Abstract: BACKGROUND: Chlorogenic acid has antibacterial, antiviral, and osteogenic properties. However, its limited solubility in water restricts its biological activity.
OBJECTIVE: To observe the osteogenic effect of chlorogenic acid protein microspheres/polycaprolactone electrospinning membranes.
METHODS: (1) Chlorogenic acid, bovine serum albumin and chitosan were used as raw materials to prepare chlorogenic acid protein microspheres by desolventization method and electrostatic adsorption technology. The micromorphology, particle size, Zeta potential, and drug encapsulation efficiency of chlorogenic acid protein microspheres were characterized. MC3T3-E1 cells in the logarithmic growth phase were divided into three groups for culture: the control group was not treated with any treatment; the chlorogenic acid group was added with 10 mg/L chlorogenic acid; the chlorogenic acid protein microsphere group was added with 50 mg/L chlorogenic acid protein microspheres. Cell proliferation, alkaline phosphatase activity after osteogenic induction and reactive oxygen levels under hydrogen peroxide-induced oxidative stress were detected. (2) Polycaprolactone electrospinning membrane and chlorogenic acid protein microsphere/polycaprolactone electrospinning membrane were prepared by electrospinning technology. The microscopic morphology of the electrospinning membrane was observed by scanning electron microscopy. MC3T3-E1 cells in the logarithmic growth period were divided into four groups for culture: the control group was not treated with any treatment; the chlorogenic acid group was added with 10 mg/L chlorogenic acid; the polycaprolactone group was added with polycaprolactone electrospinning membrane; the microsphere spinning membrane group was added with chlorogenic acid protein microsphere/polycaprolactone electrospinning membrane. After 7 days of osteogenic induction, RT-qPCR was used to detect the mRNA expression of Runt-related transcription factor 2 (RUNX2) and alkaline phosphatase. Western blot assay was used to detect the protein expression of RUNX2 and alkaline phosphatase.
RESULTS AND CONCLUSION: (1) The chlorogenic acid protein microspheres exhibited a double-layer structure with a smooth surface and relatively uniform particle size, averaging (322.38±8.39) nm. The Zeta potential of the microspheres was measured at (42.85±2.11) mV and the drug encapsulation efficiency was found to be (57.16±7.32)%. Both chlorogenic acid and chlorogenic acid protein microspheres could promote the proliferation and early osteogenic differentiation of MC3T3-E1 cells and reduce the level of reactive oxygen species in oxidative stress response. (2) Both groups of electrospun membranes were 3D mesh structures with more pores. The surface of polycaprolactone electrospun membranes was smoother and the spinning diameter was more consistent. The surface of chlorogenic acid protein microspheres/polycaprolactone electrospun membranes showed more spherical protrusions and the spinning diameter was quite different. Both chlorogenic acid and chlorogenic acid protein microspheres/polycaprolactone electrospun membranes could increase the mRNA and protein expression of RUNX2 and alkaline phosphatase in MC3T3-E1 cells. The results show that chlorogenic acid protein microspheres/polycaprolactone electrospinning membranes can promote the osteogenic differentiation of MC3T3-E1 cells.

Key words: chlorogenic acid, MC3T3-E1 cell, osteogenesis, protein microsphere, electrospinning, cell proliferation, cell differentiation, engineered bone material