Collagen fibril diameter and alignment promote the quiescent keratocyte phenotype

Abstract

In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment – 30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison to collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin - transketolase (TKT), the myofibroblast marker - α-smooth muscle actin (SMA), and four matrix proteins - collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN) and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared to the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared to the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury.

Liquid Crystalline Ordered Collagen Substrates for Applications in Tissue Engineering

“…This automated fabrication method has also been further adapted to manufacture bilayer transparent films for use as corneal substitutes 20 . Our results suggest that by using a higher starting concentration of collagen we can significantly reduce the complexity of the system, making the production of these aligned fibres far more available to researchers.…”

Aligned nanofibrillar collagen scaffolds – Guiding lymphangiogenesis for treatment of acquired lymphedema

“…In brief, purified monomeric bovine type I collagen solution in a liquid crystal state [16, 17] was sheared onto a plastic substrate [18] to produce one micron thin membrane formed of about 30 nm diameter parallel-aligned fibrils. The membrane was delaminated from the plastic substrate and converted into a scaffold using liquid-air surface tension [19].…”

Effect of Collagen Nanotopography on Keloid Fibroblast Proliferation and Matrix Synthesis: Implications for Dermal Wound Healing

“…Color images available online at www.liebertpub.com/tea Fibroblast morphology on collagen nanofibrillar substrates Previously, we have shown that fibril arrangement affects corneal fibroblast morphology and adhesion to nanopatterned collagen scaffolds. 17 To determine whether this effect is also seen in fibroblasts related to dermal wound healing, KF, SF, and HDF were grown on the nanostructured scaffolds and controls and stained to image f-actin (phalloidin) and the nucleus (Hoechst). As seen in Figure 3, fibroblasts on the SA substrate exhibited a more dendritic and elongated morphology compared with cells on the other nanofibrillar substrates and FC control, which were larger, flatter, and more spread out with the appearance of numerous stress fibers ( Supplementary Fig.…”

“…16 Additionally, previous work in our group using corneal fibroblasts cultured on collagen nanofibrillar substrates has shown that both fibril diameter and alignment promote the quiescent keratocyte phenotype and downregulate matrix synthesis. 17 The objective of this study was to evaluate the effect of collagen fibril topography on KF proliferation, morphology, and matrix synthesis, all of which are indicators of wound healing in the skin. To this end, we cultured human fibroblasts from the following types of tissue: normal skin (human dermal fibroblasts [HDF]), normal scar (scar fibroblasts [SF]), and keloid scar (KF) on four collagen nanofibrillar substrates similar to those used in our previous study.…”

“…To this end, we cultured human fibroblasts from the following types of tissue: normal skin (human dermal fibroblasts [HDF]), normal scar (scar fibroblasts [SF]), and keloid scar (KF) on four collagen nanofibrillar substrates similar to those used in our previous study. 17 The four substrates selected for this study were glass slides coated with nanopatterned bovine collagen type I (Advanced BioMatrix). The collagen coatings have the following average fibril diameter and orientation: small diameter aligned (SA; 55 -14 nm), small diameter random (SR; 74 -12 nm), large diameter aligned (LA; 165 -31 nm), and large diameter random (LR; 192 -23 nm) fibrils, respectively.…”