Experimental main material and reagents
BMSCs, MSC growth medium, and trypsin were purchased from Cyagen Biosciences Inc. (Guangdong, China). The n-HA/PLGA and β-TCP/PLGA scaffolds (4 mm × 15 mm, cylinder) were provided by The Shandong Province Key Laboratory of Medical Polymer Materials (Jinan, China).
Cell culture
BMSCs were obtained from Cyagen at passage 2. The BMSCs were thawed and cultured in a growth medium from Cyagen in a humidified atmosphere containing 5% CO2 at 37 °C. The growth medium contained 440 ml MSC basal medium, 50 ml 10% FBS, and penicillin/streptomycin mixture. The growth medium was changed every 2–3 days. The cells were passaged 3 times at approximately 80% confluence. Passage 5 BMSCs were digested and collected for determination and culture with the scaffold.
Cell seeding into the scaffold
The PLGA/n-HA and gelatin/n-HA were treated with 75% alcohol and then washed 3 times using phosphate-buffered saline (PBS). BMSCs at 5 passage were suspended in a growth medium at 2 × 107 cells/ml. Two hundred microliters of cell suspension was dropped on the top of the scaffold. The scaffold with cells was cultured in cutie for 2 h and then set in a 24-well plate with the growth medium. The medium was changed every 2–3 days.
Animal
Sixty New Zealand White rabbits (weighing 2.0–3.0 kg) were obtained from the animal experiment center of Anhui Medical University (Hefei, China). Before the experiments, approval was obtained from the Ethical Committee for animal experiments of Anhui Medical University. The rabbits were anesthetized by ear vein of 3% pentobarbital sodium (30 mg/kg). Disinfection was done with iodine and 75% alcohol. A 3–5-cm incision was created in the middle of the radius. The tissue overlying the radius was dissected. A 15-mm bony defect was made in the middle of the radius. The forelimbs with 15-mm bone defect were immersed in seawater for 3 h. The rabbits with seawater-immersed bone defect were divided into 4 groups (group A, group B, group C, and group D), with 15 rabbits in each group (n = 15). Group C was implanted with n-HA/PLGA/BMSCs, group D was implanted with β-TCP/PLGA/BMSCs, group B was implanted with autograft obtained from the iliac crest, and group A was implanted with anything. After implantation, the rabbits were treated with 2.4 ATA 100% HBO for 90 min/day for 2 weeks. The rabbits were injected intramuscularly with penicillin every day for 3 days.
Radiographic examination
Radiographs of rabbit radius were examined at 4, 8, and 12 weeks after surgery under anesthesia. Radiological evaluation was done using the Lane and Sandhu scoring system [13]. The radiographs were evaluated by orthopedists. And the evaluation was under a double-blinded study.
Histopathologic examination
After 4, 8, and 12 weeks, the rabbits were killed by euthanasia. Samples harvested from radius defect sites were fixed in 10% neutral buffered formalin for 48 h. The samples were decalcified with 10% EDTA solution for 30 days, dehydrated in graded ethanol, and embedded in paraffin. Five-micrometer sections were cut and stained with hematoxylin-eosin.
Immunohistochemistry stain of osteocalcin
Immunohistochemical examination of OCN was performed by the slide deparaffinized in xylene I–II respectively for 15 min and dehydrated in grade alcohol from 90 to 70% for 3 min. After that, blocking was done with 0.5% H2O2 in methanol for 30 min and washed with water for 5 min. Pretreatment of the slide was performed with citrate buffer in microwave cook I and cook II for each 5 min followed by blocking background target to block non-specific antigens and then incubation for 15 min. Then, it was given primary antibody to OCN and incubated for 1 h. The slide was given a universal ink secondary antibody to bind to the primary antibody for 15 min. Counterstaining was performed with hematoxylin for 1–2 min.
Bone mechanical strength test
Twelve weeks after surgery, the mechanical strength of the radius was tested by the three-point bending test. The test was performed using a universal tensile testing machine (Instron, London, UK). The ultimate force in the bending test is until the specimen was broken. The bones were placed horizontally on two rounded supporting bars located at a distance of 30 mm, and the bending load was applied at the midpoint of the defect at the loading speed of 10 mm/min until the specimen fracture. The biomechanical properties of the specimens were determined by ultimate loading (N). The data were recorded as the mean plus standard error of the mean.
Statistical analysis
All data are presented as mean ± SD. Comparisons between groups were done by one-way analysis of variance (ANOVA). The SPSS 19.0 was used for statistical analysis. The differences were considered as statistical significance at the level of P < 0.05.
