Donor Corneal Stroma Research Articles

Corneal stromal regeneration

AbstractCorneal grafting is one of the most common forms of human tissue transplantation. The corneal stroma is responsible for many characteristics of the cornea. For these reasons, an important volume of research has been made to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation techniques. There is an increasing interest today in cell therapy of the corneal stroma using induced pluripotent stem cells or mesenchymal stem cells since these cells have shown to be capable of producing new collagen within the host stroma and even to improve its transparency. The first clinical experiment on corneal stroma regeneration in advanced keratoconus cases has been reported and included. Fourteen patients were randomized and enrolled into 3 experimental groups: (1) patients underwent implantation of autologous adipose‐derived adult stem cells alone, (2) patients received decellularized donor corneal stroma laminas, and (3) patients received implantation of recellularized donor laminas with adipose‐derived adult stem cells. Clinical improvement was detected with all cases in their visual, pachymetric, and topographic parameters of the operated corneas.

Full-field Optical Coherence Microscopy for Histology-like Analysis of Stromal Features in Corneal Grafts.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

Preimplantation dehydration for corneal allogenic intrastromal ring segment implantation.

Corneal allogenic intrastromal ring segments (CAIRS) are semicircular pieces of donor corneal stroma, which may be surgically implanted to flatten keratoconic corneas. These segments can be trimmed to different thicknesses; whereas thicker segments confer greater flattening, their bulk renders them more technically challenging to insert. Consequently, thinner segments are often preferred, especially for starting surgeons. Here, we describe a technique for transiently thinning CAIRS to facilitate easy insertion, thereby permitting the use of thicker segments to achieve the maximal flattening effect.

Flattening the curve: manual method for corneal allogenic intrastromal ring segment implantation.

Corneal allogenic ring segments are semicircular pieces of donor corneal stroma that may be surgically implanted to flatten keratoconic corneas. Conventionally, these donor segments are inserted into channels created using femtosecond laser dissection. However, access to femtosecond technology is not universal. In this study, an alternate, manual technique for channel creation, which is femtosecond laser independent, is described.

Corneal Stroma Regeneration: New Approach for the Treatment of Cornea Disease.

Corneal grafting is one of the most common forms of human tissue transplantation. The corneal stroma is responsible for many characteristics of the cornea. For these reasons, an important volume of research has been made to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation techniques.There is an increasing interest today in cell therapy of the corneal stroma using induced pluripotent stem cells or mesenchymal stem cells since these cells have shown to be capable of producing new collagen within the host stroma and even to improve its transparency.The first clinical experiment on corneal stroma regeneration in advanced keratoconus cases has been reported and included. Fourteen patients were randomized and enrolled into 3 experimental groups: (1) patients underwent implantation of autologous adipose-derived adult stem cells alone, (2) patients received decellularized donor corneal stroma laminas, and (3) patients received implantation of recellularized donor laminas with adipose-derived adult stem cells. Clinical improvement was detected with all cases in their visual, pachymetric, and topographic parameters of the operated corneas.Other recent studies have used allogenic SMILE implantation lenticule corneal inlays, showing also an improvement in different visual, topographic, and keratometric parameters.In the present report, we try to summarize the available preclinical and clinical evidence about the emerging topic of corneal stroma regeneration.

Biobanking of Dehydrated Human Donor Corneal Stroma to Increase the Supply of Anterior Lamellar Grafts.

To investigate the effect of dehydration on human donor corneal stroma for biobanking. Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties. No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 μm) and after rehydration (563.6 ± 15.77 μm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 μm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa). Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.

Treatment of donor corneal tissue with immunomodulatory cytokines: a novel strategy to promote graft survival in high-risk corneal transplantation

Antigen-presenting cells (APCs) play an important role in transplant rejection and tolerance. In high-risk corneal transplantation, where the graft bed is inflamed and vascularized, immature APCs in the donor corneal stroma quickly mature and migrate to lymphoid tissues to sensitize host T cells. In this study, using a mouse model of corneal transplantation, we investigated whether enrichment of tolerogenic APCs (tolAPCs) in donor corneas can enhance graft survival in corneal allograft recipients with inflamed graft beds. Treatment of donor corneas with interleukin-10 (IL-10) and transforming growth factor-β1 (TGFβ1) altered the phenotype and function of tissue-residing APCs. Transplantation of these tolAPC-enriched corneas decreased frequencies of interferon gamma (IFNγ)+ effector T cells (Teffs), as well as allosensitization in the hosts, diminished graft infiltration of CD45+ and CD4+ cells, and significantly improved corneal allograft survival compared to saline-injected controls. These data provide a novel approach for tolAPC-based immunotherapy in transplantation by direct cytokine conditioning of the donor tissue.

New parameters in assessment of human donor corneal stroma.

To provide quantitative parameters for assessment of human donor corneal stroma by imaging stromal features of diseased and normal human corneas with full-field optical coherence microscopy (FFOCM), using confocal microscopy (CM) and histology as reference techniques. Bowman's layer (BL) thickness and keratocyte density were assessed exvivo in 23 human donor corneas and 27 human pathological corneas (keratoconus and other corneal disorders) with FFOCM, CM and histology. Stromal backscattering was assessed with FFOCM. Additionally, 10 normal human corneas were assessed invivo with CM. In FFOCM, the logarithm of the normalized stromal reflectivity was a linear function of stromal depth (R2 =0.95) in human donor corneas. Compared with keratoconus corneas, human donor corneas featured higher BL thickness (p=0.0014) with lower coefficient of variation (BL-COV; p=0.0002), and linear logarithmic stromal reflectivity with depth (higher R2 , p=0.0001). Compared with other corneal disorders, human donor corneas featured lower BL-COV (p=0.012) and higher R2 (p=0.0001). Using the 95% confidence limits of the human donor cornea group, BL thickness<6.5μm (sensitivity, 57%; specificity, 100%), BL-COV>18.6% (79%; 100%) and R2 <0.94 (93%; 71%) were revealed as indictors of abnormal cornea. In CM, keratocyte density decreased with stromal depth (r=-0.56). The mean overall keratocyte density (cells/mm2 ) was 205 in human donor corneas, 244 in keratoconus, 176 in other corneal disorders and 386 in normal corneas. Full-field optical coherence microscopy (FFOCM) provides precise and reliable parameters for non-invasive assessment of human donor corneal stroma during storage, enabling detection of stromal disorders that could impair the results of keratoplasty.

Cornea preservation time study: methods and potential impact on the cornea donor pool in the United States.

The aim of this study was to describe the aims, methods, donor and recipient cohort characteristics, and potential impact of the Cornea Preservation Time Study (CPTS). The CPTS is a randomized clinical trial conducted at 40 clinical sites (70 surgeons) designed to assess the effect of donor cornea preservation time (PT) on graft survival 3 years after Descemet stripping automated endothelial keratoplasty (DSAEK). Eyes undergoing surgery for Fuchs endothelial corneal dystrophy or pseudophakic/aphakic corneal edema were randomized to receive donor corneas stored ≤7 days or 8 to 14 days. Donor and patient characteristics, tissue preparation and surgical parameters, recipient and donor corneal stroma clarity, central corneal thickness, intraocular pressure, complications, and a reading center-determined central endothelial cell density were collected. Surveys were conducted to evaluate pre-CPTS PT practices. The 1330 CPTS donors were: 49% >60 years old, 27% diabetic, had a median eye bank-determined screening endothelial cell density of 2688 cells/mm, and 74% eye bank prepared for DSAEK. A total of 1090 recipients (1330 eyes including 240 bilateral cases) had: median age of 70 years, were 60% female, 90% white, 18% diabetic, 52% phakic, and 94% had Fuchs endothelial corneal dystrophy. Before the CPTS, 19 eye banks provided PT data on 20,852 corneas domestically placed for DSAEK in 2010 to 2011; 96% were preserved ≤7 days. Of 305 American Academy of Ophthalmology members responding to a pre-CPTS survey, 233 (76%) set their maximum PT preference at 8 days or less. The CPTS will increase understanding of factors related to DSAEK success and, if noninferiority of longer PT is shown, will have great potential to extend the available pool of endothelial keratoplasty donors.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01537393.

Characterization, isolation, expansion and clinical therapy of human corneal epithelial stem/progenitor cells.

Stem cells can be defined as cells that have the capacity to self-renew and the ability to generate differentiated progeny or multiple cell lineages. True stem cells can turn into any type of cells, while progenitor cells are more or less committed to becoming cell types of a particular tissue. Human corneal epithelial stem cells (CESCs) represent a great example and model of adult stem or progenitor cells. Human CESCs have been identified to locate in the basal epithelial layer of the limbus, and thus also referred as to limbal stem cells. We would like to use the both terms, stem and progenitor cells in this chapter based on previous use in the literature for more than two decades. Although the CESCs have been identified to reside at the limbus and many stem cell markers have been proposed, there is no consensus to date regarding the definitive markers for CESCs, and identification and isolation of these cells are still challenging. Based on evaluation of a variety of proposed markers, we have characterized that the CESCs located in the basal layer of human limbal epithelium are small primitive cells expressing three patterns of molecular markers, which represent a unique phenotype of putative corneal epithelial stem or progenitor cells. Based on adult stem cell criteria and the putative limbal stem cell phenotype, our group has attempted to enrich for human CESCs through novel approaches including cell-sizing, adhering to extracellular matrix collagen type IV, and cell sorting for side population or for expression of ABCG2 or connexin 43 cell surface markers. The 5 clonogenic populations isolated from limbal epithelium and its cultures by different methods show the properties that are characteristics of adult stem/progenitor cells: 1) relatively undifferentiated, 2) high proliferative potential, 3) self-renewal. Expansion and cultivation of corneal epithelial progenitor cells have been achieved using different methods, such as limbal tissue explant culture, and limbal epithelial cell suspension co-culture with mouse 3T3 fibroblast feed layer. To avoid the use of xeno-components, two cell lines of commercial human fibroblasts have been identified that support human corneal epithelial regeneration, and have potential use in replacing mouse 3T3 cells for corneal tissue bioengineering. The concept of CESCs has formed the basis for identifying a class of blinding diseases that display features of corneal epithelial stem cell deficiency or limbal stem cell deficiency (LSCD), where the limbal epithelium is damaged. LSCD is characterized by persistent or recurrent epithelial defects, ulceration, corneal vascularization, chronic inflammation, scarring, and conjunctivalization (conjunctival epithelial ingrowth). Only transplantation of CESCs can restore vision. Due to an increasing shortage of corneal donors, corneal tissue engineering is becoming an important discipline that holds great promise for corneal reconstruction. CESCs and optical substrates are known to be the most important factors for corneal tissue bioengineering in regenerative medicine. Our team has recently explored the utilization of natural donor corneal stroma in corneal tissue engineering. In combination with fresh limbal epithelium containing stem cells, and the donor corneal stroma, a great source of natural optical substrate, we developed a native-like corneal equivalent construct with proliferative potential. This corneal construct provides a new clinical cell therapy for corneal reconstruction.

Donor corneal stroma and host–donor interface vascularization after Descemet’s membrane stripping with automated endothelial keratoplasty

Donor corneal stroma and host–donor interface vascularization after Descemet’s membrane stripping with automated endothelial keratoplasty

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-β1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-β1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.

Light microscopic evaluation of human donor corneal stroma during organ culture.

To try to facilitate evaluation of corneal stroma during organ culture by means of light microscopy. Corneal stroma of 53 consecutive organ-cultured corneas was studied by means of light microscopy during endothelial quality control. Out of 9 corneas with bad stromal evaluation, 2 were studied by means of transmission electron microscopy, and 7 were grafted. From the remaining 44 corneas with a normal light microscopic appearance, 35 were grafted. Stromal abnormalities consisted of bright visible structures with a cell-like shape corresponding to keratocyte injuries (i.e. cellular edema, light and dark vacuoles, cell membrane disruption and, finally, internal cytolysis) as observed by TEM. At 3 months postoperatively no clinical differences between the two groups of transplants were observed. Corneal stroma can be evaluated qualitatively and easily by means of light microscopy during organ culture. Further studies are needed to investigate whether the presence of lysed keratocytes in the graft's stroma actually influences the outcome of transplantation.

Corneal Neovascularization After Penetrating Keratoplasty

The purpose of this study was to delineate the patient and surgical factors that contribute to the development of corneal neovascularization (CNV) after penetrating keratoplasty (PK). Thirty-six eyes of 36 patients with no antecedent CNV were enrolled in the study. Grafts were sutured to the host with 16 10-0 nylon sutures with the knots buried alternately in either the host or donor corneal stroma. Multiple perioperative factors were recorded for each patient, and at each postoperative visit systematic corneal drawings were used to follow the development of neovascularization. The stroma adjacent to each suture of each graft was given a neovascularization score based on the extent of vessel growth toward the wound interface. Univariate and multivariate analyses were performed, including generalized estimating equations logistic regression where each eye is considered a cluster of observations. Thirty-four patients without preoperative CNV or inflammation were followed prospectively for 6-9 (mean, 7) months after PK. Fourteen eyes (41%) developed some degree of CNV. Indication for keratoplasty, age, gender, phakic status, and size of donor button were not risk factors for CNV development. The most significant risk factor identified for any degree of CNV was placement of the suture knot in the host stroma (p = 0.00007), with the overall relative risk of CNV associated with these knots over 2 (95% confidence interval, 1.1-4.2). Furthermore, the mean recipient size in eyes with postoperative CNV was larger than eyes that did not develop neovascularization (p = 0.015), and active blepharitis was associated with a fivefold increase in the risk of developing CNV to the wound edge (p = 0.008). Embedding suture knots in the host stroma, active blepharitis, and a large recipient bed are significantly associated with postkeratoplasty CNV.

Real‐time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

AbstractA modified tandem scanning confocal microscope was used for real‐time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Mooren's Ulcer After Penetrating Keratoplasty

Seventeen months after penetrating keratoplasty for pseudophakic bullous keratopathy, a patient developed severe pain and a peripheral corneal ulcer that had the characteristic clinical appearance of a Mooren's ulcer. We performed a 10-mm penetrating keratoplasty that extended from the superior margin of the previously placed graft to the inferior corneoscleral limbus to encompass the ulcerated cornea. Histopathologic examination of tissue removed at surgery disclosed that the peripheral corneal ulcer extended nearly through the entire stromal thickness and that the donor corneal stroma adjacent to the ulcer was infiltrated with lymphocytes, plasma cells, and neutrophils. The conjunctiva adjacent to the peripheral corneal ulcer was packed with plasma cells and also showed some lymphocytes and neutrophils.