Human Wharton's jelly stem cells (HWJSCs) are able to differentiate into skin and oral mucosa epithelial-like cells. In this work, we demonstrate for the first time the capability of HWJSCs to ...differentiate in vitro into cornea epithelial-like cells in a three-dimensional model.
First, primary cell cultures of HWJSCs, corneal epithelial cells, and corneal keratocytes were cultured and three-dimensional orthotypic and heterotypic human cornea models were generated with fibrin-agarose scaffolds. Then, in vitro differentiation of HWJSCs and corneal epithelial cells was performed with keratinocytic inductive medium in a three-dimensional system that allowed interaction between stromal and epithelial compartments. Histological, histochemical, and immunofluorescence analyses were used to determine the differentiation status of each sample.
Our results demonstrated that HWJSCs were able to differentiate into corneal epithelial-like cells, with results similar to the native cornea. Heterotypical corneas generated with HWJSCs showed adequate differentiation of the epithelium and stroma, and were similar to orthotypic and native corneas in the expression of epithelial markers (CK3/12, PKG, ZO1, and CX43) and extracellular matrix components (proteoglycans, collagen, elastic and reticular fibers). Immunofluorescence analysis confirmed the presence of crystallins Cry-αA, Cry-αB, Cry-β, and Cry-ζ with moderate or weak expression of Cry-βγ3 and Cry-λ1 (key proteins involved in cornea transparency) in both models.
Our findings suggest that HWJSCs can be considered an alternative cell source for cornea regeneration and may offer a solution for patients with limbus stem cell deficiency.
Abstract Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they ...intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.
Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue‐engineered anterior human cornea, which is currently being tested in a Phase I/II ...clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin–agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical‐grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.
The Boston keratoprosthesis (B‐KPro) is globally the most commonly implanted artificial cornea for patients with severe corneal diseases, particularly those with multiple allograft failures. Despite ...providing a good visual recovery, the poor adhesion between the poly(methyl methacrylate) (PMMA)‐made stem and the donor tissue poses a challenge, impacting the clinical outcome of the B‐KPro. Using single‐molecule covalent bonding, PMMA surface is functionalized with l‐3,4‐dihydroxyphenylalanine (l‐DOPA) and its chemical, optical, mechanical, and biological properties are studied. The functionalization process significantly improves biocompatibility of PMMA, without affecting its optical and mechanical properties. Human corneal fibroblasts (HCF) and human corneal epithelial cells (HCEp) seeded on l‐DOPA surface both exhibit greater confluency and metabolic rate compared to those of PMMA during 7‐day cell culture. Moreover, HCF cultured on l‐DOPA demonstrates a higher expression of ALDH3A1, Ki67, Integrin 1, and FAK with no expression of α‐SMA, compared to those of PMMA, which instead show greater expression of α‐SMA. These suggest that l‐DOPA surface fosters cellular adhesion, proliferation, and migration, without adversely impacting the phenotype of the cells. This study offers an inexpensive and efficient tactic to modify the surface of materials with l‐DOPA to achieve the optimal biocompatibility and biointegration of medical devices.
Covalently functionalized poly(methyl methacrylate) (PMMA) surface with l‐3,4‐dihydroxyphenylalanine (l‐DOPA) is stable, and significantly improves the biocompatibility of the PMMA surface. l‐DOPA‐functionalized PMMA also preserves the phenotypic characteristics of human corneal fibroblast, supporting cellular adhesion, proliferation, and migration, which may lead to better biointegration of the Boston keratoprosthesis, the most commonly implanted artificial cornea for patients with severe corneal diseases.
Electron beam (E‐beam) irradiation is an attractive and efficient method for sterilizing clinically implantable medical devices made of natural and/or synthetic materials such as poly(methyl ...methacrylate) (PMMA). As ionizing irradiation can affect the physicochemical properties of PMMA, understanding the consequences of E‐beam sterilization on the intrinsic properties of PMMA is vital for clinical implementation. A detailed assessment of the chemical, optical, mechanical, morphological, and biological properties of medical‐grade PMMA after E‐beam sterilization at 25 and 50 kiloGray (kGy) is reported. Fourier transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry studies indicate that E‐beam irradiation has minimal effect on the chemical properties of the PMMA at these doses. While 25 kGy irradiation does not alter the mechanical and optical properties of the PMMA, 50 kGy reduces the flexural strength and transparency by 10% and 2%, respectively. Atomic force microscopy demonstrates that E‐beam irradiation reduces the surface roughness of PMMA in a dose dependent manner. Live‐Dead, AlamarBlue, immunocytochemistry, and complement activation studies show that E‐beam irradiation up to 50 kGy has no adverse effect on the biocompatibility of the PMMA. These findings suggest that E‐beam irradiation at 25 kGy may be a safe and efficient alternative for PMMA sterilization.
Electron beam (E‐beam) irradiation (25 kiloGray (kGy)) has a minimal impact on the chemical, mechanical, and optical properties of poly(methyl methacrylate) (PMMA) and no apparent adverse effect on its biocompatibility with human corneal cells. These findings suggest that 25 kGy E‐beam irradiation is a feasible sterilization method of PMMA used in medical implants.
Corneal diseases are among the main causes of blindness, with approximately 4.6 and 23 million patients worldwide suffering from bilateral and unilateral corneal blindness, respectively. The standard ...treatment for severe corneal diseases is corneal transplantation. However, relevant disadvantages, particularly in high-risk conditions, have focused the attention on the search for alternatives.
We report interim findings of a phase I-II clinical study evaluating the safety and preliminary efficacy of a tissue-engineered corneal substitute composed of a nanostructured fibrin-agarose biocompatible scaffold combined with allogeneic corneal epithelial and stromal cells (NANOULCOR). 5 subjects (5 eyes) suffering from trophic corneal ulcers refractory to conventional treatments, who combined stromal degradation or fibrosis and limbal stem cell deficiency, were included and treated with this allogeneic anterior corneal substitute.
The implant completely covered the corneal surface, and ocular surface inflammation decreased following surgery. Only four adverse reactions were registered, and none of them were severe. No detachment, ulcer relapse nor surgical re-interventions were registered after 2 years of follow-up. No signs of graft rejection, local infection or corneal neovascularization were observed either. Efficacy was measured as a significant postoperative improvement in terms of the eye complication grading scales. Anterior segment optical coherence tomography images revealed a more homogeneous and stable ocular surface, with complete scaffold degradation occurring within 3–12 weeks after surgery.
Our findings suggest that the surgical application of this allogeneic anterior human corneal substitute is feasible and safe, showing partial efficacy in the restoration of the corneal surface.
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•NANOULCOR human bioartificial corneas can be surgically implanted in human patients.•Bioartificial human corneas are highly biocompatible and free from side effects.•Preliminary signs of efficacy were found in the first cases grafted with NANOULCOR.
Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and ...physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Collagen scaffolds, one of the most used biomaterials in corneal tissue engineering, are frequently crosslinked to improve mechanical properties, enzyme tolerance, and thermal stability. Crosslinkers ...such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are compatible with tissues but provide low crosslinking density and reduced mechanical properties. Conversely, crosslinkers such as glutaraldehyde (GTA) can generate mechanically more robust scaffolds; however, they can also induce greater toxicity. Herein, we evaluated the effectivity of double-crosslinking with both EDC and GTA together with the capability of sodium metabisulfite (SM) and sodium borohydride (SB) to neutralize the toxicity and restore biocompatibility after crosslinking. The EDC-crosslinked collagen scaffolds were treated with different concentrations of GTA. To neutralize the free unreacted aldehyde groups, scaffolds were treated with SM or SB. The chemistry involved in these reactions together with the mechanical and functional properties of the collagen scaffolds was evaluated. The viability of the cells grown on the scaffolds was studied using different corneal cell types. The effect of each type of scaffold treatment on human monocyte differentiation was evaluated. One-way ANOVA was used for statistical analysis. The addition of GTA as a double-crosslinking agent significantly improved the mechanical properties and enzymatic stability of the EDC crosslinked collagen scaffold. GTA decreased cell biocompatibility but this effect was reversed by treatment with SB or SM. These agents did not affect the mechanical properties, enzymatic stability, or transparency of the double-crosslinked scaffold. Contact of monocytes with the different scaffolds did not trigger their differentiation into activated macrophages. Our results demonstrate that GTA improves the mechanical properties of EDC crosslinked scaffolds in a dose-dependent manner, and that subsequent treatment with SB or SM partially restores biocompatibility. This novel manufacturing approach would facilitate the translation of collagen-based artificial corneas to the clinical setting.
This study aims to benchmark the training programs of European ophthalmology residents.
An online survey, aimed at European ophthalmology residents in training and those within two years of ...completion, was sent through the national representatives of the European Society of Ophthalmology, Young Ophthalmologists section (SOE-YO). The study involved 214 subjects representing 36 of the 44 European countries offering ophthalmology training programs.
Among the surveyed, 74.8% of participants had an official national curriculum; 55.8% had a national specialty examination to accredit their training as ophthalmologists. 45.8% were satisfied or very satisfied with the clinical skills acquired, while 42.1% were completely dissatisfied with the surgical skills achieved. Considering the 4th year residents (mean duration of the residency), many of them did not perform phacoemulsification surgery (34%), pterygium excision (46.9%), or repair of eyelid laceration (31.3%).
There is great heterogeneity in the competencies achieved by residents in training according to their country of origin, especially in terms of surgical competences.
To evaluate long-term visual and anatomical outcomes in neovascular age-related macular degeneration (nAMD) patients treated with anti-vascular endothelial growth factor (VEGF) agents depending on ...the time delay from confirmed diagnosis to treatment initiation.
Seventy-three nAMD patients (73 eyes) treated with anti-VEGF agents for 12 months using the pro re nata regimen were included in this retrospective longitudinal study. Patients were split into 3 groups according to the time from diagnosis to first anti-VEGF injection: < 48 h (group 1); 48 h-7 days (group 2); > 7 days (group 3). Decimal best-corrected visual acuity (VA) and macular thickness (MT) were recorded at baseline and 1–2-, 3–4-, 6- and 12-month later. Furthermore, age, gender as well as the applied treatment and number of injections after 12 months of treatment were also registered and compared.
Long-term effect of the treatment demonstrated enhanced VA in group 1 patients compared with the rest of groups after 1–2-, 6-, and 12-month follow-up (P < 0.05). Positive effects of early treatment were additionally corroborated by the augmented percentage of patients with normal VA in the group 1 respect to the rest of groups over studied time points (P < 0.05). Moreover, the VA gain in nAMD at group 1 was obtained with a mean of 3.7 intravitreal injections over 1-year follow-up period. Regarding MT, non-significant difference was observed among groups.
An early initial treatment with VEGF inhibitors is critical to achieve the best functional benefits of this therapy in new-onset nAMD patients.
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•Anti-VEGF therapy within 48 h from diagnosis leads to long-term visual gain in nAMD.•Visual gain is achieved with low IV injection number if early treatment is applied.•A prompt first anti-VEGF injection promotes long-term anatomical benefits in nAMD.
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