Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories, Inc (cat. no. 8000). HUVECs were grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and cultured in a cell incubator at 95% humidity at 37˚C with 5% carbon dioxide. Ethics approval for the use of HUVECs was not required according to our institute's guidelines (http://www.gdmuah.com/info/1851/8954.htm).

CCL2 (cat. no. M1203-03S) was purchased from United States Biological. Antibodies against rho-associated coiled-coil-containing protein kinase (Rock)1 (cat. no. #4035), N-cadherin (cat. no. #13116), c-Myc (cat. no. #18583), phosphorylated (p-)ERK1/2 (cat. no. #4370), p-AKT (cat. no. #13038), AKT (cat. no. #4691), Wnt5a/b (cat. no. #2530), low-density lipoprotein receptor related protein 6 (LRP-6; cat. no. #3395), β-catenin (cat. no. #8480), p-PI3K (cat. no. #17366), PI3K (cat. no. #4257), GAPDH (cat. no. #5174) and α-tubulin (cat. no. #2125) and the secondary antibodies goat anti-rabbit IgG (cat. no. #7074) and anti-mouse IgG (cat. no. #7076) were purchased from Cell Signaling Technology, Inc. VEGFR2 (cat. no. ab134191) and MMP9 (cat. no. ab228402) antibodies were obtained from Abcam. Antibodies against Rock2 (cat. no. sc-398519), ERK1/2 (cat. no. sc-514302) and VEGF (cat. no. sc-57496) were obtained from Santa Cruz Biotechnology, Inc.

HUVECs (2x10³ cells per well) were seeded into 96-well plates in ~100 µl total medium, incubated at 37˚C for 24 h, and then treated with various concentrations (0, 25, 50, 75, 100 and 150 ng/ml) of CCL2 at 37˚C for 0-96 h. Following addition of 10 µl Cell Counting Kit-8 (Beyotime Institute of Biotechnology) solution per well and incubation at 37˚C for 2 h, the OD value was determined by a microplate reader (MK3; Thermo Fisher Scientific, Inc.) at OD 450 nm.

The fresh serum-free DMEM (100 µl) containing 2x10⁴ HUVECs and various concentrations (0, 50, and 100 ng/ml) of CCL2 was added to the upper chamber. The lower chamber was filled with 500 µl of medium containing 10% FBS and cells were incubated at 37˚C for 24 h. Subsequently, cells that remained in the upper chamber without successfully migrating were wiped away. Cells on the membrane were fixed in methanol at room temperature for 15 min and stained with crystal violet staining solution at 37˚C for 15 min. The cells were imaged in six random microscopic fields (magnification, x100) under a light microscope (Olympus Corporation). Migrated cells (% of control)=(migrated cells treated with 50 or 100 ng/ml CCL2﹣migrated cells treated with 0 ng/ml CCL2)/migrated cells treated with 0 ng/ml CCL2 x100.

HUVECs were cultured for 24 h in 6-well plates with lines at the bottom of the plate. When the cell fusion rate approached 100%, a 100-µl pipette tip was used to draw a line perpendicular to the mark line in the culture plate, and PBS was used to clean the culture well twice. Cells that had come loose were removed, 2 ml serum-free medium with various concentrations of CCL2 (0, 50 and 100 ng/ml) was added and cells were placed back in the incubator for further culture. Subsequently, the migration distance of HUVECs imaged under a light microscope (Olympus Corporation) at 0 and 24 h was compared, the migration area was calculated and the effect of different treatments on cell migration was analyzed. Migration area (%)=(migration area at 0 h﹣migration area at 24 h)/migration area at 0 h x100.

HUVECs (1.5x10⁴) were cultured with different doses of CCL2 (0, 25, 50 and 100 ng/ml) in DMEM in Matrigel-precoated (37˚C; 30 min) angiogenesis slides at 37˚C with 5% CO₂. After 4 h of incubation, tube formation was observed under a light microscope (Olympus Corporation) and images were captured for five randomly selected microscopic fields and tube formation was analyzed using ImageJ software (v1.8.0; National Institutes of Health).

Total protein was collected with an appropriate amount of protein RIPA lysis buffer (PMSF:RIPA=1:100; Beyotime Institute of Biotechnology). BCA protein assay kit (Beyotime Institute of Biotechnology) was used to measure protein concentration and each sample adjusted to the same concentration. Proteins (20 µg/lane) were separated by SDS-PAGE on 10 and 12% separation gels. After gel electrophoresis (100 V; 2 h), membrane transfer (250 V; 2.5 h) and blocking (5% skimmed milk; room temperature; 2 h), the 0.2 µm pore size PVDF membranes (Merck KGaA, Darmstadt, Germany) were incubated with primary antibodies (diluted 1:1,000) overnight at 4˚C. Subsequently, the membranes were washed with TBS with 0.1% Tween-20 (three times; 10 min) before addition of the secondary antibody (diluted 1:3,000) at room temperature for 1 h. Finally, the immunoreactive membranes were imaged using a Chemiluminescent and Fluorescent Imaging System (Tanon 5200; Tanon Science and Technology Co., Ltd.), and the gray levels of the protein bands were determined using ImageJ software (v1.8.0, National Institutes of Health).

All animal experiments were approved by the Laboratory Animal Ethics Committee of Guangdong Medical University (Zhanjiang, China; ID Number: GDY1902126; date: 25.05.2019). The SD rats (Guangdong Medical Laboratory Animal Center) were maintained at 2 or 3 rats/cage under a 12-h light/dark cycle with adequate water and standard food, at temperatures of 22-24˚C and relative humidity of ~45%. Depending on the types of intervention, 12-week-old male SD rats (n=32) weighing 350-400 g were selected and randomly divided into four groups: Group A (simple bone defect not filled in), Group B [poly (lactic-co-glycolic acid)/tricalcium phosphate (PLGA/TCP) porous scaffold; 5x5x5 mm³ filled in], Group C (PLGA/TCP porous scaffold + Gelma hydrogel filled in) and Group D (PLGA/TCP porous scaffold + Gelma hydrogel + 1 µg CCL2 filled in). The number of rats in each group was eight. The rats were anesthetized by intraperitoneal injection with pentobarbital sodium at a concentration of 1% at a dose of 30 mg/kg. The appropriate degree of anesthesia was assessed by the characteristics of stable breathing, sluggish corneal reflex, generalized muscle relaxation and loss of skin pinch response. After group assignment, the left femur of each rat was subjected to 3 mm midline osteotomy under anesthesia, and the bone defect area was filled or not filled according to the experimental group allocation. Furthermore, the left femur was fixed using an external fixator (Fig. S1). The bone defects establishment of each SD rat lasted ~40-60 min. The specific criteria for SD rat sacrifice were that rats were close to succumbing, with persistent dyspnea, inability to ingest food, significant loss of appetite, weight loss of >20% (the maximum percentage of body weight loss observed in the present study was ~10%), severe ulceration, heavy bleeding and limb paralysis. Penicillin sodium (80,000 units/kg/d;) was intramuscularly injected after surgery to prevent infection for 1 week and the surgical incision was disinfected with iodophor every day. The rats were monitored daily for surgical incision, mental status, diet, body weight, activity and excretion. Subsequently, each group was observed for 4 weeks, at which point half of the specimens were collected, and 8 weeks, at which point the other half of the specimens were collected. The SD rats were sacrificed with 10% sodium pentobarbital at a dose of 200 mg/kg by intraperitoneal injection. Mortality was verified by confirming the arrest of breathing, the disappearance of heartbeat and light reflex, and the dilation of pupils in SD rats. Finally, the femurs of the left leg were removed and fixed in 10% neutral formalin solution for 48 h and then stored in 75% ethanol.

After 10% formalin fixation at 4˚C for 48 h and EDTA solution (Beijing Solarbio Science & Technology Co., Ltd.) decalcification (2 months), bone tissue was dehydrated with gradient ethanol and xylene (75% ethanol for 1 h; 85% ethanol for 1 h; 95% ethanol for 1 h; 100% ethanol for 1 h; xylene I for 30 min; xylene II for 1 h) at room temperature. Subsequently, the bone tissue was embedded in paraffin (65˚C). After tissue sectioning (4 µm), bone callus sections were hydrated, blocked with 5% BSA (Beyotime Institute of Biotechnology) for 1 h, and incubated with the VEGF primary antibody (diluted 1:200) at 4˚C overnight. After subsequent secondary antibody (diluted 1:200) incubation (room temperature; 30 min), 3,3'-diaminobenzidine dyeing solution (5 min) and hematoxylin (2 sec) were added for staining at room temperature. The images were captured under a light microscope (Olympus Corporation).

All experimental results in the present study were obtained from experiments repeated in at least three replicates. All data are expressed as the mean ± standard deviation. ImageJ (National Institutes of Health), SPSS Statistics 19.0 software (IBM Corp.) and GraphPad Prism 8.0 software (GraphPad Software, Inc.) were used to analyze the data and create the graphs, respectively. Unpaired Student's t-test was used for comparisons between groups. Statistical differences between ≥3 groups were determined using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Cell proliferation assays revealed that CCL2 increased HUVEC proliferation in a concentration- and time-dependent manner (Fig. 1A).

The migration of HUVECs was measured using Transwell migration assay and wound healing assay. After treatment with different doses of CCL2 (0, 50 and 100 ng/ml) for 24 h, the cells that pierced the polycarbonate membrane and the coalescing wound areas were observed under a microscope at a magnification of x100. The results of the cell migration assay revealed that the numbers of migrated cells (% of control) of HUVECs were 100, 223±7 and 304±6% respectively (Fig. 1B). As shown in Fig. 1C, the migration area of HUVECs was compared among different treatment conditions. In addition, the results demonstrated that the migration areas of HUVECs were 30±5, 62±3, and 76±2%, respectively. The experiments revealed that CCL2 boosted the migration of HUVECs.

One of the most essential steps in the processes of angiogenesis is endothelial cell proliferation, and the regeneration of new blood vessels is required for bone tissue growth. Vessel formation serves a key role in tissue rebuilding (11). HUVECs exposed to different concentrations of CCL2 were cultured in Matrigel, and then the construction of capillary-like structures was imaged by microscopy at a magnification of x100. Tube formation was assessed based on the number of branch points and capillary length indexes. Following treatment with different doses of CCL2 (0, 25, 50 and 100 ng/ml) for 4 h, the branch points of tube formation were 23±3, 38±2, 47±2 and 57±2. In addition, the capillary lengths (100% of control) of tube formation were 100, 132±9, 153±6, and 162±9% respectively (Fig. 1D). The results revealed that CCL2 at a suitable concentration had a positive effect on tube formation.

A number of studies have reported that Rock1, Rock2, N-cadherin and c-Myc are closely associated with proliferation and migration, and VEGFR2 is associated with vascularization (12-15). According to the results of western blotting, HUVECs treated with CCL2 exhibited marked expression of these markers (Fig. 2A). Therefore, we hypothesized that CCL2 serves a significant role in the proliferation, migration and vascularization of HUVECs.

The PI3K/AKT, ERK1/2 and β-catenin signaling pathways are related to cell proliferation and migration (16-18). To gain further insights into the molecular mechanism whereby CCL2 alters HUVEC behavior, the present study investigated the effect of CCL2 on the PI3K/AKT, ERK1/2 and β-catenin signaling pathways. Western blotting revealed that CCL2 treatment of HUVECs increased the levels of p-AKT, p-PI3K, p-ERK1/2, MMP9, Wnt5a/b, β-catenin and LRP-6 (Fig. 2B-D). Therefore, it was hypothesized that CCL2 may exert a positive regulatory effect on proliferation and migration in HUVECs by inducing these signaling pathways.

Femoral samples collected at 4 and 8 weeks after the bone defect operation underwent EDTA decalcification and immunohistochemical staining. The results revealed that there was an increase in the expression area of VEGF in CCL2-treated groups (Fig. 3).