Study Of The Hierarchical Structure And Component Regulation Of Calcium Phosphate And Its Osteogenic Performance

Objective:The natural extracellular matrix of bones is a porous tissue with a hierarchical porous structure,formed by the delicate assembly of calcium phosphate and organic matrix in three-dimensional（3D）space.In tissue engineering,scaffold materials are used to mimic the extracellular matrix-like function and facilitate bone repair.An ideal bone tissue engineering scaffold should mimic the structure and function of the bone extracellular matrix to efficiently promote bone repair,following the bionic concept.This thesis utilizes the natural polysaccharide guar gum（GG）and calcium phosphate to create ultra-long calcium phosphate fibrous hydrogel in a specific environment.These hydrogels are then used as structural substrates to obtain 3D interconnected bionic scaffolds with a hierarchical porous structure.The present investigation has implemented a compositional modulation strategy on the scaffold,resulting in remarkable enhancement of its osteogenic properties,thereby rendering it highly conducive for bone regenerative therapeutics.Methods:（1）Preparation of guar gum hydrogel.A mixture of guar gum and copper nitrate was used to gel rapidly in an alkaline environment to form hydrogel spheres.The surface and internal microstructure,composition and interaction processes of the hydrogel spheres were systematically analyzed to obtain their gel regulation mechanisms.（2）Study of calcium phosphate hierarchical structure microspheres and their drug release performanceA mixture of guar gum,calcium nitrate,copper nitrate and diammonium hydrogen phosphate in different proportions was used to form hydrogel spheres rapidly in alkaline environment,and the hydrogel spheres were dried and calcined to obtain copper-doped calcium phosphate microspheres with different structures.The microstructure and phase composition of the microsphere surface and interior were analyzed,and tetracycline was used as a drug model to study the drug-carrying and drug-release behavior of calcium phosphate microspheres.（3）Construction of hierarchically structured calcium phosphate fibrous porous scaffolds and their osteogenic and angiogenic performance.A mixture of guar gum,calcium nitrate,different ratios of copper nitrate and diammonium hydrogen phosphate was used to form hydrogel fibers by rapid gelation in an alkaline environment to obtain hierarchically structured fibrous porous scaffolds with adjustable components.The microstructure and physicochemical properties of the scaffolds were characterized,and mouse bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were used as models to investigate the osteogenic and angiogenic activities of the scaffold materials.Results:（1）Ultrafine copper hydroxide is the key to promote the rapid gelling of guar gum.Copper hydroxide was the crosslinking agent of Cu/GG hydrogel,and GG inhibits the aggregation of copper hydroxide and guarantees the formation of ultrafine copper hydroxide.Meanwhile,copper hydroxide nanoclusters promote a rapid gelling of GG in a short time.（2）GG modulation leads to hollow hierarchical structure of calcium phosphate microspheres,and the microspheres exhibit good drug loading and release performance.GG plays a key role in maintaining the sheet-like structure of the building blocks.h HAp owns the highest SSA and pore volume among the as-obtained three groups before and after calcination,and h HAp exhibits higher drug loading capacity than d HAp and d TCP;We can conclude that the h HAp granule is a promising carrier of drug delivery for bone tissue engineering application.（3）Copper-doped calcium phosphate fibrous scaffold has a three-dimensional hierarchical porous structure of bone-like tissue,and the scaffold exhibits good osteogenic and angiogenic effects,while the copper ion content is negatively correlated with osteogenic activity.Micro-CT results showed that the pore size distribution of the scaffold macropores was between 8-500μm,most of the pore sizes were concentrated in 50-300μm,and the fiber surface contained a large number of nanopores with a hierarchical structure of macropore-mesopore-microporous-nanopore structure,and the overall porosity of the porous scaffold was about 60%,and the increase of copper doping did not affect the microstructure and porosity of the scaffold,but would slightly decrease the compressive strength of the scaffold.The Cu HAp scaffold was highly crystalline and pure,with no other phases existing.The release of Cu²⁺from the scaffold was abrupt within 24 h and slowed down after 24 h.The 0.8Cu HAp and1.2Cu HAp scaffolds had good cytocompatibility,and both m BMSCs and HUVECs were able to spread and proliferate well on the scaffold;compared with the1.2Cu HAp and 1.6Cu HAp scaffolds,the 0.8Cu HAp scaffold was more conducive to promote the growth of m BMSCs and HUVECs.The 0.8Cu HAp scaffold was more favorable to promote the expression of osteogenic differentiation-related proteins（ALP,RUNX2,COL-I,OCN）in m-BMSCs compared with 1.2Cu HAp and1.6Cu HAp scaffolds.In addition,Cu²⁺release from 0.8Cu HAp and 1.2Cu HAp fiber scaffolds also promoted the cell migration and vascular network formation of HUVECs.Conclusion:In this paper,calcium phosphate fibrous porous scaffold system with bone-like matrix was constructed based on the rapid gelling strategy,and the scaffolds possess a hierarchical pore structure of macro-medium-micro-nano pores;furthermore,the hierarchical structure and components of the calcium phosphate scaffold were synergistically regulated to achieve better osteogenic and angiogenic performance.In conclusion,the calcium phosphate fibrous porous scaffold can significantly exhibit excellent osteogenic and angiogenic properties,which is expected to provide some theoretical and practical references for the construction of new bone tissue engineering scaffolds.