[Experimental study on ectopic bone formation of chitosan/phosphonic chitosan sponge combined with human umbilical...

To investigate the ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro.

Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan; 2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 : 1 and freeze-dried to build the complex sponge, and then was put in the simulated body fluid for biomimetic mineralization in situ. The hUCMSCs were isolated by enzyme digestion method from human umbilical cord and were cultured. The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3, and the cell-scaffold composite was cultured in osteogenic medium. The growth and adhesion of the cells on the scaffolds were observed by light microscope and scanning electron microscope (SEM) at 1 and 2 weeks after culturing, respectively. The cell proliferation was detected by MTT assay at 1, 2, 3, 4, 5, and 6 days, respectively. Bilateral back muscles defects were created on 40 New Zealand rabbits (3-4 months old, weighing 2.1-3.2 kg, male or female), which were divided into groups A, B, and C. In group A, cell-scaffold composites were implanted into 40 right defects; in group B, the complex sponge was implanted into 20 left defects; and in group C, none was implanted into other 20 left defects. The gross and histological observations were made at 4 weeks

The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl, and the proton type and chemical shifts intensity were conform to its chemical structure. The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous, and the wall of the pore was thinner; the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineralized with crystal particles; the cells grew well on the surface of the chitosan/phosphonic chitosan sponge. The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the proliferation of hUCMSCs. The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A, the size of the complex sponge gradually reduced in group B, and the muscle defects wound healed with a little scar tissue in group C. The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A, the circular cavity and residual chitosan scaffolds were observed in group B, and the wound almost healed with a small amount of lymphocytes in group C.

The chitosan/phosphonic chitosan sponge has good biocompatibility, the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.