Liu Guomin,Li Yalong,Yang Shuting,Zhao Yi’an,Lu Tiancheng,Jia Wenyuan,Ji Xuan,Luo Yungang
To explore the ability of dihydroxyphenylalanine-insulin-like growth factor-1(DOPA-IGF-1) coated hydroxyapatite/poly(lactic-co-glycolic acid)(HA/PLGA) microspheres to promote the proliferation and osteoclastic differentiation of rabbit bone mesenchymal stem cells(rBMSCs), HA/PLGA microspheres with different HA content (10%, 30%, 50%, mass fraction) were prepared by electrospinning method and HA/PLGA microspheres with 50% HA were coated with IGF-1 and DOPA-IGF-1, respectively. They were co-cultured with rBMSCs, respctively. Cell counting kit-8(CCK-8) detection, confocal laser scanning microscopy(CLSM), alkaline phosphatase(ALP) detection and osteogenesis related genes COL1A1 , Runx2 and bone morphogenetic protein-2( BMP-2 ) detection were conducted to detect the proliferation activity, cell morphology, differentiation ability and the expression level of osteogenesis-related genes of cells cultured on all microspheres groups. The results showed that rBMSCs proliferation increased in an HA content dependent manner, and cells proliferated more in the IGF-1 coated and DOPA-IGF-1 coated groups, in particular in DOPA-IGF-1 coated group, and the differences were more remarkable over time ( P <0.05). HA/PLGA microspheres promoted the proliferation and osteogenic differentiation of rBMSCs, and DOPA-IGF-1 coating enhanced the proliferation and osteogenic differentiation of rBMSCs.
Polymer microspheres have recently shown outstanding potential for bone tissue engineering due to their large specific surface area, good porosity, injectable property, good biocompatibility, and biodegradability. Their good load-release function and surface modifiability make them useful as a carrier of drugs or growth factors for the repair of bone defects in irregularly injured or complex microenvironments, such as skull defects. In this study, berberine (BBR)-encapsulated poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) microspheres were fabricated using electrified liquid jets and a phase-separation technique, followed by modification with the 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth–factor-1 (DOPA-IGF-1). Both the BBR and the IGF-1 exhibited sustained release from the IGF-1@PLGA/HA-BBR microspheres, and the composite microspheres exhibited good biocompatibility. The results of the alkaline phosphatase (ALP) activity assays showed that the BBR and IGF-1 in the composite microspheres synergistically promoted the osteogenic differentiation of MC3T3-E1 cells. Furthermore, it was confirmed that immobilized IGF-1 enhances the mRNA expression of an osteogenic-related extracellular matrix and that BBR accelerates the mRNA expression of IGF-1-mediated osteogenic differentiation and cell mineralization. Further cellular studies demonstrate that IGF-1 could further synergistically activate the IGF-1R/PI3K/AKT/mTOR pathway using BBR, thereby enhancing IGF-1-mediated osteogenesis. Rat calvarial defect repair experiments show that IGF-1@PLGA/HA-BBR microspheres can effectively promote the complete bony connection required to cover the defect site and enhance bone defect repair. These findings suggest that IGF-1@PLGA/HA-BBR composite microspheres show a great potential for bone regeneration.Keywords:polymer microspheres;berberine (BBR);IGF-1;bone regeneration;osteogenic differentiation
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