Controlled Release of TGF-β3 for Effective Local Endogenous Repair in IDD Using Rat Model

Figures & data

Table 1 Primers for qPCR

Gene	Forward (5’-3’)	Reverse (5’-3’)
SOD1	CGGCTTCTGTCGTCTCCTTGC	AACTGGTTCACCGCTTGCCTTC
SOD2	GCTGGAGGCTATCAAGCGTGAC	TTAGAGCAGGCGGCAATCTGTAAG
CAT	GGCCTGACTGACGCGATTGC	CTGCTCCTTCCACTGCTTCATCTG
Adamts5	TCCTCTTGGTGGCTGACTCTTCC	TGGTTCTCGATGCTTGCATGACTG
MMP3	CAGTCCTGCTGTGGCTGTGTAC	AACCTCCATGCCAGCATCTTCTTC
BAX	CACCAGCTCTGAACAGATCATGA	TCAGCCCATCTTCTTCCAGATGGT
Col-II	ACGCTCAAGTCGCTGAACAACC	ATCCAGTAGTCTCCGCTCTTCCAC
iNOS	GAGACGCACAGGCAGAGGTTG	CAGGAAGGCAGCAGGCACAC
COX-2	TTCCAGTATCAGAACCGCATTGCC	CCGTGTTCAAGGAGGATGGAGTTG
ACAN	GGCGT CCAAA CCAAC CCGAG	GGCGT CCAAA CCAAC CCGAG
MCL1	TCATCTCCCGCTACCTGC	ACTCCACAAACCCATCCC
BCL2	CACCCCTGGCATCTTCTCCTT	AGCGTCTTCAGAGACAGCCAG
GAPDH	GACATGCCGCCTGGAGAAAC	AGCCCAGGATGCCCTTTAGT

Table 2 Classification of Disc Degeneration

Grade	Structure	Distinction of Nucleus and Annulus	Signal Intensity	Height of Intervertebral Disc
I	Homogeneous, bright white	Clear	Hyperintense, isointense to cerebrospinal fluid	Normal
II	Inhomogeneous with or without horizontal bands	Clear	Hyperintense, isointense to cerebrospinal fluid	Normal
III	Inhomogeneous, gray	Unclear	Intermediate	Normal to slightly decreased
IV	Inhomogeneous, gray to black	Lost	Intermediate to hypointense	Normal to moderately decreased
V	Inhomogeneous, black	Lost	Hypointense	Collapsed disc space

Table 3 Histological Grading Scale

Category	Grade
I. Cellularity of the annulus fibrosus	1. Fibroblasts comprise more than 75% of the cells 2. Neither fibroblasts nor chondrocytes comprise more than 75% of the cells 3. Chondrocytes comprise more than 75% of the cells
II. Morphology of the annulus fibrosus	1. Well-organized collagen lamellae without ruptured or serpentine fibers 2. Inward bulging, ruptured or serpentine fibers in less than one third of the annulus 3. Inward bulging, ruptured or serpentine fibers in more than one third of the annulus
III. Border between the annulus fibrosus and nucleus pulposus	1. Normal, without any interruption 2. Minimal interruption 3. Moderate or severe interruption
IV. Cellularity of the nucleus pulposus	1. Normal cellularity with stellar shaped nuclear cells evenly distributed throughout the nucleus 2. Slight decrease in the no. of cells with some clustering 3. Moderate or severe decrease (>50%) in the number of cells with all the remaining cells clustered and separated by dense areas of proteoglycans
V. Morphology of the nucleus pulposus	1. Round, comprising at least half of the disc area in midsagittal sections 2. Rounded or irregularly shaped, comprising one quarter to half of the disc area in midsagittal sections 3. Irregularly shaped, comprising less than one quarter of the disc area in midsagittal sections

Figure 1 Synthesis and characterization of MnO₂ NPs. (A) Digital picture and TEM images of MnO₂ NPs. (B) EDS analysis of MnO₂ NPs. (C) Relative viabilities of NPCs treated with various concentrations of MnO₂ NPs for 24 h. *, p<0.05.

Figure 2 Nanoparticle decomposition and drug behaviors of TGF-β3/MnO₂. In vitro release of TGF-β3 from MnO₂ NPs at different pH values (7.4 and 6.5) in the absence or presence of 100 μM H₂O_2.

Figure 3 Representative images of NPC uptake of BSA/MnO₂ and FITC-BSA/MnO₂ for 12 h. Green, red and blue colors represent FITC-BSA/MnO₂, F-actin, and DAPI fluorescence, respectively.

Figure 4 Antioxidant activity of TGF-β3/MnO₂ NPs. (A) Representative fluorescent images of NPCs stained with DCFH-DA in the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. (B–D) qPCR analyses of the relative expression of the SOD1, SOD2, and CAT genes of the NPCs in Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. *, p<0.05.

Figure 5 Anti-apoptotic and proliferative effects of TGF-β3/MnO₂. qPCR analyses of the relative expression of apoptotic (BAX) (A) and anti-apoptotic (MCL1 and BCL2) (B and C) genes of NPCs in the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. (D) Representative images of EdU staining of NPCs in the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. Green represents EdU-labeled proliferating cells and blue represents nuclei. (E) Quantification of the percentage of EdU-positive cells in the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. *, p<0.05.

Figure 6 TGF-β3/MnO₂ regulating the ECM metabolic balance of NPCs in vitro. qPCR analyses of the relative expression of anabolic (Col-II and ACAN) (A and B), pro-inflammatory (COX-2 and iNOS) (C and D), and catabolic (MMP3 and Adamts5) (E and F) genes in the NPCs of the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. *, p<0.05.

Figure 7 Immunofluorescent analysis of Col-II and iNOS expression in NPCs. Representative images of the immunofluorescence of Col-II (A) with semi-quantitative analysis (B) and iNOS (C) with semi-quantitative analysis (D) in the NPCs of the Ctrl, H₂O₂, MnO₂, TGF-β3, and TGF-β3/MnO₂ groups, respectively. Red, green and blue colors indicate Col-II protein, iNOS protein and DAPI fluorescence, respectively. *, p<0.05.

Figure 8 Evaluation of disc degeneration with T2-weighted MRI and gross appearance. Representative images of disc gross appearance (A) and MRI signal intensity (B) at 4 and 8 weeks. Changes in MRI grade at 4 and 8 weeks after surgery (C and D). *, p<0.05.

Figure 9 Histopathological evaluation in vivo. H&E (A) and Safranin O-Fast Green (B) images of IVDs in the rat caudal vertebrae at 4 and 8 weeks. The histopathological scores of different groups at 4 and 8 weeks after surgery (C and D). *, p<0.05.

Figure 10 Immunohistochemical analysis of iNOS and Col-II expression in each group. Representative images of the immunohistochemical staining of iNOS (A) and Col-II (B) at 4 and 8 weeks after surgery, and the semi-quantitative analysis of iNOS (C) and COL-II (D) at these timepoints. *, p<0.05.