| Bone tissue engineering offer a new promising approach to dissolve the problemof bone regeneration which is often caused by trauma, tumor, and bone diseases.Three-dimensional porous scaffold play a key role in tissue engineering. A perfectscaffold for tissue engineering should mimic bone morphology, structure andfunction.Chitosan (CS) and hydroxyapatite (HA) are materials with great potential inbone tissue engineering. HA is one component of bone. Especially, nano-HA possessmore excellent physical,chemical and biological properties than normal HA. Acomposite of HA and chitosan therefore is developed in order to combine twomaterials advantages. But nano-HA intend to congregate in CS matrix with traditionalmethod mixing HA powder with CS solution. A nano-HA/CS rode has been made viain situ hybridization which nano-HA powder scattered in the CS homogenously.However this composite has no porosity and could not be loaded with cells.In this study a novel nano-HA/CS scaffolds with high porosity were fabricatedthrough a simple and effective technique combining with in situ hybridization andfreeze-drying method. Based on this, tissue engineered bone were constructed andcytocompatibility of the composite scaffolds were estimated1. Prepare the three dimensional nano-HA/CS scaffolds through in situhybridization-freeze-drying method. Scanning electron microscopy(SEM),transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fouriertransformed infrared spectroscopy (FTIR) allowed to determine morphologyand component of the composite scaffolds.2. Study the property of scaffolds containing different content and ratio of CS andHA; fabricated through different concentration of NaOH and prefreezetemperature.3. To test the bioactivity, nano-HA/CS scaffolds were immersed in the SBF and theapatite simulated bone formed on the scaffold were detect by TEM, XRD, FTIR.4. Calvarial osteoblasts were isolated and identified from neonatal wistar.Study the proliferation, differentiation process of the osteoblasts cultured in vitro.Passage cells for the construction of tissue engineered bone.5. The passaged cells (passage number 3) were seeded onto nano-HA/CS scaffoldsto construct tissue engineered bone in vitro. Determine the cell-adhesion ratioand cell growth curve on the scaffolds, their morphology were observed byhistological staining technique and SEM.Results The macroporous nano-HA/CS scaffolds with high porosity were formed.The pore size ranged from 100μm to about 500μm, mostly from 300μm to400μm. With the increased content of CS and HA, the density of the scaffold increased, the porosity decreased and the compressive strength raised correspondingly. Thecompressive strength of scaffold (CS 3%, CS-HA 3/1)reach 1.4MPa. Pore size andporosity of nano-HA/CS scaffolds can be controlled through different prefreezetemperature. The higher prefreeze temperature result in the smaller pore and lowerporosity. SEM, TEM show that HA with nanometer size were synthesized anddispersed on the pore walls homogeneously and consecutively, binding with eachother, as "road metal" XRD, FTIR proved that the HA crystal werecarbonate-substituded and not well crystallized. There were three process duringnano-HA/CS scaffolds soaking in SBF: gradually degradation of CS, resolving ofnano-HA and the deposition of apatite simulated bone.Osteoblasts isolated from wistar grow rapidly and can keep function stably.Osteoblasts seeded onto nano-HA/CS attached and proliferated on the scaffoldwell. Cells can produce extracellular matrix containing I collagen and then mineralizeit. Nano-HA/CS scaffolds were better cytocompatible than pure chitosan scaffolds bycell-adhesion ratio assay after co-cultured for 2,4,6,8h.Conclusion The nano-HA/CS scaffolds fabricated through in situhybridization-freeze-drying with proper physical,chemical properties andcytocompatibility, can serve as a promising scaffold for bone tissue engineering.
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