绿脓杆菌对角膜基质细胞介导的胶原降解作用的实验研究

目的 探讨绿脓杆菌性角膜炎组织破坏机制,为临床治疗提供理论依据。方法 Ⅰ型胶原凝胶,混悬及不混悬角膜基质细胞,与绿脓杆菌培养液共同培养２４ｈ,超滤去除天然胶原纤维,超滤液经过水解,分光光度计测定其羟脯氨酸含量。同时检测基质金属蛋白酶抑制剂Ｇａｌａｒｏｄｉｎ对胶原降解作用的影响。结果 绿脓杆菌培养液可直接降解Ⅰ型胶原,如同时存在角膜基质细胞此作用增强。无论 角膜基质细胞存在与否,Ｇａｌａｒｄｉｎ可明     

Effect of galardin on collagen degradation by Pseudomonas aeruginosa

The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P. aeruginosa culture broth and/or Galardin for 24 hr. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline. The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P. aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by keratocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P. aeruginosa were also examined. P. aeruginosa broth by itself induced collegen gel degradation. When keratocytes were present, P. aeruginosa broth increased the amount of degraded collagen even further. Galardin significantly reduced the amounts of collagen degraded by P. aeruginosa culture broth, whether keratocytes were present or absent in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed that inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P. aeruginosa broth. Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P. aeruginosa. The present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P. aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs.     

Collagenolytic Activity of Keratocytes Cultured in a Collagen Matrix

To study the mechanism of collagen degradation by keratocytes, we developed the new in vitro model in which keratocytes were cultured three-dimensionally in a collagen matrix. Subcultured rabbit keratocytes were embedded in a type I collagen matrix and cultured in serum-free medium. Collagenolytic activity of the cells was determined by measuring the amount of hydroxyproline released into the medium from degraded collagen. Activities of collagenase in the medium were also measured, using collagen labeled with fluorescein isothiocyanate as a substrate. The presence of plasminogen was required for collagen degradation by keratocytes. In the presence of plasminogen, the amount of collagen degradation depended on both the cultivation period and the number of cells. The addition of interleukin-1 (IL-1) stimulated the collagen degradation in a dose-dependent manner. This stimulatory effect of IL-1 was completely inhibited by the addition of IL-1 receptor antagonist (IL-1ra). Collagenase activity in the medium was stimulated by the addition of IL-1, and IL-1ra antagonized this stimulatory effect. These findings indicate that our present model may be useful for investigating the mechanism of collagen degradation by keratocytes.     

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.     

Differential regulation of collagen degradation by rabbit keratocytes and polymorphonuclear leukocytes

PURPOSE: Both activated keratocytes and infiltrated polymorphonuclear leukocytes (PMNs) contribute to corneal ulceration by degrading stromal collagen. The regulation of such collagen degradation by inflammatory cytokines was investigated with rabbit keratocytes and PMNs cultured in three-dimensional collagen gels. METHODS: Rabbit keratocytes or PMNs were cultured for 24 h in three-dimensional gels of type I collagen in the presence of plasminogen and various concentrations of either interleukin (IL)-1alpha, IL-6, IL-8, or tumor necrosis factor-alpha (TNF-alpha). Degradation of collagen during culture was assessed by measurement of released hydroxyproline. RESULTS: IL-1alpha increased the amount of collagen degraded by keratocytes or PMNs in a dose-dependent manner, whereas IL-6 had no effect on collagen degradation by either cell type. IL-8 increased the extent of collagen degradation by PMNs but not that by keratocytes, and TNF-alpha promoted collagen degradation by keratocytes but not that by PMNs. CONCLUSION: Inflammatory cytokines regulate collagen degradation by rabbit keratocytes and PMNs in culture in a differential manner, and therefore may contribute to the roles of these cells in corneal ulceration.     

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.     

Induction of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs correlates with outcome of acute experimental pseudomonal keratitis

This study aimed to investigate expressions and sources of matrix metalloproteinases (MMP)-2 and MMP-9, and of tissue inhibitors of MMP (TIMP)-1 and TIMP-2 in experimental Pseudomonas aeruginosa keratitis in rabbits. Pseudomonal keratitis was induced in New Zealand white rabbits, and macroscopic and microscopic examinations were performed at appropriate time points (3, 9, 12, 18, 24, 72 h). Expressions and sources of MMP-2, 9, and TIMP-1, 2 were determined using immunohistochemistry, gelatin zymography, ELISA, and RT-PCR. A typical corneal ulcer with a ring abscess was observed 12-72 h post-infection (p.i.) with P. aeruginosa. In microscopic examinations, massive inflammatory cell (mostly polymorphonuclear leukocytes, PMNs) infiltration and liquefactive necrosis were characteristic features. MMP-2 was constitutively expressed in keratocytes, and its expression was not apparently enhanced after pseudomonal infection as evidenced by zymography, immunostaining, and RT-PCR. However, MMP-9 and its activated form were induced, and were significantly enhanced 12-24 h p.i. MMP-9 appeared to derive from PMNs rather than from resident corneal cells. TIMP-1 was expressed in PMNs, macrophages, and keratocytes, and its expression was enhanced 72 h p.i. Although TIMP-2 was constitutively expressed as seen by immunostaining and RT-PCR, its concentration was below detection limits during the experiments. We demonstrated that MMP-9 was one of the important factors for corneal tissue destruction, because it was induced and significantly expressed in keratocytes and inflammatory cells after pseudomonal infection. Although TIMP-1 was expressed in later stages of infection, enhancement and activation of MMP-9 were much faster and stronger than those of TIMP-1, thereby facilitating tissue destruction leading to corneal ulceration.     

Effect of growth factors on collagen lattice contraction by human keratocytes.

A three-dimensional gel contraction model was used to evaluate interactions between human keratocytes and different kinds of collagen in the presence or absence of various growth factors. Bovine collagen type I or human placental copolymerized collagen type I/III was used to create the lattices. Normal keratocytes from neonatal, aged, and insulin-dependent diabetic donors, as well as abnormal keratocytes from a donor with macular corneal dystrophy, were cultured. Growth factors included epidermal growth factor (EGF), basic fibroblastic growth factor (FGF), insulin-like growth factor (IGF-I), and platelet-derived growth factor homodimer beta beta (PDGF). Gel area and optical transmittance were determined from computerized measurements. Dose-response experiments (0.01-100 ng/ml) demonstrated that PDGF at 10 ng/ml (P less than 0.005) and EGF at 1 and 10 ng/ml (P less than 0.0001) were the most effective in promoting gel contraction, compared to IGF-I and FGF. Comparison of cell strains revealed different dose-response profiles. Cells from insulin-dependent diabetics and cells from a donor with macular dystrophy contracted lattices more rapidly than cells from normal neonates (P less than 0.0001). Lattices of copolymerized human collagen type III/I demonstrated significantly reduced contraction rates (P less than 0.0001) and increased optical transmittance, compared to bovine collagen type I lattices. Ultrastructural studies revealed that keratocytes extend processes to form a network within the collagen lattice. Specialized intercellular junctional complexes were observed by transmission electron microscopy. This model provides a useful in vitro corneal stroma-equivalent for the study of keratocyte, extracellular matrix, and growth factor interactions.     

Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels

PURPOSE: To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. CONCLUSION: These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.     

Proteolytic activation of corneal matrix metalloproteinase by Pseudomonas aeruginosa elastase.

Purified Pseudomonas aeruginosa elastase cleaved a 65 kDa gelatinase [inactive proenzyme form of matrix metalloproteinase (MMP-2)] from human corneal fibroblasts into a biologically active fragment with an approximate molecular mass of 58 kDa. However, purified pseudomonal alkaline protease did not cleave MMP-2 appreciably. Since activated MMP-2 is known to degrade native type IV, V and VII collagens, all components of the corneal basement membrane or stroma, our results suggest a new role for pseudomonal elastase in the pathogenesis of corneal infection, inflammation and ulceration.     

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures

PURPOSE: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. METHODS: Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. RESULTS: The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. CONCLUSIONS: PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.     

IGF-II and collagen expression by keratocytes during postnatal development

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.     

Matrix metalloproteinase 2: involvement in keratoconus

PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas. METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents. RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

Effect of L-ascorbic acid 2-phosphate on cultured rabbit keratocytes (the third report). The effect on production and secretion of type I collagen]

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting phosphate derivative of L-ascorbic acid, on intracellular distribution and production of type I collagen in cultured rabbit keratocytes by an immunohistochemistry and enzyme immunoassay. Exposure of 0.1 mM P-Asc for 10 hours decreased a type I collagen immunoreactivity of the cytoplasm as stained in fine granular materials. 0.1 mM P-Asc induced increase of type I collagen level in the medium. The results suggested that 0.1 mM P-Asc might increase the biosynthesis and secretion of type I collagen in keratocytes and have a therapeutic effect on corneal stromal damage.     

Changes in extracellular matrix components after excimer laser photoablation in rat cornea

PURPOSE: To learn more about corneal wound healing after excimer laser photoablation. METHODS: Observations were made on the chronological changes in type I collagen, fibronectin, laminin, and type IV collagen after excimer ablation of rat cornea. Immunohistochemical techniques were used. RESULTS: There was no obvious change in the localization of type I collagen in the ablated area, but the localization of fibronectin, laminin, and type IV collagen changed remarkably. One day after ablation, immunofluorescent staining for fibronectin increased on the ablated surface. Subsequently, the fluorescence of fibronectin, laminin, and type IV collagen increased remarkably; in particular, the localization of these extracellular matrix proteins was sustained in the shallow layer of the stroma until about 24 weeks after ablation. Hematoxylin-eosin staining indicated that keratocytes temporarily disappeared 1 day after ablation, and activated keratocytes then migrated to the ablated areas. CONCLUSIONS: These results suggest that activated keratocytes might be synthesizing the extracellular matrix components. Therefore sustained responses of keratocytes may be induced by excimer laser photoablation.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Reconstructed corneas: effect of three-dimensional culture, epithelium, and tetracycline hydrochloride on newly synthesized extracellular matrix

PURPOSE: To evaluate the influence of the 3-dimensional collagen-glycosaminogycan-chitosan (CGC 3D) scaffold, epithelialization, and the addition of tetracycline hydrochloride on the ultrastructural organization, measured by the diameter and spacing of newly synthesized collagen I fibrils. METHODS: Little is known about the role of interactions between epithelial cells and fibroblasts in controlling the extracellular matrix of the cornea. We developed a hemicornea from a CGC 3D matrix cocultured with keratocytes and human epithelial cells. The keratocytes colonized this substrate, proliferated, and synthesized the extracellular matrix, reproducing a living stroma equivalent. RESULTS: Without a 3D scaffold, the collagen fibrils produced had an average diameter that was 42.7 nm and sigma = 16.9 nm. In the CGC 3D scaffold, the fibrils had an average diameter of 33.4 nm, with little dispersion (sigma = 6.7 nm), suggesting a greater regulation. The epithelium permitted a significant reduction in fibril diameter and interfibrillar spacing. Tetracycline hydrochloride had no effect on spacing but did have a significant effect on fibril diameter. We found positive interactions between the epithelium and tetracycline hydrochloride on the regulation of collagen fibrils but not on spacing. The presence of epithelium led to the increased formation of collagens I and V in the subepithelial area of the newly formed matrix. Type VI collagen was localized around the keratocytes throughout the matrix. CONCLUSIONS: Epithelialization and the 3D scaffold had a great influence on the diameter of collagen I fibrils.