Incorporation of carboxymethyl chitosan (CMCS) for the modulation of physio-chemical characteristics and cell proliferation environment of the composite hydrogel microspheres

Hydrogels have excellent swelling properties and have been widely applied in tissue engineering because of their similarity to the extracellular matrix. Sodium alginate (SA) and carboxymethyl chitosan (CMCS) were prepared into hydrogel microspheres with Ca2+ crosslinking in our study. The morphology, inner structure, mechanical properties, water content, swelling rate and BMP-2 loading and releasing properties were characterized. Our results showed that the composite SA /CMCS hydrogel microspheres were translucent and spherical in shape with uniform particle size. The incorporation of CMCS further increased the diameters of the microspheres, internal pore structure, water content, and mechanical properties of the SA/CMCS hydrogel microspheres. At the same SA concentration, with the increase of CMSC concentration, the diameter of microspheres could be increased by about 0.4 mm, the water content can be increased about 1%-2%. As for the mechanical properties, the compressive strength can be increased by 0.04-0.1 MPa, and the modulus of elasticity can be increased by 0.1-0.15 MPa. BMP-2 was chosen as a model agent and it could be loaded into SA/CMCS microspheres, and the incorporation of CMCS increased BMP-2 loading. The encapsulated BMP-2 was sustainably released in vitro. The leaching solutions of the SA/CMCS hydrogel microspheres exhibited good cytocompatibility and could increase ALP activity, ALP expression, and biomineralization on MC3T3-E1 cells. After 7 days of co-culture, ALP activities in S2.5C2 and S2.5C3 groups was increased by 50% and 45% compared with that of the control group. When embedded in the SA/CMCS microspheres, the MC3T3-E1 cells were evenly distributed inside the hydrogel microspheres and remained viable. Transcriptomic studies showed that incorporation of CMCS induced upregulation of 1141 differentially expressed genes (DEGs) and downregulation of 1614 DEGs compared with SA microspheres. The most significantly enriched pathways were the Wnt and MAPK signaling pathways induced by the incorporation of CMCS and BMP-2. In conclusion, our results indicated that the physiochemical characteristics of the SA hydrogel microspheres could be greatly modulated by CMCS to better mimic the extracellular matrix microenvironment and induce osteo-inductive activities of MC3T3-E1 cells.