| Objectives：Bone defects and bone deformity may occur by reason ofinflammation, trauma, tumor, endocrine diseases. The abnormal formations inparticular oral and maxillofacial region influence the patient’s quality of life. Itbecomes a research focus in the plastic surgery field to find a simple andeffective method to improve the repairmen of bone defects.Nowadays there are a lot of techniques of promoting bone healing,including polymer biomaterial implants, artificial implants, autogenous bonegraft, tissue engineered bone graft, low intensity pulsed electromagnetictherapy. These techniques have not been widely applied in the clinical fielddue to some disadvantages.Over the years, some scholars have done a large number of experimentson bone healing by means of histology, histochemistry, histomorphometry andhave discussed the mechanism of bone repair. In recent years, bone woundhealing has been probed from cell biology to molecular biology level due tothe development of application of gene technology. With the development ofmolecular biology, it has been recognized that bone wound healing is relatedwith various growth factors. A lot of bone growth factors play an importantrole in bone wound healing besides the endocrine and systemic factors. Thesegrowth factors include TGF-β (TGF), VEGF (vascular endothelial growthfactor), FGF (fibroblast growth factor), BMP (bone morphogenetic protein),and nerve growth factor (nerve growth factor, NGF), and so on.Since the60’s of the last century, Experimental research of TraditionalChinese medicine on accelerating fracture healing has been reported. Thereare several achievements as follows: the traditional Chinese medicine toimprove the blood supply of fracture area; to promote absorption of hematomaand deposits of mineral; to increase osteoblasts number and enhance theirs activity. Epimedium (Herba Epimedii) is a kind of traditional Chinesemedicines and is used clinically for the treatment of fracture and manydiseases of bone metabolism. Some research results have shown thatEpimedium can improve the immune function, resist to tumor, strengthengonadal, delay aging, and influence cardiovascular system. Icariin, the activecomponent of Herba Epimedii(C33H40O15, molecular weight:676.67), is aflavonoid compound. Li Fangfang has co-cultured rat granulosa cells andadrenocortical cells with icariin and found that icariin can increase estradiolsecreted by follicular granulosa cells, that high concentration can promote thesecretion of corticosterone and adrenal cortical cells. So conclusion aboveresults suggested that Epimedium kidney play a role on hypothalamicpituitary-gonadal axis and adrenal axis, which can effectively regulate bodyhormone level, improve the bone metabolism, promote protein synthesis.Icariin play an important role in the repair of bone wound healing. Theexperiment of Icariin on osteoblastic proliferation and metabolism in vitro hasbeen completed by group of Dong Fusheng.This study set up New Zealand white rabbit skull defect models andobserves new bone mass, bone healing and bone quality of the defect area ofthe skull in different healing stages by gavage with icariin, by means ofhistology and histomorphometry method. In addition, mRNA of the bone ofskull defect areas was extracted and RT-PCR (real time-polymerase chainreaction) method was used to detect the gene change of bone formation and tofind the molecular signaling pathway of the bone.On the basis of the above research, immunohistochemical method wasused to study the expression of osteocalcin and Osteopontin in new bone area.The objective is to probe the mechanism of icariin in promoting formation ofthe bone and provide the theory basis for the clinical application of icariin inpromoting bone healing.Methods：1The effects of Icariin on healing quality of the repair of rabbit skull defectThirty-six female New Zealand white rabbits (12weeks age) were randomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The tissue samples of the rabbits were taken X-ray photograph. The sectionsof skull tissue were stained to observe bone trabecula width (Tb-Wi μ m),trabecula area ratio (Tb.Ar%), osteoblast number (OB·N), and osteoclastnumber (OC·N) of new bone by histomorphometry.2The expression of the gene RANKL, RUNX2of icariin on repair of skulldefect of rabbitsThirty-six female New Zealand white rabbits (12weeks age) wererandomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The total mRNA of two groups was extracted respectively and purified afteridentification of the purity and integrity. RT-PCR method was used to detectthe osteoblast proliferation, differentiation, osteoblasts function and bonemetabolism.3The expression of osteocalcin and osteopontin related to osteogenic ofIcariin on promoting repair of rabbit skull defectThirty-six female New Zealand white rabbits (12weeks age) wererandomly divided into two groups, treatment group and control group.Animals model of skull defect were established. One day after the operation,icariin was given orally at the dose of100mg/1Kg/day in the treatment groupand normal saline was given to the rabbits of the control group. After the4weeks,8weeks,12weeks postoperation the rabbits were killed respectively.The tissue samples were embedded in paraffin and were sectioned with athickness of4μm and were stained by using immunohistochemical method to observe the new bone, observed the expression of osteocalcin and osteopontin.Results：1The effects of Icariin on healing of the repair of rabbit skull defect1.1General observationsAt the4th week after surgery, the defect areas of the control group were alittle hollow, covered with fibrous tissue. The boundary was clear and therewas no obvious new bone formation around. The defect areas of theexperimental group were local depressions. The boundary was blurred andaround the boundary there were a lots of new bone.At the8th week after surgery, the defect areas of the control group weresurrounded by calluses and the bone of the center was thinner than that of theedge. The bone defects in the experimental group were evenly covered andthere were fusion with the surrounding.At the12th week after surgery, the surface of the defect of the controlgroup were surrounding by callus. The defect of the experimental group wascovered by mature bone tissue and boundary edge was not observed.1.2X-ray observationAt the4th week after surgery, the density of the defect in the controlgroup was low and the defect edges are clear. The density of the defect in theexperimental group was low and the defect edge was blurred.At the8th week after surgery, the density of the defect in the controlgroup was lower than that of the surrounding, and the boundaries between thedefect and normal tissues were distinct. The density of the defect in theexperimental group was slightly higher than that of control group.At the12th week after surgery, the density of the defect in the controlgroup was lower than that of the experimental group. The density of the defectin the experimental group was similar to normal tissues.1.3Histological observationAt the4th week after surgery, the control group showed that the defectedge had a small amount of osteoid formation and the central was fibroustissue and a lot of inflammatory cell. The defect of the experimental group was filling with thick collagen fiber and a few inflammatory cells. The trabeculaswere concentrated and there were a lot of osteoblasts with larger cell nucleolus.The osteoclasts were scarce.At the8th week after surgery, the control group showed trabeculas werethin and was not contacted. There were a lot of osteoblasts (OB) andosteoclasts (OC). Fibrous tissue connective and inflammatory cells stillexisted in the center part of the defect. In the boundary part, osteoid, fibrousbone tissue and a certain amount of osteoblasts could be seen. In theexperimental group, bone trabeculas were continuous, thickening, formedlamellar, and reticular, marrow cavity was obviously.At the12th week after surgery, the defects were filled with new bone inthe control group and woven bone and lamellar bone could be seen in themany newborn. Trabecular cells were less than those in the experimentalgroup. In experimental group, the newborn of the defect was mature andtrabeculae were thick and arranged in row. The marrow cavity was obviouslyand continuously.1.4Bone histomorphometryAt the4th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group wassignificantly higher than that of control group (P<0.01). Osteoclast Number(OC·N) of experimental group was significantly lower than that of controlgroup (P<0.01).At the8th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group wassignificantly higher than that of control group (P<0.01). Osteoclast Number(OC·N) of experimental group was significantly lower than that of controlgroup (P<0.01).At the12th week after surgery, Traberculae Area (Tb·Ar%), TraberculaeWidth (Tb·Wi), Osteoblast Number (OB·N) of experimental group weresignificantly higher than that of control group (P<0.01), Osteoclast Number(OC·N) were no significant in experimental group compared with control group (P>0.05).2The gene RANKL, RUNX2expression of icariin on repair of skull defect ofrabbits2.1Purity and integrity of the total mRNAThe ratio of OD260/OD280of all the total RNA was between1.9and2.0.The28S and18S strip could be obviously appeared, and the brightness of28Sstrip was approximately twice compared with18S strip.2.2quantitative analysis of Real-time PCRThe expression of Rankl of experimental group was significantly lowerthan that of control group. The expression of Runx2gene in experimentalgroup was obviously higher than that of control group at different times.3Expression of osteocalcin and osteopontin related to osteogenic of icariin onpromoting repair of rabbit skull defect3.1The expression of osteocalcinAt the4th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix in control group showed negative.At the8th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.At the12th week after treatment, the expression of osteocalcin (OC) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.3.2The expression of osteopontinAt the4th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed negative.At the8th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive while expression of OC in osteoblasts and matrix of control group showed weakpositive.At the12th week after treatment, the expression of osteopontin (OPN) inosteoblasts and matrix of experimental group showed strong positive whileexpression of OC in osteoblasts and matrix of control group showed weakpositive.Conclusions：1Icariin can increase the quantity of the repair area about skull defects onrabbit and improve the quality of bone healing.2Icariin can speed up the rate of repair about skull defects on rabbit.3Icariin can provide estrogen-like function and promote osteogenesis byreducing expression of gene Rankl, lessening the number of osteoclasts,inhibiting the differentiation, activation and maturity of osteoclast.4Icariin can up-regulate gene Runx2and promote the repair of defectthrough the Wnt/β-catenin pathway.5Icariin can increase the quality of osteogenesis by augment secretion ofosteocalcin in osteoblasts.6Icariin can increase the quality of osteogenesis by augment secretion ofosteopontin in osteoblasts. |
PDF Full Text Request