Corneal Guttae After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The aim of this study was to report on the occurrence of corneal guttae after Descemet membrane endothelial keratoplasty (DMEK). METHODS: In this retrospective case series, 13 eyes of 13 patients who underwent DMEK at 2 tertiary referral centers between 2007 and 2021 (average available follow-up 73 ± 52 months, range 18-174 months) and showed corneal guttae during postoperative examinations were included. Eye bank images were retrospectively reviewed. RESULTS: Occurrence of guttae was observed by specular microscopy in 13 eyes. In 11 cases, presence of guttae was confirmed by confocal microscopy and in 1 case by histology. Five eyes showed an increase in guttae density during the postoperative course. Surgery indications were Fuchs endothelial corneal dystrophy (n = 11), pseudophakic bullous keratopathy (n = 1), and DMEK graft failure after allograft rejection (n = 1); the latter eye had shown no signs of guttae after primary DMEK. Two eyes with guttae required a repeat DMEK due to graft failure. At the last available follow-up, all 11 remaining eyes had clear corneas and 10 eyes had a best-corrected visual acuity of ≥0.9 (decimal). During donor cornea processing in the eye bank, no guttae were observed on the donor tissue. CONCLUSIONS: Corneal guttae can occur after DMEK including in eyes operated for indications other than Fuchs endothelial corneal dystrophy and most likely guttae were present on the donor graft but were not detectable by routine slit-lamp and light microscopy evaluation in the eye bank. Postoperative guttae density varies among patients and especially small isolated guttae do not seem to affect clinical outcomes.

Early and late-onset cell migration from peripheral corneal endothelium.

In this study we describe peripheral corneal endothelial cell migration in vitro in the absence and presence of a ROCK-inhibitor. For this study, 21 corneal endothelial graft rims, with attached trabecular meshwork (TM), were prepared from Descemet membrane-endothelial cell sheets by 6.5 mm trepanation. For the initial proof-of-concept, 7 outer graft rims were cultured in a thermo-reversible hydrogel matrix for up to 47 days. To assess the effect of a ROCK-inhibitor, 14 paired outer rims were cultured either with or without ROCK-inhibitor for up to 46 days. At the end of culture, tissue was retrieved from the hydrogel matrix and examined for cell viability and expression of different endothelial cell markers (ZO-1, Na+/K+-ATPase, NCAM, glypican, and vimentin). All cultured rims remained viable and displayed either single regions (n = 5/21) or collective areas (n = 16/21) of cell migration, regardless of the presence or absence of ROCK-inhibition. Migration started after 4±2 days and continued for at least 29 days. The presence of ROCK-inhibitor seemed to contribute to a more regular cell morphology of migrating cells. In addition, 7 outer rims demonstrated a phenotypically distinct late-onset but fast-growing cell population emerging from the area close to the limbus. These cells emerged after 3 weeks of culture and appeared less differentiated compared to other areas of migration. Immunostaining showed that migrated cells maintained the expression patterns of endothelial cell markers. In conclusion, we observed 2 morphologically distinct migrating cell populations with the first type being triggered by a broken physical barrier, which disrupted contact inhibition and the second, late-onset type showing a higher proliferative capacity though appearing less differentiated. This cell subpopulation appeared to be mediated by stimuli other than loss of contact inhibition and ROCK-inhibitor presence. Further exploration of the differences between these cell types may assist in optimizing regenerative treatment options for endothelial diseases.

Corneal endothelial wound healing: understanding the regenerative capacity of the innermost layer of the cornea.

Currently, there are very few well-established treatments to stimulate corneal endothelial cell regeneration in vivo as a cure for corneal endothelial dysfunctions. The most frequently performed intervention for a damaged or dysfunctional corneal endothelium nowadays is corneal endothelial keratoplasty, also known as lamellar corneal transplantation surgery. Newer medical therapies are emerging and are targeting the regeneration of the corneal endothelium, helping the patients regain their vision without the need for donor tissue. Alternatives to donor tissues are needed as the aging population requiring transplants, has further exacerbated the pressure on the corneal eye banking system. Significant ongoing research efforts in the field of corneal regenerative medicine have been made to elucidate the underlying pathways and effector proteins involved in corneal endothelial regeneration. However, the literature offers little guidance and selective attention to the question of how to fully exploit these pathways. The purpose of this paper is to provide an overview of wound healing characteristics from a biochemical level in the lab to the regenerative features seen in the clinic. Studying the pathways involved in corneal wound healing together with their key effector proteins, can help explain the effect on the proliferation and migration capacity of the corneal endothelial cells.

17†Cell viability after DMEK preparation.

PURPOSE: To evaluate the effect of graft preparation and organ-culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts. METHODS: DMEK grafts (n=27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the COVID-19-related cancellation of elective surgeries. Cell viability (by Calcein-AM staining) and ECD of 5 grafts originally scheduled for transplantation, were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post-preparation or after 3-7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein-AM staining (Calcein-ECD) RESULTS: Light microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein-ECD for the 5 grafts initially allocated for transplantation was 18% (range 9-73%) lower than median LM ECD. For the paired DMEK grafts, Calcein-ECD determined by Calcein-AM staining on the day of graft preparation and after 3-7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3-7 days of graft storage was 88% and 92%, respectively. CONCLUSIONS: Cell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation with insignificant additional ECD changes during 3-7 days of graft storage. Implementing an additional post-preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications.

Endothelial Cell Viability after DMEK Graft Preparation.

Aim: To evaluate the effect of graft preparation and organ-culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts.Materials and methods: DMEK grafts (n = 27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the Covid-19-related cancellation of elective surgeries. Cell viability (by Calcein-AM staining) and ECD of five grafts originally scheduled for transplantation were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post-preparation or after 3-7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein-AM staining (Calcein-ECD).Results: Light microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein-ECD for the five grafts initially allocated for transplantation was 18% (range 92-73%) lower than median LM ECD. For the paired DMEK grafts, Calcein-ECD determined by Calcein-AM staining on the day of graft preparation and after 3-7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3-7 days of graft storage was 88% and 92%, respectively.Conclusion: Cell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation, with insignificant additional ECD changes during 3-7 days of graft storage. Implementing an additional post-preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications.

Preclinical testing of small diameter Descemet membrane endothelial keratoplasty grafts to increase tissue availability.

In this study, we describe a process of preparing, surgically manipulating, and validating a novel "small diameter" 4mm circular Descemet membrane endothelial keratoplasty (DMEK) graft in vitro. Three small diameter DMEK grafts can be prepared from a single donor endothelium and could, therefore, potentially expand the donor pool. Prior to clinical use, however, we aimed to examine each step of the process to determine the effect on the endothelial cell loss and whether or not cells retained their capacity to migrate uniformly. For this study, circular small diameter grafts, obtained from twelve corneas of ten donors deemed ineligible for transplantation, were included. Small diameter DMEK graft preparation was successful in all cases (n = 36). Endothelial cell density (ECD), determined in the eye bank on seventeen grafts, showed an average decrease from 2413 (±189) cells/mm2 before to 2240 (±413) cells/mm2 after preparation. Twenty-four grafts were used to simulate DMEK-surgery in vitro and were successfully stained with 0.06% trypan blue, loaded into a straight DMEK-injector, unfolded, positioned, and centered within the circular ~ 4mm descemetorhexis. The estimated % area populated by viable cells on the grafts decreased from on average 92 (±3) % before to 78 (±10) % (n = 4) after in vitro surgery. Cells displayed a capacity for uniform cell migration from all edges of the graft (n = 4) when embedded in the 3D hydrogel system. Our data show, that by using an in vitro model of DMEK-surgery it was possible to test the 4mm circular DMEK grafts from eye bank preparation to surgical implantation. The cell loss after in vitro surgery was comparable with the in vivo ECD decline early after DMEK and the capacity of the cells to migrate to potentially cover bare stroma indicates that these small diameter grafts may be a viable clinical option to treat central endothelial disease.

New developments in corneal endothelial cell replacement.

Corneal transplantation is currently the most effective treatment to restore corneal clarity in patients with endothelial disorders. Endothelial transplantation, either by Descemet membrane endothelial keratoplasty (DMEK) or by Descemet stripping (automated) endothelial keratoplasty (DS(A)EK), is a surgical approach that replaces diseased Descemet membrane and endothelium with tissue from a healthy donor eye. Its application, however, is limited by the availability of healthy donor tissue. To increase the pool of endothelial grafts, research has focused on developing new treatment options as alternatives to conventional corneal transplantation. These treatment options can be considered as either 'surgery-based', that is tissue-efficient modifications of the current techniques (e.g. Descemet stripping only (DSO)/Descemetorhexis without endothelial keratoplasty (DWEK) and Quarter-DMEK), or 'cell-based' approaches, which rely on in vitro expansion of human corneal endothelial cells (hCEC) (i.e. cultured corneal endothelial cell sheet transplantation and cell injection). In this review, we will focus on the most recent developments in the field of the 'cell-based' approaches. Starting with the description of aspects involved in the isolation of hCEC from donor tissue, we then describe the different natural and bioengineered carriers currently used in endothelial cell sheet transplantation, and finally, we discuss the current 'state of the art' in novel therapeutic approaches such as endothelial cell injection.

Improving Endothelial Explant Tissue Culture by Novel Thermoresponsive Cell Culture System.

AIM: Studying cell migration of corneal endothelial cells in vitro is challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, we report a thermoresponsive culture technique designed to maintain cellular viability, and to reduce tissue handling in order to analyze in vitro endothelial cell migration from corneal grafts. MATERIALS AND METHODS: As a test tissue, fifteen Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts were used that were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2-3 weeks in a humidified atmosphere at 37°C and 5% CO2. RESULTS: All grafts could be successfully cultured inside the thermoresponsive polymer solution for periods of up to 21 days. Using this system, cell migration could be assessed by light microscopy at fixed time intervals. At the end of the culture period, the gel could be removed from all grafts and immunohistochemistry analysis showed that endothelial cells were able to maintain confluence, viability, and junctional integrity. Some problems were encountered when using the thermoresponsive cell culture system. These were mostly structural inconsistencies during the sol-to-gel transition phase that resulted in the formation of tiny bubbles in the matrix. Additionally, areas with different viscosity resulted in optical distortions showing up as folds throughout the matrix which can persist even after several cycles of culture medium exchange. These effects had impact on the imaging quality but did not affect the viability of the explant tissue. CONCLUSION: This study proves that temperature-reversible hydrogel is a very useful matrix for studying in vitro corneal endothelial cell migration from explant grafts and allows for subsequent biological investigation after gel removal.

Clinical and morphological outcomes after percutaneous needle fasciotomy in Dupuytren's disease according to the contracture severity.

BACKGROUND AND OBJECTIVES: Dupuytren's disease (DD) is a chronic progressive disorder causing contractures in the palmar and digital fascia. The primary aim of management is to correct the deformity while reducing the risk of recurrence and avoiding complications. The purpose of our retrospective study was to validate the efficacity, safety and the rate of recurrence of DD after percutaneous needle fasciotomy (PNF). PATIENTS, MATERIALS AND METHODS: We present results for a population of 40 patients, divided into three groups, based on the severity of the contracture according to Tubiana staging. We analyzed patient demographic data, encountered complications and the degree of release achieved both post-procedure and at one-year follow-ups. RESULTS: From a total of 98 joints we treated, we obtained satisfactory release [passive extension deficit (PED) ≤10°] in 85% of joints, while full release was obtained in 70% of joints. Full release was obtained mostly in metacarpophalangeal (MCP) joint (95%) rather than proximal interphalangeal (PIP) joint (50%) or distal interphalangeal (DIP) joint (60%). Our patient preferred PNF in comparison to open fasciectomy because of its minimum invasive approach and the quick recovery time. CONCLUSIONS: The study shows that PNF has great short-term results and it is suitable for patients who prefer minimally invasive care.

The influence of preparation and storage time on endothelial cells in Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) grafts in vitro.

Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) has been introduced as a modification of the standard DMEK technique to increase the pool of endothelial grafts. In this study, we evaluated in vitro changes in endothelial cell distribution, viability and morphology of Quarter-DMEK grafts when stored in organ-culture medium. Quarter-DMEK grafts were prepared from 5 corneas and stored in organ-culture medium for 4, 7 and 11 days. Endothelial cell re-distribution was investigated by light microscopy, cell viability by a Calcein-AM assay, and expression of endothelial and non-endothelial markers by immunohistochemistry. Three standard DMEK-grafts were used as controls. After preparation, all Quarter-DMEK grafts showed a band with no viable endothelial cells along the radial cut graft edges [average width 190 (± 20) µm]. Endothelial cell density in the central graft area decreased by 12%, 23% and 26% after 4, 7, and 11 days of storage, respectively. At the same time, empty bands along the cut edges were re-populated and some cells migrated to the stromal side of the Descemet membrane (DM). These cells showed an altered phenotype, as indicated by expression of migration marker CD73 and fibroblast marker αSMA. Majority of migration occurred within the first 4 days of storage. Our data suggest that endothelial cells on Quarter-DMEK grafts re-distribute during organ-culture storage to re-populate preparation-induced empty bands and after re-distribution, cells may show further migration to the stromal DM side during storage.

In Vitro Evaluation and Transplantation of Human Corneal Endothelial Cells Cultured on Biocompatible Carriers.

Corneal transplantation is currently the only effective treatment option for dysfunctional corneal endothelial cells (CEC). In this study, we test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), LinkCell™ bioengineered collagen sheets of 20-µm thickness (LK20), and denuded Descemet membrane (dDM) as tissue-engineered grafts for Descemet membrane (DM) endothelial keratoplasty (DMEK) to bypass the problem of donor tissue availability. Primary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96% ± 4%). hCEC on HALC and LK20 showed unremarkable expression of zonula occludens-1 (ZO-1) and Na+/K+-adenosine triphosphatase (ATPase), while Na+/K+-ATPase expression of cells seeded on dDM was mainly cytoplasmic. All hCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using a human donor cornea without DM mounted on an artificial anterior chamber (AC) and a regular DMEK-graft used as a surgical reference model. During in vitro surgery, hCEC-HALC constructs behaved most similarly to a DMEK-graft during implantation and unfolding, showing good adhesion to the bare stroma. On the other hand, hCEC-LK20 and hCEC-dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC-dDM constructs showed similar graft adherence as hCEC-HALC constructs, while adherence of hCEC-LK20 constructs was less effective. After the in vitro surgery, the estimated area populated by viable cells on the hCEC-HALC and hCEC-LK20 constructs was ∼83% and ∼67%, respectively. Overall, hCEC-HALC constructs behaved most similarly to a DMEK-graft during in vitro DMEK surgery, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC-LK20 and hCEC-dDM constructs.

Göttingen Minipig is not a Suitable Animal Model for in Vivo Testing of Tissue-Engineered Corneal Endothelial Cell-Carrier Sheets and for Endothelial Keratoplasty.

AIM: To test the feasibility of implanting human anterior lens capsules (HALCs) with porcine corneal endothelial cells (pCEC) in vivo in Göttingen minipigs and at the same time test the suitability of Göttingen minipig as model for endothelial keratoplasty. MATERIALS AND METHODS: Cell-carrier constructs of decellularized HALC with cultured (pCEC) were created for implementation in vivo. Eight Göttingen minipigs (6 months old) underwent surgery with descemetorhexis or removal of endothelium by scraping and implementation of HALC without (animal 1-4) and with (animal 5-8) pCEC. Follow-up examinations included optical coherence tomography (OCT) imaging (1,2 and 3 months) and slit-lamp examination (<1 week as well as 1,2 and 3 months). RESULTS: Intraoperative challenges included difficulties in maintaining an anterior chamber due to soft tissue and vitreous pressure, development of corneal edema and difficulties removing Descemet's membrane because of strong adhesion to stroma. Therefore, descemetorhexis was replaced by mechanical scraping of the endothelium in animal 4-8. HALCs without pCEC were implanted in animal 1-4. Apposition to the back surface was not achieved in animal 1 and 3 because of corneal edema and poor visibility. Animal 5 was sacrificed because of a lens capsule tear. HALCs with pCEC were implanted in animal 6-8. Slit-lamp examination the first week revealed corneal edema in all animals, although mild in animals 4. One-month examination showed retrocorneal membranes with overlying corneal edema in all animals. Histology showed fibrosis in the AC and on the back surface of the cornea, compatible with the clinical diagnosis of retrocorneal membrane. CONCLUSIONS: In conclusion, the minipig is not suitable for corneal transplantation studies in vivo because of intraoperative challenges and development of retrocorneal membrane postoperatively. For in vivo testing of the surgical handling and the therapeutic potential of tissue-engineered endothelial cell-carrier constructs other animal models are required.

Effect of Surgical Indication and Preoperative Lens Status on Descemet Membrane Endothelial Keratoplasty Outcomes.

PURPOSE: To analyze 6-month results of 1000 consecutive Descemet membrane endothelial keratoplasty (DMEK) cases, and to evaluate if outcomes are influenced by surgical indication and preoperative lens status. DESIGN: Retrospective, interventional case series. METHODS: A series of 1000 eyes (738 patients) underwent DMEK mainly for Fuchs endothelial corneal dystrophy (FECD; 85.3%) or bullous keratopathy (BK; 10.5%). Main outcome measures were best-corrected visual acuity (BCVA), endothelial cell density, postoperative complications, and retransplantations. RESULTS: At 6 months after DMEK, there was no difference in BCVA outcome between FECD and BK eyes (P = .170), or between phakic and pseudophakic FECD eyes (P = .066) after correcting for patient age and preoperative BCVA. Endothelial cell loss at 6 months postoperatively was similar for phakic and pseudophakic FECD eyes (39%; P = .852), but higher for BK eyes than for FECD eyes (46% vs 39%, P = .001). Primary and secondary graft failure occurred in 3 (0.3%) and 2 eyes (0.2%), respectively, and 7 eyes developed allograft rejection (0.7%). Eighty-two eyes (8.2%) received rebubbling for graft detachment and retransplantation was performed in 20 eyes (2.0%). Rebubbling was more often required in eyes treated for BK vs FECD eyes (12.4% vs 7.4%, P = .022). CONCLUSION: DMEK consistently provides excellent short-term results, with similar high visual acuity levels for both FECD and BK eyes. As preoperative lens status did not influence DMEK outcomes, for phakic FECD eyes with a still relatively clear crystalline lens, lens preservation may be preferable in a selected group of younger patients, who may still benefit from their residual accommodative capacity.

In vitro endothelial cell migration from limbal edge-modified Quarter-DMEK grafts.

Endothelial cell migration plays a crucial role in achieving corneal clearance after corneal transplantation when using smaller-sized endothelial grafts to increase the donor pool. In this study we investigated how different strategies of Quarter-Descemet Membrane Endothelial Keratoplasty (Quarter-DMEK) limbal graft edge modification influence peripheral endothelial cell migration in an in vitro culture environment. For this study, 15 Quarter-DMEK grafts, prepared from 7 corneas deemed ineligible for transplantation but with intact and viable endothelial cells, were embedded in a cooled biocompatible, thermoresponsive matrix for culture. The limbal edge of ten Quarter-DMEK grafts were modified, either by using a small diameter punch or by peripheral radial cuts. All Quarter-DMEK grafts showed substantial collective endothelial cell migration from the radial cut graft edges, as observed by light microscopy at standardized time intervals. Grafts were retrieved from the polymer matrix after the two-week culture for immunohistochemistry analyses of the newly formed cell monolayers; this demonstrated the presence of tightly packed and viable cells that showed higher migratory ability at the leading edge. Peripheral endothelial cell migration, however, was not triggered by increasing cell exposure to free space through surgical modifications of the far periphery. Our data suggest that alterations in the far peripheral area of Quarter-DMEK grafts were insufficient to triggering cell migration from the limbal graft edge. This may be due to transient-amplifying cells that reside in the far periphery and which lack cytokinetic directional cues. Understanding the migration capacity of the peripheral endothelium could unlock cells' therapeutic potential which are, at present, routinely discarded from transplantation. Encouraging peripheral cell migration may also improve clinical outcomes from Quarter-DMEK, but a more effective solution is required prior to clinical implementation of modified grafts.

Evaluation of the Suitability of Biocompatible Carriers as Artificial Transplants Using Cultured Porcine Corneal Endothelial Cells.

Purpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK). MATERIALS AND METHODS: pCECs were isolated, cultured up to P2 and seeded onto LinkCell™ bioengineered matrices of 20 µm (LK20) or 100 µm (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM). RESULTS: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 ± 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a "taco" for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. CONCLUSIONS: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.

Asymmetrical endothelial cell migration from in vitro Quarter-Descemet membrane endothelial keratoplasty grafts.

PURPOSE: To investigate in vitro central and peripheral corneal endothelial cell (EC) migration from Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) grafts. METHODS: Quarter-DMEK grafts were obtained from 10 corneas ineligible for transplantation but with intact and viable ECs. Ten Quarter-DMEK grafts were 'sandwiched' between two glass slides and cultured over 1 week in a humidified atmosphere at 37 °C and 5% CO2 . Cell migration was evaluated by light microscopy at standardized time intervals. In addition, immunohistochemistry analyses were performed to assess the detailed structural organization of ECs in the corneal centre and far periphery. RESULTS: Endothelial cell (EC) migration occurred from the radial cut graft edges, but not from the far peripheral area. Cell migration followed three different migration patterns: (1) individual cell migration, (2) uncoordinated cell migration of cell clusters and (3) collective migration in which ECs moved as a sheet. Immunostaining showed the presence of ECs up to the far periphery but with different expression patterns of phenotypical markers ZO-1, Na+ /K+ -ATPase and vimentin compared to central ECs. CONCLUSION: In vitro EC migration from Quarter-DMEK grafts occurs along the radial cut edges with a decrease in migration activity towards the corneal far periphery. No migration occurred along the outer peripheral corneal edge possibly due to a different anatomical matrix in the far periphery. Hence, ECs from the far periphery may not contribute to corneal clearance of the adjacent bare area after Quarter-DMEK surgery, but these cells may constitute a valuable cellular reserve on the graft.

In Vivo Endothelial Cell Density Decline in the Early Postoperative Phase After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate endothelial cell density (ECD) in the first 6 months after Descemet membrane endothelial keratoplasty (DMEK) by eliminating method error as a confounding variable. METHODS: From 24 DMEK eyes operated for Fuchs endothelial corneal dystrophy, from which specular microscopy images could be taken at 1 day and 6 months postoperatively, ECD values were compared between these 2 time points. RESULTS: Using the 1-day ECD measurement as baseline, mean ECD decreased from 1913 (±326) cells/mm to 1524 (±393) cells/mm at 6 months, a decline of -18 (±19)%. With the 1-week ECD as baseline [1658 (±395) cells/mm], the decline at 6 months was -6 (±19)% and when using preoperative ECD as baseline [2521 (±122) cells/mm], the decline was -39 (±16)% at 6 months. CONCLUSIONS: After DMEK, ECD shows an in vivo decline of 18% from 1 day to 6 months postoperatively, with a sharp 13% drop in the first week, and a slower decrease thereafter. The remaining difference of 20% from preoperative ECD values may be attributed to a measurement error in the eye bank with an overestimation of the graft's viable endothelial cell population and/or intraoperative trauma to the graft.

A case of severe corneal flattening after Descemet stripping endothelial keratoplasty.

PURPOSE: To report a case of extreme corneal flattening after initially uneventful Descemet stripping endothelial keratoplasty (DSEK). METHODS: A 76-year-old man underwent uneventful DSEK for pseudophakic bullous keratopathy (PBK) in his left eye. Postoperative evaluations were performed using anterior segment optical coherence tomography and biomicroscopy. RESULTS: The DSEK graft showed full adherence to the host stroma. Two weeks after surgery, the transplanted cornea developed pronounced flattening of the central corneal curvature accompanied by graft folds and remained unchanged until the most recent follow-up 20 weeks after surgery. CONCLUSIONS: Severe corneal flattening may be an undescribed complication occurring after uneventful DSEK, without any concomitant ocular pathology or detectable cause.

An Evaluation of the Pedestrian Classification in a Multi-Domain Multi-Modality Setup.

The objective of this article is to study the problem of pedestrian classification across different light spectrum domains (visible and far-infrared (FIR)) and modalities (intensity, depth and motion). In recent years, there has been a number of approaches for classifying and detecting pedestrians in both FIR and visible images, but the methods are difficult to compare, because either the datasets are not publicly available or they do not offer a comparison between the two domains. Our two primary contributions are the following: (1) we propose a public dataset, named RIFIR , containing both FIR and visible images collected in an urban environment from a moving vehicle during daytime; and (2) we compare the state-of-the-art features in a multi-modality setup: intensity, depth and flow, in far-infrared over visible domains. The experiments show that features families, intensity self-similarity (ISS), local binary patterns (LBP), local gradient patterns (LGP) and histogram of oriented gradients (HOG), computed from FIR and visible domains are highly complementary, but their relative performance varies across different modalities. In our experiments, the FIR domain has proven superior to the visible one for the task of pedestrian classification, but the overall best results are obtained by a multi-domain multi-modality multi-feature fusion.

Sulfated hyaluronan influences the formation of artificial extracellular matrices and the adhesion of osteogenic cells.

The aim of this study is to compare differentially sulfated hyaluronan (sHA) derivatives and chondroitin sulfate (CS) with respect to their ability to influence the formation of artificial extracellular matrices (aECMs) during in vitro-fibrillogenesis of collagen type I at high- and low-ionic strength. Analysis is performed using turbidity, biochemical assays, atomic force (AFM), and transmission electron microscopy (TEM). In general, high-sulfated glycosaminoglycans (GAGs) associate to a higher amount with collagen than the low-sulfated ones. The addition of GAGs prior to fibrillogenesis at low-ionic strength results in a dose-dependent decrease in fibril diameter. At high-ionic strength these effects are only obtained for the sHA derivatives but not for CS. Likewise, increasing concentrations and degree of GAG sulfation strongly affected the kinetics of fibrillogenesis. The impact of sulfation degree on F-actin location and fiber formation in SaOS-2 cells implies that adhesion-related intracellular signaling is influenced to a variable extent.