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Arnalich-Montiel F.,Ramon y Cajal Hospital | Hernandez-Verdejo J.L.,Vissum Madrid Eye Hospital | Oblanca N.,Ramon y Cajal Hospital | Munoz-Negrete F.J.,Ramon y Cajal Hospital | De Miguel M.P.,La Paz Hospital Research Institute
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2013

Background: Descemet membrane endothelial keratoplasty (DMEK) is being proposed as the procedure of choice in corneal endothelial disease as it achieves better visual and refractive outcomes than Descemet stripping automated endothelial keratoplasty (DSAEK). Nevertheless, primary graft failure is frequent, especially during the learning curve, and secondary back-up procedure consists on DSAEK. We aim to compare corneal haze and visual acuity of patients undergoing primary DSAEK vs. patients undergoing DSAEK as a back-up procedure after primary DMEK failure. Methods: This study is a comparative case series that included 19 eyes from 16 patients with early stages of corneal failure and limitation of daily activities after primary DSAEK or secondary DSAEK. A control group of non-operated corneas included 10 aged-matched normal eyes. The study was conducted at University Hospital Ramón y Cajal and Vissum Hospital, Madrid, Spain. Corneal densitometry readings and postoperative best-corrected visual acuity in subjects with primary and secondary DSAEK were recorded 6 months after the surgery using the Pentacam Scheimpflug system (Oculus, inc.,Wetzlar, Germany). Results: In primary DSAEK median densitometry values (range) were statistically significantly higher (p < 0.05) than normal subjects for the full thickness, posterior and anterior part of the paracentral cornea; and the anterior part of the central cornea. In secondary DSAEK, median densitometry values were statistically significantly higher than normal subjects at all levels of the central and paracentral cornea. In secondary DSAEK, median densitometry values (range) were statistically significantly higher than in primary DSAEK in the full-thickness, anterior part and interface of the central cornea and in the full-thickness and posterior part of the paracentral cornea. Median visual acuity between groups (p = 0.47) was statistically better for the primary DSAEK group, which also had a higher percentage of patients achieving BCVA of ≥ 20/40 and ≥20/25 than the secondary DSAEK group (100 % vs. 62 % and 60 % vs. 0 % respectively). Conclusions: There is an increase in central corneal light scattering after secondary DSAEK performed after a failed DMEK as compared to primary DSAEK. This has a negative impact on final visual acuity that needs to be considered in each patient when starting DMEK surgery. © 2013 Springer-Verlag Berlin Heidelberg.


Alio del Barrio J.L.,Ramon y Cajal Hospital | Chiesa M.,La Paz Hospital Research Institute | Garagorri N.,Tecnalia | Garcia-Urquia N.,Tecnalia | And 8 more authors.
Experimental Eye Research | Year: 2015

Purpose: To evaluate the invivo biocompatibility of grafts composed of sheets of decellularized human corneal stroma with or without the recellularization of human adipose derived adult stem cells (h-ADASC) into the rabbit cornea. Methods: Sheets of human corneal stroma of 90μm thickness were decellularized, and their lack of cytotoxicity was assayed. The recellularization was achieved by the injection of 2×105 labeled h-ADASC in the graft followed by five days of cell culture. The grafts were implanted invivo into a stromal pocket at 50% depth. After a triple-masked three-month follow-up, the animals were euthanized and the biointegration of the graft, the viability of the stem cells and the expression of keratocan (human keratocyte-specific protein) were assessed. Results: The decellularized stromal sheets showed an intact extracellular matrix with a decellularization rate of 92.8% and an excellent recellularization capacity invitro with h-ADASC. A complete and stable graft transparency was observed during the full follow-up, with absence of any clinical sign of rejection. The postmortem analysis demonstrated the survival of the transplanted human stem cells inside the graft and their differentiation into functional keratocytes, as assessed by the expression of human keratocan. Conclusions: We report a new model of lamellar keratoplasty that requires only a simple and safe procedure of liposuction and a donor allogeneic cornea to provide an optically transparent autologous stromal graft with excellent biocompatibility and integration into the host tissue in a rabbit model. © 2015 Elsevier Ltd.


Fuentes-Julian S.,La Paz Hospital Research Institute | Arnalich-Montiel F.,Ramon Y Cajal Hospital Research Institute | Jaumandreu L.,Ramon Y Cajal Hospital Research Institute | Leal M.,Ramon Y Cajal Hospital Research Institute | And 5 more authors.
PLoS ONE | Year: 2015

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the nor-mal- risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice. © 2015 Fuentes-Julián et al.


de Miguel M.P.,La Paz Hospital Research Institute | Fuentes-Julian S.,La Paz Hospital Research Institute | Blazquez-Martinez A.,La Paz Hospital Research Institute | Pascual C.Y.,Infanta Sofia Hospital | And 3 more authors.
Current Molecular Medicine | Year: 2012

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-β, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory. © 2012 Bentham Science Publishers.


Del Barrio J.L.A.,Ramon y Cajal Hospital | Chiesa M.,La Paz Hospital Research Institute | Ferrer G.G.,Polytechnic University of Valencia | Ferrer G.G.,CIBER ISCIII | And 17 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2015

Currently available keratoprosthesis models (non-biological corneal substitutes) have a less than 75% graft survival rate at 2 years. We aimed at developing a model for keratoprosthesis based on the use of poly(ethyl acrylate) (PEA)-based copolymers, extracellular matrix-protein coating and colonization with adipose-derived mesenchymal stem cells. Human adipose tissue derived mesenchymal stem cells (h-ADASC) colonization efficiency of seven PEA-based copolymers in combination with four extracellular matrix coatings were evaluated in vitro. Then, macroporous membranes composed of the optimal PEA subtypes and coating proteins were implanted inside rabbit cornea. After a 3-month follow-up, the animals were euthanized, and the clinical and histological biointegration of the implanted material were assessed. h-ADASC adhered and survived when cultured in all PEA-based macroporous membranes. The addition of high hydrophilicity to PEA membranes decreased h-ADASC colonization in vitro. PEA-based copolymer containing 10% hydroxyethyl acrylate (PEA-HEA10) or 10% acrylic acid (PEA-AAc10) monomeric units showed the best cellular colonization rates. Collagen plus keratan sulfate-coated polymers demonstrated enhanced cellular colonization respect to fibronectin, collagen, or uncoated PEAs. In vivo implantation of membranes resulted in an extrusion rate of 72% for PEA, 50% for PEA-AAc10, but remarkably of 0% for PEA-HEA10. h-ADASC survival was demonstrated in all the membranes after 3 months follow-up. A slight reduction in the extrusion rate of h-ADASC colonized materials was observed. No significant differences between the groups with and without h-ADASC were detected respect to transparency or neovascularization. We propose PEA with low hydroxylation as a scaffold for the anchoring ring of future keratoprosthesis. © 2014 Wiley Periodicals, Inc.

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