The Preparation. Characterization, Biological Safety Evaluation Of SiO₂Mesoporous Materials With Oxytocin

Tissue engineering is an approach to rebuild tissues or organs by use of bioactivesubstances in vitro culture, which is called "regenerative medicine". When tissues ororgans is damaged in case of physical, chemical, mechanical or pathological factors,and get lost in integrity and function completely or incompletely, autologoustransplantation used to be the traditional method for repairation. Through it iseffective in some extend, but secondary injury accompany with operation is frustrated.While immune rejection is also unavoidable in allografts. New hope comes from aconception named “tissue engineering” that may pull through the dilemma aboveperfect.There are three keys as tissue engineering.(1) cells: they are the basic units toconstitute organisms.It is essential for seed cells to own abilitise that can proliferateactively and differentiate in to specific tissue or organ directionally.(2) scaffolds: itcan provide a integrated framework in which cells may proliferate and differentiateinto tissues or organs.Besides cells induction and biocompatibility,carrying signalsincluding drug molecules, proteins, cytokines is also indispensable.(3) signal: theyare going to regulate cells proliferate, differentiate and other activities. The are mainlyrefer to some bioactive proteins and active factors in narrow sense, but also includingsound, light, telecom, stress, etc all influence factors that can interfere the cells grow.Our experiment was supported by natural science foundation youth project ofscience department of Jilin province China and PhD interdisciplinary projects of Jilinuniversity. Human adipose mesenchymal stem cells were used as seeding cells fortheir widely used in the tissue engineering. Mesoporous material SBA-15wasprepared and characterized. After comfirmed osteogenesis promotion of OT toh-ADSCs, we did some loading and releasing tests of OT into SBA-15. Finally,biological toxicity and biological compatibility of the materials was evaluated in our experiment. Hope that our researches may have a contribution to further studies.h-ADSCs was used in this study, which possess multiple differentiationfunction as an autologous stem cell. It originated from ectoderm ectomesenchymaland existed in adipose tissue in quantity. For its abudance and convenience, especiallynon-legal and ethical issue, h-ADSCs would be the most popularize in the future.SiO2ordered mesoporous materials have been used as drug carrier for more than10years. The more widely useage than traditional materials mainly came from twofeatures of this biological ceramic material drug carrier system: highly controllableproperties in physical and chemical and the potential in terms of material stimulus andresponse. The excellent characteristic of the material that can bond with specificbioactive molecules by covalent to stimulate target cells grow is just the demand ofthe composite tissue engineering material, especially in bone tissue engineeringmaterial. We perpared SiO2ordered mesoporous materials SBA-15by way oforganic template methods, then did some material characterization and biologicalactivity test in vitro.Oxytocin is a kind of important estrogen, its functional receptor belongs to Gprotein coupled receptor family. Evidence have shown that it’s a key hormone of bonemetabolic regulation.OT was evaluated in this experiment in stimulating h-ADSCsproliferation and osteogenic differented. At the same time, the drug loading andreleasing tests of OT in SBA-15system were done to lay the foundation of animalstudies for further.1Isolation, cultures and Identification of hADSCsObjective: To isolate, culture and identify hADSCs.Method: Obtain abandoned adipose tissue from healthy people legally. Aftersome processing, incubate cells in DMEM with10%FBS,100U/ml double antibody,changed medium every two days, then subculture with trypsin, collect3~4passagecells for use. Observe the cell morphology with microscope. Cell surface markersincluding CD44, CD29, CD34and CD45were tested with FCM(Flow Cytometry).Inducted cells with osteogenesis medium and evaluated its osteogenic differentiation potential by way of Alizarin red staining. Inducted cells with adiposed medium andevaluated its adiposed differentiation potential by way of Red oil O staining.Result: Cells of3~4passage grew in good condition in long shuttle shape, clearnucleolus and obvious boundary which is similar with fibroblasts. During FCM test,CD44and CD29were high positive that belong to mesenchymal cell surface markers,CD34was negative that belong to hematopoietic stem cell and endothelial cell surfacemarkers, CD45was negative which belong to white blood cells characteristic surfacemarkers. The results above proved these were pure h-ADSCs. In Alizarin red stainingcalcified nodules were observed clearly, proving that cells were of osteogenicpotential. In red oil O staining lipids were observed clearly, proving that cells were ofadiposed potential.2Influence of OT to h-ADSCs on osteogenic avtivity.Objective: To evaluate the influence of OT to h-ADSCs on proliferation andALP activity, to test the minimal effective concentration of OT to h-ADSCs.Method: WST-1: the samples were divided into four groups, high-concentrationgroup (100nmol.L-1), mid-concentration group (50nmol.L-1), low-concentration group(10nmol.L-1) and blank group, to observe the cell proliferation in1d,7d and14d.ELISA: to set groups as above, to tset ALP activity by Elisa Kit in7d and14d.Result: WST-1have shown that, in low concentration team proliferation activitywas a little higher than bank group at1d,7d and14d, but without significantdifference; mid and high teams both show significant difference at the first day oncells proliferation, and this effect came obvious gradually over time to peak at14d, atthe same time the influence show a dose dependent. ELISA: the test revealed that theALP activity was a little higher than blank group but without significant difference inlow group, while the ALP activity of mid and high groups were increasing high topeak at14d, and show a dose dependent.3Synthesis and characterization of silica mesoporous materialSBA-15Objective: Synthesis of SBA-15mesoporous materials, and characterization of the structure and properties.Method: P123was dissolved in an appropriate amount of deionized water, HCLand TEOS was added into system in the35~40degrees, then magnetic, stirrer formore than24hours of intense mixing. Put system in ethylene bottles crystallizationfor72hours, filtered, washed, dried, roasted at550℃, washed repeatedly with anorganic solvent to remove the organic template,. Finally, obtained white powdermaterial SBA-15by filtering, washing and drying, grinding. Analysis andcharacterized sample with Elemental Analyzer，field emission scanning electronmicroscopy, transmission electron microscope, X-ray diffraction and so on.Result: Element analysis showed that the synthetic material of high purity Si-Oinorganic compounds. Scanning electron microscope showed that powder was20~50microns in diameter particles, particle surface is rough nanostructure. Transmissionelectron microscopy showed particles within a large number of nanopore structure.XRD test showed that samlpe has a homogeneous structure. Nitrogen adsorption testshowed IV type adsorption curve.Finally the sample was high purity SiO2mesoporousmaterial SBA-15.4. Loading and releasing test of OT in SBA-15Objective: Use OT as a model drug, assembly of OT/SBA-15system and releasedynamics characteristics.Method: Drug assembly and release experiments were performed with OT, themaximum loading capacity, the optimum concentration and time, release kinetics wastested.Result: OT reached maximum loading capacity about35%mass at theconcentration of0.06mol/L for6hours. In releasing test, OT/SBA-15powder materialsystem showed burst release phenomenon about more than40%in the first4hours.Then the releasing rate was slow down, until32hours, with nearly90%of the drugrelease.5. SBA-15biological activity in vitroObjective: Detecting the cytotoxicity and biocompatibility of SBA-15material. Method: Preparation of different concentrations of SBA-15extraction, testedthe cell toxicity by MTT. Did hemolysis test in vitro, direct contact with experimentalwith MG-63human bone cell line. Set SBA-15group, OT/SBA-15group and theblank control group, detected intoosteogenic differentiation effect in human adiposetissue derived mesenchymal stem cells. Detect the biocompatibility in vivo by injectdrug loading system into bone defect area, make imaging detection andhistomorphology detection.Result: In the cytotoxicity assay,2times to20times the concentration ofSBA-15material extract showed no cytotoxicity. In vitro hemolysis experiment thehemolysis rate was3.1%, lower than5%, did not cause acute hemolytic reaction.Direct contact experiments showed that the cells grow well on the surface of thematerial. In Elisa, SBA-15material showed no effect on human adipose tissuederived mesenchymal stem cells Osteoblastic differentiation, and OT/SBA-15drugcarrier system obviously promote human adipose tissue derived mesenchymalstem cells of the osteoblastic differentiation andenhanced alkaline phosphataseactivity. In vivo experiment show that SBA-15has a good biocompatibility andOT/SBA-15may promote bone formation.