修复不规则骨缺损 z-bo 2020-10-22 655

　　Background: In previous studies, long bone defects were successfully repaired through tissue engineered periosteum (TEP), which is formed by combining rabbit bone marrow mesenchymal stem cells with the submucosa of the small intestine. This study explored the feasibility of TEP to repair allogeneic irregular bone defects.

　　Method: Perform subtotal scapula resection on 36 rabbits to establish a large-area irregular bone defect model. The rabbits were randomly divided into 3 groups (12 in each group), and TEP (group 1), allogeneic deproteinized bone (group 2), or a mixture of TEP and DPB (group 3) were used to treat bone defects. The rabbits were sacrificed 4, 8, and 12 weeks after the operation, and the implants were taken out. Perform X-ray and histological examinations to detect bone healing. The vascularization of TEP engineered new bone was qualitatively analyzed by the formaldehyde ink perfusion method.

　　Results: The repair methods of scapular defects in all groups were different, and imaging and histological examination showed that. In the 8th and 12th weeks, the imaging scores of the first and third groups were significantly higher than those of the second group. The histological score further proved that compared with the third group, the first group had more new bone formation, and the second group had the lowest bone formation rate at all time points. Formaldehyde ink perfusion showed that there are abundant microvessels in the new bone of TEP.

　　Conclusion: Our conclusion is that TEP is promising in repairing large-scale irregular bone defects. As a three-dimensional bracket, DPB can provide mechanical support and forming guidance when combined with TEP.

　　Introduction: Huge bone defect is still a challenge facing orthopedic surgeons. Bone tissue engineering is a promising method for repairing bone defects. The classic BTE method is to select a biomaterial scaffold that provides structural support for the formation of 3D bone tissue. However, this leads to limited regeneration of bone tissue, which is mainly due to insufficient delivery of nutrients and oxygen and removal of metabolic waste in the 3D stent. Seeding cells on the outer surface of the 3D scaffold can provide the cells with sufficient nutrients, but the cells located in the scaffold are likely to undergo necrosis, thereby hindering bone regeneration. In addition, without the capillary network in the 3D implant, the maximum thickness of the engineered tissue can only reach 150-200 mm. A size larger than this threshold may cause hypoxia inside the biological material. Periosteum plays an indispensable role in bone formation and bone defect healing through endogenous repair methods. Some papers described the use of bone marrow mesenchymal stem cell (MSC) slices or periosteum for bone healing. The development of a bionic periosteal substitute that can adapt to bone defects of any size and shape is a promising method for bone defect repair. Based on the principles of tissue engineering, in previous studies, we have developed a flexible cell structure that serves as the "periosteal" for bone formation and angiogenesis, a self-made tissue engineering periosteum (TEP), which is a rabbit induced by bone formation Bone marrow mesenchymal stem cells are combined with the small intestinal submucosa (SIS) scaffold. In previous studies, it has been successfully used to reconstruct critical size defects (CSD) of long bones. In this study, we hypothesized that TEP can repair large irregular bone defects in the rabbit model.

　　Animals: 46 New Zealand White Rabbits (NZWR) consist of 36 adult rabbits (2 months old, about 2.0 kg) and 10 newborn rabbits (2 weeks old, about 0.40 kg). Cell culture: In short, bone marrow (5 mL) was collected from the ventral side of newborn NZWR (2 weeks old, approximately 0.40 kg). The MSCs isolated from bone marrow were then inoculated into plastic culture flasks containing Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2. Observe the primary MSC under a microscope. When MSCs reach 80-90% confluence, they are separated with 0.25% trypsin, transferred to a new culture flask at a density of 2×106 L-1, and passaged twice after reaching 90% confluence.

　　Osteogenic induction: The third generation of bone marrow mesenchymal stem cells undergo osteogenic differentiation in standard DMEM. DMEM is supplemented with 50 mg/L ascorbic acid, 10 mmol/L β-glycerophosphate sodium and 10-8 mol/L dexamethasone. Incubate for 3 weeks in a 5% CO2 humidity incubator at 37°C. The osteogenic differentiation of bone marrow mesenchymal stem cells was observed by modified Gomori staining method and Alizarin red staining method. After identification, the osteogenic induced MSCs were collected as inoculated cells.

　　The small intestines collected from healthy pigs within 4 hours after slaughter are cut into lengths of about 10 cm. The submucosa is obtained by mechanically removing the serosa and muscle layer. The remaining submucosa is treated with a series of chemical decellularization steps, treated with detergents, lyophilized and sterilized. Finally, all samples were freeze-dried in a freeze dryer at -70°C, sealed in airtight packaging, and then sterilized with Co-60 gamma rays. Cut the SIS into a square (5××5 cm) and sterilize it again under ultraviolet light for 2 h, and then soak it in DMEM containing 20% FBS for 1 day before seeding the cells. The suspension of bone-induced bone marrow mesenchymal stem cells (2.0 × 109/L) was slowly dropped onto the SIS box in the petri dish, and incubated at 37°C for 3 hours. Then add an appropriate amount of DMEM containing 10% FBS to each complex, and continue to culture for 7 days.

　　Scanning Electron Microscope (SEM): Take part of the TEP cultured for 15 days for scanning electron microscopic observation. Simply put, the sample was fixed in 2.5% glutaraldehyde at room temperature for 7 days, and then washed 3 times in PBS for 15 minutes each time, and then the sample was critically dried.

　　Preparation of deproteinized bone: Fresh scapula was removed by subtotal resection of fresh scapula. After soft tissue resection, protein was removed from the entire bone block. Drill a row of dense vertical holes (each hole is 1.5 mm in diameter) in each block for good deproteinization. Treat the bone mass with H2O2, NaN3, NaOH, protease, methanol/chloroform mixture, ether, ethylenediamine and absolute ethanol to prepare DPB. The samples were dried in a dry box at 50°C, sealed in a sealed package, and then sterilized with Co-60 gamma rays.

　　Irregular bone defect production and treatment: 36 NZWRs (2 months old, about 2.0 kg) were intraperitoneally anesthetized with 3% pentobarbital solution (40 mg/kg). Peel and sterilize the rabbit's unilateral shoulder. Then, directly separate and expose the scapular body attached to the periosteum, and remove the scapular glenoid and part of the scapular neck, upper and lower corners of the scapula, and establish a subtotal scapula resection model. The purpose of this model is to preserve the function of the shoulder-brachial joint and prepare triangular anchor points for the attachment of the implant. Remove the excised bone and the attached periosteum. After each animal’s scapula caused a unilateral segmental irregular bone defect, 36 rabbits were randomly divided into three groups (12 in each group), and TEP (group 1) and allogeneic DPB (group 2) ) Or TEP-DPB mixture (group 3) for treatment. In the first group, spread the TEP on the defect area and trim the edges to fit the size and shape of the bone defect, and then suture it to three prepared bone anchor points with 7-0 microsurgical sutures. These The anchor points are pre-drilled with several holes to allow the K-line suture to pass through. In the second group, the allogeneic DPB block was fixed to three bone fixation points with steel wires (0.5 mm in diameter). Group 3, TEP wraps DPB. In short, wrap the TEP on the surface of the DPB and fix it with 7-0 microsurgical sutures through the vertical holes of the DPB. Then, the hybrid implant (TEP-covered-DPB) was fixed on the three bone anchor points with steel wire (0.5mm diameter). The incision was closed layer by layer with 1-0 nylon suture. 400,000 units of penicillin were injected preoperatively and on the first day/second day after surgery. The forelimbs and shoulders of each animal were fixed with plaster for 4 weeks. Three to four rabbits in each group were sacrificed at 4, 8, and 12 weeks after surgery, and the entire scapula (including implants) was taken for analysis.

　　Histological evaluation: After X-ray examination, take the middle part of the scapula at the experimental site as a specimen. The specimens were fixed with 4% neutral buffered formalin for 3 days, and decalcified with 10% EDTA-2Na solution at 4°C for 4 weeks. They are then dehydrated with a series of ethanol solutions and cut into continuous paraffin sections by conventional methods. The specimens were stained with HE and Masson trichrome for histological analysis. All sections of each sample were evaluated by optical microscope. Use image analysis software to evaluate all slices of each sample.

　　Formaldehyde ink perfusion: At the 12th week, one rabbit was randomly selected from the four rabbits in the first group for ink perfusion to distinguish the blood vessel formation of TEP-mediated bone regeneration. In short, rabbits received general anesthesia by intraperitoneal injection of 3% pentobarbital solution (40 mg/kg body weight). Subsequently, systemic heparinization was completed by intramuscular injection of 1000 U/kg heparin. Cut the skin and subcutaneous tissue in the axillary area to expose the axillary arteries and veins. Then the axillary artery is ligated and cannulated distally. The axillary vein was severed, bleeding distally. A large amount of heparin-saline (12,500 U of heparin; 500 mL of normal saline) was injected from the distal end of the axillary artery until the blood flowing out of the axillary vein was cleared. The perfusate mixed with 30% ink, 10% formaldehyde, and 60% normal saline is injected into the axillary artery through the cannula until the axillary vein outflow becomes black and the skin and hoof are black. Ligate the femoral axillary vein. The rabbits were sacrificed, the shoulder blades were removed, stored at 4°C for 24 hours, and then fixed with formaldehyde (40 g/L) for 1 week. The bone specimens were sliced transversely and longitudinally, and the slides were stained with HE to observe the formation of microvessels in the newly formed bone.

　　Result: Osteogenic differentiation of bone marrow mesenchymal stem cells: Under light microscope, the primary MSCs were triangular or polygonal, and at the third generation they showed a uniform elongated fibroblast-like morphology. After 3 weeks of bone fusion induction, refractive intracellular particles were visible under the light microscope. Alizarin red staining further confirmed that the cells tend to aggregate and form calcified nodules. Modified Gomori staining showed that ALP expression was enhanced in MSCs induced by osteogenic formation. These signs indicate the osteogenic differentiation of induced MSCs.

　　A. Primary bone marrow mesenchymal stem cells; B The third generation of bone marrow mesenchymal stem cells have uniform morphology; C fusion MSCs induced by osteogenic culture medium can show intracellular refractive particles; D 3 weeks after induction, MSCs fuse to form calcified nodules; E Alizarin red staining confirmed that osteogenic-induced MSCs formed calcified nodules; F modified Gomori staining method to observe the ALP expression of osteogenic differentiated MSCs. Scale bar = 100μm

　　The characteristics of SIS and SEM: The self-made SIS stent is a white flexible film with a thickness of 100±20μm. The DPB implant is shaped into an irregular triangle with a thickness of 5± 2mm with multiple holes. In the petri dish, the TEP implant maintains the properties of soft tissue and is roughly membranous. HE staining showed multiple cells on TEP. Under the scanning electron microscope, SIS is composed of collagen fibers intertwined, and a large number of cells can be seen attached to TEP on SIS.

　　Bone defect implants. A Macroscopic appearance of SIS; B Macroscopic appearance of DPB derived from scapula block; C Macroscopic appearance of TEP cultured; D Seed cells attached to TEP under light microscope, HE staining is visible (scale bar  = 100μm); E Scanning electron microscope observation SIS;

　　Postoperative animal behavior: The animal recovers quickly within 1 hour after surgery, can stand normally within 24 hours, the wound is closed within 1 week, and the incision has no obvious infection. After about two weeks, you can move freely. During the entire experiment, all animals remained healthy. There are no animal infections or other complications.

　　Analyze the progress of bone defect repair with x-ray films, and compare them in parallel with groups 1, 2, and 3 at 4, 8, and 12 weeks. In the first group, low-density callus was seen in the bone defect area 4 weeks after operation. At the 8th week, more callus appeared. At the 12th week, the amount of newly formed bone and bone mineral density increased greatly.

　　X-ray and macro observation. The first row (A-C) represents the X-rays of the scapular defect repaired in the first group at 4, 8 and 12 weeks, respectively. The second row (D-F) is the X-ray film of the scapular defect repaired at 4, 8 and 12 weeks in the second group. Row 3 (A-C) are X-rays of scapular defect repaired at 4, 8 and 12 weeks in group 3. The fourth column (J-L) represents the macroscopic view of the repair of the scapula defect in the first group, the second group and the third group at 12 weeks.

　　Histological findings: The samples taken from the scapula area are very similar in macroscopic view, and form a shape similar to the scapula at 12 weeks. Under light microscope, mild mononuclear cell infiltration was seen in each group after HE staining at 4 weeks.

　　Use HE (rows 1, 3, and 5) and Masson's tricolor (rows 2, 4, and 6) for histological examination under an optical microscope. In group 1 (row 1, row 2), TEP formed island callus in week 4 (A, B). New bone tissue increases, blood vessels are irregular or the bone marrow cavity is immature, and TEP disappears (may be degraded) at 8 weeks (C, D). At the 12th week, all new bones developed into mature cancellous bone (E, F). In group 2 (rows 3 and 4), in the 4th week (G, H) DPB is mainly surrounded by scar tissue and infiltrating lymphocytes, and in the 8th week (I, J) or 12th week (K, L) accompanied by a small amount of new Bone tissue formation. In group 3 (rows 5 and 6), a small amount of new bone was formed between TEP and DPB, which was accompanied by scar tissue degradation at 4 weeks (M, N). The new bone tissue forms woven bone at the 8th week (O, P), and at the 12th week tends to form mature laminar bone (Q, R) with osteoblasts embedded in the mineral matrix. The triangle represents TEP, the black arrow represents newly formed bone tissue, and the white arrow represents the remnants of DPB. Scale bar = 1 mm.

　　TEP-mediated vascularization of bone regeneration: After the axillary artery is perfused with formaldehyde ink, the entire bone block turns black. With the naked eye, small blood vessels and mesh anastomosis can be seen on the surface of the bone.

　　Formaldehyde ink pouring. A The black arrow of the axillary artery perfusion surgery points to the axillary artery; B the macroscopic appearance of the bone after the ink infusion. C Black capillaries, filled with black ink, stripes are visible in the longitudinal area, and round dots in the cross section. D "#" represents the capillaries filled with ink; scale bar  = 1mm

　　In this study, there are several limitations that require further study. First, we did not test the mechanical properties and degradation curves of TEP and TEP/DPB mixtures. Second, the seeding efficiency or cell density of TEP has not been calculated, which may affect the standardization of TEP manufacturing. Third, in addition to the SIS implant group (negative control group), it may be necessary to design a sham operation group without any treatment for bone defects to more clearly confirm the osteogenic potential of TEP in vivo.