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PURPOSE: We evaluated the IOP-lowering efficacy and safety of latanoprostene bunod (LBN) ophthalmic solution 0.024% (Vyzulta®), the first topical nitric oxide-donating prostaglandin analog (PGA), in clinical practice. MATERIALS AND METHODS: A retrospective medical chart review from July 2021 to July 2023 of patients with open-angle glaucoma receiving LBN with at least 1 year follow-up was conducted. All included patients received LBN 0.024% as a replacement for a PGA, with examinations at 1-, 3-, 6-and 12-months follow-up. Main outcome measures were IOP, retinal nerve fiber layer thickness, visual fields before/after LBN use and adverse effects. Subgroup analysis with glaucoma types and PGA use were performed for additional IOP reduction after LBN use. RESULTS: Among 78 included patients, 47 patients (81 eyes), 60% with open-angle glaucoma (OAG) remained on LBN throughout 12-month follow-up. Baseline IOP was 18.2±4.2 mm Hg, and Prostaglandin analog (PGA)-IOP was 14.4 ± 3.0 mm Hg (21% mean IOP reduction). After switched to LBN, mean additional IOP reduction was 1.0 mm Hg at month 1, and the greatest reduction was 1.6 mm Hg (8.8% additional mean IOP reduction) at month 12 (P<0.0001). Subgroup analysis (NTG, 73%) showed that mean additional IOP reduction at month 12 was 1.3±2.0 mm Hg in NTG group and 2.1±3.2 mm Hg in POAG group (7.7% vs. 8.7% additional IOP reduction rates, P = 0.23). Subgroup analysis of PGA use at month 12 was 1.8±2.3 mm Hg in tafluoprost group and 0.5±1.7 mm Hg in travoprost group (9.5% vs.2.6% additional IOP reduction rates, P = 0.02). Tolerable ocular adverse effects included irritation (n = 16, 19.8%), mild conjunctival hyperemia (n = 11, 13.6%), dark circles (n = 4, 4.9%) and blurred vision (n = 2, 2.5%). There were no significant visual field and retinal nerve fiber layer thickness changes after 12 months of treatment with LBN 0.024%. CONCLUSIONS: Although high intolerable adverse effects including conjunctival hyperemia and eye irritation happened in the first month, remaining sixty percent of patients exhibited statistically significant additional IOP reductions in the replacement of other PGAs during 12 months of clinical use of LBN 0.024%.
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Glaucoma de Ángulo Abierto , Presión Intraocular , Soluciones Oftálmicas , Prostaglandinas F Sintéticas , Humanos , Femenino , Masculino , Estudios Retrospectivos , Anciano , Presión Intraocular/efectos de los fármacos , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Persona de Mediana Edad , Prostaglandinas F Sintéticas/uso terapéutico , Prostaglandinas F Sintéticas/administración & dosificación , Prostaglandinas F Sintéticas/efectos adversos , Antihipertensivos/uso terapéutico , Antihipertensivos/efectos adversos , Antihipertensivos/administración & dosificación , Anciano de 80 o más Años , Resultado del Tratamiento , Estudios de SeguimientoRESUMEN
Ocular disorders, encompassing both common ailments like dry eye syndrome and more severe situations for instance age-related macular degeneration, present significant challenges to effective treatment due to the intricate architecture and physiological barriers of the eye. Polysaccharides are emerging as potential solutions for drug delivery to the eyes due to their compatibility with living organisms, natural biodegradability, and adhesive properties. In this review, we explore not only the recent advancements in polysaccharide-based technologies and their transformative potential in treating ocular illnesses, offering renewed optimism for both patients and professionals but also anatomy of the eye and the significant obstacles hindering drug transportation, followed by an investigation into various drug administration methods and their ability to overcome ocular-specific challenges. Our focus lies on biological adhesive polymers, including chitosan, hyaluronic acid, cellulose, cyclodextrin, and poloxamer, known for their adhesive characteristics enhancing drug retention on ocular surfaces and increasing bioavailability. A detailed analysis of material designs used in ophthalmic formulations, such as gels, lenses, eye drops, nanofibers, microneedles, microspheres, and nanoparticles, their advantages and limitations, the potential of formulations in improving therapeutic outcomes for various eye conditions. Moreover, we underscore the discovery of novel polysaccharides and their potential uses in ocular drug delivery.
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Celulosa , Quitosano , Ciclodextrinas , Oftalmopatías , Ácido Hialurónico , Poloxámero , Humanos , Quitosano/química , Quitosano/uso terapéutico , Ácido Hialurónico/química , Ácido Hialurónico/uso terapéutico , Celulosa/química , Celulosa/uso terapéutico , Poloxámero/química , Oftalmopatías/tratamiento farmacológico , Ciclodextrinas/química , Ciclodextrinas/uso terapéutico , Sistemas de Liberación de Medicamentos , Animales , Portadores de Fármacos/química , Soluciones Oftálmicas/química , Soluciones Oftálmicas/uso terapéutico , Administración OftálmicaRESUMEN
Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.
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Células Ependimogliales , Glaucoma , Inhibidores de Histona Desacetilasas , Histona Desacetilasas , Ratones Endogámicos C57BL , Estrés Oxidativo , Animales , Estrés Oxidativo/efectos de los fármacos , Glaucoma/tratamiento farmacológico , Glaucoma/metabolismo , Glaucoma/patología , Inhibidores de Histona Desacetilasas/farmacología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Ratones , Histona Desacetilasas/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacología , Masculino , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & controlRESUMEN
Dry eye disease (DED) is a common multifactorial disease affecting a substantial proportion of the population worldwide. Objective tests and subjective symptoms evaluation are necessary to assess DED. Although various treatments have been introduced, accurately evaluating the efficacy of those treatments is difficult because of the disparity between diagnostic tests and patient-reported symptoms. We reviewed the questionnaires used to evaluate DED and the improvements of quality of life with various treatments. In addition, we highlighted the importance of patient-reported outcomes (PRO) assessments for evaluating the effect of DED treatments. Given that the assessment of DED treatment effectiveness substantially relies on individual ocular experiences, acquiring qualitative PRO data is essential for comprehensive evaluation and optimal treatment management. Clinicians should not only focus on improving objective symptoms but also prioritize the well-being of patients in clinical management.
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Intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents have been increasingly applied in the treatment of retinal neovascular diseases. Concerns have arisen that these intravitreal agents may be associated with a potential risk of arterial thromboembolic (ATE) events. We conducted a retrospective, nationwide population-based cohort study to analyze the risks for ATE events in patients receiving intravitreal ranibizumab (IVR) or intravitreal aflibercept (IVA). Data (2011-2018) were obtained from Taiwan's National Health Insurance Research Database. Cox proportional-hazards model was used to identify the risk factors for ATEs. Of the total 3,469 patients, 1393 and 2076 patients received IVR and IVA, respectively. In our result, 38 ATEs occurred within 6 months after IVR or IVA. The risk of ATEs was lower in patients receiving IVR than in those receiving IVA (adjusted hazard ratio [aHR], 0.27; 95% confidence interval [CI], 0.11-0.66). Patients with coronary artery disease (CAD) exhibited a higher risk of ATEs than did those without CAD (aHR, 3.47; 95% CI, 1.41-8.53). The risk of ATEs was higher in patients with an event of acute myocardial infarction (AMI) or ischemic stroke (IS) within 6 months prior to index IVI than in those without recent AMI/IS events (aHR, 23.8; 95% CI, 7.35-77.2 and IS: aHR, 290.2; 95% CI, 103.1-816.4). In conclusion, compared with IVA, IVR was associated with a lower risk of ATEs. When strategies for anti-VEGF agents are devised, risk factors, such as CAD and a history of AMI or IS within 6 months should be considered. Further large-scale studies are warranted to elucidate the safety of anti-VEGF injections.
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Inhibidores de la Angiogénesis , Ranibizumab , Humanos , Ranibizumab/efectos adversos , Inhibidores de la Angiogénesis/efectos adversos , Estudios Retrospectivos , Estudios de Cohortes , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/efectos adversos , Factores de Crecimiento Endotelial Vascular , Medición de Riesgo , Inyecciones IntravítreasRESUMEN
The prevalence of myasthenia gravis (MG), an autoimmune disorder, is increasing among all subsets of the population leading to an elevated economic and social burden. The pathogenesis of MG is characterized by the synthesis of autoantibodies against the acetylcholine receptor (AChR), low-density lipoprotein receptor-related protein 4 (LRP4), or muscle-specific kinase at the neuromuscular junction, thereby leading to muscular weakness and fatigue. Based on clinical and laboratory examinations, the research is focused on distinguishing MG from other autoimmune, genetic diseases of neuromuscular transmission. Technological advancements in machine learning, a subset of artificial intelligence (AI) have been assistive in accurate diagnosis and management. Besides, addressing the clinical needs of MG patients is critical to improving quality of life (QoL) and satisfaction. Lifestyle changes including physical exercise and traditional Chinese medicine/herbs have also been shown to exert an ameliorative impact on MG progression. To achieve enhanced therapeutic efficacy, cholinesterase inhibitors, immunosuppressive drugs, and steroids in addition to plasma exchange therapy are widely recommended. Under surgical intervention, thymectomy is the only feasible alternative to removing thymoma to overcome thymoma-associated MG. Although these conventional and current therapeutic approaches are effective, the associated adverse events and surgical complexity limit their wide application. Moreover, Restivo et al. also, to increase survival and QoL, further recent developments revealed that antibody, gene, and regenerative therapies (such as stem cells and exosomes) are currently being investigated as a safer and more efficacious alternative. Considering these above-mentioned points, we have comprehensively reviewed the recent advances in pathological etiologies of MG including COVID-19, and its therapeutic management.
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PURPOSE: The purpose of this study is to evaluate the association between lipid-lowering agent use and the risks of diagnosed dry eye disease (DED). METHODS: This retrospective, case-control study included 780 786 patients who received lipid-lowering agents in 2002-2016, of which 17 409 were newly diagnosed with DED during a ≥2-year follow-up period. These patients were matched 1:4 with control participants for age, sex, and comorbidities. Separate odds ratios (OR) were calculated for DED and each of statin and fibrate use. RESULTS: Statin users had significantly higher odds of DED (adjusted OR = 1.12; 95% confidence interval (CI) = 1.08-1.16, p < 0.0001) than nonusers. Fibrate users did not show higher odds of DED than nonusers (adjusted OR = 1.04; 95% CI = 0.99-1.10, p = 0.125). The lipophilic statin users did not show higher odds of DED compared with the hydrophilic statin users (adjusted OR = 0.99, 95% CI = 0.93-1.06, p = 0.729). Among statin users, the odds of DED did not differ significantly between patients receiving statin therapy for >180 days vs. ≤90 days or patients receiving statin therapy for 91-180 days vs. ≤90 days (adjusted OR = 1.00, p = 0.922; adjusted OR = 0.94, p = 0.541, respectively). The odds of DED were not statistically different among patients receiving low-intensity, moderate-intensity, and high-intensity of statin therapy. CONCLUSIONS: Patients receiving statin therapy had a higher DED risk than patients not receiving statin therapy. The type of statin, the duration, and the intensity of statin use were not significantly associated with DED risks. Further studies are required to identify the relevant factors related to DED risks with statin.
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Síndromes de Ojo Seco , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Humanos , Estudios de Casos y Controles , Estudios Retrospectivos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Taiwán/epidemiología , Lípidos , Síndromes de Ojo Seco/inducido químicamente , Síndromes de Ojo Seco/diagnóstico , Síndromes de Ojo Seco/tratamiento farmacológico , Ácidos Fíbricos , Factores de RiesgoRESUMEN
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are uncommon but life-threatening diseases mostly caused by drugs. Although various systemic immunomodulating agents have been used, their therapeutic efficacy has been inconsistent. This study aimed to provide an evidence-based review of systemic immunomodulating treatments for SJS/TEN. We reviewed 13 systematic review and meta-analysis articles published in the last 10 years. The use of systemic corticosteroids and IVIg is still controversial. An increasing number of studies have suggested the effectiveness of cyclosporine and biologic anti-TNF-α in recent years. There were also some promising results of combination treatments. Further large-scale randomized controlled trials are required to provide more definitive evidence of the effectiveness of these treatments. The pathogenesis of SJS/TEN has been elucidated in recent years and advances in the understanding of SJS/TEN may inspire the discovery of potential therapeutic targets.
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Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.
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Materiales Biocompatibles , Córnea , Células Endoteliales , Vesículas Extracelulares , Regeneración , Materiales Biocompatibles/metabolismo , Cadherinas/metabolismo , Cromatografía Liquida , Mezclas Complejas , Córnea/citología , Vesículas Extracelulares/metabolismo , Humanos , Proteómica , Espectrometría de Masas en TándemRESUMEN
There has been great interest in identifying the biological substrate for light-cell interaction and their relations to cancer treatment. In this study, a near-infrared (NIR) laser is focused into the nucleus (nNIR) or cytoplasm (cNIR) of a single living cell by a high numerical aperture condenser to dissect the novel role of cell nucleus in mediating NIR effects on mitochondrial dynamics of A549 non-small cell lung cancer cells. Our analysis showed that nNIR, but not cNIR, triggered mitochondrial fission in 10 min. In contrast, the fission/fusion balance of mitochondria directly exposed to cNIR does not change. While the same phenomenon is also triggered by single molecular interactions between epidermal growth factor (EGF) and its receptor EGFR, pharmacological studies with cetuximab, PD153035, and caffeine suggest EGF signaling crosstalk to DNA damaging response to mediate rapid mitochondrial fission as a result of nNIR irradiation. These results suggest that nuclear DNA integrity is a novel biological target for cellular response to NIR.
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Daño del ADN , Receptores ErbB , Neoplasias Pulmonares , Células A549 , Núcleo Celular/metabolismo , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/metabolismo , Humanos , Dinámicas Mitocondriales , RadiaciónAsunto(s)
Malformaciones Arteriovenosas Intracraneales/complicaciones , Quiasma Óptico/irrigación sanguínea , Arteria Retiniana/anomalías , Oclusión de la Vena Retiniana/etiología , Vena Retiniana/anomalías , Dexametasona/administración & dosificación , Implantes de Medicamentos , Femenino , Glucocorticoides/administración & dosificación , Humanos , Inyecciones Intravítreas , Oclusión de la Vena Retiniana/diagnóstico , Trastornos de la Visión/diagnóstico , Trastornos de la Visión/etiología , Agudeza Visual/fisiología , Adulto JovenRESUMEN
Human platelet lysate (HPL) is a complex mixture of potent bioactive molecules instrumental in tissue repair and regeneration. Due to their remarkable safety, cost-effective production, and availability at global level from collected platelet concentrates, HPLs can become a powerful biotherapy for various therapeutic applications, if standardized and carefully validated through pre-clinical and clinical studies. In this work, the possibility to use a tailor-made HPL as a corneal transplant alternative to treat the gradual decrease in the number of corneal endothelial cells (CECs) associated with aging, was evaluated. The HPL preparation was thoroughly characterized using various proteomics tools that revealed a remarkable richness in multiple growth factors and antioxidants. Treatment of B4G12 and BCE C/D-1b CECs with the HPL increased their viability, enhanced the wound closure rate, and maintained cell growth and typical hexagonal morphology. Besides, this HPL significantly protected against tert-butyl hydroperoxide (TBHP)-induced oxidative stress as evidenced by increasing CEC viability, decreased cell death and reactive oxygen species formation, and enhanced antioxidant capacity. Proteomics analysis of treated CECs confirmed that HPL treatment triggered the corneal healing pathway and enhanced oxidative stress. These data strongly support further pre-clinical evaluation of this tailor-made HPL as a novel CEC regeneration biotherapy. HPL treatment may eventually represent a pragmatic and cost-effective alternative to corneal transplant to treat damages of the corneal endothelium which is a major cause of blindness worldwide.
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Antioxidantes/metabolismo , Productos Biológicos/farmacología , Plaquetas/metabolismo , Endotelio Corneal/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Animales , Bovinos , Línea Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Endotelio Corneal/citología , Endotelio Corneal/patología , Voluntarios Sanos , Humanos , Factores de Crecimiento Nervioso/aislamiento & purificación , Regeneración/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , terc-Butilhidroperóxido/toxicidadRESUMEN
Fuchs endothelial corneal dystrophy is one of the most common indications for corneal transplantation, and impaired anti-oxidative function is observed in corneal endothelial cells (CECs). Curcumin is well-known for its anti-oxidative property; but, no study has examined the effect of curcumin on anti-oxidative therapeutic roles in corneal endothelial disease. In our experiments, oxidative stress 0.25 mM tert-butyl hydroperoxide for 2 h was induced in immortalized human CECs pretreated with curcumin. Cell behavior and viability, reactive oxygen species production, and the protein expression of the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element (ARE) pathway were examined; the Keap1/Nrf2/ARE pathway is crucial anti-oxidative pathway of curcumin. The results showed that pretreatment with 12.5 µM curcumin significantly reduced the ROS production and improved the survival of CECs under oxidative stress. In addition, curcumin pretreatment significantly increased the expression of nuclear Nrf2, and the productions of superoxide dismutase 1 and heme oxygenase-1, which were the target anti-oxidative enzymes of the Keap1/Nrf2/ARE pathway. Our findings showed that curcumin enhanced the growth and differentiation of CECs under oxidative stress. The activation of Keap1/Nrf2/ARE pathway by curcumin was crucial for CECs to improve their anti-oxidative capacity.
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Elementos de Respuesta Antioxidante/efectos de los fármacos , Antioxidantes/farmacología , Pérdida de Celulas Endoteliales de la Córnea/prevención & control , Curcumina/farmacología , Células Endoteliales/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch/agonistas , Factor 2 Relacionado con NF-E2/agonistas , Proteínas de Transporte Vesicular/agonistas , Línea Celular/efectos de los fármacos , Córnea/citología , Córnea/efectos de los fármacos , Humanos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Surface modification on microporous polyethylene (PE) membranes was facilitated by plasma polymerizing with two hydrophilic precursors: ethylene oxide vinyl ether (EO1V) and diethylene oxide vinyl ether (EO2V) to effectively improve the fouling against mammalian cells (Chinese hamster ovary, CHO cells) and proteins (bovine serum albumin, BSA). The plasma polymerization procedure incorporated uniform and pin-hole free ethylene oxide-containing moieties on the filtration membrane in a dry single-step process. The successful deposition of the plasma polymers was verified by Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) analyses. Water contact angle measurements and permeation experiments using cell and protein solutions were conducted to evaluate the change in hydrophilicity and fouling resistance for filtrating biomolecules. The EO1V and EO2V plasma deposited PE membranes showed about 1.45 fold higher filtration performance than the pristine membrane. Moreover, the flux recovery reached 80% and 90% by using deionized (DI) water and sodium hydroxide (NaOH) solution, indicating the efficacy of the modification and the good reusability of the modified PE membranes.
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INTRODUCTION: Adipose-derived stem cells (ASCs) are potential cell sources for cartilage tissue engineering. Chitosan has been shown to enhance the stemness and differentiation capability of ASCs, and the native extracellular matrix (ECM) derived from articular cartilage has been also reported to induce chondrogenic differentiation of ASCs. Here we tested the hypothesis that a porous three-dimensional (3D) hybrid scaffold composed of chitosan and cartilage ECM can provide a better environment to induce ASC chondrogenesis. METHODS: Mixed solution composed of chitosan and cartilage ECM was frozen and lyophilized to form a composite construct. The porous 3D scaffolds were further crosslinked by genipin and used for ASC culture. RESULTS: Cultivation of ASCs in the chitosan/cartilage ECM composite 3D scaffolds induced the formation of cell spheroids with profound glycosaminoglycan production after 14 and 28 days culture. Chondrogenesis of ASCs seeded in the 3D scaffolds was also evident by mRNA expressions of cartilage-specific gene COL2A1 and ACAN on day 14. Histology and immunohistochemistry on day 28 also showed abundant cartilage-specific macromolecules, namely collagen type II and proteoglycan, deposited in a surface layer of the composite scaffold with tangential layer, transitional layer, and lacunae-like structures. Otherwise, hypertrophic markers collagen type I and X were concentrated in the area beneath the surface. CONCLUSION: Our findings demonstrated spatial chondrogenic differentiation of ASCs in the chitosan-cartilage ECM composite scaffolds. This 3D hybrid scaffold exhibits great potentials for ASC-based cartilage tissue engineering.
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This study aimed to investigate the underlying mechanisms of corneal endothelial cells (CECs) differentiation and identify the extracellular matrix (ECM) compositions using chitosan/polycaprolactone (PCL) blended membrane, hence exploring the potential use of chitosan/PCL blends in tissue engineering of CECs. We utilized the chitosan/PCL blends named as PCL25 consisting of PCL at 25% by weight. The surface characteristics of PCL25 were confirmed by using Fourier Transform Infrared Spectroscopy (FTIR) and Atomic Force Microscope (AFM). Bovine CECs were cultured on the blends, compared with TCPS and pure chitosan membrane. Cell behaviors in terms of cell attachment, proliferation, differentiation phenotype and expression of differentiation proteins were examined. Furthermore, ECM protein productions were also analyzed. From the experiments, we found the topography (roughness) of PCL25 membrane examined by AFM was greater than pure chitosan membrane. FTIR results confirmed the functional groups of C=O bond of PCL. The CECs displayed hexagonal morphology and similar proliferation rate on both PCL25 membrane and TCPS. In addition, the immunofluorescence evidence showed well-localized ZO-1 and Na+/K+ ATPase expression of membrane proteins. ECM protein productions of CECs on PCL were no inferior to TCPS. Moreover, western blot results verified the higher amount of collagen type IV, and reduced TGF-ß2 expression on PCL25 membrane compared to TCPS substrate. In conclusions, chitosan/PCL blends membrane provided a favorable environment for CECs in terms of ECM compositions, therefore enhancing the growth and differentiation. Accordingly, for CEC tissue engineering applications, PCL 25 might be a suitable alternative for cadaveric cornea transplantation in the near future.
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Diferenciación Celular/fisiología , Quitosano/química , Células Endoteliales/citología , Proteínas de la Matriz Extracelular/metabolismo , Membranas Artificiales , Poliésteres/química , Animales , Materiales Biocompatibles , Western Blotting , Bovinos , Células Cultivadas , Quitosano/farmacología , Colágeno Tipo IV/metabolismo , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Microscopía de Fuerza Atómica , Poliésteres/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta2/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
There is accumulating experimental evidence that human platelet lysate (HPL) made from platelet concentrates can replace fetal bovine serum (FBS) as a xeno-free clinical-grade supplement of growth media to expand mesenchymal stromal cells (MSCs). However, uncertainties exist in regard to impacts that various manufacturing methods of HPL can exert on the expansion and differentiation capacity of MSCs. In particular, there is a need to evaluate the possibility of implementing virus-inactivation treatment during HPL production to ensure optimal safety of industrial HPL pools. Expired human platelet concentrates from four different donors were pooled and subjected to freeze-thaw cycles (-80/+37 °C), followed or not by serum-conversion by calcium chloride, heat-treatment at 56 °C for 30 min, or solvent-detergent (S/D) virus inactivation. The concentrations of total proteins, growth factors and fibrinogen, and the chemical compositions of the HPLs were characterized. The impact of HPL supplementation on the cell morphology, doubling time, immunophenotype and trilineage differentiation capacity of Wharton jelly MSCs (WJMSCs) were compared over five passages, using FBS as a control and normalizing the protein content. Data showed that WJMSCs expanded equally well, exhibited a typical fibroblast morphology, had short doubling times, maintained their immunophenotypes, and differentiated into chondrocyte, osteocyte, and adipocyte lineages in all HPL-supplemented media, all of which were more effective than FBS. In conclusion, we found minimal detectable impact of the HPL manufacturing process, including S/D virus inactivation, on the suitability of expanding WJMSCs in vitro.
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Plaquetas/metabolismo , Detergentes/farmacología , Células Madre Mesenquimatosas/citología , Solventes/farmacología , Gelatina de Wharton/citología , Fibrinógeno/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacosRESUMEN
IMPACT STATEMENT: This study successfully developed decellularized corneal scaffolds that were prepared by organic acid, which safely exist in animal tissues and plants. The results showed the highly efficient removal of cell debris from porcine corneas, and excellent preservation of optical properties, extracellular matrix (ECM) architecture, and biomolecules. In addition, decellularized corneal scaffolds revealed excellent biocompatibility and recellularization potential in vitro. In an animal model, the transplanted corneas were completely epithelialized, clear, showed no signs of immune response, and effectively supported stromal keratocytes growth. Hence, this could be a promising scaffold material for corneal tissue engineering applications.
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Córnea/cirugía , Animales , Córnea/citología , Enfermedades de la Córnea/cirugía , Trasplante de Córnea/métodos , Epitelio Corneal/citología , Epitelio Corneal/cirugía , Matriz Extracelular , Porcinos , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
PURPOSE: To determine the relationship between mechanical behavior in cross-linked corneas and changes in the corneal ultrastructure after corneal cross-linking (CXL). METHODS: Porcine corneas were treated following the "Dresden" protocol, the current gold standard for clinical treatment, consisting of dropwise application of 0.1% riboflavin in 20% dextran followed by 30 minutes of ultraviolet-A (UVA) irradiation. The effect of CXL was assessed using uniaxial tensile testing, transmission electron microscopy, and Fourier transform infrared spectroscopy, with results compared against corneas treated with each of the treatment solution components individually. RESULTS: UVA/riboflavin cross-linked corneas displayed 28% ± 17% increase in the material tangent modulus compared with dextran treatment alone, and altered collagen architecture within the first 300 µm of stromal depth consisting of 5% increase in the thickness of collagen fibrils, no significant changes to interfibrillar spacing, and an 8% to 12% decrease in number of fibrils per unit area. Fourier transform infrared spectroscopy confirmed formation of interfibrillar bonds (P = .012) induced by UVA-mediated CXL. CONCLUSIONS: The data support a model wherein collagen fibril diameter and structural density are fundamental parameters in defining tissue stiffening following UVA/riboflavin CXL and provide benchmarks against which modifications to the Dresden CXL protocol can be evaluated. [J Refract Surg. 2018;34(4):264-272.].
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Colágeno/metabolismo , Córnea/ultraestructura , Reactivos de Enlaces Cruzados , Queratocono/tratamiento farmacológico , Fármacos Fotosensibilizantes/uso terapéutico , Riboflavina/uso terapéutico , Animales , Fenómenos Biomecánicos , Córnea/fisiopatología , Sustancia Propia/metabolismo , Queratocono/metabolismo , Queratocono/fisiopatología , Microscopía Electrónica de Transmisión , Espectroscopía Infrarroja por Transformada de Fourier , Porcinos , Rayos UltravioletaRESUMEN
BACKGROUND: Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity. MATERIAL AND METHODS: Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56°C for 30min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting. RESULTS: BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture. CONCLUSION: CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.