Irisin-loaded electrospun core-shell nanofibers as calvarial periosteum accelerate vascularized bone regeneration by activating the mitochondrial SIRT3 pathway

Abstract

The scarcity of native periosteum poses a significant clinical barrier in the repair of critical-sized bone defects. The challenge of enhancing regenerative potential in bone healing is further compounded by oxidative stress at the fracture site. However, the introduction of artificial periosteum has demonstrated its ability to promote bone regeneration through the provision of appropriate mechanical support and controlled release of pro-osteogenic factors. In this study, a poly (l-lactic acid) (PLLA)/hyaluronic acid (HA)-based nanofibrous membrane was fabricated using the coaxial electrospinning technique. The incorporation of irisin into the core-shell structure of PLLA/HA nanofibers (PLLA/HA@Irisin) achieved its sustained release. In vitro experiments demonstrated that the PLLA/HA@Irisin membranes exhibited favorable biocompatibility. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was improved by PLLA/HA@Irisin, as evidenced by a significant increase in alkaline phosphatase activity and matrix mineralization. Mechanistically, PLLA/HA@Irisin significantly enhanced the mitochondrial function of BMMSCs via the activation of the sirtuin 3 antioxidant pathway. To assess the therapeutic effectiveness, PLLA/HA@Irisin membranes were implanted in situ into critical-sized calvarial defects in rats. The results at 4 and 8 weeks post-surgery indicated that the implantation of PLLA/HA@Irisin exhibited superior efficacy in promoting vascularized bone formation, as demonstrated by the enhancement of bone matrix synthesis and the development of new blood vessels. The results of our study indicate that the electrospun PLLA/HA@Irisin nanofibers possess characteristics of a biomimetic periosteum, showing potential for effectively treating critical-sized bone defects by improving the mitochondrial function and maintaining redox homeostasis of BMMSCs.

Keywords: critical-sized bone defect; irisin; mitochondrial function; periosteum; redox homeostasis.

Graphical abstract

Figure 1.

Schematic diagram of the preparation of biomimetic irisin-loaded electrospun nanofibers (PLLA/HA@Irisin) using coaxial electrospinning and the therapeutic application in a critical-sized calvarial defect.

Figure 2.

Morphology and characterizations of different membranes. (A) Representative images of SEM. (B) Representative images of TEM. (C) The pore size distribution of the PLLA membranes. (D) The pore size distribution of the PLLA/HA membranes. (E) The images of water contact angles of PLLA and PLLA/HA membranes. (F) The quantification of water contact angles, n = 3. (G, H) The stress-strain curve and the tensile strength of the PLLA and PLLA/HA membranes, n = 3. (I) Irisin release profile at each time point within 30 days, n = 3. (J) Degradation rate of PLLA and PLLA/HA membranes within 8 weeks, n = 3. Statistically significant differences were indicated by *P < 0.05 or **P < 0.01.

Figure 3.

The biocompatibility of PLLA/HA membranes loaded with irisin. (A) The cell viability of BMMSCs cultured on electrospun nanofibers was determined using the live/dead cell staining. Scale bar = 1 mm. (B) Quantification of the live cells cultured on electrospun nanofibers, n = 3. (C) The cytoskeleton and nuclear staining of BMMSCs cultured on the nanofibrous membranes. Scale bar = 50 μm. (D) Cell proliferation rate was determined by CCK-8 assays, n = 4. Statistically significant differences were indicated by *P < 0.05.

Figure 4.

PLLA/HA@Irisin membranes promoted the in vitro osteogenic differentiation of BMMSCs. (A, B) The expression level of COL I was evaluated by immunofluorescence staining, n = 3. Scale bar = 50 μm. (C) After 7 days of osteogenic induction, ALP staining was performed on BMMSCs scale bar = 100 μm. (D) Quantitative results of ALP activity of BMMSCs, n = 3. (E) Representative images of bone mineral deposition in BMMSCs stained by ARS. Scale bar = 100 μm. (F) Quantification of the stained bone mineral deposition in BMMSCs cultured on different groups of membranes, n = 3. (G) The gene expression of osteogenic makers, including Col1a1, Sp7, and Runx2, was determined by RT-PCR, n = 4. (H, I) The protein levels of COL I, SP7 and RUNX2 were examined by western blot, n = 3. Statistically significant differences were indicated by *P < 0.05 or **P < 0.01.

Figure 5.

PLLA/HA@Irisin membranes improved the mitochondrial function of BMMSCs. (A, B) JC-1 staining indicated the MMP of BMMSCs, n = 3. Scale bar = 5μm. (C) ATP production of BMMSCs, n = 4. (D) The gene expression of mitochondrial respiratory chain factors, including Atp5a, Nd4 and Sdha, was determined by quantitative RT-PCR, n = 4. (E, F) The protein levels of ATP5A, ND4 and SDHA were examined by western blot, n = 3. Statistically significant differences were indicated by *P < 0.05 or **P < 0.01.

Figure 6.

PLLA/HA@Irisin membranes enhanced the antioxidant functions of BMMSCs. (A) Mitochondrial and intracellular ROS were detected through MitoSOX and DCFH-DA staining, respectively. Scale bar = 50 μm. (B) Quantification of mitochondrial ROS in BMMSCs, n = 3. (C) Quantification of intracellular ROS in BMMSCs, n = 3. (D) The gene expression of Sirt3, Sod2 and Gpx1 was determined by RT-PCR. (E, F) Western blot was used to determine the protein levels of SIRT3, SOD2 and GPX1. Statistically significant differences were indicated by *P < 0.05 or **P < 0.01.

Figure 7.

Micro-CT evaluation of in vivo bone regeneration of critical-sized calvarial defects. Two full-thickness defects (4 mm in diameter) were drilled on rat calvarium. PLLA/HA and PLLA/HA@Irisin nanofibrous membranes were in situ implanted. The untreated defects served as the defect group. (A) Schematic diagram of surgery and implantation process. (B) At 4 and 8 weeks post-surgery, the new bone formation in the calvarial defect was evaluated by micro-CT imaging and 3D reconstruction. Quantitative analysis of (C) BMD, (D) bone volume ratio (BV/TV) and (E) Tb.Th in the defect area after 4 and 8 weeks of implantation, n = 3. Statistically significant differences were indicated by *P < 0.05 or **P < 0.01.

Figure 8.

Histological and immunohistochemical analysis of the newly formed bone tissue in the calvarial defects. (A) H&E images of the defect area at 4 and 8 weeks post-surgery. (B) MTS images of the defect area at 4 and 8 weeks post-surgery. (C) IHC images of COL I in the defect area. (D) IHC images of CD31-positive cells that represented the newly formed blood vessels. Scale bar = 100 μm. (E) Quantification of the COL I-positive area, n = 3. (F) Quantification of the CD31-positive cells, n = 3. Statistically significant differences are indicated by *P < 0.05 or **P < 0.01.