| Objective To study the effect of lycium barbarum polysaccharides （LBP） on theexpression of BMP-2and TGF-β1during the process of fracture healing in rats andinvestigate the manifestation of histopathology and imaging during the process of fracturehealing.In order to explore the effect of LBP on fracture healing and the possible mechanism.Methods1403-months-old male rats were randomly divided into four groups: LBPhigh-dose group （H）, LBP low dose group （L）, Shangkejiegu tablets group （S） and distilledwater group （W）,35rats in each group. The femoral fracture healing models were establishedby internal fixation with Kirschner wires. The postoperative corresponding to each groupwere given the LBP160mg.（Kg.d）-1, LBP40mg.（kg.d）-1, Shangke Jiegu tablets320mg.（kg.d）-1and equal volume of distilled water foe intervention. Selected randomly10rats ineach group on2,4,6weeks of postoperative, To evaluate whether they have significantdifferences of bone healing by gross specimen observation of femur, measurement of thevolume of osteotylus, radiograph and HE staining. Observed the expression of BMP-2andTGF-β1in bone osteotylus by immunohistochemistry.Results1. imagic manifestations of fracture healing: At2weeks postfracture, eachgroup show visible osteotylus formation, less, mainly located in the the polyline proximal anddistal periosteum, fracture lines were visible. At4weeks, most of the specimens had beenfully formed Fibrocartilaginous osteotylus, some continuous osteotylus was across thefracture line, the fracture line was blurred and reached clinical healing standard. At6weeks,all groups achieved the standard of bone healing and fracture line disappears. There were nosignificant differences in the groups throughout the period of the study（P＞0.05）. 2. Gross observation and measurement of the volume of osteotylus: Each group of thesecond weeks after the fracture were seen obvious osteotylus formation, bulky, slightly softtexture, mild actived after removing the Kirschner wires. there were no distinction amonggroups by naked eyes. At4weeks, volume osteotylus were smaller, hard texture, static whenremoving the Kirschner wires, the texture of osteotylus was no difference between all groups.The volume of osteotylus in H group and S group were lager than L group and W group; At6weeks, when the volume of osteotylus in each groups further reduced, some specimensosteotylus has disappeared, osteotylus were alterated and shapedr, and there were nodistinction by eye view. Measure osteotylus volume,4weeks after fracture: S group, H groupcompared with the L group, W group were statistically significant （P＜0.05）.3. Histological observation:2weeks after fracture, fracture line were formed visibly atouter and inner membrane which in both ends of the bone, visible cartilage island formed bya large number of cartilage cells in its peripheral, fiber connection were also connected withthe fracture fragments. The bone formation and cartilage islands were larger in H and Sgroups compared with L and W groups. By4weeks postfracture, the trabecular bonecontinued to increase thickening and by arrangement. L group and W group is relativelysmall size of osteotylus, trabecular bone thin, sparse, part of the specimen between thefracture fragments remained fibrous tissue connection. The H and S groups showed betterhealing in the form of dense trabecular bone and osteotyluses size as compared to the L andW groups.6weeks after fracture, bone trabecula between the two fracture fragments oftrabecular bone of group S and Group H completely into lamellar bone marrow cavityrecanalization.Compared to the L and W groups, the trabecular was completely into lamellarbone and marrow cavity recanalization. Score based on the NILSSON histological gradingstandards, statistical processing, each of the S group, H group and L group, W group werestatistically significant （P＜0.05）, the second weeks and the sixth weeks was significantdifference（P＜0.01）. 4. The expression of BMP-2and TGF-β1in osteotylus: The expression of BMP-2waspositive in the cell cytoplasm of fibroblasts, chondrocytes, osteoblasts and bone trabeculas.The positive expression was in the same cell types, but their intensity is different. The majorcomponents of initial stage were osteoblasts, trabecular bone cells and outer peripheralportion of the osteotylus immature chondrocytes, and osteoblast cells in the late stage duringthe process of the fracture healing. The cytoplasm coloring depth was greater in S and Hgroups compared to the L and W groups. The MOD values of BMP-2expression in eachgroup continue to rise on2,4,6weeks after fracture. By2and6weeks postfracture, MODvalues are expressed as follows：S group＞H group＞L group＞W group，and H group＞Sgroup＞L group＞W group after4weeks.2weeks after fracture: S group compared with theH group, L group, W group and H group compared with the W group were statisticallysignificant （P＜0.05）, where S group and W group with significant difference （P＜0.01）;4and6weeks after fracture: S group, H group compared with the L group, W group and Lgroup compared with the W groups at6weeks were statistically significant （P＜0.05）.The expression of TGF-β1was similar with BMP-2in addition to the fibroblasts. Theexpression of MOD values in each group continued to rise at2,4weeks and decline at6weeks after fracture. The cytoplasm coloring depth and MOD values were expressed asfollows：S group＞H group＞L group＞W group. There were significant differences in S andH groups compared to the L and W groups（P＜0.05）.Conclusion1. LBP can promote the expression of BMP-2and TGF-β1on the fracturehealing process which exists dose-dependence relationship, and promote the expression ofBMP-2exists duration-effect relationship;2. LBP does not alter the cell types of expressBMP-2and TGF-β1, which has a significant influence in the expression of expressionalintensity.3. Compared to Shangkejiegu tablets, the effect of LBP on the expression ofBMP-2was weaker in the early fracture healing, the effect of LBP on the expression of TGF-β1was similar with Shangkejiegu tablets;4. LBP can accelerate the formation of osteoblast and promote the remodeling of early osteotylus in membrane bone formation andosteogenesis. However, this was only a manifestations in the micro-structure, which was notobvious on the macro-structure;5. LBP can promote the fracture healing, which are relatedwith promotion of the expression of BMP-2and TGF-β1. |
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