Klotho enhances bone regenerative function of hPDLSCs via modulating immunoregulatory function and cell autophagy

Cell culture and treatment

hPDLSCs were isolated and cultured as previously described [19]. They were obtained from 12 healthy premolars with orthodontic demands (12–20 years old, 6 males and 6 females). This study was approved by the Ethics Committee of the Affiliated Stomatological Hospital of Sun Yat-sen University (ERC-[2016]-46). For this study, hPDSLCs were acquired from passages 3–5. The hPDLSCs were seeded in 6-well plates at a density of 1 × 105 cells/well. Recombinant Klotho protein (PeproTech, Rocky Hill, NJ, USA) was added to the culture medium and incubated for 24 h. According to a previous study, 100 ng/mL Klotho was used in this study for the optimum protective effect. The hPDLSCs were pretreated with 100 ng/mL Klotho for 24 h.

Transplantation of cells in a rat model of cranial bone defect

The animal experiments were conducted in accordance with the approved principles and procedures of the Ethics Committee of Zhongshan School of Medicine on Laboratory Animal Care (SYSU-IACUC-2022-001065). Male Sprague Dawley (SD) rats, weighing 180–220 g and aged 8 weeks, were procured from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The rats were divided into groups of six for the experiment. Anesthesia was induced with 1% pentobarbital administered through intraperitoneal injection. The skin on the rats' heads was prepared and incised, and the periosteum was carefully dissected to expose the cranial bone. Two critical-size cranial defects with a diameter of 6 mm were created on each side of the cranium to evaluate new bone formation. The hPDLSCs were pretreated with 100 ng/mL Klotho for 24 h and then cultured with fresh medium before collected for implantation. hPDLSCs pretreated with or without Klotho were transplanted into the defects using Matrigel scaffolds (Corning, NYC, USA), simultaneously. 2 × 106 cell/50 µL cell suspension was mixed with 100 µL Matrigel on ice for each defect and can be used after changing into solid phase in room temperature. The cranial defects treated with only Matrigel scaffolds but no cells were defined as Blank control. Finally, absorbable sutures were used to suture the incisions (n = 6).

Evaluation of cell viability after transplantation

To evaluate the viability of hPDLSCs in vitro, the expression of c-Caspace-3 and total Caspase-3 were detected using immunofluorescence staining at 0 and 24 h cultured with or without Klotho treatment. To evaluate the viability of hPDLSCs after transplantation in vivo, tissue samples from cranial bone defects were collected 3 days after the operation. The soft tissue samples were fixed in a 10% formalin solution and embedded in paraffin. Paraffin sections were dewaxed at 58 °C for 20 min and hydrated with gradient concentration of ethanol. The sections were soaked in citrate repair solution for antigenic thermal repair. 0.1% TritonX-100 (prepared in PBS) was added to cover the tissues for 30 min, and then the sections were closed in 5% BSA solution for 2 h at 25 °C. Rabbit anti-human Ki-67 (1:400; Cell Signaling Technology, Waltham, MA, USA)/mouse anti-human HNA antibody (HNA) (1:200; Millipore, Bedford, MA, USA) and rabbit anti-human c-Caspase-3 antibody (1:400; Cell Signaling Technology, Waltham, MA, USA)/mouse anti-human HNA antibody were added separately, and incubated overnight at 4 °C. The samples were then conjugated to secondary antibodies containing fluorescent of both species for 1 h at 25 °C. Finally, the nuclei were stained with Hoechest (Life Technologies, USA) and observed in laser confocal microscope (Carl Zeiss, Oberkochen, Germany).

Autophagy activity evaluation of transplanted cell

Tissues in cranial bone defects were collected 3 d after the operation to assess PDLSC autophagy activity after transplantation. The samples were treated via the method mentioned in the “Evaluation of cell viability after transplantation” section, after being blocked with 5% (w/v) bovine serum albumin/PBS for 2 h at 25 °C, and incubated with LC3 antibody (1:200; Abcam, Cambridge, MA, UK) and HNA (1:200; Millipore, Bedford, MA, USA) overnight at 4 °C, and further conjugated to Alexa Fluor555 or Alexa Fluor488 (1:500; EMAR, Beijing, China) as secondary antibodies for 1 h at 25 °C. Finally, the nuclei were counterstained using DAPI (1:100; Abcam, Cambridge, MA, UK). Fluorescent images were observed and recorded using a laser confocal microscope (Carl Zeiss, Oberkochen, Germany).

Micro-computed tomography (CT) examination

The tissues of the cranial defect area were collected from each group 12 week post-transplantation and Micro-CT (SCANCO μCT50, Muttenz, Switzerland) scans were performed to observe new bone formation in rat cranial defects. Mimics software (version 17.0, Materialise, Leuven, Belgium) was used to analyze the selected region of bone defect and export the analytical outcome including the measurement of bone volume and bone density of the newly formed bone.

Histological evaluation

The specimens were evaluated histologically after undergoing μCT scanning. To perform histological analysis, the specimens were fixed in a 10% formalin solution, decalcified with a 10% EDTA solution (pH 7.4), and subsequently embedded in paraffin. 5-μm-thick sections were stained with hematoxylin and eosin (H&E) as well as Masson's trichrome stains to facilitate histological evaluation. To assess the local macrophage polarization, iNOS (1:400; Cell Signaling Technology, Waltham, MA, USA) and Arginase1 (Arg1) (1:400; ProteinTech Group, Chicago, IL, USA) antibodies were applied at the bone defect site. Additionally, immunohistochemical staining was performed using antibodies against BSP, IL-6, IL-1β and IL-10. The digital slice scanner (Leica, Wetzlar, Germany) was used to capture images. Tartrate-resistant acid phosphatase (TRAP) staining was performed on cryosections using the Acid Phosphatase, Leukocyte (TRAP) Kit (Millipore Sigma, USA) according to the manufacturer's instructions. Percent of the positive area was measured by ImageJ (Germany).

Macrophage culture and conditioned medium treatment

To explore the effect of Klotho on the immunoregulatory capacity of hPDLSCs, macrophages were cultured in different hPDLSC-conditioned medium (PDLSC-CM). At first, the hPDLSCs were seeded onto 10 cm dishes at a density of 5 × 105 cells/well and treated with or without 100 ng/mL Klotho protein for 24 h. Then the supernatants were collected and mixed with fresh DMEM (Gibco, GrandIsland, USA) containing 10% FBS in a 1:1 ratio, named as PDLSC-CM-control and PDLSC-CM-Klotho. To test the effect of Klotho and CMs on the viability of Raw264.7 macrophages, cells were treated with different concentrations of Klotho or CMs for 24 h, and then CCK8 assay was performed. The murine RAW264.7 macrophage line was cultured in DMEM, supplemented with 10% heat-inactivated FBS. After the adherent culture using DMEM for 16 h, the RAW264.7 macrophages were cultured in different PDLSC-CM for 24 h, and then with M1 inducers (1 µg/mL LPS and 20 ng/ml IFN-γ) for another 24 h at 37 °C. Meanwhile, the macrophages that were only cultured in DMEM throughout the course were used as negative controls (uninduced group), and macrophages that were treated without PDLSC-CM but only with M1 inducers for the last 24 h were made as positive controls (induced group).

Flow cytometric assessment of surface markers

The RAW264.7 macrophages (3 × 105) were cultured in 24-well plates in DMEM overnight (16 h), and then cultured via the method mentioned in the “Macrophage culture and conditioned medium treatment” section. The macrophages were then washed twice with PBS, harvested using a scraper, collected through centrifugation, and maintained on ice. Subsequently, the cells were resuspended in 100 µL PBS, in tubes, blocked with CD16/32 Fc block (1:200; BioLegend, CA, USA) for 15 min on ice. The cells were then washed with PBS, collected, and maintained on ice. Then, a mixture of 0.2 µL anti-mouse BV421-CD163 and 0.2 µL anti-mouse APC/cyanine7 IA/IE antibodies (BioLegend, CA, USA) in 100 µL PBS was added to each tube, and rested for 30 min, in darkness, on ice. The cells were then washed and resuspended in 250 µL PBS and recorded using a flow cytometer (BD LSRFortessa flow cytometry, State of New Jersey, USA). The data were analyzed using the FlowJo 10.8.1 software.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA samples were isolated from the hPDLSCs and RAW264.7 macrophages, using an Ultrapure RNA kit (CWBIO, Beijing, China). Reverse transcription was performed using a Reverse Transcriptase M-MLV Kit (Takara, Shiga, Japan), according to the manufacturer’s instructions. PCR was performed using the SYBR Green Kit (GenStar, Beijing, China) and the LightCycler® 480 Real-Time PCR System (Roche, Basel, Switzerland). The amplification conditions were set as follows: 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and final extension at 72 °C for 20 s. The gene expression levels were calculated using the 2−ΔΔCt method (n = 3). The primer sequences used are listed in Table 1.

Table 1 Primer sequences used in quantitative real-time reverse transcription polymerase chain reactionWestern blot analysis

The cells were lysed and quantified using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Then, the protein samples were separated using 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were then blocked by 5% skimmed milk for 1 h and then incubated with primary antibodies at 4 °C, overnight. Finally, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Emar, Beijing, China) or fluorescent secondary antibodies (LI-COR, Biosciences, Lincoln, NE, USA) for 1 h at 25 °C. Antibodies against iNOS (1:1000; Cell Signaling Technology, Waltham, MA, USA), Arg1 (1:5000; ProteinTech Group, Chicago, IL, USA), LC3 (1:1000; Abcam, Cambridge, UK), Autophagy-related protein 5 (ATG5) (1:1000; Abcam, Cambridge, UK), Beclin-1 (1:1000; Cell Signaling Technology, Waltham, MA, USA), PINK1 (1:600, ProteinTech Group, Chicago, IL, USA) and GAPDH (1:1000; Zenbio, Chengdu, China) were used in this study. The relative density of the protein bands was analyzed using the ImageJ software and normalized to GAPDH (n = 3).

Statistical analysis

Statistical analysis was performed using the SPSS 20.0 software package (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was conducted, and the post hoc Bonferroni test was performed for multiple comparisons. Statistical significance was set at P < 0.05.

The experiments were performed in triplicate, and the data were expressed as mean ± standard deviation.

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