| Background:In recently, orthopedic technology and equipment has made great progress, many new technologies and achievements are available. Fracture and bone nonunion caused by trauma, infection still represents the medical and socioeconomic challenge. In order to improve the treatment outcome fundamentally, exploration of the pathophysiology and molecular mechanisms of bone formation, reconstruction and remodeling is the foundation and the first step. Bone is a complex tissue with a dynamic processional of extracellular matrix mineralizing and the ability to adjust to its functional demands, self-healing. Bone fracture healing is a complex process involving a well-defined cascade of cellular and molecular events, which lead to bone repair and fracture union. This complex sequence of biological processes is orchestrated by a variety of local factors, cytokines, growth factors and systemic hormones. It is known that fracture repair process include the synergistic effects of cytokines, body fluids and various tissues, which were closely related to blood supply and innervation. Adequate blood supply is a prerequisite for the healing of bone tissue. The stability of biological environment is supported by the synergies and feedback effect of accurate innervation. Many researchers have been confirmed that reconstruction blood supply is important for maintaining biological activity in the bone and bone grafts. In addition, the nervous regulation also plays an important role in the process of bone regeneration. However, there is less research report regarding of the biological mechanisms of neural factors in bone tissue regeneration. Some researcher have confirmed that the long bone in the human exist sensory and sympathetic nerve fibers, which were related to bone regeneration. In 1960, Gibson first described in femur fracture patients with central nervous system injuries, their fracture sites with more callus than simple femur fracture, and callus were constructed in the shorter time. In addition, foreign studies also have confirmed that neural factors play a regulatory role in fracture healing. In summary, neural factors play an important biological role in the regulation of bone metabolism.Some research results show that there are many neuropeptides nerve fibers in bone. The nutrition and metabolism of bone are regulated by neuropeptides secreted neuropeptide that these neuropeptides are mostly accompanied with blood vessels, and there are a few free nerve endings extend beyond the periosteum lining layer of cells, bone marrow cells and other groups,its target cells are osteoblasts and osteoclasts.Neuropeptide is a peptide-like substances, produced by the activity of the nervous system with the function of neuromodulator, SP, CGRP, NPY are the representative substance. SP and CGRP are the two main neuropeptide secreted by sensory nerve fibers; NPY is the most widely distributed in the central nervous system and one of the highest level of neuropeptides in this area. Neuropeptide SP, as an important neurotransmitter secreted by bone sensory neurons, plays an important role in the regulation of bone physiology. Many researches have clarified CGRP mechanism in bone regulation, while less research regarding SP and NPY. Neuropeptide SP is a kind of tachykinin secreted by sensory neurons. It plays an important role in the physiological regulation of bone through three kinds SP receptors:NK1 receptor, NK2 receptor, NK3 receptor, NK1 receptor is the most important one. NPY is mainly synthesized by the arcuate nucleus, and its positive nerve fibers are widely distributed in the bone marrow, metaphysea, periosteum and other metabolically active areas, and also be observed around blood vessels. NPY exerts their biological effect through five receptors (Y1,Y2,Y4,Y5 and Y6). These five receptors are G protein-coupled receptors. Y1 and Y2 receptor is closely related to osteoblasts differentiation and proliferation, recent research finds that Y4 is also involved in bone metabolism.Our previous studies showed that bone marrow stromal cells (BMSCs) express CGRP, SP, NPY receptors, and had confirmed that additional added neuropeptides could promote BMSC proliferation, differentiation through its associated receptors in vitro. Abroad research also shows that a neuropeptide receptor is expressed on bone cells, and neuropeptides involved in fracture repairing and bone metabolism.There was little reason to ask many questions about it, what the relationship between the expression of these marks and bone fracture healing, and which specific part is the neuropeptides expression in fracture end. This has not been reported. To investigation the relationship between the secretion of neuropeptides and fracture healing needs a good animal fracture model. In recent years, rats housing is easier and less expensive, breeding cycles are substantially shorter. Further, rats used as laboratory animals are, in general, genetically well defined. When using mice, genetically manipulated animals may allow an elegant approach to study the effect relationship among the secretion of neuropeptides and fracture healing process. Large array of monoclonal antibodies are available for mice, which provides excellent tool to target individual molecules in vivo. The main factors limiting rats were lack of stability of animal fracture model fixation device. Locking plates, external fixators, intramedullary screws, the locking nail and the pin-clip device were used for fracture fixation devices in animal fracture model. Each implant has strengths and weaknesses. The advantages of use common pins are the simple less invasive surgical technique,but this technique of that do not provide rotational and axial stability during fracture stabilization should be discouraged in the future. Although Locking plate、interlocking nails could provide some axial and rotational stability, but including the induction of damage to the intramedullary cavity,thus may effects the mechanism of fracture healing., plate system may impact on the local fracture broken ends, and increasing experimental bias due to it fixed through the ends of fracture. The external fixator consists of a fixator element that resides outside of the animal’s limb and four skin-penetrating screws that are drilled into the bone fragments. The major advantage of this fixation system is the fact that the fracture zone is totally unaffected by the presence of an implant. The advantage of using an external fixator is the high axial and rotational stability, the simple adjustment of the fixation stability. Therefore,in this study,we selected the rats as the experimental object, and established a femoral fracture model in rat, made four skin-penetrating screws that are drilled into the bone fragments, we evaluate the repeatability and reliability of complex fracture model in rats by the X-ray, Micro-CT, histological staining, to achieve standardized and defined study conditions. The animals need to be sacrificed at different time points in the period of the fracture healing process. Then we detected the expression of the neuropeptide SP, NPY and their receptors by Q-PCR and Immunohistochemistry. The experiment as the foundation and basis for the animal experiment of neuropeptides, but also provides new ideas and theoretical basis for treatment of fracture and bone metabolic diseases.Objective:1. Establish a reliable and reproducible femur fracture model in rats2. To explore the relationship between the expression of neuropeptide SP, NPY and its receptor NK1, NPYR1 in the fracture healing process of ratsPart 1 To establish a rat femoral fracture modelObjective:to establish a simple, reliable, reproducible rat fracture model.Methods:1. Design and fabrication of the External fixator:External fixator with reference to clinical practice application of unilateral external fixator for design. External fixation is made up of the titanium alloy material of the frame, the lateral needle clamp block, four stainless steel self-tapping screw Kirschner-wire. The clamps block were connected by a central threaded bar, which allowed for adjustable and controlled distraction and compression. The Kirschner-wire diameter is 1.2 mm.2. To establishment the rat femur fracture model:twelve 480～550 g healthy adult male SD rat, all animal were provided by the experimental animal center of Southern Medical University. Operation process:after anesthesia, intramuscular injection of antibiotics to prevent infection, shave off the hair of surgical field, the lateral decubitus position, alcohol gauze sterilization, drapes, make a incision along the lateral of the right lower limb, separate anterior lateral and posterior muscular, expose the femur fully, two threaded Kirschner wires were screwed into the in femoral ends respectively, and fix the main installation frame on Kirschner wire, make a femoral osteotomy slowly with miniaturesaw, physiological saline flushing the fragments, the wound is washed after osteotomy, close the wound. Operation process is carried out under strict aseptic conditions. After intramuscular injection of antibiotics again, wrappe the external fixator with alcohol gauze, put animals in thecage.3. Postoperative general observation:the postoperative observation of wound healing, gait, activity, diet, with or without needle infection, as well as the external fixator presence of loose4.Take X-ray films to observation the fracture of the registration and callus growth situation, micro-CT scan and quantitative assessment of the bone fracture area of BV/TV value, histological staining to observe osteogenesis.Results:General condition:experimental animals all survived, and into the analysis of experimental results. General observation:all animal postoperative incision healing, no infection, postoperative external fixator and fixed good, no loosening and withdrawing needle etc. Animal health in good condition, external fixator had no adverse effect on the animals. X-ray showed postoperative fracture counterpoint is good, the callus increased gradually. Micro-CTResults:Four weeks Micro-CT scan BV/TV (49+4)%, eight weeks BV/TV for (61 +6)%(p<0.05).Histological observation:4 weeks fracture end connected to a portion of the callus,8 weeks of fracture end bridge, marrow cavity began to mutually, peripheral callus decrease.Conclusion:the custom-made external fixator can provide good stability on rat femur fracture, this experiment provides a simple, reliable and reproducible rat femoral fracture model.Part 2 To explore the relationship between the expression of neuropeptide SP, NPY and its receptor NK1, NPYR1 in the fracture healing process of ratsObjective:To explore the relationship between the expression of neuropeptide SP, NPY and its receptor NK1, NPYR1 in the fracture healing process of ratsMethods:1. Establish animal fracture model:choose fifty-five 480～550 g healthy male adult SD rats. Fracture model is established with the first part of the process.2. Eight rats were sacrificed after 2,4,6,8,12 weeks respectively, Obtain the right femoral diaphysis, evaluate of fracture healing by HE, Masson staining, X-ray and Micao-CT. Observation the express of SP, NPY, NK1 and NPYR1 of the end of fracture by Immunohistochemistry, and for Real-time PCR detect the transcription of SP, NPY, NK1 and NPYR1 of total RNA of the fracture ends tissue by Real-time PCR.Results:X-rays Observation:2 weeks after the operation, the Fracture lines were blurred, bone callus through has formed in 4 weeks, but can still be seen in the fracture line, fracture healing were obtained in mostly rats at 6 weeks, bone marrow cavity is communicated at 8 weeks, fracture healing completely and bone remodeling at 12 weeks.HE staining and Masson staining:Fracture line is visible in 2 week on the end of the fractures, the cartilage callus were connected in 4 weeks, fracture healing mostly in 6 weeks, the marrow cavity is begin communicated and peripheral callus are decreased in 8 weeks, marrow cavity completely connected and fundamental fracture healing completely in 12 weeks.Immunohistochemistry staining:the expression of SP, NK1, NPY exist in the osteoblasts、chondroblasts around, around blood vessels and peripheral of new bone and periosteum. In the end of the fractures, the positive expression is maximum after 4 weeks and 6 weeks, and is gradually reduced in 8 weeks and 12 weeks. immunohistochemistry staining of NPYR1 showed that a small amount of expression in osteoblasts, chondroblasts, and under the periosteum after 2,4 weeks of NK1 in the end of the fracture, after and a large number of expression around subperiosteal new bone, neovascularization in 6 weeks, the expression relative reduction in 8 weeks and 12 weeks.Postoperative immunohistochemical staining SP, NK1, NPY in osteoblast on the fracture end, into cartilage cells, around the new blood vessels and around the new bone and periosteum were expressed,4 weeks,6 weeks after the most amount of positive expression, with the passage of time 8 weeks and 12 weeks, gradually reduce the amount of positive expression.NPYR1 immunohistochemical staining in 2,4 weeks after NK1 osteoblasts on the fracture end, into cartilage cells, decreases and the expression of a small amount of around 6 weeks after surgery in osteoblast, periosteum, new blood vessels and new bone surrounded by a large number of expression, as time goes by express less amount of 8 and 12 weeks.Conclusion:the levels of neuropeptide SP, NPY and their receptors expression were higher than that of normal bone formation in the process of fracture healing, the expression of SP, NK1 was significantly increased in stage of inflammatory reaction. And the expression trend is approximately accordance, indicating that SP may through the NK1 promoting fracture healing in fracture early metaphase. The mRNA transcription of SP, NK1 in the later stage was higher than that of normal bone tissue, suggesting that SP and NK1 may play a role in the remodeling of bone callus; the expression of NPY increased in early and middle fracture healing, suggesting that it may promote the healing of fracture. But the transcription and expression of NPYR1 are not obvious in the early stage, suggesting that NPY may regulate bone formation through other receptors. The expression of NPYR1 was low in the early time, while increased in the middle and end stage, suggesting that NPYR1 may be related to intermediate the bone callus remodeling in advanced stage. |
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