Phosphorylated Modification Of Chitosan For Biomimetic Synthesis Of Bone Regeneration Materials

IntroductionNature's mineralizers use small amounts of organic macromolecules as template to manipulate inorganic crystal nucleation, growth, microstructure, and consequently the properties of their mineral-based materials. These macromolecular structures contain highly repetitive patterns of anionic groups; these anionic groups lead to interactions between organic chains and mineral precursor ions, and concentrate the inorganic cations creating local supersaturation followed by oriented nucleation of the crystal. In the present study, by the theory of biomineralization, polyelectrolyte complex (PEC) hydrogel composited of polycationic chitosan (CS) and polyanionic phosphorylated chitosan (PCS), was self-organized, and the PEC hydrogel with hierarchical porosity was used as the templet for hydroxyapatite (HA) growth. One kind of method for biomimetic synthesis of bone generation materials was developed, and the materials were fabricated according to biological principles and processes of self-assemblyMaterial and methodFirstly, chitosan phosphorylation was carried out in ethanesulfonic acid with phosphorus pentoxide (P₂O₅). By changing the reaction time and the dosage of P₂O₅, PCSs with different degree of substitution (DS) were obtained. To evaluate the property of biomimetic mineralization, two kind of insoluble phosphorylated chitosan were soaked in simulated body fluid (SBF), and the biocompatibility of PCS and its product of biomimetic mineralization were evaluated by rat osteoblasts culture test. Secondly, polyelectrolyte complex (PEC) hydrogel composited of polycationic chitosan (CS) and phosphorylated chitosan (PCS), was self-organized, then PEC hydrogel with hierarchical porosity was soaked in supersaturated calcium phosphate solution to form a composite hydrogel of PEC and HA, and the composite was co-cultured with osteoblasts. Finally, by combination of the technique of forming PEC hydrogel and microencapsulation for drug release, the formation of PEC and the controlled HA crystal growth were realized by one step, and the microencapsule of "PEC-HA" resembling matrix vesicles formed in bone mineralization was synthesized.ResultsThe property of PCS is much affected by the degree of phosphate group substitute (DS) in chitosan molecules The solubility of PCS is related to DS, as the P content increases, phosphorylated chitosan becomes into water-soluble, but above a threshold value, it becomes insoluble once more and can dissolve in the alkaline aqua. P content is a crucial factor for PCS to stimulate the biomimetic mineralization, a lower DS promoted higher extent ofmineralization than a higher DS. In the cytocompatibility study in vitro, PCS inhibited osteoblast proliferation and differentiation, and its product of biomineralization interacted with osteoblast as the same as sintered HA ceramicsPEC hydrogels showed a three-dimensional porous and anastomosing, sponge-like network structure with hierarchical porosity. The macropores werein the range of ⁵ - 100 jjl m diameters (average ⁶0 ijl m); The walls of the macropores contained many micropores with diameter in the range of ¹00 -120nm; The fibrils defining the micropores were about 0.1 - 5 |x m in width.When PEC hydrogel was soaked in supersaturated calcium phosphate solution, HA crystals preferential nucleated and deposited on the fibers of the PEC hydrogels, formed a three-dimensional hydrogel structure with its physicochemical properties and constitution resembling those of extracellular matrix (ECM) of bone. Rat osteoblasts were seeded in the three-dimensional scaffold of HA-PEC composite, and the three-dimensional scaffold of HA-PEC hydrogel revealed excellent biocompatibility compared to sintered HA ceramics. The microencapsule of "PEC-HA" resembled matrix vesicles formed in theprocess of bone mineralization. Its size ranged from 1 to 20 |x m and theinorganic phase constituted by 70% in weight resembling bone. The inorganic crystals were carbonate-contained nano-apatite with lower crystallization, and evenly distributed in organic matrix. The microencapsule had complexes porosity.ConclusionPolyelectrolyte complex (PEC) hydrogel with hierarchical porosity, composited of polycationic chitosan (CS) and phosphorylated chitosan (PCS), can be used as the templet for hydroxyapatite (HA) nucleation and growth. The new type biomaterials of "PEC-HA" composite synthesized by biomimetic method is degradable, can promote osteoblast proliferation and differentiation, It is a promise biomaterials for hard tissue replacement, scaffold of tissue engineering, vehicle for cell, gene, drug and growth factor.