| Bone tissue engineering scaffolds are very important in treatment of bone defects and other bone diseases. Hydroxyapatite is the main calcium and phosphorus compound in human body, and it is one of the most familiar material for bone tissue engineering. However, single hydroxyapatite scaffolds can not possess neither biological properites of bone, such as hematopoiesis, osteogenesis, and so on, nor function of promoting vascularization and inhibiting immune rejection, so it can not meet the requirements of human bone tissue. Previous studies found that Angelica polysaccharides(APS) have activities of cell proliferation promoting, vascularization promoting and anti-inflammatory. Therefore, we prepared bone tissue engineering scaffolds from the two kinds of materials and studied its application possibility in bone tissue engineering. First of all, we prepared Angelica polysaccharide by traditional water extraction and alcohol precipitation, then APS fractions was isolated and purified for antioxdation research and scaffold preparation. Secondly, hydroxyapatite was prepared by method of precipitation, and Angelica polysaccharide/chitosan/hydroxyapatite composite microspheres were prepared by reverse phase emulsion method for slow-releasing of Angelica polysaccharide release-slow. The hydroxyapatite and composite micropheres were made into calcium phosphate cement, and we optimized the preparation process, characterized the cement. Finally, hiscompatibility was studied with cortical defect and tibial bone defect of animal models. The results showed, that the extraction yield of Angelica polysaccharide was 12.6%. APS1 a, APS1 b and APS2 c were purified by DEAE-cellulose 52 cellulose column and Sephadex G-50 column. The hydroxyl radical and DPPH radical scavenging rate of APS2 c were 63.08 ± 0.962% and 93.31 ± 1.587% respectively concentration of 0.5 mg/ mL. DPPH radical scavenging rate of APS was 95.92 ± 0.936%, the total reducing force at absorbance measurement was 1.332 ± 0.136 at 0.5 mg/mL. The antioxidant capacities of APS and APS2 c were better than others, and APS was selected for follow-up studies at last. The composite microspheres were regular in shape and uniform in size, when stirring speed of 500 r/min was set, and its drug loading capacity and encapsluation rate were 32.9% and 78.4%. When releasing rate in vitro was tested, CMs released the highest amount of polysaccharide 1.028μg at first day, and reached close to stabilization after 2-14 days. The compressive strength of HAp-CMs and HAp after coagulation were tested 17 ± 3.5MPa and 18 ± 1.1MPa. After immersed in PBS 28 days, degradation rate of HAp-CMs was 11.54 ± 1.03 %, a litter faster than that of HAp 8.66 ± 0.41% at day 28. Before implantation, the composite scaffold showed irregular sheets and close structure by SEM. After implantation, the composite scaffold created large pore structure, increasing specific surface area, helped attachment of cells. In the fourth week of cortical defect experiment, we found that the cell proliferation and degradation of HAp-CMs was faster than that of HAp. The releasing polysaccharides of microspheres were like a gathering point, inducing a large number of cells attached. At the same time, a certain number of blood vessels were formed in the HAp-CMs scaffolds, and double-concave-disc shaped red blood cells appeared in the lumen. In the tibial bone defect experiment, blood vessels formation and bone cell growth promoting were also found in HAp-CMs. In summary, Angelica polysaccharide/hydroxyapatite composite material could be used for bone tissue engineering with very good prospect. |
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