MN
M. Nouri Goushki
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Towards Direct Co-culture Models for Osteoimmunodulatory Studies
An exploratory study on the effects of nanopatterns and shear stress on cellular crosstalk
Master thesis
(2021)
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B.I.M. Eijkel, A.A. Zadpoor, E.L. Fratila-Apachitei, M. Nouri Goushki, M. Mirzaali Mazandarani
In the last two decades, it has been generally accepted that the interaction between bone and immune cells is critical in the process of bone healing and regeneration. Steering this immune response with the development of biomaterials could lead to implant acceptance in the long term. The understanding of osteoimmunological responses is of high importance in the development of new biomaterials. In order to validate these biomaterials, better in vitro techniques are required that could mimic this inflammatory response immediately after implantation in more detail. This study investigated the effect of a direct co-culture model on a prototype nanopattern. In addition the effect of microflows, that are normally present in vivo, induced inside a microfluidic chip, on mono and co-cultures was investigated in order to improve in vitro research.
Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials.
The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices. ...
Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials.
The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices. ...
In the last two decades, it has been generally accepted that the interaction between bone and immune cells is critical in the process of bone healing and regeneration. Steering this immune response with the development of biomaterials could lead to implant acceptance in the long term. The understanding of osteoimmunological responses is of high importance in the development of new biomaterials. In order to validate these biomaterials, better in vitro techniques are required that could mimic this inflammatory response immediately after implantation in more detail. This study investigated the effect of a direct co-culture model on a prototype nanopattern. In addition the effect of microflows, that are normally present in vivo, induced inside a microfluidic chip, on mono and co-cultures was investigated in order to improve in vitro research.
Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials.
The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices.
Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials.
The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices.
Master thesis
(2019)
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Katerina Isaakidou, Lidy Fratila-Apachitei, Mahdiyeh Nouri Goushki, Amir Zadpoor
Objective: Advances in the field of biomaterials have positively affected implant acceptance and nowadays nanostructured surfaces are known to have a positive effect on bone regeneration. The development of the field of osteoimmunology has contributed to a steadily increasing interest towards the investigation of the immunomodulatory effect of nanopatterned surfaces. Luckily, advances in nanofabrication methods have resulted in fabricating relatively large nanostructured areas with high resolution. This study is taking advantage of the 3D printing technique called two-photon polymerization (2PP) for the generation of 3D submicron pillar structures (patterns) to systematically investigate their effect on murine macrophages regarding cell migration, viability, cytoskeletal organisation and phenotype polarization. Methods: Six different pillar patterns were fabricated using a Photonic Professional GT (Nanoscribe, Germany) system. The pillars were characterized with scanning electron microscopy (SEM). Cell viability of J774A.1 macrophages on the nanofabricated patters was assessed by live/dead staining with calcein AM and ethidium homodimer-1 and fluorescently analyzed. Cell migration to the patterns was studied using DAPI staining. The cytoskeletal organization of the macrophages was investigated by actin staining and analyzed both through fluorescent microscopy as well as SEM. Macrophage expression of nitric oxide (NO) was tested with Griess assay. The phenotype polarization of the macrophages on the patterns was explored by dual immunofluorescent staining with iNOS/ARG-1 and CCR7/CD206.
Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch. ...
Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch. ...
Objective: Advances in the field of biomaterials have positively affected implant acceptance and nowadays nanostructured surfaces are known to have a positive effect on bone regeneration. The development of the field of osteoimmunology has contributed to a steadily increasing interest towards the investigation of the immunomodulatory effect of nanopatterned surfaces. Luckily, advances in nanofabrication methods have resulted in fabricating relatively large nanostructured areas with high resolution. This study is taking advantage of the 3D printing technique called two-photon polymerization (2PP) for the generation of 3D submicron pillar structures (patterns) to systematically investigate their effect on murine macrophages regarding cell migration, viability, cytoskeletal organisation and phenotype polarization. Methods: Six different pillar patterns were fabricated using a Photonic Professional GT (Nanoscribe, Germany) system. The pillars were characterized with scanning electron microscopy (SEM). Cell viability of J774A.1 macrophages on the nanofabricated patters was assessed by live/dead staining with calcein AM and ethidium homodimer-1 and fluorescently analyzed. Cell migration to the patterns was studied using DAPI staining. The cytoskeletal organization of the macrophages was investigated by actin staining and analyzed both through fluorescent microscopy as well as SEM. Macrophage expression of nitric oxide (NO) was tested with Griess assay. The phenotype polarization of the macrophages on the patterns was explored by dual immunofluorescent staining with iNOS/ARG-1 and CCR7/CD206.
Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch.
Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch.