No abstract

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.

Human corneal fibroblasts stimulated by VitC and TGF-beta1 appear to generate a model that resembles processes observed in human corneal fibrosis. This model should be useful in examining matrix deposition and assembly in a wound-healing situation.

Purpose Corneal tissue engineering has attracted the attention of many researchers over the years in part due to the cornea’s avascularity and relatively, straight forward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model where human corneal fibroblasts (HCFs) are stimulated by a stable Vitamin C (VitC) derivative to self-assemble an extracellular matrix (ECM). Addition of TGF-β1 enhanced the assembly of ECM; however, it was accompanied by the upregulation of specific fibrotic markers. In this study, we tested the effects of all three TGF-β isoforms (-β1, -β2 and -β3) on ECM production, as well as, expression of fibrotic markers. Methods HCFs were grown in four media conditions for 4 weeks: (Control) VitC only; (T1) VitC+TGF-β1; (T2) VitC+TGF-β2; or (T3) VitC+TGF-β3. Cultures were analyzed with Western Blots, TEM and indirect-immunofluorescence (IF). Results Compared to controls, all TGF-β isoforms stimulated matrix production by ~3 times. IF showed the presence of type III collagen and smooth muscle actin (SMA) in T1 and T2; however, T3 showed little, to no, expression. In western blots, T3 stimulated a lower type III/type I collagen ratio when compared to the other conditions. In addition, TEM indicated that T3 stimulated a higher level of matrix alignment and organization. Conclusions HCFs stimulated by VitC and TGF-β3 appear to generate a matrix that mimics the normal adult or developing human cornea; whereas, TGF-β1 and -β2 are driving the constructs toward a more fibrotic path.

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.