| Calcium phosphate is the main formulation of hard tissue and has been used as the good carrier of various drugs. It has good biocompatibility, osteoconductivity and forms the bone bonding with bone. The most representative are hydroxyapatite(HA) and biological activities Calcium phosphate cement (CPC).However, the limited osteoconductivity of calcium phosphate biomaterials is difficult to satisfy the requriment in clinic use. Therefor, usually different drugs or biological factors were added in calcium phosphate biomaterials to promote bone healing. There are a large number reportes of calcium calcium phosphate loaded drugs. Traditional Chinese medicine (TCM) has been used to promote the healing of bone in China for a long time. In recent years, calcium phosphate loaded TCM have been widely reported. In our previous study, calcium phosphate loaded TCM were preparated and characterizated, furthermore, TCM in vitro release from calcium phosphate biomaterials has been studied. However, the orthopedic disorders often require the participation of multiple drugs. Therefore, based on the theories of blood circulation, tonifying and bone health, three kinds of Chinese medicine herbal extracts was chosed:Salvianolic acid B (Sal B), Astragalus polysaccharides (APS) and Naringin respectively. HA and CPC were used as the carriers for loading the three drugs respectively. The aim of this study was to determine the active ingredients of three kinds of Chinese medicine and their effective concentration on osteoblasts, the amount of the drug loaded in HA and CPC, the cell compatibility and the in vivo osteogenesis of CPC loaded three kinds of Chinese medicine.Hydroxyapatite (HA) powders containing different amounts of Astragalus polysaccharides (APS), Naringin and salvianolic acid B (Sal B) were synthesized by a wet chemical method. The effect of APS, Naringin and Sal B on the crystal structure, crystallinity and crystal size of HA was characterized by X-ray Diffraction (XRD), the particle size and specific surface area of HA were detected by Laser Partical Size Distribution Analyzer and Automated Surface Area and pore size Analyzer respectively. Furthermore, the effective concentration of APS, Naringin and Sal B on MC3T3-E1 osteoblasts. HA/APS, HA/Naringin and HA/Sal B on osteoblastic activity and differentiation were evaluated by Alamar blue, Methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase assay (ALP). Cell morphology was observed by light microscopy and scanning electron microscope (SEM). CPC was composed of HA loading with three kinds of Chinese medicine and a-TCP powder at a mass ratio of 1:1, the ratio of solid to liquid was 1 g:0.6 ml, and which were noted as CPC/APS. CPC/Naringin and CPC/Sal B. respectively. XRD was used to analyze the crystal structure before and after CPC hydration. Setting time and compressive strength were measured. The osteoblastic activity was evaluated by Alamar blue. The CPC was implanted in the femur of rabbits and remove the samples after 2,4 and 8 weeks, respectively. Diet and activities were evaluated after animal experiments, X-ray and Marco observation were carried out. Hematoxylin-eosin (HE) and Masson staining were used to evaluate the osteogensis of CPC containing medicine in vivo.It was showed that the crystal structure, crystallinity and crystal size of HA were not obviously affected when the amount of APS from 100 to 600μg, Naringin from 100 to 400μg and Sal B from 50 to 200 fig. respectively. The average particle size of HA was 1.17μm and specific surface area was 132.194 m2/g. The effective concentrations of APS, Naringin and Sal B on osteoblasts were time and dose-dependent. The concentrations within 80-200μg/mL of APS and 50-150μg/mL of Naringin enhanced the cells activity and ALP expression but Sal B did not have obviously effect. Osteoblasts had integrated morphology. HA/APS, HA/Naringin and HA/Sal B enhanced osteoblastic activity, proliferation and ALP. Osteoblasts cultured on HA/APS, HA/Naringin and HA/Sal B appeared the integrated morphology, in particular, it was obvious for HA/APS in cell culture.The results of CPC loaded different medicines showed that the adding of Naringin and Sal B did not influenced on the transformation of a-TCP to HA but APS had inhibited the transformation of a-TCP to HA. The CPC had the relative lower compressive strength. The initial setting time and final setting time were about 5 and 13 min respectively;CPC/APS mainly promote osteoblast proliferation in the early stage, whereas CPC/Naringin and CPC/Sal B promote osteoblast proliferation both in the early and late stage. In vivo animal experiments showed that the activities and diet rabbits had reduced at the early stage and recovery later. X-ray showed that the defect of the bone gradually heals as the time prolonged. Histological observation showed that the CPC implantate loaded with medicine could enhance the bone repair. CPC/APS and CPC/Naringine resulted in a certain inflammatory response, but there was no effect on bone repair. CPC/Sal B did not result in the inflammation. Masson staining showed that three kinds of Chinese medicine could promote the osteoblast activity and lead to expression of more collagen. This phenomenon was remarkable after implanted for two weeks. The bone defects were gradually repaired, which was in agreement with those of HE staining.It was summarized that it is feasible to prepare calcium phosphoric loading with three different kinds of Chinese medicine. They could not only enhance osteoblasts activity, proliferation and differentiation but also could promote the defects repair certainly. It was suggested that calcium phosphoric loading traditional Chinese medicine was a potential bone replacement in clinic use. |
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