Experimental Research Of Poly(Lactic-co-glycolic Acid)/ Mesoporous Silica(SBA-15)Artificial Bone Scaffold Based On Electrospinning Technique

BACKGROUNDMany clinical bone trauma disease causes local bone defect including calcaneal collapsed fractures, compression fractures of tibial plateau and so on. Long bone defect occur after tumor resection, postoperative infection debridement of open fractures or Chronic osteomyelitis osteotomy. At present, the method of clinical healing of bone defect is mainly by autologous bone graft and allograft. However, a person’s autologous bone volume is limited and cannot meet a lot of bone graft or long bone defect repair. Although allograft sources is abundant, but expensive. And although reduce the processing of antigen, but there are still a few patients with rejection reaction. And after implanted absorption may occur, bone healing time is usually is longer than autogenous bone. Patients of bone substitute transplantation therapy is increasing year by year in domestic, the number is increasing along with the diversification of transportation, as well as the development of the construction and manufacturing, people health consciousness constantly improving after injury. Therefore, the use of tissue engineering technology to build a good bioactivity and biocompatibility of bone graft substitutes will have great social significance and great application prospect. Speeding up the study of new artificial bone not only can provides patients with economic, security, enough bone graft material, and is expected to be really conquer a lot of clinical bone defect repair problems.OBJECTIVEThis research use the electrostatic spinning technology, prepare composite scaffolds by PLGA compounding with mesoporous silica (SBA-15). Through the relevant testing and analysis including Surface morphology, microstructure and biocompatibility and osteogenetic activity, verify the biological activity of PLGA/SBA-15 composite scaffolds. To provide a good new artificial bone scaffold material for bone defect repair.MATERIALS & METHODSSBA-15 powder is prepared by three block polymer surfactant. SBA-15 samples surface morphology and pore size distribution is analyzed by transmission electron microscopy (TEM) and small Angle X-ray diffraction (SAXRD). The BET method/BJH method was used for the determination of SBA-15 specific surface area, pore volume. The SBA-15 powder is mixed with PLGA according to different mass fraction, then PLGA/SBA-15 composite scaffolds and pure PLGA scaffold were prepared by electrostatic spinning equipment. Using scanning electron microscopy (SEM) respectively observe four groups of artificial bone scaffolds containing the different content of SBA-15, and observe content include the surface morphology and microscopic pore structure. The four groups of artificial bone scaffolds are respectively analyzed by the water contact Angle tester and spectrometer (EDS), including hydrophilicity and distribution of element. adsorption quantity and the amount of slow release of the bone morphogenetic protein-2 (rhBMP-2) is determined by ELISA kit. Cell morphological observation, cell adhesion, cell proliferation rate is determined by HE staining, fluorescence staining, electron microscopy and cytoskeleton staining. Verify the biocompatibility of PLGA/SBA-15 composite scaffolds. Immerse four groups of scaffolds in simulated body fluid (SBF) and observe its biological mineralization, calcium salt deposition is through the alizarin red staining. Osteogenesis inductive effect is observed by alkaline phosphatase activity analysis of four groups of scaffolds.RESULTSUnder the electron microscope, the SBA-15 samples of synthesis has the distribution of uniform pore structure, its aperture size is about 6 nm. Small Angle X-ray diffraction results verify the effectiveness of the synthetic SBA-15 with high purity. synthetic SBA-15 has a larger specific surface area(732 m2/g) and specific volume(1.2 cm3/g). Scanning electron microscopy results show that the fibers are interlaced between these PLGA/SBA-15 composite scaffolds, and form a random felt sample and beaded structure. scaffolds have formed a uniform and random distribution of fiber mesh structure and interconnected three-dimensional channel With the increasing of SBA-15 content in scaffolds. Water contact Angle results show the water contact Angle reduced in a certain range as the scaffold of the improvement of SBA-15 percentage points, and increasing SBA-15 can improve hydrophilicity in a certain range. Energy spectrum analysis show Si element content of PLGA/10%SBA-15 scaffolds is significantly higher than other scaffolds. Silicon is evenly distributed in each sample, suggesting that SBA-15 is evenly distributed in the scaffolds. Soaking in SBF solution after 7 days, all scaffold surface is covered by layered or block of apatite. Compared with the pure PLGA scaffolds, the PLGA/2.5% SBA-15, PLGA/5% SBA-15, PLGA/10% SBA-15 scaffolds surface are covered with more apatite. PLGA/10% SBA15 scaffolds surface formed a more uniform distribution of layered apatite, and show improved scaffolds biomineralization by the introduction of SBA-15. ELISA results of rhBMP-2 adsorption amount f showed that PLGA/2.5% SBA-15, PLGA/5% SBA-15, PLGA/10% SBA-15 scaffolds show higher rhBMP-2 adsorption than pure PLGA scaffolds and PLGA/10% SBA-15 scaffold show the highest adsorption quantity. Scaffold adsorption quantity of BMP-2 is improved by the introduction of SBA-15. At the same time, PLGA/ 2.5% SBA-15, PLGA/5% SBA-15, PLGA/10% SBA-15 scaffolds show more slowly BMP-2 released rate than pure PLGA scaffolds. PLGA/10% SBA-15 scaffold show the most slow release rate and show scaffold slow-release effect of BMP-2 is improved by the introduction of SBA-15. DAPI staining fluorescence microscope results show that cells in each group scaffold survived well. PLGA/10% SBA-15 scaffold has higher cell density, and the join of SBA-15 is good for hMSCs cells proliferation on the scaffolds. MTT results showed that the quantity of cells on scaffolds was no significant different after 1 or 3 day cell culture. But after 3 day cell culture, cells in the PLGA/10% SBA-15 is more than pure PLGA scaffold and show the join of SBA-15 is good for hMSCs cells proliferation on the scaffolds. Cell morphology and spread is observed by scanning electron microscope. After 3h cell culture, Cell morphology of pure scaffold is a class of circular form. PLGA/SBA-15 scaffolds have more elongated cells. After 24h cell culture, PLGA/SBA-15 scaffolds have a wider range of cell spreading and more flat cells, PLGA/10% SBA-15 scaffold showed good cell adhesion and spreading, cells of spreading on the PLGA and PLGA/SBA-15 scaffolds of under actin microfilament fluorescent microscope present a random polygon and show cells in the scaffolds surface has good spreading. The number of pseudopodia form of cells in PLGA/ 10%SBA-15 scaffold is more than the pure PLGA scaffold and show the introduction of SBA-15 enhances the adsorption force of cell, and is conducive to cell adhesion and spreading on the scaffolds. Live/Dead staining results can be observed that PLGA/SBA-15 scaffolds have more alive cells than pure PLGA scaffolds. Cells survive on the PLGA/10% SBA-15 scaffold is relatively good and maintain a fibroblast sample form in the process of cultivating, show PLGA/SBA-15 scaffolds no cytotoxicity. ALP staining results show each group scaffolds perform low alkaline phosphatase activities on the 7 day, ALP concentration of each group scaffolds have obviously improved on the 14 day, concentration of ALP of PLGA/10% SBA-15 group than pure PLGA group, show the introduction of SBA-15 to hMSCs to osteoblast differentiation and osteogenic induction. Compared with the blank control group and pure PLGA group, ALP activity of PLGA/10% SBA-15 scaffolds loading with rhBMP-2 increased obviously. Compared with cell culture for 4 or 7 days, ALP activity of PLGA/10% SBA-15 scaffolds unloading with rhBMP-2 are increased in cell culture for 14 days. ALP activity of scaffolds loading with rhBMP-2 increased than before and show the introduction of SBA-15 enhance hMSCs osteoblast differentiation and rhBMP-2 controlled-release. Alizarin red staining results show calcium nodule distribution of PLGA/SBA-15 compounds scaffolds are more dense than that of blank control group and pure PLGA scaffold and the addition of SBA-15 enhance calcium salt deposition and biomineralization.CONCLUSIONSPrepared PLGA/SBA-15 composite scaffolds by the electrostatic spinning technology. SBA-15 evenly distributed in this kind of compound scaffold. This kind of scaffold has connectivity of three-dimensional network structure. The interconnected pore structure encourages the growth of cells and spreading. Adding SBA-15 can improve scaffold hydrophilicity in a certain range and enhance hMSCs osteoblast differentiation and rhBMP-2 controlled-release. Cells growing in such a complex scaffold showed good cell proliferation and adhesion, show this scaffold is no cytotoxicity and have a good biocompatibility. PLGA/SBA-15 composite scaffolds is more advantageous than pure PLGA scaffolds on mineral deposits, improve osteogenesis induction activity. the PLGA/SBA-15 composite scaffolds have great application potential in the field of bone defect repair.