46 New Zealand white rabbits (NZWRs), including of 36 adults (2months, about 2.0 kg) and 10 neonatals (2 weeks, about 0.40 kg), were invovled in this study. All the experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Lanzhou University, China.
Cell culture and osteogenic induction
Cell culture and osteogenic induction were performed as previous studies [17, 18]. Briefly, bone marrow (5 mL) was aspirated from the ventral ilium of neonatal NZWRs (2 weeks, about 0.40 kg). Marrow mesenchymal stem cells (MSCs) isolated from bone marrow were collected and then planted in a plastic culture flask with Dulbecco’s modified Eagle medium (DMEM, Invitrogen, USA) containing 10% fetal bovine serum (FBS, Sijiqing, China) incubating at 37 0C with 5% CO2. The primary passage MSCs were observed under microscopy and further confirmed with Giemsa staining .When MSCs reached 80–90% confluence, they were detached with 0.25% trypsin (Gibco, USA) and transferred to new culture flasks at a density of 2 × 106L−1 and subcultured 2 times when the confluence reached 90%. The passage 3 MSCs were used for osteogenic differentiation with a standard DMEM supplemented with 50 mg L− 1 ascorbic acid (Sigma, USA), 10 mmol L − 1 sodium β-glycerophosphate (Sigma, USA) and 10− 8 mol L− 1 dexamethasone (Sigma, USA) at 37 0C in a humidified 5% CO2 incubator for 3 weeks. Successfully osteogenic induction was determined by formation of calcified colliculus from induced MSCs, by which can be inspected alizarin red staining. The osteogenically induced MSCs were collected as seeding cells.
The porcine small intestinal was cut in length of approximately 10 cm each, which was collected from healthy porcine (Lanzhou slaughter factory, Gansu Province) within 4 hours since sacrifice. Submucosa was obtained by mechanical removal of the tunica serosa and muscularis. Then the remaining submucosa layer was treated with a series of chemical decellularization, detergent treatment, lyophilization and sterilization . Finally, all the samples were frozen-dried under − 70 0C with a lyophilizer, sealed into hermetic packages, and then sterilized by using Co-60 gamma irradiation (25–35 kGy).
The SIS was clipped into square (5 cm x 5 cm) and sterilized again under ultraviolet for 2 hours, then were soaked in DMEM containing 20% FBS for 1 day before cells seeding. The osteoblasts (osteoinduced for 2–3 weeks, 2.0 × 109 L− 1 ) was slowly dripped onto SIS and incubated for 3 hourss at 37 0C. Then an appropriate volume of DMEM with 10% FBS was added to each composites, the incubation period lasted for 7 days.
Scanning electron microscopy (SEM)
Some of the tissue-engineered periosteum (TEP), which was cultured for 15 days, was collected for scanning electronmicroscopy (SEM JSW-680LA, Japan) inspection. Briefly, they were fixed in 2.5% glutaraldehyde for 7 days at room temperature, followed by washing thrice in PBS for 15 min each. After that, the specimens were subjected to critical point drying, gold sputter coating and then reviewed under SEM.
Preparation of deproteinized bone (DPB)
Fresh NZWR’s scapular body harvested from surgical procedure of subtotal scapulectomy, which would be described below in detail, were deproteined in whole bone blocks after soft tissue removed. Densely vertical holes (each hole was 1.5 mm in diameter) were drilled into each block to make well deproteinization. The bone blocks were treated sequently with H2O2, NaN3, NaOH, protease, methanol/chloroform mixture, ether, ethanediamine and absolute alcohol to produce DPB . The samples were dried under 50 0C with dry oven, sealed into hermetic packages, and then sterilized by using Co-60 gamma irradiation (25–35 kGy).
Animal surgery and treatment
In strict accordance with the regulations of medical animal experiments, 36 NZWRs (2 months, about 2.0 kg) were anesthetized intraperitoneally with an injection of 3% pentobarbital solution (40 mg/kg, body weight). The unilateral shoulder of the rabbits was skinned and disinfected.The scapular body with periosteum attached on it was exposed by buntly separating of the muscles, and resected off except for the glenoid and part of the scapular neck, angulus superior and inferior to establish a subtotal scapulectomy model. This model is aimed to preserve glenohumeral joint function for animal movement and prepare triangle anchoring points for implants attachment. Bone block from resection was removed together with the periosteum attached on it. The segmentally irregular bone defect was then created at unilateral shoulder blade of animal.
36 rabbits /defects were divided randomly into 3 groups with different treatment. The scapula defects were treated respectively with TEP (Group 1, n = 12), allogenic DPB (Group 2, n = 12), and TEP hybrid DPB (Group 3, n = 12).
In Group 1, TEP was spreaded over the defect area and trimmed the margins to fit the size and shape of the bone defect, then sutured with 7–0 microsurgical suture respectively to three bony anchoring points (mentioned above in bone resection part), which was pre-drilled several holes with K-wire for sutrure passing through. In Group 2, allogenic DPB block was fixed with steel wire (0.5 mm in diameter) respectively to three bony anchoring points. In Group 3, TEP enveloped DPB. Briefly, TEP wrapped on the surface of DPB and fixed with 7–0 microsurgical suture through vertical holes in DPB by a puerperal suture manner. Then the hybrid implants (TEP covered DPB) was fixed with steel wire (0.5 mm in diameter) respectively to three bony anchoring points. After implants were fixed tightly to the bony anchoring points with tension suture or K-wire, the incision was closed layer by layer with 1–0 nylon suture. The rabbits received 400,000 units penicillin preoperatively and at the first/next postoperative day. The forelimbs and shoulders of animals at surgical sides were immobilized with plaster casts for 4 weeks. The surgical procedure described is depicted using a schematic drawing, as seen in Fig. 1.
At 4, 8, and 12 weeks after surgery, four rabbits in each group were sacrificed under anesthesia and the whole scapula involved of implants were harvested as samples.
Radiological analysis of was performed at 4, 8 and 12 weeks postoperatively (DR3000 Dryview8900, Koda, Japan). The anteroposterior view of the scapula was obtained by X-ray. All radiographic results were evaluated in randomized and double-blind conditions. The Lane-Sandhu scoring system was applied for radiographic outcomes . A defect was considered healed if the area of the newly formed bone exceeded 25% of the defect area according to bone healing critera of literature . Radiographic scores were compared between the three groups.
Middle area tissue in experimental sites of scapula was excised out as specimens after X-ray radiographic examination. The specimens were fixed with 4% neutral-buffered for 3 days and decalcified with10% EDTA-2Na solution for 4 weeks at 4°C. Then they were dehydrated with an ascending series of ethanol solutions, followed by serial paraffin sections by conventional method. Specimens were stained with hematoxylin & eosin (HE) and Masson for histological analysis.
All sections from each specimen were evaluated by light microscopy. Image analysis software (Pro-image Analysis System) was used to evaluate all sections per specimen. A region of interest (ROI) for quantitative evaluation of new bone formation was defined as the area of the tissue within the defect. The results were analyzed according to a histological numerical scoring system .
Statistical analysis was performed with SPSS 15.0 software. All quantitative data were expressed as the mean ± standard deviation (M ± SD). Statistical comparison
was performed by one-way analysis of variance (ANOVA) test. Statistical significance was considered at a probability < 0.05.