Attenuation of osteoarthritis progression via locoregional delivery of Klotho-expressing plasmid DNA and Tanshinon IIA through a stem cell-homing hydrogel

Abstract

Background: Osteoarthritis (OA) is an aging-related degenerative joint disorder marked by joint discomfort and rigidity. Senescent chondrocytes release pro-inflammatory cytokines and extracellular matrix-degrading proteins, creating an inflammatory microenvironment that hinders chondrogenesis and accelerates matrix degradation. Targeting of senescent chondrocytes may be a promising approach for the treatment of OA. Herein, we describe the engineering of an injectable peptide-hydrogel conjugating a stem cell-homing peptide PFSSTKT for carrying plasmid DNA-laden nanoparticles and Tanshinon IIA (pPNP + TIIA@PFS) that was designed to attenuate OA progression by improving the senescent microenvironment and fostering cartilage regeneration.

Results: Specifically, pPNP + TIIA@PFS elevates the concentration of the anti-aging protein Klotho and blocks the transmission of senescence signals to adjacent healthy chondrocytes, significantly mitigating chondrocyte senescence and enhancing cartilage integrity. Additionally, pPNP + TIIA@PFS recruit bone mesenchymal stem cells and directs their subsequent differentiation into chondrocytes, achieving satisfactory chondrogenesis. In surgically induced OA model rats, the application of pPNP + TIIA@PFS results in reduced osteophyte formation and attenuation of articular cartilage degeneration.

Conclusions: Overall, this study introduces a novel approach for the alleviation of OA progression, offering a foundation for potential clinical translation in OA therapy.

Keywords: Cartilage rejuvenation; Klotho; Osteoarthritis; Senescent; Stem cell-homing hydrogel.

Fig. 1

Schematic illustration of locoregional generation of Klotho protein to mitigate OA. (A) Chemical structure of the NPs featuring a nuclear localization sequence (NLS) peptide as the hydrophilic moiety and stearic acid (SA) as the hydrophobic domain. (B) Schematic illustration of the preparation of plasmid DNA (pDNA)-laden peptide NPs (pPNPs). (C) Schematic illustration of the pPNP coating on hydrogel. (D) The pPNP-gel generates Klotho protein to ameliorate the aging microenvironment and delay arthritis progression. In the anterior cruciate ligament transection (ACLT) rat model, this approach ameliorates arthritis and promotes osteogenesis

Fig. 2

Design and characterization of the pPNP coating. (A) Gel retardation assay of pPNPs. (B) TEM images of pPNPs with a weight ratio of SA-peptide: pDNA of 10:1. Scale bar, 100 nm. (C) Zeta potential and size distributions of pPNPs with a weight ratio of SA monomers: pDNA of 10:1. (D, E) FTIR spectra (D) and circular dichroism spectra (E) of the hydrogels. (F) Photos of peptide solutions and the hydrogels. (G, H) SEM (G; scale bar, 20 μm) and TEM (H; scale bar, 500 nm) images of the hydrogels. (I) Changes in shear viscosity with the increased shear rate of the hydrogels. (J) Rheology traces of the hydrogels. (K) Representative fluorescence images of hydrogels with PFS@Cy5 and PFS@Cy5-pPNP collected via a live imaging system at 0, 10, 30, and 60 min. Scale bar, 600 μm. (L) Quantitative analysis of a time course of fluorescence radiant efficiency of PFS@Cy5 and PFS@pPNP hydrogels (n = 3). Data are presented as the mean ± SD

Fig. 3

pPNP-mediated KL programming in chondrocytes. (A) Confocal images of chondrocytes treated with free pDNA or pPNP. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (B, C) Cellular uptake of free pDNA or pPNP by chondrocytes, as measured through flow cytometry analysis (n = 3). (D) Representative microscopy image of EGFP-positive chondrocytes. Scale bars, 25 μm. (E) Typical confocal images of chondrocytes incubated with pPNPs for 1–4 h at 37 °C. Nuclei nuclei were stained with DAPI (blue), endosomes/lysosomes were stained with LysoTracker Green (green), and pDNAs were labeled with Cy5 (red). Scale bars, 10 μm. (F–I) Quantitative analysis of the co-localization of Cy5-labeled pDNA with LysoTracker Green-labeled endosomes/lysosomes (n = 3). (J–K) Percentage of EGFP-positive chondrocytes treated with free pDNA or pPNP (n = 3). (L) Volcano plot of genes differentially expressed in untreated versus pPNP-treated chondrocytes. (M) KEGG pathway analysis of the differentially expressed genes. (N) Heatmap of differentially expressed senescence- and inflammatory-related genes between untreated and pPNP-transduced chondrocytes (n = 3). Data are presented as the mean ± SD. ***P < 0.001

Fig. 4

Klotho-mediated alleviation of senescence and rescue of OA cartilage degeneration in rat chondrocytes. (A) DCFH-DA fluorescence staining to detect chondrocytes with oxidative stress damage and measure ROS scavenging. Scale bar, 100 μm. (B) Quantitative analysis of ROS clearance (n = 3). (C) SA-β-galactosidase staining images of cultured chondrocytes in the Control, pPNP, T II A, and pPNP + T II A treatment groups. Scale bar, 500 μm. (D) Quantification of SA-β-galactosidase positivity (n = 3). (E) Immunofluorescence staining of Col2A1, MMP13, and P16^INK4a in rat chondrocytes. Scale bar, 40 μm. (F) Quantitative analysis of fluorescence intensity (n = 3). (G) Expression of Col2A1, MMP13, CCN1, ADAMTS5, Klotho, P16^INK4a, and P21 at 2 days post-transfection of rat chondrocytes with different cultures, as determined by WB (n = 3). (H) qPCR evaluation of the expression of Col2A1, MMP13, Klotho, and P16^INK4a at 2 days post-transfection of rat chondrocytes with different cultures (n = 3). Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001. NS, not significant

Fig. 5

PFS@pPNP-mediated induction of BMSC recruitment and promotion of chondrogenic differentiation. (A) Schematic illustration of the recruitment process pattern of BMSCs during cartilage repair. (B, C) WB and qRT-PCR analyses of protein (B) and mRNA (C) levels of Sox9, Aggrecan, and Col2A1 in BMSC pellets in the indicated treatment groups (n = 3). (D) Immunofluorescence staining of Aggrecan and Col2A1 in BMSCs cultured on KLDL or PFS for 7 days. Scale bar, 40 μm. (E) Representative images of the Transwell bottom membrane stained with crystal violet after 24-h culture in the indicated treatment groups. Scale bar, 500 μm. (F) Quantification of cells observed at the bottom of the membrane (n = 3). (G) Alcian blue staining in BMSC pellets after 7-day culture in the indicated treatment groups. Scale bar, 250 μm. (H) Quantification analysis of chondrocyte area following Alcian blue staining (n = 3). (I) Alexa Fluor 488 phalloidin and DAPI staining showing morphology of BMSCs and chondrocytes grown under KLDL or PFS. Scale bar, 200 μm. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant

Fig. 6

pPNP + T II A@PFS-mediated rescue of OA cartilage degeneration post-ACLT surgery in rats. (A) Schematic illustration of the experimental design. (B) Representative 2D images in the sagittal and coronal planes, and 3D images of subchondral bone reconstructed by micro-CT. (C) Three-dimensional images of rat knee joints showing the abnormal growth of osteophytes in the control, Blank PFS, pPNP@PFS, T II A@PFS, and pPNP + T II A@PFS groups at 7 and 10 weeks post-ACLT. (D, E) Quantitative statistics of the percentage of BV/TV (D) and total BMD (E) on micro-CT (n = 4). (F) Representative rat knee joint images stained with safranin O/Fast Green and H&E at 7 and 10 weeks. Scale bar, 200 μm. (G, H) OARSI scores of rat joints (G) and relative cartilage thickness (H) at 7 and 10 weeks (n = 4). (I) Representative immunohistochemistry staining images of P21 (Scale bar, 850 μm) and MMP13 (Scale bar, 200 μm) in rat knee joints from the control, Blank gel, pPNP@PFS, T II A@PFS, and pPNP + T II A@PFS groups at 7 and 10 weeks. (J, K) Quantification of cells with histological positivity for P21 (J) and MMP13 (K) (n = 4). Data are presented as the mean ± SD. *P < 0.05 versus pPNP + TIIA@PFS group at 7 weeks, ^#P < 0.05 versus pPNP + TIIA@PFS group at 10 weeks, **P < 0.01, ***P < 0.001. NS, not significant

Fig. 7

pPNP + TIIA@PFS-mediated promotion of chondrogenic differentiation in vivo. (A) In vivo biodegradation of pPNP + TIIA@PFS labeled with Cy5, visualized using in vivo fluorescence imaging of knee at day 14 post-injection (n = 4). (B, C) Quantification of cells with histological positivity for Col2A1 (B) and Aggrecan (C) (n = 4). (D) Representative images of immunohistochemistry staining of Col2A1 and Aggrecan in rat knee joints at 7 and 10 weeks post-ACLT surgery. Scale bar, 200 μm. (E) Immunofluorescence staining of CD90 in rat knee joints at 7 and 10 weeks. Nuclei were stained with DAPI. Scale bar, 500 μm. Data are presented as the mean ± SD. *P < 0.05 versus pPNP + TIIA@PFS group at 7 weeks, ^#P < 0.05 versus pPNP + TIIA@PFS group at 10 weeks