Characterization of the Immediate and Delayed Biomechanical Response to UV-A Crosslinking of Human Corneas.

PURPOSE: Keratoconus leads to visual deterioration due to irregular astigmatism and corneal thinning. Riboflavin-based corneal UV-A crosslinking (CXL) induces novel intramolecular and intermolecular links resulting in corneal tissue stiffening, thereby halting disease progression. The purpose of this study was to analyze the immediate and delayed biomechanical responses of human donor corneas to CXL. METHODS: CXL was performed according to the Dresden protocol to corneas not suitable for transplantation. Biomechanical properties were subsequently monitored by measuring the Young modulus using nanoindentation. The immediate tissue response was determined after 0, 1, 15, and 30 minutes of irradiation. Delayed biomechanical effects were investigated with follow-up measurements immediately and 1, 3, and 7 days after CXL. RESULTS: Young's modulus indicated a linear trend in direct response to increasing irradiation times (mean values: total 61.31 kPa [SD 25.53], 0 minutes 48.82 kPa [SD 19.73], 1 minute 53.44 kPa [SD 25.95], 15 minutes 63.56 kPa [SD 20.99], and 30 minutes 76.76 kPa [SD 24.92]). The linear mixed model for the elastic response of corneal tissue was 49.82 kPa + (0.91 kPa/min × time [minutes]); P < 0.001. The follow-up measurements showed no significant delayed changes in the Young modulus (mean values: total 55,28 kPa [SD 15.95], immediately after CXL 56,83 kPa [SD 18.74], day 1 50.28 kPa [SD 14.15], day 3 57.08 kPa [SD 14.98], and day 7 56.83 kPa [SD 15.07]). CONCLUSIONS: This study suggests a linear increase of corneal Young modulus as a function of CXL timing. No significant short-term delayed biomechanical changes posttreatment were observed.

Cataract Surgery-Indications, Techniques, and Intraocular Lens Selection.

BACKGROUND: Opacification of the lens of the eye (cataract) is usually due to aging. It is a painless, progressive condition that affects contrast and color perception and alters refraction, leading to visual loss that may be total. In cataract surgery, the turbid lens is replaced by an artificial lens. An estimated 600 000 to 800 000 such procedures are performed in Germany each year. METHODS: This review is based on pertinent publications retrieved by a selective search in PubMed, including meta-analyses, Cochrane reviews, and randomized controlled clinical trials (RCTs). RESULTS: Cataract is the most common reversible cause of blindness around the world (approximately 95 million people). The surgical replacement of a turbid lens with an artificial lens is usually carried out under local anesthesia. The standard technique for fragmentation of the nucleus of the lens is ultrasonic phacoemulsification. RCTs have not shown the superiority of the femtosecond laser over phacoemulsification for this purpose so far. The spectrum of artificial intraocular lenses, aside from the conventional type with a single focus, include lenses with multiple foci, extended-depth-of-focus (EDOF) lenses, and astigmatism-correcting lenses. CONCLUSION: In Germany, cataract surgery is usually performed on an outpatient basis under local anesthesia. Artificial lenses with various additional functions are available nowadays; the choice of lens depends on the needs of the individual patient. Patients must be adequately informed about the advantages and disadvantages of the different lens systems.

Outcomes and complications of cataract surgery in patients with chronic ocular graft-versus-host-disease-a multicenter, retrospective analysis.

PURPOSE: To evaluate the outcome of phacoemulsification in patients with chronic ocular Graft-versus-host disease (oGVHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). METHODS: Retrospective, observational multicenter study from 1507 oGVHD patients. From the patient files, data were collected including best-corrected visual acuity (BCVA), intraocular pressure (IOP), Schirmer's test I, tear film break-up time (TFBUT), corneal fluorescein staining score, postoperative complications, and pre- and post-operative topical therapy. RESULTS: Seventy-three patients underwent cataract surgery in 104 eyes. In n = 84 eyes, the oGVHD NIH grade was documented; 12% (n = 12) of analyzed eyes were staged oGVHD NIH grade 1, 31% (n = 32) NIH 2 and 39% (n = 41) NIH 3. The mean BCVA improved in 82% of the eyes (n = 86 eyes). BCVA significantly increased from 0.7 ± 0.5 to 0.4 ± 0.4 LogMAR after surgery independent from oGVHD severity. The mean IOP decreased from 14 ± 4 to 13 ± 4 mmHg after surgery. Visual acuity was moderately correlated to the pre-operative degree of corneal staining (Pearson p = 0.26, p = 0.002, Cohen's effect size f = 0.29). The visual acuity decreased by 0.078 LogMar units (95% CI = 0.027-0.141) with each increase of corneal staining by one grade (p = 0.05). After surgery, corneal epitheliopathy increased significantly in 42% (n = 44) of the eyes. Postoperative complications included corneal perforation (n = 6, 6%), cystoid macular edema (n = 4, 4%), and endophthalmitis (n = 1, 1%). CONCLUSION: Phacoemulsification in patients with chronic oGVHD significantly improves visual acuity, but is associated with an increased risk of complications in particular corneal epitheliopathy and corneal perforations.

Quality Assurance in Cataract and Lens Surgery with Special Consideration of Subjective Patient Reported Outcome Measures and Clinical Reported Outcome Measures. / Qualitätssicherungen in der Katarakt- und Linsenchirurgie unter besonderer Berücksichtigung der subjektiven Patientenbeurteilung und der refraktiven Ergebnisse.

BACKGROUND: Results of medical interventions must be documented and evaluated. In studies, this is done with clinical outcomes data (clinician/clinical reported outcome measure, CROM). In the past, less weight has been given to patient surveys with questionnaires (patient reported outcome measure, PROM). PATIENTS/MATERIALS AND METHODS: This retrospective study included 104 eyes from 53 patients. Of these, 35 patients had cataract surgery and 15 patients had a refractive lens exchange. The implanted lenses included 62 trifocal IOLs (Asphina trifiocal 839, Zeiss), 34 trifocal toric IOLs (Asphina trifocal toric 939, Zeiss) and 8 bifocal IOLs (Asphina 808, Zeiss) with the same IOL platform. Patients completed a modified questionnaire before surgery and one year after surgery. We made changes to the CatQuest-9SF questionnaire so as to also document side effects. RESULTS: The effort required by the patients to answer the questionnaire was a burden. Transcribing the data into electronic files so as they could be saved and analyzed was a lot of work for the staff. Among the patients, 88.7% were spectacle-independent in everyday life, and 77.5% for reading. 44.4% had a halo problem. 92% reported the operation as a success. 100% had a prediction error of ≤ ± 0.75 dpt. CONCLUSION: There is a high rate of patient satisfaction with the outcome of the intervention. New questionnaires are needed for new IOLs. The Catquest-9SF is from 2009. Accordingly, revisions and new validation is necessary. Beyond that, only automatic data transfer will reduce the amount of work involved in data input.

[Back-calculation of the keratometer index-Which value would have been correct in cataract surgery?] / Rückrechnung des Keratometerindex ­ Welcher Wert wäre bei der Kataraktchirurgie richtig gewesen?

BACKGROUND AND PURPOSE: In the clinical routine the conversion of corneal radii into corneal refractive power using a keratometer index is rarely discussed. The purpose of this study was to back-calculate the keratometer index in pseudophakic eyes based on the refractive power of the lens, biometric measurements and refraction, and to compare it to clinically established values. PATIENTS AND METHODS: In this retrospective case series 99 eyes of 99 patients without pathological alterations, previous diseases, comorbidities or history of ocular surgery apart from the uneventful cataract surgery were enrolled. In all eyes a CT Asphina 409M(P) (Carl-Zeiss Meditec, Berlin, Germany) had been implanted by two surgeons (EF and PE). For calculation we used shape and power data of the intraocular lens and data from optical biometry (axial length, pseudophakic anterior chamber depth, lens thickness, corneal radius; IOLMaster 700, Carl-Zeiss Meditec, Jena, Germany). The refraction was derived manually with a trial frame (measurement distance 5 m) and autorefractometry (iProfiler, Carl-Zeiss, Jena, Germany). For this three model eyes were used: a thin lens with the nominal refractive power positioned in the equatorial plane (model A) or in the secondary principal plane of the thick lens (model B) as well as a model considering the intraocular lens as a thick lens located at its measured position (model C). RESULTS: Back-calculation of the keratometer index using vergence formulas resulted in a keratometer index based on subjective refraction measurements considering lane distance correction of 1.3307 ± 0.0026/1.3312 ± 0.0026/1.332 ± 0.0027 for model A/model B/model C, respectively. Based on objective refraction measurements (autorefraction calibrated to infinity object distances) resulted in a keratometer index of 1.3301 ± 0.0021/1.3306 ± 0.0021/1.3315 ± 0.0021, for model A/model B/model C, respectively. The keratometer index did not show any trend in linear regression for axial length or corneal radius for any of the three models or for any refraction method. CONCLUSION: The keratometer index derived from back-calculation matched with the Zeiss index (1.332) but was much lower compared to other established indexes, e.g. the Javal index (1.3375). The missing trend for axial length or corneal radius implies that simple vergence formulas for intraocular lens refractive power calculation without correction terms or fudge factors perform best with a keratometer index slightly below 1.332, if the biometrically measured position of the intraocular lens is used as the effective lens position.

Semifluorinated Alkane Eye Drops in Chronic Ocular Graft-versus-Host Disease: A Prospective, Multicenter, Noninterventional Study.

PURPOSE: Ocular graft-versus-host disease (oGvHD) following allogeneic hematopoietic stem cell transplantation develops as severe dry eye disease (DED) and is initially treated with lubricants, although no clinical trials are available using artificial tears in oGvHD. This trial was set up to test perfluorohexyloctane (NovaTears®) as nonpreserved layer-forming agent for the treatment of DED in oGvHD. METHODS: 25 patients with severe DED due to oGvHD received 1 drop perfluorohexyloctane 4 times daily during a prospective, multicenter, observational 12-week study on top of established topical therapy. Clinical parameters included Schirmer test, tear film breakup time, corneal staining, meibum secretion and ocular surface disease index. Adverse events, visual acuity and intraocular pressure were key safety parameters. RESULTS: From 25 patients recruited, 23 presented for the second visit. Perfluorohexyloctane treatment did not lead to any changes in clinical or safety parameters but led to fast relief in symptoms in 57% of the patients. One adverse reaction occurred. CONCLUSIONS: This study showed no change in clinical signs in severe DED due to oGvHD, which was not unexpected due to the underlying pathomechanisms. However, the study showed improvement of symptoms in individual patients allowing application of perfluorohexyloctane as an additional symptomatic therapy in oGvHD.

Influence of Postoperative Intraocular Pressure on Graft Detachment After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) seems to be the most promising technique for treating endothelium-related disorders of the cornea. However, graft detachment is a frequent complication. It often can be treated by rebubbling procedures. Unfortunately, this increases the risk of subsequent graft failure. It is therefore essential to define risk factors for graft detachment in DMEK. In this study, postoperative intraocular pressure (IOP) is investigated in this regard. METHODS: In total, 1047 DMEK procedures in patients with Fuchs endothelial dystrophy or bullous keratopathy were reviewed. All rebubbling procedures were recorded as well as postoperative IOP values (first measure 2 hours postoperatively and highest and lowest value within 3 days after surgery), donor factors (donor age, organ culture time, and endothelial cell count), and whether DMEK surgery had been combined with cataract surgery (triple-DMEK). Incidence rates were estimated with the Kaplan-Meier method. Cox regression was used to assess statistical significance. RESULTS: The overall rebubbling rate was 24% after 8 weeks. Relative dips in IOP may be associated with a higher risk for graft detachment (hazards ratio = 0.41; P < 0.001). Graft detachment was neither significantly influenced by donor factors, first or maximum IOP, nor by simultaneous cataract surgery (triple-DMEK). CONCLUSIONS: Avoiding dips in IOP may reduce the risk for graft detachment after DMEK. To prevent lowering of IOP values in the early postsurgical period, it may be advisable to achieve tight surgical wounds at the end of DMEK surgery and optimal filling of the anterior chamber with air and optionally with gas.

Nanofibered Gelatin-Based Nonwoven Elasticity Promotes Epithelial Histogenesis.

Regarding tissue regeneration, mechanics of biomaterials gains progressive importance. Therefore, this study reports on in situ crosslinked electrospun gelatin nonwoven mats (NWMs) whose distinct modulus of elasticity (ME) promotes epithelial tissue formation in a graded manner. NWMs, comprising fiber diameters in various distributions, yield an ME of about 2.1, 3.2, and 10.9 kPa. A two-step approach of preclinical in vitro validation identifies the elasticity of 3.2 kPa as superior to the other, regarding the histogenetic epithelial outcome. Hence, this 3.2 kPa candidate NWM is colonized with oral mucosal epithelial keratinocytes in the absence or presence of mesenchymal fibroblasts and/or endothelial cells. Evaluation of epithelial histogenesis at days 1 to 10 occurs by colorimetric and fluorescence-based immunohistochemistry (IHCH) of specific biomarkers. These include cytokeratins (CK) 14, CK1, and involucrin that indicate different stages of epithelial differentiation, as well as the basement membrane constituent collagen type IV and Ki-67 as a proliferation marker. Intriguingly, histogenesis and IHCH reveal the best resemblance of the native epithelium by the NWM alone, irrespective of other cell counterparts. These findings prove the gelatin NWM a convenient cell matrix, and evidence that NWM mechanics is important to promote epithelial histogenesis in view of prospective clinical applications.

Use of Donor Corneas From Pseudophakic Eyes for Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The prevalence of pseudophakia increases with age and affects more than 30% of people older than 80 years. Because grafts from older donors seem to be more suitable for Descemet membrane endothelial keratoplasty (DMEK) surgery rather than grafts from younger donors, eye bank technicians and corneal surgeons have to deal with corneas from pseudophakic donors for DMEK graft preparation. However, graft preparation from corneas of pseudophakic donors can be more complicated. METHODS: We retrospectively analyzed our donor registry for the prevalence of pseudophakia. We compared grafts from phakic and pseudophakic donors concerning graft survival, rebubbling rates, and endothelial cell loss after DMEK surgery. RESULTS: More than 20% of our donors were pseudophakic, of which 35% were primarily suitable for transplantation. Regarding the comparison of grafts from phakic and pseudophakic donors used for DMEK, we observed comparable survival rates (90% after 3 years) and similar endothelial cell loss (30% with an endothelial cell density above 1000 cell/mm after 3 years) but significantly lower rebubbling rates for grafts from pseudophakic donors (18% vs. 28%). CONCLUSIONS: Our data strongly suggest that pseudophakic persons should not be excluded from corneal donation because success rates after DMEK with grafts from pseudophakic eyes are comparable to those with grafts from phakic eyes. However, in the eye bank, grafts from pseudophakic donors have a higher primary discard rate because of low endothelial cell counts.

Graft dislocation and graft failure following Descemet membrane endothelial keratoplasty (DMEK) using precut tissue: a retrospective cohort study.

PURPOSE: Although the functional results following DMEK are better than after DSAEK, DSAEK still seems to be the standard procedure, as surgeons fear losing grafts during DMEK graft preparation. Therefore, eye banks offer precut DMEK grafts for direct use by the surgeon. Here, we analysed the functional results and complication rates for a precut technique compared to fresh preparations. METHODS: We retrospectively compared 453 standard to 11 precut (1-2 days before surgery) DMEK cases. Precut preparations did not differ from the standard preparation, except that the preparation was stopped after trephination with the graft still attached in a small central area. The preparation had to be completed before surgery by peeling off the centrally attached part of the graft. This technique was first tested in an experimental series of 14 grafts, which were unsuitable for transplantation. All surgeries were performed in the same standardized way. RESULTS: There was no significant endothelial cell loss during the storage period following precut preparation. We found a statistically significantly higher endothelial cell loss and graft failure rate for the precut grafts. CONCLUSION: Because of the statistically significant higher graft failure rate in the precut group, we stopped using this technique, so the numbers in this retrospective case series are small. The higher graft failure rate may be explained by (ultra)structural changes in the DMEK graft (e.g. accumulation of dextran) during the second storage period. Therefore, careful evaluation is recommended before using precut DMEK grafts stored in dextran containing media in order to avoid early primary graft failures.

Comparison of Long-Term Outcomes of Femtosecond Laser-Assisted Keratoplasty with Conventional Keratoplasty.

PURPOSE: Astigmatism is a significant problem in penetrating keratoplasty. Lower astigmatism and better visual outcomes had been expected from laser-assisted penetrating keratoplasty, that is, from the top-hat, mushroom, or zig-zag cutting profiles. We report the long-term outcomes of 141 femtosecond laser-assisted penetrating keratoplasties retrospectively. We compare these outcomes with those of penetrating keratoplasty using the guided trephine system (GTS). METHODS: In all, 141 femtosecond laser-assisted penetrating keratoplasties had been performed in 119 patients. The results were compared with those of conventional keratoplasty (n = 1254; visual and refractive outcomes, graft rejections, and graft failure). RESULTS: Follow-up averaged 33 months. In eyes with keratoconus, the time to achieve a visual acuity of 10/20 (Snellen) was shorter in the mushroom than in the GTS group. However, there was no relevant long-term difference. Graft astigmatism was higher in the laser groups [keratometric astigmatism at the end of follow-up: -4.5 ± 4 (GTS), -5.8 ± 3.3 (top-hat), -5.9 ± 3.2 D (mushroom); P < 0.01]. In eyes with keratoconus, rates of graft rejection were highest in the mushroom group (55%). In the other groups, top-hat keratoplasty resulted in lower rates of rejection than GTS keratoplasty (31%). CONCLUSIONS: There is no significant difference in the refractive and visual outcomes after femtosecond laser-assisted penetrating keratoplasty compared with GTS keratoplasty. The benefits from the use of the femtosecond laser may be limited and should be weighed against an increased risk of immune reactions, higher surgical complexity, and higher costs.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events.

Simultaneous transplantation of limbal stem cells may reduce recurrences of granular dystrophy after corneal transplantation: 2 long-term case reports.

To present 2 cases with long-term relapse-free intervals only after limbo-keratoplasty but not after conventional penetrating keratoplasty in granular dystrophy.Retrospective review of the patient charts and photographs taken during long-term follow-up of 2 cases with granular dystrophy, in which 1 eye received penetrating keratoplasty and the fellow eye received penetrating limbo-keratoplasty.In the first patient, 1 eye showed extensive recurrence of granular deposits 17 years after penetrating keratoplasty was performed while in the second eye two-thirds of the corneal transplant adjacent to the transplanted limbal area remained clear 12 years after the limbo-corneal transplant. In the second patient, 1 eye showed no signs of recurrence 5 years after limbo-keratoplasty, whereas a recurrence of granular corneal deposits occurred 18 months after surgery in the fellow eye.These cases show that the simultaneous transplantation of healthy donor limbus when performing penetrating keratoplasty may prolong recurrence in granular corneal dystrophy. Although we were unable to prove it on the molecular level, these clinical courses may support the hypothesis that a limbal transplant helps prevent a recurrence.

Cystoid macular oedema following Descemet membrane endothelial keratoplasty.

BACKGROUND: To determine the incidence and potential risk factors of cystoid macular oedema (CMO) following Descemet membrane endothelial keratoplasty (DMEK) with or without simultaneous cataract surgery. METHODS: In this study, 155 eyes of 88 patients suffering from Fuchs endothelial dystrophy (81%), bullous keratopathy (17.6%) or other corneal diseases (1.4%) underwent DMEK. 52% were pseudophacic (DMEK) and 48% received simultaneous cataract surgery (DMEK combined with cataract surgery (Triple-DMEK)) at the Eye Center at Albert Ludwigs University of Freiburg between May 2011 and June 2013. Spectral-domain optical coherence tomography (SD-OCT) was performed 6 weeks, 3 months and 6 months following (Triple-)DMEK and in unscheduled visits due to limited or decreased visual acuity. The medical records were reviewed for pre-existing comorbidities limiting visual acuity. Patients with a history of macular oedema were excluded. We estimated the incidence of CMO using the Kaplan-Meier method. Potential risk factors for CMO were analysed with a Cox regression analysis and Pearson's correlation. The Cox model included the following variables: patient age and axial length, simultaneous cataract surgery, rate of rebubbling, donor age and donor endothelial cell density. RESULTS: 13% of all eyes developed a single episode of CMO at the end of the follow-up. After 6 months, 13.3% of eyes following Triple-DMEK and 12.5% of eyes following DMEK showed CMO. There was a statistically significant correlation between CMO development and best spectacle corrected visual acuity. Long axial length had a protective effect on CMO development (HR=0.3; p=0.03). Under medical therapy, central foveal thickness decreased in all patients. CMO did not have a relevant effect on long-term visual acuity. CONCLUSIONS: CMO is a frequent complication following DMEK in phacic and pseudophacic eyes. The prognosis is excellent given medical treatment. We recommend regular SD-OCT monitoring during the first 6 months following DMEK.

Are entry criteria for cataract surgery justified?

PURPOSE: The German Ophthalmological Society (GOS) recently proposed surgical entry criteria, i.e. 300 cataract surgeries. We herein correlate the surgical hands-on experience with the risk of posterior capsule ruptures in order to assess whether this number is appropriate. METHODS: We identified all cataract operations that had been performed at the University Eye Hospital Freiburg since 1995. For each surgeon, we assigned a running number to his/her procedures in the order they had been performed. Thereafter, we excluded all combined procedures and the second eyes. We then selected the 5475 surgical reports between November 2008 and November 2012 for detailed review. We additionally classified each surgery into low- vs. high- à priori risk for posterior capsule ruptures. We fitted a multifactorial logistic regression model to assess the GOS recommendation of 300 surgeries under supervision. In the low-risk group, we additionally visualized the 'typical' learning curve by plotting the posterior capsule ruptures against the respective rank numbers. RESULTS: The odds ratio for posterior capsule ruptures of 'learning-mode' (one of the respective surgeon's 300 first procedures) vs. the non-learning-mode was 3.8 (p<0.0001). By contrast, classification into the low-risk group lowered the risk of posterior capsule ruptures three fold (p<0.0001). According to the low-risk plot, the surgeons started with a complication rate of 4% and continuously improved towards 0.5% after 1500 operations. Thereafter, the rate increased again and stabilized around one percent. CONCLUSION: The learning curve with respect to posterior capsule ruptures is surprisingly flat. The GOS entry criterion of 300 cataract procedures is therefore most likely justified. Careful selection of low-risk patients for the training surgeons may help in reducing the rate of posterior capsule ruptures during training.

Influence of donor characteristics on descemet membrane endothelial keratoplasty.

PURPOSE: Penetrating keratoplasty is being replaced by posterior lamellar techniques like Descemet stripping automated endothelial keratoplasty or Descemet membrane endothelial keratoplasty (DMEK) for the surgical treatment of patients with endothelial insufficiency. Although DMEK leads to the best visual results, Descemet stripping automated endothelial keratoplasty is still the standard procedure for many surgeons because it is technically more standardized. Here, we investigated how donor characteristics may influence DMEK surgery. METHODS: After in vitro preparation of DMEK grafts (n = 28), we measured the width of the graft roll, which we correlated to various donor characteristics. In 31 DMEK cases, we measured the intraoperative time from implantation to attachment of the graft, which we correlated to the respective donor characteristics and endothelial cell loss. We used Pearson's method and a multifactorial linear model for the statistical assessments. RESULTS: We found a statistically significant correlation between donor age (P < 0.001) and endothelial cell density (P < 0.05), and the width of the DMEK rolls. That is, older donors and grafts with higher endothelial cell densities formed broader graft rolls. Donor age also showed a trend to directly influence the unfolding time that took longer using younger grafts. Furthermore, the relative endothelial cell loss increased with longer unfolding times. CONCLUSIONS: We found that donor age and endothelial cell density influence the properties of DMEK grafts, and thereby the duration of the surgical procedure. Increased unfolding times result in higher endothelial cell loss. Therefore, it seems reasonable to accept preferably older donors with high endothelial cell densities for DMEK, which may be particularly true for inexperienced surgeons or complex clinical situations.

Natural corneal cell-based microenvironment as prerequisite for balanced 3D corneal epithelial morphogenesis: a promising animal experiment-abandoning tool in ophthalmology.

To achieve durable recognition as a promising animal experiment-abandoning tool in ophthalmology, in vitro engineered tissue equivalents of the human cornea should exhibit proper morphogenesis. Regarding this issue, we were seeking for the natural cell microenvironment fulfilling the minimum requirements to allow human corneal keratinocytes to develop a balanced epithelial morphology with regular spatial appearance of tissue homeostatic biomarkers. Hence, we established cocultures of 3D cell-based collagen scaffolds comprising immortalized corneal keratinocytes combined with a gradual cornea-derived in vivo-like cell microenvironment, together with immortalized stromal fibroblasts alone (nonholistic) or fibroblasts and immortalized endothelial cells (holistic). With matched non-holistic microenvironments revealing mostly flattened cells and putative apical cell ablation foci at day 6, and 9 in HE stains, holistic counterparts yielded proper epithelial stratification with cell flattening restricted to apical layers. Concordantly, RT(2)-PCR showed a tremendous increase in gene expression for progressive and terminal biomarkers of corneal keratinocyte differentiation, cytokeratin (CK) 12, and filaggrin (FIL), in response to nonholistic environments, while involucrin (INV) was moderately but significantly upregulated. Although visible, this increase was moderate in corneal keratinocytes with a holistic environment. On the protein level, indirect immunofluorescence revealed that only epithelia of holistic environments showed diminishment in CK19, counteracted by CK12 rising over time. This time-dependent progression in differentiation coincided with declined proliferation and tissue-regular focus of differentiation biomarkers inv and fil to suprabasal and apical cell layers. Our novel findings suggest the interplay of native tissue forming cell entities, important for balanced corneal epithelial morphogenesis. In addition, they provide evidence for a holistic cell microenvironment as a prerequisite for development of an in vitro engineered corneal epithelial tissue equivalent, exhibiting a regular appearance of tissue homeostatic biomarkers. Such equivalents will be promising tools in ophthalmology, for example, for mechanistic studies in basic research and/or testing of generics or preclinical validation of innovative cornea-tailored biomaterials, desired for regenerative strategies.

Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications.

Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease.

PURPOSE: To assess the expression of human leucocyte antigen (HLA)-DR in epithelial cells and cluster of differentiation (CD8)-positive lymphocytes as possible markers of chronic ocular graft versus host disease (cGvHD) after hematological stem cell transplantation (HSCT). METHODS: Twenty-seven consecutive patients with dry-eye symptoms following HSCT (24 [89%] with peripheral blood stem cell transplantation and 3 [11%] with bone marrow transplants; 17 [63%] familiar allogenic grafts) and 19 age-matched controls were included. Conjunctival impression cytology specimens were stained for HLA-DR, cytokeratin 19, and CD8. Oxford grading scale, blinking frequency, Schirmer test, tear film break-up time (TBUT), and Ocular Surface Disease Index (OSDI) were also recorded. Wilcoxon nonparametric testing was used to compare controls and HSCT recipients and to assess HSCT recipient subgroups with and without clinical cGVHD. RESULTS: Eighteen patients showed clinical signs of ocular cGVHD. TBUT and Schirmer test scores were significantly lower in patients, while Oxford grades and OSDI were significantly higher than in controls. Epithelial HLA-DR expression was generally higher in HSCT recipients than in controls, but it did not correlate with ocular cGVHD status. CD8-positive lymphocytes were identified in five patients with ocular cGvHD and one control. CONCLUSIONS: A strong HLA-DR expression as detected by impression cytology appears to indicate a general HSCT response and fails to predict ocular cGVHD. However, the detection of CD8-positive lymphocytes using impression cytology was frequently associated with ocular cGvHD. Our data warrant further evaluation of CD8 expression in impression cytology, along with comparison to conjunctival biopsies and brush cytology, as impression cytology may offer a less invasive strategy for assessing cGVHD status.