Injectable heat-sensitive nanocomposite hydrogel for regulating gene expression in the treatment of alcohol-induced osteonecrosis of the femoral head

Abstract

For repairing lesions, it is important to recover physiological and cellular activities. Gene therapy can restore these activities by regulating the expression of genes in lesion cells; however, in chronic diseases, such as alcohol-induced osteonecrosis of the femoral head (ONFH), gene therapy has failed to provide long-term effects. In this study, we developed a heat-sensitive nanocomposite hydrogel system with a secondary nanostructure that can regulate gene expression and achieve long-term gene regulation in lesion cells. This nanocomposite hydrogel exists in a liquid state at 25 °C and is injectable. Once injected into the body, the hydrogel can undergo solidification induced by body heat, thereby gaining the ability to be retained in the body for a prolonged time period. With the gradual degradation of the hydrogel in vivo, the internal secondary nanostructures are continuously released. These nanoparticles carry plasmids and siRNA into lesion stem cells to promote the expression of B-cell lymphoma 2 (inhibiting the apoptosis of stem cells) and inhibit the secretion of peroxisome proliferators-activated receptors γ (PPARγ, inhibiting the adipogenic differentiation of stem cells). Finally, the physiological activity of the stem cells in the ONFH area was restored and ONFH repair was promoted. In vivo experiments demonstrated that this nanocomposite hydrogel can be indwelled for a long time, thereby providing long-term treatment effects. As a result, bone reconstruction occurs in the ONFH area, thus enabling the treatment of alcohol-induced ONFH. Our nanocomposite hydrogel provides a novel treatment option for alcohol-related diseases and may serve as a useful biomaterial for other gene therapy applications.

Scheme 1.

Schematic diagram of the nanocomposite hydrogel for the treatment of alcohol-induced ONFH. (a) Schematic diagram of the heat-sensitive hydrogel synthesis. (b) Schematic diagram of the gene-loaded nanoparticle synthesis. (c) Pathophysiological mechanism of chronic alcohol consumption that leads to ONFH. (d) Nanocomposite hydrogels inhibit the apoptosis and adipogenic differentiation of MSCs, thus treating ONFH.

FIG. 1.

Characterization of gene-loaded nanoparticles. (a) Schematic diagram of the synthesis of nanoparticles loaded with plasmids and siRNA, respectively. (b) TEM image of blank nanoparticles. (c) Particle size distribution of gene-loaded nanoparticles. (d) Zeta potential values of gene-loaded nanoparticles. (e) The mapping energy spectrum of the nanoparticles.

FIG. 2.

Characterization of heat-sensitive hydrogels. (a) Schematic diagram of the synthesis and mechanism of action of the heat-sensitive hydrogel. (b) Particle size distribution of nanomicelles. (c) Phase transitions in the water systems of triblock polymers. (d) Change in the modulus of the water system of triblock polymer varies with temperature. (e) Change in the modulus of the nanocomposite hydrogels varies with temperature. (f) Photographs of polymer hydrogels and nanocomposite hydrogels achieving solid and liquid transitions at different temperatures.

FIG. 3.

Biological effects of the nanocomposite hydrogels in the treatment of ONFH. (a) Schematic diagram of the mechanism by which nanocomposite hydrogels regulate gene expression and inhibit MSC apoptosis and adipogenic differentiation. (b) Flow cytometry was performed to detect the apoptosis of MSCs. (c) Statistical analysis of the MSC apoptosis rate. (d) Expression of Bcl-2 was detected using western blotting. (e) Quantitative analysis of Bcl-2 expression. (f) Expression of PPARγ was detected using western blotting. (g) Quantitative analysis of PPARγ expression. (h) Quantitative analysis of triglyceride content in MSCs. NS: nonsignificant; ^*P < 0.05, ^**P < 0.01, ^***P <0.001.

FIG. 4.

Animal experiments using the nanocomposite hydrogel for treating ONFH. (a) Schematic illustration of the generation and treatment of the rat model of alcohol-induced ONFH. (b) The liquid nanocomposite hydrogel was injected into the hip joint using a syringe. (c) Fluorescence residue test verified the indwelling of the nanocomposite hydrogel in the rats. (d) Quantitative analysis of the fluorescence residue assay. (e) Micro-CT was performed to detect femoral head necrosis in the rats. (f) Statistical analysis of BMD. (g) Statistical analysis of BV/TV. (h) Statistical analysis of Tb.Th. ns: nonsignificant; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

FIG. 5.

Section staining of the rat femoral head. (a) Hematoxylin and eosin staining of the rat femoral head. (b) Quantitative analysis of the number of vacuoles in the femoral head. (c) Immunohistochemical staining for cleaved caspase-3 in the femoral head. (d) Quantification of cleaved caspase-3 expression. (e) Immunohistochemical staining for OPN in the femoral head. (f) Immunohistochemical staining for OCN in the femoral head. (g) Quantification of OPN expression. (h) Quantification of OCN expression. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.