Mechanism Of Chitin Derivatives On Fracture Healing In Rats

OBJECTIVE To establish rat tibial fracture model and investigated the effects and mechanisms of three kind of different chitin derivative (glycose, oligarsaccharide and solution Zhuanggu) on the rat fracture healing. METHODS Establish rat tibial fracture and the rats were divided into four groups:chitin derivative treatment group (glycose, oligarsaccharide and solution Zhuanggu) and control model group.7day,14day and 21 days after the fracture operation and treatment, the X-ray radiography and Alcian blue/HE staining were performed to observe the histological changes of fracture healing, and proliferating cell nuclear antigen (PCNA) immunohistochemical staining were used for dectect the the ability of proliferation of osteocytes. And the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was employed for the detection of apoptotic cells using in situ cell death detection kit. The content of calcium and phosphorus and the activity of alkaline phosphatase (ALP) in the blood were measured by enzyme biochemistry method. Mouse calvaria-derived osteoblastic clone MC3T3-E1 cell line were incubated with different concentrations of oligarsaccharide Cconcertration range from 200pg/ml to 1000pg/ml) inα-MEM culture medium. Cell proliferation and apoptosis was determined by MTT assay and Hoest 33258 staining. The expression of ALP mRNA and osteocalcin mRNA were detected by RT-PCR assays. RESULTS Representative radiographs obtained on days 7,14, and 21 after fracture showed that fracture healing is accelerated in groups of oligarsaccharide and solution Zhuanggu treated mice, especially in solution Zhuanggu treated group. Representative histologic sections stained by Alcian blue/hematoxylin showed that fracture healing in oligarsaccharide and solution Zhuanggu treated groups showed earlier bone formation, higher total callus area, less fibrous tissue and cartilage formation than the normal and glycose treated groups (p<0.05). A peak level of cell proliferation, as revealed by PCNA labeling, appeared in the undifferentiated mesenchymal cells, inflammatory cells at the fractured edges, cells in primitive connective tissue and chondrocytes on day 7, and declined at later stages of fracture healing. At this time, a high proliferation activity was detectable in mesenchymal cells and inflammatory cells in normal control group. Whereas in groups treated with oligarsaccharide and solution Zhuanggu, cartilage islands were detectable with proliferating chondrocytes. Fractures healing after 14 day, a large number of PCNA-positive osteoblasts were visible in the newly formed woven trabecular bone in groups treated with oligarsaccharide and solution Zhuanggu than normal controls groups (p<0.05). High PCNA-positive chondrodytes was detected in cartilage islands in control group.21 days after fracture, cell proliferation was observed only in the fibrous layer of the periosteum near the fracture site and in the endochondral ossification front, and the number of PCNA-positive osteoblasts in groups treated with oligarsaccharide and solution Zhuanggu were less than normal controls groups(p<0.05). Positive TUNEL-labeled cells were seen in the fibrous tissues especially in normal control and glycose treated groups and newly formed callus near the fracture gap especially in oligarsaccharide and solution Zhuanggu treated groups on day 7 after fracture healing. The number of the TUNEL-labeled cells in chondrocytes and osteoblasts in the newly formed cartilage and callus peaked on day 14 after fracture healing and significantly declined on day 21, where the TUNEL-labeled cells were mainly seen in the remaining cartilage islands and in the newly remodeled lamellar bone. The number of the TUNEL-labeled cells in groups treated with oligarsaccharide and solution Zhuanggu were less than normal controls groups (p<0.05). On day 14 and 21, the higher concentration of blood calcium and the activity of ALP and lower concentration of blood phosphorus were detected in oligarsaccharide and solution Zhuanggu treated group than normal controls (P<0.05). In vivo studies showed that over a concentration range of 400-1000μg·mL-1 oligarsaccharide could promote MC3T3-E1 cells proliferation, inhibit cell apoptosis, increase the expression of ALPmRNA and osteocalcin mRNA in a dose-dependent manner (P<0.05). CONCLUSION Three different chitin derivative (glycose, oligarsaccharide and solution Zhuanggu) could promote rat tibial fracture healing by promoting osteoblasts proliferation, inhibiting cell apoptosis, increasing blood calcium and the activity of ALP and decreasing blood phosphorus. Oligarsaccharide could promote MC3T3-E1 cells proliferation, inhibit cell apoptosis, increase the expression of ALPmRNA and osteocalcin mRNA in a dose-dependent manner and further promote cell division.