Characterization of a hierarchical network of hyaluronic acid/gelatin composite for use as a smart injectable biomaterial

Abstract

Hybrid HA/Ge hydrogel particles are embedded in a secondary HA network to improve their structural integrity. The internal microstructure of the particles is imaged through TEM. CSLM is used to identify the location of the Ge molecules in the microgels. Through indentation tests, the Young's modulus of the individual particles is found to be 22 ± 2.5 kPa. The overall shear modulus of the composite is 75 ± 15 Pa at 1 Hz. The mechanical properties of the substrate are found to be viable for cell adhesion. The particles' diameter at pH = 8 is twice that at pH = 5. The pH sensitivity is found to be appropriate for smart drug delivery. Based on their mechanical and structural properties, HA-Ge hierarchical materials may be well suited for use as injectable biomaterials for tissue reconstruction.

Figure 1

a) Schematic of thiol-modified HA and Ge crosslinked by PEGDA.^[27] b) Microparticle of crosslinked HA and Ge. c) Hierarchical network of HA–Ge microgels with HA network in water.

Figure 2

SEM of synthesized HA–Ge particles at different magnifications. a) Cluster of particles (scale bar: 5 μm), and b) a single particle (scale bar: 500 nm). The particles are spherical with a smooth surface. The concentrated electron beam from the SEM caused wrinkles on the surface during prolonged imaging. The inset shows the surface of the particle with the micellar structure after electron bombardment which resulted in particle shrinkage. The scale bar in the inset is 100 nm.

Figure 3

a) TEM image of filtered particles suspended in acetone and air-dried. The scale bar is 500 nm. The particles were significantly shrunk in the absence of water. b) Magnified TEM picture of a hybrid HA–Ge particle. The original size of the particle in water was around 800 nm. The scale bar is 20 nm.

Figure 4

CLSM of the HA–Ge particles stained with fluorescamine to illuminate the location of Ge in the intraparticle structure: a) bright field image, and b) fluorescent image of the particles superimposed on the bright field image. c) Bright field and d) fluorescent images of a single particle. e) Superimposed image of fluorescent and bright field pictures (c and d). The scale bar for (a) and (b) is 5 μm, and for (c) (d), and (e) is 1 μm.

Figure 5

a) Normalized particle size distribution based on intensity for different pH values with constant ionic strength. From right to left, Dark blue line pH = 8; pink line pH = 7.4; green line pH = 7; blue line pH = 6; red line pH = 5. b) Variation of average particle diameter as a function of pH. The error bars indicate standard deviation of the mean value.

Figure 6

Indentation response of a 1 μm sized HA–Ge particle obtained by AFM. The experiments were carried out under water and at room temperature. The Hertzian indentation model regressed over the first 25 nm, where the shell of the particle obeys linear elasticity. The Poisson’s ratio of the sample particle was assumed to be 0.5, the effective contact radius was 1 μm, and the calculated Young’s modulus was 22 kPa. Dark Blue: indentation force (pN); pink line: Hertz force (pN).

Figure 7

CLSM of the HA–Ge particles stained with mBBr to illuminate the free thiol groups remaining after the fabrication of HA–Ge. a) fluorescent image of the particles, b) bright field image, and c) superimposed image of the bright field and fluorescent pictures. Free thiols are still available on the particles for further crosslinking. The scale bar is 10 μm.

Figure 8

Shear elastic (G′) and viscous moduli (G″) of bulk HA and HA/Ge composite network. Solid dark blue line: elastic modulus of composite; dashed dark blue line: viscous modulus of composite; solid pink line: elastic modulus of bulk; dashed pink line: bulk viscous modulus.