Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.     

Morphological characteristics and intercellular connections of corneal keratocytes

PURPOSE: To investigate the morphological characteristics of keratocytes and the interconnection of keratocytes with adjacent keratocytes using the flat preparation method and scanning electron microscopy with a frontal section of the human corneal stroma. METHODS: The thin, corneal collagen lamellae were carefully dissected from the cornea (n=7), which had been stained by the flat preparation method. The remaining tissue was fixed in 3% glutaraldehyde and observed by transmission electron microscopy following the frontal section. RESULTS: The flat preparation revealed the corneal fibroblasts between the lamellae of the collagen fibers and showed that the ramifying cellular processes of the keratocytes were in contact with the cytoplasmic processes or cell bodies of neighboring fibroblasts. Two types of discrete subpopulations of keratocytes were identified: a smaller, cellular type of keratocyte with spindle-shaped nucleus with heterochromatin, and a larger, cellular type with a large indented nucleus with relatively scanty cytoplasm. Collagen fibers ran parallel to each other toward the fenestration of the cytoplasmic wall of the keratocyte. CONCLUSIONS: These flat preparation method results showed that the keratocytes within the corneal stroma are interconnected with the adjacent keratocytes, which indicates the presence of a functional communicating network through the keratocyte circuits within the stroma. A smaller, cellular type of keratocyte with spindle-shaped nucleus was morphologically differentiated from a larger, cellular type with a large, indented nucleus by flat preparation and transmission electron microscopy.     

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea

PURPOSE: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. METHODS: Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. RESULTS: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. CONCLUSIONS: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.     

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.     

Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes

PURPOSE: To evaluate the anti-ganglioside monoclonal antibody 3G5 as a marker of corneal keratocytes. METHODS: 3G5 expression on keratocytes was investigated by immunofluorescence microscopy. Studies were performed on frozen sections of normal human, bovine, porcine, rabbit, rat, and mouse corneas and on repairing rabbit cornea. In vitro studies were performed on tissue-cultured human, bovine, porcine, mouse, and rabbit keratocytes. RESULTS: 3G5 stained frozen sections of human, bovine, porcine, rat, and rabbit cornea but not mouse cornea and the staining pattern followed the distribution of stromal keratocytes but did not stain epithelium or endothelium. Subconfluent human and bovine keratocyte cultures were 3G5 negative. Almost 100% of the human and bovine cells that were maintained at confluence without replacement of serum-containing culture medium for 2 weeks became 3G5 positive. The 3G5 antigen was constitutively expressed on cultured rabbit and porcine keratocytes under all conditions examined. Mouse keratocyte cultures did not express 3G5. The 3G5 antigen was not present on myofibroblastic cells in the repairing area of a full-thickness wound in rabbit cornea that had been healing for 20 days. The area surrounding the healing wound expressed 3G5 antigen in an altered distribution, whereas 3G5 antigen was distributed in the expected pattern in areas that were distant from the wound. When rabbit keratocytes were induced to express the myofibroblast marker alpha-smooth muscle actin by treatment with TGFbeta1 in vitro, the pattern of 3G5 staining was altered. CONCLUSIONS: The 3G5 antigen is a useful marker for the identification of corneal keratocytes and for documenting their response to environmental stimuli associated with wound repair.     

Comparison of stromal remodeling and keratocyte response after corneal incision and photorefractive keratectomy

PURPOSE: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy to understand the histophysiological and immunohistochemical differences between these two types of surgery. METHODS: Corneal incision or photorefractive keratectomy was performed in rabbits or rats. Then we chronologically observed the histological changes and the changes in the localization of extracellular matrix proteins. RESULTS: In both types of surgery, the keratocyte population at the damaged stroma became sparse, and the cells began undergoing apoptosis immediately after the surgical procedure. Subsequently, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. We observed deposition of amorphous substances between keratocytes that had migrated, and stromal remodeling appeared to start. Three months after surgery, the corneal structure had recovered to a near-normal condition at the corneal incision. After photorefractive keratectomy, however, extracellular matrix proteins were strongly immunoreactive at the subepithelial regions. CONCLUSIONS: These results suggest that the stromal wound-healing processes are similar after corneal incision and photorefractive keratectomy. A corneal incision may induce a transient keratocyte response during stromal remodeling, but photorefractive keratectomy may induce a sustained keratocyte response.     

Mesenchymal stem cells derived from human limbal niche cells

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.     

New cryoprotectant for cryorefractive surgery

Cryorefractive surgeries, keratomileusis, keratophakia, and epikeratophakia cause destruction of keratocytes, which may result in postoperative corneal haze. We examined the effects of two cryoprotectants on keratocyte survival following freeze injury. We compared the ability of CPTES and the standard cryoprotectant KM-26 to prevent keratocyte death by altering the length of time corneal tissue was exposed to the cryoprotectant. When corneal stroma was immersed in CPTES for five minutes prior to freezing, 66.5% of the keratocytes survived; when tissue was immersed in KM-26 for the same length of time, 27.5% survived (P less than .01). Immersion for one to 30 minutes in CPTES prior to freezing produced keratocyte viabilities that were 40% to 80% of those of fresh, unfrozen tissue; immersion in KM-26 produced keratocyte viabilities of 20% to 60%. We compared the ability of these cryoprotectants to reduce corneal haze following freeze injury using our rabbit model of lamellar keratoplasty. The postoperative data were comparable to those in the cell culture experiments. Based on our findings in rabbit corneas, a cryoprotective medium such as CPTES may promote cell survival and thereby speed recovery from cryorefractive procedures in humans.     

Long-term storage of endocytosed latex beads in keratocytes in vivo.

Endocytosis by keratocytes (corneal fibroblasts) is an important part of the host defense system. To investigate the long-term fate of endocytosed materials, we injected polystyrene latex beads into the corneal stroma of four rabbits. The corneal stroma was observed under a transmission electron microscope 4 and 800 days after the injection. After 4 days, the beads were found not only between the collagen fibers of the stroma, but also in some keratocytes. After 800 days, no extracellular beads were seen, but endocytosed beads remained, surrounded by limiting membranes, in the cytoplasm of keratocytes. These observations demonstrate that keratocytes endocytose latex beads and store them for a long time, isolating these foreign materials from the corneal stroma. These observations suggest that keratocytes, like some other fibroblasts perform a noninflammatory and nonimmunological defense function.     

IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.     

Comparison of stromal remodeling and keratocyte response after corneal incision and photorefractive keratectomy]

PURPOSE: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy, respectively to learn the difference between the two surgeries histophysiologically and immunohistochemically. METHODS: We performed corneal incision or photorefractive keratectomy in rabbits or rats, and then we chronologically observed the histological changes and the changes in localization of extracellular matrix proteins. RESULTS: In both types of surgery, the keratocyte population in the damaged stroma became sparse, and the cells were undergoing apoptosis immediately after the procedures. After that, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. Deposition of amorphous substances was observed between migrated keratocytes, and stromal remodeling began. Three months after the surgery, corneal structure had recovered to near normal condition in the corneal incisions. In photorefractive keratectomy, however, strong immunoreactivity of extracellular matrix proteins was observed in the subepithelial regions. CONCLUSIONS: These results suggested that stromal wound healing processes were similar in both corneal incision and photorefractive keratectomy. Corneal incision may induce transient keratocyte response during the stromal remodeling, but photorefractive keratectomy may induce sustained keratocyte response.     

Collagen and glycosaminoglycan synthesis in aging human keratocyte cultures

Human corneal keratocytes were serially subcultured and the synthesis and secretion of collagen and glycosaminoglycans (GAGs) were studied as a function of increasing culture age in five different keratocyte strains. With repeated passage these cultures showed a significant decrease in the synthesis and secretion of total protein, collagen, and GAGs into the culture medium. These events were observed as gradual changes over a significant portion of the keratocyte's lifespan. Cultures at all ages studied synthesized and secreted type I procollagen. At all passage levels studied hyaluronic acid, dermatan sulfate, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and non-sulfated chondroitin were present in the culture medium. Sepharose 6B chromatography of the GAG fractions from young and old keratocyte cultures showed similar size ranges with no age associated decrease in GAG chain length to account for the observed decrease in GAG synthesis. Thus, cellular aging is an important factor in the evaluation of the synthetic potential of human keratocytes in culture. However, in our system the phenotypic expression of the major extracellular matrix components seems to be independent of aging in culture.     

Isolation of a putative keratocyte activating factor from the corneal stroma

Fetal bovine serum has commonly been used to expand the population of keratocytes in culture. Tissue extracts, however, have also been used to grow other cell types. We prepared a DMEM/F12 extract of corneal stroma and compared the growth and morphology of collagenase-isolated keratocytes cultured in DMEM/F12, or DMEM/F12 containing either stromal extract or fetal bovine serum. Cell proliferation was measured by 3H-thymidine and BrdU incorporation as well as by DNA quantitation. The extract was fractionated by gel filtration. Cell morphology was assessed by phase-contrast microscopy. Culture in both extract and serum stimulated keratocytes to proliferate, but keratocytes cultured in the extract grew more slowly due to a longer cell cycle and to a lower final density because of greater sensitivity to contact inhibition. Keratocytes cultured in serum became fibroblastic while those cultured in extract retained the dendritic morphology of quiescent keratocytes. The stimulating factors in the corneal extract were more sensitive to heat inactivation and of higher molecular weight than the stimulating factors in serum. These results indicate that the mitogenic activity in extract and serum are different and that the phenotypes resulting from growth in serum and extract are also different. Keratocytes cultured at low cell densities in the corneal extract may mimic keratocyte activation, an initial and crucial event for keratocytes during the corneal wound healing process.     

Characterization of the major matrix degrading metalloproteinase of human corneal stroma. Evidence for an enzyme/inhibitor complex

The human cornea is an avascular, highly organized tissue with the unique property of transparency. While the extracellular matrices of this tissue are composed of a variety of collagen types, proteoglycans and glycoproteins, little is known of the normal degradation and remodeling of these components. We examined the capacity of organ cultured human ocular tissues to produce and secrete metalloproteinases, a family of related enzymes capable of digesting a variety of extracellular matrices. We demonstrated that while enzymatic activities similar to type I collagenase and stromelysin are produced, the predominant activities of the corneal stroma and keratocyte cultures are a 68-kDa gelatinase. In our hands, this enzyme does not appear to be induced significantly by phorbol esters in vitro. In addition, this enzyme appears to be secreted as a complex with a 21-kDa protein that functions as an enzymatic inhibitor. Moreover, the keratocytes also produce a 28-kDa inhibitor which has similar properties to tissue inhibitor of metalloproteinase (TIMP).     

Formation of capillary-like tubes by vascular endothelial cells cocultivated with keratocytes.

This report describes an in vitro system that could be useful for investigating corneal neovascularization. When isolated bovine capillary endothelial cells (CEC) were cocultivated with rabbit corneal keratocytes, capillary-like cords extended actively from the endothelial cells in the multilayers of the keratocytes. The developing cords continued to elongate, branch out, and anastomose with each other, forming a capillary-like network by the 12th day of coculturing. Electron microscopic observation revealed that the cords were tubular structures composed of three to seven endothelial cells in the multilayers of keratocytes, and that a basal lamina-like matrix was situated at the abluminal face of the endothelial cells. The growth of cellular cords from the cloned CEC also occurred when CEC were seeded onto a cell-free extracellular matrix that had been synthesized by keratocytes. Keratocyte-conditioned medium alone did not stimulate proliferation of CEC. These observations clearly indicate that the formation of capillary-like structures by CEC depended upon the presence of an extracellular matrix produced by keratocytes, rather than upon the keratocytes themselves or any other keratocyte byproduct. This simple in vitro experimental system is proposed as a useful tool for studying corneal neovascularization.     

Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model

PURPOSE: To study keratocyte activation and cellular apoptosis in transplanted human corneas during the early postoperative period. METHODS: Ten human donor corneas preserved for 6 days at 4 degrees C were transplanted into the eyes of 10 adult cats. After confocal and specular microscopy in vivo 1 week after keratoplasty, the cats were killed, and the fixed corneas were examined by TUNEL assay and by scanning (SEM) and transmission electron microscopy (TEM). RESULTS: Abnormal keratocytes, in which portions of cell bodies and processes as well as nuclei were visible, were present in all corneas and occupied the anterior 16 to 562 microm of the stroma. By TEM in the same corneas, these abnormalities represented keratocytes that were activated to a repair phenotype. Only 0% to 1% of all corneal cells were apoptotic by TUNEL assay, except for the donor keratocytes near the wound, where 7% were apoptotic. The midstromal keratocyte density was decreased at 13,936 +/- 5,910 cells/mm(3) (mean +/- SD), and the endothelial cell density was 2,298 +/- 688 cells/mm(2), representing an endothelial cell loss of 7% +/- 16%. CONCLUSIONS: Substantial keratocyte activation and low levels of cellular apoptosis occur 1 week after human corneal transplantation. The human-to-cat xenograft model of corneal transplantation demonstrated endothelial cell loss and other clinical findings similar to human allografts. The model will be useful for preclinical testing of new methods of long-term corneal preservation and of donor endothelial cell augmentation, as well as the study of human corneal wound healing and keratocyte replacement during the early postoperative period.     

Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts

PURPOSE: To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. METHODS: Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy. RESULTS: Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27-51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deep-etch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures. CONCLUSIONS: Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.