Photoacoustic stimulation promotes the osteogenic differentiation of bone mesenchymal stem cells to enhance the repair of bone defect

Abstract

The aim of this study was to evaluate the direct photoacoustic (PA) effect on bone marrow mesenchymal stem cells (BMSCs) which is a key cell source for osteogenesis. As scaffold is also an indispensable element for tissue regeneration, here we firstly fabricated a composited sheet using polylactic-co-glycolic acid (PLGA) mixing with graphene oxide (GO). BMSCs were seeded on the PLGA-GO sheets and received PA treatment in vitro for 3, 9 and 15 days, respectively. Then the BMSCs were harvested and subjected to assess alkaline phosphatase (ALP) activity, calcium content and osteopontin (OPN) on 3, 9 and 15 days. For in vivo study, PLGA-GO sheet seeded with BMSCs after in vitro PA stimulation for 9 days were implanted to repair the bone defect established in the femoral mid-shaft of Sprague-Dawley rat. PLGA-GO group with PA pretreatment showed promising outcomes in terms of the expression of ALP, OPN, and calcium content, thus enhanced the repair of bone defect. In conclusion, we have developed an alternative approach to enhance the repair of bone defect by making good use of the beneficial effect of PA.

Figure 1

(A) Schematic diagram showing the process for manufacturing the PLGA-GO scaffold. (B) Schematic diagram showing the setup of PA treatment platform. (C) The images showing the establishment of bone defect model at the femoral mid-shaft of rat.

Figure 2

Characterization of the fabricated scaffolds in vitro. (A) The microstructures of PLGA-GO scaffolds (a1) and PLGA scaffolds (a2) under the scanning electron microscope. (B) Raman spectra of GO, pristine PLGA film and PLGA-GO film. (C) Under the 10 mJ pulsed laser stimulating, graph (c1) showed the PA signal producing by PLGA-GO scaffolds; graph (c2) showed the PA signal of PLGA-GO scaffolds with different intensities of Laser. (D) Scanning electron microscopy images showed that BMSCs attached and spread well on the surface of PLGA-GO scaffold after cultivating 72 h. White arrows indicated the BMSCs. (E) Proliferation tests of BMSCs growing on PLGA-GO scaffolds with or with PA stimulation at 24 h and 48 h. All quantitative data were presented as mean ± SD, n = 4. No statistical difference was found between group as indicated by unpaired two-tailed Student’s t test.

Figure 3

PA-pretreatment enhances the osteogenic differentiation of BMSCs in vitro. (A) The time course expression profiles of total protein in the indicated groups. The osteogenic markers of each experimental group, including osteopontin (B), alkaline phosphatase activity (C) and calcium content (D) were used to evaluate the osteogenic differentiation of BMSCs by quantitative analysis. All quantitative data are presented as mean ± S.D, n = 4; *represents statistical difference as compared with Control group; ^#represents statistical difference as compared with PA group. *P < 0.05, **P < 0.01, ***P < 0.001; ^# P < 0.05, ^## P < 0.01; ^### P < 0.001 from One-way ANOVA with Student–Newman-Keuls post hoc test.

Figure 4

Alizarin red staining and its quantitative measurement further support that the osteogenic differentiation of BMSCs is enhanced by PA treatment. (A) The Alizarin red staining show calcium deposition in each experimental group (Control, GO, OS, GO + OS, Light, PA). (B) The mineralization of BMSCs were quantified by measuring the absorbance at 590 nm wavelength at 3, 9 and 15 days. All quantitative data were presented as mean ± SD, n = 4; *represents statistical difference as compared with Control group; ^#represents statistical difference as compared with PA group at 15 days. *P < 0.05, **P < 0.01, ***P < 0.001; ^## P < 0.01 from One-way ANOVA with Student–Newman-Keuls post hoc test.

Figure 5

PA-pretreated PLGA-GO seeding with BMSCs significantly enhances the repair of bone defect in vivo. (A,B) Radiographs (A) and micro-CT 3D images (B) show new bone formation in bone defects of Control, PLGA-GO, PLGA-GO + BMSCs and PLGA-GO + BMSCs~PA groups at weeks 4 and 8 after implantation. The white arrows identify the bone defects. (C) BV/TV was quantified to analyze new bone formation within the bone defects. All quantitative data were presented as mean ± SD, n = 5; *represents statistical difference as compared with Control group; ^#represents statistical difference as compared with PA group. *P < 0.05, **P < 0.01, ***P < 0.001; ^# P < 0.05, ^## P < 0.01 from One-way ANOVA with Student–Newman-Keuls post hoc test.

Figure 6

Histological and immunohistochemical results indicate best outcome in animals implanting with PA-pretreated PLGA-GO combined with BMSCs. H.E staining (A) and immunohistochemical staining of type I collagen (C) and OPN (E) for analyzing the new bone formation within the sites of bone defects. The red dashed line marked the edge of the bone defect while the red arrows pointed to the GO oxide particles. Panels B, D, F are quantitative data from panels A, C, E, respectively. All quantitative data were presented as mean ± S.D, n = 5; *represents statistical difference as compared with Control group at the same time point; ^#represents statistical difference as compared with PA group at the same time point. *P < 0.05, **P < 0.01, ***P < 0.001; ^# P < 0.05, ^## P < 0.01 from One-way ANOVA with Student–Newman-Keuls post hoc test.

Figure 7

Both Runx2 and Osterix are significantly increased at the bone defect sites of animals implanting with PA-pretreated PLGA-GO combined with BMSCs. (A,C) Immunohistochemical staining of Runx2 and Osterix for estimating the new bone formation within the bone defects. The red dashed line marked the edge of the bone defect while the red arrows pointed to GO particles. Panels B and D were quantitative data from panels A and C, respectively. All quantitative data were presented as mean ± S.D, n = 5; *represents statistical difference as compared with Control group at the same time point; ^#represents statistical difference as compared with PA group at the same time point. *P < 0.05, **P < 0.01, ***P < 0.001; ^# P < 0.05, ^## P < 0.01 from One-way ANOVA with Student–Newman-Keuls post hoc test.

Figure 8

PA treatment significantly induces neovascularization within at the bone defect sites of animals implanting with PA-pretreated PLGA-GO combined with BMSCs. (A) Immunofluorescence assays show the α-SMA (Red color) and VEGF (Green color) expression in PLGA-GO, PLGA-GO + BMSCs and PLGA-GO + BMSCs~PA groups after implantation 2 weeks and 4 weeks. White arrows point to the α-SMA positive micro-vessels. (B) Mean microvessel density at the bone defect were quantified for statistical analysis. (C) The VEGF expression was quantified by average fluorescence intensity. All quantitative data were presented as mean ± S.D, n = 5; **P < 0.01, as compared with PA group at the same time point, from One-way ANOVA with Student–Newman-Keuls post hoc test.