Dataset Information

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

ABSTRACT: In a fibroblast colony model of corneal stromal development, we asked how physiological tension influences the patterning dynamics of fibroblasts and the orientation of deposited extracellular matrix (ECM). Using long-term live-cell microscopy, enabled by an optically accessible mechanobioreactor, a primary human corneal fibroblast colony was cultured on three types of substrates: a mechanically biased, loaded, dense, disorganized collagen substrate (LDDCS), a glass coverslip, and an unloaded, dense, disorganized collagen substrate (UDDCS). On LDDCS, fibroblast orientation and migration along a preferred angle developed early, cell orientation was correlated over long distances, and the colony pattern was stable. On glass, fibroblast orientation was poorly correlated, developed more slowly, and colony patterns were metastable. On UDDCS, cell orientation was correlated over shorter distances compared with LDDCS specimens. On all substrates, the ECM pattern reflected the cell pattern. In summary, mechanically biasing the collagen substrate altered the early migration behavior of individual cells, leading to stable emergent cell patterning, which set the template for newly synthesized ECM.

SUBMITTER: Zareian R

PROVIDER: S-EPMC5073220 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

Zareian Ramin R Susilo Monica E ME Paten Jeffrey A JA McLean James P JP Hollmann Joseph J Karamichos Dimitrios D Messer Conor S CS Tambe Dhananjay T DT Saeidi Nima N Zieske James D JD Ruberti Jeffrey W JW

Tissue engineering. Part A 20160929 19-20

In a fibroblast colony model of corneal stromal development, we asked how physiological tension influences the patterning dynamics of fibroblasts and the orientation of deposited extracellular matrix (ECM). Using long-term live-cell microscopy, enabled by an optically accessible mechanobioreactor, a primary human corneal fibroblast colony was cultured on three types of substrates: a mechanically biased, loaded, dense, disorganized collagen substrate (LDDCS), a glass coverslip, and an unloaded, d ...[more]

PMID: 27600605

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Project description:Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G'). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-? levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-? was significantly greater for HMFs encapsulated in stiff hydrogels.Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn't support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Project description:PurposeTo determine corneal cross-linking (CXL) efficacy and chromophore penetration after excimer laser-assisted patterned de-epithelialization.MethodsTwo-hundred-twenty porcine eyes were de-epithelialized ex vivo, either fully (mechanical; n = 88) or patterned (excimer laser; n = 132). Consecutively, corneas were impregnated with hypo- or hyperosmolar riboflavin (RF; n = 20, RF-D; n = 40, respectively) or water-soluble taurine (WST11; n = 40, and WST-D; n = 40, respectively), or kept unimpregnated (n = 80). Sixty corneas were subsequently irradiated, inducing CXL, with paired contralateral eyes serving as controls. Outcome measurements included strip extensiometry to assess CXL efficacy, and spectrophotometry and fluorescence microscopy to determine stromal chromophore penetration.ResultsAll tested chromophores induced significant CXL (p < 0.001), ranging from 7.6% to 14.6%, with similar stiffening for all formulations (p = 0.60) and both de-epithelialization methods (p = 0.56). Light transmittance was significantly lower (p < 0.001) after full compared with patterned de-epithelialization. Stromal chromophore penetration was comparable between fully and patterned de-epithelialized samples, with full penetration in RD and RF-D samples and penetration depths measuring 591.7 ± 42.8 µm and 592.9 ± 63.5 µm for WST11 (p = 0.963) and 504.2 ± 43.2 µm and 488.8 ± 93.1 µm for WST-D (p = 0.669), respectively.ConclusionsExcimer laser-assisted patterned de-epithelialization allows for effective CXL. Stromal chromophore concentration is, however, reduced, which may have safety implications given the need for sufficient UVA attenuation in RF/UVA CXL. The different safety profile of near-infrared (NIR) may allow safe WST11/NIR CXL even with reduced stromal chromophore concentration values. In vivo studies are needed to evaluate the benefits and further assess safety of excimer laser-assisted patterned de-epithelialization for corneal CXL.

Dataset Information

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

Publications

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

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