The rheological properties of hydrogel precursor of HA/ND were determined with viscosity change and shear rate. To prepare nanocomposite hydrogel precursors, 0.02 wt % carboxylated or hydroxyl ND were mixed with 2 wt % of MeHA at pH 7 and pH 8. After the full pre-sheared state of the nanocomposite hydrogel precursor, the shear rate decreased significantly and the viscosity change is recorded during the subsequent 100 sec. As shown in Figure 1, the shear-thinning effect of all HA, HA/ND-COOH, and HA/ND-OH nanocomposite hydrogel precursor generated low viscosities (approximately 2 Pa·s) in the pre-sheared zone. After the shear rate was decreased and fluids were fully structured, the viscosity of the nanocomposite hydrogel precursors increased rapidly with a stiff gradient to approximately 33.24 Pa·s on average in 2/s. The viscosity of the nanocomposite hydrogel precursors with nanodiamonds was 35.74 Pa·s for ND-COOH and 27.5 Pa·s for ND-OH at pH 7, respectively. In addition, the viscosity of MeHA was 13.34 Pa·s at pH 7 and 32.29 Pa·s in 2/s. It is noted that nanocomposite HA hydrogel containing NDs showed higher viscosity and shorter response time, compared to HA only hydrogels, indicating a pH controllable filament shape in the 3D-printing process. The nanocomposite HA hydrogel precursors showed higher viscosity at pH 8, compared to those at pH 7. Rheological characteristics such as viscosity may vary depending on the degree of polymerization of HA, but by using ND, it was possible to form a viscosity suitable for printing despite the use of a lower content of HA. Compared to the results obtained by measuring the viscosity using a commercially available product (Cellink®) and pentenoate-functionalized HA, viscosity in 2/s was 52.8 Pa·s for HA/ND-COOH (at pH 8), about 100 Pa·s for Cellink®, and 120 Pa·s for 3% pentanoate functionalized HA [35]. Compared to the results of other studies, the viscosity was relatively low, but concentration of hyaluronic acid (2 wt %) was lower than other hydrogel (at least 3%), and HA/ND hydrogel showed stronger shear thinning effect than other materials. As the nanodiamond was added, the increased viscosity increased up to 270% compared to when HA was used alone, but it was confirmed that the viscosity decreased very rapidly with increasing shear rate. HA/ND-COOH (at pH 8) showed the highest viscosity 15.33 Pa at 10/s, which is a 70% decreased value from initial viscosity. Weak shear-thinning originates from high molecular weight hyaluronic acid itself [48], but nanodiamonds appear to induce shear-thinning more strongly. Shear-thinning hydrogels were researched by blending shear-thinning materials to hyaluronic acid [13, 36] and nanodiamonds performed a similar role in hyaluronic acid. As the negatively charged end groups of HA and ND increased, electrostatic interaction between nanophase ND and HA resulted in the augmented viscosity, which was further influenced by the 3D-printing manufacturing process.
The change of zeta potential and hydrodynamic diameter of ND-OH and ND-COOH agglutinates depending on pH were fully characterized in previous study [42]. In brief, the zeta potentials of ND−COOH were negative −22.5 ± 0.2 mV and −27.0 ± 0.4 mV, at pH 7 and pH 8, respectively. ND−COOH dispersion showed the decrease in zeta potential as the increased pH from 7 to 8 due to deprotonation of carboxyl groups. ND−OH produced by the reduction of ND−COOH showed stable zeta potentials in the wide pH ranges, ranged from −51.4 ± 0.9 mV to −44.5 ± 0.7 mV at the pH 4 - 10. The zeta potentials of ND−COOH and ND−OH were negative zeta potential of −22.5 ± 0.2 mV and −42.7 ± 1.2 mV at pH 7, respectively, suggesting good colloidal stability. The diameters of the dispersed ND−OH and ND−COOH agglutinate were also affected by environmental pH. Depending on the pH, the average sizes of ND−COOH were changed into 105.2 nm at pH 7 and 77.9 ± 1.6 nm at pH 8. The average sizes of ND−OH were 61.5 ± 0.3 nm at pH 7 to 67.2 ± 3.3 nm at pH 8, showing less pH sensitivity and high dispersion stability. These pH dependence by surface functional group. pH dependent size changes of ND agglutinates are closely related with the dissociation constants (pKa) of carboxylic acids 5.0 and the alcohol 14−16, as reported. The degree of ionization might determine colloidal stability and resulting agglutinate size due to interparticle interaction.
Regarding with these, pH dependent colloidal stability and negative zeta potentials of ND-COOH and ND-OH might have repulsive electrostatic interaction between negative HA. The agglutinate size resulted from environmental pH dependent charge effect might also contribute the significantly increased shear thinning effect. Considering that size of ND-COOH became significantly increased at pH 7, increased shear thinning effect should be considered with colloidal size. ND-OH with smaller stabilized particle size and higher negative zeta potentials showed less shear thinning effect, compared to the ND-COOH. Resulting shear thinning effect by addition of NDs might be complex of factors including colloidal size, surface charge, surface functionality. The resulting higher shear thinning effect by addition of ND, compared to HA only gained the processible high quality of printing ink.
Shear thinning was observed using nanocomposite hydrogel precursors, which can be attributed to the electrostatic interactions from the COO- and hydrogen bonding (-OH) between ND surface and HA. The rheological properties of the nanocomposite hydrogel precursors estimated as storage modulus G′ and loss modulus G″ revealed that the HA/ND-COOH and HA/ND-OH hydrogel increased by 1.18 (185.4 Pa) and 1.13 (177.6 Pa) fold (G′), 1.10 (67.86Pa) and 1.02 (63.24 Pa) fold (G′′) at pH 7, respectively, compared to HA only hydrogel at 62.83 rad/s (10 Hz) (Figure 2a). This result suggests that the existence of physical interaction, i.e., electrostatic interaction between −COO− on the ND surface and HA chains, contributes to the increase in G′ and G″ in HA/ND hydrogel precursors. Remarkably, G′ and G″ in HA/ND-COOH hydrogel precursor at pH 8 was increased 1.4 fold (260.5 Pa) and 1.26 fold (85.26 Pa), respectively, compared with those of HA/ND-COOH hydrogel at pH 7. This proves that the deprotonated ND-COOH surface became hydrophilic and was interacting with the HA chains. In a pH dependent manner, the shear-thinning behavior of the HA/ND hydrogels precursor occurred through quick absorption and desorption of HA chains from ND surface, affecting the 3D printing structure and filament stability.
The filament diameter is generally controlled by operating parameters including the inner diameter of the needle, nozzle path speed, and standoff distance on the filament width. When deposited as a liquid filament of nanocomposite hydrogel precursor, filament structure and stability are critical during the extrusion. In general, filament widths can be controlled by varying the pressure of the cylinder, the inner diameter of the nozzle, the path speed, and the standoff distances [11, 49]. A 26 gauge needle was used to extrude stable filaments of the HA/ND-COOH and HA/ND-OH hydrogel nanocomposites, demonstrated in Figure 3. The same operating conditions were used to explore the dependence of mechanical properties on pH between the different NDs.
The 3D-printed nanocomposite hydrogels with spatiotemporal 3D printing construction were visualized both optically and with SEM. The HA nanocomposite hydrogel revealed that the 3D-dimensional porous networked structure was uniformly distributed with an average pore size of 128 μm (Figure 4a and 4b). Reproducibility in size, shape, and fidelity of structure are shown as optical images in Figure 4c and 4d.
The compressive mechanical stress of nanocomposite hydrogels containing ND-COOH and ND-OH were compared with HA only hydrogels. Nanocomposite hydrogel precursors of HA/ND-COOH and HA/ND-OH were prepared using either 0.02 wt % or 0.04 wt % ND with 2 wt % of MeHA and printed using predetermined printing conditions. The highest level of compressive stress was observed in the vicinity of 90-93% of the compressive strain, and a gel breakage was observed when strain was higher than 90% of the strain. The 3D structure of HA/ND-COOH (0.02 wt %) and HA/ND-OH(0.02 wt %) nanocomposite hydrogels showed maximum compressive stresses of 152.82 kPa, and 161.59 kPa at pH 7, showing 2.2 fold and 2.35 fold, respectively, higher than the HA only hydrogels (68.72 kPa) (Figure 5a). With the addition of higher composition of ND-COOH (0.04 wt %), the compressive stress was increased by 163 %, compared to that of the lower composition ND-COOH hydrogel (0.02 wt %). Moreover, the 0.04 wt % composite ratio of ND-OH resulted in the stiffest gradient and dramatically enhanced peak compressive force of 296% (478.03 kPa), compared to that of hydrogel for ND-COOH (0.02 wt %). Similar compressive stress was observed for HA/ND-COOH and HA/ND-OH nanocomposite hydrogels when processed at pH 8 (Figure 5b). HA nanocomposite hydrogels with ND-OH at pH 8 showed an increase of 1.40 fold (236.18 kPa) and 1.37 fold (616.72 kPa) compressive stress at the composition of 0.02 wt % and 0.04 wt %, respectively, compared to those of ND-COOH (168.31 kPa (0.02wt% ND-COOH), 449.59 kPa(0.04 wt% ND-COOH)) at the same pH 8. Moreover, the compressive stress of HA/ND-OH (0.04 wt %) at pH 8 was mechanically enhanced 1.29 fold, compared to that of HA/ND-OH (0.04 wt %) at pH 7.
The results indicate that 3D printed HA hydrogels filled with hydrophilic surface functionalized nanodiamond boost mechanical properties even at low weight ratios of ND. The enhanced mechanical properties can be attributed to the high surface-to-volume ratio and hydrophilic ND surface which generated sufficient nanophase distribution within hydrogel matrix [50].
The comparison of pH dependence and the effect of surface functional groups are also apparent in the gel peak breaking force. 3D-printed HA/ND nanocomposite hydrogels with (Figure 5c) and without (Figure 5d) post-UV exposure were used to investigate the impact of pH on mechanical integrity. Both HA nanocomposite hydrogels containing ND-COOH or ND-OH showed higher peak forces at pH 8, compared to the same nanocomposite hydrogels prepared at pH 7. The difference in pH at the stage of nanocomposite hydrogel precursor resulted in the high peak force 0.905 N of HA/ND-OH (0.02 wt %) at pH 8, compared to 0.511 N of HA/ND-OH (0.02 wt %) at pH 7, revealing a 177 % boost in peak force. pH dependence became even more apparent when the composite amount was increased to 0.04 wt %, which showed the enhancement of the peak force with an increase in pH. The peak force 1.920 N of HA/ND-OH (0.04 wt %) at pH 8 showed 155 % amplified peak force, compared to 1.236 N of HA/ND-OH (0.04 wt %) at pH 7. The boosted mechanical properties of HA/ND-COOH showed the same pH dependent tendencies. Without post-UV exposure, the gel peak breaking force of the nanocomposite hydrogels at varied pH was not critically different from any sample when compared to HA only hydrogels (Figure 5d). A change in the gel peak breaking force post-UV crosslinking confirmed that NDs within an HA matrix were spread out in a nanophase, thus concerting molecular environments that promoted interactions between the ND and HA. Hydrogels containing ND-OH showed the best performance at pH 8 and pH 7, compared to those with ND-COOH. The agglomerate stability of the ND varied depending on the pH and surface functionality in the aqueous dispersion [42]. State of ink precursors was a viscous dispersion of HA and ND before extrusion processing, and settled 3D firm hydrogels allowed for post-UV exposure, allowing for stable ND dispersion without confinement at local foci. Agglomerates of hydroxylated NDs are stable at both pH 7 and 8, while similar agglomerates of carboxylate ND are critically enhanced at pH 8 due to the deprotonation of the carboxyl group resulting in a hydrophilic surface. These intrinsic properties of NDs affect the distribution within the HA matrix, intermolecular electrostatic interaction, and hydrogel bonding through physical contact of gel network. Without chemical crosslinking, the physical network created by the addition of ND-COOH or ND-OH was able to achieve the mechanically stabilized 3D-printed hydrogel construction.