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Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly.

ABSTRACT: In this work, the gelation of three-dimensional collagen and collagen/hyaluronan (HA) composites is studied by time sweep rheology and time lapse confocal reflectance microscopy (CRM). To investigate the complementary nature of these techniques, first collagen gel formation is investigated at concentrations of 0.5, 1.0, and 1.5 mg/mL at 37 degrees C and 32 degrees C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (t(c)), the slope of the growth phase (k(g)), and the arrest time (t(a)). The first two measures are determined by rheology, and the third by CRM. A frequency-independent rheological measure of gelation, t(g), is also measured at 37 degrees C. However, this quantity cannot be straightforwardly determined for gels formed at 32 degrees C, indicating that percolation theory does not fully capture the dynamics of collagen network formation. The effects of collagen concentration and gelation temperature on k(g), t(c), and t(a) as well as on the mechanical properties and structure of these gels both during gelation and at equilibrium are elucidated. Composite collagen/HA gels are also prepared, and their properties are monitored at equilibrium and during gelation at 37 degrees C and 32 degrees C. We show that addition of HA subtly alters mechanical properties and structure of these systems both during the gelation process and at equilibrium. This occurs in a temperature-dependent manner, with the ratio of HA deposited on collagen fibers versus that distributed homogeneously between fibers increasing with decreasing gelation temperature. In addition to providing information on collagen and collagen/HA structure and mechanical properties during gelation, this work shows new ways in which rheology and microscopy can be used complementarily to reveal details of gelation processes.

SUBMITTER: Yang YL

PROVIDER: S-EPMC2717242 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly.

Yang Ya-li YL Kaufman Laura J LJ

Biophysical journal 20090201 4

In this work, the gelation of three-dimensional collagen and collagen/hyaluronan (HA) composites is studied by time sweep rheology and time lapse confocal reflectance microscopy (CRM). To investigate the complementary nature of these techniques, first collagen gel formation is investigated at concentrations of 0.5, 1.0, and 1.5 mg/mL at 37 degrees C and 32 degrees C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (t(c)), the slope of the g ...[more]

PMID: 19217873

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Project description:Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (?200 mg ml?¹). Young's modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg ml?¹) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ?5-200 mg ml?¹). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R²=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R²=0.83, exponents of -0.7 and -1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

Dataset Information

Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly.

Publications

Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly.

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