2.1. Establishment of the T2DM rat model
All animal care and experimental protocols were strictly carried out according to international standards of animal welfare and in accordance with the Animal Ethics Committee of Shandong University (Jinan, China). Thirty male Sprague-Dawley rats (obtained from the Experimental Animal Center of Pengyue, Jinan, China) aged 10-11 weeks old with 200±20 g body weight were chosen for this study. They were offered standard food and water ad libitum, and were housed under a stabilized environment at a temperature of 25°C, humidity of 55%, and with a 12:12-h light/dark cycle. After 7 days of acclimation to laboratory conditions, all rats were randomly divided into the control group (n = 6) or the type 2 diabetic groups (n = 24). Type 2 diabetic rats were induced by a four-week high-fat and high-carbohydrate diet (53% carbohydrate, 31% total kcal of fat, and 16% protein; Beijing Ke’ao Xieli Feed Co. Ltd., China), followed by one intraperitoneal (IP) injection of streptozotocin (STZ, Sigma, USA) dissolved in 0.1 M citrate buffered saline (pH 4.2) at a dose of 30 mg/kg 36. The control rats received regular food and citrate buffer injection. Fasting blood glucose above 11.1 mmol/L steady for one week indicated successful establishment of the T2DM model.
2.2. Implantation procedure
The diabetic rats were randomly assigned into the following groups (with 6 rats per group): T2DM group, insulin-treated T2DM group (T2DM-Insulin), genipin-treated T2DM group (T2DM-Genipin), genipin and insulin combination-treated T2DM group (T2DM-Genipin + Insulin), and normal rats were defined as the control group (n = 6 for every group). The chemical structure of genipin is displayed in Figure 1A 37. Animals were anesthetized by intraperitoneal injections of pentobarbital sodium (40 mg/kg body weight, Sigma, USA). Afterwards, every rat received two devised sand-blasted and acid-etched (SLA) titanium implants (1 mm in diameter and 10 mm in length) in the distal femurs as previously described 38. By using a scalpel, a 1-cm-long incision was made along the medial side of the knee joint, and the extensor mechanism with the knee joint was laterally dislocated to expose the distal femoral metaphysis. With the knee in flexion, an implant bed was prepared from the intercondylar notch of the femur to access the medullary cavity with a rotary drill. Then, the implant was introduced into the femoral medullary canal until the implant end was below the articular surface (Fig. 1B). The extensor mechanism was relocated, and the surgical incisions were closed in layers. All the rats received intramuscular injection of antibiotic immediately after surgery and for three postoperative days.
2.3. Treatment regimens
Three days after implantation, neutral protamine Hagedorn insulin (10 IU/kg, Novolin, Denmark) was administered by subcutaneous injection every day for the T2DM-Insulin group during the entire experiment 39. The T2DM-Genipin rats were treated for 12 weeks with genipin (Nanjing Dilger Medical Technology Co. Ltd., China, 98% purity), which was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline solution to a final concentration of 1% DMSO, at a dose of 50 mg/kg/d by oral gavage. The dosage and timing of genipin administration were determined based on previous studies 40, 41. Rats in the T2DM-Genipin + Insulin group were treated with genipin and insulin daily. In the control group and T2DM group, DMSO and saline solution were administered using the same volume and route as those of the genipin treatment group. Body weight and fasting blood glucose determined from caudal vein blood in different groups were regularly monitored. Three months after surgery, all rats were euthanized, and the double-sided femora with implants were further evaluated.
2.4. Microscopic computerized tomography (micro-CT) reconstruction
After soft tissue was removed, the non-demineralized specimens were scanned on a micro-CT system (60 kV/5 W, Quantum GX2, PerkinElmer, Japan), and three-dimensional (3-D) images were reconstructed from the microtomographic slices. The region of interest (ROI) was defined as an annular domain of the 200-μm region around the implant. The parameters of bone volume per total volume (BV/TV), the mean trabecular number (Tb.N), and percentage of osseointegration (OI%) which was represented as the percentage of bone voxels to total voxels in direct contact with the implant, were analyzed within the ROI zone in order to assess osteogenesis associated with the implants.
2.5. Pull-out test
The pull-out test 42 can evaluate the biomechanical properties of the implant-bone interfaces. After specimens were collected, the test was performed using a universal mechanical testing machine (Shimadzu, Japan) at a loading rate of 1 mm/min until the implant-bone interface ruptured. The maximum retention force gained by the implant in the bone, which is referred to as the maximal pulling force, was recorded.
2.6. Histology and histomorphometric analysis
2.6.1. Hard tissue slicing
Specimens including implants were dehydrated through a series of graded ethanol solutions, embedded in methylmethacrylate without decalcification, and sawn using a rotary diamond saw (SP1600, Leica, Germany). Thereby, we obtained undecalcified 50-μm-thick sections, which were stained in methylene blue-acid fuchsin. Bone-to-implant contact (BIC, the length percentage of the direct interface of bone and implant to the total implant’s surface in the cancellous bone) was calculated using Image-Pro Plus 6.0 software 43.
2.6.2. Preparation and staining of decalcified sections
We obtained the specimens whose soft tissue was removed after the rats were euthanized. After fixation in 4% polyformaldehyde for 48 h and washing under flowing water, the femora were decalcified in 10% EDTA solution for 6–8 weeks until a needle was able to pierce the femur without resistance. Then, without damaging the interface tissue, we carefully removed the implants along the direction of the major axis, after which the femurs were dehydrated in gradient ethanol and embedded in paraffin. A slicer was used to produce 5-μm sections around the implants, in order to evaluate the formation and maturity of new bone by separately staining with hematoxylin-eosin (HE) and modified Masson.
2.6.3. Immunohistochemistry (IHC) examinations
For prepared decalcified sections, IHC staining was processed with primary antibodies against phospho-AMPK (CST, 2535T, 1:100 dilution) and 8-OHdG (8-hydroxy-20-deoxyguanosine, a marker of DNA damage in oxidative stress, Santa Cruz Biotechnology, sc-393871, 1:200 dilution) as previously reported 32, 44. For quantitative valuation, three slices from each group were observed using an optical microscope and analyzed by Image-Pro Plus 6.0. The mean integrated optical density (IOD) for pAMPK and 8-OHdG were also calculated around the implants.
2.7. Statistical analysis
The statistical analyses are presented as the mean ± standard deviation (SD) of at least three independent measurements. One-way ANOVA was used to analyze group-to-group statistical differences. The significance level was set at p < 0.05. All calculations were performed using SPSS software (SPSS 23.0).