Data collection and preprocessing
We investigated the development of DMT1 in osteoporotic and non-osteoporotic bone marrow mesenchymal stem cell samples by analyzing human osteoporotic and non-osteoporotic bone marrow mesenchymal stem cell samples in an online database. The data GSE147287 was downloaded from Gene Expression Omnibus database. This database included single cell sequencing of osteoporosis tissue (GSE147287) and osteoarthritis tissue (GSM4423511). Further down-clustering analysis, selection of differentially expressed genes, differential analysis and acquisition of marker genes were performed using R software (version 4.2.0) and its single-cell data analysis software set Seurat package (version 4.0) developed by Satija Lab. DMT1 was found to have some regularities in the development of osteoporosis by this analysis, and pseudo-time analysis revealed up-regulation of this gene expression in cells developing osteoporosis.
Pseudo time analysis
The single-cell pseudotime analysis using Monocle2 (http://cole-trapnell-lab.github.io/monocle-release) with DDR-Tree and default parameter was applied. Before Monocle analysis, marker genes of the Seurat clustering result and raw expression counts of the cells that passed filtering was selected. Based on the pseudo time analysis, branch expression analysis modeling was applied for the branch fate–determined gene analysis.
Cell culture
The MC3T3-E1 cell line derived from mice (iCell, China) was cultured in α-MEM (WISENT, China) supplemented with 10% fetal bovine serum and incubated at 37℃ in a 5% CO2 incubator. Before seeding, MC3T3-E1 Cells were cultured and transferred using MEM medium supplemented with 10% FBS (Gibco, USA) and 100 µ/mL penicillin–streptomycin-amphotericin B (Biotech, China), with daily medium replacement.
Cell viability assay
Cell viability assay was used to detect the cytotoxicity of MT (Sigma-Aldrich, China) using the CCK-8 method.MC3T3-E1s were placed in 96-well plates at a density of 1 *10^4/well. They were then treated with varying amounts of MT (0, 5, 10, 25, 50, 100 µmol/L) for a duration of 3 days. Afterward, 10 µl of CCK-8 cell viability buffer was added to each well, followed by incubation at 37℃ for an additional hour. Finally, the absorbance at 450 nm was determined using a microplate reader.
Preparation of PAP, CS and CS@PAP
Materials-CS (Guangzhou Yunmei Technology China).The amino group of p-Phenylenediamine and N-phenyl-1,4-phenylenediamine, which was shielded with butane diacid anhydride, were dissolved in a solution containing DMF and hydrochloric acid (HCl). Toluene was used to azeotropically distill the hydroxyl-capped PLA (PLA). Next, a flame-dried glass reactor was filled with 2 mmol of purified PLA, 1 mmol of emeraldine carboxyl-capped aniline pentamer (EM AP), 5 mmol of DCC, 5 mmol of DMAP, and 15 ml of NMP.Following the reaction, filtration was employed to eliminate dicyclohexylurea. The copolymer present in the filtrate was precipitated using ethanol and subsequently dissolved in CHCl3. PAP was then dried at room temperature under vacuum.Preparation of CS Complex Coated PAP Granules. Initially.Ethyl acetate was used to disperse PAP (10 g). Water was added to the system to agglomerate the dispersed particle mixtures, which were then agitated. After the separation process, the resulting particles were dried. The granules that were screened were dispersed in the CS solution and subsequently coated with CS. Following 30 minutes of stirring, the granules that had been coated were isolated, rinsed with water, and subsequently dried in a desiccator. Sieve analysis was used to determine the size distribution of the desiccated granules.
Cytotoxicity assessment of CS, PAP and CS@PAP
Cell viability was assessed using the CCK-8 assay (Biosharp, China) as per the instructions provided by the manufacturer to confirm the cytotoxic effects of CS, PAP, and CS@PAP. In short, the MC3T3-E1s were placed in a 96-well dish with a concentration of 3 × 10^4 cells /ml and left to incubate for a period of 1 day. The MC3T3-E1s were exposed to CS, PAP, and CS@PAP in a culture medium for a duration of 3 days. Subsequently, 10 ul of CCK-8 was introduced into the culture and incubated at 37°C in the absence of light for 3 hours. A microplate reader was used to measure the absorbance at 450 nm.
Quantitative RT-PCR analysis
MC3T3-E1 cells were cultured in 6-well plates with medium at a density of 10^5 cells per well.MC3T3-E1 cells were subjected to treatment with MT at concentrations of 0, 5, 10, 25, 50, and 100 umol/L. After a period of 3 days, the RNA from the cells was obtained by utilizing a rapid extraction kit designed specifically for RNA. Subsequently, it was converted into cDNA through the utilization of a Reverse Transcription Kit. Next, SYBR Green PCR Master Mix was utilized to conduct real-time quantitative PCR. To ensure data accuracy, 40 cycles of PCR were performed with the following conditions: 10 minutes at 94℃, 15 seconds at 95℃, and 60 seconds at 60℃. Additionally, 6 replicate wells were used for all reactions.
In vitro release of CS@MT and CS@MT/PAP
After the gel formed, 100 microliters of a liquid solution with a concentration of 50 micromoles per liter of MT were added to CS/CS@PAP and placed in a 24-well plate with 500 microliters of PBS.The well plates were placed in a shaking incubator at 100 RPM and incubated for 14 days at a temperature of 37°C. The supernatant was collected with fresh PBS (0.1 M) at specific intervals (0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (1 week), 10 days, and 14 days (2 weeks)). Afterwards, the liquid above was preserved at a temperature of -80°C until it was ready for the ELISA test. After the conclusion of the trial, the thawing process was applied to all gathered supernatants. The concentration of MT in each sample was evaluated using a Human MT ELISA Kit (Fine Test, China) following the manufacturer's instructions, at a wavelength of 450nm. The measurement was conducted six times for each time period. The total quantity of discharged MT was computed and graphed over time.
Microparticle characterization
The microparticle's morphology was analyzed using scanning electron microscopy (SEM; Zeiss SEM Gemini 2, German). To prepare the samples, a small amount of the microparticle mixture was placed on the surface and left to dry for a whole night. The microparticles were then analyzed using a Bruker INVENIO-R spectrometer Fourier transform infrared spectroscopy (FTIR). The dried hydrogels were scanned using a Micro CT (American, PE Quantum GX2) and the software was utilized to calculate the percentage of total porosity.
Immunohistochemistry analysis
Immunohistochemistry was performed for RUNX-2 proteins detection. Following a 2-week co-culture period, MC3T3-E1 cells were placed on slides at a concentration of 5 × 104 cells per milliliter. Next, the medium was supplemented with Osteogenic induction medium and incubated for a duration of 48 hours. Following fixation of cells in 4% paraformaldehyde for 30 minutes, slides were treated with goat serum to prevent non-specific binding. Subsequently, they were incubated with the primary antibody (RUNX-2, 1:200) overnight at 4 degrees Celsius. Next, the slides were treated with RUNX-2 conjugated secondary antibody (at a dilution ratio of 1:50) for 30 minutes at ambient temperature. Then take the photos under the microscope.
Immunofluorescence staining
The MC3T3-E1 cells were cultured in a 6-well plate with a density of 10^5 cells per well. They were then divided into four groups: Control, CS, CS@PAP, CS@MT, and CS@MT/PAP group.Following a week, the MC3T3-E1 cells were rinsed using PBS, then treated with 4% paraformaldehyde for 30 minutes, and subsequently permeabilized with Triton X-100 for a duration of 10 minutes. Next, the cells were cultured with the primary anti-RUNX-2 antibody at a temperature of 4℃ for the entire night. Afterward, they were stained with RUNX-2 and Molecular Probes Alexa Fluor 488 Phalloidin in the absence of light for a duration of 1 hour.Following a 10-minute fixation period using mounting fluid containing DAPI, the cells were captured using the EVOS M5000 cell imaging system.
Build osteoporosis mouse model and In vivo experiments
The animal experiments in this study were approved by the Animal Ethics and Welfare Committee (AEWC) of Guangdong Medical University Hospital. A total of 32 C57BL/6 mice, 8 weeks old and weighing approximately 20g each, were purchased from Ruige, China. These mice were then randomly assigned to four groups: Control group, CS@PAP group, MT group, and CS@MT/PAP group.After administering anesthesia, all the mice were surgically excised, and the incisions were closed following the removal of both ovaries and the surrounding adipose tissues. After a period of four weeks, the CS@PAP/MT group received a single injection of 10 mg/kg CS@MT/PAP. In contrast, the CS@PAP group was injected with the same dose as the CS@MT/PAP group. Similarly, the MT group received the same dose of MT, and the control group received the same dose of saline. Following a 4-week intervention, the mice were executed by receiving overdose desflurane anesthesia and the femur was extracted and immobilized in a 4% paraformaldehyde solution. To examine the bone microstructure, samples of mouse femur were dissolved in PBS at room temperature for the purpose of observing the bone microstructure. The femur bones underwent hematoxylin-eosin (HE) staining and micro-CT analyses. Micro-CT was utilized for quantitative determination in both two-dimensional (2D) and three-dimensional (3D) formats, following previously established protocols. The Siemens Preclinical Imaging System was employed to measure microstructure parameters of cancellous bone, including trabecular number (Tb.N), trabecular thickness (Tb.Th), bone mineral density (BMD), and bone volume/total volume (BV/TV). These measurements were conducted using the multi-slice mode in standard resolution.
Statistical analysis
The mean ± standard deviation of quantitative data obtained from a minimum of six biological replicates is presented in this study. Statistical significances were evaluated using SPSS 20.0 software. Student's t-test and analysis of variance methods were used to determine the distinctions between 2 and > 2 groups. Groups were considered significantly different if theP value was less than 0.05.