1.1 Main experimental materials, reagents and instruments: DMEM/F12 medium, fetal calf serum, 500U/ml penicillin, 500μg/ml streptomycin (Gibco Life Technologies, USA), CCK-8 cell proliferation and toxicity assay kit (KeyGEN BioTECH, Nanjing, China). TRIzol@Reagent (Invitrogen, USA). Porous Ta-Nb material (Powder Metallurgy Research Institute of Central South University) was disc-shaped, with the diameter of 11 mm and the thickness of 4 mm (see Figure 1). This material was featured by a 3-D porous structure, with the porosity of 60% - 65% [2]. The uniform porosity and the interconnections between pores were confirmed under SEM (MIRA3 TESCAN, Research Institute of Tsinghua University in Shenzhen). The surface of the material was rough and uneven after local magnification (see Figure 2). The porous Ta-Nb material was soaked and rinsed with distilled water, sterilized with autoclave and dried prior to use.
1.2 Separation and culture of rabbit osteoblasts (OB): Three fetal rabbits younger than 10-day old (Animal Experimental Center of Guangzhou Medical University) were killed by intravenous injection of excess anaesthetics (3% pentobarbital sodium) from the edge of the ear. After cleaning, the rabbits were soaked in 75% ethanol for 5 min for sterilization. The sterilized rabbit was placed on the clean bench, and the residual ethanol was removed. Under aseptic condition, the skin was sliced and the skull (parietal bone and frontal bone) was harvested with scissors. The skull was rinsed with PBS containing 500 U/ml penicillin and 500 μg/ml streptomycin for several times. The adhesive connective tissues and blood vessels were removed with another set of scissors and pincers and then sliced into small pieces of about 1 mm3. These small pieces were digested with 0.25% trypsin at 37°C for 20 min, and the digestate was discarded to eliminate fibroblasts. Then the digestion solution containing 0.2% collagenase type I and 0.1% hyaluronidase was added for digestion for 5 cycles on a shaker at 37°C for 20 min. The cell suspensions at the 3rd, 4th and 5th cycles were centrifuged at 1000 rbp for 10 min, with the supernatant discarded. The precipitated cells were rinsed with serum-free F-12 medium, centrifuged and resuspended in F-12 medium containing 10% fetal calf serum. After gentle blowing, the cells were inoculated into the culture bottle with the density of 1×105/ml and then cultured in an incubator containing 5% CO2 at 37°C. The culture medium was refreshed every 3d. The cells were digested with trypsin when the cells grew to 70% - 80% confluence, and cell passage was performed with the ratio of 1:2-1:3. The third-generation osteoblasts were co-cultured with the porous Ta-Nb material.
1.3 Co-culture of osteoblasts and porous Ta-Nb material Osteoblasts were inoculated to the porous Ta-Nb material placed in the F-12 medium containing 10% fetal calf serum under aseptic condition. The experiment included two groups: experimental group (Group A), with the co-culture of osteoblasts and porous Ta-Nb material, and control group (Group B), with single culture of osteoblasts. The culture was performed using 24-well plates in an incubator containing 5% CO2 at 37°C, with the inoculum density of 5×105/well. The medium was refreshed every 3d.
1.4 Detection indicators
1.4.1 Detection of cell proliferation by CCK-8 assay Five replicates were set up for group A and B, respectively. Each well was added with 50µL of CCK-8 solution on day 1, 3, 5, 7 and 9. After incubation at 37°C for 4h, the liquid in each well was transferred into 96-well plate, with equal liquid volume in each well. The absorbance (A) was detected by ELIASA at 490 nm and the cell proliferation curve was plotted.
1.4.2 SEM observation The porous Ta-Nb material was taken out after being co-cultured for 1, 2 and 3 weeks, respectively. The porous Ta-Nb material was rinsed with PBS for 2 times, fixed with 2.5% glutaraldehyde for 2h, and dehydrated with graded series of ethanol. Then it was dried at the critical point in liquid CO2. After metal spraying, the cell adhesion was observed under SEM.
1.4.3 RT-PCR assay The assay was conducted by referring to the method of Saqomonyants et al. [3]. After being co-cultured for 1, 2 and 3 weeks, 1×106 osteoblasts of group A were collected, respectively. When the primary rabbit osteoblasts (P0) grew to 90% confluence, the total RNA was extracted as blank control. The cells collected at different time points were treated with 1 mL of Trizol reagent (Invitrogen) for total RNA extraction. The RNA was quantified with ultraviolet spectrophotometer (WPA UV1101) at A260/280>1.8. Two-step RT-PCR method was employed. The first-strand cDNA synthesis was performed according to the kit instruction (Fermentas). 25µL reaction system: 10x Taq buffer 2.5 μL, 25 mmol/L MgCl2 3.0 μL, 10 mmol/L dNTP 2.0 μL, Taq DNA Polymerase (1 U/μL) 1.0 μL, ddH2O 18.5 μL, template cDNA 1.0μL; forward primer and reverse primer (20 μmol/L), 1.0 μL each. PCR amplification program: 95°C predegeneration for 3 min, 1 cycle; annealing for 45 s (the annealing temperature was as follows), 72°C extension for 1 min, 30 cycles; final extension at 72°C for 7 min. Type-I collagen (Col-I): upstream primer 5'-CCCAACCAAGGATGCACTAT-3', downstream primer 5'-TGTTCTGAGAGGCGTGATTG-3', annealing temperature 52°C, amplified fragment 259 bp. Osteocalcin (OCN): upstream primer 5'-CATGAGAGCCCTCACA-3', downstream primer 5'-AGAGCGACACCCTAGAC-3', annealing temperature 59°C, amplified fragment 310 bp. Reference primer GAPDH: upstream primer 5'-ACGGATTTGGTCGTATTGGG-3', downstream primer 5'-CGCTCCTGGAAGATGGTGAT-3', annealing temperature 56°C, amplified fragment 239 bp. 4 μL of type-I collagen, osteocalcin and 8 μL of GAPDH amplification product were stained with ethidium bromide and treated with 2% agar gel electrophoresis, respectively. The gray value of different bands was determined with Quantity One, and semi-quantitative comparison was performed.
1.5 Statistical analysis Measurement data were expressed as x±s. One-way ANOVA was performed on the data of each group using SPSS 13.0, and SNK-q test was performed for intergroup comparison.