Stimulation of Collagen Synthesis by Insulin and Proteoglycan Accumulation by Ascorbate in Bovine Keratocytes In Vitro

Musselmann, Kurt; Kane, Bradley; Alexandrou, B.L.; Hassell, John R.

doi:10.1167/iovs.06-0612

Cited by 42 publications

(39 citation statements)

References 55 publications

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“…This indicates that keratocytes cultured in IGF-II can proliferate and maintain their native phenotype similar to that shown for keratocytes cultured in 10 micrograms of insulin/ml (Musselmann et al 2005;Musselmann et al 2006;Guerriero et al 2007). IGF-II and high concentrations of insulin can bind IGF-IR, the receptor for IGF-I, to activate cell proliferation [for review see (Lelbach et al 2005)].…”

Section: Discussionsupporting

confidence: 70%

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

Musselmann

Kane

Alexandrou

et al. 2008

Experimental Eye Research

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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-β/ml over a 7-day culture period. We found that IGF-II and TGF-β, alone or combined, increased both 3 Hthymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to α-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no α-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-β expressed α-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-β, however, expressed α-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-β 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-β.

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Section: Discussionsupporting

confidence: 70%

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

Musselmann

Kane

Alexandrou

et al. 2008

Experimental Eye Research

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“…In our study, we used a combination of insulin and ascorbate to encourage collagen synthesis. This combination however has been shown recently to drive the accumulation of SLRPs in keratinocytes (Musselmann et al, 2006). Whether such a combination has a similar effect in chondrocytes remains to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Lumican inhibits collagen deposition in tissue engineered cartilage

et al. 2008

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“…Vitamin C also promotes the processing of procollagen to collagen and extracellular deposition of collagen [Levene and Bates, 1975;Hata and Senoo, 1989]. In the absence of vitamin C, a nonhydroxylated form of collagen is synthe- sized, which is less resistant to degradation due to the decrease in triple-helix stability [Peterkofsky, 1972;Levene and Bates, 1975;Musselmann et al, 2006]. Picrosirius red staining was used to quantify the amount of collagen secreted.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Vitamin C-Induced Cell Sheets Formed from Primary and Immortalized Human Corneal Stromal Cells for Tissue Engineering Applications

Grobe

Reichl²

2013

Cells Tissues Organs

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The purpose of this study was to compare the ability of primary human corneal stromal cells (HuFib cells) and SV40-immortalized human corneal keratocytes (HCK cells) to synthesize their own extracellular matrix induced by vitamin C supplementation. Therefore, the amount of collagen secreted and resulting biomechanical properties based on the culture duration were assessed. Cells were cultivated for several weeks with or without vitamin C. The amount of collagen secreted by the cells was quantified based on the culture duration. Cell viability was simultaneously determined via the MTT assay. Collagen secretion was increased as a result of vitamin C supplementation. The effect was stronger in primary cells. In addition, vitamin C supplementation had a positive effect on HuFib cell viability. Vitamin C supplementation induced the formation of detachable cell sheets in both primary and immortalized cells. The biomechanical properties of the sheets were evaluated using a static material testing machine, and the ultrastructure of the cell sheets was examined using scanning electron microscopy. The cell sheets formed from HuFib cells had a higher percentage of light transmission between 400 and 800 nm and were superior in terms of E-modulus and ultimate strength testing. Indirect immunofluorescence and Western blot confirmed the presence of collagen type I in the HuFib and HCK cell cultures. Stimulating secretion of the extracellular matrix in corneal stromal cells is a promising approach for corneal stroma reconstruction for tissue engineering applications.

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Stimulation of Collagen Synthesis by Insulin and Proteoglycan Accumulation by Ascorbate in Bovine Keratocytes In Vitro

Cited by 42 publications

References 55 publications

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

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

Lumican inhibits collagen deposition in tissue engineered cartilage

Characterization of Vitamin C-Induced Cell Sheets Formed from Primary and Immortalized Human Corneal Stromal Cells for Tissue Engineering Applications

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