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.

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.

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.

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.

The relative immunogenicity of corneal epithelium, stroma, and endothelium. The role of major histocompatibility complex antigens.

Corneal allografts have been shown to give rise to immune responses, but the role and relative importance of individual corneal cell populations in evoking such responses remain unclear. We dissected ACI (RT1a) rat corneas into separate epithelial, stromal, and endothelial components by a method that yields pure cell populations in tissue culture, and grafted these components separately to groups of fully allogeneic PVG (RT1c) recipients). Grafts of corneal stroma elicited strong cellular cytotoxic immune responses in a cell-mediated lymphocytotoxic assay, but corneal epithelium failed to generate any detectable response. Grafts of corneal endothelium alone, however, evoked a potent cellular cytotoxic response. Using congenic rats, it was found that grafts from PVG.1A (RT1a) donors to PVG (RT1c) recipients (which differ at both the RT1.A and B loci) yielded identical results. However, no corneal component graft from PVG.R1 (RT1rl) donors to PVG recipients (which differ only at RT1.A) generated a detectable immune response. Use of target lymphoblasts from congenic strains established that at least part of all responses detected were directed against class I (RT1.A) major histocompatibility complex antigens. These findings indicate that there is differential immunogenicity of specific corneal tissue components in the rat that may be further influenced by the degree of MHC disparity between donor and recipient.

Morphology of transplanted corneal endothelium derived from tissue culture.

Rabbit corneal endothelial cells grown in vitro on glutaraldehyde-fixed gelatin membranes were examined by light and electron microscopy both before and after use as donor material in corneal endothelial transplants with autologous stroma and epithelium. The cultured cells had many of the morphologic characteristics of normal native endothelium, including polygonal cell shape stainable with silver nitrate, typical apical and lateral junctional complexes, terminal web, and cytoplasm dominated by profiles of granular endoplasmic reticulum and mitochondria. However, several distinct structural dissimilarities were also noted. These included intracellular accumulation of autophagocytic vacuoles and residual bodies, frequent pronounced swelling of intercellular spaces with retention of junctional complexes, excessive overlapping of cytoplasmic processes from two to four cells over long distances, and absence of normal cellular interdigitation. In contrast, the grafting of endothelial cell cultures into rabbit corneas resulted in clearing of residual bodies, appearance of normal intercellular spacing, elimination of abnormal cell overlapping, acquisition of complex basal and lateral cell interdigitation, and production of new, normal Descemet's membrane. The transplanted cells were morphologically indistinguishable from their native counterparts, except for the retention of the gelatin membrane, which showed no signs of degeneration. The reacquisition of normal cell morphology coincided with the re-establishment of normal endothelial cell function in the transplants. It was concluded that cultured endothelial cells possess the principal structural attributes of native endothelium but that their intercellular associations are incompletely developed.

Transplantation of tissue-cultured corneal endothelium.

Cultured endothelial cells have been shown to regain their physiological function when replaced in the rabbit eye. Corneas were wiped free of native endothelium and seeded with cultured cells. After an incubation period, full-thickness buttons were cut from these corneas and transplanted into recipient animals. Clear grafts were obtained only when the donor cells were derived from cultures less than a month old. Light and scanning electron microscopy showed the endothelial cells of these grafts to be present as a slightly irregular monolayer on the posterior surface of the cornea. In corneas made edematous by benzalkonium chloride, the clear graft remained surrounded by thick and cloudy host tissue. In those grafts with 3H-thymidine--labeled cells, radioactivity was limited to the host tissue.

A method for preparing a viable corneal endothelial layer, completely denuded of overlying stroma.

A method for denuding Descemet's membrane by dissection of all overlying stroma, while preserving the corneal endothelial layer, was developed and evaluated. The technique consists in treating the stroma with trypsin and surgically removing the softened stromal layers. With an automated thickness-measuring technique, the endothelium-Descemet preparation was found to range from 23 to 42 micron in thickness. Endothelial cell morphology was normal under specular and light-transmission microscopic examinations. Under electron microscopy, the endothelial cells appeared intact, except for an increase in the number of intracellular vacuoles. Occasionally, small portions of an intercellular space were found to be mildly dilated, but the over-all integrity of the junctional complex was intact. In vitro, the viability of the preparation was comparable with that of a cornea with all layers intact. Endothelial resting membrane potentials, measured with intracellular microelectrodes, were found to be within the normal (33 +/- 2 mV) range. It is thus possible to obtain a viable endothelial layer, completely stripped of stroma.

Transplantation of cultured bovine corneal endothelial cells to species with nonregenerative endothelium. The cat as an experimental model.

The in vivo transplantation of cultured bovine corneal endothelial cells has been attempted in the cat. Cat corneas denuded of their endothelium were coated with bovine corneal endothelial cells previously maintained in tissue culture. When grafted back into cat recipients, the corneal buttons remained clear with no edema. Alizarin red staining of the endothelial side of the corneal transplant demonstrated that the coated bovine corneal endothelial cells reorganized themselves into a highly organized cell monolayer within eight days in vivo. In contrast, corneas denuded of their endothelium became opaque and edematous within seven days and remained so thereafter. These results demonstrate that cultured corneal endothelial cells remain functional in vitro and can replace a damaged or nonfunctional endothelium i

Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results.

Autologous endothelial seeding of thin-walled ePTFE vascular prostheses (I.D.4 mm), interposed in the carotid artery, was performed in 10 dogs and 14 sheeps. Aspirin (250 mg/day) and dipyridamole (75 mg/twice daily) were given throughout the study as antiplatelet therapy. Animals were killed 2 and 5 weeks after surgery. Patency rates for seeded grafts in dogs were 75% (6:8) and 83% (10:12) at 2 and 5 weeks, respectively. In control grafts the patency rates were identical. Patency rates for seeded grafts in sheep were 0% (0:5) and 11% (1:9) at 2 and 5 weeks, respectively. Control grafts in sheep had a patency rate of 40% (2:5) and 0% (0:9) at corresponding times. Scanning electron microscopy showed an almost complete endothelialization of seeded grafts in dogs after 5 weeks. Platelet deposition was studied in the dogs by means of chromium-51-labeled autologous platelets. Significantly fewer platelets accumulated on seeded grafts, and the ratio of 6-keto-PCF1 alpha to thromboxane B2 was significantly higher, compared with unseeded grafts, which indicated the presence of a functionally active endothelial lining in seeded grafts. Differences in the hemostatic system could account for the high clotting incidence in sheep, compared with that in dogs. Such species differences make extrapolations to the clinical situation from autologous endothelial seeding in experimental animals hazardous.

Endothelial cell seeding of small-caliber synthetic grafts in the baboon.

Endothelial cell (EC) seeding has been proposed as a method to improve the performance of small-caliber synthetic vascular prostheses. Seeding experiments to date have all been carried out in the dog. This study investigates EC seeding of small-caliber Dacron carotid interposition grafts compared with contralateral control grafts in the baboon. Surface thrombogenicity was assessed at 24 hours, 2 weeks, and 4 weeks after implantation with indium 111-labeled autologous platelets. Morphologic and immunohistochemical techniques were used to assess the identity and homogeneity of the EC inoculum before seeding and to identify cell types on the harvested grafts. There was no significant difference in patency rates between seeded and control grafts at 5 weeks. Platelet accumulation on seeded grafts was significantly less (p less than 0.05) than on paired controls at 2 and 4 weeks after implantation. The luminal lining of seeded grafts had more cellular ingrowth, less adherent thrombus, and more surface cells with the morphologic and histochemical characteristics of EC than did the lining of controls. EC seeding reduces the platelet reactivity and accelerates EC coverage of small-caliber grafts in the baboon.

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.

Corneal endothelium under various storage conditions.

Three rabbit corneas each were stored in McCarey-Kaufman (M-K) medium, rabbit serum, and in a moist chamber at 4 degrees C refrigeration for various lengths of observation. The endothelial cells appeared normal under all conditions for the first 24 hours as compared with control corneas processed concurrently with each experimental group. After 48 hours of storage the specimens in the moist chamber showed isolated endothelial cell damage. The endothelia in M-K medium or rabbit serum appeared viable up to six days without significant differences although those stored in rabbit serum showed a better preservation of microvilli on individual endothelial cells. Under all conditions a mild shrinkage of the cells seemed to have taken place as indicated by the more pronounced cell boundaries. We incubated an equal number of control rabbit corneas at 37 degrees C with 5% CO2 and moist air in M-K medium, serum, and minimal essential medium (MEM) with 10% fetal calf serum and 100 units/ml of a penicillin and streptomycin mixture. In serum, the endothelia showed rapid destruction with swelling of the entire cornea. Those stored in M-K medium maintained a normal endothelial covering of the cornea up to six days. At nine days of storage, marked cellular changes were observed with dehiscence of the cellular layer. When stored in the MEM mixture, the endothelial cells showed a normal layer without obvious cell damage when compared with those stored in M-K medium up to four days. However, after six and nine days of storage, cellular destruction was greater in these specimens than in those stored in M-K medium. In addition, there was considerable swelling of the whole cornea under this storage condition.

Transplantation of cultured human neonatal corneal endothelium.

Human neonatal corneal endothelial cells were successfully maintained in tissue culture, morphologically resembling adult corneal endothelium. Eyebank donor corneas were obtained, denuded of their native endothelium and seeded with a suspension of the cultivated neonatal endothelial cells. After 48 hours, the eye-bank tissue was then transplanted into the eyes of Rhesus monkeys. Over a five month period, five of eight transplants cleared, with a mean central corneal thickness of 0.480 mm and endothelial cell densities ranging from 560 to 1650 cells/mm2. All control eyes without donor endothelium remained cloudy. In the experimental group three eyes initially thinned but subsequently became edematous. Further studies are needed to improve the seeding procedure and to assess the long-term viability of transplanted endothelium.

A corneal model for studying the interaction between the endothelium and sensitized lymphocytes.

We wished to develop a simple model for morphological studies on the interaction between the corneal endothelium and specifically sensitized lymphocytes. Rabbits were transplanted with orthotopic allogeneic skin or corneal grafts. After the onset of the transplantation reaction, lymphoid cells from the recipient's spleen or preauricular lymph nodes were harvested and placed on the endothelial surfaces of the donor and recipient corneas which were incubated in tissue culture medium at 37 degree C for 4 hours before being examined by vital staining or scanning electron microscopy. Compared to the recipient, the donor endothelium contained more lymphoid cell aggregates and more corneal cells were damaged. The in vitro model seemed suitable for studying the cytotoxic behaviour of sensitized lymphocytes confronting the corneal endothelium.

[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.

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.