IM
I. Mooij
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Bone regeneration and osseointegration are important factors for orthopedic implant success. Osteoimmunomodulation (OIM) has emerged as a design strategy to guide macrophage polarization through biomaterial surface properties, promoting the immune conditions that favours osseointegration. While discrete topographic features have been extensively studied, substrate curvature as a continuously variable geometric cue remains underexplored. This thesis investigated whether curvature orientation influences human THP-1-derived macrophage behaviour, using polystyrene substrates with convex (cvPS) and concave (ccPS) unduloid geometries compared to flat controls (fPS).
Although a decrease of M1 macrophages, both M0 and M1 are found viable on all substrates. Curvature orientation influenced spatial distribution: macrophages on ccPS preferentially localised within concave unduloid regions, while on cvPS cells accumulated relatively more in the flat interspaces by day 3. Three morphological categories were identified across all surfaces, with a general temporal shift toward rounded morphologies by day 3 independent of substrate geometry. M0 macrophages on curved substrates showed upregulation of TNFA, CD86 and CD206, while M1 macrophages mainly showed trends toward increased anti-inflammatory gene expression with high inter-experimental variability.
These results provide the first evidence that unduloid surface curvature influences macrophage spatial organisation and gene expression, supporting substrate curvature as a relevant and tunable parameter for osteoimmunomodulatory biomaterial design. ...
Although a decrease of M1 macrophages, both M0 and M1 are found viable on all substrates. Curvature orientation influenced spatial distribution: macrophages on ccPS preferentially localised within concave unduloid regions, while on cvPS cells accumulated relatively more in the flat interspaces by day 3. Three morphological categories were identified across all surfaces, with a general temporal shift toward rounded morphologies by day 3 independent of substrate geometry. M0 macrophages on curved substrates showed upregulation of TNFA, CD86 and CD206, while M1 macrophages mainly showed trends toward increased anti-inflammatory gene expression with high inter-experimental variability.
These results provide the first evidence that unduloid surface curvature influences macrophage spatial organisation and gene expression, supporting substrate curvature as a relevant and tunable parameter for osteoimmunomodulatory biomaterial design. ...
Bone regeneration and osseointegration are important factors for orthopedic implant success. Osteoimmunomodulation (OIM) has emerged as a design strategy to guide macrophage polarization through biomaterial surface properties, promoting the immune conditions that favours osseointegration. While discrete topographic features have been extensively studied, substrate curvature as a continuously variable geometric cue remains underexplored. This thesis investigated whether curvature orientation influences human THP-1-derived macrophage behaviour, using polystyrene substrates with convex (cvPS) and concave (ccPS) unduloid geometries compared to flat controls (fPS).
Although a decrease of M1 macrophages, both M0 and M1 are found viable on all substrates. Curvature orientation influenced spatial distribution: macrophages on ccPS preferentially localised within concave unduloid regions, while on cvPS cells accumulated relatively more in the flat interspaces by day 3. Three morphological categories were identified across all surfaces, with a general temporal shift toward rounded morphologies by day 3 independent of substrate geometry. M0 macrophages on curved substrates showed upregulation of TNFA, CD86 and CD206, while M1 macrophages mainly showed trends toward increased anti-inflammatory gene expression with high inter-experimental variability.
These results provide the first evidence that unduloid surface curvature influences macrophage spatial organisation and gene expression, supporting substrate curvature as a relevant and tunable parameter for osteoimmunomodulatory biomaterial design.
Although a decrease of M1 macrophages, both M0 and M1 are found viable on all substrates. Curvature orientation influenced spatial distribution: macrophages on ccPS preferentially localised within concave unduloid regions, while on cvPS cells accumulated relatively more in the flat interspaces by day 3. Three morphological categories were identified across all surfaces, with a general temporal shift toward rounded morphologies by day 3 independent of substrate geometry. M0 macrophages on curved substrates showed upregulation of TNFA, CD86 and CD206, while M1 macrophages mainly showed trends toward increased anti-inflammatory gene expression with high inter-experimental variability.
These results provide the first evidence that unduloid surface curvature influences macrophage spatial organisation and gene expression, supporting substrate curvature as a relevant and tunable parameter for osteoimmunomodulatory biomaterial design.
The use of biomaterials for orthopedic implants has significantly increased over the past decades, offering promising solutions as bone substitutes. However, challenges such as implant-associated infections and aseptic loosening remain, as biomaterials trigger an immune response upon implantation. Recent insights have highlighted the crucial role of this immune response in bone regeneration, shifting the focus toward understanding the dynamic interplay between immune cells and osteogenic cells. Macrophages, as key regulators of inflammation and bone regeneration, closely interact with human mesenchymal stem cells (hMSCs), influencing their cellular behavior. As a result, there is increasing interest in designing biomaterials that simultaneously support osteogenesis and modulate immune responses to facilitate enhanced implant integration. However, most studies investigating 3D-printed biomaterials and their effects on hMSCs and macrophage behavior rely on monoculture or indirect co-culture models, thereby neglecting direct cell-cell interactions. This limitation creates a gap between in vitro models and the in vivo environment.
This study addresses this gap by developing a direct co-culture model of hMSCs and THP-1-derived M0 macrophages on 3D-printed Ti-6Al-4V biomaterials to investigate their interactions at the implant-tissue interface. By examining cell adhesion, morphology, and cytokine secretion, this research provides insights into how direct cell-cell interactions and biomaterial properties influence early-stage cellular responses.
To interpret the results of the co-culture model, both cell types were first characterized in monoculture to assess their proliferation, adhesion, and morphology affected by the titanium substrate using immunofluorescence staining and scanning electron microscopy (SEM). In addition, TNF-a and IL-6 cytokine secretion by M0 macrophages was assessed via Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate macrophage polarization. Following this, the mixed culture medium was optimized, and seeding densities and ratios were determined to ensure the viability of both the hMSCs and macrophages in co-culture. M0 macrophages were seeded first to allow attachment before introducing hMSCs, mimicking in vivo conditions where macrophages are the first to arrive at the implant site. Co-culture effects on cell morphology and cytokine secretion were analyzed using immunofluorescence staining, SEM, and ELISA. Additionally, hMSCs were cultured on both dense and porous 3D-printed Ti-6Al-4V substrates in monoculture to assess the effects of substrate porosity on early-stage cell adhesion and morphology.
The findings of this study showed the significant influence of the 3D-printed Ti-6Al-4V substrate on cell morphology, suggesting that it can override the effects of cell-cell interactions and paracrine signaling. As a result, macrophages adopted a more pro-inflammatory morphology, while hMSCs exhibited a more spread-out shape, which may be indicative of early osteogenic differentiation. In addition, cytokine secretion profiles in monoculture showed a trend toward an M1-like macrophage phenotype when the cells were cultured on titanium. In contrast, co-culture conditions led to a shift toward a more pro-repair environment, characterized by reduced TNF-asecretion and increased IL-6 production. This suggests that hMSCs modulate the macrophage response toward a more pro-repair phenotype.
A comparison between dense and porous 3D-printed Ti-6Al-4V substrates revealed no statistically significant differences in hMSC morphology after 7 days, indicating that substrate geometry has only a minor effect on early-stage cell adhesion and morphology.
Overall, these findings highlight the potential of the developed co-culture model for studying osteoimmunomodulation on titanium biomaterials, contributing to bridging the gap between in vitro models and in vivo conditions.
...
This study addresses this gap by developing a direct co-culture model of hMSCs and THP-1-derived M0 macrophages on 3D-printed Ti-6Al-4V biomaterials to investigate their interactions at the implant-tissue interface. By examining cell adhesion, morphology, and cytokine secretion, this research provides insights into how direct cell-cell interactions and biomaterial properties influence early-stage cellular responses.
To interpret the results of the co-culture model, both cell types were first characterized in monoculture to assess their proliferation, adhesion, and morphology affected by the titanium substrate using immunofluorescence staining and scanning electron microscopy (SEM). In addition, TNF-a and IL-6 cytokine secretion by M0 macrophages was assessed via Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate macrophage polarization. Following this, the mixed culture medium was optimized, and seeding densities and ratios were determined to ensure the viability of both the hMSCs and macrophages in co-culture. M0 macrophages were seeded first to allow attachment before introducing hMSCs, mimicking in vivo conditions where macrophages are the first to arrive at the implant site. Co-culture effects on cell morphology and cytokine secretion were analyzed using immunofluorescence staining, SEM, and ELISA. Additionally, hMSCs were cultured on both dense and porous 3D-printed Ti-6Al-4V substrates in monoculture to assess the effects of substrate porosity on early-stage cell adhesion and morphology.
The findings of this study showed the significant influence of the 3D-printed Ti-6Al-4V substrate on cell morphology, suggesting that it can override the effects of cell-cell interactions and paracrine signaling. As a result, macrophages adopted a more pro-inflammatory morphology, while hMSCs exhibited a more spread-out shape, which may be indicative of early osteogenic differentiation. In addition, cytokine secretion profiles in monoculture showed a trend toward an M1-like macrophage phenotype when the cells were cultured on titanium. In contrast, co-culture conditions led to a shift toward a more pro-repair environment, characterized by reduced TNF-asecretion and increased IL-6 production. This suggests that hMSCs modulate the macrophage response toward a more pro-repair phenotype.
A comparison between dense and porous 3D-printed Ti-6Al-4V substrates revealed no statistically significant differences in hMSC morphology after 7 days, indicating that substrate geometry has only a minor effect on early-stage cell adhesion and morphology.
Overall, these findings highlight the potential of the developed co-culture model for studying osteoimmunomodulation on titanium biomaterials, contributing to bridging the gap between in vitro models and in vivo conditions.
...
The use of biomaterials for orthopedic implants has significantly increased over the past decades, offering promising solutions as bone substitutes. However, challenges such as implant-associated infections and aseptic loosening remain, as biomaterials trigger an immune response upon implantation. Recent insights have highlighted the crucial role of this immune response in bone regeneration, shifting the focus toward understanding the dynamic interplay between immune cells and osteogenic cells. Macrophages, as key regulators of inflammation and bone regeneration, closely interact with human mesenchymal stem cells (hMSCs), influencing their cellular behavior. As a result, there is increasing interest in designing biomaterials that simultaneously support osteogenesis and modulate immune responses to facilitate enhanced implant integration. However, most studies investigating 3D-printed biomaterials and their effects on hMSCs and macrophage behavior rely on monoculture or indirect co-culture models, thereby neglecting direct cell-cell interactions. This limitation creates a gap between in vitro models and the in vivo environment.
This study addresses this gap by developing a direct co-culture model of hMSCs and THP-1-derived M0 macrophages on 3D-printed Ti-6Al-4V biomaterials to investigate their interactions at the implant-tissue interface. By examining cell adhesion, morphology, and cytokine secretion, this research provides insights into how direct cell-cell interactions and biomaterial properties influence early-stage cellular responses.
To interpret the results of the co-culture model, both cell types were first characterized in monoculture to assess their proliferation, adhesion, and morphology affected by the titanium substrate using immunofluorescence staining and scanning electron microscopy (SEM). In addition, TNF-a and IL-6 cytokine secretion by M0 macrophages was assessed via Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate macrophage polarization. Following this, the mixed culture medium was optimized, and seeding densities and ratios were determined to ensure the viability of both the hMSCs and macrophages in co-culture. M0 macrophages were seeded first to allow attachment before introducing hMSCs, mimicking in vivo conditions where macrophages are the first to arrive at the implant site. Co-culture effects on cell morphology and cytokine secretion were analyzed using immunofluorescence staining, SEM, and ELISA. Additionally, hMSCs were cultured on both dense and porous 3D-printed Ti-6Al-4V substrates in monoculture to assess the effects of substrate porosity on early-stage cell adhesion and morphology.
The findings of this study showed the significant influence of the 3D-printed Ti-6Al-4V substrate on cell morphology, suggesting that it can override the effects of cell-cell interactions and paracrine signaling. As a result, macrophages adopted a more pro-inflammatory morphology, while hMSCs exhibited a more spread-out shape, which may be indicative of early osteogenic differentiation. In addition, cytokine secretion profiles in monoculture showed a trend toward an M1-like macrophage phenotype when the cells were cultured on titanium. In contrast, co-culture conditions led to a shift toward a more pro-repair environment, characterized by reduced TNF-asecretion and increased IL-6 production. This suggests that hMSCs modulate the macrophage response toward a more pro-repair phenotype.
A comparison between dense and porous 3D-printed Ti-6Al-4V substrates revealed no statistically significant differences in hMSC morphology after 7 days, indicating that substrate geometry has only a minor effect on early-stage cell adhesion and morphology.
Overall, these findings highlight the potential of the developed co-culture model for studying osteoimmunomodulation on titanium biomaterials, contributing to bridging the gap between in vitro models and in vivo conditions.
This study addresses this gap by developing a direct co-culture model of hMSCs and THP-1-derived M0 macrophages on 3D-printed Ti-6Al-4V biomaterials to investigate their interactions at the implant-tissue interface. By examining cell adhesion, morphology, and cytokine secretion, this research provides insights into how direct cell-cell interactions and biomaterial properties influence early-stage cellular responses.
To interpret the results of the co-culture model, both cell types were first characterized in monoculture to assess their proliferation, adhesion, and morphology affected by the titanium substrate using immunofluorescence staining and scanning electron microscopy (SEM). In addition, TNF-a and IL-6 cytokine secretion by M0 macrophages was assessed via Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate macrophage polarization. Following this, the mixed culture medium was optimized, and seeding densities and ratios were determined to ensure the viability of both the hMSCs and macrophages in co-culture. M0 macrophages were seeded first to allow attachment before introducing hMSCs, mimicking in vivo conditions where macrophages are the first to arrive at the implant site. Co-culture effects on cell morphology and cytokine secretion were analyzed using immunofluorescence staining, SEM, and ELISA. Additionally, hMSCs were cultured on both dense and porous 3D-printed Ti-6Al-4V substrates in monoculture to assess the effects of substrate porosity on early-stage cell adhesion and morphology.
The findings of this study showed the significant influence of the 3D-printed Ti-6Al-4V substrate on cell morphology, suggesting that it can override the effects of cell-cell interactions and paracrine signaling. As a result, macrophages adopted a more pro-inflammatory morphology, while hMSCs exhibited a more spread-out shape, which may be indicative of early osteogenic differentiation. In addition, cytokine secretion profiles in monoculture showed a trend toward an M1-like macrophage phenotype when the cells were cultured on titanium. In contrast, co-culture conditions led to a shift toward a more pro-repair environment, characterized by reduced TNF-asecretion and increased IL-6 production. This suggests that hMSCs modulate the macrophage response toward a more pro-repair phenotype.
A comparison between dense and porous 3D-printed Ti-6Al-4V substrates revealed no statistically significant differences in hMSC morphology after 7 days, indicating that substrate geometry has only a minor effect on early-stage cell adhesion and morphology.
Overall, these findings highlight the potential of the developed co-culture model for studying osteoimmunomodulation on titanium biomaterials, contributing to bridging the gap between in vitro models and in vivo conditions.