Icariin Promotes The Maturation And Differentiation Of Osteoblasts Cultured In Three-Dimensional Collagen Hydrogels
Keywords
Extracellular Matrix Epimedium Brevicornum/Administration & Dosage Icariin Hydrogel Collagen Osteoblasts/Drug Effects Cells, Cultured
Abstract
Objective: To study the effect of icariin on the differentiation and maturation of neonatal rat calvarial osteoblasts (ROB) cultured in three-dimensional collagen hydrogel. Methods: The skulls of neonatal SD rats were taken, and ROBs were cultured by enzymatic digestion method. The ROBs of the first passage were embedded in 2 mg/mL type I rat tail collagen for three-dimensional culture. After 48 hours, fluorescein diacetate/iodine was performed. Cells were stained with propidium chloride (FDA/PI) and hematoxylin-eosin (HE), and the cell growth status was observed with a scanning electron microscope. Icariin with final concentrations of 1¡Á10-4, 1¡Á10-5, 1¡Á10-6 and 1¡Á10-7 mol/L was used for intervention respectively, and the cells were detected after 3, 6 and 9 days of culture. Alkaline phosphatase activity in the three-dimensional gel was used to screen the optimal drug concentration; after intervening ROB in the three-dimensional gel with the optimal concentration of icariin for 12, 24, 36, and 48 hours, real-time quantitative PCR and Western blotting were used to detect bone morphology. The expression of BMP-2, Runt-related transcription factor 2 (RUNX-2), Osterix mRNA and proteins. Results: FDA/PI staining, HE staining and scanning electron microscopy of ROB cultured in three-dimensional collagen hydrogel showed that ROB were evenly distributed in collagen hydrogel and were in good growth status. Icariin with final concentrations of 1¡Á10-5, 1¡Á10-6, and 1¡Á10-7 mol/L can significantly increase the alkaline phosphatase activity of ROB in three-dimensional gels, among which 1¡Á10-6 The alkaline phosphatase activity of ROB was the highest after intervention with mol/L concentration of icariin. 1¡Á10-6 mol/L icariin can significantly increase the expression of BMP-2, RUNX-2, Osterix mRNA and protein. Conclusion: Icariin can significantly promote the expression of osteogenesis-related factors in three-dimensional cultured ROB, suggesting that icariin has strong osteogenesis activity on ROB three-dimensionally cultured in collagen hydrogel. Abstract: Objective: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. Methods: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1¡Á10-4, 1¡Á10 -5, 1¡Á10-6 and 1¡Á10-7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2(BMP-2), Runt-related transcription factor 2(RUNX-2) and Osterix were detected by real time RT -PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. Results: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1¡Á10-5, 1¡Á10-6 and 1¡Á10-7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1¡Á10 -6 mol/L was the optimal concentration. Besides, icariin(1¡Á10-6 mol/L) increased mRNA and protein expression of BMP-2, RUNX-2 and Osterix compared to control group. Conclusion: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly. Key words: Extracellular matrix Epimedium brevicornum/administration & dosage Icariin Hydrogel Collagen Osteoblasts/drug effects Cells, cultured
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Original research was done by Xie Yanfang, Wang Minggang, Chen Keming, Shi Wengui, Zhou Jian, Gao Yuhai
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