Reprogramming mitochondrial metabolism of macrophages by miRNA-released microporous coatings to prevent peri-implantitis

Abstract

Although various new biomaterials have enriched the methods for peri-implant inflammation treatment, their efficacy is still debated, and secondary operations on the implant area have also caused pain for patients. Recently, strategies that regulate macrophage polarization to prevent or even treat peri-implantitis have attracted increasing attention. Here, we prepared a laser-drilled and covered with metal organic framework-miR-27a agomir nanomembrane (L-MOF-agomir) implant, which could load and sustain the release of miR-27a agomir. In vitro, the L-MOF-agomir titanium plate promoted the repolarization of LPS-stimulated macrophages from M1 to M2, and the macrophage culture supernatant promoted BMSCs osteogenesis. In a ligation-induced rat peri-implantitis model, the L-MOF-agomir implants featured strong immunomodulatory activity of macrophage polarization and alleviated ligation-induced bone resorption. The mechanism of repolarization function may be that the L-MOF-agomir implants promote the macrophage mitochondrial function and metabolism reprogramming from glycolysis to oxidative phosphorylation. Our study demonstrates the feasibility of targeting cell metabolism to regulate macrophage immunity for peri-implantitis inhibition and provides a new perspective for the development of novel multifunctional implants.

Keywords: Dental implants; Macrophages; Mitochondrial metabolism; Peri-implantitis.

Fig. 1

Characterization of MOF-agomir particles and L-MOF-agomir plates/implants. A Transmission electron microscopy images of MOF and MOF-agomir particles. B Energy dispersive spectroscopy images of N, Zn, P elements in MOF-agomir particles. C Encapsulation efficiency of MOF at different concentration of agomir (n = 3). D Zeta potential of MOF-agomir particles with various agomir inputs. (n = 3). E Agomir release from L-MOF-agomir plates at pH 5.5, pH 6.5 or pH 7.4 over time (n = 3). F Zinc ion release rate of L, L-MOF and L-MOF-agomir titanium plates in 10% FBS-containing DMEM. G Scanning electron microscope images of L, L-MOF and L-MOF-agomir implants. H Energy dispersive spectroscopy analysis of element composition in L, L-MOF and L-MOF-agomir implants

Fig. 2

L-MOF-agomir titanium induced repolarization of macrophages from M1 into M2 phenotype after LPS stimulus in vitro. A Immunofluorescence images of macrophages incubated on different titanium plates (live/dead assay); live cells were stained with calcein-AM (green fluorescence), while dead cells were stained with PI (red fluorescence). B CCK8 assay was performed to measure the number of living cells. C Expression of miR-27a in macrophages incubated on different titanium plates. D Macrophages were stained with antibodies against the M1 surface marker CD86 and M2 surface marker CD206, followed by flow cytometric analysis. Representative histogram graphs are presented. E Statistical analysis of flow cytometry. F Expression of M1 polarization-related genes (inos, tnf-α) and M2 polarization-related genes (arg1, il-10) in macrophages incubated on different titanium plates (qRT-PCR). G Representative images of immunofluorescent staining showing M2 polarization-related marker CD206 and M1 polarization-related marker iNOS in macrophages. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 3

L-MOF-agomir titanium promotes immune osteogenesis by inducing macrophages M2 repolarization. A Representative images and distribution map of immunofluorescent staining showing osteogenesis marker RUNX2 in BMSCs cultured with macrophages supernatant in different groups. B Representative images showing the mineralization extracellular matrix and ALP staining of BMSCs in different groups. C The quantification of Alizarin Red and ALP staining. D Expression of osteogenesis-related genes (alp, runx2, ocn, col-1) in BMSCs (qRT-PCR). Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 4

L-MOF-agomir titanium enhanced the mitochondrial function and promoted the metabolic transition from glycolysis to OXPHOS. A Representative TEM images showing the number and structure of mitochondria in macrophages after LPS stimulus incubated on different titanium plates. B Representative images and distribution map of immunofluorescent staining showing ROS in macrophages. C Representative images and fluorescence intensity histogram of immunofluorescent staining showing mitochondrial membrane potential in macrophages. D ROS and ATP production in macrophages. E Real-time ECARs of macrophages in different groups response to glucose, Oligo and 2-DG in 120 min. F Quantification of glycolysis, glycolytic capacity and glycolytic reserve. G Real-time OCRs of macrophages in different groups response to Oligo, FCCP and ROT/AA in 120 min. H Quantification of basal respiration, ATP production and respiration capacity. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 5

Incubated on the L-MOF-agomir titanium plates with OXPHOS inhibitor abrogated the macrophage repolarization function. A Representative images of immunofluorescent staining showing M2 polarization-related marker CD206 and M1 polarization-related marker iNOS in macrophages. B Mean gray value of immunofluorescent intensity. C Expression of M1 polarization-related genes (inos, tnf-α) and M2 polarization-related genes (arg1, il-10) in macrophages (qRT-PCR)

Fig. 6

L-MOF-agomir implants promotes recruited macrophages toward M2 polarization in vivo. A Representative immunofluorescent staining of M1 polarization-related markers (iNOS/CD68) and M2 polarization-related markers (Arg1/CD68) in macrophages around the implant after 2-week ligation. B Quantification and statistical analysis of immunofluorescent positive area. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 7

L-MOF-agomir implants inhibited ligation induced bone resorption around the implant. A Representative of 3D reconstruction of the bone around the implant after two weeks of ligation. B Distance from the implant top to the highest point of alveolar bone around the implant. C Quantitative analysis of BV/TV, BMD, Tb.Th and Tb.Sp at 2 weeks after ligation based on micro-CT scanning. D Representative images of H&E, Masson, and anti-RUNX2 immunofluorescence staining of tissue around implants. The implant is indicated by a dotted line. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01