动物实验 z-bo 2020-09-03 525

　　Background: The treatment of delayed union, nonunion, malunion, and bone loss are major challenges for orthopedic surgeons. Since the second operation is to stabilize the fracture and stimulate bone growth, impaired fracture healing will negatively affect the patient's quality of life and increase medical expenses. Often, auxiliary means such as bone grafts are needed to stimulate bone healing and fill bone defects. Although the gold standard for repairing bone defects is autologous transplantation, its use is affected by morbidity and the limited supply of donor sites. The limitations of allogeneic transplantation include the host's immunogenic response to transplantation and the possibility of disease transmission. To overcome these limitations, more and more biomaterials are being developed to help treat bone defects. The replacement bone substitute is autologous or allogeneic cancellous or cortical bone, demineralized bone matrix, calcium phosphate bone graft substitute or autologous bone marrow. In addition to bone grafts and bone substitutes, plastic surgeons now also use steroid drugs (such as teriparatide and strontium tripartate) to promote fracture healing and improve bone quality. Due to the combination of bone remodeling and bone balance, anabolic drugs increase bone mass, thereby increasing bone strength. Strontium ranelate promotes the filling of bone defects and improves bone healing in rats. The core of strontium is about the same size as calcium, and it is easily absorbed by the human body and absorbed by the enamel of bones and teeth. Strontium ranelate improves the microstructure of bones through the dual action of bone formation and absorption. The drug is approved to prevent postmenopausal osteoporosis and hip fractures. Preclinical studies have shown that strontium ranelate can enhance fracture healing and osseointegration, and improve bone microstructure. The ulna of humans and limbs have similar functions. Ulnar fractures usually occur at the joint between the ulna and the ulna, or at the joint between the ulna and the wrist. Ulnar fractures can cause severe pain. When the fracture is healed, it is difficult to move the affected joint and even deform the arm. The maximum duration of natural fracture healing is about 6 weeks. Compared with the control group, strontium strontium treatment is expected to heal faster.

　　Method: Use New Zealand white rabbits (n = 5) for 28-32 weeks. The body weight at the time of surgery is 2 to 2.5 kg. Animals can eat and drink freely. The temperature of the breeding room is 26-28°C and the humidity is 60%. All operations are performed in the operating room of the animal room. All rabbit ears are permanently marked. Weigh the animals before surgery. Calculate the anesthetic dose (ketamine 20 mg/kg IM) containing the sedative and muscle relaxant xylazine (3 mg/kg). Intramuscular injection of antibiotics (kombitrim240, 1ml/10kg). Scrape the left front leg of the rabbit. During the entire experiment, all rabbits gained an average weight of 0.1–0.3 kg.

　　Surgery: After the anesthesia is completed, scrape off the left front leg and place the rabbit's back on the operating table. After disinfection with iodophor, spread out disposable sterile paper. Perform aseptic operations during surgery. Identify the ulnar center of the diaphysis, mark the skin surface, and apply local anesthesia through the planned incision. Make a 1.5 cm longitudinal incision. Identify the intramuscular septum and expose the central axis of the ulna. Separate the soft tissue to expose the bone, and make a 4 cm longitudinal incision at the skin boundary of the inner surface of the left ulna, marking 2 cm in the center of the existing bone and 2 cm at the end. Then cut the fascia and use a blunt tool to dissect the ulnar muscle and expose the ulna. The planned location of the osteotomy is marked on the bone and measured with a small stainless steel ruler. Then use a vibrating saw for osteotomy, and take care to avoid damaging the periosteum. Rinse with saline during osteotomy. Each ulna has a 5 mm defect. All rabbits underwent surgery and returned to normal activities within a few days. No wound infection. One day after the operation, all animals can roam freely. No neuropathy such as paralysis, cramps, difficulty breathing or pain. Strontium treatment: Each rabbit in the treatment group received 5 ml strontium ranelate solution (2 g strontium ranelate in 10 ml distilled water) for 6 consecutive weeks.

　　Macro evaluation: After autopsy, remove soft tissue to obtain radius bone and ulna bone. Visually observe bone defects and take pictures. X-ray and CT scan: X-ray and CT scan were used to evaluate the bone defect healing of each group of rabbits. Each rabbit was scanned at 1, 3, and 6 weeks to see the progress of defect repair in each group. Under general anesthesia, position the rabbit precisely on the right side of the CT scanner, and then lift the left front leg of the sample holder. The radius and the long axis of the ulna are aligned perpendicular to the X-ray axis. The image layer is 0.6mm thick and the resolution is 2048 x 2048 pixels. The scanned image will be saved in DICOM format. Senior plastic surgeons assess calcification, bone call and continuity of cancellous bone.

　　Bone growth quality assessment: assess the formation of initial and bridging callus. The acquisition of 3D reconstruction images is suitable for observing and evaluating bone regeneration. In the rabbit ulna model, if bridge callus is observed in the bone section, it can be considered that the fusion is successful. The bridging callus must appear in the three cortexes. Because the bone is intact and the rabbit can quickly support body weight, clinical evaluation of fracture healing is not possible. The bone growth pattern and behavior of each group were evaluated and analyzed by X-ray film. Biochemical study: Serum osteocalcin, alkaline phosphatase, calcium and phosphate were obtained from the ear vein at 1, 3 and 6 weeks. Centrifuge at 10000xg for 10 minutes. Then use the corresponding kit to analyze the levels of osteocalcin, ALP, calcium and phosphate. Measure the optical density of each parameter with a microplate reader.

　　Histopathology: After autopsy, the radius bone and ulna bone are excised. The specimen is used for histopathological sectioning. The samples were stored in 10% neutral formalin solution for 4 days and decalcified with 10% formic acid for 5 days. The samples were embedded in paraffin, cut longitudinally and stained with HE. The section was enlarged and inspected with an optical microscope. The pathologist examined the histopathological section. According to the relative ratios of lamellar bone, braided bone, cartilage, fibrous tissue, intrachondral bone formation and intramembranous ossification, the progress of fracture healing in each specimen was quantitative. X-rays and CT scans are used to collect bone growth data. Results: At the end of the study, the strontium-treated rabbits were examined with radius and ulnar X-rays. At the end of the 6-week study, none of the samples showed radiation cure. Excessive callus formation was observed in the bone and ulna, but no excessive callus formation was observed in the fracture cavity. The above findings indicate that strontium treatment cannot significantly improve the healing of rabbit ulnar fractures. In the experimental group and the control group, X-ray bone healing and insufficient osseointegration were tested by Cheung method. The healing of ulnar fractures has nothing to do with strontium. The control group had a higher cure rate. All rabbits were examined and CT scans were performed within 1 week after surgery. No signs of callus formation were observed, and 0.5 cm gaps were clearly visible. The smallest callus formation was observed in about 3 weeks. The callus is formed from the end of the osteotomy. More bones appear between the ulna and the bones, but not in the skeletal space. At the 6th week, only 2 samples showed radiation healing. At the end of the experiment, the figure shows CT scans of the forelimbs of rabbits in group A (strontium) and group B (without strontium). CT scans were performed in both groups within the first week after surgery. No evidence of callus formation was found. Three weeks after surgery, flaky calcification was found in the center of the defect and the edge of the bone. Lamellar calcification moves to the center of the bone defect. The callus gradually expanded in the defect. About 4 weeks after the operation, the proximal callus grew faster and reached the central callus. After that, it grew slowly and the distal bone call was seen within about 6 weeks. At this time, there is no gap. Bone growth is used to fill the entire osteotomy site. The fracture healed within 6 weeks. The diameter of the new bone is smaller than the actual defect. The P value of Fisher's exact test was 0.52. In the control group, the X-ray cure rate was 100%, and the CT scan cure rate was 80%. The biochemical parameters of serum alkaline phosphatase, serum calcium, serum phosphorus and serum osteocalcin were measured at 1, 3, and 6 weeks after surgery. Compare the serum markers of callus age. There were no changes in serum calcium and phosphate at 1, 3, and 6 weeks. However, there were statistical differences in serum ALP and osteocalcin levels in the 6th week. Histological examination showed the grade and percentage of lamellar bone, braided bone, cartilage and fibrous tissue. In discontinuous bone specimens, the presence of lamellar bone may be caused by the radius. However, there was no significant difference in healing between the strontium treated group and the untreated group.

　　Conclusion: The rabbit model of strontium ranelate treatment has not been shown to promote bone healing, but bone formation can be delayed, especially in the case of bone cavity in the early stage of fracture healing. Further research is needed to determine the role of strontium ranelate in larger animal models.