RESUMEN
Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.
Asunto(s)
Acanthamoeba castellanii , Supervivencia Celular , Células Epiteliales , Humanos , Acanthamoeba castellanii/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Línea Celular , L-Lactato Deshidrogenasa/metabolismo , Células Cultivadas , Proliferación Celular/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Caspasa 3/metabolismo , Tiazoles/metabolismo , Sales de Tetrazolio , OxazinasRESUMEN
There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.
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Células Endoteliales , Células Madre Mesenquimatosas , Humanos , Células Endoteliales/metabolismo , Córnea/metabolismo , Técnicas de Cocultivo , Fenotipo , Antígenos CD34/metabolismo , Células Cultivadas , Proliferación Celular , Diferenciación CelularRESUMEN
The eggshell membrane (ESM) is an abundant resource with innate complex structure and composition provided by nature. With at least 60 million tonnes of hen eggs produced globally per annum, utilisation of this waste resource is highly attractive in positively impacting sustainability worldwide. Given the morphology and mechanical properties of this membrane, it has great potential as a biomaterials for wound dressing. However, to date, no studies have demonstrated nor reported this application. As such, the objective of this investigation was to identify and optimise a reproducible extraction protocol of the ESM and to assess the physical, chemical, mechanical and biological properties of the substrate with a view to use as a wound dressing. ESM samples were isolated by either manual peeling (ESM-strip) or via extraction using acetic acid [ESM-A0.5] or ethylenediaminetetraacetic acid, EDTA [ESM-E0.9]. Energy dispersive X-ray spectroscopy (EDS) confirmed that there were no traces of calcium residues from the extraction process. Fourier transform infrared (FTIR) spectroscopy revealed that the extraction method (acetic acid and EDTA) did not alter the chemical structures of the ESM and also clarified the composition of the fibrous proteins of the ESM. Scanning electron microscopy (SEM) analyses revealed a three-layer composite structure of the ESM: an inner layer as continuous, dense and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane) and the outer layer (outer shell membrane) of larger fibres. Material properties including optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical profiling of the ESM samples were performed and demonstrated suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. Taken together, these results document the development of a novel sustainable biomaterial that may be used for ophthalmic wounds and/or other biomedical therapies.
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Materiales Biocompatibles/química , Lesiones de la Cornea/terapia , Cáscara de Huevo/química , Cicatrización de Heridas , Animales , Vendajes , Biomimética , Técnicas de Cultivo de Célula , Pollos , Microscopía Electrónica de Rastreo , Espectroscopía Infrarroja por Transformada de Fourier , Ingeniería de TejidosRESUMEN
Ocular surface inflammatory disorders (OSIDs) are a group of highly prevalent, heterogeneous diseases that display a variety of aetiologies and symptoms and are risk factors for serious complications, including ocular and cornea impairment. Corneal inflammation is a common factor of all OSIDs, regardless of their cause or symptoms. Current medications include over-the-counter lubricating eye drops, corticosteroids, and ciclosporin, which either do not treat the corneal inflammation or have been associated with multiple side effects leading to alternative treatments being sought. Regenerative medicine cell therapies, particularly mesenchymal stem cells (MSCs), have shown great promise for immunosuppression and disease amelioration across multiple tissues, including the cornea. However, for successful development and clinical translation of MSC therapy for OSIDs, significant problems must be addressed. This review aims to highlight considerations, including whether the source of MSC isolation impacts the efficacy and safety of the therapy, in addition to assessing the feasibility of MSC topical application to the cornea and ocular surface through analysis of potential scaffolds and cell carriers for application to the eye. The literature contains limited data assessing MSCs incorporated into scaffolds for corneal administration, thus here we highlight the necessity of further investigations to truly exploit the potential of an MSC-based cell therapy for the treatment of OSIDs.
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Oftalmopatías , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Córnea , Oftalmopatías/terapia , Humanos , Inflamación/terapiaRESUMEN
Amniotic membrane transplantation is an established therapeutic and biological adjunct for several clinical situations, including treatment of diabetic foot ulcers and ocular surface disease. However, poorly standardized and validated clinical preparation and storage procedures can render the final product highly variable and an unpredictable biomaterial. We have therefore developed a novel, standardized method for processing and dry-preserving amniotic membrane, minimizing biochemical, compositional, and structure damage to produce a potentially superior membrane suitable for clinical use. The intellectual property associated with this methodology was patented by the University of Nottingham and licensed to NuVision® Biotherapies which formed the basis of the Tereo® manufacturing process which is used to manufacture Omnigen®.
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Amnios/trasplante , Córnea/crecimiento & desarrollo , Enfermedades de la Córnea/terapia , Regeneración/genética , Córnea/patología , Epitelio Corneal/trasplante , Oftalmopatías/patología , Oftalmopatías/terapia , HumanosRESUMEN
Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and -negative bacteria.
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Amnios/efectos de los fármacos , Antibacterianos/farmacología , Descontaminación , Vacio , Amnios/microbiología , Recuento de Colonia Microbiana , Humanos , Pruebas de Sensibilidad Microbiana , Rafinosa/farmacología , Reproducibilidad de los Resultados , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/crecimiento & desarrollo , EsterilizaciónRESUMEN
BACKGROUND: An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells (hCEC). AIM: To investigate whether corneal-stroma derived stem cells (CSSC) seeded on an amniotic membrane (AM) construct manifests an anti-inflammatory, healing response. METHODS: Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20% (v/v) ethanol for 30 s with 1 ng/mL interleukin-1 (IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs. The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1B, TNF, IL6, and CXCL8 mRNA expression were assessed. RESULTS: Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface. CONCLUSION: CSSC were shown to have a potentially therapeutic anti-inflammatory effect when treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.
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The advent of innovative surgical procedures utilizing partial thickness corneal grafts has created a need for the development of synthetic implants to recreate corneal stromal tissue. This work evaluates electrospun gelatin and polycaprolactone (PCL) scaffolds as a potential biomaterial suitable for use in regeneration of corneal stromal tissue. Electrospun gelatin has been used for many years in tissue engineering; however, post-production modification, such as crosslinking, is usually required to mechanically strengthen such scaffolds. This article aims therefore to compare glutaraldehyde (GA) crosslinked electrospun gelatin scaffolds with electrospun blends of gelatin and PCL at different ratios. Scaffolds were fabricated using electrospinning and characterized by scanning electron microscopy, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy, and tensile testing. To evaluate biocompatibility, primary human corneal stromal cells (hCSC) were seeded upon the scaffolds to assess adherence, proliferation, and phenotype. Results demonstrated that scaffolds fabricated from mixtures of gelatin and PCL showed increased mechanical strength and plasticity compared to scaffolds fabricated from GA crosslinked gelatin alone. In addition, scaffolds fabricated from PCL and gelatin showed comparable support of hCSC adhesion and proliferation. In conclusion, blended mixtures of gelatin and PCL can be considered as an option in the selection of corneal repair materials in the future© 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 828-838, 2019.
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Córnea/metabolismo , Gelatina/química , Poliésteres/química , Andamios del Tejido/química , Córnea/citología , Humanos , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Keratocytes of the corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a corneal MSC (cMSC) phenotype is achieved, regaining the keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of keratocytes to cMSC and compared different supplements in their ability to return cMSC to a keratocyte phenotype. Immunofluorescence and quantitative reverse transcription polymerase chain reaction demonstrated in vivo keratocyte expression of aldehyde dehydrogenase 3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not keratocyte markers. Differentiating the cMSC back to a keratocyte phenotype using nonsupplemented, serum-free medium restored keratocyte markers but did not maintain cell viability or support corneal extracellular matrix production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2, transforming growth factor-ß3 and retinoic acid maintained viability, restored expression of CD34, aldehyde dehydrogenase 3A1 and keratocan, and facilitated production of abundant extracellular matrix as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with retinoic acid, fibroblast growth factor-2 and transforming growth factor-ß3 and can enhance this effect. This is relevant for development of corneal regenerative strategies that require the production of a keratocyte phenotype. Copyright © 2016 John Wiley & Sons, Ltd.
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Diferenciación Celular/efectos de los fármacos , Queratocitos de la Córnea/citología , Medio de Cultivo Libre de Suero/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Mesenquimatosas/citología , Factor de Crecimiento Transformador beta3/farmacología , Tretinoina/farmacología , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Queratocitos de la Córnea/efectos de los fármacos , Queratocitos de la Córnea/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In recent years, there has been increased research interest in generating corneal substitutes, either for use in the clinic or as in vitro corneal models. The advancement of 3D microfabrication technologies has allowed the reconstruction of the native microarchitecture that controls epithelial cell adhesion, migration and differentiation. In addition, such technology has allowed the inclusion of a dynamic fluid flow that better mimics the physiology of the native cornea. We review the latest innovative products in development in this field, from 3D microfabricated hydrogels to microfluidic devices.
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Materiales Biomiméticos/química , Córnea/metabolismo , Células Epiteliales/metabolismo , Hidrogeles/química , Dispositivos Laboratorio en un Chip , Andamios del Tejido/química , Animales , Adhesión Celular , Córnea/citología , Células Epiteliales/citología , HumanosRESUMEN
BACKGROUND: There is a clinical need for biomimetic corneas that are as effective, preferably superior, to cadaveric donor tissue. Decellularized tissues are advantageous compared to synthetic or semi-synthetic engineered tissues in that the native matrix ultrastructure and intrinsic biological cues including growth factors, cytokines and glycosaminoglycans may be retained. However, there is currently no reliable, standardized human corneal decellularization protocol. METHODS: Corneal eye-bank tissue unsuitable for transplantation was utilized to systematically compare commonly used decellularization protocols. Hypertonic sodium chloride; an ionic reagent, sodium dodecyl sulphate; a non-ionic detergent, tert-octylphenol polyoxyethylene (Triton-X); enzymatic disaggregation using Dispase; mechanical agitation; and the use of nucleases were investigated. Decellularization efficacy, specifically for human corneal tissue, was extensively evaluated. Removal of detectable cellular material was evidenced by histological, immunofluorescence and biochemical assays. Preservation of macroscopic tissue transparency and light transmittance was evaluated. Retention of corneal architecture, collagen and glycosaminoglycans was assessed via histological, immunofluorescence and quantitative analysis. Biocompatibility of the resulting scaffolds was assessed using cell proliferation assays. RESULTS: None of the decellularization protocols investigated successfully removed 100% of cellular components. The techniques with the least residual cellular material were most structurally compromised. Biochemical analysis of glycosaminoglycans demonstrated the stripping effects of the decellularization procedures. CONCLUSION: The ability to utilize, reprocess and regenerate tissues deemed "unsuitable" for transplantation allows us to salvage valuable tissue. Reprocessing the tissue has the potential to have a considerable impact on addressing the problems associated with cadaveric donor shortage. Patients would directly benefit by accessing greater numbers of corneal grafts and health authorities would fulfill their responsibility for the delivery of effective corneal reconstruction to alleviate corneal blindness. However, in order to progress, we may need to take a step back to establish a "decellularization" criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality.
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Córnea/citología , Trasplante de Córnea , Donantes de Tejidos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Proliferación Celular , Córnea/cirugía , Bancos de Ojos , HumanosRESUMEN
PURPOSE: Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC. METHODS: Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin), and MSC markers (CD73, CD90, and CD105) were compared in CEC and CSC by immunocytochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton's jelly (WJ-MSC). RESULTS: In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12, and CK19, but not other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC expressed lower levels of CK3, CK12, and CK19, but also stained for pan cytokeratin and expressed KRT5. CONCLUSIONS: Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way in which differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognized.
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Sustancia Propia/metabolismo , ADN/genética , Regulación de la Expresión Génica , Queratinas/genética , Células Madre Mesenquimatosas/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Sustancia Propia/citología , Humanos , Inmunohistoquímica , Queratinas/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AIMS: The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. METHODS: Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS: All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. CONCLUSIONS: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.
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Proliferación Celular/efectos de los fármacos , Sustancia Propia/citología , Medios de Cultivo/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Pluripotentes/citología , Animales , Antígenos CD34/metabolismo , Bovinos , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/metabolismo , Humanos , Fenotipo , Antígenos Embrionarios Específico de Estadio/metabolismoRESUMEN
Toll-like receptors (TLRs) are a family of proteins that initiate the innate immune response in reaction to invading microbes. Studies confirm the expression of TLRs in a variety of ocular tissues and cells, and it has also been suggested that selected TLRs may be associated with geographic atrophy and neovascularisation in age-related macular degeneration, diabetic retinopathy and other vascular and inflammatory diseases of the ocular posterior segment. However, TLR expression and localisation in the retinal and choroidal vasculature has not been defined. A better understanding of differential TLR expression in the choroid and retina, particularly in endothelial cells would improve our knowledge of vascular and inflammatory diseases in the posterior segment of the eye. In this study the gene (mRNA) expression of TLRs 1-10 was investigated using RT-PCR and comparative qPCR and the protein expression and localisation of selected TLRs (3, 4, 6 and 9) were examined using western blotting, flow cytometry and immunofluorescent staining. PCR showed gene expression of TLR1-6 and 9 in human choroidal endothelial cells (hCEC) and TLR2-6, 9 and 10 in human retinal endothelial cells (hREC). Western blotting detected TLR3, 4 and 9 proteins in both hCEC and hREC with higher levels in hCEC, whilst TLR6 protein was not detectable in either endothelial cell type. Flow cytometry detected all four TLRs (3, 4, 6 and 9) on the cell surface and intracellularly, TLR6 expression was detectable but low. The expression and localisation of TLR3, 4 and 9 were confirmed by immunofluorescent staining in endothelial cells and whole tissue sections and their functionality tested by expression of IL-6 (ELISA) in response to stimulation with specific TLR ligands. This study has, for the first time, identified the differential expression and localisation of TLRs in intraocular endothelial cells. This profiling will help inform our understanding of different retinal and choroidal vascular diseases, as well as the development of future treatments for intraocular vascular diseases.
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Coroides/irrigación sanguínea , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Vasos Retinianos/fisiología , Receptores Toll-Like/genética , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Perfilación de la Expresión Génica , Humanos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS: To study ex vivo the difference in endothelial cell density (ECD) in tissue harvested by pneumodissection for pre-Descemet's endothelial keratoplasty (PDEK) and Descemet's membrane endothelial keratoplasty (DMEK). METHODS: Tissue for PDEK and DMEK were obtained from 10 eye bank sclerocorneal discs by trephination after air injection into corneal stroma and big bubble (BB) formation. PDEK tissue was prepared in five corneas after achieving a type 1 BB and DMEK after a type 2 BB in five corneas. Five sclerocorneal discs for each group were used as controls. Endothelial cell counts were obtained from all samples before and after injection using phase-contrast microscopy with an eyepiece reticle. We used paired t test to analyse the results using the GraphPad Prism V.6 software. RESULTS: The range of change of ECD before and after injection in the PDEK sample group varied from -9% to +0.2% with an average of -5.36% ±3.8%. The difference was not statistically significant (p=0.0512). On the other hand, the range of change of ECD of the DMEK groups before and after injection varied from -0.4 to -20.6, with an average of -12.44% ±8.11%. This difference was statistically significant (p=0.0456). Also, there was a significant difference between DMEK test samples (postinjection) and their controls (p=0.028). CONCLUSIONS: Corneal endothelial cell loss in PDEK tissue preparation is no worse, if not slightly better than, in DMEK tissue prepared by pneumodissection. PDEK preparation by pneumodissection represents a viable graft preparation technique.
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Pérdida de Celulas Endoteliales de la Córnea/diagnóstico , Queratoplastia Endotelial de la Lámina Limitante Posterior , Endotelio Corneal/patología , Recolección de Tejidos y Órganos/métodos , Anciano , Anciano de 80 o más Años , Recuento de Células , Bancos de Ojos , Femenino , Humanos , Masculino , Microscopía de Contraste de Fase , Persona de Mediana Edad , Manejo de Especímenes , Donantes de TejidosRESUMEN
Nonunion fractures and large bone defects are significant targets for osteochondral tissue engineering strategies. A major hurdle in the use of these therapies is the foreign body response of the host. Herein, we report the development of a bone tissue engineering scaffold with the ability to release anti-inflammatory drugs, in the hope of evading this response. Porous, sintered scaffolds composed of poly(D,L-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were prepared with and without the anti-inflammatory drug diclofenac sodium. Analysis of drug release over time demonstrated a profile suitable for the treatment of acute inflammation with â¼80% of drug released over the first 4 days and a subsequent release of around 0.2% per day. Effect of drug release was monitored using an in vitro osteoblast inflammation model, comprised of mouse primary calvarial osteoblasts stimulated with proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Levels of inflammation were monitored by cell viability and cellular production of nitric oxide (NO) and prostaglandin E2 (PGE2). The osteoblast inflammation model revealed that proinflammatory cytokine addition to the medium reduced cell viability to 33%, but the release of diclofenac sodium from scaffolds inhibited this effect with a final cell viability of â¼70%. However, releasing diclofenac sodium at high concentrations had a toxic effect on the cells. Proinflammatory cytokine addition led to increased NO and PGE2 production; diclofenac-sodium-releasing scaffolds inhibited NO release by â¼64% and PGE2 production by â¼52%, when the scaffold was loaded with the optimal concentration of drug. These observations demonstrate the potential use of PLGA/PEG scaffolds for localized delivery of anti-inflammatory drugs in bone tissue engineering applications.
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Diclofenaco/uso terapéutico , Sistemas de Liberación de Medicamentos , Inflamación/tratamiento farmacológico , Osteoblastos/patología , Polietilenglicoles/química , Poliglactina 910/química , Andamios del Tejido/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diclofenaco/administración & dosificación , Diclofenaco/farmacología , Dinoprostona/biosíntesis , Humanos , Inflamación/patología , Interferón gamma/farmacología , Interleucina-1beta , Ratones , Modelos Biológicos , Óxido Nítrico/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Cráneo/patología , Factor de Necrosis Tumoral alfaRESUMEN
CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34-positive (CD34(+) ) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34(+) cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34(+) cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34(+) cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34.
Asunto(s)
Antígenos CD34/metabolismo , Células Madre/metabolismo , Animales , Anticuerpos/metabolismo , Antígenos CD34/química , Biomarcadores/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Humanos , Células Madre/citología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-1beta/metabolismo , Osteogénesis/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/citología , Ratones , Osteoblastos/citologíaRESUMEN
The worldwide limited availability of suitable corneal donor tissue has led to the development of alternatives, including keratoprostheses (Kpros) and tissue engineered (TE) constructs. Despite advances in bioscaffold design, there is yet to be a corneal equivalent that effectively mimics both the native tissue ultrastructure and biomechanical properties. Human decellularized corneas (DCs) could offer a safe, sustainable source of corneal tissue, increasing the donor pool and potentially reducing the risk of immune rejection after corneal graft surgery. Appropriate, human-specific, decellularization techniques and high-resolution, non-destructive analysis systems are required to ensure reproducible outputs can be achieved. If robust treatment and characterization processes can be developed, DCs could offer a supplement to the donor corneal pool, alongside superior cell culture systems for pharmacology, toxicology and drug discovery studies.