Enhancement of lacrimal gland cell function by decellularized lacrimal gland derived hydrogel.

Sustainable treatment of aqueous deficient dry eye (ADDE) represents an unmet medical need and therefore requires new curative and regenerative approaches based on appropriatein vitromodels. Tissue specific hydrogels retain the individual biochemical composition of the extracellular matrix and thus promote the inherent cell´s physiological function. Hence, we created a decellularized lacrimal gland (LG) hydrogel (dLG-HG) meeting the requirements for a bioink as the basis of a LG model with potential forin vitroADDE studies. Varying hydrolysis durations were compared to obtain dLG-HG with best possible physical and ultrastructural properties while preserving the original biochemical composition. A particular focus was placed on dLG-HG´s impact on viability and functionality of LG associated cell types with relevance for a futurein vitromodel in comparison to the unspecific single component hydrogel collagen type-I (Col) and the common cell culture substrate Matrigel. Proliferation of LG epithelial cells (EpC), LG mesenchymal stem cells, and endothelial cells cultured on dLG-HG was enhanced compared to culture on Matrigel. Most importantly with respect to a functionalin vitromodel, the secretion capacity of EpC cultured on dLG-HG was higher than that of EpC cultured on Col or Matrigel. In addition to these promising cell related properties, a rapid matrix metalloproteinase-dependent biodegradation was observed, which on the one hand suggests a lively cell-matrix interaction, but on the other hand limits the cultivation period. Concluding, dLG-HG possesses decisive properties for the tissue engineering of a LGin vitromodel such as cytocompatibility and promotion of secretion, making it superior to unspecific cell culture substrates. However, deceleration of biodegradation should be addressed in future experiments.

Influence of Storage Conditions on Decellularized Porcine Conjunctiva.

Porcine decellularized conjunctiva (PDC) represents a promising alternative source for conjunctival reconstruction. Methods of its re-epithelialization in vitro with primary human conjunctival epithelial cells (HCEC) have already been established. However, a long-term storage method is required for a simplified clinical use of PDC. This study investigates the influence of several storage variants on PDC. PDC were stored in (1) phosphate-buffered saline solution (PBS) at 4 °C, (2) in glycerol-containing epithelial cell medium (EM/gly) at -80 °C and (3) in dimethyl sulfoxide-containing epithelial cell medium (EM/DMSO) at -196 °C in liquid nitrogen for two and six months, respectively. Fresh PDC served as control. Histological structure, biomechanical parameters, the content of collagen and elastin and the potential of re-epithelialization with primary HCEC under cultivation for 14 days were compared (n = 4-10). In all groups, PDC showed a well-preserved extracellular matrix without structural disruptions and with comparable fiber density (p ≥ 0.74). Collagen and elastin content were not significantly different between the groups (p ≥ 0.18; p ≥ 0.13, respectively). With the exception of the significantly reduced tensile strength of PDC after storage at -196 °C in EM/DMSO for six months (0.46 ± 0.21 MPa, p = 0.02), no differences were seen regarding the elastic modulus, tensile strength and extensibility compared to control (0.87 ± 0.25 MPa; p ≥ 0.06). The mean values of the epithelialized PDC surface ranged from 51.9 ± 8.8% (-196 °C) to 78.3 ± 4.4% (-80 °C) and did not differ significantly (p ≥ 0.35). In conclusion, all examined storage methods were suitable for storing PDC for at least six months. All PDC were able to re-epithelialize, which rules out cytotoxic influences of the storage conditions and suggests preserved biocompatibility for in vivo application.

Descemet Membrane Endothelial Keratoplasty and Triple Descemet Membrane Endothelial Keratoplasty in Eyes With Macular Comorbidity.

PURPOSE: The aim of this study was to evaluate results after Descemet membrane endothelial keratoplasty (DMEK) and DMEK with cataract surgery (triple DMEK) in eyes with endothelial dysfunction and concomitant macular pathology. METHODS: A monocentric, prospective clinical observational study of patients who underwent DMEK or triple DMEK surgery at the Department of Ophthalmology, University Hospital Düsseldorf, Germany, from June 2013 to February 2016 was conducted. Sex, age, best-corrected visual acuity (BCVA), central corneal thickness, endothelial cell density, and central retinal thickness in the 1 millimeter zone were analyzed. RESULTS: A total of 209 eyes were included in the study. Forty-two eyes (20.1%) had vision-limiting concomitant maculopathies. These were age-related macular degeneration (n = 17, 8.1%), epiretinal gliosis (n = 13, 6.2%), chronic macular edema (n = 7, 3.3%), macular holes (n = 3, 1.4%), and macular scarring (n = 2, 1.0%). BCVA significantly increased in patients without maculopathy from 0.6 ± 0.33 logMAR to 0.1 ± 0.15 logMAR ( P < 0.001) and also in patients with maculopathy from 0.9 ± 0.38 logMAR to 0.27 ± 0.23 logMAR 12 months postoperatively ( P < 0.001). There was a significant central retinal thickness increase of 34.1 µm 6 weeks after triple DMEK in the central 1-mm zone ( P = 0.011). This increase was insignificant after DMEK. Postoperative macular edema occurred in 5.9% of cases after DMEK and 8.1% after triple DMEK, which was not significantly different. CONCLUSIONS: DMEK and triple DMEK significantly increase BCVA in eyes with concomitant maculopathy. Postoperative macular edema is a common disorder after lamellar keratoplasty; therefore, prophylactic treatment with nonsteroidal antiinflammatory drugs should be considered. Maculopathies did not predispose the development of postoperative macular edema.

DMEK outcome after one year - Results from a large multicenter study in Germany.

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) accounts for >50% of all corneal transplants in Germany. So far, no data from such a large multicenter study have been published. METHODS: This retrospective study included 3200 DMEKs at seven departments performed for Fuchs endothelial corneal dystrophy (FECD) or bullous keratopathy (BK). We evaluated best corrected visual acuity (BCVA, logMAR), endothelial cell density (ECD, cells/mm2 ), minimal corneal thickness (CT, µm), rebubbling-, primary transplant failure- and immune reaction-rate. Changes over time were evaluated by linear mixed models for repeated measures and correlation with case number by center by weighted linear regression. RESULTS: For patients without vision-limiting comorbidities (74% of all analysed eyes, n = 2270), mean BCVA improved from 0.6 ± 0.4 logMAR to 0.2 ± 0.2 logMAR 6 months (p < 0.001, n = 1441) and 0.1 ± 0.2 logMAR 12 months (p = 0.001, n = 1402) postoperatively. BK- had a worse BCVA compared to FECD-patients (0.3 ± 0.5 vs. 0.1 ± 0.2 logMAR [p < 0.001] at 1 year). ECD declined from 2465 ± 259 cells/mm2 (n = 2876 preoperatively) to 1587 ± 433 cells/mm2 after 12 months (p < 0.001, n = 1237). Mean rebubbling rate was 0.4 ± 0.7/eye. 784 eyes (25%) received at least one rebubbling. More rebubblings correlated with a lower ECD, a worse BCVA, a higher CT, and higher transplant failure and rejection rates (p < 0.001, p = 0.013 for BCVA at 12 months). A single rebubbling did not influence the BCVA (p = 0.785). Graft failure rate was 3% (n = 67), rejection rate 1.5% (n = 48). CONCLUSION: Descemet membrane endothelial keratoplasty increases visual acuity with low transplant failure- and rejection-rates. FECD has a better outcome than BK. Since a quarter of all patients need a rebubbling, this should be included in the informed consent. Remarkably, one rebubbling has no influence on the outcome.

Preserflo MicroShunt: Efficacy and Endothelial Cell Density.

PRCIS: A main safety concern associated with new glaucoma implants is corneal endothelial cell loss, which could even be observed in already established procedures. PURPOSE: The purpose of this study was to evaluate the efficacy and corneal safety, especially corneal endothelial cell loss (ECL), after Preserflo MicroShunt implantation. METHODS: A monocentric, prospective clinical observational study of patients with a follow-up of at least 12 months who underwent Preserflo MicroShunt implantation at the Department of Ophthalmology, University Hospital of Düsseldorf, Germany, between January 2020 and October 2021. Endothelial cell density (ECD), the distance between the tip of the implant and the corneal endothelium, and the success of the surgery were analyzed. RESULTS: In total, 14 eyes of 12 patients were included. After a mean follow-up of 20±2.7 months intraocular pressure was significantly reduced by 8.2 mm Hg (P=0.0041); in 28.6% of patients, a revision surgery was necessary. Reduction of preoperative and follow-up ECD was not statistically significant (ECL of 45 cells/mm2, ECDpreoperative=2074±703.6 cells/mm2, ECDlast follow-up=2029±742.3 cells/mm2, P=0.42). A longer intracameral shunt tube length correlated significantly with a higher distance between the shunt tip and corneal endothelium (r=0.61, P=0.036). CONCLUSIONS: Preserflo MicroShunt effectively lowered intraocular pressure without substantial ECL after a minimum follow-up period of 17 months.

[Conjunctival reconstruction-State of the art of regenerative treatment forms beyond the limbus]. / Bindehautrekonstruktion ­ Status quo regenerativer Therapieformen jenseits des Limbus.

BACKGROUND: The demands on conjunctival replacement tissues are high: they need to be elastic, clinically compatible, surgically feasible and support goblet cell growth. OBJECTIVE: This article provides an overview of currently applied conjunctival replacement tissues and those under investigation. METHOD: Current publications on clinically applied conjunctival replacement tissues and substrates which are the subject of scientific research and those already tested in animal models are presented and discussed. RESULTS: Replacement tissues in clinical use are autologous and allogenic conjunctiva, nasal and oral mucous membranes, amniotic membrane and decellularized tissues. Autologous conjunctiva shows good results but is not suitable for large defects due to limited availability. In these cases autologous nasal and oral mucous membranes can be used; however, success is limited in cases of autoimmune diseases. Amniotic membranes are frequently applied clinically but goblet cell growth is limited. Different decellularized tissues are used clinically and goblet cell growth was found in vivo. Robust comparative studies are not yet available. Biological matrices such as fibrin, collagen, elastin, gelatin or hyaluronate and synthetic tissues from the group of polyesters are being investigated in the laboratory and in animal models. These studies show good epithelialization and goblet cell growth in vivo. CONCLUSION: Transplantation of conjunctiva, nasal and oral mucous membranes and amniotic membranes show satisfactory clinical results but exhibit individual weaknesses. Further studies in animal models and clinical settings are required to further evaluate the benefits of other matrices, such as cell-free tissues or other biological and synthetic matrices.

[Regenerative medicine for the corneal epithelium : Cell therapy from bench to bedside]. / Regenerative Medizin für das Hornhautepithel : Zelltherapie in Wissenschaft und Klinik.

In the case of thermal or caustic burns of the ocular surface, loss of limbal epithelial stem cells leads to compromised self-renewal of the corneal epithelium. This results in permanent loss of vision. In these situations, transplantation of cultured limbal epithelial cells on an amniotic membrane or fibrin gel as substrate (Holoclar®) can help to regenerate the corneal surface. The required cells are obtained from the healthy partner eye, if available. Adult stem cells from other parts of the body potentially serve as alternative cell sources: hair follicles, oral mucosa, mesenchymal stromal cells, or induced pluripotent stem cells (originally, e.g., skin fibroblasts). The reprogramming of such cells can be achieved with the help of transcription factors. In addition, work is being done on biosynthetic or synthetic matrices, which not only serve as substrate material for the transplantation but also support the functional properties of these cells (self-renewal, corneal epithelial-typical phenotype).

Iris Obstruction Following PRESERFLO Microshunt Implantation and Surgical Management.

PURPOSE: The aim was to present a case of lumen obstruction after implantation of microshunt with subconjunctival drainage and subsequent management. CASE REPORT: A 56-year-old male with primary open angle glaucoma underwent uneventful PRESERFLO MicroShunt (PMS) implantation in both eyes. Five months after surgery the patient presented with an intraocular pressure (IOP) of 26 mm Hg in the right eye because of iris obstruction. RESULTS: The iris fibers were severed and removed surgically to free the lumen. Follow-up showed a normalized IOP of 10 mm Hg and a free lumen. CONCLUSION: Uncontrolled IOP because of iris obstruction following PMS was addressed by surgical removal of iris fibers at the lumen entrance without the need for tube repositioning.

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva.

PURPOSE: The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC). METHODS: PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells. RESULTS: From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva. CONCLUSION: Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Evaluation of Plastic-Compressed Collagen for Conjunctival Repair in a Rabbit Model.

IMPACT STATEMENT: Conjunctival integrity is crucial for a healthy ocular surface and visual acuity. In severe cases of inflammatory surface disorders or after trauma, thermal or chemical burns as well as after ocular surgery, a surgical reconstruction using conjunctival substitutes is required. Due to limitations of currently used substitutes, such as the amniotic membrane, there is a need for the development of new scaffolds of consistent quality for conjunctival reconstruction. This study explored the biocompatibility and surgical usability of plastic-compressed collagen as an alternative conjunctival substitute in a rabbit model.

Generation and characterisation of decellularised human corneal limbus.

PURPOSE: Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC. METHODS: Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay. RESULTS: In the DHL, the DNA content was reduced from 1.5 ± 0.3 µg/mg to 0.15 ± 0.01 µg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity. CONCLUSIONS: The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

Decellularised conjunctiva for ocular surface reconstruction.

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE: Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (ß-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

Purpose: Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. Methods: To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (ß-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Results: Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and ß-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. Conclusions: SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

Descemet-membrane endothelial keratoplasty in patients with retinal comorbidity-a prospective cohort study.

AIM: To investigate indications, surgical challenges, and outcome of Descemet-membrane endothelial keratoplasty (DMEK) in patients with retinal comorbidities (RC). METHODS: In a prospective cohort study, 8 eyes of 8 DMEK-patients with known RC were compared to 38 eyes of 38 DMEK-patients without RC. The duration of surgery, the degree of difficulty graded by the surgeon, and the complications through DMEK-surgery were analyzed for each patient. The best-corrected visual acuity (BCVA), the endothelial cell count, the intraocular pressure, and the subjective satisfaction was evaluated after a 6-month follow-up. Data were compared applying the non-parametric Wilcoxon-, Chi-square- and Fishers-exact-test with P≤0. 05 as level of significance. RESULTS: RC-patients had dry age-related macular degeneration (n=4) or history of pars-plana vitrectomy (n=4). The main indication for DMEK was pain due to bullous keratopathy for the RC-patients (n=7, 88%) and visual impairment due to Fuchs endothelial keratoplasty for the non-RC-patients (n=33, 87%). The BCVA increased for both groups (P=0.01, P<0.001) and all corneas cleared. For the RC-patients, the subjective satisfaction improved significantly (P=0.02). Oil-filling and missing support of the vitreous body complicated surgery in vitrectomized eyes. CONCLUSION: DMEK is a favorable technique to treat endothelial disorders even if patients suffer from a retinal comorbidity. By enhancing the corneal clarity, it enables retinal examination or intraocular surgery and increases the patients satisfaction. However, in vitrectomized or silicone-oil filled eyes, the duration of surgery and degree of complexity are increased. An experienced surgeon should perform DMEK in these patients. CLINICAL TRIAL REGISTRATION NUMBER: DRKS00007566.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells.

PURPOSE: The application of lacrimal gland-derived mesenchymal stem cells (LG-MSC) for the regeneration of lacrimal gland tissue could result in a novel therapy for dry-eye syndrome. To optimize the culture conditions, the purpose of this study was to evaluate the influence of low oxygen on phenotype, differentiation potential, proliferative, and regenerative capacity of murine LG-MSC. METHODS: Murine LG-MSC were cultured in 21% and 5% oxygen and characterized by flow cytometry, cell sorter assisted proliferation-, and colony forming unit-assays. Reactive oxygen species (ROS) levels as well as lineage differentiation were evaluated. The effect of conditioned medium of LG-MSC from both oxygen conditions (CM MSC 21%, respectively, CM MSC 5%) on lacrimal gland epithelial cells (LG-EC) was examined in wound healing and proliferation assays. RESULTS: Cells under both culture conditions revealed differentiation potential and presented a MSC-specific flow cytometric phenotype. In 5% oxygen, cells yielded less ROS, showed a stable morphology, higher colony forming potential, and an increased proliferation capacity. Five percent oxygen significantly increased the number of CD44+ LG-MSC. Furthermore, CM MSC 5% significantly enhanced migration and proliferation in LG-EC. CONCLUSIONS: In vitro expansion in low oxygen preserves the proliferation capacity and differentiation potential of LG-MSC and increases the effects of conditioned medium on migration and proliferation in LG-EC. Therefore, expansion in low oxygen seems to be an excellent method, to obtain vital MSC. Also, an increased number of LG-MSC expressing CD44 was observed under low oxygen, which might be a valuable marker to identify a potent MSC subpopulation.

Engineering of a Secretory Active Three-Dimensional Lacrimal Gland Construct on the Basis of Decellularized Lacrimal Gland Tissue.

Lacrimal gland (LG) insufficiency is a main cause for severe dry eye leading to pain, visual impairment, and eventually loss of sight. Engineering of transplantable LG tissue with secretory capacity is a desirable goal. In this study, a three-dimensional decellularized LG (DC-LG) scaffold with preserved LG morphology was generated by treatment with 1% sodium deoxycholate and DNase solution using porcine LG tissue. To address clinical applicability, the primary in vitro culture of secretory active LG cells from a small tissue biopsy of 1.5 mm diameter was introduced and compared with an established isolation method by enzymatic digestion. Cells from both isolation methods depicted an epithelial phenotype, maintained their secretory capacity for up to 30 days, and exhibited progenitor cell capacity as measured by aldehyde dehydrogenase-1 activity, side population assay, and colony-forming units. Cells from passage 0 were reseeded into the DC-LG and secretory active cells migrated into the tissue. The cells resembled an LG-like morphology and the constructs showed secretory activity. These results demonstrate the possibility of engineering a secretory competent, three-dimensional LG construct using LG cells expanded from a small tissue biopsy and DC-LG as a matrix that provides the native structure and physiological niche for these cells.

Urrets-Zavalia syndrome after Descemet membrane endothelial keratoplasty.

PURPOSE: To report a case of Urrets-Zavalia syndrome (UZS) after Descemet membrane endothelial keratoplasty (DMEK). METHODS: A 74-year-old woman with Fuchs endothelial dystrophy and inconspicuous ocular history developed UZS after DMEK surgery. The intraoperative and postoperative course is presented. RESULTS: After uneventful DMEK surgery, intraocular pressure was elevated up to 40 mm Hg on the first postoperative day. A small bleed from the peripheral wide-open iridectomy in the 12 o'clock position in the otherwise deep anterior chamber was observed. On the sixth postoperative day, a 4-mm-wide pupil, nonreactive to light, was noted. One year after surgery, the fixed medium mydriasis (4 mm) persisted and best-corrected vision was 0.1 logMAR. No pupillary reaction was noted after application of 0.2% or 2% pilocarpine. CONCLUSIONS: Filling the anterior chamber with air to secure fixation of a grafted Descemet membrane carries the risk of early acute postoperative ocular hypertension. This can lead to iris sphincter defects resulting in a fixed dilated pupil after DMEK surgery. Large patent iridectomy in the 12 o'clock position is insufficient to prevent this. Patients undergoing DMEK surgery should be informed about this potential complication.

Association between congenital nasolacrimal duct obstruction and delivery by cesarean section.

OBJECTIVE: Congenital nasolacrimal duct obstruction (CNDO) is the most common cause of neonatal epiphora. Persistence can lead to chronic dacryocystitis and amblyopia. This study analyzed the association between the incidence of CNDO and delivery by cesarean section. STUDY DESIGN: This was a retrospective cohort study of 386 children with CNDO (born between 2000 and 2008). The incidence of the delivery mode in patients with CNDO was compared with data from a corresponding population derived from annual birth statistics. RESULTS: There was no statistically significant association between the overall cesarean section rate and the incidence of CNDO, but primary cesarean section was significantly more frequent among patients with CNDO (73.15%, p < 0.05). The difference was significant for both genders for the period from 2000 to 2008 (p < 0.05%). The relative risk for CNDO was 1.7-fold increased in children delivered by primary cesarean section. CONCLUSION: Primary cesarean section may be a risk factor for CNDO.