Topography Construction And Cellular Responses Of Polymeric Microsphere/Scaffold For Bone Repair

Bone defect is a commonly seen surgical disease. The traditional approaches of bone graftshave their own problems. For instance, although the autograft has an ideal repair outcome, ithas to sacrifice the patient’s normal tissue. And the allograft is accompanied by the risk ofinfection and immune rejection and limited source. The advent of bone tissue engineeringprovides a new choice for the bone defect therapy. The surface of bone repair implantcontacted directly with cells, which is an important approach to regulate the cell behaviors.Numerous studies have proven that the surface topography can affect cellular adhesion,migration, proliferation, differentiation and so on. The aim of this project is to constructmacropores topographies on composite microspheres and scaffolds, and to investigate thecorresponding cellular responses. This may enhance the function of these substrates,providing guidance for the design of novel repair device.(1) We prepared PLGA/PCL blend microspheres with island-sea topography using phaseseparation technique. In emulsion, the surface of the oil droplet solidified fast, allowing littletime for the movement and assembly of polymer chains. PLGA and PCL had to separate inlateral way, producing PLGA islands on the surface. Polymers in the internal part of the oildroplet had more time to move and assemble. So PLGA and PCL separated in stratified way,producing the PLGA core and PCL shell. The existence of PLGA islands could improve thehydrophilicity of the microsphere, generating a surface with zones of different hydrophilicity.Therefore, the stem cells on microspheres tended to adhere onto the more hydrophilic PLGAislands, which could be used to control the distribution of cells on the microsphere vehicle.(2) We used inorganic particles as pore-forming agent to obtain PLGA/HA/CC compositeporous microspheres without using any additional porogens. The microspheres had bothsurface macropores and internal cavities. The action of HA to attract water was the key factorin the formation of surface macropores and internal cavities; while CC contributed to theopening of the macropores via gas release. The macropores improved the hydrophilicity of themicrosphere, while the internal cavities improved the suspension. Moreover, the inorganiccomponents could neutralize the acidic products from PLGA degradation. Pig synovialmesenchymal stem cells adhered and proliferated well on the surface of the PLGA/HA/CCmicrosphere, implying its potential as cell vehicle in bone repair.Then we simplified the formulation of the microsphere and obtained PLGA/HA compositeporous microspheres. HA was confirmed to induce the formation of the macropores. Theeffect of starting formulation on the topography of the microsphere was studied in detail. It was found out that the topography was not sensitive to the contents of HA and concentrationof PLGA, but to the size of HA. We could obtain topographies with different densities ofmacropores via using different sizes of HA particles. The macropores of these microspheresfaded with degradation, accompanied by comparable ion release. Fetal mesenchymal stemcells were cultured on these microspheres and the adhesion, proliferation and osteogenicdifferentiation were evaluated. It turned out that topography with medium density ofmacropores (0.0070ml/g) benefited best the osteogenic performance of the cells.Based on the role of HA playing in the macropores formation, we tried to preparePLGA/CC composite porous microsphere. The size of CC was found to be the key factor inpore formation. This PLGA/CC microsphere was advantageous over traditional microspheresin degradation pH and mechanics. The topography of PLGA/CC microsphere could beflexibly regulated by adjusting the starting formulation. After qualitative and quantitativecomparison, we chose three groups of microspheres different in the number and size ofsurface macropores. The three microspheres had specific evolution of topography and Ca2+release with degradation. Mouse mesenchymal stem cells were cultured on the mircospheresto figure out the cellular responses. It was found that cells adhered and proliferated best onmicrospheres with lest macroproes.(3) We prepared PLGA/CC scaffolds with multi-level pores via low temperature fusingtechnique. There were two types of pores in the scaffold: pores between microspheres andsurface macropores of the microspheres. Since the scaffold was obtained under mildtemperature (37oC), the loaded drug (BMP-2and vancomycin) had higher activity whencompared with counterparts in scaffold prepared via heating. The surface of the PLGA/CCscaffold had slight hydrolysis, which didn’t influence the cellular performance. The effect ofpore structure of the scaffold on cell function was invesitaged. It turned out that scaffolds withsmaller pores (54.2μm, porosity53.8%) had a relatively higher proliferation, while theosteogenic differentiation of stem cells was better on scaffolds with larger pores (72.3μm,porosity46.6%). The cellular response to the size of surface macropores of the scaffold werealso investigated. The results showed that cell proliferation was not sensitive to themacropores size (8.77μm vs.5.44μm), while cells differentiated better on large macropores.In summary, the topography design on substrates can, to some extent regulate the behaviorsof stem cells, providing a new choice and scientific reference to design bone repair devices.