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Maximum intensity projections of F-actin (green) and collagen fibrils (red) at the interface between the inner and outer matrices. (A) Migrating cells in basal media maintained a stellate morphology with dendritic processes, and collagen fibrils appeared to be randomly oriented without significant compaction or alignment. (B) After culture in 10% FBS, migrating cells developed a bipolar morphology with occasional stress fibers along the cell body. Collagen fibrils were compacted and aligned parallel to the long axes of pseudopodia. (C) After culture in TGF2, cells developed a broad morphology, and intracellular stress fibers were observed. Collagen fibrils were compacted both around and between the cells. (D) Migrating cells in PDGF appeared more elongated with branching processes. Collagen fibrils remained more randomly aligned around the cells.
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To evaluate a novel 3D culture model of the corneal stroma and apply it to investigate how key wound-healing growth factors regulate the mechanics of corneal keratocyte migration.
Rabbit corneal keratocytes were seeded within collagen matrices that were compacted using external compression. Six-millimeter-diameter buttons were then incubated in med...
Contexts in source publication
Context 1
... assess the local pattern of cell-induced matrix reorganiza- tion during migration, confocal reflection microscopy was per- formed. Reflected light imaging allows detailed visualization of the cells and the fibrillar collagen surrounding them. 41,63,64 Cell-matrix interactions can best be appreciated in movies showing maximum intensity projection images over a range of projection angles (Movies S1-S3, http://www.iovs.org/cgi/ content/full/51/2/864/DC1). Migrating cells in basal media maintained a stellate morphology with dendritic processes, and collagen fibrils appeared to be randomly oriented without compaction or alignment ( Fig. 9A; Movie S1). After culture in 10% FBS, cells developed a bipolar morphology with occa- sional stress fibers along the along the cell body ( Fig. 9B; Movie S2). Collagen fibrils were compacted and aligned parallel to the long axis of pseudopodia, and there was often an enhancement of cell-induced collagen reorganization between the migrating cells. Cells cultured in TGF developed a broad morphology, and stress fibers were observed within the cell. Collagen fibrils were compacted around the cells and between the cells (Fig. 9C). Migrating cells in PDGF appeared more elongated with branching processes. Collagen fibrils remained more randomly aligned around the cells, and there was less ECM compaction than with FBS or TGF ( Fig. 9D; Movie ...
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... assess the local pattern of cell-induced matrix reorganiza- tion during migration, confocal reflection microscopy was per- formed. Reflected light imaging allows detailed visualization of the cells and the fibrillar collagen surrounding them. 41,63,64 Cell-matrix interactions can best be appreciated in movies showing maximum intensity projection images over a range of projection angles (Movies S1-S3, http://www.iovs.org/cgi/ content/full/51/2/864/DC1). Migrating cells in basal media maintained a stellate morphology with dendritic processes, and collagen fibrils appeared to be randomly oriented without compaction or alignment ( Fig. 9A; Movie S1). After culture in 10% FBS, cells developed a bipolar morphology with occa- sional stress fibers along the along the cell body ( Fig. 9B; Movie S2). Collagen fibrils were compacted and aligned parallel to the long axis of pseudopodia, and there was often an enhancement of cell-induced collagen reorganization between the migrating cells. Cells cultured in TGF developed a broad morphology, and stress fibers were observed within the cell. Collagen fibrils were compacted around the cells and between the cells (Fig. 9C). Migrating cells in PDGF appeared more elongated with branching processes. Collagen fibrils remained more randomly aligned around the cells, and there was less ECM compaction than with FBS or TGF ( Fig. 9D; Movie ...
Context 3
... assess the local pattern of cell-induced matrix reorganiza- tion during migration, confocal reflection microscopy was per- formed. Reflected light imaging allows detailed visualization of the cells and the fibrillar collagen surrounding them. 41,63,64 Cell-matrix interactions can best be appreciated in movies showing maximum intensity projection images over a range of projection angles (Movies S1-S3, http://www.iovs.org/cgi/ content/full/51/2/864/DC1). Migrating cells in basal media maintained a stellate morphology with dendritic processes, and collagen fibrils appeared to be randomly oriented without compaction or alignment ( Fig. 9A; Movie S1). After culture in 10% FBS, cells developed a bipolar morphology with occa- sional stress fibers along the along the cell body ( Fig. 9B; Movie S2). Collagen fibrils were compacted and aligned parallel to the long axis of pseudopodia, and there was often an enhancement of cell-induced collagen reorganization between the migrating cells. Cells cultured in TGF developed a broad morphology, and stress fibers were observed within the cell. Collagen fibrils were compacted around the cells and between the cells (Fig. 9C). Migrating cells in PDGF appeared more elongated with branching processes. Collagen fibrils remained more randomly aligned around the cells, and there was less ECM compaction than with FBS or TGF ( Fig. 9D; Movie ...
Context 4
... assess the local pattern of cell-induced matrix reorganiza- tion during migration, confocal reflection microscopy was per- formed. Reflected light imaging allows detailed visualization of the cells and the fibrillar collagen surrounding them. 41,63,64 Cell-matrix interactions can best be appreciated in movies showing maximum intensity projection images over a range of projection angles (Movies S1-S3, http://www.iovs.org/cgi/ content/full/51/2/864/DC1). Migrating cells in basal media maintained a stellate morphology with dendritic processes, and collagen fibrils appeared to be randomly oriented without compaction or alignment ( Fig. 9A; Movie S1). After culture in 10% FBS, cells developed a bipolar morphology with occa- sional stress fibers along the along the cell body ( Fig. 9B; Movie S2). Collagen fibrils were compacted and aligned parallel to the long axis of pseudopodia, and there was often an enhancement of cell-induced collagen reorganization between the migrating cells. Cells cultured in TGF developed a broad morphology, and stress fibers were observed within the cell. Collagen fibrils were compacted around the cells and between the cells (Fig. 9C). Migrating cells in PDGF appeared more elongated with branching processes. Collagen fibrils remained more randomly aligned around the cells, and there was less ECM compaction than with FBS or TGF ( Fig. 9D; Movie ...
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Citations
... Secreted growth factors, such as transforming growth factor-beta 1 (TGF-β1) and platelet derived growth factor-BB (PDGF-BB), among others, are released into the stromal space and have been shown to initiate this response (13)(14)(15). Among these soluble factors, TGF-β1 is thought to promote myofibroblast differentiation (16)(17)(18), while PDGF-BB stimulates keratocyte proliferation and motility (19). In addition, PDGF signaling has been shown to act synergistically with TGF-β1 to regulate the myofibroblast differentiation (20,21). ...
During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-β1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-β1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-β1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-β1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-β1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of ⍺-SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of ⍺-SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of ⍺-SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-β1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes.
Statement of Significance
In vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-β1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated ⍺-SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-β1 and PDGF-BB.
... A suitable natural polymer for bioink preparation is collagen, which is abundant in the extracellular matrix and thus is very natural to use and is also highly available in the connective tissues of organisms [4]. Collagen has a high affinity for adherent cells, which is the reason why these hydrogels are widely used in biomedical applications, from testing materials for 3D bioprinting to general tissue models for in vitro cell studies, drug testing, and specialized tissue models, especially when using cell-filled hydrogels [5]. ...
It is believed that 3D bioprinting will greatly help the field of tissue engineering and regenerative medicine, as live patient cells are incorporated into the material, which directly creates a 3D structure. Thus, this method has potential in many types of human body tissues. Collagen provides an advantage, as it is the most common extracellular matrix present in all kinds of tissues and is, therefore, very natural for cells and the organism. Hydrogels with highly concentrated collagen make it possible to create 3D structures without additional additives to crosslink the polymer, which could negatively affect cell proliferation and viability. This study established a new method for preparing highly concentrated collagen bioinks, which does not negatively affect cell proliferation and viability. The method is based on two successive neutralizations of the prepared hydrogel using the bicarbonate buffering mechanisms of the 2× enhanced culture medium and pH adjustment by adding NaOH. Collagen hydrogel was used in concentrations of 20 and 30 mg/mL dissolved in acetic acid with a concentration of 0.05 and 0.1 wt.%. The bioink preparation process is automated, including colorimetric pH detection and adjustment. The new method was validated using bioprinting and subsequent cultivation of collagen hydrogels with incorporated stromal cells. After 96 h of cultivation, cell proliferation and viability were not statistically significantly reduced.
... Several studies have reported that its signaling is involved in corneal fibroblast migration and proliferation, which are fundamental processes for corneal wound healing [23][24][25][26] . In addition, treatment with PDGF induces cell elongation in rabbit corneal keratocytes 27 . Similarly, human corneal stromal cells treated with PDGF have a very elongated spindle shape 28 . ...
Corneal astigmatism is reportedly associated with polymorphisms of the platelet-derived growth factor receptor alpha (PDGFRA) gene region in Asian populations of Chinese, Malay, and Indian ancestry and populations of European ancestry. In this study, we investigated whether these PDGFRA polymorphisms are associated with corneal astigmatism in a Japanese population. We recruited 1,535 cases with corneal astigmatism (mean corneal cylinder power across both eyes: ≤ − 0.75 diopters [D]) and 842 controls (> − 0.75 D) to genotype 13 single-nucleotide polymorphisms (SNPs) in the PDGFRA gene region. We also performed imputation analysis in the region, with 179 imputed SNPs included in the statistical analyses. The PDGFRA SNPs were not significantly associated with the cases with corneal astigmatism ≤ − 0.75 D. However, the odds ratios (ORs) of the minor alleles of SNPs in the upstream region of PDGFRA, including rs7673984, rs4864857, and rs11133315, tended to increase according to the degree of corneal astigmatism, and these SNPs were significantly associated with the cases with corneal astigmatism ≤ − 1.25 D or ≤ − 1.50 D (Pc < 0.05, OR = 1.34–1.39). These results suggest that PDGFRA SNPs play a potential role in the development of greater corneal astigmatism.
... However, little is known whether Fn fiber tension might correlate with or even regulate the K-F/M transition, or its reversal. Treatment with wound healing-associated growth factors like PDGF, FGF, and TGFβ1 initiates K-F/M phenotype transition and stimulates Fn fibrillogenesis, and matrix remodeling and contraction 5,27,28 . Because cellular forces can stretch Fn 19,21 , we asked here to what extent the treatment with and deprivation of wound healing-associated growth factors affects the ability of corneal keratocytes to generate forces, assemble a Fn matrix and stretch the Fn fibrils within it. ...
... A remodeling of the contractile actomyosin cytoskeleton is crucial for cell phenotype transitions (Supplementary Figs. S1 and S5) 28,35,36 . To quantify how this relates to changes in cell contractility during the K-F/M transition and reversal, Fn coated SU-8 nanopillar arrays were used to directly measure cellular contractility on a single cell level in our K-F/M transition model (Fig. 2a) 30 . ...
... The growth factor concentrations represent the lowest concentrations with a maximal effect on cell morphology and F-actin organization. These concentrations were adopted from previous studies 28,59 . For interventional experiments, cells were seeded onto glass cell culture substrates with adsorbed unlabeled Fn. ...
Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K–F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFβ1) induce the K–F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K–F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K–F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.
... In the corneal stroma, keratocytes are quiescent, dendritic cells that are activated (corneal fibroblasts) upon injury [103]. In numerous cell culture studies, primary keratocytes isolated from corneal stromal tissue are maintained with 10% FBS to stimulate proliferation [104][105][106]. However, this can transform them into fibroblasts or myofibroblasts phenotypes. ...
Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70-90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFβ1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFβ1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFβ1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, thirteen were responsive to CMS, and six were responsive to TGFβ1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFβ1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFβ1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFβ1 treatment under CMS, suggesting a potential role of TGFβ1 and biomechanical stretch in KC development.
... Upon injury to the epithelial basement membrane, large amounts of PDGF-BB are released into the corneal stroma and bind to PDGF receptors on corneal keratocytes [7]. The binding of PDGF-BB has been shown to modulate several keratocyte behaviors including proliferation and migration [8][9][10][11]. In addition, the treatment of keratocytes with PDGF induces them to adopt a more elongated morphology [11,12]. ...
... The binding of PDGF-BB has been shown to modulate several keratocyte behaviors including proliferation and migration [8][9][10][11]. In addition, the treatment of keratocytes with PDGF induces them to adopt a more elongated morphology [11,12]. ...
During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.
... Besides, fibroblastic features of keratocytes when cultured in high-glucose condition were associated with more pronounced cell migration and contraction (Foster, Gouveia and Connon, 2015). The enhanced migratory behavior of activated keratocytes with a fibroblastic phenotype compared to the quiescent state has also been shown in three-dimensional (3D) compressed collagen matrices (Kim et al., 2010). On the other hand, myofibroblastic keratocytes migrated less than fibroblastic cells, but caused more contraction and reorganization of the ECM (Kim et al., 2010). ...
... The enhanced migratory behavior of activated keratocytes with a fibroblastic phenotype compared to the quiescent state has also been shown in three-dimensional (3D) compressed collagen matrices (Kim et al., 2010). On the other hand, myofibroblastic keratocytes migrated less than fibroblastic cells, but caused more contraction and reorganization of the ECM (Kim et al., 2010). Cell migration behavior and force generation appear to be dependent in a dose-dependent manner on the cellular pathways that are stimulated upon exposure to specific cytokines. ...
... Here we used the HCK cell line in combination with TGFβ supplementation to have corneal fibroblastic and myofibroblastic cell culture models. The experimental protocols have been optimized and validated in a previous study for the HCK cell line (Manzer et al., 2009) and is similar to those used in another study with primary keratocytes (Kim et al., 2010). Curvature was imposed using 2.5D chips containing cylindrical structures of varying dimensions, coated with fibrillar collagen to mimic corneal stromal tissue architecture. ...
Functional tissue repair after injury or disease is governed by the regenerative or fibrotic response by cells within the tissue. In the case of corneal damage, keratocytes are a key cell type that determine the outcome of the remodeling response by either adapting to a fibroblast or myofibroblast phenotype. Although a growing body of literature indicates that geometrical cues in the environment can influence Myo(fibroblast) phenotype, there is a lack of knowledge on whether and how differentiated keratocyte phenotype is affected by the curved tissue geometry in the cornea. To address this gap, in this study we characterized the phenotype of fibroblastic and transforming growth factor β (TGFβ)-induced myofibroblastic keratocytes and studied their migration behavior on curved culture substrates with varying curvatures. Immunofluorescence staining and quantification of cell morphological parameters showed that, generally, fibroblastic keratocytes were more likely to elongate, whereas myofibroblastic keratocytes expressed more pronounced α smooth muscle actin (α-SMA) and actin stress fibers as well as more mature focal adhesions. Interestingly, keratocyte adhesion on convex structures was weak and unstable, whereas they adhered normally on flat and concave structures. On concave cylinders, fibroblastic keratocytes migrated faster and with higher persistence along the longitudinal direction compared to myofibroblastic keratocytes. Moreover, this behavior became more pronounced on smaller cylinders (i.e., higher curvatures). Taken together, both keratocyte phenotypes can sense and respond to the sign and magnitude of substrate curvatures, however, myofibroblastic keratocytes exhibit weaker curvature sensing and slower migration on curved substrates compared to fibroblastic keratocytes. These findings provide fundamental insights into keratocyte phenotype after injury, but also exemplify the potential of tuning the physical cell environments in tissue engineering settings to steer towards a favorable regeneration response.
... Another advantage of compressed collagen is the simplicity of the production and ease of scalability. Based on compressed collagen, a variety of tissue-engineering constructs are already suggested: for regeneration of skin [107][108][109], bone [110][111][112], cornea [113][114][115], bladder [116,117], blood vessels [118]. ...
Sometimes, to move forward, it is necessary to look back. Collagen type I is one of the most commonly used biomaterials in tissue engineering and regenerative medicine. There are a variety of collagen scaffolds and biomedical products based on collagen have been made, and the development of new ones is still ongoing. Materials, where collagen is in the fibrillar form, have some advantages: they have superior mechanical properties, higher degradation time and, what is most important, mimic the structure of the native extracellular matrix. There are some standard protocols for the formation of collagen fibrils in vitro, but if we look more carefully at those methods, we can see some controversies. For example, why is the formation of collagen gel commonly carried out at 37 °C, when it was well investigated that the temperature higher than 35 °C results in a formation of not well-ordered fibrils? Biomimetic collagen materials can be obtained both using culture medium or neutralizing solution, but it requires a deep understanding of all of the crucial points. One of this point is collagen extraction method, since not every method retains the ability of collagen to reconstitute native banded fibrils. Collagen polymorphism is also often overlooked in spite of the appearance of different polymorphic forms during fibril formation is possible, especially when collagen blends are utilized. In this review, we will not only pay attention to these issues, but we will overview the most prominent works related to the formation of collagen fibrils in vitro starting from the first approaches and moving to the up-to-date recipes.
... However, within hydrated 3D collagen matrices, FGF2 stimulates ruffling of keratocyte processes without inducing major changes in cell morphology, formation of stress fibers, or collagen matrix organization [51]. When corneal keratocytes are seeded within compressed 3D collagen matrices, fibroblast transformation is again observed [52]. Various studies are aimed at understanding the mechanisms involved in tissue contraction after corneal stromal wounding; Hibino et al. showed rabbit keratocytes cultured in a collagen gel, which contracted in the presence of Fetal Calf Serum (FCS) [53]. ...
Diabetic keratopathy is a corneal complication of diabetes mellitus (DM). Patients with diabetic keratopathy are prone to developing corneal haze, scarring, recurrent erosions, and significant wound healing defects/delays. The purpose of this study was to determine the contractility profiles in the diabetic human corneal stromal cells and characterize their molecular signatures. Primary human corneal fibroblasts from healthy, Type 1 DM (T1DM), and Type 2 DM (T2DM) donors were cultured using an established 3D collagen gel model. We tracked, measured, and quantified the contractile footprint over 9 days and quantified the modulation of specific corneal/diabetes markers in the conditional media and cell lysates using western blot analysis. Human corneal fibroblasts (HCFs) exhibited delayed and decreased contractility compared to that from T1DMs and T2DMs. Compared to HCFs, T2DMs demonstrated an initial downregulation of collagen I (day 3), followed by a significant upregulation by day 9. Collagen V was significantly upregulated in both T1DMs and T2DMs based on basal secretion, when compared to HCFs. Cell lysates were upregulated in the myofibroblast-associated marker, α-smooth muscle actin, in T2DMs on day 9, corresponding to the significant increase in contractility rate observed at the same time point. Furthermore, our data demonstrated a significant upregulation in IGF-1 expression in T2DMs, when compared to HCFs and T1DMs, at day 9. T1DMs demonstrated significant downregulation of IGF-1 expression, when compared to HCFs. Overall, both T1DMs and T2DMs exhibited increased contractility associated with fibrotic phenotypes. These findings, and future studies, may contribute to better understanding of the pathobiology of diabetic keratopathy and ultimately the development of new therapeutic approaches.
... The influence of growth factors on keratocytes is mainly investigated in wound healing studies 10 and studies investigating the composition of the ECM 12 or the phenotype of the cells. 7,11,[30][31][32] In these studies, the cells are cultivated for a maximum of a few days with the respective media. To obtain tissue-engineered stroma without the use of scaffolds, cultivation for several weeks would be necessary. ...
... 65 Furthermore, TGF-β1 promotes differentiation into myofibroblasts, a keratocyte phenotype that plays a role in wound healing. 7,30,31,63,66 Myofibroblasts increasingly synthesize ECM and are responsible for its reorganization and for wound edge contraction. They are characterized by the presence of α-SMA. ...
... They are characterized by the presence of α-SMA. 30,31,63,64 In our study, compared to cultivation with vitamin C alone, cultivation with TGF-β1 and vitamin C resulted in a reduction of the cell count (Figure 3(a)). Since all sheets were generated with medium containing FBS, it can be assumed that TGF-β1 is present in the serum and that some of the cells are differentiated into α-SMApositive myofibroblasts. ...
The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-β1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-β1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.
