PPAR-γ activation promotes xenogenic bioroot regeneration by attenuating the xenograft induced-oxidative stress

Study approval

All animal experiments and procedures in this study were in accordance with institutional guidelines on animal welfare and were approved by the Institutional Laboratory Animal Care and Use Committee of Sichuan University (permit no. WCHSIRB-D-2019-063).

pTDM preparation

Xenogeneic teeth, harvested from the freshly extracted deciduous incisor teeth of porcine, were made into pTDM (porcine derived treated dentin matrix) following gradient demineralization by 17%, 10% and 5% ethylene diamine tetraacetic acid (EDTA, Sigma-Aldrich, St. Louis, MO, USA) as previously reported.2 For pNDM (porcine derived natural dentin matrix), no demineralization was proceeded. All the obtained xECM were prepared for later experiment after performing ethylene oxide disinfection following freeze-dried for 8 h.

Biophysical properties of pTDM

SEM was used to determine the surface morphology of pTDM or pNDM at 15 kV. pTDM extracts were collected at different times after culturing in the saline with the weight/volume ratio of pTDM to solvent 1:5 (g: mL) for protein release detection. At each time-point, equal volume of fresh saline were added into the dishes after the extracts were totally taken away.

Cell culture and identification

rDFC (rat derived DFC) was isolated from the mandibular 1st molar of 3-5 days old SD rats and cultured in a complete α-MEM medium containing 10% FBS (HyClone) and 1% of 100 U of penicillin and 100 μg·mL−1 streptomycin at 37 °C with 5% CO2 incubator. rDFC of 2-5 passage were seeded on 12-well plates at the number of 1 × 104 for following experiments. For rDFC sheets formation, 50 mg·mL−1 ascorbic acid was extra added into the medium to promote more extracellular matrix secretion when rDFC reached 70% confluence. For osteogenesis, 5 mmol·L−1 L-glycerophosphate (Sigma-Aldrich), 100 nmol·L−1 dexamethasone (Sigma-Aldrich), and 50 mmol·L−1 ascorbic acid were supplemented into the medium for 21 days’ culturing. Subsequently, alizarin red S (ARS) staining was carried out after the cells were fixed with 4% paraformaldehyde (PFA). For neurogenesis, 2%DMSO (Amresco), 200 μmol·L−1 Butylated hydroxyanisole (Sigma-Aldrich), 25 mmol·L−1 KCl(bdhg.company, China), 2 mmol·L−1 Valporic acid sodium salt (Sigma-Aldrich), 10 mmol·L−1 forskolin (Sigma-Aldrich), 1 mmol·L−1 hydrocortisone(aladdin), and 5 μg·mL−1 insulin (Novo Nordisk) were additionally imparted for culturing 2 hours. Afterwards, IF staining with βIII Tubulin (abcam, ab78078) was performed. Briefly, cells were fixed with 4% PFA at room temperature for 15 minutes after washing with PBS for three times, then permeabilized with 0.25% Triton-X and blocked with 5% BSA. Primary antibody against βIII Tubulin at a dilution of 1:100 was added onto the fixed cells and incubated overnight at 4 °C. DAPI was used to stain nuclei finally. All the IF staining in this study were all in accordance with this procedure (primary antibodies: anti-Vimentin (santa cruz, sc-6260), anti-CK14 (millipore, MAB3232), anti-Alex Fluor 555 Phalloidin (CST,#8953)).

Cell proliferation, grouping and migration

CCK-8 test was carried out to assess the cells’ proliferation. Different concentrations of H2O2 and RSG were separately added into the 96-well plates to culture the cells with the counting number of 4 000 per well initially. To detect the protection effect of RSG on the H2O2-induced damaged cells, rDFC was pretreated with 10 μmol·L−1 RSG for 4 h before 25 μmol·L−1 H2O2 was supplemented into the medium. With another 24 h culture, 10% (v/v) CCK-8 medium was added into each wells to culture the cells for 1.5 h at 37 °C for detecting the cell viability under different induced conditions. Cell viability was calculated based on the optical density (OD) value measured at 450 nm by absorbance microplate reader following the formula: (OD value in control - OD value in blank) / (OD value in experiment - OD value in blank). Cell migration was assessed through scratch test when the cells were fusion with different chemical regents stimulation. For cell grouping, rDFC in rDFC/H2O2 group was treated with 25 μmol·L−1 H2O2 for 24 hours while cells in rDFC/RSG/H2O2 group was pretreated with 10 μmol·L−1 RSG for 4 hours before cultured with 25 μmol·L−1 H2O2. Pipette tip was used to make a scrape on the monolayer cell and the non-adherent cells were removed from the plates with PBS washed for three times. The cell morphology was recorded under light microscopy (Nikon, Japan) at 0 h, 24 h, and 48 h for cell proliferation and migration evaluation.

ARS staining, RT-PCR and Western Blot

For evaluating the mineral nodule formation, ARS staining was performed after the cells were fixed with 4% PFA with culturing in the osteogenic medium for 21 days. After being cultured in the osteogenic medium for 7 days, rDFC was collected for assessing the expressions of the osteogenic and odontogenic differentiation-related genes or proteins as previously described.2 Briefly, real-time PCR was conducted by mixing the cDNA, which was at the final concentration of 100 ng·μL−1, with SYBR Premix Ex Taq II (Tli RNaseH Plus) and primers (synthesized by Sangon Biotech) under the procedure of denaturing at 105 °C for 5 minutes followed by 40 cycles of PCR (95 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s). Relative expression levels were calculated using the 2−ΔΔCT method and normalized to the internal control GAPDH gene. The specific primer information was shown as follows (5’-3’): GAPDH (Forward: TATGACTCTACCCACGGCAAG; Reverse: TACTCAGCACCAGCATCACC); COL-1 (Forward: TGCTGCCTTTTCTGTTCCTT; Reverse: AAGGTGCTGGGTAGGGAAGT); Runx2 (Forward: TCATTTGCACTGGGTCACAT; Reverse: TCTCAGCCATGTTTGTGCTC); ALP (Forward:CGTTGACTGTGGTTACTGCTGA; Reverse: CTTCTTGTCCGTGTCGCTCAC); Periostin (Forward:TTCGTTCGTGGCAGCACCTTC; Reverse: TCGCCTTCAATGTGGATCTTCGTC); IL-1β (Forward:GGGATGATGACGACCTGCTA; Reverse: TGTCGTTGCTTGTCTCTCCT); TGF-β (Forward:GACCGCAACAACGCAATCTATGAC; Reverse: CTGGCACTGCTTCCCGAATGTC); TNF-α (Forward:GCACGGAAAGCATGATCCGA; Reverse: AAGAGGCTGAGGCACAGACA); PPARγ (Forward:GCCCTTTGGTGACTTTATGGAG; Reverse:GCAGCAGGTTGTCTTGGATGT). For western blot (WB) analysis, proteins were transformed into 0.22 μm polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk at room temperature for 2 hours and then incubated with the primary antibodies of anti-IL-1β (Abcam, ab9787) and anti-NF-κB (CST, #8242) at a dilution ratio of 1:1 000 and anti-β actin (HUABIO, EM21002) at a dilution ratio of 1:5 000 at 4 °C overnight. WB signals were normalized to the internal control β-actin

Intracelluar ROS detection

For detecting intracelluar ROS level and inflammation expression, 2‘, 7’ - dichlorofluorescein diacetate (DCFH-DA, Sigma, America) staining, JC-1 (Beyotime, China) staining, transmission electron microscope (TEM) test and Elisa assay were conducted as previously described.2 In brief, cells were stained with JC-I or 10 μmol·L−1 DCFH-DA in dark at 37°C for 20 minutes for confocal microscope observation. For TEM detection, cells were fixed with 0.5% glutaraldehyde, dehydrated in gradient alcohol and permeated and embedded with resin. Consecutive 60-um-thick horizontal sections were obtained and double stained with uranium acetate and lead nitrate for 15 min for ultrastructural observation of mitochondria. Cell supernatants were collected for elisa test.

Animal models and surgery

pTDM/rDFCs group and pTDM-RSG/rDFCs group were included in animal experiments. pTDM was cultured in 200 ng·μL−1 RSG solution at 37°C with 5% CO2 for 2 days and wrapped with the 5 ng·μL−1 RSG pretreated rDFCs, composed of pTDM-RSG/rDFCs group.

Transplant the xenogeneic bioroots into an orthotopic implant model of SD rats

pTDM was prepared into a uniform size of 1 mm × 1 mm × 2 mm for orthotopic implant after ethylene oxide disinfection. A total of 48 Sprague-Dawley rats (8 weeks old, females, 230–260 g), purchased from Chengdu Dossy Biological Technology Company, were performed surgery. After anesthetization with 2% pentobarbital sodium (3 mL·kg−1) and Zoletil 50 (50 mg·kg−1), skin, muscles and mucous membrane were blunt separated from the corner of the mouth until the bilateral 1st molars could be directly seen. The xenogeneic bioroots, pretreated with or without RSG, were transplanted into the mesiobuccal roots of the freshly extracted 1st molars. Then, the buccal and palatal mucosa were sutured to ensure that the implanted site could be completely covered. The muscle and skin in the open wounds were subsequently sutured in layers with 6–0 silk. At last, rats were injected with penicillin (1 U per 200 g) and fasted for 10 h post-surgery. 3–5 rats in each group were randomly selected and sacrificed at 1 week, 1 month and 2 months post-implantation. Maxilla, including the first molar extraction socket, was harvested and fixed in 4% PFA at 4°overnight, and then demineralized with 10% EDTA for 2 months. Paraffin sections and frozen sections were prepared for subsequent HE, Masson, trap staining and IF staining as previously described.2 Briefly, frozen sections were washed by PBS for 3 times, repaired with antigen repair solution for 10 min, penetrated with 0.25% triton-100 for 10 minutes, blocked with 5% BSA for 1 h, separately incubated with the primary antibody anti-4 Hydroxynonenal (abcam, ab48506) and anti-TNFα(santa cruz, sc-52746) at a dilution of 1:200 overnight at 4°C, subsequently cultured with the corresponding second fluorescent antibody for 1 h, and finally stained with DAPI.

Tissue homogenates extraction

This experiment was carried out as previously described.2 Briefly, SD rats were sacrificed at 7 days post-surgery under general anesthesia and subsequently, maxilla was separated; then, the transplantation along with the surrounding fibrous tissues were obtained for further experiments. Precooled normal saline was used to rinse the samples for removing the red blood cells as much as possible. After, the 1:100 diluted PMSF was mixed with the tissue at the volume ratio of 9:1 to grind into tissue homogenate. Supernatants of the specimens were collected by centrifugation at 12 000  r·min−1 for 15 min at 4 °C for subsequent Elisa analysis (8-OHdG/3-NT Elisa kit, Deco, Cat. #3455/ 3305).

Transplant the xenogeneic bioroots into a subcutaneous implant model of SOD1−/− C57 mice

pTDM was shaped into a hollow circular column (3 mm × 2 mm × 4 mm) for subcutaneous implant after ethylene oxide disinfection. Shanghai Model Organisms Center, Inc. was entrusted to establish an animal disease model of B6;129S-Sod1tm1LebJ (SOD1−/−) of C57 mice. After anesthetized with isoflurane, the prepared xenogeneic bioroots with or without RSG pretreated were implanted subcutaneously on the back of 4-month-old SOD1−/− C57 mice. The mice were killed 2 months after implantation and the samples were fixed at 4°C in 4% PFA overnight, and then demineralized with 10% EDTA for 4 months. Paraffin sections were prepared for subsequent histological staining as previously described.2 Anti-TGFβ1 (abcam, ab92486), anti-Periostin (H-300) (santa cruz, sc-67233) and anti-MMP2 (abcam, ab92536) primary antibodies were applied at a dilution of 1:200.

Statistical analysis

All data are presented as the mean ± standard deviation (M ± SD). The Student’s t test was used to assess differences between two groups. One-way analysis of variance (ANOVA) was performed to evaluate the discrepancies among multiple groups. All in vitro or in vivo work was represented at least three biological replicates by independent experiments.

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