2.1. Synthesis of MHAP scaffolds
All of the chemical reagents were purchased from sigma. The synthetic process of the MHAP scaffolds is as follows. Weighing 0.251 CTAB (mCTAB/(theoretical output mCa5(PO4)3.OH)*100 = 5%) and adding 100 ml deionized water, stirring for 10 minutes, dissolving completely, and then putting into 90℃ oil bath. Next, weighing 4.0018 g (0.03 mol) (NH4) 2HPO4 and dissolving in CTAB solution, stirring for 20 minutes, 280r/min to make 0.05M solution. After 20 minutes, adjusting the pH value of solution with ammonia water is 10.5. And then 11.9268 g (0.05 mol) Ca (NO3) 2.4H2O was dissolved in pure water to prepare 100 ml, 0.3M solution. The pH value of the solution was adjusted by ammonia water to be 10.5. The solution in previous step was slowly dripped into the solution of step 2 for about 40 minutes. The whole process keeps the solution PH = 10.5 unchanged, stirring for 2 hours, 280 r/min, and aging at 90 ℃ for 12 hours. Then, Wash with deionized water to neutral then with ethanol for 1–2 times. Next, drying at 80 ℃ for 24 hours; grinding. Finally, Calcination, 2 degree/minute heating, 900 degree heat preservation for 4 hours, natural cooling to room temperature, grinding.
2.2. Synthesis of MHAP-CS hybrid scaffolds
Preparation of Chitosan (CS) Solution: 2 g Chitosan is dissolved in 50 ml 2 vol% acetic acid solution (1:25), stirred mechanically for 3 hours until completely dissolved, 320r/min. Next, weighing 2 g mesoporous hydroxyapatite powder (MHAP) and slowly adding it into CS solution, stirring and dispersing for 2 h (mass ratio: MHAP: CS = 1:1). Then, transfer the dispersed and uniform solution to 24 mesh cell culture medium, transfer it to the magnetic field of − 20 C refrigerator for 24 hours, and then transfer it to freeze-dryer for freeze-drying after freezing: cooling to − 56 C (taking about 4 hours), freeze-drying with vacuum less than 10 Pa (about 96 hours in winter and 72 hours in summer); the formed samples were immersed in 10 wt% NaOH solution for 1 day, washed repeatedly to PH = 7 (to wash for 5 days, change water three times a day), and then freeze-dried again (about 1 day). Finally, the preparation of mesoporous hydroxyapatite/chitosan composite scaffolds can be obtained by cutting brittle fracture in liquid nitrogen.
2.3. Drug loading-release tests of MHAP-CS-UA hybrid scaffolds
First, ursolic acid powder was formulated into a solvent of 1 mg/ml. Next, 2.2835 mL of the above ursolic acid solution was taken up and placed in a 50 mL volumetric flask. And then anhydrous ethanol was added to the volumetric flask to prepare a 100 umol ursolic acid solution. The prepared ursolic acid scaffold material(1 µM) is used for sustained drug release. The scaffold material (5 µM, 10 µM) operates as described above.
The release test of the MHAP-CS-UA (1 µM, 5 µM, 10 µM) drug scaffold material was carried out after immersing in 5.0 ml of phosphate buffer solution (PBS), and the above operations were carried out under normal temperature and an oscillation atmosphere of 80 rpm. We extracted 1.0 ml of the above solution at different time points (3, 6, 9, 12, 24, 48 and 72 h) while adding an equal volume of PBS solution. Finally, the corresponding drug concentration was analyzed by high performance liquid chromatography (HPLC, Agilent 1100, US).
2.4. Material characterization
The morphologies of MHAP microspheres and MHAP-CS composite scaffolds were studied by scanning electron microscopy (SEM) and characterized by energy dispersive spectroscopy (EDS). The study used transmission electron microscopy to detective nano-mesoporous structures of MHAP microspheres. Also analyzed the porous structure of MHAP by automatic surface area and porosity analyzer at 80K. The pore size distribution of MHAP was calculated by Barrett-Joyner-Halenda (BJH) method. The phase compositions of MHAP-CS and MHAP-CS-UA composite scaffolds were analyzed by X-ray powder diffraction (XRD; D/Max III C, Japan). At last, Fourier transform infrared spectroscopy identified the functional groups in MHAP-CS and MHAP-CS-UA composite scaffolds.
2.5. Cell viability and attachment
MC3T3-E1 cells were purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Human bone marrow mesenchymal stem cells from Shanghai Rochen Biotechnology Co., Ltd. (Shanghai, China). The cells were cultured in 10% fetal bovine serum and 1% penicillin/streptomycin medium at 37 °C, and 1 × 104 MC3T3-E1 cells were seeded into 96 well plated. After 24 hours, the medium was replaced with MHAP-CS or MHAP-CS-UA scaffold extract. The cell counting kit 8 was cultured for 1 day, 2 days, and 3 days, respectively. hBMSC was used as a cell model to detect the adhesion of cells on different scaffolds. Each scaffold was inoculated with 1 × 104 h BMCs into 24-well plates. hBMSCs were cultured for 12 hours and washed with phosphate. Buffered saline (PBS) was then fixed with 2.5% glutaraldehyde for 20 minutes. The HBMSCs were washed 3 times with phosphate buffered saline (PBS). The samples were dehydrated with ethanol at a concentration gradient of 75, 85, 95 and 100. Finally, the morphology of hBMSCs was characterized by scanning electron microscopy (SEM; FEI; Thermo Fisher Scientific, Inc.) on the scaffold with a magnification of 10 kV.
2.6. In vitro tests of osteogenic capability
The detection of differentiation of hBMSCs is based on alkaline phosphorylation (ALP, Renbao, Shanghai, China) and alizarin red staining (AR, Sigma-Aldrich). 1 × 104 hBMSCs were inoculated into 24-well plants for 24 hours and cultured with MHAP-CS, MHAP-CS-UA scaffolds. After 7 and 14 days of culture, hBMSCs were immobilized with 4% paraformaldehyde. Wash hBMSC 3 times with phosphate buffer saline (PBS). Next, stained hBMSC with ALP kit and alizarin red kit. Next, the residual substance of the above reagent is washed away with PBS. Finally, the samples stained with the above alkaline phosphatase and alizarin red were photographed by an inverted phase contrast microscope.
The expression levels of osteogenesis-related genes, including alkaline phosphorylation (ALP), collagen 1 (COL1) and osteoprotegerin (OPG) were detected by real-time quantitative polymerase chain reaction RT-PCR. 4 × 106 MC3T3-E1 cells were seeded in a 6-well plate containing the extract of the above scaffold material. Seven days later, total RNA was collected by RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) and reverse transcribed into cDNA (Takara Bio, Inc., Otsu, Japan). The SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) and an ABI 7500 Sequencing Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as a standard reference, the following thermocycling conditions were used: 40 cycles of denaturation at 95˚C for 5 s and amplification at 60˚C for 24 s. And the data were calculated by 2ΔΔCt method. All the above steps are in accordance with the instructions of the reagent manufacturer. The PCR primers were designed as follows:
GAPDH forward 5′-CACCACCATGGAGAAGGCCG-3′
and reverse 5′-ATGATGTTCTGGGCAGCCCC-3′
OPG forward 5'-CGAGCGCAGATGGATCCTAA-3'
And reverse 5'-CCACATCCAACCATGAGCCT-3'
Col1 forward 5′- GCTCCTCTTAGGGGCCACT-3′
And reverse 5′- CCACGTCTCACCATTGGGG-3′
ALP forward 5′- CATCATCATGTTCCTGGGAG-3′
And reverse 5′- GACCTGAGCGTTGGTGTTGT-3′
MC3T3-E1 cells were cultured in the medium of MHAP/CS or MHAP-CS-UA scaffolds to measure the expression of osteoblast-related proteins (BMP-2, COL1, RUNX2, Smad1/5). Protein was extracted from the radioimmunoprecipitation assay (RIPA) lysis buffer (cat. no. C500005; Sangon Biotech Co., Ltd.) containing 1 µM protease inhibitor. Then centrifuge at a speed of 12,000 rpm for 10 minutes. The bicinchoninic acid assay (BCA) method was used to detect the corresponding protein concentration. Gel electrophoresis was carried out on SDS-PAGE and transferred to PVDF membrane. Close the film with 5% milk for 1 hour. The membrane and the first antibody were incubated overnight at 4 degrees. After 24 hours, the membrane was washed with PBS three times and incubated with the second antibody coupled with horseradish peroxidase for 1 hour. Finally, the horseradish peroxidase-conjugated secondary antibodies reactivity was detected by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
2.7. In vivo tests of osteogenic capability
The Animal Research Committee of the Ninth People's Hospital Affiliated to the Medical College of Shanghai Jiao Tong University approved all the animal experiments in this experiment. Fifteen Sprague-Dawley female rats (200–250 g) were selected. A bilateral critical size skull defect model was used to evaluate bone regeneration. The diameter and height of bone defect were 5 mm and 2 mm respectively. MHAP-CS and MHAP-CS-UA stents (n = 5) were filled in the defect area and the scalp was sutured. Multicolor continuous fluorescence labeling was used to characterize the formation and mineralization of new bone. Alizarin red (30 mg/kg, Sigma-Aldrich) and calcein (30 mg/kg, Sigma-Aldrich) were injected intraperitoneally to label the animals 3 and 21 days before euthanasia. Twelve weeks later, the animals were euthanized. The bilateral critical size skull defects were isolated from the surrounding tissues. Samples were immersed in formalin solution buffered by 4% phosphate for 7 days, and then detected by micro-CT system (Skyscan 1072; Skyscan, Aartselaar, Belgium). The parameters are set to 90KV voltage, 88uA current and 28um voxel size. After the scanning is completed, the 3D image is reconstructed. Bone healing was evaluated by calculating bone mineral density (BMD) and new bone mass/tissue volume (BV/TV). Undecalcified samples were embedded in polymethyl methacrylate. The sagittal section of the skull was cut 150 mm thick with a slicer (Leica, Hamburg, Germany). Multicolor continuous fluorescence labeling (Leica, Heidel-berg, Germany; alizarin red: 543/580–670 nm, calcein: 488/500–550 nm) was observed under confocal laser scanning microscopy. The mineralization rate is quantified by brightness analysis system based on different color bands. Soft tissue around the skull was removed and immersed in 10% EDTA for 30 days. And then embedded in paraffin. Histological sections were prepared for Masson staining and immunohistochemistry of BMP-2, COL-1, RUNX2 and OPG protein. Finally, they were observed in a light microscopy (magnification, x10). Images were analyzed using Image-Pro Plus 6.0(Media Cybernetics, Inc., Rockville, MD, USA).
2.8. Statistical analysis
All the data were expressed as means ± standard deviation (SD) and were analyzed using one-way ANOVA. *P < 0.05 was considered statistically significant.