| Aim:1. To find out a way to prepare controlled release microspheres with ideal drug loading and encapsulating rate.2. To study the drug release function of Dex-GMA microspheres. Try to find the best match of drug release of the microspheres and the need of growth factors of seed cells.3. Try to get seed cells with enhanced adhesion and proliferation ability by adding controlled-release microspheres with certain compound growth factor.Method:1. Rats bone marrow stromal cells of separation, cultivate and phenotypic appraisal: We separate MSCs from bone marrow of SD rats by percoll gradient method. And we use flow cytometric to analysis cell cycle and determination MSCs surface antigen CD34, CD44 and CD45. 2. The test of bFGF and PDGF’s stimulating function for MSCs proliferation and adhesion: By adding 3.125, 0, to 6.25, 12.5, 25, 50, 100, 200 ng ? mL- 1 of bFGF, PDGF or bFGF and PDGF compound growth factor. Test MSCs proliferation and adhesion at 24, 48, 96 and 144h.Then find out an optimal concentration ratio between bFGF and PDGF.3. Preparation of microsphere controlled-release system: Prepare a series of particle size of Dex-GMA controlled-release system （DG-NPs） by the method of improved emulsion polymerization. And searching for the best reaction conditions for the preparation of microspheres. Select the optimized reaction conditions which can achieve satisfied controlled-release stability and drug loadings.4. The proliferation and adhesion effects study of microsphere controlled-release system loaded with compound growth factor: The groups are bFGF microspheres, PDGF microspheres and bFGF + PDGF composite microsphere groups. We add 0, 3.125, 625, 12.5, 25, 50, 100, 200 ng?mL- 1 bFGF microspheres, PDGF microspheres, bFGF+PDGF composite microspheres into the medium. After 24, 48, 96, 144h couture at the condition of 37℃, to 5% CO2 and saturated humidity, we make comparison of cell proliferation and adhesion by counting cell quantity and CCK-8 determination. Use Weston blot method to determine collagen typeⅠandⅢthat MSCs secreted. Use flow cytometric to analysis cell surface antigen characteristics.Results:1. The bone marrow stem cells were taken from femur bone of SD rats. After three times of subcultring the cell quantity can achieve 107. The magnitude of cell can satisfy the demand of request of seed cells of tissue engineering. 2. We found bFGF and PDGF had great effect on promoting MSCs proliferation: At 24h and 3.125 ng·mL-1 bFGF group （value: 0.180.01） vs. blank group （value: 0.12 0.02） had significant difference （p < 0.05）, and the concentration of 100ng·mL-1 of bFGF and PDGF have the most significant proliferation result （p < 0.01）. And compare to bFGF PDGF with same dose bFGF and PDGF compound growth factors have a greater proliferation result （p < 0.05）.3. When we set the stirring speed at 20rpm and emulsifier at 1/25 dose of total drug use. We acquire microspheres with smooth surface and the sizes of the spheres were between 20-40μm. The encapsulation efficiency was 88.2 % and controlled-release effect reaches 30 d, and no side effect to cells.4. BFGF controlled-release microspheres compare to bFGF: At 24h with 25ng·mL-1 concentration, the cell proliferation function of bFGF（0.170.01） and bFGF（0.19 0.02） microspheres groups had significant difference compared to the control group（0.13 0.02） （p<0.05）. At 48h bFGF microspheres group compared to bFGF group in the proliferation effect with no statistically significant difference （p>0.05）. At 96h dose above 25 ng·mL-1 bFGF microspheres had greater MSCs proliferation effect than pure bFGF group （p < 0.05）.5. PDGF controlled-release microspheres compare to PDGF: At 24h with 6.25ng·mL-1 concentration, the cell proliferation function of PDGF（0.150.01） and PDGF microspheres（0.16 0.01） groups had significant difference compared to the control group（0.13 0.02） （p<0.05）. At 48h PDGF microspheres group compared to PDGF group in the proliferation effect with no statistically significant difference （p>0.05）. At 96h dose above 6.25ng·mL-1 PDGF microspheres had greater MSCs proliferation effect than pure PDGF group （p < 0.05）. 6. bFGF and PDGF controlled-release microspheres compare to bFGF and PDGF: At 50ng·mL-1 after 144 h, bFGF and PDGF microspheres had greater MSCs proliferation effect than bFGF and PDGF group （p < 0.05）. Each concentration of bFGF + PDGF controlled-release microspheres has greater proliferation effect compare to bFGF controlled-release microspheres group （p < 0.05）. 24h, above 12.5 ng·mL-1, and above 48h all concentrations bFGF + PDGF controlled-release microspheres have greater proliferation effect compare to PDGF controlled-release microspheres group （p < 0.05）.7. Controlled-release microspheres compare to non-microspheres group: At 24 h a concentration between 6.25 ng·mL-1 and 100 ng·mL-1 cell adhesion count, both controlled-release microspheres and non-microspheres group have significant higher cell adhesion effect （p < 0.05）. But there is no significant difference between controlled-release microspheres and non-microspheres group （p > 0.05）. BFGF + PDGF composite controlled-release microspheres compare to PDGF microspheres and bFGF microspheres have significant cell adhesion effect （p < 0.05）.8. The compare of collagen type I and type III secretion: bFGF + PDGF composite controlled-release microsphere group （collagen secretion type I 26.79, type III 26.54） compare to bFGF + PDGF composite group （collagen secretion type I 13.18, type III 16.82） were significantly higher （p < 0.05）. And both group had significantly higher collagen type I and type III secretion compare to the counter control group （p < 0.05）.Conclusion:1. The improved emulsion polymerization method we used to prepare controlled-release microspheres can have satisfactory encapsulation efficiency, slow-release effect drug-loading rate. The microspheres prepared under 200RPM had satisfactory drug delivering characteristics for tissue engineering seed cells.2. The compound growth factor microspheres of bFGF and PDGF had an obvious simulative effect on MSCs proliferation and secretion. Continued low doses of growth factors stimulate doesn’t cause MSCs phenotypes change.3. bFGF controlled-release microsphere and PDGF controlled-release microspheres both contribute to cell proliferation and adhesion of MSCs. At 96h dose above 25 ng·mL-1 bFGF controlled-release microsphere had higher proliferation effect than pure bFGF. At 96h dose above 6.25 ng·mL-1 PDGF controlled-release microsphere had higher proliferation effect than pure PDGF group.4. At 144h above 50 ng·mL-1, compound growth factor controlled-release microspheres compare to non-controlled-release growth factor group had significantly higher proliferation effect of MSCs. At all concentration bFGF + PDGF controlled-release microspheres had significantly higher proliferation effect compare to bFGF controlled-release microsphere group. Above 24h and 12.5 ng·mL-1 and all concentration above 48h bFGF + PDGF controlled-release microspheres had significantly higher proliferation effect compare to PDGF controlled-release microsphere group.5. bFGF + PDGF controlled-release microspheres had significantly higher adhesion effect compare to bFGF controlled-release microsphere group and bFGF controlled-release microsphere group at all test points.6. The collagen type I and type III secretion of bFGF + PDGF composite controlled-release microsphere group compare to bFGF + PDGF composite group were significantly higher. And both group had significantly higher collagen type I and type III secretion compare to the counter control group. |
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