Tunable Collagen I Hydrogels for Engineered Physiological Tissue Micro-Environments

<div><p>Collagen I hydrogels are commonly used to mimic the extracellular matrix (ECM) for tissue engineering applications. However, the ability to design collagen I hydrogels similar to the properties of physiological tissues has been elusive. This is primarily due to the lack of quantitative correlations between multiple fabrication parameters and resulting material properties. This study aims to enable informed design and fabrication of collagen hydrogels in order to reliably and reproducibly mimic a variety of soft tissues. We developed empirical predictive models relating fabrication parameters with material and transport properties. These models were obtained through extensive experimental characterization of these properties, which include compression modulus, pore and fiber diameter, and diffusivity. Fabrication parameters were varied within biologically relevant ranges and included collagen concentration, polymerization pH, and polymerization temperature. The data obtained from this study elucidates previously unknown fabrication-property relationships, while the resulting equations facilitate informed <i>a priori</i> design of collagen hydrogels with prescribed properties. By enabling hydrogel fabrication by design, this study has the potential to greatly enhance the utility and relevance of collagen hydrogels in order to develop physiological tissue microenvironments for a wide range of tissue engineering applications.</p></div

Compression modulus of collagen hydrogels at 0.1%/s deformation rate.

<p>Blue bars with dashed lines represent hydrogels polymerized at 23°C while red bars with solid lines represent hydrogels polymerized at 37°C. pH is indicated by color saturation. Data shown are mean + SE with N = 4–16 per bar. Significance was calculated for pH-averaged groups (N = 12–48) as indicated by horizontal black bars. Temperature means comparison is indicated with # while hydrodynamic radius means comparison is indicated with *.</p

Analysis of compression data from four representative hydrogel samples.

<p>All data shown is 4 mg/ml, pH 8.4, 37°C. a) Identification of contact position, marked by a filled circle. b) Robust loess smoothing. c) Linear regression of smoothed data from 5–15% strain. Sample 1 (green) is discarded after step (a) because the sample thickness, determined from the contact position, is 6.9% (585 μm) less than the nominal thickness. Sample 2 (blue) is discarded after step (c) because E = -8617 Pa < 0. Sample 3 (red) is also discarded after step (c) because the regression R² = 0.065 < 0.5. Sample 4 (black) is considered valid and returns a compression modulus of 3320 Pa which contributes to the mean compression modulus calculated for this fabrication condition.</p

Sensitivity of key hydrogel material properties to fabrication parameters.

<p>Sensitivities are obtained from the multiple regression coefficients, normalized to the largest coefficient in each model. The R² of the multivariate fit is indicated for each property. Analysis was performed on transformed (nondimensionalized) parameters. Only factors with p<0.01 (ANOVA) are shown.</p

Fabrication parameters varied in experiments.

<p>Fabrication parameters varied in experiments.</p

Pore and fiber diameter of collagen hydrogels.

<p>Blue symbols with dashed lines represent hydrogels polymerized at 23°C while red symbols with solid lines represent hydrogels polymerized at 37°C. Data shown are mean + SE with N = 12. Significance was calculated for pH-averaged groups. At each concentration, the difference between means at T = 23°C and T = 37°C is significant at p<0.0001 for both pore diameter and fiber diameter.</p

Rate of diffusion of dextran in collagen hydrogels.

<p>Blue square symbols with dashed lines represent hydrogels polymerized at 23°C while red triangle symbols with solid lines represent hydrogels polymerized at 37°C. Data shown are mean + SE with N = 72. Each measurement is averaged over pH and concentration. Significance was calculated for pH- and concentration-averaged groups: temperature means comparison is indicated with # while hydrodynamic radius means comparison is indicated with *.</p

Kinetics of collagen hydrogel polymerization.

<p>Blue square symbols with dashed lines represent hydrogels polymerized at 23°C while red triangle symbols with solid lines represent hydrogels polymerized at 37°C. As the effect of pH was statistically non-significant (p>0.05), data is averaged over all three pH groups. Data shown are mean + SE with N = 6–12. Significance was calculated for pH-averaged groups: temperature means comparison is indicated with # while concentration means comparison is indicated with *. At 37°C (solid red lines), concentration had no significant effect on either polymerization half-time or polymerization lag time.</p

Fiber structure images obtained from confocal reflectance.

<p>Images shown are for hydrogels polymerized at pH 7.4. Scale bar 25 μm.</p

Representative FRAP image sequence.

<p>Data shown is for 40 kDa dextran in 10 mg/ml collagen hydrogel polymerized at pH 7.4 and 23°C. Left to right: Prebleach; t = 0 s, t = 6 s, t = 12 s, t = 18 s. Scale bar 50 μm.</p