Abstract:Barrier
membranes for guided tissue regeneration are essential
for bone repair and regeneration. The implanted membranes may trigger
early inflammatory responses as a foreign material, which can affect
the recruitment and differentiation of bone cells during tissue regeneration.
The purpose of this study was to determine whether immobilizing interleukin
4 (IL4) on plasma immersion ion implantation (PIII)-activated surfaces
may alter the osteo-immunoregulatory characteristics of the membranes
and produce pro-os… Show more
“…Macrophages were classically polarized into inflammatory phenotypes by LPS stimulation in vitro. 21 In this study, we observed that inflammatory macrophages treated with MDs-IL4 displayed markedly elongated cell morphologies compared to non-treated and MDs treated groups ( Figure 4A ), similar to the reports by other groups. 22 , 23 Figure 4 Immunoregulatory role of bioactive MDs-IL4 on inflammatory macrophages.…”
Section: Resultssupporting
confidence: 89%
“…Macrophages were classically polarized into inflammatory phenotypes by LPS stimulation in vitro. 21 In this study, we observed that inflammatory macrophages treated with MDs-IL4 displayed markedly 4A), similar to the reports by other groups. 22,23 Next, we used qPCR to determine the relative gene expression of proinflammatory markers in different groups.…”
Section: Immunoregulatory Role Of Bioactive Mds-il4supporting
confidence: 91%
“… 27 It has been demonstrated that modulation of early osteoimmune environment will have significant impact for fracture healing. 20 , 21 In general, bone healing involves four specific biological phases: 1) initial inflammation phase, 2) soft callus phase, 3) hard callus formation, and 4) bone remodeling phase. The initial inflammatory phase is characterized by the formation of fracture hematoma, activation of the innate immune system, recruitment of immune and mesenchymal stem cells, and release of various cytokine and growth factor, etc.…”
Section: Discussionmentioning
confidence: 99%
“… 42 Furthermore, Wei et al recently developed plasma immersion ion implantation (PIII)-activated polycaprolactone (PCL) surfaces with IL4-immobilization and found its unique osteo-immunoregulatory role of IL-4/PIII surface with significant pro-osteogenic effects when compared to bio-inert PCL surface. 21 Therefore, fabrication of bioactive materials capable of enhancing M1 to M2 phenotype transition, decreasing prolonged M1 macrophages or inflammation, and promoting pro-healing M2 macrophage populations have gained an increasing attention in recent years.…”
Section: Discussionmentioning
confidence: 99%
“…Macrophages were activated with LPS according to a previous study. 21 Briefly, macrophages were seeded on 24-well tissue culture coverslip overnight, then stimulated with 1000 ng/mL of Lipopolysaccharide (LPS, Escherichia coli 0111: B4, Sigma, China) for 12 h. Cells were rinsed with PBS three times, then treated with different formulation of MDs for 12 h. Conditioned medium (CM) from RAW264.7 cells stimulated with or without different formulation of MDs were collected for subsequent analysis. CM were centrifuged at 2000 g for 10 min 4°C, then filtered using a 0.2 µm filter to remove cell debris.…”
Background
Despite the inherent regenerative ability of bone, large bone defect regeneration remains a major clinical challenge for orthopedic surgery. Therapeutic strategies medicated by M2 phenotypic macrophages or M2 macrophage inducer have been widely used to promote tissue remodeling. In this study, ultrasound-responsive bioactive microdroplets (MDs) encapsulated with bioactive molecule interleukin-4 (IL4, hereafter designated MDs-IL4) were fabricated to regulate macrophage polarization and potentiate the osteogenic differentiation of human mesenchymal stem cells (hBMSCs).
Materials and Methods
The MTT assay, live and dead staining, and phalloidin/DAPI dual staining were used to evaluate biocompatibility in vitro. H&E staining was used to evaluate biocompatibility in vivo. Inflammatory macrophages were further induced via lipopolysaccharide (LPS) stimulation to mimic the pro-inflammatory condition. The immunoregulatory role of the MDs-IL4 was tested via macrophage phenotypic marker gene expression, pro-inflammatory cytokine level, cell morphological analysis, and immunofluorescence staining, etc. The immune-osteogenic response of hBMSCs via macrophages and hBMSCs interactions was further investigated in vitro.
Results
The bioactive MDs-IL4 scaffold showed good cytocompatibility in RAW 264.7 macrophages and hBMSCs. The results confirmed that the bioactive MDs-IL4 scaffold could reduce inflammatory phenotypic macrophages, as evidenced by changing in morphological features, reduction in pro-inflammatory marker gene expression, increase of M2 phenotypic marker genes, and inhibition of pro-inflammatory cytokine secretion. Additionally, our results indicate that the bioactive MDs-IL4 could significantly enhance the osteogenic differentiation of hBMSCs via its potential immunomodulatory properties.
Conclusion
Our results demonstrate that the bioactive MDs-IL4 scaffold could be used as novel carrier system for other pro-osteogenic molecules, thus having potential applications in bone tissue regeneration.
“…Macrophages were classically polarized into inflammatory phenotypes by LPS stimulation in vitro. 21 In this study, we observed that inflammatory macrophages treated with MDs-IL4 displayed markedly elongated cell morphologies compared to non-treated and MDs treated groups ( Figure 4A ), similar to the reports by other groups. 22 , 23 Figure 4 Immunoregulatory role of bioactive MDs-IL4 on inflammatory macrophages.…”
Section: Resultssupporting
confidence: 89%
“…Macrophages were classically polarized into inflammatory phenotypes by LPS stimulation in vitro. 21 In this study, we observed that inflammatory macrophages treated with MDs-IL4 displayed markedly 4A), similar to the reports by other groups. 22,23 Next, we used qPCR to determine the relative gene expression of proinflammatory markers in different groups.…”
Section: Immunoregulatory Role Of Bioactive Mds-il4supporting
confidence: 91%
“… 27 It has been demonstrated that modulation of early osteoimmune environment will have significant impact for fracture healing. 20 , 21 In general, bone healing involves four specific biological phases: 1) initial inflammation phase, 2) soft callus phase, 3) hard callus formation, and 4) bone remodeling phase. The initial inflammatory phase is characterized by the formation of fracture hematoma, activation of the innate immune system, recruitment of immune and mesenchymal stem cells, and release of various cytokine and growth factor, etc.…”
Section: Discussionmentioning
confidence: 99%
“… 42 Furthermore, Wei et al recently developed plasma immersion ion implantation (PIII)-activated polycaprolactone (PCL) surfaces with IL4-immobilization and found its unique osteo-immunoregulatory role of IL-4/PIII surface with significant pro-osteogenic effects when compared to bio-inert PCL surface. 21 Therefore, fabrication of bioactive materials capable of enhancing M1 to M2 phenotype transition, decreasing prolonged M1 macrophages or inflammation, and promoting pro-healing M2 macrophage populations have gained an increasing attention in recent years.…”
Section: Discussionmentioning
confidence: 99%
“…Macrophages were activated with LPS according to a previous study. 21 Briefly, macrophages were seeded on 24-well tissue culture coverslip overnight, then stimulated with 1000 ng/mL of Lipopolysaccharide (LPS, Escherichia coli 0111: B4, Sigma, China) for 12 h. Cells were rinsed with PBS three times, then treated with different formulation of MDs for 12 h. Conditioned medium (CM) from RAW264.7 cells stimulated with or without different formulation of MDs were collected for subsequent analysis. CM were centrifuged at 2000 g for 10 min 4°C, then filtered using a 0.2 µm filter to remove cell debris.…”
Background
Despite the inherent regenerative ability of bone, large bone defect regeneration remains a major clinical challenge for orthopedic surgery. Therapeutic strategies medicated by M2 phenotypic macrophages or M2 macrophage inducer have been widely used to promote tissue remodeling. In this study, ultrasound-responsive bioactive microdroplets (MDs) encapsulated with bioactive molecule interleukin-4 (IL4, hereafter designated MDs-IL4) were fabricated to regulate macrophage polarization and potentiate the osteogenic differentiation of human mesenchymal stem cells (hBMSCs).
Materials and Methods
The MTT assay, live and dead staining, and phalloidin/DAPI dual staining were used to evaluate biocompatibility in vitro. H&E staining was used to evaluate biocompatibility in vivo. Inflammatory macrophages were further induced via lipopolysaccharide (LPS) stimulation to mimic the pro-inflammatory condition. The immunoregulatory role of the MDs-IL4 was tested via macrophage phenotypic marker gene expression, pro-inflammatory cytokine level, cell morphological analysis, and immunofluorescence staining, etc. The immune-osteogenic response of hBMSCs via macrophages and hBMSCs interactions was further investigated in vitro.
Results
The bioactive MDs-IL4 scaffold showed good cytocompatibility in RAW 264.7 macrophages and hBMSCs. The results confirmed that the bioactive MDs-IL4 scaffold could reduce inflammatory phenotypic macrophages, as evidenced by changing in morphological features, reduction in pro-inflammatory marker gene expression, increase of M2 phenotypic marker genes, and inhibition of pro-inflammatory cytokine secretion. Additionally, our results indicate that the bioactive MDs-IL4 could significantly enhance the osteogenic differentiation of hBMSCs via its potential immunomodulatory properties.
Conclusion
Our results demonstrate that the bioactive MDs-IL4 scaffold could be used as novel carrier system for other pro-osteogenic molecules, thus having potential applications in bone tissue regeneration.
Biomaterials encounter considerable challenges in extensive bone defect regeneration. The amelioration of outcomes may be attainable through the orchestrated modulation of both innate and adaptive immunity. Silicon‐hydroxyapatite, for instance, which solely focuses on regulating innate immunity, is inadequate for long‐term bone regeneration. Herein, extra manganese (Mn)‐doping is utilized for enhancing the osteogenic ability by mediating adaptive immunity. Intriguingly, Mn‐doping engenders heightened recruitment of CD4+ T cells to the bone defect site, concurrently manifesting escalated T helper (Th) 2 polarization and an abatement in Th1 cell polarization. This consequential immune milieu yields a collaborative elevation of interleukin 4, secreted by Th2 cells, coupled with attenuated interferon gamma, secreted by Th1 cells. This orchestrated interplay distinctly fosters the osteogenesis of bone marrow stromal cells and effectuates consequential regeneration of the mandibular bone defect. The modulatory mechanism of Th1/Th2 balance lies primarily in the indispensable role of manganese superoxide dismutase (MnSOD) and the phosphorylation of adenosine 5′‐monophosphate‐activated protein kinase (AMPK). In conclusion, this study highlights the transformative potential of Mn‐doping in amplifying the osteogenic efficacy of silicon‐hydroxyapatite nanowires by regulating T cell‐mediated adaptive immunity via the MnSOD/AMPK pathway, thereby creating an anti‐inflammatory milieu favorable for bone regeneration.
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