Animals
All experiments involving animals were carried out in accordance with the Guidelines of the Animal Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology Institutional Review Board (IRB ID: 20171019). The study complied with the ARRIVE guidelines for preclinical animal studies [19]. Twenty-four healthy New Zealand white rabbits of 3 months old in healthy condition, each weighing 2.5-3 kg, male, were obtained from the animal care center of Tongji Medical College. All animals were kept in separate cages under identical standard conditions. General anesthesia of ketamine (10 mg/kg) and xylazine (3 mg/kg) was taken before operations. Adequate measures were implemented to minimize the pain or discomfort of animals during all surgical procedures [20].
ASCs isolation and the multi-lineage differentiation inducement
Subcutaneous adipose tissue was harvested from the inguinal fat pad of all rabbits. After thoroughly washing with PBS, the well-minced adipose tissue was digested by 0.1% collagenase at 37 ˚C for one hour (type II-S; Sigma–Aldrich, St. Louis, MO, USA). Digestion activity was terminated by supplemented Dulbecco’s modified Eagle’s medium (DMEM) (10% fetal bovine serum, 100U/mL penicillin, and 100mg/mL streptomycin) (Gibco Biocult Co., Paisley, Strathclyde, U.K.). The obtained chyle-like tissue was centrifuged at 1000 rpm for 10 min to discard the supernatant tissue. The residual was filtered by 200µm pores cell strainer before another 10 min centrifugation. Finally, the cell suspension was cultured in supplemented DMEM at 37 ℃ in a humidified 5% CO2 incubator. Non-adherent cells were removed three days later, and the adherent cells were propagated twice weekly [21]. Cells in passages 2–5 were used [22].
Osteogenic, chondrogenic, and adipogenic lineage differentiation tests were conducted to identify the multi-differentiation potentials of ASCs. One group of cells cultured in osteogenic medium (DMEM, 10% FBS, 1% penicillin/streptomycin, 50 µg/mL L-ascorbic acid, 10 mM glycerophosphate and 100 nM dexamethasone) (Sigma Aldrich) for four weeks. Alizarin red S staining (Sigma Aldrich) was conducted for calcified nodules detection. One group was induced in adipogenic medium (0.5 mM isobutylmethylxanthine, 0.5 mM hydrocortisone and 60 mM indomethacin) for 21 days according to the company description (Cyagen Biosciences Inc., Guangzhou, Guangdong, China) before oil red O staining [23]. One group induced in a chondrogenic medium (Cyagen) containing 10% FBS, 1% penicillin/streptomycin, 1% ITS, 0.1 mM L-ascorbate-2-phosphate, 0.4 mM proline, 100 nM dexamethasone and 10 ng/ml transforming growth factor-β3 (TGF-β3) for four weeks. Chondrogenic differentiation was detected by Alcian blue staining [24]. The cell-surface antigens CD34, CD44, CD45, CD105, and CD11b were assayed by flow cytometry, as described previously [25].
Ad-BMP-2 gene transduction
Based on our previous study, the recombinant replication-defective adenovirus with BMP-2 transfected at a multiplicity of infection (MOI) of 50 plaque-forming units (pfu)/cell. The recombinant replication-defective adenovirus with an enhanced green fluorescent protein (EGFP) was transfected under the identical method as the control [22].
In vitro expression of BMP-2
In vitro secretion of BMP-2 by BMP-2 gene-enhanced ASCs was assessed by the enzyme-linked immunosorbent assay (ELISA). BMP-2/ASCs were plated in a 24-well plate at a density of 5×104 cells/well before BMP-2 transduction. The transduction was performed according to our previous protocols [22]. ASCs prepared at the same time for the control. The culture medium was collected at 1, 4, 7, 10, 14, 21, and 28 days after transduction. The collected media froze at -80 ℃ before the final analysis. A commercial ELISA kit (Quantikine BMP-2 microplate, R&D systems, Minneapolis, MA, USA) coated with the mouse monoclonal antibody against BMP-2 was adopted for BMP-2 concentration measurement. All experiments were performed in triplicate [26, 27].
Osteogenic Differentiation
Immunofluorescence staining of OPN was applied 14 days after transfection. BMP-2 overexpressing ASCs were seeded on sterile glass coverslips loaded on the bottom of a 6-well plate at a density of 1 × 102 cells/well. After osteogenic medium culture for 14 days, the cells were washed twice with cold PBS and fixed in 4% paraformaldehyde for 15 min at 4 ˚C. Cells were then treated with 0.3% Triton-X100 for 30 min and blocked in 3% BSA at room temperature for 30 min. Specific primary antibodies targeting rabbit OPN (Abcam, Cambridge, MA, USA) were added to the fixed cells at a dilution of 1:100 and incubated at 4 ˚C overnight. A fluorescent labeling phycoerythrin (PE) goat anti-mouse secondary antibody (Abcam, USA) was incubated with the cells at a dilution of 1:500 in blocking buffer at 37 ˚C in the dark for 1 h. Nuclei were stained with DAPI in the dark for another 5 min. The specimens were examined under a confocal laser scanning microscope (CLSM; Leica TCS Sp2 AOBS, Germany).
Tissue-engineered bone constructs preparation (TEBCs)
Untreated and transfected ASCs were digested by trypsin/EDTA (Sigma–Aldrich) three days after gene transduction. The cell suspensions were concentrated to a density of 2×107 cells/ml in DMEM. The proper volume of the cell suspension was slowly pipetted onto 150 mg β-TCP without spilling. The complexes were then incubated for an additional 4 h for cell attachment. Four groups of tissue-engineered bone grafts were prepared as: A: β-TCP (n = 12), B: ASCs/β-TCP (n = 12), C: EGFP/ASCs/β-TCP (n = 12), and D: BMP-2/ASCs/β-TCP (n = 12).
Implant surgery
All rabbits were anesthetized with 0.5 mg/kg sodium pentobarbital intravenously. Then 0.5 ml of 1% lidocaine with epinephrine (1:100,000) was injected subcutaneously for local anesthesia. After anesthetization, a 6 cm long incision was made along the distal end of the femur with a NO. 15 blade. The skin, the subcutaneous tissue, and the muscle were drawn back to expose the bone surface. An implant bed was prepared stepwise through the double cortex and perpendicular to the femur shaft without entering the distal femoral condyle (Fig. 1). After the last drill, a slow-speed burr was used to enlarge the double cortical defects to the diameter of 8 mm [28]. Once made the implant bed, the prepared TEBC was stuffed into the space. The implant (10 mm height, 3.8 mm diameter of the implant body, China Dental Implantology Center, Sichuan, China) was inserted into the TEBC without touching the rest cortical bone border. The prepared TEBC was pushed into the space between the implant surface and the residual bone wall to keep the implant’s stability within the oversized site. The TEBCs were placed in a randomized order. The surgeons were blinded regarding TEBC type. The implant insertion depth was controlled, which sets the implant neck well below the upper edge of the cortical bone. The implant insertion torque was measured at the time of placement employing the Surgic XT Plus™ (NSK, Kanuma, Japan) device [3]. The incision was closed in layers, leaving the implants submerged.
Trichromatic fluorescent labeling
The trichromatic sequential fluorescent labeling was implemented to reflect the active process of new bone formation [29]. Two, four, and eight weeks after the operation, the animals were intraperitoneally administered 25 mg/kg hydrochloride tetracycline (TE), 30 mg/kg alizarin red s (AL), and 20 mg/kg calcein (CA, Sigma), respectively.
Animal Sacrifice and perfusion
Animals were sacrificed at eight weeks post-operation (3 days after the injection of CA) and perfused with 10% buffered formalin. The femora were cut into single blocks with one implant before storage in a 4% neutral formaldehyde solution at 4 ℃ [20] [30, 31].
Histological and histomorphological analysis
Calibration between examiners (LX, XQ) was performed prior to the histological analysis. The examiners were blinded regarding TEBC type and healing time. Half samples were gradually dehydrated and embedded in methyl methacrylate-based resin (Technovit 7200 VLC, Kulzer, Friedrichsdorf, Germany). The implant blocks were cut along the mesiodistal direction (ExaktA, Parenteau, Norderstedt, Germany). Three central sections were prepared of one implant and subsequently polished to 200 µm for trichrome fluorescent labeling observation. Five sites adjacent to the implant surface were chosen.
The areas of single and total trichrome fluorescent labeling were evaluated. Trichrome fluorescent labeling was observed under CLSM on all sections. Four sites adjacent to the implant surface were chosen. The excitation/emission wavelengths were 405/580 nm (TE), 543/617 nm (AL), and 488/517 nm (CA). The bone formation indices were evaluated on a picture-analysis system (Image Pro 5.0, Media Cybernetic, MD, USA), and the areas of trichrome fluorescent labeling (TFL%) were measured by calculating the mean value of the images taken around the implant surface on the sections.
These sections were then polished to 40 µm. Van Gieson's picro-fuchsin (VG) staining was performed on these finely polished sections. Histomorphometric measurements were performed blinded and carried out using Image Pro 5.0. The following parameters were measured: new bone area (NB, %): the percentage of new bone area to the region of interest (ROI) on the central section crossing the mesiodistal direction of the implant (Fig. 5b). The ROI area was the one cm width zone extending from the implant surface to concentration on the new bone deposition on the implant. The area encompassed the peri-implant tissue in both the cortical and medullar regions at the distal side of the implant. Residual material (RM, %): the percentage of residual material area to the ROI (Fig. 6b). Bone contact with implant (BIC, %): the portion of new bone contact to the implant surface: summation of the lengths of contact between the implant and the host bone/implant length corresponding to the width between the double cortical bone. Sections were imaged with an Olympus BX-51 microscope, and cross-sections were analyzed using the Image J software package.
Removal torque tests
The removal torque test was applied to assess the SS of the integrated implant in the bone [32]. The removal torque value (RTV, N/cm) reflects the interfacial shear strength. Static torque was applied to the implant by a machine-run gradual increase at a linear rate of 9.5 Ncm/s. A rotational unscrewing force was applied, and the strength was determined as the peak force applied to loosen the implant from the bone as measured with a digital torque meter (MGT20Z, Mark-10 Corp., New York, NY).
Statistical analysis
The data are presented as mean ± standard deviation (SD) from at least three independent experiments. Sample size was calculated using PASS 11 software with 5% significance (α = 0.05) and 80% power (β = 0.2). Statistically significant differences (p < 0.05) between the different groups were measured using a one-way analysis of variance with Tukey post hoc analysis when indicated. All statistical analysis was completed by a SAS 6.12 statistical software package (SAS, Cary, NC, USA).