Effect of improved preservation solution with methoxy polyethylene glycol succinimidyl propionate on rat cornea.

To observe the effect of DMEM/F12 pegylated with methoxy polyethylene glycol succinimidyl propionate (mPEG-SPA) on the biophysical and immune characteristics of the rat cornea preserved in it. Corneal grafts were harvested from Wistar rat and preserved in the DMEM/F12 plus mPEG-SPA, DMEM/F12 without mPEG-SPA, and standard Optisol-GS solution at 4 °C for 14 days, referred as plus-PEG, minus-PEG, and Optisol grafts, respectively. The biophysical properties of those grafts, including transmittance, thickness, water content, and biomechanics were investigated. The survival of those grafts was observed in the high-risk corneal transplantation model. Transmittance and biomechanics did not show any differences among those grafts. Thickness and water content of plus-PEG grafts were slightly improved. Proliferation and activation of lymphocytes were lower while they were incubated with plus-PEG grafts, compared with minus-PEG grafts and Optisol grafts. The mean survival time was significantly prolonged in plus-PEG grafts. DMEM/F12 solution plus mPEG-SPA improved the survival of corneal grafts and maintained the comparative biophysical characteristics of them, compared with the standard preservation solution.

Translational issues for human corneal endothelial tissue engineering.

Corneal endothelial disorders collectively represent a significant healthcare burden in most developed nations, and corneal transplantation is currently the only treatment available for patients with poor visual acuity and corneal blindness secondary to endothelial failure. Although vision in these patients can be restored by transplantation, the global demand for donor human corneas is far in excess of what can be provided for by eye banks around the world, and this deficit is set to increase with an ageing global population. As such, there has been a pressing need to explore novel and more sustainable options for the treatment of corneal endothelial diseases. In recent years, significant progress has been made not only in the development of corneal endothelial cell culture techniques, but also in the exploration of various translational strategies. Considered together, we are now much closer to attaining success in the treatment of corneal endothelial diseases via a cell-based, tissue-engineering approach. The aim of this review article is to provide an update of the translational issues currently facing human corneal endothelial cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.

La estandarización en el trasplante endotelial de membrana de Descemet: resultados de las primeras 450 cirugías / Standardization of the Descemet membrane endothelial keratoplasty technique: Outcomes of the first 450 consecutive cases

OBJETIVOS: Analizar los resultados de 450 casos con diferentes patologías endoteliales intervenidos mediante trasplante endotelial con la técnica de recambio de la membrana de Descemet (DMEK, por sus siglas en inglés: Descemet membrane endothelial keratoplasty) y evaluar las consecuencias de la estandarización de esta técnica. MATERIAL Y MÉTODOS: Se compararon 3 subgrupos consecutivos: el subgrupo I (casos 1-125) representaba la extensión de la curva de aprendizaje; el subgrupo II (casos 126-250) la transición a la estandarización de la técnica y el subgrupo III (casos 251-450) la estandarización propiamente dicha. Se registraron los resultados de agudeza visual mejor corregida pre- y postoperatorios, la densidad de células endoteliales, la paquimetría y las complicaciones. RESULTADOS: A los 6 meses de la cirugía, el 79% de los pacientes alcanzaron una agudeza visual mejor corregida ≥ 0,8 y el 43% ≥ 1. La densidad de células endoteliales media preoperatoria fue 2.530 ± 220 células/mm2, y 1.613 ± 495 células/mm2 al sexto mes poscirugía. La paquimetría era 668 ± 92 μm y 526 ± 46 μm pre- y postoperatoria a los 6 meses, respectivamente. No se encontraron diferencias en cuanto a la agudeza visual mejor corregida, la densidad de células endoteliales o la paquimetría entre los subgrupos (p > 0,05). El desprendimiento del injerto se observó en el 17,3% de los ojos. La tasa de desprendimientos disminuyó del 24 al 12%, y el número de reintervenciones, del 9,6 al 3,5%, del subgrupo I al III respectivamente. CONCLUSIONES: Los resultados visuales y la densidad de células endoteliales tras DMEK son independientes de la estandarización de la técnica quirúrgica. Sin embargo, la estandarización de la técnica podría haber contribuido con un descenso en el número de desprendimientos y con un relativamente bajo número de intervenciones secundarias. A la vista de estos resultados, DMEK podría convertirse en el tratamiento de elección para las enfermedades del endotelio corneal

OBJECTIVES: To evaluate the clinical outcome of the first 450 consecutive cases after Descemet membrane endothelial keratoplasty (DMEK), as well as the effect of standardization of the technique. MATERIAL AND METHODS: Comparison between 3 groups: Group I: (cases 1-125), as the extended learning curve; Group II: (cases 126-250), transition to technique standardization; Group III: (cases 251-450), surgery with standardized technique. Best corrected visual acuity, endothelial cell density, pachymetry and intra- and postoperative complications were evaluated before, and 1, 3 and 6 months after DMEK. RESULTS: At 6 months after surgery, 79% of eyes reached a best corrected visual acuity of≥0.8 and 43%≥1.0. Mean preoperative endothelial cell density was 2,530±220 cells/mm2 and 1,613±495 at 6 months after surgery. Mean pachymetry measured 668±92 μm and 526±46 μm pre- and (6 months) postoperatively, respectively. There were no significant differences in best corrected visual acuity, endothelial cell density and pachymetry between the 3 groups (P > .05). Graft detachment presented in 17.3% of the eyes. The detachment rate declined from 24% to 12%, and the rate of secondary surgeries from 9.6% to 3.5%, from group I to III respectively. CONCLUSIONS: Visual outcomes and endothelial cell density after DMEK are independent of the technique standardization. However, technique standardization may have contributed to a lower graft detachment rate and a relatively low number of secondary interventions required. As such, DMEK may become the first choice of treatment in corneal endothelial disease

Long-term outcome and predictors of adverse cardiac events in patients undergoing percutaneous coronary interventions with Genous endothelium predecessor cell coated stents compared to drug eluting stents.

This clinical research paper summerazed the 3-year long study, performed by interventional specialists and scientists as well and designated to assess the efficacy the percutaneous coronary intervention with the different types of coronary stents.

Aligned nanofibrillar collagen regulates endothelial organization and migration.

AIM: Modulating endothelial cell (EC) morphology and motility, with the aim to influence their biology, might be beneficial for the treatment of vascular disease. We examined the effect of nanoscale matrix anisotropy on EC organization and migration for vascular tissue engineering applications. MATERIALS & METHODS: We developed a flow processing technique to generate anisotropic nanofibrillar collagen. Human ECs were cultured on aligned or on randomly oriented collagen, and their cellular alignment and cytoskeletal organization were characterized by immunofluorescence staining and time-lapse microscopy. RESULTS: ECs were elongated along the direction of aligned collagen nanofibrils and had organized focal adhesions. Cellular protrusion migrated with greater directionality and higher velocity along the anisotropic nanofibrils compared with cells on random nanofibrils. The flow technique can be adapted to fabricate vascular grafts that support the endothelial phenotype. CONCLUSION: Aligned nanofibrillar collagen regulates EC organization and migration, which can significantly contribute to the development of vascular grafts.

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction.

BACKGROUND: Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI. METHODS AND RESULTS: In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone. CONCLUSION: Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.

Qualitative assessment of connective tissue graft with epithelial component. A microsurgical periodontal plastic surgical technique for soft tissue esthetics.

Connective tissue grafts have been used successfully in the treatment of gingival recession. In the mid 80s and late 90s, the periodontal literature presented various techniques such as free gingival grafts, pedicle flaps, subepithelial connective tissue grafts, acellular dermal matrix grafts, and guided tissue regeneration to cover denuded root surfaces. Currently, connective tissue grafting is a reliable treatment for esthetic root coverage. This paper presents a qualitative assessment of a surgical technique that uses a connective tissue graft, including a portion of epithelium in the shape of the defect. This procedure enhances the healing of the covered root surface, increases the thickness of the soft tissue and improves esthetics. The criteria used for evaluation were: color, volume, texture, and blending. This evaluation demonstrated encouraging results from an esthetic viewpoint.

Cryopreservation of composite tissue flaps: experimental studies in the rat.

Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylin & eosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylin & eosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.

Administration of ex vivo-expanded bone marrow-derived endothelial progenitor cells attenuates focal cerebral ischemia-reperfusion injury in rats.

OBJECTIVE: This study aimed to examine early effects of ex vivo-expanded bone marrow-derived endothelial progenitor cells (EPCs) on focal cerebral ischemia-reperfusion injury. METHODS: EPCs were obtained from mononuclear cells of autologous bone marrow of a rat. After culture on fibronectin-coated dishes for 10 to 14 days, 2.5 x 10 cells of EPCs were administered transarterially after 90 minute occlusion of the middle cerebral artery. RESULTS: Administration of EPCs significantly reduced both the infarct volume and the scores of neurological deficits at 24 and 48 hours. EPCs administered 2 hours after insult did not reduce infarct volume, but attenuated neurological deficits at 24 hours. Administration of EPCs significantly reduced the number of myeloperoxidase-immunoreactive cells in the ischemic lesion at 24 hours and increased regional cortical blood flow at 48 hours. EPCs were observed in the ischemic hemisphere and around the endothelial layer of the pial arteries. Most of them expressed endothelial nitric oxide synthase. CONCLUSION: Administration of ex vivo-expanded bone marrow-derived EPCs reduced infarct volume and neurological deficits in acute focal brain ischemia-reperfusion injury caused, at least in part, by attenuation of endothelial dysfunction.

Transplantation of corneal endothelium with Descemet's membrane using a hyroxyethyl methacrylate polymer as a carrier.

AIMS: To evaluate the histology and function of Descemet's membrane transplanted with intact endothelium. METHODS: Japanese white rabbits and human eyebank eyes were used as donors and recipients of Descemet's membrane transplantation. Donor endothelium was hydrodissected by injecting indocyanine green from a limbal incision, and then processed as a corneal scleral button. A 6 mm diameter donor sheet was trephined, and folded in half using a 6 mm diameter polymer as a carrier. Recipient endothelium was also hydrodissected from the limbus using trypan blue to stain the Descemet's membrane. Continuous curvilinear descemetorhexis (CCD) was performed to remove a circular section of the Descemet's membrane using a 27 gauge cystotome. Donor tissue was inserted into the anterior chamber through a 5 mm limbal incision and apposed to the host stroma. Polymers were removed following transplantation. Similar surgical procedures were performed in both rabbits and eyebank eyes. Haematoxylin eosin stains were performed after 28 days in rabbits, and eyebank eyes were fixed immediately following surgery for endothelial cell counts. RESULTS: Rabbit control eyes demonstrated stromal oedema caused by loss of Descemet's membrane, whereas transplanted eyes had clear corneas. The mean (standard deviation) pachymetry of operated eyes was 376.6 (SD 32.5) mum compared with 389.6 (SD 25.1) mum in the unoperated eye. Mean endothelial density immediately following surgery in eyebank eyes was 2749 (SD 288) cells/mm(2). CONCLUSIONS: Transplantation of Descemet's membrane by CCD produces a functional graft with an optically clear interface similar to control cornea.

[Relative quantitative analysis of corneal immunogenicity].

OBJECTIVE: To quantitate the relative immunogenicity of three major corneal cell layers (epithelium, stroma, and endothelium) among the whole corneal immunogenicity respectively. METHODS: Cellular immunity: Three porcine major corneal cell layers were heterotopically allografted to subcutaneous layer of 40 BALB-c mice respectively. Twelve days later, the peripheral white blood cells of recipients were double directly stained with anti-mouse IgG-fluorescein conjugate mAb and analyzed by flow cytometry. Humoral immunity: The suspension of porcine cornea or corneal epithelium was used as antigen to produce anti-porcine immune sera in C57BL-6 mice. With the anti-porcine immune sera, enzyme linked immunosorbent assay was performed. RESULTS: On cellular immunity detection, the immunogenicities of intact endothelium, epithelium and stroma, equal in thickness, were 70.75%, 27.63%, and 1.62% respectively. On humoral immunity detection, the immunogenicities were 62.11%, 31.77% and 6.12% in the three respective layers of equal thickness. CONCLUSION: These findings indicate that the immunogenicity of corneal stroma is the lowest among the three corneal cell layers of equal thickness. The preliminary results provide the experimental basis for clinical application of xenogenic graft of corneal stroma.

Distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product.

Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding beta-galactosidase (Ad beta Gal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of Ad beta Gal-transduced DC, EC, or myoblasts and were then monitored for anti-beta-galactosidase (anti-beta-Gal) antibody production, induction of gamma interferon-secreting CD8(+) T cells, and protection against melanoma tumor cells expressing beta-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8(+) T-cell response at a level of 50% of the specific response obtained with the Ad beta Gal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a beta-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.

Clinical autologous in vitro endothelialization of 153 infrainguinal ePTFE grafts.

BACKGROUND: Over the past 17 years, our group has developed and clinically applied an in vitro endothelialization procedure whereby infrainguinal expanded polytetrafluoroethylene (ePTFE) prostheses are confluently lined with cultured autologous endothelial cells before implantation. After a successful randomized pilot study from 1989 to 1993, the procedure was adopted for routine operations. METHODS: Since June 1993, 153 endothelialized ePTFE grafts were implanted in the infrainguinal position in 136 patients (102 above knee (AK) and 51 below knee (BK), 89 men and 47 women, mean age 64.7+/-9.4 years). Seventeen patients received an endothelialized prosthesis bilaterally. Autologous endothelial cells were harvested from 4- to 5-cm segments of a subcutaneous vein (in 86% the cephalic vein), grown to first-passage mass cultures and confluently lined onto 6- (n = 113) or 7-mm (n = 40) inner diameter (ID) ePTFE grafts, precoated with fibrin glue. The observation period for 6-mm grafts was 7 years, and for 7-mm grafts was 4 years. Patency assessment for Kaplan-Meier survivorship analyses was based on duplex sonography and angiography. RESULTS: Kaplan-Meier survivorship function revealed a primary patency rate of 62.8% after 7 years (SE = 0.05) for all infrainguinal reconstructions (60% AK/70.8% BK). The primary patency for stage II and III patients was 64.4% after 7 years. The more recent group of 7-mm ID grafts showed a primary patency of 83.7% after 4 years. CONCLUSIONS: Our data provide strong evidence that autologous endothelial cell lining distinctly improves the patency of small diameter vascular grafts.

Tissue engineering of vascular bypass grafts: role of endothelial cell extraction.

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5-6 mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patients' own endothelial cells. This review examines the various techniques and technologies used to date in order to extract endothelial cells for such graft engineering.

Gene transfer to the central nervous system by transplantation of cerebral endothelial cells.

A cerebral endothelial immortalized cell line was used in transplantation experiments to deliver gene products to the adult rat brain. Survival of grafted cells was observed for at least 1 year, without any sign of tumor formation. When genetically modified to express bacterial beta-galactosidase and transplanted into the striatum, these cells were shown, by light and electron microscope analysis, to integrate into the host brain parenchyma and microvasculature. Following implantation into the striatum and nucleus basalis of adult rats, endothelial cells engineered to secrete mouse beta-nerve growth factor (NGF) induced the formation of a dense network of low-affinity NGF receptor-expressing fibers near the implantation sites. This biological response was observed from 3 to 8 weeks after engraftment. The present study establishes the cerebral endothelial cell as an efficient vector for gene transfer to the central nervous system.

Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type.

PURPOSE: Endothelial cell transplantation has been suggested as a method to improve the patency of prosthetic grafts used for vascular reconstruction. A major technical concern of all cell transplantation studies has been the purity of cells in the primary isolate used for subsequent transplantation. Accordingly we have evaluated the cellular constituents of liposuction-derived human fat with immunocytochemistry and scanning electron microscopy. METHODS: Samples of liposuction-derived human fat were processed for immunohistochemistry and subsequently stained for the presence of von Willebrand factor (vWF), alpha-smooth muscle cell actin, cytokeratin (peptide 18), and the endothelial cell-specific marker EN4. We also performed histochemistry studies on the cells derived from this fat after collagenase dispersion of the liposuction far. RESULTS: Immunohistochemistry revealed that 86.1% of the cells in intact, liposuction-derived fat express vWF, whereas 5.7% of the cells exhibited alpha-smooth muscle cell actin, and 1.0% expressed the mesothelial cell-related antigen, cytokeratin peptide 18. Expression of EN4 was found in 89.6% of the cells counted in intact far. After digestion of fat with collagenase and centrifugal separation of adipocytes from vascular and stromal cells, the expression of vWF, alpha-smooth muscle cell actin, and cytokeratin was 77.5%, 5.8%, and 2.1%, respectively. EN4 expression was observed in 74.6% of the isolated cells. Thus most cells present in liposuction-derived fat, even before tissue digestion and cell isolation, were characterized as endothelium. Although other cells common to mesodermally derived tissue were identified (e.g., adipocytes, smooth muscle cells, and mesothelium), they represented a minor fraction of the total cells present. On isolation, the number of cells expressing vWF- and EN4-specific antigens was less than that observed in intact fat. CONCLUSIONS: This finding suggests that a portion of cells reacting with antibodies in situ lose vWF and EN4 staining during the isolation procedure. Unlike omentum, liposuction-derived fat predominantly contains adipocytes and endothelial cells. On digestion of liposuction-derived fat and separation of cells, vascular endothelial cells represent the major cellular component.

Robust survival of isolated bovine adrenal chromaffin cells following intrastriatal transplantation: a novel hypothesis of adrenal graft viability.

Previous investigations have demonstrated that adrenal chromaffin cells survive poorly when grafted into the striatum of rodents, nonhuman primates, and patients with Parkinson's disease. This poor survival has been attributed to the low levels of endogenous NGF within the striatum. However, chromaffin cells isolated from the nonchromaffin constituents of the adrenal medulla (fibroblasts and endothelial cells) have recently been demonstrated to survive grafting into a number of CNS sites. The present study determined whether nonchromaffin constituents of the adrenal medulla may be responsible for poor graft survival. We compared the survival of intrastriatally grafted isolated bovine chromaffin cells with that observed following implantation of either perfused adrenal medullary suspensions containing all adrenal medullary cell types or isolated chromaffin cells that were then reseeded with autologous fibroblasts and endothelial cells. Implants of perfused adrenal medullary cells survived poorly and most graft sites were infiltrated with macrophages. The chromaffin cells in this group that did survive appeared to be in the process of degeneration. In contrast, large numbers of isolated chromaffin cells survived for up to 2 months following transplantation. These cells maintained their endocrine phenotype and stained for all enzymatic markers of catecholamine synthesis as well as chromogranin A. Morphologically, these cells resembled chromaffin cells seen in situ and the perigraft region was essentially devoid of macrophages. When isolated chromaffin cells were reseeded with autologous fibroblasts and endothelial cells, the implants degenerated and few, if any, surviving chromaffin cells were observed. Interestingly, these latter grafts induced a host-derived sprouting response of tyrosine hydroxylase-immunoreactive fibers. These data demonstrate that large numbers of adrenal chromaffin cells can survive intrastriatal implantation in the absence of exposure to exogenous NGF. Rather, the nonchromaffin cells of the adrenal medulla (fibroblasts and endothelial cells) appear to compromise the viability of grafted chromaffin cells. Once they are eliminated from the graft, robust survival of chromaffin cells occurs. If clinical trials employing adrenal medullary grafts are still to be considered for the treatment of Parkinson's disease, isolation of the chromaffin cells should be considered to enhance graft viability.

Mesothelial seeding of knitted Dacron.

Bilateral superficial femoral artery replacement using knitted Dacron was performed in 38 dogs. One side was seeded with omental mesothelium and the other acted as an unseeded control. 111In-labelled platelet accumulation on grafts was measured at 5 days and 2 months and the thrombogenicity index of seeded and unseeded grafts calculated. Patency was monitored for 2 months, at which time grafts were removed and luminal thrombus, ultrastructural cell cover and prostacyclin release were measured. Cell seeding did not influence the mean(s.e.m.) thrombogenicity index of 0.95(0.25) and 0.88(0.24) at 5 days in control and seeded grafts respectively; nor was there any difference between the groups at 2 months. Occlusion occurred in six control and four seeded grafts. Seeding did not significantly improve the percentage thrombus-free area or luminal cell cover. Neither did it enhance mean(s.e.m.) luminal 6-keto-prostaglandin F1 alpha release of 2.58(0.80) pg cm-2 in controls and 2.63(0.78) pg cm-2 in seeded grafts. Further studies demonstrated that only a mean(s.e.m.) of 4.4(1.9) per cent of the seeded inoculum was present on grafts 48 h after implantation, providing too few cells to achieve confluent cover. Mesothelial cell seeding might be useful in promoting a healed graft surface but critical levels of seeding density must be achieved before the technique can be properly evaluated.