Effect of Sheng-Ji liquor on in vitro osteogenesis of bone marrow-derived mesenchymal stem cells in rabbits / 中国组织工程研究

ABSTRACT

BACKGROUND: The Chinese medicine, Sheng-Ji Ointment, is utilized to cure bone defect due to infectious open fractures in the clinical field. So it was imagined that it was a new inductive factor of osteogenesis and its ingredients could acceleate the proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) and induce them into osteoblasts. In our primary experiment, the Sheng-Ji liquor, which was extracted from the same Chinese herbs with Sheng-Ji Ointment, had been identified that it affected the proliferation of BMSCs with its different concentration.OBJECTTVE To investigate the effect of Sheng-Ji liquor on the osteogenesis of in vitro cultured BMSCs from rabbit tibia.DESTGN An observation in single kind of sample.SETTING: Orthopaedic Research Institute of Tianjin Hospital.MATERIALS The experiment was carried out in the Tissue Engineering Laboratory of Tianjin Orthopadic Institute of Integrated Chinese and Western Medicine from October 2004 to December 2005. BMSCs were obtainedfrom male healthy Japanese long-eared rabbits of 3 months old, (3.5±0.5) kg. Sheng-Ji liquor (5×102 g/L) was extracted from the Chinese herbs with Sheng-Ji Ointment (Tianjin Factory of Chinese Crude-drug and Cut Crude-drug), including the crude drugs of angelica, rehmannia dride rhizome, carapax testudinis, corium elephatis and crinis carbonisatus.METHODS: The Sheng-Ji liquor was put to BMSCs in different concentrations of 25, 8.3, 5 g/L. Basic medium without Sheng-Ji liquor was taken as blank control, while the standard-controlled medium contained the ingredients of the following blank-controlled medium, dexamethasone 10-8 mol/L, vitamin C 0.05 g/L and β-sodium glycerophosphate 10 mmol/L. The BMSCs passaged in different conditions were seeded in plate and cultured for 2-3 weeks, then the mineralized nodes were observed under inverted fluorescence microscope, and the cell expressions were observed with acheomycin labeling and collegan Ⅰ immunohistochemical staining. The culture medium was collected to determine the contents of extracellular and intracellular alkaline phosphatase (ALP), osteocalcin (OCN) and lactate dehydrogenase (LDH), then the ratios of ALP/LDH and OCN/LDH were calculated.MAIN OUTCOME MEASURES: After 2 and 3-week culture, the mineralized nodes, results of acheomycin labeling and collegan Ⅰ immunohistochemical staining, and the ratios of ALP/LDH and OCN/LDH were observed.were prolonged, proliferated assembly and overlapped. The inter-cell limits disappeared gradually and sporadic cellular Results of acheomycin labeling and collegan Ⅰ immunohistochemical staining There were positive cells and mineralized nodes in all of groups with Sheng-Ji liquor and standard-controlled group. BMSCs of the blank-controlled group were negative. The positive cells and mineralized nodes were green with yellowish color under irrigation of fluorescence; The results of immunocytochemistry for collagen Ⅰ were positive in all of groups with Sheng-Ji liquor and standard-controlled group. The result of blank-controlled group was negative. The positive induced BMSCs had the cytoplasm with granula of highest as compared with the other groups (P ＜ 0.05). The ratios of ALP/LDH and OCN/LDH in the other groups were close (P ＞ 0.05).