Biodegradable magnesium implant enhances angiogenesis and alleviates medication-related osteonecrosis of the jaw in rats

Abstract

Background: Medication-related osteonecrosis of the jaw (MRONJ) is a serious complication associated with antiresorptive and antiangiogenic medications, of which impaired angiogenesis is a key pathological alteration. Since Magnesium (Mg)-based implants possess proangiogenic effects, we hypothesized that the biodegradable Mg implant could alleviate the development of MRONJ via enhancing angiogenesis.

Methods: MRONJ model was established and divided into the Veh ​+ ​Ti group (Vehicle-treated rat, with Titanium (Ti) implant), BP ​+ ​Ti group (Bisphosphonate (BP)-treated rat, with Ti implant), BP ​+ ​Mg group (BP-treated rat, with Mg implant), BP ​+ ​Mg ​+ ​SU5416 group (BP-treated rat, with Mg implant and vascular endothelial growth factor (VEGF) receptor-2 inhibitor), BP ​+ ​Mg ​+ ​BIBN group (BP-treated rat, with Mg implant and calcitonin gene-related peptide (CGRP) receptor antagonist), and BP ​+ ​Mg ​+ ​SU5416+BIBN group (BP-treated rat, with Mg implant and VEGF receptor-2 inhibitor and CGRP receptor antagonist). The occurrence of MRONJ, alveolar bone necrosis, new bone formation and vessel formation were assessed by histomorphometry, immunohistochemistry, and micro-CT analysis.

Results: Eight weeks after surgery, the BP ​+ ​Mg group had significantly reduced occurrence of MRONJ-like lesion and histological osteonecrosis, increased bone microstructural parameters, and increased expressions of VEGFA and CGRP, than the BP ​+ ​Ti group. By simultaneously blocking VEGF receptor-2 and CGRP receptor, the vessel volume and new bone formation in the BP ​+ ​Mg group were significantly decreased, meanwhile the occurrence of MRONJ-like lesion and histological bone necrosis were significantly increased.

Conclusion: Biodegradable Mg implant could alleviate the development of MRONJ-like lesion, possibly via upregulating VEGF- and CGRP-mediated angiogenesis. Mg-based implants have the translational potential to be developed as a novel internal fixation device for patients with the risk of MRONJ.

The translational potential of this article: This work reports a biodegradable Mg implant which ameliorates the development of MRONJ-like lesions possibly due to its angiogenic property. Mg-based implants have the potential to be developed as a novel internal fixation device for patients at the risk of MRONJ.

Keywords: Angiogenesis; Biodegradable implants; Bisphosphonate; Magnesium; Medication-related osteonecrosis of the jaw.

Fig. 1

BP ​+ ​Mg group presented alleviated MRONJ occurrence and enhanced bone defect repair as compared to the BP ​+ ​Ti group (A) Schematic and intraoperative images of surgical procedures for establishing the MRONJ model with implant fixation, bone defect creation and dental extraction. A Mg rod (with a purity of 99.99%, 3 ​mm in length, and 1.3 ​mm in diameter) or a Ti rod (pure titanium, the same size with Mg rod) was implanted into the mandible; a 1 ​mm-wide bone defect was cut inferior to the implantation site; the mandibular 1st molar was extracted. Scale bar: 3 ​mm (B) Representative photos and coronal micro-CT images of extraction sites (Ext) at 8 weeks after surgery, showing complete healing mucosae (Left and Right) and a MRONJ-like lesion with exposure of necrotic bone and sequestrum (Middle). Scale bar: 3 ​mm (C) Occurrence rate of MRONJ-like lesion in three groups at 8 weeks after surgery. Veh ​+ ​Ti group n ​= ​5, BP ​+ ​Ti group n ​= ​9, and BP ​+ ​Mg group n ​= ​10, by Chi-square test (D) Representative sagittal micro-CT images and 3D reconstructed coronal images of mandibular bone defect (red dotted box) in three groups, 4 weeks and 8 weeks postoperatively. Scale bar: 1 ​mm (E) Bone micro-architectural morphometric assessment on the newly formed bone in the defect at 4 weeks and 8 weeks after surgery by micro-CT. Column and bar indicate mean and SD, respectively. n ​= ​5 for each group, by One-way ANOVA with Bonferroni post hoc test. ∗, p ​< ​0.05. ∗∗, p ​< ​0.01. ∗∗∗, p ​< ​0.001.

Fig. 2

Comparison of histological osteonecrosis and TRAP-positive osteoclasts at MRONJ-like lesions (A) Representative H&E staining and TRAP staining images of extracted tooth socket 8 weeks after removal in three groups. Imp, implant. M, molar. Nec, necrotic bone. Yellow arrow points the empty lacunae. Black arrow points the TRAP-positive stained osteoclast. Scale bar in H&E stain: 1 ​mm. Scale bar in TRAP stain: 100 ​μm (B) Histomorphometric assessments of H&E staining on the degree of alveolar bone necrosis, and TRAP staining on the number of TRAP-positive cells among groups. Column and bar indicate mean and SD, respectively. Veh ​+ ​Ti group n ​= ​5, BP ​+ ​Ti group n ​= ​9, and BP ​+ ​Mg group n ​= ​10, by One-way ANOVA with Bonferroni post hoc test. ∗, p ​< ​0.05. ∗∗, p ​< ​0.01. ∗∗∗, p ​< ​0.001.

Fig. 3

VEGFA and CGRP expression compared among Veh ​+ ​Ti, BP ​+ ​Ti and BP ​+ ​Mg groups (A) Representative images of IHC staining of CD31, VEGFA and CGRP in three groups, respectively. Black arrow points the positive stained cell. Scale bar: 400 ​μm (B) Corresponding quantitative data of VEGFA and CGRP. Column and bar indicate mean and SD, respectively. n ​= ​5 for each group, by One-way ANOVA with Bonferroni post hoc test. ∗∗∗, p ​< ​0.001.

Fig. 4

Mg enhanced angiogenesis and coupled osteogenesis in MRONJ, which could be inhibited by blocking VEGF receptor-2 and CGRP receptor simultaneously, as indicated by micro-CT based angiography and bone histomorphometry analysis (A) Representative 3D reconstructed images of micro-CT based angiography showing the differences of vessel volume compared among the BP ​+ ​Ti, BP ​+ ​Mg, and BP ​+ ​Mg ​+ ​inhibitor/antagonist groups at 8 weeks after surgery. Scale bar: 5 ​mm (B) Quantitative analysis of angiography in different groups at 8 weeks after surgery (C) Representative 3D reconstructed images showing the differences of newly formed bone in the defect compared among groups at 8 weeks after surgery. Scale bar: 1 ​mm (D) Quantitative analysis of newly formed bone in different groups at 8 weeks after surgery. SU5416, VEGF receptor-2 inhibitor; BIBN, CGRP receptor antagonist. Column and bar indicate mean and SD, respectively. n ​= ​4–5 for each group, by One-way ANOVA with Bonferroni post hoc test. ∗, p ​< ​0.05; ∗∗, p ​< ​0.01; ∗∗∗, p ​< ​0.001.

Fig. 5

Mg-alleviated MRONJ-like lesion development, possibly due to angiogenesis and osteogenesis (A) Occurrence rate of MRONJ-like lesion in the BP ​+ ​Ti, BP ​+ ​Mg, and BP ​+ ​Mg ​+ ​inhibitor/antagonist groups at 8 weeks after surgery (B) Histomorphometric assessments on the degree of alveolar bone necrosis among groups (H&E staining) (C) IHC staining of VEGFA (brown) and CGRP (brown) in the alveolar bone region in each group at 8 weeks after surgery. Scale bar: 100 ​μm (D) Corresponding quantitative data from (C). Column and bar indicate mean and SD, respectively. n ​= ​4–5 for each group, by Chi-square test (A), and One-way ANOVA with Bonferroni post hoc test (B and D). ∗∗, p ​< ​0.01; ∗∗∗, p ​< ​0.001 (E) Illustration of the underlying mechanism of biodegradable magnesium implant on attenuating the development of MRONJ via VEGF- and CGRP-mediated angiogenesis.