Study On P Material Loaded From Calcium Phosphate Composite Of Bone Marrow Mesenchyme Stem Cells In Vitro

Objective:By inflammation, trauma and other causes of the alveolar bone defect and missing in clinical practice is very common, serious impact on patients with oral function and appearance can be. The use of regenerative medicine and tissue engineering alveolar bone defect reconstruction theory and technology is a research hotspot at present. Calcium phosphate cement(CPC) is recognized as having good biocompatibility and bone regeneration guided bone graft material, with mechanical strength, suitable for bone defect in high strength mechanical non load region filling, but its lack of biological activity of bone induction in vivo degradation and transformed to take a long time for bone organization. This study by loading the synthetic neural polypeptide substance P(SP) CPC composite materials, and type I collagen, simulating the composition and structure of natural bone, constructed with composite scaffolds for bone inducing activity of SD rat bone marrow mesenchymal stem cells cultured in vitro(BMSCs), to observe the material effect on the biological behavior of BMSCs the.Methods: 1. Material: buy the clinical use of calcium phosphate materials, artificial synthetic peptide substance P, rat tail tendon type 3%I collagen solution, divided into 3 groups: the preparation of composite scaffold. Group A pure CPC group; group B + type Icollagen group CPC; group C + CPC + P type I collagen material group. Using scanning electron microscopy(SEM) to observe the surface morphology of the sample holder.2. SD rats, BMSCs culture and identification: 4 week old female rats were decapitated after anesthesia, anatomy of femoral and tibial metaphysis, excision of the stem to the net soft tissue after PBS buffer out of the bone marrow, after centrifugation, transferred to the culture flask, adding alpha cell medium(containing DMEM 10% fetal bovine serum), CO2 cells were cultured in the incubator. The next day PBS washed and fresh medium to culture, then was changed every 2 days for 1 times. By measuring the osteogenic and adipogenic identification of BMSCs cell proliferation, logarithm.3. Effects of substance P on the biological behavior of BMSCs by BMSCs: the primary cultured P4, seeded in 96 well plates, were randomly divided into 2 groups, the experimental group culture fluid added substance P, were measured in 1, 3, 5, 6, 7 days cell proliferation logarithm, the difference between the two groups and drawing.4. Each material on the biological behavior of BMSCs by BMSCs: the primary cultured P4 generation were inoculated on each surface, SEM morphology, adhesion and proliferation of BMSCs, fluorescence staining of the proliferation and adhesion of BMSCs quantitative analysis, ALP assay for cell ALP activity, alizarin red staining of calcium deposits, osteogenic differentiation a comparison of BMSCs.Results: 1. Material: A, B, C synthesis of three groups of material surface morphologies were observed in SEM, A group visible material surface of a large number of nano apatite crystal structure, pore diameter is several microns; B, C two groups of materials and CPC collagen close association, coating on apatite crystal surface material, large pores. The funicular, up to tens of microns.2. SD rats, BMSCs culture and identification: the primary BMSCs vigorous growth, the growth curve of composite typical "S" shape, culture 3 days accounted for about 50% of bottom of the bottle, bottle about 5 days 70%, 7 days can be subcultured. After osteogenic and adipogenic induction medium for 3 weeks, alizarin red and oil red O staining, optical microscope were calcified nodules and lipid droplet formation, good BMSCs differentiation ability.3. Effects of substance P on the biological behavior of BMSCs cells: in 96 pore plate was 1, 3, 5, 6, 7 days of the logarithm of the proliferation of BMSCs, the experimental group(added substance P) BMSCs proliferation was significantly better than the control group(P<0.05).4. Each material on the biological behavior of BMSCs SEM under observation, the surface of A group BMSCs B, group C is less than the quantity of adhesion, the surface of BMSCs cells in A group were spindle shaped cell synapse is short; B, C group BMSCs cells showed irregular polygon, star, especially in the number of cells in collagen surface the long-term and mutually connected, and good cell spreading. After fluorescence staining, observed by fluorescence microscope, A, B, C three groups of material surface 1, 7, 14 days BMSCs the number of viable cells gradually increased, the 14 day has basically covered the dead, or the proportion of apoptotic cells decreased gradually, C group of living cells was slightly higher than that of A and B two groups. After 14 days of osteogenic differentiation by alizarin red staining, eye view group C darker, ALP detection of C group BMSCs ALP group was higher than that in A and B two.Conclusion:CPC collagen composite scaffold composite and loaded P material preparation, adhesion and proliferation of rat BMSCs have promoted BMSCs osteogenic differentiation, the expression of ALP was significantly increased, cell toxicity, has a good application prospect in bone regeneration by tissue engineering materials.