Fig. 1. Schematic illustration for the principles of bone defects filling and repair by MMSC. The MMSs are not only of the desired physicochemical properties to meet the requirement of injectable bone cement for clinical application, but also can be cured into a 3D interconnected porous scaffold conducive to cells and tissue ingrowth in situ. Moreover, the controllable biodegradation of MMSC continuously provides increasing space while the release of magnesium ions induces a tissue repair favourable immunoregulation via the M2 phenotype polarization of macrophages for the consecutive vascularization and new bone formation.
Fig. 2. Preparation and characterization of MMSs. (a) Schematic diagram of the MMSs fabrication process. (b) SEM showed that micropores distributed on the surface and in the interior of the MMSs. (c) BET results indicated that the pore diameters of MMSs mainly ranged from 2~60 nm. (d) The elemental mapping results displayed two distribution areas: Ca-P and Mg-Si-O. (e) XRD patterns showed that the main phase of MMSs was MgO, and the secondary phases were Ca7Mg2P6O24 and Mg2SiO4 compared with JCPDS#45-0946, JCPDS#20-0348, and JCPDS#34-0189, which corresponded to MgO, Ca7Mg2P6O24, and Mg2SiO4, respectively.
Fig. 3. Setting process of MMSC. (a) Digital photography and SEM images exhibited interconnected porous scaffolds with different 3D shapes cured by MMSC. (b) XRD results indicated that MgO within MMSs had reacted completely, and a new phase, NH4MgPO4.6H2O, formed after setting for 12 h compared with JCPDS#15-0762. (c) SEM image at a high magnification showed lamellar crystals formed between microspheres after setting for 20 min. (d) Element line scan results indicated that the phase composite of the lamellar crystals was NH4MgPO4.6H2O; 1, 2, and 3 pointed to the phases of MgO/Mg2SiO4, Ca7Mg2P6O24 and NH4MgPO4.6H2O, respectively. (e) Schematic diagram of the MMSC setting process.
Fig. 4. Physiochemical properties of MMSC compared with those of the MPC and CPC. (a) The rheological curve showed that the MMSC pastes had a constant and low viscosity, which suggested good rheological properties and practicable injectability. (b) The heat release of MMSC was well-controlled in comparison with MPC suggesting that the microspheres effectively reduced the exothermic reaction (c) The setting time was approximately 10 min in the MMSC, which well-met the requirement of clinical application. (d) Mechanical strength results showed that the compressive strength of the MMSC was stable after setting for 1 h. (mean ± SD; n = 3; *significant difference compared with the MPC group, *p < 0.05, **p < 0.01; # significant difference between groups, #p < 0.05). (e) MMSC had a moderate degradation rate with a weight loss of approximately 8 wt% after 21 days (mean ± SD, n=5). (f, g) Calcium and magnesium ions can be released from the MMSC simultaneously at a moderate rate (mean ± SD, n=5).
Fig. 5. In vitro cytocompatibility, immunoregulation and osteogenic induction. (a, b) CCK-8 assay showed all cement extracts with concentration ≤ 3.125 mg/mL were of no cytotoxicity to RAW264.7 cells. B: DMEM; 128, 64, 32, and 16 represent 1/128, 1/64, 1/32 and 1/16 of the original extract concentration (200 mg/mL), respectively. (c) Cement extracts with a concentration of 3.125 mg/mL had no cytotoxicity on rbMSCs. (d, e) RT-PCR results showed that the relative expression levels of CD206 and IL-10 significantly upregulated in RAW264.7 cells treated with MMSC extract at a concentration of 3.125 mg/mL, whereas the relative expression levels of IL-6 were significantly higher than IL-10 in the MPC and CPC groups. (f) ELISA tests revealed that the concentration of IL-10 in the MMSC group was significantly higher than that in the B and MPC group in the coculture system of RAW264.7 cells and rbMSCs. (g) RT-PCR results demonstrated that the relative expression level of COL1 in the MMSC group was upregulated and significantly higher than that in the B group in the coculture system (mean ± SD; n = 3; *significant difference compared with the B group, *p < 0.05, **p < 0.01, ***p < 0.001; # significant difference between groups, #p < 0.05).
Fig. 6. Short-term immune response in vivo. Masson staining of MPC, CPC and MMSC subcutaneously implanted for 3 days (a, b, c) and 7 days (d, e, f). White dash lines portray the material boundaries. Green pentagrams indicate the cells infiltrated into the materials via the biodegradation of MPC and MMSC. White arrows point out the newly formed blood vessels in the gap of the MMSs. Scale bars are 50 μm. (g-l) SEM with elemental mapping images indicated the tissue ingrowth after 7 days of material implantation through the distribution of carbon elements (marked in red). White dotted lines describe the material boundaries. Scale bars are 500 μm. (m, n, o) HE staining of MPC, CPC and MMSC after 14 days of implantation. Aggregation of massive immune cells was observed surrounding the incompletely degraded MPC, while the CPC with no obvious degradation was encapsulated by compact fibrous tissues. By contrast, continuous ingrowth of cells and blood vessels took the space provided by the degradation of the microspheres in MMSC.
Fig. 7. Immunostaining of CD68, F4/80, CD86 and CD163 in the (a) MPC, (b) CPC and (c) MMSC after 14 days of material implantation. Red arrows pointed out the CD163-positive stained cells. CD68, F4/80, CD86 and CD163 are the cell markers of the immune cells, macrophages, M1 macrophages and M2 macrophages, respectively. The positive staining of these markers was found surrounding the MPC and CPC while inside the MMSC, demonstrating the porous structure of MMSC was beneficial to the inward migration of immune cells and macrophages. More importantly, CD163 was much more positive than CD86 in MMSC, which suggested a predominant M2 phenotype polarization of macrophages conducive to anti-inflammation and tissue repair induced by the MMSC.
Fig. 8. Subcutaneous ectopic osteogenesis after 12 weeks of cements implantation. (a-f) No obvious new bone formation was found within the MPC and CPC, while they were encapsulated by dense fibrous membranes. (g-k) There was not only no obvious fibrous membrane encapsulating the MMSC, but also massive new bone formation was observed within the MMSC. Osteoblasts were also noticed at the boundaries of the incompletely degraded MMSs. M: material; NB: new bone; OB: osteoblast; Dotted circle: microspheres with new bone