal burn presented two months after the acute episode of chemical injury to his right eye (OD) and was diagnosed with severe limbal stem cell deficiency and with vascularized corneal opacity OD. Limbal cell transplantation and penetrating keratoplasty (PKP) was performed. The autologous and allograft limbal tissue included peripheral cornea, limbus and conjunctiva obtained from contralateral eye and cadaveric eye bank cornea. Central corneal button was used for a PKP with 32 intermittent sutures. One year after the procedure, the corneal surface remains clear with a best corrected visual acuity of 6/12 (-2.00 DS / -2.75 DC-/ 150°. Eighteen sutures are still in place; no vascularization has extended beyond the host graft junction. Ocular surface is wetting well with no filamentary keratitis.Combined autologous and allograft limbal cell transplant can be performed for severe deficiency of corneal stem cells in a patient with chemical corneal burn.

Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns

To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoring transparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation in arresting corneal neovessel ingrowth. Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, Buenos Aires, Argentina. Limbal stem-cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbal cell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layer grown on autologous clotted platelet-poor plasma or commercial fibrin for 21 days. Anterior keratectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vessels occurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90, 180, and 360 days and the corneas processed for histopathology and inmunohistochemistry. A small (2.5 mm(2)) limbal biopsy achieved stem-cell replication in vitro. Corneal clarity and epithelial defects evolved with a trend toward improvement. There was a significant reduction in corneal neovascularization. Histology showed a multilayered stratified epithelium including several epithelial-like cells with clear cytoplasm in the deepest part. There were no signs of intraepithelial mucin cells on the implanted corneas. Immunohistochemical results showed expression of cytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19. Autologous limbal epithelial cell transplantation improved the corneal surface in eyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1-year follow-up. Results may facilitate the application of this technique in patients.

Histopathologic Limbus Evolution After Alkaline Burns

To study the histopathologic evolution of the corneal limbus after alkaline burns according to the clinical severity and therapy used. A prospective study of 15 eyes from 12 patients (9 men and 3 women) with moderate and severe alkaline burns was performed. All patients were divided into 2 groups in accordance with the clinical ocular severity and the therapy that was used: medical therapy, amniotic membrane transplantation (AMT), autologous limbal transplantation (ALT), and ALT combined with AMT (ALT + AMT). Biopsies were obtained from affected limbal areas immediately after the ocular burn and 9 months later. Limbal regeneration was limited to small areas in patients with moderate burns treated with medical therapy; in contrast, the limbal structure showed significant stromal and epithelial regeneration in patients with moderate burns treated with AMT. There was an important stromal regeneration with an incomplete reepithelialization in patients with severe burns treated with AMT. Patients treated with ALT showed a good reepithelialization with a defective stromal regeneration. Epithelial and stromal regeneration was notable in patients with severe burns treated with ALT + AMT. In patients with moderate alkaline burns, AMT improved both limbal stromal and epithelial regeneration more effectively than medical therapy. In patients with severe burns, the best reepithelialization and stromal regeneration were obtained with ALT + AMT.

Deep Anterior Lamellar Keratoplasty Combined With Autologous Limbal Stem Cell Transplantation in Unilateral Severe Chemical Injury

To evaluate the efficacy of deep anterior lamellar keratoplasty combined with autologous limbal stem cell transplantation for ocular surface reconstruction and visual rehabilitation in eyes with unilateral, late-stage, severe chemical injury. This was a retrospective, noncomparative, interventional case series that included 7 eyes of 7 patients, with severe unilateral late stage chemical injury, exhibiting corneal vascularization, conjunctivalization, and extensive corneal scarring were treated at the C. J. Shah Cornea Service, Sankara Nethralaya, a tertiary care center. Surgical procedures included releasing symblepharon adhesions, excising epibulbar fibrous tissue, superficial keratectomy to remove fibrovascular tissues over cornea, deep anterior lamellar dissection, grafting a lamellar corneal button, and transplanting autologous limbal graft, with or without amniotic membrane transplantation. The main outcome measures were relief of patient symptoms, postoperative recovery of the ocular surface, corneal clarity, corneal epithelial stability, and best corrected visual acuity. The mean duration between the injury and surgery was 24.4 +/- 21.8 months. No intraoperative complications were noted. Successful epithelialization was achieved in all eyes. The reconstructed corneal surface remained stable during the entire follow-up period (mean follow-up, 16.57 +/- 5.12 months). All patients had resolution of ocular symptoms. Remarkable improvement in vision was noted in all (85.7%) except 1 eye in which recovery was limited due to amblyopia. The average best corrected visual acuity at last follow-up was 20/50. No complications were noted in the donor fellow eye. DALK combined with autologous limbal transplantation can restore a healthy, stable ocular surface, besides providing a clear cornea that remarkably improves the visual acuity, in patients with unilateral, late stage, severe chemical injury.