Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, light junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.     

Ocular surface reconstruction by tissue engineering

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation modality. In order to treat bilaterally affected, severe ocular surface disorders, we investigated the transplantation of two types of cultivated mucosal epithelia: allogenic corneal epithelial stem cells, and autologous oral mucosal epithelial cells. For this, first, we summarized the clinical results of allogenic keratoepithelioplasty and limbal transplantation. In addition, we showed that the immunological shift from Th1 to Th2 by using keyhole limpet hemocyanin was effective in suppressing the incidence of immunological rejection. Second, we investigated the transplantation of cultivated human corneal epithelial stem cells onto amniotic membrane. The cultivated sheet was created by co-culture with 3T3 fibroblasts, using the air-lift method, in cultivating the corneal epithelial stem cell on the amniotic membrane. These cultivated cells demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, tight junction related proteins, and telomerase activity. The transplanted allogenic human corneal epithelial sheet survived on the corneal surface in all cases, and was quite effective for achieving ocular surface stability in the acute phase of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, or chemical injury. However, a few cases developed immunological rejection or opportunistic infection. Third, to establish the transplantation of the autologous cultivated oral mucosal epithelial sheet, we performed animal experiments using rabbits. In vitro oral mucosal epithelial sheet showed histology similar to that of in vivo corneal epithelial sheet. It expressed positive keratin 3 as well. Since the autologous transplantation of this sheet survived on the ocular surface with the recovery of corneal transparency, a cultivated oral mucosal epithelium may become a substitute for corneal epithelium. Fourth, we created a cultivated human corneal endothelial cell sheet on amniotic membrane using a similar technique, and transplanted it to a rabbit eye as a xenograft. The transplanted corneal endothelial cell density was over 3,000 cells/mm2, and it was actively functioning even after the transplantation. Lastly, to explore cell markers for corneal epithelial stem cells, we established a technique using laser micro-capture, and introduced amplified fragment length polymorphism (AFLP), identifying several candidate molecules as stem cell markers.     

A fibrin-based bioengineered ocular surface with human corneal epithelial stem cells

PURPOSE: The purpose of the investigation was to prepare a bioengineered ocular surface tissue replacement consisting of (presumed) human corneal epithelial stem cells in a cross-linked fibrin gel for potential transplant. METHODS: Presumed human epithelial stem cells were harvested, isolated, and cultivated as previously described from adult donor corneas obtained from a tissue and organ bank. The cultured corneal epithelial stem cells were suspended in a fibronectin/fibrin gel cross-linked by factor XIII. Plasma components were derived from a fibrinogen-rich cryoprecipitate of human plasma. Suspended cells proliferated in the fibrin gel, giving rise to colonies that eventually coalesced to near confluence over the 15 days of cultivation. The gels were sectioned and immunostained for keratin 3 (AE5) and keratin 19. RESULTS: The fibrin gel product with corneal stem cells was easily manageable and maneuverable. Addition of the protease inhibitor aprotinin to the incubation medium prevented gel degradation; once it was removed, gels disintegrated within 24 hours. All of the cells cultivated in the fibrin gel stained positively for keratin 3 (AE5), indicating differentiation along the corneal epithelium lineage. Cells located in the center of the colonies were keratin 19-positive, suggesting a more primitive cell type. Growth kinetics were documented. CONCLUSIONS: A bioengineered ocular surface with a combination of presumed corneal epithelial stem cells in a cross-linked fibrin gel represents a potential improvement in current attempts to create a transportable, pliable, and stable tissue replacement. Since both the cells and the plasma components of the fibrin gel are of human origin, this technique provides the potential for a totally autologous bioengineered replacement tissue.     

Actin filament localization in developing and pathologic human corneas

Actin is associated with motility, cell morphology, and cell-substrate adhesion. The molecular probe NBD phallacidin, which reacts with filamentous actin, was used to study the distribution of actin filaments in the corneal and conjunctival epithelium, stroma, and endothelium. Frozen sections of human fetal eyes from 8 weeks to 40 weeks of gestation were reacted with NBD phallacidin. Pathologic tissues included keratoplasty specimens from patients with hereditary posterior polymorphous corneal dystrophy (PPMD) and surgically excised tissues removed for treatment of epithelial down-growth. Normal human cornea was used as a control. Immunofluorescent staining disclosed actin filament distribution in corneal epithelium as early as 9-10 weeks of gestation. Staining increased with maturation until term. Adult human corneal epithelium showed more pronounced staining of the surface layers. Stromal staining was more extensive in earlier stages of gestation and decreased in later stages of gestation, after 20-21 weeks. In pathologic corneas with posterior polymorphous dystrophy, there was localization of actin, as well as keratin, in the abnormal epithelial-like layers lining the posterior cornea. In epithelial downgrowth, actin and keratin were demonstrated in multilayered squamous epithelium on the anterior iris surface. Actin appears to be involved in migration of corneal epithelial and endothelial cells.     

The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.     

Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface

PURPOSE: Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces. METHODS: Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined. RESULTS: All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium. CONCLUSIONS: Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.     

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.     

Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia

PURPOSE: To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. METHODS: Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. RESULTS: The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. CONCLUSION: EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.     

Oberfl?chenrekonstruktion bei Limbusstammzellinsuffizienz

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.     

Concept and clinical application of cultivated epithelial transplantation for ocular surface disorders

Corneal epithelial replacement using a tissue engineering technique holds much promise for ocular surface reconstruction in cases of corneal epithelial stem cell deficiency. However, even though an autologous cultivated corneal epithelial stem cell sheet is the safest and most reliable form of sheet, bilaterally affected ocular surface disorders cannot be treated by this method. To treat bilateral cases, we must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. In the case of the former, immunological reaction is a threat. Thus, understanding of the immunological background of ocular surface reconstruction using allogeneic tissues is essential. In the case of the latter, the transplanted sheet is not exactly the same as corneal epithelium, and understanding ocular surface epithelial biology is important. In this review, we summarize and explain the concept and clinical application of cultivated mucosal epithelial transplantation for ocular surface disorders.     

组织工程角膜上皮移植重建眼表面的临床研究

目的 观察组织工程角膜上皮移植重建眼表面治疗完全性角膜上皮干细胞缺乏的短期临床效果.方法 系列病例研究.6例(6眼)单眼全角膜缘干细胞缺乏患者,包括碱烧伤3例、爆炸伤2例、热烧伤1例.采用去上皮羊膜组织作为载体,体外扩增患者自体健眼角膜上皮干细胞,构建组织工程角膜上皮.然后进行组织工程角膜上皮移植重建眼表面.术前及术后检查指标包括裸眼视力、裂隙灯显微镜、超声生物显微镜、泪液分泌试验.结果 患者自体来源的角膜上皮干细胞在去上皮羊膜上培养3周后均可形成直径15 mm的复层上皮片.组织工程角膜上皮均成功移植于所有受体眼表面.移植术后3个月,所有患者角膜上皮完整光滑,角膜瘢痕及纤维血管翳明显减轻,6例患者视力均有不同程度的提高.结论 患者自体来源的角膜上皮干细胞构建的组织工程角膜上皮移植可以成功重建全角膜缘干细胞缺乏患者的眼表面,为这类患者的复明带来希望.     

Corneal epithelial diseases related to limbal stem cell deficiency

Treatment of corneal epithelial diseases induced by limbal stem cell deficiency is an important challenge in ocular surface reconstruction. Since the 1990s, corneal stem cells have been localized in the limbus. This new concept completely changed the way we consider ocular surface reconstruction, with new diseases now found to be isolated in the ocular surface. Limbus insufficiency syndromes are specific depending on their origin (congenital or acquired), their expression (unilateral or bilateral, partial or total), their progression (acute or chronic), and the mechanism involved (burn, infection, chronic inflammation, etc.). Some of these diseases are local diseases and others are systemic diseases. Clinically, limbus insufficiency is a switch of the normal corneal epithelial phenotype (expression of a specific keratin, avascularity, and transparency of the corneal matrix) in an opaque and fibrovascularized cornea. In terms of cellular biology, a phenotype is a terminal expression of a cell differentiation process. This process is the outcome of the interaction between the genome of a cell or a group of cells with their microenvironment. In limbus insufficiency, epithelial cells and corneal matrix are destroyed, and it is the destruction of these two components that leads to limbus insufficiency syndrome.     

Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease

PURPOSE: Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction. METHODS: The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM. RESULTS: At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects. CONCLUSIONS: We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders

PURPOSE: To study the short-term clinical results of transplanting of cultivated corneal/limbal epithelial cells on human amniotic membrane (AM) for limbal deficiency. DESIGN: Noncomparative, retrospective interventional case series. PARTICIPANTS: Thirteen eyes of 13 patients with severe limbal deficiency (Stevens-Johnson syndrome in eight eyes, ocular cicatricial pemphigoid in three eyes, and chemical burns in two eyes) were treated at the department of Ophthalmology, Tokyo Dental College, Japan. INTERVENTION: Cultivated allo-limbal epithelium was transplanted onto the ocular surface of patients with severe limbal deficiency. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, changes in visual acuity, and postoperative complications were studied. Histologic examinations were also performed on cultivated epithelium. RESULTS: Cultivated corneal epithelium on AM formed two to three layers with the formation of basement membrane-like structures. After the surgery, the epithelium regenerated and covered the ocular surface in eight eyes (61.5%). However, three of the eight eyes developed partial conjunctival invasion, and two eyes later developed epithelial defects. At last examination, corneal epithelialization was achieved in six eyes (46.2%). Five eyes had conjunctivalization, one eye had dermal epithelialization, and one eye was not epithelialized. Complications were corneal perforation in four eyes and infectious keratitis in two eyes. CONCLUSIONS: This study demonstrates that the success rate for transplanting cultivated allo-limbal epithelium on the AM is not different from the conventional limbal and AM transplantation for the treatment of severe limbal stem cell dysfunction.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF,and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease

BACKGROUND: Patients with severe unilateral ocular surface disease require reconstruction of the damaged ocular surface. We succeeded in culturing primary corneal limbal epithelial cells taken from minimal biopsy and, once grown, transplanting them on denuded amniotic membrane (AM). METHODS: Autologous corneal limbal epithelial cells from a 3 mm(2) biopsy of the uninjured eye were grown for 3 weeks on a denuded AM carrier. The resultant sheet was then transplanted onto the unilateral severely chemically injured eye. RESULTS: Minimal biopsy showed the autologous cultivated corneal epithelial cells to have 4-5 layers of sufficient stratification and to be well differentiated. At 19 months post-transplantation, the ocular surface epithelium was stable and there were no epithelial defects. CONCLUSION: We document that it is possible to produce sufficiently stratified, well differentiated, autologous cultivated corneal limbal epithelium on AM from a minimal biopsy of the donor eye and to transplant it onto the injured eye.     

Association of preoperative tear function with surgical outcome in severe Stevens-Johnson syndrome

OBJECTIVE: To retrospectively study the surgical outcome in severe Stevens-Johnson syndrome (SJS). DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Twenty-nine eyes of patients with severe SJS associated with total conjunctivalization were examined. INTERVENTION: Preserved human amniotic membrane grafting and keratolimbal allograft transplantation was performed. Intensive immunosuppression and epithelial management were continued postoperatively. MAIN OUTCOME MEASURES: Successful ocular surface reconstruction was determined by epithelialization with corneal epithelium. The association between surgical outcome and preoperative tear function was studied. RESULTS: The ocular surface was successfully covered by corneal epithelium in 13 eyes (44.8%). The mean corrected visual acuity recovered from 0.0039 to 0.017. Persistent epithelial defect was the most common complication. Eyes with successful ocular surface reconstruction had significantly better preoperative Schirmer's test values (P = 0.025) and tear clearance rates (P = 0.043) than those that failed. CONCLUSIONS: In patients with severe SJS, preoperative tear function significantly influenced surgical outcome. Eyes with Schirmer's test value of >10 mm have a greater chance of successful ocular surface reconstruction.     

Nerve growth factor and corneal wound healing in dogs

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.