N-3Polyunsaturated Fatty Acids Promote Fracture Healing Through Arachidonic Acid Arachidonic Acid Metabolism Pathwa

1. BackgroundFracture healing is a complex biological process and is marked by the cellular events of inflammation, intramembranous ossification, chondrogenesis, endochondral ossification and remodeling. The reparative phase of fracture healing is initiated by proliferation and initial chondroblastic differentiation of bone marrow stromal cell, resulting in a hyaline cartilage callus around the wounded bone. Then, chondrocytes experience the initial differentiation, proliferation, and terminal differentiation, including non-hypertrophic condrocytes, and hypertrophic condrocytes. At a later stage of healing, the cartilage callus calcifies in the process of enchondral ossification. The callus could be divided into four zones:resting zone, proliferating zone, mature zone and hypertrophic zone. The arrangement of chondrocytes in callus was irregular and the boundary between the neighboring zones not as clear as that in growth plate.Animal studies represent an essential tool to analyze the biology of fracture healing. There are many reasons why mice models have become of increasing interest for fracture healing studies. It is of utmost importance that a broad spectrum of antibodies and target gene are available for mice, allowing mechanistic studies on fracture healing. Thus, the murine model is considered as the ideal animal model for fracture healing studies. Compared with the tibia, the mouse femur is a relatively straight long tubular bone, so that the femur fracture model are established easily and considered to be ideal for fracture model. In order to investigate endochondral ossification in fracture healing, we establish a closed femur fracture model fixed with intramedullary nailing in mice.The essential polyunsaturated fatty acids (PUFAs) comprise two main classes: n-3and n-6PUFAs. Both n-3and n-6PUFAs are main composition of membrane phospholipids. n-6PUFAs mainly include linoleic acid (LA) and arachidonic acid (AA). n-3PUFAs mainly include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic (DHA). The n-3PUFAs are essential for normal growth and development. The n-3PUFAs are beneficial for various diseases. Specifically, their beneficial effects have been shown in the prevention and management of coronary artery disease, hypertension, diabetes, rheumatoid arthritis, ulcerative colitis, other inflammatory and autoimmune disorders, and cancer. The n-3PUFAs have critical roles in the regulation of a variety of biological processes including bone metabolism.With regards to bone health, n-6PUFAs (AA) and n-3PUFAs (EPA and DHA) are the most important fatty acids. Prostanoids (PGs) and leukotrienes (LTs), which are metabolites of AA, are potent inflammatory mediators, and can have positive or negative effects on bone and cartilage tissues. Recently, beneficial effects of n-3PUFAs on bone metabolism have been reported in mice, rats and humans. But until now, the effect of n-3PUFAs on endochondral ossification during fracture healing is still unknown. Therefore, it becomes important to study the effect and mechanism of n-3PUFAs on fracture healing. 2. Objectives(1) Study on the effect of n-3polyunsaturated fatty acids on fracture healing.(2) Study on the mechanism of n-3polyunsaturated fatty acids on fracture healing.3. Materials and Methods(1) Establishing of closed femur fracture fixed with intramedullary naling in miceWe improve the previous device used for generating fractures in a murine model. This improved murine femur fracture device, a gravity-driven three-point bending design, is easy to construct, operate, and maintain. Based on fracture mechanics and bone biomechanics, the ideal parameters for creating a transverse fracture in the mouse femur are tested and set, including impact mass, impact height, and impact depth. The small size of mice femur is challenging for the development of a fracture model, during which the experienced surgical skills are required. The characteristics of the femur were analyzed through animal and radiographic anatomy. The surgical skills used in the retrograded intramedullary nail were taken to fix the fractures produced by the fracture device. The fractures were examined using radiographic and histological methods. The closed femur fracture model in mice established in this experiment was typically endochondral ossification.(2) Analysis methodsHistological analysis:The fractured femora were decalcified, dehydrated, rinsed and embedded. Sections of6μm were prepared sagittally through the fracture site. The sections were stained by Safranin O/fast green staining and examined by light microscope. Radiographic analysis:At the end of the0,7,10,14,18,21,28and35days post-fracture, lateral X-rays (Kodak, DirectView DR3500, Rochester NY) of the healed femora were taken to observe the fracture type and healing. Soft X-ray radiography:Anteroposteriorly and laterally, soft X-ray radiographs of the fractured femora were taken (GE Senographe2000D, Singapore) to observe the fracture healing. Micro-CT:For micro-CT scan, a desktop micro-CT system (ZKKS-MCT-Ⅲ micro-CT, Guangzhou Zhongke Kaisheng Medical Technology Company Ltd., Guangzhou, China) was employed to analyze bone remodeling.(3) The fat-1transgenic mice are used as a tool for studying the effects of n-3PUFAs on fracture healingThe fat-1gene from Caenorhabditis elegans encodes for an n-3desaturase enzyme, which can convert n-6fatty acids into n-3fatty acids. Unfortunately, mammals cannot naturally produce n-3PUFAs from the abundant n-6PUFAs, and so they must rely on a dietary supply. Meanwhile, the source of n-3PUFAs is limited. The mice engineered to carry a fat-1gene can convert n-6PUFAs to n-3PUFAs endogenously. This results in an abundance of n-3PUFAs and a reduction in n-6PUFAs in the organs and tissues of these mice. This fat-1transgenic mouse model presents an opportunity to investigate the roles played by n-3PUFAs in the body. Therefore, we used the fat-1transgenic mice to investigate the effects of endogenously produced n-3PUFAs on fracture healing in a mice femoral fracture model. The fat-1transgenic mice (Fat-1) and wild type C57BL/6mice (WT) were compared after closed midshaft femur fractures were created and stabilized with intramedullary fixation.(4) The exogenous arachidonic acid (AA) alters the ratio of n-6/n-3PUFAsAA oil application increases the ratio of n-6/n-3PUFAs, while endogenous produced n-3PUFAs and reduced n-6PUFAs decreases the ratio of n-6/n-3PUFAs. After closed femur fractures model were established, mice were divided into four groups(C、C+AA、F and F+AA), group C for C57BL/6J mice, group C+AA for C57BL/6J mice fed with AA oil, group F for fat-1transgenic mice, and F+AA for fat-1transgenic mice fed with AA oil.(5) Celecoxib inhibits COX pathway and reduces PGE2productionCelecoxib, a COX-2inhibitor, reduced the production of PGE2through AA metabolism pathway. To investigate whether the COX pathway participate in fracture healing, after closed femur fractures model were established, mice were divided into two groups (F and F+Cel), group F for fat-1transgenic mice, and group F+Cel for fat-1transgenic mice fed with celecoxib.(6)Statistical analysisAll data were expressed as mean±standard deviation (x±s) and analyzed with SPSS13.0. Differences were considered as statistically significant for P-value<0.05.4. Results(1) n-3PUFAs promote bone healing and remodeling and are beneficial for fracture healingCompared with wild type C57BL/6mice (WT), the fat-1transgenic mice (Fat-1) endogenously increased n-3PUFAs. On postoperative days7,10,14,18,21,28and35, sections from Fat-1showed that callus formation, endochondral ossification, and remodeling occurred earlier and faster than those from WT. Histological analysis indicated that n-3PUFAs promote bone healing and remodeling.Serial radiographs at various time points showed that calcified callus and evidence of bone union were observed at21and28days after fracture respectively in WT, but were promoted at18and21days after fracture in Fat-1. Furthermore, remodeling of the calcified callus also occurred earlier in Fat-1. In WT, limited evidence of radiographic remodeling was observed28days after fracture. In Fat-1, by21days after fracture, clear evidence of remodeling was observed, and radiographic remodeling was nearly complete by35days.Soft X-ray images at21days after fracture showed that the fracture line was still visible in the WT, but was barely detectable in the Fat-1. Two out of12in WT and7out of12in Fat-1completely healed which demonstrated a significant difference statistically.The three-dimension reconstruction images with micro-CT demonstrated that the remodeling of the calcified callus occurred earlier and faster in Fat-1. At49days after fracture, the callus was larger and filled with woven bone in WT, while the callus in the Fat-1was remodeled better with lamellar bone bridging the fracture site. The cortex and the medullary cavity in the Fat-1group showed greater remodeling than that of the WT group. Additionally, the cortical bone mass has been shown to increase in the Fat-1group.(2) n-3PUFAs alter the ratio of n-6/n-3PUFAs, and promote fracture healing through arachidonic acid metabolism pathwayCompared with C and F, C+AA and F+AA increased n-6PUFAs and the ratio of n-6/n-3PUFAs. Histological analysis at7,10, and14days after fracture showed that, chondrocytes primary differentiation and proliferation were more but terminal differentiation and endochondral ossification were less in C+AA and F+AA than in C and F. Compared with C+AA increasing the ratio of n-6/n-3PUFAs, F decrease the ratio of n-6/n-3PUFAs. Histological sections showed chondrocytes terminal differentiation and endochondral ossification were inhibited in C+AA but promoted in F, indicating that the ratio of n-6/n-3PUFAs was negatively related to endochondral ossification.On the serial radiographs at7,10,14,18,21,28and35days after fracture and soft X-ray images at21and49days after fracture, fracture healing and bone remodeling showed a evident slowdown in C+AA and F+AA compared with C and F, indicating that AA inhibited endochondral ossification. Compared with C+AA, F decreased the ratio of n-6/n-3PUFAs evidently. The endochondral ossification and bone remodeling were promoted in F but inhibited in C+AA, indicating that the ratio of n-6/n-3PUFAs was negatively related to endochondral ossification.The three-dimension reconstruction images with micro-CT demonstrated that the remodeling of the calcified callus were delayed in C+AA and F+AA compared with C and F, indicating that AA inhibited bone remodeling. Histological and radiological analysis demonstrated that the ratio of n-6/n-3PUFAs was negatively related to endochondral ossification, bone healing and remodeling. n-3PUFAs promote fracture healing through arachidonic acid metabolism pathway by lowering the ratio of n-6/n-3PUFAs.Celecoxib reduced the production of PGE2through inhibiting COX pathway in AA metabolism. Compared with histological sections from F, F+Cel with less PGE2have smaller callus with less chondrocytes, indicating that the COX pathway participate in early stage fracture healing. Serial radiographs at various time points, soft X-ray images at21and49days after fracture, and the micro-CT images at49days after fracture showed that bone healing and remodeling during later stage fracture healing were not evidently affected although the callus from F+Cel were smaller than those from F. Histological and radiological analysis demonstrated that the COX pathway participate in early stage fracture healing.5. Conclusions(1) This study demonstrated that endogenous n-3PUFAs in fat-1transgenic mice could promote bone healing and remodeling in a femoral fracture model when compared to that of wild type mice, indicating that n-3PUFAs is beneficial for fracture healing through promoting endochodral ossification.(2) AA increased the ratio of n-6/n-3PUFAs and inhibited endochondral ossification and bone remodeling, while n-3PUFAs decreased the ratio of n-6/n-3PUFAs and promoted cndochondral ossification and bone remodeling, indicating that the ratio of n-6/n-3PUFAs was negatively related to endochondral ossification, bone healing and remodeling.(3) One of the mechanisms that n-3PUFAs promote fracture healing was to lower the ratio of n-6/n-3PUFAs, through AA metabolism pathway to promote the endochondral ossification and bone remodeling.