| Objective:Bone defects resulting from various reasons are very common and seriously influence the quality of patients life. Bone healing is an extremely complex biological repair process and has always been a difficulty in clinical. How to promote the repairing of bone defect has been paid much attention. Over the years, multiple studies have been done to discuss the mechanism of bone defect repair by means of bone biomechanics and ultrastructure and so on.Epimedium is a traditional Chinese pharmic plant, in which icariin(ICA) is the main active ingredients. The characteristics of Epimedium is gump, moderate and sheen. It belongs to the liver and kidney and has multiple function. Numerous studies have confirmed that ICA can induce marrow mesenchymal stem cells differentiation, thereby increasing bone cell activity to promote bone healing.Concentrated Growth Factors(CGF), a new extract from plasma,contains a large amount of fibrin and growth factors. It can promote tissue healing, reduce scarring formation, and can be injected alone or with other biological materials into hard tissue defect to repair the defect,induced growth, accelerate the local wound healing, and improve the quality of healing.In this study, we produced a rabbit skull defect model. The rabbit with skull defect were treated with icariin by administration or CGF fibrin gel membrane by covering the skull defect surface. At different time points after damage, general observation, X-ray, histological observation, and bone morphology metrology were performed to explore the role of icariin and CGF in promoting bone defect repair. A rabbit whole genome microarray analysis was also performed to observe the gene expression profile variance. At last, real-time PCR and Western blot analysis were performed to explore the signaling pawthway by which icariin or CGF promotes rabbit skull defect repair.Methods:The first part Studies about the effect of ICA and CGF on the rabbit skull defect repair.1 Animals and grouping: 72 rabbits(12 weeks old, 1.8 ~ 2.0 kg)were randomly divided into control group(n=24), icariin-treated group(n=24) and CGF-treated group(n=24), numbered, and housed separately.Control group: the periosteum and skin were sutured after the rabbit skull defect was made. No treatment was done until the bone defect was healed naturally. The rabbits were administrated with 1 ml/kg normal saline the each day after surgery;Icariin Group: the periosteum and skin were sutured after the rabbit skull defect was made. The rabbits were administrated with moderate icariin the each day after surgery;CGF Group: the CGF fibrin gel membrane was covered on the defect region after the rabbit skull defect was made. The periosteum and skin were sutured;2 The process of surgery: animals were anesthetized with 10%chloral hydrate and made a 15 mm diameter defect in the skull. The control rabbits were administrated with 1 ml/kg normal saline the each day after surgery; the icariin rabbits were administrated with moderate icariin the each day after surgery; the CGF rabbits were covered with CGF fibrin gel membrane on the skull. The animals fed water freely after surgery. The animals were sacrificed 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the surgery. The newborn bone tissue were cut and general observation and X-ray were performed to observe the skull defect healing.3 histological observation: HE and immunohistochemical staining were performed with 4 μm paraffin cross-sections from the bone defect region to observe the healing of bone defect.4 bone metrology observation: the number and characteristics of osteoclasts and osteoblasts were observed with a microscopic. The average width and area of bone trabecular, as well as the bone forming speed and quality were also observed.5 statistical analysis: Data are presented as bar graphs(means ±SEM) of at least three independent experiments. Statistical analyses were performed using Student’s t test. The results were considered statistically significant at P < 0.05.The second part Analysis of gene expression profile of icariin,autologous CGF promote rabbit skull defect repair.1 Animal model producing and grouping were the same as described in the first part.2 Total RNA extraction and purification: Animals were executed 2 or4 weeks after surgery, new tissue in the bone defect region were cut under aseptic conditions and grinded in liquid nitrogen. Total RNA were extracted and purificated. The concentration, purity and integrity of RAN were determined.3 Affymetrix genome-wide m RNA expression profile chip experiments.4 Bioinformatics software Analysis: Robust Multichip Analysis(RMA) was used to screen the differently expressed genes. Function classification analysis were used to analyze genes differently expressed 2times greater or 2 times lower.The third part Discussion on the main signal path icariin,autologous CGF promote rabbit skull defects of conduction.1 animal groups, animal experiments with the first part.2 skull defect new tissue m RNA extracted and purified with the second part.3 The second part of the experiment screened icariin comparison with the control group differences in gene HGF and CGF comparison with the control group differences in gene Runx2 and reverse primers were synthesized.4 use of M-MLV reverse transcriptase denatured RNA and primer,and added to the reaction solution, first strand c DNA synthesis.5 Real-Time PCR, first strand c DNA was synthesized in the above as a template by Real-Time PCR amplification with 2-( △ Ctsample- △Ctcontrol) method calculated to detect icariin group and the control group HGF m RNA expression, detection and control groups CGF Runx2 m RNA expression.6 extracts of skull defects newborn tissue protein, by Western blot analysis to detect and control groups icariin HGF protein expression,CGF and control groups Runx2 protein expression.Results:The first part Studies about the effect of ICA and CGF on the rabbit skull defect repair.1 General observation2 weeks after treatment, these groups showed no significant difference;4 weeks later, in icariin and CGF groups, central local depression and blunt boundaries were seen and there were small amount of new bone growth. The central part of the fibrous connective tissue filling. No significant new bone formed in the control group;After 8 weeks, In ICA and CGF groups, the rabbit skull defects were covered with hard tissue and merge with the surrounding normal bone tissue. In the control group, there were just a little callus formation;12 weeks after surgery, in the ICA and CGF groups, rabbit skull defects were covered with newly formed bone tissue, red, hard, with no obvious boundaries in the edge, and integrated well with the surrounding normal bone tissue. In the control group, there were some callus formation and the peripheral portion is not fully formed with bone. There were no normal physiological curvature of the skull.2 X-ray observation After2 weeks, there were low and uniform density in the bone defect region;After 4 weeks, in the ICA and CGF groups, there were low and cloudy density in the bone defect region with edges slightly blurred; In the control group, there were low density in the bone defect region;After 8 weeks, in the ICA and CGF groups, there were high density in the bone defect region. The defect region became smaller. The border of the bone defect were blur. In the control group, density of the defect is lower than the regional surrounding normal bone tissue, with a clear boundary;12 weeks after surgery, in the ICA and CGF groups, there were no obvious boundary with the surrounding normal bone tissue. In the control group there were still an obvious boundary.3 HE staining After 2 weeks, in the ICA and CGF groups, there were a lot of fibrous connective tissue in the central of defects. The collagen fibers were thick and visible. There were also a large number of osteoclasts. In the control group, the defect region were filled with a lot of fibers and inflammation cells; No new bone were formed in the three groups;After 4 weeks, in the ICA and CGF groups, the number of fibrous connective tissue reduced. The collagen fibers were thick and filled with a little inflammation cells. A little new and fiber-like bone formed. In the control group, the defect region were filled with a lot of fibers and inflammation cells. The number of osteoclasts increased;After 8 weeks, in the ICA and CGF groups, a lot of new bone formed in the defect region. The trabecular became thickened and continuous. Some newly formed bone were net-like arranged and some bone with thickened density formed a parallel lamellar shape with the osteoblasts arranged regularly around them. There were obvious new bone formation in the defect region and basically filled the whole defect region.In the control group, the defect region were filled with a lot of fibers and inflammation cells. A little osteoblasts exists. There were a little osteoclasts around the bone trabeculae;After 12 weeks, in the ICA and CGF groups, the lamellar bone became more mature. There were a lot osteoblasts and little osteoclasts.The bone trabeculae arranged regularly and increasing number of collagen fibers were observed.In the control group, the defect region were filled with a little newly formed bone and a lot of fibers which surrounded the bone island structure. The bone trabeculae were continues, tiny and arranged irregularly. The number of fibrous connective tissue were more than the ICA or CGF treated group.4 measurement of bone observation2 weeks, 4 weeks, and 8 weeks after surgery, in the ICA and CGF groups, the bone mass, the trabecular width, and the number of osteoblasts were significantly higher than the control group; the number of osteoclasts was significantly lower than the control group. These indexes between the ICA-treated group and the CGF-treated group showed no differences.12 weeks after surgery, in the ICA and CGF groups, the bone mass, trabecular width, and the number of osteoblasts were higher than that in the control group; No significant difference of the number of osteoclasts among the three groups were observed. These indexes between the ICA-treated group and the CGF-treated group showed no differences.5 Expression of BMP-2 and TGFβ1 were detected by Immunohistochemical staining.Immunohistochemical staining for BMP-2: in the ICA and CGF groups, there were new cartilage matrix formation 4-12 weeks after surgery. The BMP-2 staining were positive in the cartilage matrix,original bone cells, and granulation tissue. The staining was uniform.Immunohistochemical staining for TGFβ1: in the ICA and CGF groups, there were new cartilage matrix formation 4-12 weeks after surgery. The bone trabeculae became broad. The TGFβ1 staining were positive and uniform in the granulation tissue.The second part Analysis of gene expression profile of icariin,autologous CGF promote rabbit skull defect repair.1 m RNA purity and integrity identificationOD260/OD280 ratio of the total RNA in each group was between1.9-2.0, indicating that the purity and concentration of RNA were accorded with the experimental requirements. Agrose gel electrophoresis showed that three bands were seen in each group and the brightness of28 S band was about twice than that of the 18 S band, indicating that the integrity of RNA in each group was favourable.2 through PARTEK analysis software, variance analysis comparison between groups were selected signal up- and down-regulated gene 2-fold difference in the entire probeset.2.1 The comparison between icariin group and control groupIn 2 weeks, 23 differential gene expression were observed with a2-fold difference(10 genes were up-regulated and 13 genes were down-regulated)In 4 weeks, 35 differential gene expression were observed with a2-fold difference(10 genes were up-regulated and 25 genes were down-regulated)2.2 The comparison between CGF group and control groupIn 2 weeks, 25 differential gene expression were observed with a2-fold difference(5 genes were up-regulated and 20 genes were down-regulated)In 4 weeks, 40 differential gene expression were observed with a2-fold difference(17 genes were up-regulated and 23 genes were down-regulated)2.3 The comparison between CGF group and icariin groupIn 2 weeks, 20 differential gene expression were observed with a2-fold difference(6 genes were up-regulated and 14 genes were down-regulated)In 4 weeks, 42 differential gene expression were observed with a2-fold difference(5 genes were up-regulated and 37 genes were down-regulated)3 Summary of 5 before the most regulated and down-regulated genes significantly different, and were compared between groups. Which icariin group and the control group in 2 weeks and 4 weeks have appeared HGF gene expression; CGF group and the control group in 2 weeks and 4weeks have appeared Runx2 gene expression; icariin group and CGF Group 2 weeks ZIC5 gene expression occurs, but in the 4 weeks showed not present.4 In Pathway analysis, ten different signaling pathways were involved:protein digestion and absorption, 5- HT synapses, ECM-receptor interaction, adhesion, tumor protein polysaccharides, PI3K-Akt signaling pathway, calcium signaling pathways, dilated cardiomyopathy, vascular smooth muscle contraction and nerve activity-receptor interactions.The third part Discussion on the main signal path icariin,autologous CGF promote rabbit skull defects of conduction.1 HGF m RNA expression in icariin and control group was detected by real-time PCR.Totla RNA in the control and Icariin group was reverse transcribed into c DNA. Real-time PCR was then performed using the specific primers. The melting curves were seen as a single peak, indicating that the primers were well desgined. The results showed that the HGF m RNA expression in the icariin group was significantly higher than that of control group.2 HGF and p-Akt protein expression in the icariin and control group were detected by Western blot.Total protein extracted from the icariin and control group were detected by Western blot analysis. The results showed that the protein expression of HGF and p-Akt in icariin group was significantly higher then that of control group.3 Runx2 m RNA expression in CGF and control group was detected by real-time PCR.Totla RNA in the control and CGF group was reverse transcribed into c DNA. Real-time PCR was then performed using the specific primers. The melting curves were seen as a single peak, indicating that the primers were well desgined. The results showed that the Runx2 m RNA expression in the CGF group was significantly higher than that of control group.4 Runx2 and p-ERK protein expression in the CGF and control group were detected by Western blot.Total protein extracted from the CGF and control group were detected by Western blot analysis. The results showed that the protein expression of Runx2 and p-ERK in CGF group was significantly higher then that of control group.Conclusion:1 CGF and icariin were able to increase the rabbit skull defect area into bone mass, improve healing quality.2 CGF and icariin in promoting the healing of rabbit skull defect,agreed to accelerate the healing of the skull defect rate in effect.3 In 2 weeks and 4 weeks, HGF and Runx2 factors are in icariin group, CGF group was significantly upregulated compared4 in icariin and CGF skull lesion genes are not identical, indicating that both play a role in different ways and mechanisms.5 icariin can promote the m RNA and protein expression of hepatocyte growth factor HGF;6 icariin promote HGF expression by activation of PI3 K / Akt signaling pathway, and further promote bone healing.7 CGF can promote Runx2 protein and m RNA expression.8 CGF activates ERK MAPK signaling pathway to promote Runx2 protein, play a function to promote bone healing. |
