| With the increase of aging population and transportation/sports injuries, people have an increasing demand of bone grafting materials. It’s becoming an ideal treatment to repair or replace the hard tissue with tissue engineered bones. From a clinical perspective, the use of injectable tissue engineered bone has been more and more attractive as it minimizes patient discomfort, risk of infection, scar formation and the cost of treatment.In this paper, the injectable tissue engineered bone were constructed by thermosensitive chitosan hydrogel, calcium phosphate nanoparticles and rat-derived bone marrow mesenchymal stem cells (MSCs), working as fluid scaffold, drug carrier and seed cells, respectively. The excellent loading&release properties of nCP allowed for a controlled release of drugs and growth factors in it, thus contributing to a better performance of the tissue engineered bone in treating and repairing the bone defects. The specific contents and results of the study are as follows:1. nCP particles were prepared using chemical co-precipitation method and its morphology, crystallinity and porosity were characterized. Recombinant human bone morphogenetic proteins2(rhBMP-2) were obtained by prokaryotic expression in Escherichia coli BL21. Meanwhile, rhBMP-2and dexamethasone (Dex) were loaded to nCP, determining their loading capacity, release rate and cycle. The results showed that nCP were spherical particles with good crystallinity. It had an average particle size of56.2±5.8nm. The average pore size in the particles was11.96nm. The porosity and specific surface area were47.2%and89.337m2/g, respectively. The porous structure of nCP was beneficial to the subsequent loading&release of drugs in it. It turned out that rhBMP-2could be successfully expressed in Escherichia coli. The protein yield after purification was27mg per liter of culture medium and the renaturation yield after urea gradient dialysis was only35.9%. The loading rate of rhBMP-2and Dex in per50mg nCP were56.1±0.493%and12.8±0.372%respectively. Loaded Dex and rhBMP-2could keep a sustained release from nCP for about half a month, which proved that the prepared nCP was an ideal drug carrier, meeting the application requirements of bone tissue engineering.2. Chitosan/β-glycerophosphate/nCP (CS/GP/nCP) hydrogel was prepared. Several properties of the hydrogel, such as gelation time, morphology and storage property at low temperature et al, were characterized as well. The results showed that, with the increase of GP concentration, the CS/GP/nCP system had a higher pH, shorter gelation time and could remain liquid for a longer period of time at4℃. The CS/GP/nCP hydrogel held a porous sponge-like structure. The pore size of the hydrogel was about100μm, which is suitable for cell proliferation in it, as well as the delivery and transportation of nutrients and growth factor.3.MSCs were successfully extracted from newly-born rats. The injectable tissue engineered bone was fabricated by seeding the cells into the sterilized CS/GP/nCP hydrogel and its biocompatibility was characterized. The results showed MSCs could grow well in the hydrogel, indicating that the prepared tissue engineered bone had a good biocompatibility. MSCs proliferated faster when Dex concentration in the system was around10-9M. Further increase of the Dex amount may inhibit the proliferation of MSCs. When the rhBMP-2concentration reached30μg/mL, it promoted the osteogenic differentiation of MSCs significantly. MSCs could proliferate and osteogenic differentiate better in the system with both Dex and rhBMP-2than that without or with either of the drugs. |
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