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.</p

Spontaneous full thickness macular hole development and closure in a patient with nucleus dislocation due to hypermature cataract: a case report

Spontaneous posterior capsule rupture with lens-nucleus dislocation is a very rare entity, as is the development and spontaneous closure of a full thickness macular hole (FTMH) after vitrectomy. The occurrence of these two entities in one eye has not been previously described. A 79- year-old woman was referred because of the right eye intermittent pain and progressive visual loss. Best corrected visual acuity (BCVA) with correction for aphakia was 20/20. Intraocular pressure was normal with therapy. The cornea, anterior chamber, and vitreous were clear. Gonioscopy was normal. The capsular bag was clear, with rolled-up anterior and posterior lens capsule, and the nucleus dislocated in the vitreous. As surgery waiting time was prolonged due to administrative problems, the patient’s intraocular pressure (IOP) increased and cystoid macular edema (CME) with lamellar macular hole developed. The patient underwent pars plana vitrectomy with endophacofragmentation and epiretinal membrane peeling. Postoperative optical coherence tomography was normal, BCVA was 20/40, and IOP was normal with topical therapy. One month after surgery, the eye was without signs of inflammation and IOP started rising in spite of maximum therapy. CME reoccurred and progressed to a FTMH, which started closing spontaneously in one month. One year after surgery, IOP normalized and FTMH closed completely. A dislocated crystalline lens in a quiet eye with normal BCVA, which rapidly developed into intractable glaucoma and FTMH, is an unusual finding. The deterioration was followed by spontaneous IOP normalization and macular hole closure. Such unexpected disease course, suggesting a possible autoimmune reaction, has not yet been describe

Amnijska membrana kot biološki nosilec, njena priprava in uporaba v regenerativni medicini v Sloveniji

Izhodišča: Amnijska membrana (AM) je notranja stran posteljice, ki obdaja in ščiti zarodek. AM je večplastna struktura, ki je sestavljena iz amnijskih epitelijskih celic, amnijskih mezenhimskih stromalnih celic, bazalne lamine in vezivnega tkiva. Iz njene zgradbe izhajajo tudi lastnosti AM, zaradi katerih se že vrsto let uporablja v terapevtske namene, predvsem v oftalmologiji, saj pospešuje epitelizacijo, deluje kot substrat za celice, zmanjšuje fibrozo in neovaskularizacijo tkiva ter deluje protimikrobno. Zaradi mehanskih lastnosti AM, ki so posledica predvsem molekul zunajceličnega matriksa bazalne lamine in vezivnega tkiva, se AM v zadnjih letih vedno pogosteje uporablja tudi kot biološki nosilec v regenerativni medicini.   Zaključki: Regenerativna medicina je interdisciplinarno področje raziskav in kliničnih aplikacij, ki uporablja načela bioloških in inženirskih znanosti za razvoj živih tkivnih ali organskih nadomestkov. V regenerativni medicini ločimo tri pristope: 1) vsaditev funkcionalnih celic, med drugim tudi matičnih celic, v poškodovano ali okvarjeno tkivo, 2) uporaba različnih sintetičnih materialov ali materialov naravnega izvora, ki pomagajo pri ponovnem oblikovanju poškodovanega ali okvarjenega tkiva in 3) tkivno inženirstvo, tj. uporaba ustreznih nosilcev, ki spodbujajo rast tkivno specifičnih celic in oblikovanje novega tkiva. V prispevku predstavljamo tudi uporabo amnijske membrane kot biološkega nosilca v regenerativni medicini v Sloveniji

Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures.

To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67

Morphological and flow cytometry profile results of prolonged limbal explant cultures cultivated without amniotic membrane.

<p>Limbal explant outgrowth of cells cultured on plastic culture plates in the absence of feeder layer cells and in medium supplemented with only human serum showed after 3 weeks of culture two morphologically distinct cell population types, a predominantly epithelial-like (black arrow) and a more fibroblast-like cell population (white arrow) (A). A sphere-shaped formation with spindle shaped cells around it was observed (B). (A,B: Magnification:4x). A limbal explant culture after 2 months of culturing without any passaging showing a translucent corneal stromal-like 3D tissue (C) (the total time of observation in culture was 6 months). A plot representing the mean percentages ± SEM (n = 7, paired two-sample t-test) of flow cytometry profiles, red bars representing the primary limbal cultures and white bars the transfered secondary limbal cultures, the differences not being statistically significant (p > 0.05) (D). (*) means limbal explant; (**) means translucent tissue. Bar, 100 μm.</p

Results of flow cytometry profiles of confluent primary limbal explant cultures cultivated on plastic culture plates in only 10% or 20% human serum supplemented medium.

<p>A plot of the mean percentages of positive cells ± SEM for the tested markers in both tested HS concentration groups is shown (Student two-tailed t-test; * p < 0.05).</p

Limbal explant outgrowth of cells cultured on amniotic membrane after 3 days of culture and after 3 weeks of culture.

<p>The cells adjacent to the limbal explants were more uniform, smaller and had larger nuclei (A). In the first week of culture an epithelial line (B) was observed on AM (**), which after 3 weeks of culture reached the AM border (C) (◆) (A,B: Magnification:10x; C: Magnification:4x). Light microscopy showed limbal epithelial cells cultivated on AM (stromal side) produced a well-stratified cell layer (D); (HE: 10x). (*; explant; AM: amniotic membrane; HE: hematoxylin and eosin). Bars, 100 μm.</p

Immunohistochemical comparison of primary and secondary limbal explant cultures cultivated on the epithelial or stromal side of the AM in a human serum supplemented medium.

<p>In primary limbal explant cultures cultivated for 14 days, limbal epithelial growth was observed on both sides of the AM on HE sections (A), similarmorphologywas observed in secondary limbal explant cultures on both sides of the AM after the limbal explants were cultured for 40 days on plastic culture plates and stained positive for pan-cytokeratinmarker after 21 days of culture on AM (B). Cells from primary and secondary cultures on both sides of the AM (explants transferred after 14 days, from the same donor) expressed p63 and Ki67 marker, however in the secondary cultures a slight decrease was observed for Ki67 marker expression (C). (Magnification A:20x, B:10x; C:20x). (AM: amnioticmembrane; epithelial side, + stromal side; HE: hematoxylin and eosin). Bars, 50 μm.</p