(94d) Synergistically Enhance Bone Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds Using Two-Dimensional Black Phosphorus and Graphene Oxide Nanosheets
AIChE Annual Meeting
2019
2019 AIChE Annual Meeting
Regenerative Engineering Society
Regenerative Engineering: Substrate-Derived Control of Cellular Behavior
Monday, November 11, 2019 - 9:10am to 9:30am
text-align:center;line-height:115%">Synergistically
Enhance Bone Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds Using Two-Dimensional
Black Phosphorus and Graphene Oxide Nanosheets text-align:center;line-height:115%"> 115%;font-family:" times new roman> text-align:center;line-height:115%"> 115%;font-family:" times new roman>Xifeng Liuab, A. Lee
Miller IIb, Sungjo Parkc, Matthew N. Georgea,
Brian E. Waletzkib, Haocheng Xua, Andre Terzicc,
and Lichun Lu*ab text-align:center;line-height:115%"> 115%;font-family:" times new roman> text-align:center;line-height:115%"> 115%;font-family:" times new roman>a Department of Physiology and
Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA text-align:center;line-height:115%"> 115%;font-family:" times new roman>b Department of Orthopedic Surgery,
Mayo Clinic, Rochester, MN 55905, USA. text-align:center;line-height:115%"> 115%;font-family:" times new roman>c Department of Cardiovascular
Diseases and Center for Regenerative Medicine, Mayo Clinic, Rochester,
Minnesota 55905, USA text-align:center;line-height:115%"> 115%;font-family:" times new roman>* Corresponding author. Email
address: Lu.Lichun@mayo.edu; Phone: 507-284-2267; Fax: 507-284-5075. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">Keywords:
2D Materials; 3D-Printed Scaffolds; Bone Cell Osteogenesis justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">1.
Introduction: line-height:115%;font-family:" times new roman> Incorporation of two©\dimensional
(2D) materials to tissue scaffolds for improved biocompatibility and cell
affinity has emerged as a promising new strategy in tissue engineering. For
example, 2D graphene oxide (GO) has been widely reported to enhance cell
adhesion and proliferation. Recently, a newly emerged black phosphorus (BP) 2D
material has attracted attention in biomedical applications due to its unique
mechanical and electrochemical characteristics. In this
study, we investigated the synergistic effect of these two types of 2D materials
on cell osteogenesis on three-dimensional (3D) printed poly(propylene fumarate)
(PPF) scaffolds for bone tissue engineering (Fig. 1a). justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">2.
Materials and Methods: 115%;font-family:" times new roman> Graphene oxide sheets were oxidized
and exfoliated from natural graphite (~150 mm flakes) through improved Hummers¡¯
method.[1] BP nanosheets were synthesized using a liquid exfoliation method.
Poly(propylene fumarate) was synthesized and used as the cross-linkable polymer
for 3D printed scaffolds.[2] The 3D scaffold model was designed using computer-aided
design (CAD) software (SolidWorks Corp., Concord, MA) and printed using the
Viper si2 stereolithography system (3D Systems, Valencia, CA).[3] The
ammonolyzed 3D-PPF-Amine scaffolds were coated with 2D materials by soaking in solution
containing BP nanosheets, GO nanosheets or both BP and GO nanosheets. Sterilized
functionalized 3D scaffolds were adhered to the bottom of 48-well tissue
culture polystyrene (TCPS) plates and MC3T3 pre-osteoblast cells were seeded. Upon
harvest, cells were fixed in paraformaldehyde (PFA, 4% solution) and stained
with anti-vinculin−FITC antibody (Sigma-Aldrich Co., Milwaukee, WI) to label
vinculin and rhodamine-phalloidin (RP, Cytoskeleton Inc, Denver, CO, USA) to
stain cellular filaments. The immune-fluorescence stained cells on the
functionalized 3D Scaffolds were immediately scoped on an inverted laser
scanning confocal microscope (Carl Zeiss, Germany). justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">3.
Results and Discussion: 115%;font-family:" times new roman> All 3D scaffolds after coating with both
types of 2D materials showed good biocompatibilities (Fig. 1b). In addition, the
increased surface area provided by GO sheets enhanced cell attachment at the initial
stage. Afterward, cells were further stimulated by phosphate, which was continuously
released from slow oxidation of BP nanosheets and played an important role in
osteoblast differentiation. Through the use of 3D confocal imaging, unique
interactions between cells and BP nanosheets were observed, including a stretched
cell shape and development of filaments around the BP nanosheets, along with
increased cell proliferation when compared with scaffolds incorporating only
one of the 2D materials (Fig. 1c). All these results indicate that the incorporation
of 2D BP and GO materials could effectively and synergistically stimulate cell
proliferation and osteogenesis on 3D tissue scaffolds. text-align:center;line-height:115%"> 115%">
justify;text-justify:inter-ideograph;line-height:115%">
12.0pt;line-height:115%;font-family:" times new roman>Fig. 1 a) Fabrication
process of 3D printed scaffolds functionalized with 2D GO and BP nanosheets. b)
Live/dead staining of ammonolyzed 3D scaffolds as well as scaffolds functionalized
with BP, GO and GO@BP nanosheets. c) Immunofluorescence images of MC3T3
pre-osteoblasts growing on functionalized 3D scaffolds. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">4.
Conclusions: font-family:" times new roman> We fabricated a series of 3D-printed
scaffolds functionalized with 2D materials for use in tissue engineering. Our
results showed that the addition of 2D BP and GO nanosheets to scaffolds
improved cell adhesion, proliferation, and differentiation. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">Acknowledgments: This
work was supported by National Institutes of Health grant R01 AR56212. justify;text-justify:inter-ideograph;line-height:115%">References justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[1]. X. Liu et
al, Journal of Materials Chemistry B 2016, 4 (43), 6930-6941. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[2]. X. Liu et
al, RSC Advances 2015, 5 (27), 21301-21309. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[3] K.
W. Lee et al, Biomacromolecules 2007, 8 (4), 1077-1084.
Enhance Bone Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds Using Two-Dimensional
Black Phosphorus and Graphene Oxide Nanosheets text-align:center;line-height:115%"> 115%;font-family:" times new roman> text-align:center;line-height:115%"> 115%;font-family:" times new roman>Xifeng Liuab, A. Lee
Miller IIb, Sungjo Parkc, Matthew N. Georgea,
Brian E. Waletzkib, Haocheng Xua, Andre Terzicc,
and Lichun Lu*ab text-align:center;line-height:115%"> 115%;font-family:" times new roman> text-align:center;line-height:115%"> 115%;font-family:" times new roman>a Department of Physiology and
Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA text-align:center;line-height:115%"> 115%;font-family:" times new roman>b Department of Orthopedic Surgery,
Mayo Clinic, Rochester, MN 55905, USA. text-align:center;line-height:115%"> 115%;font-family:" times new roman>c Department of Cardiovascular
Diseases and Center for Regenerative Medicine, Mayo Clinic, Rochester,
Minnesota 55905, USA text-align:center;line-height:115%"> 115%;font-family:" times new roman>* Corresponding author. Email
address: Lu.Lichun@mayo.edu; Phone: 507-284-2267; Fax: 507-284-5075. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">Keywords:
2D Materials; 3D-Printed Scaffolds; Bone Cell Osteogenesis justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">1.
Introduction: line-height:115%;font-family:" times new roman> Incorporation of two©\dimensional
(2D) materials to tissue scaffolds for improved biocompatibility and cell
affinity has emerged as a promising new strategy in tissue engineering. For
example, 2D graphene oxide (GO) has been widely reported to enhance cell
adhesion and proliferation. Recently, a newly emerged black phosphorus (BP) 2D
material has attracted attention in biomedical applications due to its unique
mechanical and electrochemical characteristics. In this
study, we investigated the synergistic effect of these two types of 2D materials
on cell osteogenesis on three-dimensional (3D) printed poly(propylene fumarate)
(PPF) scaffolds for bone tissue engineering (Fig. 1a). justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">2.
Materials and Methods: 115%;font-family:" times new roman> Graphene oxide sheets were oxidized
and exfoliated from natural graphite (~150 mm flakes) through improved Hummers¡¯
method.[1] BP nanosheets were synthesized using a liquid exfoliation method.
Poly(propylene fumarate) was synthesized and used as the cross-linkable polymer
for 3D printed scaffolds.[2] The 3D scaffold model was designed using computer-aided
design (CAD) software (SolidWorks Corp., Concord, MA) and printed using the
Viper si2 stereolithography system (3D Systems, Valencia, CA).[3] The
ammonolyzed 3D-PPF-Amine scaffolds were coated with 2D materials by soaking in solution
containing BP nanosheets, GO nanosheets or both BP and GO nanosheets. Sterilized
functionalized 3D scaffolds were adhered to the bottom of 48-well tissue
culture polystyrene (TCPS) plates and MC3T3 pre-osteoblast cells were seeded. Upon
harvest, cells were fixed in paraformaldehyde (PFA, 4% solution) and stained
with anti-vinculin−FITC antibody (Sigma-Aldrich Co., Milwaukee, WI) to label
vinculin and rhodamine-phalloidin (RP, Cytoskeleton Inc, Denver, CO, USA) to
stain cellular filaments. The immune-fluorescence stained cells on the
functionalized 3D Scaffolds were immediately scoped on an inverted laser
scanning confocal microscope (Carl Zeiss, Germany). justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">3.
Results and Discussion: 115%;font-family:" times new roman> All 3D scaffolds after coating with both
types of 2D materials showed good biocompatibilities (Fig. 1b). In addition, the
increased surface area provided by GO sheets enhanced cell attachment at the initial
stage. Afterward, cells were further stimulated by phosphate, which was continuously
released from slow oxidation of BP nanosheets and played an important role in
osteoblast differentiation. Through the use of 3D confocal imaging, unique
interactions between cells and BP nanosheets were observed, including a stretched
cell shape and development of filaments around the BP nanosheets, along with
increased cell proliferation when compared with scaffolds incorporating only
one of the 2D materials (Fig. 1c). All these results indicate that the incorporation
of 2D BP and GO materials could effectively and synergistically stimulate cell
proliferation and osteogenesis on 3D tissue scaffolds. text-align:center;line-height:115%"> 115%">
justify;text-justify:inter-ideograph;line-height:115%">
12.0pt;line-height:115%;font-family:" times new roman>Fig. 1 a) Fabricationprocess of 3D printed scaffolds functionalized with 2D GO and BP nanosheets. b)
Live/dead staining of ammonolyzed 3D scaffolds as well as scaffolds functionalized
with BP, GO and GO@BP nanosheets. c) Immunofluorescence images of MC3T3
pre-osteoblasts growing on functionalized 3D scaffolds. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">4.
Conclusions: font-family:" times new roman> We fabricated a series of 3D-printed
scaffolds functionalized with 2D materials for use in tissue engineering. Our
results showed that the addition of 2D BP and GO nanosheets to scaffolds
improved cell adhesion, proliferation, and differentiation. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman> justify;text-justify:inter-ideograph;line-height:115%">Acknowledgments: This
work was supported by National Institutes of Health grant R01 AR56212. justify;text-justify:inter-ideograph;line-height:115%">References justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[1]. X. Liu et
al, Journal of Materials Chemistry B 2016, 4 (43), 6930-6941. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[2]. X. Liu et
al, RSC Advances 2015, 5 (27), 21301-21309. justify;text-justify:inter-ideograph;line-height:115%"> 12.0pt;line-height:115%;font-family:" times new roman>[3] K.
W. Lee et al, Biomacromolecules 2007, 8 (4), 1077-1084.