Nucleic Acid-Based Dual Cross-Linked Hydrogels for in Situ Tissue Repair via Directional Stem Cell Migration

Abstract

In situ tissue repair holds great potential as a cell-free regenerative strategy. A critical aspect of this approach is the selection of cell instructive materials that can efficiently regulate the defect microenvironment via the release of chemoattractant factors to mobilize and recruit endogenous stem cells toward the site of implantation. Here we report the design of a DNA-based hydrogel as a drug delivery platform for the sustained release of a promising chemoattractant, SDF-1α. The hydrogel is composed of chemically cross-linked DNA strands, which are bridged via silicate nanodisks (nSi). Silicate nanodisks electrostatically interact with the negatively charged DNA backbone resulting in the formation of a dual cross-linked nanocomposite hydrogel with a combination of chemical and physical cross-link points. The formulated nanocomposites display enhanced elasticity and mechanical toughness as compared to their nonsilicate containing counterparts. Moreover, the electrostatic interaction between nSi and SDF-1α leads to sustained release of the chemokine from the hydrogels. The in vitro bioactivity assays confirm the retention of chemotactic properties of the protein after its release. Overall, the dual cross-linked DNA-based hydrogel platform could be potentially used as a cell-instructive material for the recruitment of host stem cells to guide the process of in situ tissue repair.

Keywords: DNA; dual-cross-linked; nanosilicate; regenerative medicine; stem cell recruitment; sustained release.

Figure 1.

Design of nanoengineered dual cross-linked DNA-based hydrogels for in situ tissue repair. The amine groups present in the DNA nucleotides can react with the epoxide functionalities of the cross-linker polyethylene glycol diglycidyl ether (PEGDE) to form a chemically conjugated DNA network. Silicate nanodisks (nSi) act as physical cross-linkers which provides additional bridging points by establishing electrostatic interactions between their positively charged edges and the negatively charged phosphate groups of the DNA. Under compression, these additional network points enable the material to resist deformation, resulting in pronounced elastomeric properties with minimum permanent deformation. The nanoengineered hydrogels with encapsulated stromal cell-derived factor-1 alpha (SDF-1α) can function as stem cell recruiting materials for tissue repair applications.

Figure 2.

Effect of nSi on the strength and toughness of the nanocomposite hydrogels. (a) Frequency sweep profiles from 0.01–10 Hz illustrate an enhancement in the storage moduli (G’) of the hydrogels as the concentrations of nSi is increased. (b) Stress vs strain plots obtained under uniaxial compression tests up to 0.5 mm/mm strain. (c) Comparison between the compressive moduli of the formulated hydrogels. An increase in compressive strength was observed with the incorporation of nSi. Compressive modulus was determined from the slope of the stress strain plot in the region of 0.1–0.2 mm/mm strain. Results are shown as mean ± standard deviation (n = 3) (***p < 0.001). (d) Optical images depicting the capability of the nanocomposite gels to sustain compressive forces without permanent deformation. The black arrow indicates the area where the gels without nSi breaks under the compression with a spatula. In contrast the nanocomposites bend and maintain structural integrity after the removal of the force, thus displaying a higher mechanical toughness and flexibility. (e) Comparative plots of the toughness as a function of the nSi concentration. The results demonstrate an increase in this parameter as the concentration of nSi is increased. Toughness was estimated from the area under the stress vs strain plot. Results are shown as mean ± standard deviation (n = 3) (***p < 0.001).

Figure 3.

Effect of nSi on the elasticity of the formulated hydrogels. Stress vs strain plots under cyclic compression tests for (a–c) 0% nSi and (d–f) 0.5% nSi, at the strain amplitudes of 0.4, 0.6, and 0.8 mm/mm. (g) Comparison in the energy dissipated at different strain amplitudes, calculated from the area from the hysteresis loop. A reduction in the dissipated energy at 0.8 mm/mm strain reveals in the nSi 0.5% group is indicative of enhanced elastomeric properties compared to the 0% nSi group. Results are shown as mean ± standard deviation (n = 3) (**p < 0.01). (h) Stress relaxation behavior of the hydrogels under 5% of strain. Results confirm a higher network stability for the 0.5% nSi group as compared to 0% nSi hydrogel. (i) Representative creep curves obtained stressing the hydrogels at 100 Pa. The results indicate superior resistance to external forces for the 0.5% nSi group.

Figure 4.

Effect of nanosilicate on the in vitro biocompatibility and immunogenicity of the hydrogels. (a) MTS assay at 24 and 72 h on hASCs grown along with the hydrogels containing different concentrations of nSi. Results are shown as mean ± standard deviation (n = 3) (*p < 0.05). (b) Fluorescent images of live/dead assay of hASCs after growing them for 72 h along with the formulated hydrogels. The results support high cell viability, thereby confirming minimum cytotoxicity of the nanocomposite hydrogels (scale bar = 1000 μm). (c) Relative expression levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and inteleukin-6 (IL6) secreted by RAW 264.7 macrophages which are grown in contact with the hydrogels for 24 h. Cytokines in the culture media were analyzed by multi-analyte ELISA. Results are shown as mean ± standard deviation (n = 3) (***p < 0.001). (d) Quantification of TNF-α concentrations in the culture media, released by RAW 264.7 macrophages which are grown in contact with the hydrogels for 24 h. Quantification was performed by TNF-α ELISA kit. Results are shown as mean ± standard deviation (n = 3) (*p < 0.05, ***p < 0.001). (e) Relative gene expression of inducible nitric oxide synthase (iNOS) and TNF-α in RAW 264.7 macrophages seeded for 24 h in contact with the designated hydrogels. Nonsignificant differences in the gene expressions for experimental groups as compared to negative control highlight the noninflammatory response of the hydrogels. Results are shown as mean ± standard deviation (n = 3). For all the experiments with RAW 264.7 macrophages, negative control refers to cells grown without being in contact with any gel or LPS, whereas positive control is denoted for cells treated with LPS with a concentration of 1 μ/mL.

Figure 5.

Analysis of physical properties and the extended release of encapsulated SDF-1α from the nanocomposite hydrogels (a) Scanning electron microscope images of the formulated hydrogels displaying a highly porous morphology (scale bar = 100 μm). (b) Quantification of the pore size with ImageJ analysis. A significant increase in average pore diameter with the incorporation of nSi is observed from the comparison of pore sizes. Results are shown as mean ± standard deviation (n = 50, from five different images) (***p < 0.001). (c) Comparison of swelling analysis of the gels with and without nSi in phosphate buffer (pH = 7.4) demonstrates higher swelling ratios of 0.5% nSi as compared to 0% nSi at all time points. Results are shown as mean ± standard deviation (n = 4). (d) Degradation profiles of 0% nSi and 0.5% nSi in phosphate buffer (pH = 7.4) confirms enhanced structural stability of the nanocomposite hydrogels. Results are shown as mean ± standard deviation (n = 5) (***p < 0.001). (e) Schematic illustration of the extended drug release properties of the nanocomposite hydrogels leading to enhanced retention of the drug for a longer period of time. (f) Comparative release profiles of the entrapped SDF-1α displays a sustained release of the chemokine from the nanocomposite hydrogels in contrast to 0% nSi. Results are shown as mean ± standard deviation (n = 3).

Figure 6.

Confirmation of chemotaxis properties of the released SDF-1α. (a) Schematic representation of the experimental setup for the scratch assay. (b) Bright field images of hASCs displaying more migration in the groups containing SDF-1α (scale bar = 200 μm). (c) Quantification and comparison of the uncovered area after 6 and 8 h. The results reveal enhanced scratch closure for + nSi (hASCs treated with the SDF-1μ released from 0.5% nSi) and positive control (hASCs treated with fresh SDF-1α at the same concentration) as compared to the negative control (hASCs grown in basal media without any SDF-1α). Results are shown as mean ± standard deviation (n = 6) (**p < 0.01). (d) Schematic representation of the experimental setup for the transwell migration assay. (e) Fluorescent images of hASCs which are migrated to the bottom of the transwell insert membrane after 12 h (scale bar = 200 μm). (f) Quantification of migrated cells confirm the retention of the chemotaxis properties of SDF-1α after release. Cell numbers in all the groups are normalized by those in the negative control. Results are shown as mean ± standard deviation (n = 9) (***p < 0.001).