A Magnesium-Enriched 3D Culture System that Mimics the Bone Development Microenvironment for Vascularized Bone Regeneration

Abstract

The redevelopment/regeneration pattern of amputated limbs from a blastema in salamander suggests that enhanced regeneration might be achieved by mimicking the developmental microenvironment. Inspired by the discovery that the expression of magnesium transporter-1 (MagT1), a selective magnesium (Mg) transporter, is significantly upregulated in the endochondral ossification region of mouse embryos, a Mg-enriched 3D culture system is proposed to provide an embryonic-like environment for stem cells. First, the optimum concentration of Mg ions (Mg²⁺) for creating the osteogenic microenvironment is screened by evaluating MagT1 expression levels, which correspond to the osteogenic differentiation capacity of stem cells. The results reveal that Mg²⁺ selectively activates the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway to stimulate osteogenic differentiation, and Mg²⁺ influx via MagT1 is profoundly involved in this process. Then, Mg-enriched microspheres are fabricated at the appropriate size to ensure the viability of the encapsulated cells. A series of experiments show that the Mg-enriched microenvironment not only stimulates the osteogenic differentiation of stem cells but also promotes neovascularization. Obvious vascularized bone regeneration is achieved in vivo using these Mg-enriched cell delivery vehicles. The findings suggest that biomaterials mimicking the developmental microenvironment might be promising tools to enhance tissue regeneration.

Keywords: 3D culture systems; biomaterials; developmental microenvironment; magnesium; vascularized bone regeneration.

Figure 1

a) Colocalization of ALP and MagT1 in mouse embryos using double immunofluorescence staining. b) DNA gel electrophoresis analysis of MagT1 expression in BMSCs incubated with or without osteogenic medium (DX). c) qPCR shows the upregulation of MagT1 during osteogenic induction (n = 3, ** p < 0.01). d) Double immunofluorescence staining of OCN and MagT1. e) Schemata of construction of the Mg‐enriched 3D culture system for bone regeneration. Mg²⁺ influx via MagT1 mediates the osteogenic differentiation of stem cells.

Figure 2

Mg²⁺ promotes osteogenic differentiation via high MagT1 expression. a) Analysis of the effect of Mg²⁺ on cell viability using IC₅₀ assays (n = 6). b) qPCR analysis of MagT1 expression in BMSCs treated with different concentrations of Mg²⁺ (n = 3, **p < 0.01). c) Immunofluorescence assay of MagT1 expression in BMSCs after 5 days of incubation. The cytoskeletal structure (green) and nuclei (blue) were stained separately. d) Flow cytometry‐based apoptosis assay using Annexin V‐FITC/PI staining. Dots in the upper right quadrant represent late apoptotic cells. e) ALP staining of BMSCs exposed to different Mg²⁺ environments for 5 days. f) Semiquantitative analysis of ALP activity after 5 days of induction (n = 3, **p < 0.01). g) Immunoblots display the increased expression of OCN in BMSCs induced by a Mg‐enriched environment. h) Detection of OCN levels by immunofluorescence staining.

Figure 3

Investigation of the mechanisms underlying the osteoinductivity of Mg²⁺. a) Western blot assay shows the phosphorylation of MAPK pathway components in BMSCs treated with 5 × 10⁻³ m Mg²⁺. b) Immunoblots display the inhibitory effects of PD98059, a specific Erk1/2 inhibitor, on the activation of Erk1/2 (60 min) induced by Mg²⁺. c) An obvious decrease in OCN levels in BMSCs treated with PD98059 was detected by western blotting. d) ALP staining of BMSCs treated with or without PD98059. e) Semiquantitative analysis of ALP activity in BMSCs (n = 3, *p < 0.05). f) Site targeted by the CRISPR/Cas9 system in the rat MagT1 gene. g) A 6 bp mutation (in yellow area) at the target site resulting from CRISPR/Cas9 targeting was confirmed by gene sequencing. h) Mg–Fura‐2 detection of Mg²⁺ entry into BMSCs. i) Inhibitory effects of MagT1 knockout on Erk1/2 activation. j) ALP staining of normal and mutated BMSCs incubated with 5 × 10⁻³ m Mg²⁺ for 5 days. k) Semiquantitative analysis shows a sharp decrease in ALP activity in mutated BMSCs (n = 3, **p < 0.01).

Figure 4

Optimization of the Mg‐enriched 3D culture system and the in vitro osteogenic differentiation of encapsulated BMSCs. a) Cell viability analysis of BMSCs inside spheroids of different sizes. Live cells (green) and dead cells (red) were detected by CLSM. b) BMSCs remained viable inside spheroids with a localized Mg‐enriched microenvironment. c) CCK‐8 assays of the viability of encapsulated BMSCs at different time points (n = 3, 50 µL of spheroids per sample). d) Sequential evaluation of encapsulated BMSC proliferation by EdU staining. e) Percentage of EdU‐positive cells encapsulated in spheroids (n = 3, *p < 0.05, **p < 0.01). f) Observation of GFP‐labeled cell migration from spheroids to culture dishes. 3D images were reconstructed by CLSM. g–j) The graphs show that the local Mg‐enriched environment in spheroids significantly stimulated the expression of Runx2 and ALP in BMSCs at the early stage of incubation (day 3), while high Osx and OCN expressions were observed at day 5 (n = 3, 50 µL of spheroids per sample. **p < 0.01).

Figure 5

In vitro and in vivo analyses of the angiogenic effects of a Mg‐enriched microenvironment. a–c) qPCR analysis of the expression levels of chemotactic cytokine genes (n = 3, **p < 0.01). d) Schemata of the transwell migration model. e) Cells that migrated to the lower chamber were visualized by microscopy after crystal violet staining (n = 3, **p < 0.01). f) Cell migration was quantified by cell counting. Five random high‐power fields (HPFs) were selected in each well (**p < 0.01). g) In vivo evaluation of neovascularization. Blood perfusion in each group was detected by LDI at different time points (n = 3). Implantation sites are circled. Relative flux intensity was calculated using LDI system software (**p < 0.01). Mice were sacrificed on day 14, and samples were prepared for immunofluorescence staining of CD31 and α‐SMA. Six random HPFs were selected in each group for the calculation of blood vessel area (n = 6, **p < 0.01).

Figure 6

In vivo evaluation of vascularized bone regeneration using the rat cranial defects model. a) The graph shows the radiological analysis of samples collected 4 weeks after implantation. The yellow area indicates the defect area (n = 6). b,c) Statistical analysis of newly formed bone in the study groups (**p < 0.01). d) Decalcified sections were stained with H&E and Masson trichrome, and newly formed bone tissues appear blue. e) Costaining of CD31 and ALP revealed significant vascularized bone regeneration in the SA‐Mg/BM group.