| Osteogenic factors, such as bone morphogenetic protein (BMP) and basic fibroblast growth factor (bFGF), are expensive and somehow lack of stability, hence, limiting the development of bone tissue engineering to some extent. Searching for a kind of safe, effective and inexpensive growth factor has become the objective requirements of the bone tissue engineering. Icariin is a kind of traditional Chinese Traditional Medicine, has high medicinal value and health care effects, and being widely used in the treatment of osteoporosis. Its low price, stable nature, tolerance of disinfection and sterilization, moreover, the existing research has proved that icariin could promote the osteogenic differentiation of mesenchymal stem cells in the bone marrow, and its mechanism of osteogenesis was clarified at the cellular and molecular level, but the application in inducing adipose-derived mesenchymal stem cells into osteoblasts studies has not been reported.Degreasing and deproteinized bone powder is a sort of natural bio-derived materials, after some physical chemic pretreatment, the protein was skimmed off, the antigen was eliminated, and the remaining key components are hydroxyapatite and calcium carbonate, polylactic acid is one tpye of synthetic polymer material, has good biocompatibility and a wide range of applications in the biomedical field. These two categories materials are plentiful, composite preparation could compensate each other’s shortcomings mutually, leading to good properties in functional adaptation, organization blending, biodegradability, mechanical properties superior to single material. In this paper, there are two main purposes, one was to study the osteogenic differentiation effect caused by traditional Chinese medicine icariin onto adipose-derived stem cells, the other one was to prepare a series of drug-releasing composite scaffold of icariin-bone powder/polylactic acid, using polylactic acid as matrix material, deproteinized bone powder as the carrier of the drug, and and their physical and chemical properties and biological properties were studied.Isolation, culture and identification of human adipose-derived stem cells (hADSCs): Adult female surgical patients after informed consent, abdominal resection fat tissue was abtained for about500mg, cultured amplificated and passaged after digested by trypsin and collagenase. Observed cell growth under microscope, and cell growth curve of the5th passage hADSCs was drew and adipogenic, chondrogenic and osteogenic induction were detected. The results showed that the primary cells began to adhere onto culture flasks after24h, at the beginning cells were short spindle and sizes varied; with the increase of passage. cell grew faster, and vortex-shaped spiral growth, the cells were gradually purified and shape changed to long spindle, cells of5th generation entered into the logarithmic growth phase at the forth day, and entered into the plateau phase at the ninth day, and hADSCs could differentiated into adipocyte, chondrocytes and osteoblasts after induction,Osteogenic differentiation effect of icariin on human adipose-derived stem cells: Prepared different concentrations icariin medium, ranging from10-9M to1.0×10-3M, and cultured5th passage hADSCs, the effect of different concentrations of icariin on cell adhesion, proliferation were studied by CCK-8method, the cytotoxicity of icariin was discussed. In order to clarify impact of icariin on hADSCs proliferation, cells were inoculated for2×103cells/well (low density) intervened with icariin medium ranging from10-9M to1.0x10-5M, cell growth curve were plot by CCK-8assay. Cells were inoculated at2×107cells/well (high density) and intervened with10-9M-1.0×10-5M icariin medium, osteogenic differentiation of icariin on hADSCs were detected. The results showed that:the cytotoxicity of icariin increased with drug concentration, cytotoxicity grade of10-3M and104M was2. and cytotoxicity grade of icariin betweenl0-9M～10-5were all level;10-9M could promote cell proliferation; concentration from10-5M to10-8M could induce adipose stem cells differentiate to osteogenic, alkaline phosphatase staining was positive, and the the best osteogenic concentration was10-7M.Preparation of bone powder/PLA scaffold and its cell adhesion characteristics: After removed periosteum and bone marrow of quarantine inspection commercially available fresh pork bone, cutting into0.5cm×0.5cm×0.5cm small pieces, skim48h with methanol and chloroform; deproteinized24h by10%hydrogen peroxide, dried in goven after repeated wash. Bone was processed into granules prepared by mechanical milling method. The particle size distribution of the bone powder was detected by laser particle size analyzer; composition was tested by infrared spectroscopy spectrometer, surface structure was observed by scanning electron microscopy. Bone powder/PLA scaffolds was prepared by materials science, adhesion rate of hADSCs on PLA and bone powder/PLA scaffolds was compared. The results showed that the main component of skim deproteinized bone powder was hydroxyapatite and calcium carbonate, the median diameter was30.77μm, the surface under SEM was not smooth, and the rate of cell adhesion in the the PLA and bone powder/PLA scaffolds were47.71%and53.22%, respectively.Preparation of icariin-bone powder/PLA scaffold and its physicochemical property: Take a certain amount of icariin accurately and were dissolved in anhydrous ethanol. and bone powder as a drug-carrier was mixed with10%PLA solution, after ultrasonic dispersion, and the resulting mixture was poured into mold covered with paraffin microspheres. centrifuged at1200rpm for10min, and dried in vacuum drying oven for24h. Then the materials were removed and cut into a thickness of1mm for the next step, and small pieces were put into the cyclohexane solution in an air bath shock for72h, the medium was changed every12h in order to wash away the paraffin wax microspheres in the scaffolds. Dried in vacuum drying oven at40℃, the drug loading of10-5M,10-6M,10"7M and10-8M of ICA-bone powder/PLA composite scaffolds were obtained. The results showed that the porosity, diameter of bone powder/PLA material were91.52%and200to300μm, meawhile, after joining the ICA, porosity of ICA-bone powder/PLA material was decreased, but still greater than85%with a pore size ranging from200to300μm; FTIR analysis showed that icariin drug loading process did not affect the chemical properties of the bone powder/PLA material.Biological compatibility of icariin-bone powder/PLA hybrid scaffold:The cytotoxicity of icariin-bone powder/PLA scaffold and its osteogenic differentiation effects of extracts on the hADSCs were investigated, the5th passage hADSCs were vaccinated into24-well plates, at the density of5×105cells/well, n=6, when hADSCs adhering to plates, the original medium were drained off, and1ml of complete medium were added, at the same time,10-5M,10-6M and10-7M ICA-bone powder/PLA material were placed in each hole, and bone powder/PLA material was control group. Cultured in incubator under37℃,5%CO2, and culture medium was changed every2d, microscope photos were taken after7d. The expression of ALP was detected by ALP staining. The results showed that cells grew well without obviously dead, floating signs after48h,7d ALP staining showed that cells grew in the three drug loading dose ICA-bone powder/PLA scaffolds became short shuttle and other irregular shapes, cell surface showed a large number of secreted extracellular matrix, and gradually gathered, the number of ALP staining cells, dark in color,10-7M ICA-bone powder/PLA material ALP staining was strongly positive,10-7M ICA-bone powder/PLA material has the best osteoinductive effect. |
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