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1.
Figure 1

Figure 1. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Schematic description of HA functionalization and crosslinking. HA-methacrylate was functionalized with an RGD peptide using the Michael-type addition reaction between the methacrylate groups on the polymer and the cysteine thiol groups on the peptide. The same addition reaction with the methacrylate groups was used to induce crosslinking via reaction with dithiothreitol (DTT) to form HA hydrogels.

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
2.
Figure 2

Figure 2. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Mechanical characterization of HA gels. Shear elastic moduli of the DTT-crosslinked HA hydrogels were measured by oscillatory rheometry. Each curve represents HA gels containing a particular HA-methacrylate polymer (HA-60 or HA-85 with 60% and 85% degree of methacrylation, respectively) and weight fraction, at varying ratios of thiols used for crosslinking. Error bars represent standard deviation; N ≥ 2 replicates.

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
3.
Figure 7

Figure 7. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Cell number on variable-stiffness HA-RGD substrates. U373-MG cells were cultured on matrices of the specified rigidity and constant ligand density for 4 days. Cell number was then measured using the WST-1 metabolic assay. Error bars represent standard error of the mean; N = 6 replicates. Statistically distinct groups (p < 0.01) determined by Tukey’s test are marked by A, B (see methods).

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
4.
Figure 6

Figure 6. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Regulation of glioma cell motility by matrix stiffness. The plot depicts the average speed of random motility of U373-MG cells cultured on RGD-functionalized HA gels of constant peptide density and varying stiffness. N ≥ 120 cells for each condition. Statistically distinct groups (p < 0.01) determined by Dunn’s test are marked by A, B, C (see methods).

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
5.
Figure 3

Figure 3. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Scanning Electron Microscopy (SEM) imaging of dehydrated HA gels (higher magnification images in insets). The gel microstructure consists of dense, folded sheets of polymer free of fibrillar structures and does not contain micron-sized pores. The apparent density of the polymer network increases with increasing polymer weight fraction and stiffness. Scale bar = 20 μm; inset scale bar = 5 μm.

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
6.
Figure 8

Figure 8. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

3D invasion of glioma spheroids through HA-RGD hydrogels. (A) Variation of extent and patterns of invasion with cell type and matrix density. U373-MG cells dispersed and invaded as single cells (open arrows) whereas U87-MG and C6 cells retained spheroid borders with cells invading at the edges (filled arrows). No cells invaded the dense 5 kPa hydrogel. (B) Time-lapse images of U373-MG cells invading the 150 Pa hydrogel. Cells exhibited distinctly non-mesenchymal motility, with dynamically extending and branching leading processes, following by abrupt movement of the cell-body forward. Scale bar = 100 μm.

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
7.
Figure 4

Figure 4. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Glioma cell morphology on 35 kPa HA gels with varying surface density of RGD peptide. (A) Morphology and cytoarchitecture of cells adhered to variable-RGD density gels after 24 hr incubation, as visualized by epifluorescence imaging of F-actin (green) and nuclear DNA (blue). Scale bar = 50 μm. (B) Quantification of projected cell spreading area. (C) Quantification of cell shape, as measured by circularity (see methods). N ≥ 55 cells for each condition. Statistically distinct groups (p < 0.05) determined by Dunn’s test are marked by A, B, C, D (see methods).

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.
8.
Figure 5

Figure 5. From: Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.

Glioma cell adhesion to variable-stiffness RGD-functionalized HA gels. (A) Morphology of cells adhered to variable-stiffness gels after 24 hr incubation. Top row: immunofluorescence imaging of vinculin (orange), F-actin (green), and nuclear DNA (blue). Bottom row: Isolated vinculin signal at higher magnification. Scale bar = 50 μm. (B) Quantification of projected cell area. (C) Quantification of circularity. N ≥ 85 cells for each condition. Statistically distinct groups (p < 0.01) determined by Dunn’s test are marked by A, B (see methods).

Badriprasad Ananthanarayanan, et al. Biomaterials. ;32(31):7913-7923.

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