Mice
BALB/c mice (~ 20 g) and SD rats (~ 200 g) were bought from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were housed in a laboratory room at 20–24°C, at 45–55% humidity, and a strict cycle of light : dark (12 h : 12 h) for 1 week. Mice were freely fed with laboratory standard chow and tap water during the acclimatization period, as well as the whole experimental period. The experiments and welfare were performed in strict conformity with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted in compliance with the relevant ethical regulations. All experiments and protocols were approved by the Animal Ethics Committee of Hebei University.All studies were performed in accordance with ARRIVE guidelines. Unless otherwise noted, animals were euthanized by carbon dioxide inhalation followed by cervical dislocation. Detailed descriptions of materials and procedures are presented in the Supplementary Information (SI).
Cell lines and reagents
MC3T3-E1 and RAW264.7 cell lines was provided by ATCC. Fetal Bovine Serum (FBS) was purchased from Sigma (USA). CCK-8 kit was purchased from Abbkine (Hubei, China), Minimum Essential Medium (MEM) was purchased from Corning (Manassas, VA, USA). Primary antibodies for western blot experiments included NFATc1, Dc-stamp, c-Fos, CTSK were purchased from Proteintech (Hubei, China). Runx2 and OPG were purchased from Affinity (Jiangsu, China), RANKL was purchased from Abcam (Cambridge, MA), Secondary antibodies were purchased from Beyotime Biotechnology Co.Ltd (Shanghai, China). Hydroxyproline assay kit, TRAP staining kit and alkaline phosphatase assay Kit were purchased from Nanjing Jiancheng Co. (Jiangsu, China). The 16 kinds of reference amino acids were obtained from Solarbio (Beijing, China). The kit for measuring Ca2+, P, BALP, TRAP, OCN, TGF-β is from FABKEW. Retinoic acid reagent obtained from Macklin (Shanghai, China). Ossotide Tablets were purchased from Hongye Pharmaceutical Co., LTD (Anhui, China).
Preparation and analysis of compound ossotide
The compound ossotide was provided by Hebei Zhitong biopharmaceutical Co., LTD and the product batch NO. is 0220904. The compound ossotide is prepared as below: healthy porcine bone was extracted with water for 1–2 cycles, and the solution was degreased at 75–85 oC to obtain the aqueous layer. Firstly, the pH value of this aqueous solution was adjusted to 3.5–4.5 and the precipitation is filtered off. Subsequently, the pH value of this supernatant was adjusted to 8.5–9.5 and the precipitation was filtered off again. The solution was ultra-filtrated to obtain the bone peptide with molecular weight ranging from 3 k to 10 k Da. The scorpion body was crushed and extracted with ethanol, and the extracting solution was concentrated, and the pH value was adjusted to 3.5–4.5. And the precipitation was filtered off to obtain the whole scorpion extracting solution. Both of the peptide stock solution and whole scorpion extracting solution were mixed to obtain the compound ossotide solution.
The compound ossotide was prepared to 1 mg/mL, and the quality was evaluated using high performance liquid chromatography (HPLC) at 220 nm detection wavelength.
Amino acid composition analysis
100 mg of compound ossotide lyophilized powder was dissolved in 10 mL HCl (6 M), and then hydrolyzed under vacuum at 110 oC for 22 h. After derivation by phenyl isothiocyanate (PITC), the composition and content of amino acids in the hydrolysis products were determined using HPLC at 254 nm detection wavelength.
Absolute Bioavailability Study
Because hydroxyproline is a unique amino acid in bone collagen, so it is used as an indicator to conduct the pharmacokinetics study of compound ossotide. After administration via oral administration and i.v. injection, the blood of SD rats was collected at different time points (0, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0 h) and the amount of hydroxyproline in the blood was measured using the hydroxyproline assay kit.
Biodistribution studies
The compound ossotide was labeled with Cy5-SE fluorophore, which reacts with the amino group to form a stable amide bond. The drug and Cy5-SE dye were mixed in a 1 : 1 ratio and centrifuged after magnetic stirring for 4 h. The fluorescently labeled compound ossotide were administered orally and intravenously, respectively, and the mice and organs were imaged using the IVIS imaging system to compare the distribution differences between oral administration and i.v. injection.
Cytotoxicity assay
The cytotoxicity of compound ossotide was analyzed using the CCK-8 kit. First, RAW264.7 cells (5×103) were separately inoculated into 96-well plates and incubated with different concentrations of compound ossotide for 48 h. Then 10 µL of CCK-8 reagent was added to each well for 4 h. The absorbance was measured at 450 nm using a microplate reader.
TRAP staining
RAW264.7 were seeded into 48-well plates at a density of 1.5×104 cells per well, After 24 h, the cells were treated with 50 ng/mL RANKL and different concentrations of compound ossotide (0.2, 0.5, 1.0, 2.0 mg/mL), and the medium was changed every other day. After that, the cells were stained with TRAP staining kit after paraformaldehyde fixation, and the number of mature osteoblasts containing three or more nuclei was counted using Image J software.
F-actin staining
RAW264.7 cells were seeded into 48-well plates as described above. After 5–7 days, the medium was removed and the cell were fixed with paraformaldehyde. The cells were permeabilized with Triton X-100 for 5 min. Finally, the configured FITC-labeled ghost pen cyclic peptide staining solution was added, incubated for 30 min away from light, and DAPI was added to re-stain the cells for 5 min. After completion, the cell morphology was observed under the microscopy.
Quantitative real-time polymerase chain reaction (qRT-PCR)
RAW264.7 cells were seeded into six-well plates with 2×105 per well as described above. Total RNA was extracted using Trizol reagent and then converted to cDNA using all-in-one 5x RT masterMix. mRNA expression of c-Fos, Acp5, and Dc-stamp was quantified using a real-time quantitative PCR system. It was first denatured at 95°C for 90 seconds and then amplified for 45 cycles at different temperatures (95°C, 15 s; 60°C, 60 s). All the primer sequences used in this work were described as follows: c-Fos, Forward 5’-CCAGTCAAGAGCATCAGCAA-3’, Reverse 5’-AAGTAGTGCAGCCCGGAGTA-3’; NFATc1, Forward 5’-GGTGCTGTCTGGCCATAACT-3’, Reverse 5’-GAAACGCTGGTACTGGCTTC-3’; Acp5, Forward 5’-ACGGCTACTTGCGGTTTCA-3’, Reverse 5’-TCCTTGGAGGCTGGTCTT-3’; Dc-stamp, Forward 5’-TCTGCTGTATCGGCTCATCTC-3’, Reverse 5’-ACTCCTTGGGTTCCTTGCTT-3’; GAPDH, Forward 5’-AACTTTGGCATTGTGGAAGG-3’, Reverse 5’-ACACATTGGGTAGGAACA-3’.
Western blot analysis
MC3T3-E1 cells were inoculated into 6-well plates (5×104) and cultured for 24 h. The cells were added with different concentrations of compound ossotide (0.1, 0.2, 0.5 mg/mL) for 72 h. RAW264.7 cells were cultured for 24 h, RANKL stimulation was added along with different concentrations of drugs (0.2, 0.5, 1.0, 2.0 mg/mL) for 72 h of treatment. After completion of treatment, RIPA cell lysis buffer was added to obtain total cell lysate, which was then centrifuged and the protein sample obtained from the supernatant was collected.
Electrophoresis was performed and then the separated proteins were transferred to PVDF membranes. After that, the membranes were closed with skim milk powder for 2 h. The sealing solution was discarded, washed with TBST and incubated for 14–16 h with the addition of primary antibodies at 4 oC. The protein bands were imaged using the chemiluminescence (ECL) detector (Tanon 5200 Multi, USA), and the grey value was analyzed by Image J software.
Proliferation and differentiation of osteoblasts
MC3T3-E1 cells were cultured with DMEM medium (10% FBS, 1% PS.). When the cells grew to 90%, they were digested with trypsin for 1 min, then centrifuged at 1500 rpm for 5 min, and inoculated into a 96-well plate (2×103). Different concentration of compound ossotide (0, 0.05, 0.2, 0.5, 1 mg/mL) were incubated, and the absorbance was measured at 450 nm using CCK-8 reagent at 48 h and 72 h after compound ossotide treatment.
The cultured cells were inoculated into 24-well plates at a density of 2 × 104, and the cell induction medium was changed every 3 d after cell attachment. Cells were then collected after 3 and 7 d and ALP activity was determined using the ALP kit. The absorbance at 520 nm was measured to quantify the enzyme activity.
Cell alizarin red staining
After 24 h of osteogenic differentiation, cells were treated with the compound ossotide at different concentrations for 21 d. The cells were fxed with 4% paraformaldehyde for 30 min after being rinsed with PBS. Cells were washed in PBS, stained for 40 min with 1% alizarin red, and then washed again to remove any remaining color. The nodules were pictured by digital camera and microscope.
Animal model construction and experiment design
Safety evaluation
Mice were randomly divided into four groups and the mice were orally administered different concentrations of the compound ossotide (250, 500, 1000, and 2000 mg/kg). The toxic symptoms and mortality rates in each group were recorded for 48 h after oral administration. The LD50 of the compound ossotide was determined using standard procedures.
The cumulative toxicity tests were performed, and the mice were dosed at 1000 mg/kg three times a day for 7 d, and the toxic symptoms and mortality rates in each group were recorded for 48 h after oral administration.
The chronic toxicity experiments were conducted, and the mice were dosed at 150 mg/kg once a day for 28 d. The body weight of the mice was recorded. The blood samples were collected at the 29th day to test the liver and kidney functions by the ALT/AST and CRE biomarkers, and the liver and kidney organs were harvested to evaluate the organ index and H&E staining.
The comparison of the treatment effect between oral administration and i.v. injection of compound ossotide
Mice were randomly divided into four groups: control group (saline administration), model group (oral retinoic acid 90 mg/kg), oral group and intravenous group (n = 10). After successful modeling, the control and model groups were injected with saline, the oral group was injected with 150 mg/kg of compound ossotide, and the intravenous group was injected with 90 mg/kg of compound ossotide for two consecutive weeks. Bone mineral density was measured by dual-energy bone density scanner.
The treatment effect of compound ossotide at different concentrations
Mice were divided into six groups: Control, Model group, Positive control, compound ossotide (50 mg/kg), compound ossotide (150 mg/kg), compound ossotide (250 mg/kg) (n = 10). The mice were fed with retinoic acid (90 mg/kg) for two weeks, except the control group. After successful modeling, the positive control group were treated with etidronate disodium (40 mg/kg). The compound ossotide were dosed with different concentration (50, 150, 250 mg/kg) with oral administration for 28 d. The bone mineral density was measured by dual-energy bone density scanner.
Serum ELISA assay
Serum concentrations of Ca2+, P, BALP, TRACP, OCN, and TGF-β were determined using an ELISA kits. Blood was collected from mice eyes at the end of treatment and used to determine changes in the concentration of relevant factors.
Statistical analysis
Each experiment was representative and repeated at least 3 times. Quantitative data were analyzed by one-way ANOVA or Student’ t-test, and expressed as the mean ± standard deviation (SD). Confidence levels for statistical significance were set at P < 0.05.