| To fabricate artificial synthetic materials based on the porous ceramic scaffolds by the technology of bone tissue engineering,which could meet the demand of bone tissue repariment in vivo,is one of the research hot spots in recent years.Many studies have found that macroporous structures of tissue engineering scaffolds affect their physiological behaviors,vascular growth and bone formation.However,it is difficult to characterize the effect of macroporous structures on cells in vitro.As a result,systematic understanding of the mechanism of osteoinduction in scaffolds remains lacking,limiting the development and application of bioceramic materials.Moreover,further modification of porous ceramic materials to impart improved biological activities is a critical task for-extending their applications in orthopaedics.This thesis investigated the roles of the macroporous structure of porous hydroxyapatite(HAp)scaffolds and the dynamic microfluidic environment during bone repair.A dynamic in vitro fluid perfusion system was developed to study the effect of scaffold macroporous structure on fluid field distribution,behaviors of cells cultured under fluid mechanical stimuli,and their relationship.The findings were expected to serve as a basis for the further optimization of the porous structure of HAp scaffolds.In addition,antimicrobial surface structures were constructed by small-molecular templating and doping of bioactive ions,which may provide preliminary insights for further enhancement of tissue engineering scaffold surfaces.The main scope and conclusions of this thesis are summarized as follows:1.A device was developed to prepare uniform sodium alginate gel beads.The size of the beads was adjusted by controlling the size of the injection needle.The mechanical properties of the beads were further enhanced by solvent exchange.Three-dimensional porous HAp scaffold with different macropore sizes were successfully prepared by one-step extrusion molding of a water-based HAp slurry using sodium alginate gel beads of different sizes as templates.Pore interconnectivity was controlled by adjusting the compression applied on the template.2.Bone marrow mesenchymal stem cells were isolated,cultured and identified by modified whole bone marrow adherence method.The proliferative capacity of primary cells was not affected after subculture.Flow cytometry results and induced differentiation experiment results showed that the multi-induced differentiation and stem cell-specific markers of different generations of cells were maintained.By optimizing the extraction steps,the controllable cell selection conditions(including cell adherence time,fluid exchange method and enzyme digestion parameters)were established to develop the isolation and culture system for bone marrow mesenchymal stem cells.3.Two types of scaffolds with different pore structure were selected as the carrier for cells.Static seeding,withdrawal seeding,and dynamic perfusion seeding were used to seed cells on scaffolds.Cell-binding rate,survival rate,distribution ability,and long-term proliferation effects on scaffolds were quantitatively evaluated.Results showed that the seeding rates of withdrawal seeding and static seeding were significantly higher than that of perfusion inoculation,and the two methods produced good cell viabilities.However,the static seeding method gave an inhomogeneous cell distribution.Subsequent batch experiments found that,the withdrawal seeding method resulted in even cell distribution unaffected by the number and pore structure of the scaffold,which could effectively improve the seeding efficiency.The three-dimensional porous HAp scaffolds and mesenchymal stem cells prepared by the withdrawal seeding method may be combined to form a cell-scaffold composite bone culture model for further in vitro study.4.HAp scaffolds of different pore sizes but same pore interconnectivity were used to construct mesenchymal stem cell-scaffolds complexes but withdrawal seeding for in vitro perfusion culture.The changes of flow field distribution and the protein/gene expression of osteogenesis differentiation on the scaffolds were studied by simulating the dynamic environment of microfluidic fluid in vivo.It was found that,the fluid shear strength in the perfusion culture promoted osteogenic differentiation of BMSCs cultured on the scaffolds compared with those cultured under static conditions.In the same interconnectivity(through hole size:macro hole size =1:4),the scaffold macro pore size(500-1300 μm)on the cell fluid perfusion stimulus had a critical dimension:when the scaffold macropores were reduced from 1300 to 800 μm,the increase of internal fluid shear force promoted the expression of ALP,Col-Ⅰ,OCN and OPN and the secretion of osteogenic protein ALP on the scaffolds.However,when the scaffold macropores were reduced from 800 to 500 μm,internal fluid shear of scaffold was so great that reduced the osteogenesis-related gene and protein level.Fluid shear stress introduced some external stimuli into the cell nucleus by activating the ERK1/2 pathway,thereby causing a cascade of biological reactions.Microfluidic flow simulation revealed that,the pore structure of the scaffold can affect the redistribution of the perfusion flow field,expanding the high velocity flow zone in the scaffold and intensifying the internal shear force distribution and shear force level.These mechanical signals influenced the biological behaviors of cells on different scaffolds even under the same level of stimulation.5.In the presence of inositol hexakisphosphate(IP6),micro/nano-structured and Cu2+-doped HAp(HAp-IP6-Cu)microspheres were successfully fabricated via a hydrothermal method.The results of scanning electron microscopy,X-ray diffraction and specific surface area showed that IP6 controlled the growth orientation of HAp crystals to produced micro-nano hybrid structure and increased the specific surface area.X-ray photoelectron spectroscopy showed that copper substituted calcium ions in the HAp lattice.Compared with the HAp micro-rods or IP6 modified HAp(HAp-IP6)microspheres,HAp-IP6-Cu microspheres had larger specific surface areas,better hydrophilicity and stronger ability to adsorbed bovine serum albumin which could promoted the adhesion,proliferation and osteogenic differentiation of osteoblasts.In addition,quantitative test by the agar disk diffusion showed that HAp-IP6-Cu microspheres were effectively against S.aureus and E.coli. |
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