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PURPOSE: Corneal collagen cross-linking (CXL) can halt corneal ectasia. Leaving corneal epithelium intact during treatment may reduce the incidence of complications. However, it is under debate whether this reduces efficacy and if oxygen supplementation may be necessary to optimize the cross-linking effect. This study aimed to investigate the impact of hyperbaric oxygenation (HBO) on intracorneal oxygen concentrations during epi-off and epi-on CXL. METHODS: CXL was performed using riboflavin and ultraviolet-A (UV-A) irradiance (3 mW/cm2 for 30 min) on porcine corneas under normobaric and hyperbaric conditions, with and without supplemented oxygen, with and without epithelium. Intracorneal oxygen concentrations were continuously monitored before and during irradiation. Biomechanical properties were assessed through tensile strength testing. RESULTS: HBO alone did not cause perceivable changes in stromal oxygen concentrations. Oxygen supplementation resulted in higher oxygen concentration in corneal stroma during CXL. HBO may cause a further increase in oxygen levels, although this was not statistically significant in this study. Notably, a tendency of oxygen levels to rise continuously during UV-irradiation was observed using HBO. Biomechanical properties showend no statistically significant differences between any groups. CONCLUSIONS: In this ex-vivo model, HBO increased stromal oxygen levels during CXL, regardless of the presence of corneal epithelium. The dynamics in oxygen concentrations in corneal stromal tissue during CXL suggest that time is an important factor to consider in modifications of established protocols. Also, we hypothesize that stromal levels of riboflavin and UV-A irradiance may be more critical to the CXL effect when oxygen is supplemented and epithelium is not removed.
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Purpose: Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. Methods: The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10 days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10 days, and EpC secretory activity was analyzed by ß-hexosaminidase assay. Results: The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25 mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5 mM after 10 days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. Conclusions: A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.
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Supervivencia Celular , Reactivos de Enlaces Cruzados , Hidrogeles , Iridoides , Ingeniería de Tejidos , Animales , Iridoides/farmacología , Iridoides/metabolismo , Porcinos , Ingeniería de Tejidos/métodos , Reactivos de Enlaces Cruzados/farmacología , Células Cultivadas , Células Madre Mesenquimatosas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Materiales BiocompatiblesRESUMEN
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.
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Aparato Lagrimal , Células Madre Mesenquimatosas , Aparato Lagrimal/metabolismo , Aparato Lagrimal/ultraestructura , Hidrogeles/química , Células Endoteliales , Ingeniería de Tejidos/métodos , Matriz Extracelular/metabolismoRESUMEN
BACKGROUND: Preliminary clinical work indicates that increasing eyelid tension improves the function of the meibomian glands. The aim of this study was to optimize laser parameters for a minimally invasive laser treatment to increase eyelid tension by coagulation of the lateral tarsal plate and canthus. METHODS: Experiments were performed on a total of 24 porcine lower lids post mortem, with six lids in each group. Three groups were irradiated with an infrared B radiation laser. Laser-induced lower eyelid shortening was measured and the increase in eyelid tension was assessed with a force sensor. A histology was performed to evaluate coagulation size and laser-induced tissue damage. RESULTS: In all three groups, a significant shortening of the eyelids after irradiation was noticed (p < 0.0001). The strongest effect was seen with 1940 nm/1 W/5 s, showing -15.1 ± 3.7% and -2.5 ± 0.6 mm lid shortening. The largest significant increase in eyelid tension was seen after placing the third coagulation. CONCLUSION: Laser coagulation leads to lower eyelid shortening and an increase in lower eyelid tension. The strongest effect with the least tissue damage was shown for laser parameters of 1470 nm/2.5 W/2 s. In vivo studies of this effect have to confirm the efficacy of this concept prior to clinical application.
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Rayos Láser , Glándulas Tarsales , Animales , Porcinos , Glándulas Tarsales/patología , Rayos Infrarrojos , Fenómenos Mecánicos , LágrimasRESUMEN
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.
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Human amniotic membrane (hAM) is usually stored at -80 °C. However, in many regions, cryopreservation at -80 °C is not feasible, making hAM unavailable. Therefore, the possibility of cryopreservation at -28 °C (household freezer) was investigated. hAMs (n = 8) were stored at -80 °C or -28 °C for a mean time of 8.2 months. hAM thickness, epithelial integrity and basement membrane were assessed histologically. The collagen content, concentration of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) were determined. Elastic modulus and tensile strength were measured. The mean thickness of hAM stored at -28 °C was 33.1 ± 21.6 µm (range 9.7-74.9); thickness at -80 °C was 30.8 ± 14.7 µm (range 13.1-50.7; p = 0.72). Mean collagen content, epithelial cell number and integrity score showed no significant difference between samples stored at -28 °C or -80 °C. Basement membrane proteins were well preserved in both groups. Mean tensile strength and elastic modulus were not significantly different. Concentration of bFGF at -28 °C was 1063.2 ± 680.3 pg/g (range 369.2-2534.2), and 1312.1 ± 778.2 pg/g (range 496.2-2442.7) at -80 °C (p = 0.11). HGF was 5322.0 ± 2729.3 pg/g (range 603.3-9149.8) at -28 °C, and 11338.5 ± 6121.8 pg/g (range 4143.5 to 19806.7) at -80 °C (p = 0.02). No microbiological contamination was detected in any sample. The cryopreservation of hAM at -28 °C has no overt disadvantages compared to -80 °C; the essential characteristics of hAM are preserved. This temperature could be used in an alternative storage method whenever storage at -80 °C is unavailable.
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There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia.
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Conjuntiva/metabolismo , Córnea/metabolismo , Cavidad Nasal/anatomía & histología , Mucosa Nasal/metabolismo , Conducto Nasolagrimal/anatomía & histología , Infecciones por Adenoviridae/patología , Animales , Bovinos , Técnicas de Cultivo Tridimensional de Células , Células Cultivadas , Conjuntivitis/patología , Células Epiteliales/metabolismo , Células Caliciformes/metabolismo , Humanos , Sarampión/patología , Cavidad Nasal/fisiología , Conducto Nasolagrimal/fisiología , Conejos , Tomografía de Coherencia ÓpticaRESUMEN
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.
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Conjuntiva , Animales , Células Epiteliales , Células Caliciformes , Humanos , Mucina 5AC , Conejos , Células Madre , PorcinosRESUMEN
Dry eye disease (DED) is a multifactorial disease characterized by a disrupted tear film homeostasis and inflammation leading to visual impairments and pain in patients. Aqueous-deficient dry eye (ADDE) causes the most severe progressions and depends mainly on the loss of functional lacrimal gland (LG) tissue. Despite a high prevalence, therapies remain palliative. Therefore, it is of great interest to develop new approaches to curatively treat ADDE. Mesenchymal stem/stromal cells (MSC) have been shown to induce tissue regeneration and cease inflammation. Moreover, an increasing amount of MSC was found in the regenerating LG of mice. Therefore, this study investigated the therapeutic effect of MSC transplantation on damaged LGs using duct ligation induced ADDE in mice. Due to the transplantation of sex-mismatched and eGFP-expressing MSC, MSC could be identified and detected until day 21. MSC transplantation significantly improved LG regeneration, as the amount of vital acinar structures was significantly increased above the intrinsic regeneration capacity of control. Additionally, MSC transplantation modulated the immune reaction as macrophage infiltration was delayed and TNFα expression decreased, accompanied by an increased IL-6 expression. Thus, the application of MSC appears to be a promising therapeutic approach to induce LG regeneration in patients suffering from severe DED/ADDE.
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Síndromes de Ojo Seco/terapia , Aparato Lagrimal , Trasplante de Células Madre Mesenquimatosas , Regeneración , Animales , Femenino , Aparato Lagrimal/patología , Aparato Lagrimal/fisiología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
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Colágeno/farmacología , Conjuntiva/patología , Plásticos/química , Cicatrización de Heridas/efectos de los fármacos , Amnios/trasplante , Animales , Modelos Animales de Enfermedad , Epitelio/efectos de los fármacos , Humanos , Miofibroblastos/efectos de los fármacos , Conejos , RatasRESUMEN
The loss of functional lacrimal gland (LG) tissue causes quantitative tear deficiency and is the most common reason for the development of severe dry eye disease (DED). The induction of LG regeneration in situ would be a promising approach to curatively treat DED, but underlying mechanisms are mainly unclear. Therefore, this study aims to comparatively evaluate the dynamic of LG damage and regeneration in two mouse models in order to study mechanisms of LG regeneration. Male C57BL/6â¯J mice were used to induce damage to the right extraorbital LG either by a single interleukin (IL) 1α injection or a ligation of the secretory duct for 7 days. Fluorescein staining (FL) and LG wet weight were assessed. In addition, the dynamic of damage and regeneration of acini structures as well as inflammation and the appearance of progenitor cells were (immuno-) histologically evaluated on day 1, 2, 3, 5, 7 after IL-1α injection and day 3, 7, 14, 21, 28 after duct ligation (DL). While LG weight was only slightly affected after IL-1α injection, DL led to a significant decrease at day 7 followed by an increase after re-opening. Additionally, DL resulted in a more pronounced inflammatory reaction than IL-1α injection. After DL the infiltration with CD3+ T cells, CD138 + plasma cells and CD68 +â¯macrophages increased, while IL-1α injection only caused an infiltration with CD68â¯+ macrophages. Furthermore, the damage of LG structures was significantly higher after DL than after IL-1α injection. Accordingly, regeneration of LG was prolonged and only partial at day 28 after DL, whilst 5 days after IL-1α injection a complete LG completely regeneration was achieved. We also found a significantly increased number of nestin + mesenchymal stem cells in both models during injury phase. Our results showed that both models induce LG damage followed by a spontaneous regeneration of acini structures. IL-1α injection caused an immediate inflammation with a transient period of slight tissue damage. However, DL caused a more distinct tissue damage followed by a prolonged period of regeneration, which might make it appear more attractive to study regenerative therapies and their effects on LG regeneration.
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Modelos Animales de Enfermedad , Síndromes de Ojo Seco/fisiopatología , Interleucina-1alfa/farmacología , Enfermedades del Aparato Lagrimal/fisiopatología , Aparato Lagrimal/fisiología , Regeneración/fisiología , Animales , Síndromes de Ojo Seco/etiología , Inflamación , Aparato Lagrimal/efectos de los fármacos , Aparato Lagrimal/cirugía , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
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Enfermedades de la Córnea/cirugía , Trasplante de Córnea , Limbo de la Córnea/ultraestructura , Trasplante de Células Madre/métodos , Ingeniería de Tejidos/métodos , Supervivencia Celular , Células Cultivadas , Enfermedades de la Córnea/patología , Humanos , Microscopía Electrónica de RastreoRESUMEN
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.