An esterase-responsive ibuprofen nano-micelle pre-modified embryo derived nucleus pulposus progenitor cells promote the regeneration of intervertebral disc degeneration

Abstract

Stem cell-based transplantation is a promising therapeutic approach for intervertebral disc degeneration (IDD). Current limitations of stem cells include with their insufficient cell source, poor proliferation capacity, low nucleus pulposus (NP)-specific differentiation potential, and inability to avoid pyroptosis caused by the acidic IDD microenvironment after transplantation. To address these challenges, embryo-derived long-term expandable nucleus pulposus progenitor cells (NPPCs) and esterase-responsive ibuprofen nano-micelles (PEG-PIB) were prepared for synergistic transplantation. In this study, we propose a biomaterial pre-modification cell strategy; the PEG-PIB were endocytosed to pre-modify the NPPCs with adaptability in harsh IDD microenvironment through inhibiting pyroptosis. The results indicated that the PEG-PIB pre-modified NPPCs exhibited inhibition of pyroptosis in vitro; their further synergistic transplantation yielded effective functional recovery, histological regeneration, and inhibition of pyroptosis during IDD regeneration. Herein, we offer a novel biomaterial pre-modification cell strategy for synergistic transplantation with promising therapeutic effects in IDD regeneration.

Keywords: Biomaterial pre-modification; Esterase-responsive nano micell; Intervertebral disc degeneration; Nucleus pulposus progenitor cells; Synergistic transplantation therapy.

Graphical abstract

Scheme 1

General schematic of synthesis of esterase-responsive ibuprofen nano-micelles (PEG-PIB) to pre-modify embryo-derived long-term expandable nucleus pulposus progenitor cells (NPPCs) for synergistic transplantation in intervertebral disc degeneration.

Fig. 1

Isolation and characterization of Tie2⁺ mouse embryo derived mNPPCs in vitro. (A) Schematic diagram showing the isolation of E8.5 S Tie2-GFP⁺mouse embryo derived mNPPCs and screening of mouse NPEM. (B) The GFP epifluorescence images of the E8.5 Tie2-GFP⁺ mice embryo on the left. Detailed images of notochord (white rectangle) and is further amplificated (red arrow). Scale bar = 100 μm. (C) Bright field (bottom row) and GFP epifluorescence (up row) images of culture mNPPCs aggregates at the Day1, 2, 3, 5, 7 after cultured in 3D format. Scale bar = 500um. (D) The FACS results of isolation Tie2-GFP⁺ cells from the dissociated notochord cells. (E) Bright field 10X images of cultured mNPPCs from embryos at different passages. Scale bar = 100 μm. Bright field 200X images of cultured mNPPCs from embryos at different passages. Scale bar = 2000 μm (F) Number of mNPPCs after 30 passages (starting from 2 × 10⁴ cells). (G) FACS analysis of indicated surface markers on mNPPCs (red line) compared with mESCs (blue line). The grey column indicated the IgG for each antibody. (H) Western blot analysis of indicated proteins between mESCs and mNPPCs. (I) Immunofluorescence results (20X) of mNPPCs with Aggrecan, Collagen II, Collagen I and SOX9 expression. Scale bar = 100 μm (J) The quantitative result of Fig. 1H. **p < 0.01, *p < 0.05 vs. mESCs (Student's t-test).

Fig. 2

Derivation and long-term culture of embryo derived hNPPCs. (A) Schematic diagram showing the isolation of human embryo derived hNPPCs. (B) The total embryo dissected and their gestation ages as well as the derivation efficiency. (C) Number of hNPPCs after 30 passages (starting from 5.4 × 10⁵ cells). (D) Bright field 10X images of cultured mNPPCs from embryos at different passages. Scale bar = 100 μm. Bright field 200X images of cultured mNPPCs from embryos at different passages. Scale bar = 2000 μm (E) FACS analysis of hNPPCs' expression of Tie2. (F) FACS analysis of indicated surface markers on hNPPCs (red line) compared with hiPSCs (blue line). The grey column indicated the IgG for each antibody. (G) Western blot analysis of indicated proteins between hiPSCs and hNPPCs. (H) Immunofluorescence results (20X) of hNPPCs with Aggrecan, Collagen II, Collagen I and SOX9 expression. Scale bar = 100 μm. (I) The quantitative result of Fig. 2G. **p < 0.01, *p < 0.05 vs. hiPSCs. (Student's t-test).

Fig. 3

Synthesis and characterization of PEG-PIB (A) Synthesis diagram of PEG-PIB. (B) SEM results of PEG-PIB (red arrow). Scale bar = 100 μm (C) 1H NMR spectrum of PEG-PIB monomer. (D) 1H NMR spectrum of PEG-PIB polymeride. (E) GPC traces of the PEG-PETTC and PEG-PIB. (F) Size distributions and the PDI of PEG-PIB. (G) ζ-potential of PEG-PIB at different pH values. p < 0.05 (one-way ANOVA test) (H) ibuprofen release rate from PEG-PIB with or without esterase at different pH values (pH 7.4, pH 6.8, pH 6.4, pH 6.2). p < 0.05 (two-way ANOVA test) (I) FACS results of rhodamine packaged PEG-PIB endocytosed by the hNPPCs. (J) Immunofluorescence results of hNPPCs treated with FDA at different pH values (pH 7.4, pH 6.8, pH 6.4, pH 6.2), and further added rhodamine packaged PEG-PIB for 30 min to observe the efficiency of endocytosis. Scale bar = 50 μm (K) The quantitative result of Fig. 3J p < 0.05 (one-way ANOVA test) (L) CCK-8 results of the PEG-PIB effect on hNPPCs proliferation. (Student's t-test) (M) ibuprofen release rate from PEG-PIB within the hNPPCs at different pH values (pH 7.4, pH 6.8, pH 6.4, pH 6.2). p < 0.05 (two-way ANOVA test).

Fig. 4

PEG-PIB inhibit hNPPCs pyroptosis in vitro (A) IHC results of ASC expression from clinical degenerated IVD sample. (B) Western blot analysis of indicated proteins between hNP and hDNP. (C) The quantitative analysis of Fig. 4B. **p < 0.01, *p < 0.05 vs. hNP. (Student's t-test) (D) Immunofluorescence results of ASC expression in hNPPCs treat with different pH values (pH 7.4 and pH 6.2) (E) Fluorescence image of live (green) and dead (red) cells at different pH circumstance (pH 7.4, pH 6.8, pH 6.4, pH 6.2). Scale bar = 100 μm (F) Immunofluorescence of ASC expression in rhodamine packaged PEG-PIB pre-modified hNPPCs at different pH circumstance (pH 7.4, pH 6.8, pH 6.4, pH 6.2). Scale bar = 100 μm (G) The quantitative analysis of Fig. 4D. **p < 0.01 vs. pH 7.4. (Student's t-test) (H) The quantitative analysis of Fig. 4E p < 0.05 (one-way ANOVA test) (I) The quantitative analysis of Fig. 4F p < 0.05 (one-way ANOVA test) (J) CCK-8 results of the proliferative potential of PEG-PIB pre-modified hNPPCs at different pH circumstance (pH 7.4, pH 6.8, pH 6.4, pH 6.2). p < 0.05 (one-way ANOVA test) (K) Western blot analysis of indicated proteins between all 3 groups (NC group, pH 6.2 group and PEG-PIB group). (L) The quantitative result of Fig. 4K. **p < 0.01, *p < 0.05 vs. NC group. ##p < 0.01, #p < 0.05, vs. PEG-PIB group. (one-way ANOVA test).

Fig. 5

Radiographs and MRI results.(A) Radiographs of all five groups, which were obtained at 2, 6, 10 and 18 weeks after modeling, both IVD segments were treated with the same procedure.(B) DHI% was calculated from digitized radiographs using Image J. (C) The quantitative analysis of DHI%. *p < 0.05. (two-way ANOVA test) (D) Representative T2 MRI scans，both IVD segments were treated with the same procedure. (E) The sagittal plane T2 MRI index of each group. *p < 0.05 (two-way ANOVA test) (F) The transverse plane T2 MRI indexes of different groups. *p < 0.05 (two-way ANOVA test).

Fig. 6

PEG-PIB pre-modified hNPPCs histopathological regeneration results in vivo. (A) Representative H&E staining of disc samples from different groups at 2 and 18 weeks after modeling. Scale bar = 1000 μm (B) Representative S–O staining of disc samples from different groups at 2 and 18 weeks after modeling. Scale bar = 1000 μm.

Fig. 7

PEG-PIB pre-modified hNPPCs histopathological and functional regeneration results in vivo. (A) Immunohistochemistry staining of Collagen Ⅱ in disc samples from different experimental groups. Scale bar = 1,000 μm (B) Histological scores of all five groups at 18 weeks after modeling. p < 0.05 (one-way ANOVA test) (C) The relative Collagen II expression quantitative analysis of Fig. 7A p < 0.05 (one-way ANOVA test) (D) The relative ASC expression quantitative analysis of Fig. 7E. (one-way ANOVA test) (E) hNPPC tracer (Green) and ASC (Red) immunofluorescence co-location results at 18 weeks after modeling. Dotted lines are the region of NP. (F) The scheme of elasticity modulus. The red arrow points at the coccygeal vertebrae Co 7/Co 8. (G, H and I) The force-displacement curve of all five groups at 6, 10 and 18 weeks after modeling.

Fig. 8

PEG-PIB pre-modified hNPPCs histopathological and Pfirrmann grading (A) Immunohistochemistry staining of Aggrecan in disc samples from different experimental groups. Scale bar = 1,000 μm (B) The relative Aggrecan I expression quantitative analysis of Fig. 8A p < 0.05 (one-way ANOVA test) (C) The Pfirrmann grading system of different experimental groups were used to quantify the structure, distinction of nucleus and annulus fibrosus, signal intensity, and height of intervertebral disc at 18 weeks. Sample numbers N = 6.