ObjectiveTo evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups: microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation (by MTT assay) and the glycosaminoglycan (GAG) contents (by Alician blue staining) were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance (A) value at 1, 3, and 7 days between 2 groups (P>0.05), but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05). Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.
ObjectiveTo evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro.MethodsNGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% (W/V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D.ResultsThe chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group (P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D (P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 (P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D (P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B (P<0.05), and there was no significant difference between groups C and D (P>0.05).ConclusionNGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.