The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome

PURPOSE: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.     

"去上皮"羊膜对体外培养结膜上皮细胞的作用

目的 :探索羊膜基底膜对结膜上皮细胞形态功能的影响。方法 :把“去上皮”羊膜按照基底膜或实质面向上分为两组 ,接种结膜上皮细胞 ,同样条件下培养。取培养 14天的两组标本进行组织学 (HE和PAS染色 ) ,透射电镜 ,以及免疫组化检查 (包括抗角蛋白和抗波形蛋白染色 )。结果 :细胞 72小时内贴附在羊膜上。基底膜面接种的上皮细胞顶端见微绒毛 ,单层排列 ,细胞间有丰富桥粒 ,基底膜完整 ,可见半桥粒 ,抗角蛋白染色阳性 ,抗波形蛋白染色阴性 ;实质面接种的上皮细胞胞浆内有大量脂滴 ,表面未见微绒毛 ,细胞松散堆积 ,与羊膜连接不紧密 ,未见桥粒和半桥粒 ,抗角蛋白染色弱阳性 ,抗波形蛋白染色强阳性。结论 :羊膜基底膜是结膜上皮细胞体外培养的良好载体 ,有利于上皮细胞生长、粘附、分化 ,并能减缓上皮细胞老化。     

Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system

PURPOSE: To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells. METHODS: Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin. RESULTS: The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RT-PCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes. CONCLUSIONS: This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.     

Autologous cultivated conjunctival transplantation for pterygium surgery

PURPOSE: To investigate the safety and efficacy of autologous cultivated conjunctival transplantation for the treatment of primary pterygium. DESIGN: Prospective nonrandomized clinical trial. METHODS: Forty patients with primary pterygium were recruited. Excision of the pterygium was followed by reconstruction using a serum-free derived autologous cultivated conjunctival equivalent in 22 patients (group A) and conventional denuded amniotic membrane transplantation in 18 patients (group B). In group A patients, conjunctival epithelial equivalents were constructed by the ex vivo expansion of conjunctival epithelial cells on human amniotic membranes (HAM). The main outcome measures were conjunctival epithelialization, recurrence, survival analysis, and incidence of complications. RESULTS: The mean follow-up was 14.1 +/- 3.9 months (range, 12 to 25 months). Complete epithelialization was achieved within five days after surgery in group A patients compared with approximately three weeks for group B patients. The proportion of patients that had true recurrences was 22.7% in group A and 25.0% in group B. The mean time to recurrence was 4.8 +/- 1.6 months in group A and 5.0 +/- 2.9 months in group B. No ocular complications were noted in group A, while one eye (6.0%) in group B developed scleral necrosis associated with a persistent epithelial defect. CONCLUSIONS: Transplantation of an autologous cultivated conjunctival epithelial sheet facilitated early postoperative epithelialization and recovery, and may aid in preventing serious complications associated with simple denuded HAM transplantation, such as scleral necrosis and secondary infection. This may provide a novel method for conjunctival epithelial replacement in the treatment of ocular surface disorders.     

The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells

AIM: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE). METHODS: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. RESULTS: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. CONCLUSIONS: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.     

"去上-皮"羊膜對體外培養的結膜上皮細胞的作用

目的探索羊膜基底膜對結膜上皮細胞形態功能的影響.方法把"去上皮"羊膜按照基底膜或實質面向上分為兩組,接種結膜上皮細胞,同樣條件下培養.取培養14天的兩組標本進行組織學(HE和PAS染色),透射電鏡,以及免疫組化檢查(包括抗角蛋白和抗波形蛋白染色).結果細胞72小時内貼附在羊膜上.基底膜面接種的上皮細胞頂端見微絨毛,單層排列,細胞間有豐富橋粒,基底膜完整,可見半橋粒,抗角蛋白染色陽性,抗波形蛋白染色陰性;實質面接種的上皮細胞漿内有大量脂滴,表面未見微絨毛,細胞鬆散堆積,與羊膜連接不緊密,未見橋粒和半橋粒,抗角蛋白染色弱陽性,抗波形蛋白染色强陽性.結論羊膜基底膜是結膜上皮細胞體外培養的良好載體,有利于上皮細胞生長、黏附、分化,并能减緩上皮細胞老化.     

The lack of extracellular laminin beta2 chain deposition correlates to the loss of conjunctival epithelial keratin K4 localization in culture

PURPOSE: To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics. METHODS: Keratin K4 and laminin beta2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin beta2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts. The synthesis and secretion of laminin beta2 chain by conjunctival fibroblasts in culture was determined by western blot analysis and immunoprecipitation. The ability of conjunctival epithelium to respond to exogenous laminin beta2 chain was assayed by culturing epithelial cells on a laminin matrix isolated from human placenta. RESULTS: In culture, conjunctival fibroblasts synthesize and secrete laminin beta2 chain but do not deposit this chain into an extracellular matrix substrate or basement membrane-like structure. The lack of extracellular deposition of this chain correlates to the gradual loss of keratin K4 protein in conjunctival epithelial cell culture. Conjunctival epithelium remains responsive to laminin beta2 chain in vitro because keratin K4 localization can be rescued in these cells by culture on a substrate of exogenous placental laminin. CONCLUSIONS: In vitro, alterations in native conjunctival epithelial-mesenchymal interactions results in aberrant basement membrane laminin isoform composition. This, in turn, leads to the loss of adult epithelial cell phenotype characteristics, suggesting that at least some aspects of conjunctival epithelial cell differentiation are regulated by the extracellular matrix.     

Biocompatibility of nanocomposites used for artificial conjunctiva: in vivo experiments

PURPOSE: To evaluate the biocompatibility of nanocomposites used for artificial conjunctiva. METHODS: Fifty New Zealand white rabbits were used for the experiments. Nanocomposites of poly -caprolactone (PCL) and of PCL coated with polyvinyl alcohol (PCL+PVA), polyvinyl pyrrolidone (PCL+PVP), or chitosan (PCL+C), and amniotic membrane (AM) as a control, were cut into small disks with a diameter of 3.5 mm. The disks were inserted underneath the conjunctiva to measure their inflammation-inducing properties. To investigate epithelial adhesion and goblet cell differentiation, the disks were transplanted after round conjunctival excision. Cultivated conjunctival epithelial cells on nanocomposite were then transplanted onto the abdomen of Balb/c athymic mice. The number of inflammatory cells and the density of goblet cells were measured using hematoxylin and eosin, periodic-acid-Schiff, and immunohistochemistry after 2 weeks. RESULTS: The number of inflammatory cells found inside of the inserts was as follows: 21 +/- 4.9 for controls, 21 +/- 15.1 for PCL, 49.6 +/- 26.0 for PCL+PVP, 40.2 +/- 17.1 for PCL+C, and 13.8 +/- 3.9 for PCL+PVA. In PCL+PVA, the accumulation of inflammatory cells was significantly lower than in the controls (p < 0.01, Mann-Whitney U). The reepithelialization of conjunctival cells was accomplished in more than 75% of all disks except for the PCL+C. In addition, we found the differentiation of goblet cells in the following order from greatest to least: amniotic membrane, PCL, and PCL+PVP. CONCLUSIONS: Nanocomposites of PCL were biocompatible in rabbit conjunctiva, suggesting that PCL may be considered as a candidate for use in the development of artificial conjunctiva.     

Ultrastructural study of autologous cultivated conjunctival epithelium.

BACKGROUND AND OBJECTIVE: This report is one of the first in the literature on the attempted cultivation and clinical application of human conjunctival epithelium. The authors investigated the possibility of restoring severely damaged ocular surface with autologous cultivated conjunctival epithelium. PATIENTS AND METHODS: The conjunctival cells needed for the experiment were harvested from six patients with oculopalpebral diseases. Confluent epithelial sheets were developed from each biopsy specimen. The new epithelium was then implanted on the patients' or donors' eye surface. RESULTS: The histologic examination showed a pluristratified squamous non-keratinized epithelium lying on a basement membrane and with a lamina propria of well-vascularized connective tissue. Normal ultrastructural characteristics were evident on electron microscopy. CONCLUSION: The cultivation of autologous conjunctival cells may be a good option for rapid and safe repair of large single or bilateral conjunctival defects, as an alternative to heterotopic or allogenic grafts.     

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.     

Preliminary serological studies comparing immunofluorescence assay with radioimmunoassay

We assayed serum from 12 patients with untreated cicatricial pemphigoid affecting the conjunctiva for circulating autoantibodies directed against the epithelial basement membrane zone. We employed a conventional indirect immunofluorescence assay, with monkey esophagus and human conjunctiva as substrates, and compared the results with those obtained employing a radioimmunoassay measuring antibasement membrane zone antibody binding to COLO-16 and to SCaBER tumor cell lines. The indirect immunofluorescence assay on normal human conjunctival substrate detected circulating antibodies to conjunctival epithelium in 6 of 12 CP patient serum specimens. Monkey esophagus failed to detect antibodies to the epithelial basement membrane zone. In contrast, autoantibodies were detected in all 12 specimens by the radioimmunoassay. Specificity, as demonstrated by appropriate controls and assay of normal human serum, was 100%. These results demonstrate that radioimmunoassay employing COLO-16 or SCaBER cells is an exquisitely sensitive and specific assay for detection of circulating antibasement membrane antibodies in patients with cicatricial pemphigoid affecting the conjunctiva.     

An investigation of removed cultivated epithelial transplants in patients after allocultivated corneal epithelial transplantation

OBJECTIVE: To investigate the ultrastructural changes of removed cultivated corneal epithelial transplants using scanning and transmission electron microscopy. METHODS: Allocultivated corneal epithelial transplantation, using an amniotic membrane carrier, was carried out on 3 patients. The primary diagnoses consisted of 1 with acute-phase chemical burn, one with drug-induced pseudopemphigoid, and 1 with Stevens-Johnson syndrome. After a period of several months the transplants were removed from these patients because of graft opacities. The removed transplants were then prepared for examination by scanning and transmission electron microscopy. RESULTS: In all 3 cases there was a similar pattern of findings: the amniotic membrane remained intact, although it had become partially vascularized and invaded by keratocytes. Inflammatory cells were present in the epithelial layer and within the amniotic membrane. Most of the amniotic membrane was covered by conjunctival epithelial cells and goblet cells. Only a few areas of normal cultivated corneal epithelial cells were found. CONCLUSIONS: We suggest that the process of allograft rejection is responsible for the corneal epithelial loss and that this is followed by conjunctival invasion onto the amniotic membrane.     

Histological study of the effect of conjunctival transplantation on rabbit cornea with alkali burned

PURPOSE: A histopathological study to investigate the efficacy of conjunctival auto-transplantation on alkaline chemical burns of the ocular surface. MATERIAL AND METHODS: An alkaline chemical burn model was developed in one eye of rabbits using 0.1 N NaOH solution. In one group conjunctival transplantation was performed. A control group did not receive conjunctival transplantation. A histological follow-up study was performed by light microscopy with hematoxylin eosin stain and periodic acid Schiff staining by fluoro-microscopy using epithelial Keratin-AE 5 antibody, and by transmission electron microscopy(TEM). RESULTS: The transplanted group showed scar formation between the transplanted conjunctival tissue and the sclera. At 20 days and 8 weeks after the transplantation, neovascularization and cell infiltration in the subepithelium of the limbus was decreased compared with the transplanted group at 10 days. The control group showed a decrease of cell infiltration in the limbal area compared with the group at 10 days, but conjunctival tissue with thick collagenous tissue and neovascularization instead of scar tissue developed on the injured cornea. AE 5 positive cells were not found in the limbus in either group. In the transplanted group, in TEM, the basement membrane of transplanted conjunctiva showed less irregularity, and degenerated fibroblasts were present at the margin of the scar tissue. In the control group, the basement membrane of the conjunctiva showed an irregular pattern and fibroblasts beneath the conjunctival epithelium had large nuclei. CONCLUSION: In the transplanted group, scar tissue developed and suppressed cell infiltration, neovascularization, and conjunctival tissue in the injured cornea and secured re-structuring of the limbal tissue.     

羊膜移植结膜囊成形术临床报告

目的：观察和评价羊膜在结膜囊成形术中的应用价值。方法：采用保存人羊膜移植于结膜囊狭窄的患者行结膜囊成形术9例9只眼。结果：9只眼无1例感染，7天后移植的羊膜变透明，缝合处结膜向羊膜向羊膜上爬行。1个月后新生的结膜上皮安全覆盖移植区，结论：保存的人羊膜无抗原性，移植后能作为结膜上皮移行生长的载体，替代球结膜用于结膜囊成形术的效果满意。     

Immunohistochemical and ultrastructural analysis of dysplastic epithelium of human ocular surface: basement membrane and intermediate filament.

PURPOSE: The dysplastic corneal epithelium is characterized by the abnormal proliferation of epithelial cells. The phenotypes of these cells have not been elucidated. We investigated whether such epithelium expresses the phenotypes of corneal or conjunctival epithelial cells. METHODS: The corneas and conjunctivae from four normal subjects and from one patient with epithelial dysplasia of the central cornea were immunostained for IV and VII collagens and for cytokeratins. Monoclonal antibodies against collagen IV reacted to the [alpha1(IV)]2alpha2(IV) or alpha5(IV) molecule. Anti-cytokeratin antibodies were used to define epithelial cell types. The ultrastructure of the basement membrane (BM) of each specimen also was examined. RESULTS: Type VII collagen immunoreactivity was detected in all the specimens of epithelial BM. The anti-collagen IV [alpha1(IV)]2alpha2(IV) antibody labeled the conjunctival BMs, not the BMs of the corneal epithelia, of each subject. The normal corneal epithelial BM, not the BM of the conjunctival or dysplastic corneal epithelium, was immunolabeled with anti-alpha5(IV) antibody. The pattern of cytokeratin expression in the corneal epithelial dysplasia resembled that seen in the normal conjunctivae. Small breaks in the BM of dysplastic corneal epithelium were ultrastructurally revealed. The number of hemidesmosomes in the dysplastic corneal epithelium was decreased as compared with that in the normal BM. CONCLUSION: The composition of collagen types within the BM and the cellular phenotype of the dysplastic epithelium in the cornea resembled those of conjunctival epithelium, not of the cornea.     

Autologous cultivated conjunctival transplantation for recurrent viral papillomata

PURPOSE: To describe the use of an autologous serum-free derived cultivated conjunctival epithelial equivalent in the treatment of extensive recurrent viral papillomata. DESIGN: Interventional case report. METHODS: A 10-year-old child with extensive recurrent viral papillomata involving the superior and inferior tarsal, forniceal, and bulbar conjunctiva underwent surgical excision of all diseased areas and double freeze-thaw cryotherapy. Autologous serum-free cultivated conjunctival equivalents were used to reconstruct the ocular surface and conjunctival fornices. RESULTS: Almost complete epithelialization was achieved within 5 days postoperatively. A good cosmetic and functional result was obtained, and no recurrences or cicatricial complications developed during 12-month follow-up. CONCLUSION: Transplantation of autologous cultivated conjunctiva was effectively used in the reconstruction of the ocular surface after extensive excision of recurrent viral papillomata. This modality of treatment may be useful in the treatment of ocular surface disorders in which extensive conjunctival and fornix reconstruction is required.     

Conjunctival epithelial cell differentiation on amniotic membrane

PURPOSE: Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation. METHODS: Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS: In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid. CONCLUSIONS: RCEs cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further.     

Ultrastructural changes in corneas of diabetic patients: an electron-microscopy study

BACKGROUND: Although diabetic retinopathy has been thoroughly studied, little attention has been given to the corneal changes of diabetic patients. Pathophysiologic and clinical findings may be related to the ultrastructural changes found in these corneas. PURPOSE: To investigate the ultrastructural corneal changes of diabetic patients. PATIENTS AND METHODS: Transmission electron microscopic ultrathin sections were prepared from corneas of 16 noninsulin-dependent diabetic patients (mean age, 65 years; range, 40-82 years) who suffered from the disease for a mean period of 22 years (range, 10-30 years). We used 16 corneas from healthy age-matched donors as normal controls. RESULTS: In addition to the epithelial changes that include accumulation of glycogen granules, occasional focal epithelial cell degeneration, and irregular thickening and multilamination of the epithelial basement membrane, unusual 120-nm wide-spaced collagen fibril bundles were observed scattered among both Descemet's membrane and stromal matrix. CONCLUSIONS: The aggregates of wide-spaced collagen fibrils, which have not been described in other basement membranes of diabetic patients, may reflect an excessive glycosylation rate.     

The surface morphology of retinal breaks and lattice retinal degeneration. A scanning electron microscopic study

In 14 of 110 eye bank eyes, lesions characteristic of peripheral retinal surface pathology were examined by scanning electron microscopy (SEM). These included operculated and flap tears, trophic round holes, lattice degeneration with holes, and paravascular retinal "pitting" degeneration. By SEM, the edges of the retinal breaks were covered by smooth cellular membranes, merging peripherally with a meshwork of vitreous fibrils. The membrane cells had poorly defined borders, a pitted surface, and variable numbers of microvilli consistent with glia. Lattice surfaces and foci of paravascular retinal degeneration were covered by similar membrane, but showed characteristic differences. It appears that breaks in the internal limiting membrane always stimulate proliferation of preretinal glial membranes. Similar cellular morphology of the membranes associated with breaks is consistent with a common cell of origin. Limited proliferation of these membranes suggests that surface gliosis is normally inhibited when the cells contact either intact basement membrane or vitreous.