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Applications of Polymer, Composite, and Coating Materials

Two-Dimensional Black Phosphorus and Graphene Oxide Nanosheets Synergistically Enhance Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds Xifeng Liu, A. Lee Miller, Sungjo Park, Matthew George, Brian E. Waletzki, Haocheng Xu, Andre Terzic, and Lichun Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04121 • Publication Date (Web): 03 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019

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ACS Applied Materials & Interfaces

Two-Dimensional Black Phosphorus and Graphene Oxide Nanosheets Synergistically Enhance Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds Xifeng Liuab, A. Lee Miller IIb, Sungjo Parkc, Matthew N. Georgea, Brian E. Waletzkib, Haocheng Xua, Andre Terzicc, and Lichun Lu*ab aDepartment

of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905,

USA. bDepartment

of Orthopedic Surgery, Mayo Clinic, Rochester, MN 55905, USA.

cDepartment

of Cardiovascular Diseases and Center for Regenerative Medicine, Mayo Clinic,

Rochester, Minnesota 55905, USA. Abstract Two ‐ dimensional (2D) materials have emerged as a new promising research topic for tissue engineering because of their ability to alter the surface properties of tissue scaffolds and thus improve their biocompatibility and cell affinity. Multiple 2D materials, such as graphene and graphene oxide (GO), have 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 for bone tissue engineering. BP was first wrapped in negatively charged GO nanosheets, which were then adsorbed together onto positively charged poly(propylene fumarate) (PPF) 3D scaffolds. The increased surface area provided by GO nanosheets would enhance cell attachment at the initial stage. In addition, slow oxidation of BP nanosheets wrapped within GO layers would generate a continuous release of phosphate, an important osteoblast differentiation facilitator designed to stimulate cell osteogenesis toward the new bone formation. Through the use of 3D confocal imaging, unique interactions between cells and BP nanosheets were observed, including a 1 ACS Paragon Plus Environment

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stretched cell shape and the development of filaments around the BP nanosheets, along with increased cell proliferation when compared with scaffolds incorporating only one of the 2D materials. Furthermore, the biomineralization of 3D scaffolds, as well as cellular osteogenic markers, were all measured and improved on scaffolds with both BP and GO nanosheets. 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. Keywords: 2D materials; Black phosphorus; Graphene Oxide; 3D-Printing; Osteogenesis

1. Introduction Two‐dimensional (2D) materials refer to ultrathin nanomaterials with high degrees of anisotropy and/or functionality.1-2 Currently, 2D materials research is mainly focused on the properties characterization of new materials, as well as the investigation of their use as electrochemical catalysts, elements in optoelectronic devices, or components in solar cell applications.3-9 In recent years, this area of research has expanded to the study of 2D nanoparticles for biomedical applications, including carbon‐based 2D materials, transition metal oxides (TMOs), and transition metal dichalcogenides (TMDs).10 Within tissue engineering, the addition of 2D materials with useful chemical, material, or electrical properties to 3D polymer scaffolds is an exciting new area of research, with the potential to improve the biocompatibility and functionality of materials used for regenerative medicine. Graphene oxide (GO), the oxidized form of graphene, is a 2D nanomaterial that has attracted substantial interest for biomedical applications due to its unique single plane nanostructure, outstanding mechanical properties (Young’s modulus > 200 GPa for monolayer GO), large surface area, and strong protein adsorption abilities.11-14 Recent work has shown that GO nanosheets enhance cell adhesion, spreading, proliferation, and differentiation,15-16 with GO coated 3D scaffolds supporting stem cell differentiation, epithelial genesis, adipogenesis, and osteogenesis,1718

as well as myoblast differentiation and osteoblast proliferation.19-20 Furthermore, the weak

electrical conductance of GO makes the material a potent dopant for the regeneration of electrically active tissues, with recent studies reporting higher proliferation of cardiomyocytes on hydrogels 2 ACS Paragon Plus Environment

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with incorporated GO nanosheets21 and improved cell proliferation and spreading in a 3D hydrogel environment.22 Similarly, previous studies from our laboratory have found that the incorporation of GO onto polymer scaffolds enhanced the proliferation, spreading, and differentiation of neuron cells.23-24 In contrast to GO, black phosphorus (BP) is an understudied 2D material, that has great potential in tissue engineering due to its in-plane anisotropy that originates from a unique puckered orthorhombic structure.25-27 This structure imbues BP nanosheets with unique optical, mechanical, electrical, and thermal characteristics that have broad applications in electronic or optoelectronic devices,28-32 while also retaining the materials’ biocompatibility, which is a highly desired trait of materials used in biomedical applications.33-35 In addition to biocompatibility, biodegradation products of BP are phosphate ions or phosphonates, which commonly exist within the blood and thus pose no safety concerns.36-38 Phosphate is required during skeleton development and bone regeneration and plays an important role in stimulating osteogenesis and osteointegration.39-40 These properties making BP an attractive 2D material for imaging formulations, biosensors, or scaffolds intended for implantation within the body. However, currently, the utilization of this biodegradation product for tissue engineering is still in its infancy, with limited reports on this topic.39 Biocompatible 2D materials with useful material properties such as BP and GO have the potential to synthetically improve the performance of 3D polymer-based scaffolds that are currently under development for tissue engineering applications. The advent of three-dimensional (3D) printing is driving advances in multiple fields including manufacturing, engineering, and medicine.41-42 For tissue engineering, the creation of supportive scaffolds using 3D printing technology is viewed as a big advance in the field,43 allowing for the precision needed to manufacture implantable structures with sophisticated architectures that promote tissue growth and integration. Synthetic polymers currently in use in 3D printing today include poly(ε-caprolactone) (PCL),44-46 poly(Llactic acid) (PLLA),47 poly(lactic-co-glycolic acid) (PLGA),48 and poly(propylene fumarate) (PPF),49 all of which have been widely explored both in vitro or in vivo. Among these materials, PPF is a biodegradable and cross-linkable polymer with favorable rigidity and biocompatibility for bone tissue formation,50-52 with previous work in our laboratory showing excellent osteoblast 3 ACS Paragon Plus Environment


proliferation and mineralization both in vitro and in vivo. We have also developed a series of 3D printed scaffolds or stents and observed excellent bone regeneration after implantation.53-54

Fig. 1 a) Schematic representation of the fabrication process of 3D printed scaffolds functionalized with 2D GO and BP nanosheets. b) GO nanosheets enhance cell adhesion to the surface of the scaffolds. c) BP nanosheets release phosphate groups as they degrade, which enhance the proliferation of pre-osteoblasts. d) Phosphate groups released from BP nanosheets also enhance scaffold mineralization and cellular differentiation, making this approach an attractive option. e) Schematic demonstration of 2D materials functionalized 3D scaffolds for bone tissue engineering application.

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In this study, we investigated the osteogenic capacity of 3D printed biodegradable scaffolds coated with 2D materials that alter their surface properties (Fig. 1a). These functionalized 3D scaffolds consist of a crosslinked biodegradable PPF matrix, superficially coated with either 2D GO nanosheets, BP nanosheets, or a GO and BP wrapped composite (GO@BP). GO was adopted to enhance initial cell adhesion to the scaffold surface utilizing its well-known protein adsorption ability (Fig. 1b). BP nanosheets were selected to investigate the effect of phosphate release and increased structural complexity on osteoblast cell behavior, such as cell proliferation, spreading, shape, filament development, and osteogenic differentiation (Fig. 1c). In addition, the modification of the material properties of 3D PPF scaffolds by each 2D material, such as surface roughness, phosphate release, and mineralization, were also investigated (Fig. 1d). After implantation, the polymeric scaffolds were designed to enhance new bone formation in-situ (Fig. 1e), coupled with biodegradation by hydrolysis of ester bonds over time, making this method an exciting candidate for bone tissue engineering applications.

2. Materials and Methods 2.1 Synthesis of 2D GO and BP nanosheets Graphene oxide nanosheets were oxidized and exfoliated from natural graphite (~150 µm flakes, Sigma Aldrich, Milwaukee, WI) through improved Hummers’ method.55-56 Briefly, graphite flakes (3 g) and KMnO4 (18 g) were added into concentrated H2SO4/H3PO4 (360 mL/40 mL) with vigorous stirring. The components were reacted at 50 °C with constant stirring for 12 h and then transferred into a glass beaker filled with ice and 3 mL of 30% H2O2. The oxidized mixture was sifted, centrifuged and washed multiple times with HCl solution. The washed products were then dialyzed for three days using cellulose dialysis bag (MWCO 2000) to remove acidic residues and metal ions and dried in vacuum. BP nanosheets were synthesized using a liquid exfoliation method as described elsewhere.57-58 Briefly, 30 mg of BP powder (ACS Material, LLC, Pasadena, CA) was mixed with 30 mL deionized (DI) H2O and sonicated for 1 hour using a probe model sonicator (Qsonica Q500) to break the large BP nanosheets into small pieces. The mixture solution was then sonicated for 5 ACS Paragon Plus Environment


another 12 hours while using an ice bath sonicator (Elmasonic S10, Elma Schmidbauer GmbH, Germany). The obtained grey suspension was centrifuged for 10 min under 5000 rpm to remove the large size unexfoliated BP powder. The supernatant with exfoliated BP nanosheets was collected and dried by lyophilization to obtain BP nanosheets in powder form. 2.2 Synthesis of PPF polymer Poly(propylene fumarate), used as the cross-linkable polymer for 3D printed scaffolds, was synthesized according to our previous references.59 In brief, diethyl fumarate (DEF, Sigma Aldrich, Milwaukee, WI) and an excess amount of 1,2-propylene glycol were polymerized together with hydroquinone (cross-linking inhibitor) and zinc chloride (catalyst) first at 100 °C for 1 h and then at 150 °C for 7 h to get the intermediate dimer. The intermediate dimer was polymerized to PPF via condensation under vacuum at 130 °C for another 4 h. 2.3 Printing of 3D scaffolds 3D printing resin preparation. To obtain UV curable resin, PPF polymer was dissolved in diethyl fumarate (DEF) (wt:wt ratio = 60:40) by stirring for 24 h at 37 °C. The PPF/DEF solution was then added with 1.5 wt% of bisacrylphosphrine oxide (BAPO, Ciba Specialty Chemicals, Tarrytown, NY) as a photoinitiator to obtain the final PPF/DEF/BAPO resin for 3D scaffolds printing. 3D scaffold design in computer-aided design (CAD). The 3D scaffold model was designed using CAD software (SolidWorks Corp., Concord, MA) with orthogonal cubic-lattice disks and square pores, as referred to our previous work.53 The external dimensions were 5 mm in length, 5 mm in width, and 5 mm in height. The wall length of the square pores is 1 mm and the thickness of the ridges is 0.5 mm. Scaffold printing by 3D stereolithography. CAD model files of the 3D scaffolds were converted to stereolithography (STL) files and imported to 3D Lightyear software to generate the 2D slice data (BFF) files with 101.6-µm layer thickness and support. BFF files were read by the Viper si2 6 ACS Paragon Plus Environment

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stereolithography system (3D Systems, Valencia, CA) to print 3D scaffolds layer-by-layer with 365 nm UV laser through photo-crosslinking of PPF/DEF polymer resins. Post-printing treatment. After printing, supports were exfoliated gently and scaffolds were washed with minimal amounts of acetone and absolute ethanol to remove excess resin. Washed scaffolds were dried and further post-cured under UV light for 2 h. The 3D printed scaffolds were further extracted in a 50/50 solution of Ethanol and tetrahydrofuran (THF) for 3 days within a Soxhlet extraction apparatus to remove toxic sol fractions and make scaffolds biocompatible. After Soxhlet scaffolds were rinsed in water for 2 days to remove excess THF. 2.4 Surface functionalization of 3D Scaffolds Ammonolysis of 3D scaffolds. The ammonolysis solution was prepared by dissolving 6.0 g of hexamethylenediamine (Sigma Aldrich, Milwaukee, WI) in 100 mL isopropyl alcohol. The 3D printed scaffolds were placed in the hexamethylenediamine/isopropyl alcohol ammonolysis solution and reacted for 30 min at 60 °C. After the reaction, the ammonolyzed 3D scaffolds were washed with excessive DI H2O for 2 days with at least 10 times water exchange to remove unreacted hexamethylenediamine on the surface. The washed products were then dried under vacuum to obtain 3D-PPF-Amine scaffolds. Surface coating of 2D materials. The ammonolyzed 3D-PPF-Amine scaffolds were pre-treated by soaking in DI H2O for 10 min to wet the surface and free the amine chains. The BP coating solution was prepared by sonicating of 2D BP nanosheets for 10 min in DI H2O to generate a final concentration of 1 mg/mL. The GO coating solution was prepared by sonicating 2D GO nanosheets for 10 min in DI H2O to generate a final concentration of 1 mg/mL. The GO@BP coating solution was prepared by sonicating of 2D BP and GO nanosheets for 10 min in DI H2O to generate a final concentration of 1 mg/mL BP and 1 mg/mL GO coating solution. The prewetted 3D-PPF-Amine scaffolds were haphazardly grouped and then soaked for 1 hour in BP solution, GO solution, and GO@BP solution, respectively. After coating, the scaffolds were dried under vacuum to generate 3D-PPF-Amine-BP, 3D-PPF-Amine-GO, and 3D-PPF-Amine-GO@BP scaffolds, respectively.



2.5 Material Characterizations Protein adsorption on 3D scaffolds. To analyze protein adsorption, the scaffolds were soaked for 2 hours in 37 °C cell culture α-Minimum Essential Medium (α-MEM, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS). Then the scaffolds were washed with phosphate-buffered saline (PBS) 3 times to remove any unabsorbed proteins. Proteins that adsorbed on the 3D scaffolds were washed off with 1% sodium dodecyl sulfate (SDS, BioRad Laboratories, Hercules, CA) and determined with MicroBCA protein assay kit (Pierce, Rockford, IL) according to provided instructions. Atomic force microscopy (AFM). The morphology and height of the GO and BP nanosheets were determined by AFM analysis using a Nanoscope IV PicoForce Multimode AFM (Bruker, Santa Barbara, CA) in an ambient environment.60 Scanning electron microscopy (SEM). The BP nanosheets, GO@BP mixture, and functionalized 3D printed scaffolds were all dried, sputter coated with gold-palladium and imaged on a Scanning electron microscope (S-4700, Hitachi Instruments, Tokyo, Japan) at a voltage of 5 kV. The amount and distribution of adsorbed black phosphorous nanosheets were characterized by energy dispersive X-ray spectrometry (EDS) element mapping. Transmission electron microscopy (TEM). The morphology of GO and BP nanosheets were observed using a transmission electron microscope (1200-EX II, JEOL Inc., Japan) at 80 kV voltage. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The 3D scaffolds before and after ammonolysis were characterized on a Nicolet Continuum Infrared Microscope (Thermo Fisher Scientific, Waltham, MA) with a wavenumber range of 400 to 4000 cm-1. The functionalized scaffolds after mineralization in simulated body fluid (SBF) were also characterized using ATR-FTIR with the same parameters.


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Coated mass determination. For mass determination, every 10 scaffolds were set into one group to increase the mass for easier determination. The weight of scaffolds in each group was recorded as W1 before functionalization. Then the independent groups went through different functionalization process including soaking in BP, GO, or GO@BP solution. After functionalization, the scaffolds were dried in vacuum and the total mass was recorded as W2. The weight change was calculated as (W1-W2)/10 to estimate the averaged mass coating on each scaffold. Phosphate release from 3D scaffolds. To determine phosphate release kinetics, the four types of scaffolds were separately immersed in 1 mL of DI H2O in a glass vial and placed under two conditions, i.e. room temperature and 37 °C. At each time point, 0.4 mL of released solution was taken out and the vials were refilled with fresh DI H2O. The phosphate concentration in the release medium was determined using a phosphate assay kit (ab65622, Abcam, Cambridge, UK) according to the kit protocol. 2.6 Cytotoxicity of 2D material functionalized 3D Scaffolds Live/dead cell viability staining. MC3T3 pre-osteoblast cells were expanded in α-MEM supplemented with 10% fetal bovine serum and 0.5% streptomycin/penicillin. The functionalized 3D scaffolds were sterilized by UV irradiation for 2 hours in cell culture hood and then adhered to the bottom of 48-well tissue culture polystyrene (TCPS) plates. MC3T3 cells were seeded to the scaffolds at a density of 30,000 cells per well. At 3 days post-seeding, the scaffolds were washed to remove unattached cells and stained with a LIVE/DEAD® Cell Imaging Kit (Thermo Fisher Scientific). The live cells (green) and dead cells (red) on the scaffolds were viewed on an Axiovert 25 Zeiss light microscope (Carl Zeiss, Germany). The number of live and dead cells from three views were counted and averaged. The morphology of live cells, including area, length, circularity and aspect ratio, were calculated using the ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA) and averaged from 20 independent cells. Cytotoxicity of leaching medium. To further evaluate whether functionalized 3D scaffolds could release cytotoxic substances, the sterilized 3D scaffolds were placed into transwell chambers 9 ACS Paragon Plus Environment


(mesh size 3 µm). The transwells with scaffolds were then placed into six-well TCPS plates seeded with MC3T3 pre-osteoblast cells at a density of 15, 000 cells per well. At 3 days post-seeding, the medium in each well was replaced by MTS solution (CellTiter 96, Promega, Madison, WI) and absorbance was measured by UV-vis absorbance microplate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA) at 490 nm. The relative cell viability was obtained by normalizing to the positive control wells without sample treatment (set as 100%). 2.7 Pre-osteoblast cell proliferation on functionalized 3D Scaffolds Sterilized, functionalized 3D scaffolds were adhered to the bottom of 48-well TCPS plates using sterile silicone-based high vacuum grease (Dow Corning, Midland, MI). MC3T3 cells were seeded at a density of 30,000 cells per well. At 6 hours post-seeding, the scaffolds were transferred to new wells in 48-well TCPS plates and washed with sterile PBS to remove unattached cells. Cell number on each scaffold was measured using MTS assay and absorbance at 490 nm was read on a UV-vis absorbance microplate reader. The relative cell attachment rate was calculated by normalizing the absorbance in each scaffold to that of the positive TCPS control wells (set as 100%). For cell proliferation study, the scaffolds after seeded with cells were cultured in ascorbic acid-free nonosteogenic α-MEM medium for 1, 3, and 6 days. The proliferated cell numbers on each type of scaffolds were measured using the MTS assay and the absorbance was read at 490 nm using the UV-vis absorbance microplate reader. Immuno-fluorescence imaging of cells on the functionalized 3D scaffolds was conducted at 1, 3, and 6 days post-seeding. Prior to staining, cells were fixed in paraformaldehyde (PFA, 4% solution), washed with PBS and permeabilized with 0.2% Triton X-100. To block non-specific binding sites, scaffolds seeded with cells were soaked for 1 hour in a PBS solution containing 1% bovine serum albumin (BSA). Permeabilized and blocked cells were then incubated with antivinculin−FITC antibody (Sigma-Aldrich Co., Milwaukee, WI; 1:50 diluted in PBS) at 37 °C for 1 h, in order to label vinculin, an important component in cellular focal adhesion. After initial staining, secondary staining with rhodamine-phalloidin (RP, Cytoskeleton Inc, Denver, CO, USA; 1:200 diluted in PBS) was conducted for an hour at 37 °C to stain cellular filaments. The immune-


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fluorescence stained cells on the functionalized 3D Scaffolds were immediately scoped on an inverted laser scanning confocal microscope (Carl Zeiss). Total collagen production. Total collagen production by pre-osteoblasts growing on functionalized 3D scaffolds was detected by the Sirius red staining method.61-62 Briefly, MC3T3 cells after 1 week of growth on 3D scaffolds were fixed by 4% PFA solution and stained by Sirius red stain solution (0.1% Direct Red 80 in saturated picric acid) for 16 hours. Stained 3D scaffolds were washed by DI water and dehydrated by 100% ethanol. After drying, the stain on 3D scaffolds was eluted by 0.2 M NaOH: methanol (v/v, 1: 1) solution for 15 min and absorbance was measured by UV-vis absorbance microplate reader at 490 nm. To normalize the collagen amount to cell numbers, another series of scaffolds were seeded with the same density of cells and cultured for 1 week under the same condition. Cells on the scaffolds were then trypsinized using 0.5% trypsin solution (Thermo Fisher Scientific, Waltham, MA) and counted under microscope. The relative collagen amount was calculated by normalizing the obtained OD value to the number of cells observed on each type of scaffold. 2.8 Biomineralization of functionalized 3D Scaffolds The accelerated biomineralization of functionalized 3D Scaffolds was evaluated using a conventional simulated body fluid (SBF) method, according to previous references.63-65 SBF solution was prepared by dissolving NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g), K2HPO4 ･3H2O (0.228 g), MgCl2 ･6H2O (0.305 g), CaCl2 (0.278 g), Na2SO4 (0.071 g), (CH2OH)3CNH2 (6.057 g) and 1M-HCl (40 mL) in DI water, with a final volume of 1 L. Scaffolds were immersed in SBF solution for 7 days to allow for adequate deposition of biominerals on the surface. Biomineralized 3D scaffolds were then washed with DI water, dried, and characterized by SEM and ATR-FTIR. To determined cell proliferation on these biomineralized 3D scaffolds, scaffolds were sterilized by UV irradiation for 2 hours, adhered to 48-well TCPS plates, and then seeded with MC3T3 cells at a density of 30,000 cells per well. After being cultured for 1, 3, and 6 days in ascorbic acid-free



non-osteogenic α-MEM, the medium was replaced with MTS solution and absorbance was measured by UV-vis absorbance microplate reader at 490 nm. Immuno-fluorescence imaging of cells on the biomineralized 3D scaffolds was conducted by fixing cells in 4% PFA solution, followed by washing with PBS and permeabilizing with 0.2% Triton X-100. Permeabilized cells were incubated with rhodamine-phalloidin for an hour at 37 °C to stain cellular filaments followed by incubation for 10 min in 4',6-diamidino-2-phenylindole (DAPI) solution to stain cellular nuclei. Fluorescence-stained MC3T3 cells on the biomineralized 3D scaffolds were scoped on an Axiovert 25 Zeiss light microscope. 2.9 Osteogenic differentiation of MC3T3 cells on functionalized 3D scaffolds Osteogenic differentiation of MC3T3 cells on functionalized 3D scaffolds and biomineralized 3D scaffolds were evaluated using two media, i.e. ascorbic acid-free non-osteogenic α-MEM medium and osteogenic medium supplemented with 10 mM β-glycerophosphate (β-GP) and 50 µg/mL ascorbic acid (AA). Alkaline phosphatase (ALP) activity. Intracellular alkaline phosphatase was obtained from cells growing on 3D scaffolds after 12 days in culture by removing cells with trypsin and centrifuging the solution for 10 min at 3000 rpm. Collected cells were repeatedly washed with sterilized DI H2O and centrifuged at least 5 times to remove potential ethylenediaminetetraacetic acid (EDTA) from trypsin solution and were then counted and lysed in 0.2% Triton X-100 (0.5 mL solution per 1 × 104 cells) at 4 °C overnight. The alkaline phosphatase activity in the cell lysate mixtures was measured by an Alkaline Phosphatase Assay Kit (QuantiChromeTM, BioAssay Systems, Hayward, CA). Osteocalcin (OCN) content. After culturing of MC3T3 cells on the 3D scaffolds for 21 days, the osteocalcin concentration released in the medium was determined using a quantitative Mouse Osteocalcin Enzyme Immunoassay Kit (Alfa Aesar, Thermo Fisher Scientific), as referred to previous reports.66 The absorbance was read on the UV-vis absorbance microplate reader at a


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wavelength of 450 nm. The concentration of osteocalcin was calculated using a standard curve method based on known concentrations, according to the provided kit protocol. 2.10 Statistical analysis Prior to statistical testing, the normality of each response variable was confirmed using the Shapiro-Wilk test. The statistical difference among data groups was analyzed by one-way analysis of variance (ANOVA) followed by Tukey post-test if necessary. Any two data groups with p-value calculated to be < 0.05 was recognized as significantly different.

3. Results and Discussion 3.1 Synthesis of 2D materials TEM and AFM images confirmed the production of thin layers of graphene oxide nanosheets after exfoliation (Fig. 2a-b), with an approximate height of 1 nm (Fig 2c). The height is consistent with the dimensions of a single nanosheet55, indicating that a single layer was deposited on scaffolds. For BP nanosheets, TEM and AFM imaging also indicated small size flakes in the nanometer range, as shown in Fig. 2d-e. Layer height analysis demonstrated a height around 16-28 nm for exfoliated BP nanosheets, which are consistent with previous reports.38, 57 Further SEM images in Fig. 2g showed clearly exfoliated layers on the BP nanosheets. Statistical analysis showed that BP nanosheets had a lateral size in a range of 0-10 µm with the highest peak emerged at around 4 µm after exfoliation (Fig. 2h). To investigate whether GO nanosheets can be grafted with BP, a GO@BP mixture was generated by mixing BP with GO under sonication (Fig. 2i). When coated to a glass surface, BP nanosheets were shown by SEM imaging to be successfully wrapped together with GO nanosheets (Fig. 2j). TEM images indicated that BP nanosheets were homogeneously dispersed within the GO material 13 ACS Paragon Plus Environment


(Fig. 2k). Detailed scoping of BP nanosheets demonstrated that they were tightly covered by a large area of GO layer (Fig. 2k). Element mapping of carbon and oxygen (main components of GO nanosheets) and phosphorous (main components of BP nanosheets) showed BP nanosheets (green area) were widely dispersed with GO nanosheets (Fig. 2l). These results indicate that BP and GO do not exclude each other and can be combined to form mixed complexes.

Fig. 2 a) TEM image, b) AFM image, and c) layer heights of 2D GO nanosheets. d) TEM image, e) AFM image, and f) layer heights of 2D BP nanosheets. g) SEM image and h) lateral size 14 ACS Paragon Plus Environment

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distribution of 2D BP nanosheets. i) Schematic demonstration of wrapping BP with GO nanosheets. Detailed j) SEM images and k) TEM images of BP nanosheets wrapped with GO nanosheets. l) Element mapping of carbon (C: red), oxygen (O: blue) and phosphorous (P: green) on mixtures of BP nanosheets wrapped by GO nanosheets (white line circles out the BP nanosheets). 3.2 3D scaffold fabrication and functionalization 3D scaffolds in desired dimensions were designed by CAD with external dimensions of 5 mm in length, width, and height (Fig. 3a). The length of the square pores and the thickness of the ridges were 1 mm and 0.5 mm, respectively. Scaffolds were printed by 3D stereolithography layer-bylayer using UV crosslinking of a PPF/DEF polymer resin. Obtained scaffolds were washed, postcured, and extracted in 50/50 Ethanol/THF to obtain the final transparent biocompatible 3D scaffolds. Photographs of fabricated 3D PPF scaffolds showed that the scaffolds were colorless with homogenous cubic pores (Fig. 3b). The microscopic images of the printed 3D scaffolds demonstrated a relatively smooth surface morphology, with ridge size of 450 µm wide and pore sizes of 1000 µm wide (Fig. 3b). This is consistent with the CAD design with slightly thinner ridge, most likely due to the shrinkage caused by the removal of uncrosslinked polymers during post-cure processing. The ammonolysis of 3D scaffolds was performed in a hexamethylenediamine/isopropyl alcohol solution at 60 ᵒC for 30 min. The molecular mechanism of ammonolysis is the introduction of amine groups in hexamethylenediamine by breaking the ester bonds in the PPF polymer chain, as demonstrated in Fig. 3c. After ammonolysis, 3D scaffolds showed a typical amine adsorption peak when characterized by ATR-FTIR, confirming the introduction of amine groups to the surface of the 3D scaffolds (Fig. 3d). After ammonolysis, scaffolds were coated in different solutions containing either 1 mg/ml of BP nanosheets, 1 mg/ml of GO nanosheets, or 1 mg/mL of both BP and GO, as demonstrated in (Fig. 3e). The images of scaffolds after coating are presented in (Fig. 3f). SEM images of the surface of the four types of scaffolds showed different morphological features at the nanoscale. For 3D-PPFAmine scaffolds without coating, the surface is relatively flat without debris. However, after 15 ACS Paragon Plus Environment


coating with BP or GO, 3D-PPF-Amine-BP and 3D-PPF-Amine-GO scaffold surfaces were observed to have a deposit of BP nanosheets or a thin layer of GO nanosheets with ridges, respectively. For the 3D-PPF-Amine-GO@BP scaffolds coated with both BP and GO nanosheets, the surfaces were covered with an abundance of BP nanosheets wrapped by a GO layer (Fig. 3f, Fig. S1). Detailed scoping was able to show that GO and BP were deposited together, with BP deposited on the GO layer, GO deposited on the BP layer, or BP deposited on GO and then on the BP layer (Fig. 3g, Fig. S2). The surface morphologies on the four types of scaffolds were concluded from the SEM observation and a schematic demonstration is presented in Fig. 3h.


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Fig. 3 a) CAD model dimensions and b) photographs of 3D printed PPF scaffolds. c) Mechanisms of surface ammonolysis of the 3D-PPF scaffolds and d) FTIR spectra confirming the surface amine groups. e) Coating of 3D scaffolds in GO, BP, and GO@BP solutions. f) Photographs and SEM images of scaffolds after coating. g) SEM images of varied morphologies of BP and GO on the surfaces of the 3D-PPF-amine-GO@BP scaffold. h) Schematic demonstration of the four types of 3D scaffolds. The i) AFM images and j) averaged roughness (n=5; *: p < 0.05) of the 3D scaffolds. The k) protein adsorption and l) total mass coated on the functionalized 3D scaffolds (n=3; *: p < 0.05). AFM testing further observed morphological features that are consistent with SEM observations (Fig. 3i). Root mean square roughness calculation showed statistically higher roughness on BP coated scaffolds compared to GO coated scaffolds (Fig. 3j). The 3D-PPF-Amine-GO@BP scaffolds coated with both BP and GO nanosheets displayed the highest roughness of the four groups studied (Fig. 3j). Numerous studies have reported that surface roughness plays a critical role in enhancing cellular adhesion and proliferation.67-70 For protein adsorption, scaffolds coated with GO or GO@BP nanosheets had significantly more protein adsorption when incubated in cell culture medium than other groups (Fig. 3k). This result is consistent with previous reports, which also observed robust adsorption of proteins to GO nanosheets.20, 71 The enhanced protein adsorption ability is mainly attributed to the ability of ionic bonding formation and the large surface area of GO nanomaterials.72-73 In addition, significantly higher amounts of GO was coated onto these scaffolds than BP (Fig. 3l). This may be due to the negative charges on the GO surface, which could create strong electric forces to allow better adhesion of GO layers to the positively charged surface of ammoniated 3D scaffolds than BP. In addition, GO has a larger surface area than BP, which makes GO easier to attach to but harder to un-attach from the scaffold compared to BP. The GO@BP mixture takes advantage of the strong adsorption ability of the GO material while at the same time brings BP nanosheets together onto the surface of 3D scaffolds. This combination allowed 3D-PPF-AmineGO@BP scaffolds to achieve the highest coating mass (Fig. 3l) on the surface, which originated from the GO mass and the BP nanosheets wrapped within the GO layer. 17 ACS Paragon Plus Environment


3.3. Biocompatibility of 3D scaffolds The biocompatibility of the 3D scaffolds after functionalization is critical for tissue regeneration. In this work, the cytotoxicity of scaffolds was evaluated using the live/dead stain directly in culture in addition to assessing cell viability using the leaching medium from scaffolds. As demonstrated in (Fig. 4a), the live/dead staining confirmed the presence of living cells on all four types of scaffolds. There was substantially higher cell growth on 3D-PPF-Amine-BP and 3D-PPF-AmineGO scaffolds functionalized with BP and GO, respectively, when compared to 3D-PPF-Amine scaffolds. For 3D-PPF-Amine-GO@BP scaffolds, a broader cell distribution was also observed, with cells attaching to the entirety of the scaffolds’ surface.

Fig. 4 a) Live/dead staining of ammonolyzed 3D scaffolds after functionalization with BP, GO and GO@BP nanosheets (green: live cells; red: dead cells). b) Numbers of live and dead cells on the 3D scaffolds after 1 day of MC3T3 cell culture (n=3). c) Cytotoxicity of the leaching medium 18 ACS Paragon Plus Environment

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from scaffolds placed in transwells during 3 days of MC3T3 cell culture (n=4). Cell viability on TCPS without scaffold treatment was set at 100%. d) Spreading area of cells after 1 day of growth on functionalized 3D scaffolds (n = 20 cells; *: p < 0.05). An enlarged view of the cross-section of scaffolds showed a clear demonstration of the cell concentration on the scaffolds. As presented in (Fig. 4a), an increased trend in cell density was observed on the scaffolds coated with BP or GO, with treatment with both BP and GO resulting in the highest live densities of confluent cells (Fig. S3a). The 3D printed scaffold itself showed autofluorescence during the imaging process. Therefore, a blank 3D-PPF-Amine scaffold with the same treatment was imaged for comparison (Fig. S3b). A quantitative calculation of the number of cells on these scaffolds showed a significantly higher number of cells on 3D scaffolds functionalized with 2D BP or GO nanosheets (Fig. 4b). In addition to the direct culture of cells on the scaffolds, we evaluated the cytotoxicity of the leaching medium from scaffolds that placed in transwells during cell culture. After 3 days, MTS assays showed there was no significant difference between wells cultured in the presence of 3D scaffolds and the positive control wells without scaffolds (Fig. 4c). No cytotoxicity was observed with further culture till 7 days (Fig. 4c). These results indicate that the scaffolds are not releasing cytotoxic components to the medium and thus are biocompatible and suitable for implantation. Besides cell viability, cell spreading on these scaffolds was also investigated. Results showed that cells could expand and spread on all four types of scaffolds. However, cells had significantly larger projection area on the three functionalized scaffolds as compared to non-coated 3D-PPF-Amine scaffolds (Fig. 4d). Further comparison demonstrated that cells had significantly more cell area on the GO coated scaffolds than BP coated scaffolds, implying that GO had a stronger ability in facilitating cell spreading than BP materials (Fig. 4d). 3.4 Phosphate release and cell responses The amount and distribution profile of BP adsorbed on scaffolds were characterized by EDS element mapping. As shown in (Fig. 5a), there is a substantially higher amount of phosphorous on 19 ACS Paragon Plus Environment


the cross-section of 3D-PPF-Amine-GO@BP scaffolds, as compared to 3D-PPF-Amine-BP scaffolds. The quantitative analysis determined a higher phosphorous content on the surface of 3D-PPF-Amine-GO@BP scaffold than 3D-PPF-Amine-BP scaffold (Fig. 5b). This indicates that the GO layer, which wrapped BP nanosheets, aided in the adhesion of BP nanosheets onto the surface of scaffolds.

Fig. 5 a) Phosphorous elemental mapping on 3D scaffolds functionalized with BP and GO@BP nanosheets and b) calculated amount of phosphorus content. c) Phosphate release kinetics from 3D scaffolds functionalized with BP and GO@BP nanosheets at room temperature and 37oC (n = 2). d) Immunofluorescence and SEM images showing the morphology of MC3T3 pre-osteoblast cells after 1 day of growth on functionalized 3D scaffolds (yellow lines indicate to the edges of 20 ACS Paragon Plus Environment

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BP nanosheets; green arrows point to GO layers). e) Cell length, f) cell circularity, and g) cell aspect ratio calculated from immunofluorescence images (n = 20 cells; *: p < 0.05). The release of phosphate ions from the 3D-PPF-Amine-BP and 3D-PPF-Amine-GO@BP scaffold were further studied at both room temperature and body temperature (37 °C). For 3D-PPF-AmineBP scaffolds, phosphate ions were released from scaffolds during the first 8 days after immersion in DI water (Fig. 5c). After 10 days, the release reached a plateau and no substantial phosphate release was detected till 21 days. For 3D-PPF-Amine-GO@BP scaffolds, the observed phosphate release was greater than that of the 3D-PPF-Amine-BP scaffolds (Fig. 5c), while the rate of release was observed to be slightly slower after immersion in DI water for 10 days. Afterward, phosphate release continued, albeit to a lesser degree. This change may be caused by the inhibited oxidation of BP nanosheets after they are wrapped and buried under the GO layer. The covering GO layer on BP nanosheets may reduce surface fluid exchange and thus limit the H2O and O2 to oxidize the buried BP nanosheets.74 Immunofluorescence imaging of MC3T3 pre-osteoblast cells showed elongated morphology on the three types of functionalized scaffolds, as compared to 3D-PPF-Amine scaffolds without 2D material coating (Fig. 5d), while SEM imaging showed that cells developed robust filaments when they came into contact with BP nanosheets (Fig. 5d, Fig. S4). This morphology may be due to the attraction of continuous phosphate release from the BP nanosheets, with numeric analysis of cellular shape indicators, including cell length, cell circularity, and cell aspect ratio revealing that cells possessed a more elongated, linear shape on the three types of functionalized scaffolds, as compared to 3D-PPF-Amine scaffolds without 2D material coating (Fig. 5e-g). 3.5 Pre-osteoblast proliferation To observe proliferation densities on these scaffolds, MC3T3-E1 pre-osteoblast cells were stained with anti-vinculin−FITC antibody and rhodamine-phalloidin to label vinculin (green) and cellular filaments (red), respectively. Immunofluorescence observation by confocal imaging showed clear cell proliferation on these scaffolds after 1, 3, and 6 days of culture (Fig. 6a and Fig. S5-7). As 21 ACS Paragon Plus Environment


presented in (Fig. 6a), cells attached and proliferated on both the sides and the cross-sections of scaffolds in all four types of scaffolds. However, the cell numbers on these scaffolds varied widely. For 3D-PPF-Amine scaffolds without 2D materials, cells proliferated sparsely, covering around half of the surface area. For 3D-PPF-Amine-GO scaffolds functionalized with GO single components, cells proliferated to greater density when compared with scaffolds without 2D materials. This is consistent to our previous study that observed enhanced cell proliferation on carbon materials functionalized substrates.75 Similar to the 3D-PPF-Amine-GO scaffolds, cells on 3D-PPF-Amine-BP scaffold also proliferated to a higher density than with scaffolds without 2D materials. For 3D-PPF-Amine-GO@BP scaffolds coated with both BP and GO nanosheets, cells proliferated to a much higher density with confluent cells covering almost all the surface area. 3D reconstruction imaging using depth scanning about the Z-axis showed that cells covered all the sides of the scaffolds’ ridges. This result indicates that GO and BP could synergistically enhance cell proliferation on the 3D scaffolds. Focal adhesions (FAs) are critical dynamic protein complexes that function to connect internal cytoskeleton with the extracellular matrix (ECM).76-77 During the initial stage of cell adhesion, FAs serve as the anchorage to promote cells to adhere to the scaffold surface. At the same time, FAs also function as signal carriers to feedback real-time external ECM details to internal cell components in order to adjust subsequent adhesion and spreading behaviors.76-77 Vinculin is one of the key components of FA and frequently used to indicate FA existence and intensity by immunofluorescence staining.77-78 In this study, as evidenced by the FITC signals resulting from vinculin staining (green), cells were shown to have developed obvious FA components on the scaffolds functionalized with 2D BP or GO materials. The adhesion of MC3T3 pre-osteoblasts on 3D scaffolds coated with 2D materials was also investigated. As shown in Fig. 6b, 3D-PPF-Amine-GO, and 3D-PPF-Amine-GO@BP scaffolds, both were coated with a layer of GO, showed significantly more cell adhesion. However, for 3D scaffolds coated with BP alone, there was no observed increase in cell adhesion, as compared to 3D-PPF-Amine scaffolds without 2D material coating, indicating that GO functions to mainly enhance initial cell adhesion.


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Fig. 6 a) Immunofluorescence and 3D reconstruction images of MC3T3 pre-osteoblasts after 6 days of growth on functionalized 3D scaffolds (green: vinculin; red: F-actin). b) Cell attachment rate at 6 hours post-seeding (n = 4 samples; *: p < 0.05). c) Cell proliferation at 1, 3, and 6 days post-seeding on functionalized 3D scaffolds (n = 4 samples; *, #, $: p < 0.05 compared to 3D-PPFAmine group at days 1, 3 and 6, respectively; &: p < 0.05). d) Total collagen production from MC3T3 cells growing for 1 week on functionalized 3D scaffolds (n = 3 samples; *: p < 0.05). Quantitative assessment of cell numbers on scaffolds 1-day post-seeding confirmed that 3D-PPFAmine-GO and 3D-PPF-Amine-GO@BP scaffolds had significantly higher cell densities (Fig 6c). At 3 days of culture, all three types of functionalized scaffolds showed significantly higher cell 23 ACS Paragon Plus Environment


densities compared to scaffolds without 2D material coating. Cell densities on 3D-PPF-AmineGO@BP scaffolds were also significantly higher than 3D-PPF-Amine-BP scaffolds (Fig 6c). At 6 days of culture, 3D-PPF-Amine-GO@BP scaffolds had the highest cell densities compared to all other scaffolds, i.e. non-functionalized, or only functionalized with a single component of BP or GO (Fig 6c). Collagen productions by the cells were also quantified in addition to cell proliferation. Cells on the 3D-PPF-Amine-GO and 3D-PPF-Amine-GO@BP scaffolds functionalized with GO layers displayed increased collagen production compared to that of 3D-PPF-Amine scaffolds without 2D materials (Fig. 6d). Additionally, 3D-PPF-Amine-GO scaffold coated with GO supported significantly more collagen production from cells than 3D-PPF-Amine-BP scaffold that coated with BP, indicating that GO is a better material in enhancing cell collagen production. Collagen is a main component of the extracellular matrix (ECM). The abundant collagen deposition may also indicate strong ECM development for cells on 3D scaffolds stimulated by the coated GO layer. The 3D-PPF-Amine-GO@BP scaffolds, functionalized by both GO and BP, showed the strongest stimulation of cell collagen production. This robust stimulation effect is believed to be mainly taking advantage of enhancement from both 2D GO and BP nanosheets (Fig. 6d). 3.6 Mineralization of 3D scaffolds To investigate whether GO and BP nanosheets could enhance scaffold mineralization, all four types of scaffolds were soaked in simulated body fluid (SBF) for 7 days to allow minerals deposition, as demonstrated in (Fig. 7a). After 7 days in solution, 3D-PPF-Amine-GO and 3DPPF-Amine-GO@BP scaffolds darkened in color, perhaps due to the partial reduction of GO nanosheets. After drying, images of all 3D scaffolds showed mineral deposition regardless of scaffold type, with scaffolds functionalized with either BP or GO, or GO@BP having relatively more minerals deposited on their surfaces (Fig. 7b). According to previous references, the minerals that BP created in the SBF solution were mainly calcium phosphate nanoparticles.39 ATR-FTIR analysis showed characteristic peaks for phosphate on all scaffolds, confirming the deposition of essential minerals on these scaffolds (Fig. 7c), with no significant difference observed between these curves. However, a close comparison of peaks points to a trend of higher phosphate peaks 24 ACS Paragon Plus Environment

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around 1060 cm-1 for 3D-PPF-Amine-BP and 3D-PPF-Amine-GO@BP scaffolds, both functionalized with BP nanosheets. This may imply that the continuous releasing of phosphate ions by BP nanosheets may slightly enhance the biomineralization of 3D scaffolds. SEM observation of the surface of scaffolds showed distinct mineral morphology and distribution profile present in each scaffold type (Fig. 7d). On 3D-PPF-Amine scaffolds without 2D materials, minerals were sparsely and randomly distributed, while on 3D-PPF-Amine-BP scaffolds with BP nanosheets there were denser mineral aggregations on the surface. Consistent with previous cell orientation results, large crystalline mineral deposits were observed to form along the edges of BP nanosheets (Fig. 7d), further confirming that BP nanosheets could enhance the scaffold’s mineralization process by facilitating the continuous release of phosphates. This result is consistent with a previous report that showed calcium phosphate nanoparticle were created by BP nanosheets after soaking in SBF solution.39 Similar mineral development was also seen on 3D-PPF-AmineGO scaffolds, which may have been enhanced by the added surface area afforded the material through the addition of layers of GO. However, 3D-PPF-Amine-GO@BP scaffolds showed the most robust mineral development, as evidenced by the thick minerals layer covering almost all of the scaffold’s surfaces (Fig. 7d). Cell behaviors on these mineralized scaffolds were investigated after washing and sterilization. Immunofluorescence images showed that 3D-PPF-Amine-GO@BP scaffold had the highest cell density on the surface (Fig. 7e). The other mineralized scaffolds also supported excellent cell proliferation with obvious cell growth. Cell attachment rate at 6 hours post-seeding showed that 3D-PPF-Amine-GO and 3D-PPF-Amine-GO@BP scaffolds with GO layers attracted more cell adhesion to the surface (Fig. 7f). MTS assays showed there were significantly higher cell numbers on the three functionalized scaffolds compared to the scaffolds without 2D materials (Fig. 7g). Within these three functionalized scaffolds, the 3D scaffolds coated with GO developed a significantly larger amount of collagen compared to that coated with BP. The 3D-PPF-AmineGO@BP scaffolds supported the best cell proliferation with the highest cell densities determined. However, the difference among groups is not so drastic compared to the proliferation results on scaffolds before mineralization (Fig. 6c). This may because minerals on the surface played a role in enhancing cell proliferation. 25 ACS Paragon Plus Environment


Fig. 7 a) Schematic demonstration of mineralization of functionalized 3D scaffolds in simulated body fluid (SBF). B) photographs of 3D scaffolds after mineralization in SBF. c) FTIR spectra and d) SEM of the mineralized scaffolds showing the presence of phosphates on surface. e) Immunofluorescence images of MC3T3 pre-osteoblasts growing for 3 days on the mineralized 3D scaffolds (red: F-actin; blue: nuclei). f) Cell attachment rate at 6 hours post-seeding (n=4; *: p < 26 ACS Paragon Plus Environment

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0.05) and g) cell proliferation at 1, 3, and 6 days post-seeding on the mineralized 3D scaffolds (n=4; *, #, $: p < 0.05 compared to 3D-PPF-Amine group at days 1, 3 and 6, respectively; &: p < 0.05). 3.7 Differentiation of pre-osteoblasts on 3D scaffolds

Fig. 8 Relative ALP activity of MC3T3 pre-osteoblasts on a) 3D scaffolds functionalized with 2D materials and b) 3D scaffolds after mineralization in SBF solution. ALP activity on each scaffold was normalized to that of 3D-PPF-Amine scaffold without mineralization (set as 1). Relative OCN content in pre-osteoblasts cultured in α-MEM and osteogenic medium on c) 3D scaffolds functionalized with 2D materials and d) 3D scaffolds after mineralization in SBF solution. OCN content on each scaffold was normalized to that of 3D-PPF-Amine scaffold without mineralization (set as 1). (n=3; *, #: p < 0.05 compared to 3D-PPF-Amine group in α-MEM medium and βGP/AA medium, respectively; &: p < 0.05).



To investigate the osteogenic differentiation of MC3T3 pre-osteoblasts on different 3D scaffolds, two typical osteogenic markers, ALP activity and OCN content, were measured and compared. Cells were cultured with either ascorbic acid-free non-osteogenic α-MEM medium, or osteogenic medium supplemented with 10 mM β-glycerophosphate (β-GP) and 50 µg/mL ascorbic acid (AA). Corresponding groups of scaffolds that were pre-mineralized in SBF solution were also studied for comparison. As presented in Fig. 8a, ALP activity was significantly elevated in cells cultured on the functionalized scaffolds in both α-MEM medium and β-GP/AA osteogenesis medium. In addition, cellular ALP activity on scaffolds coated with BP nanosheets was much stronger than on scaffolds coated with GO nanosheets (Fig. 8a). After mineralization, the functionalized scaffolds also showed higher cellular ALP activities than on scaffolds without 2D material coatings, as presented in Fig. 8b. However, the difference was minimal, perhaps due to the abundance of ions present on all scaffolds as a result of scaffold mineralization. In contrast to ALP activity, there was no significant difference observed in OCN content from cells grown on either 3D-PPF-Amine-BP or 3D-PPF-Amine-GO scaffolds, regardless of the medium used (Fig. 8c). The highest OCN content was recorded in cells growing on 3D-PPF-AmineGO@BP scaffolds while cultured in either α-MEM or β-GP/AA osteogenesis medium. After mineralization, elevated OCN content was observed for all the four types of scaffolds (Fig. 8d), with cells growing on 3D-PPF-Amine-GO@BP scaffolds exhibiting the strongest OCN content under culture in either α-MEM or β-GP/AA osteogenesis medium. Taken together, these results indicate that 2D BP and GO nanomaterial coatings have the potential to enhance the osteogenesis of pre-osteoblasts on 3D scaffolds.

4. Conclusions In summary, we fabricated a series of 3D-printed scaffolds functionalized with 2D materials for use in tissue engineering. The surface morphology, degree of mineralization, phosphate ion release, biocompatibility, and osteogenic capacity of scaffolds functionalized with either BP nanosheets, GO nanosheets, or both GO@BP were described, with the aim of identifying which surface coating was best at promoting cell attachment, proliferation, differentiation, and mineralization. Our results showed that the addition of GO nanosheets to scaffolds improved protein adsorption and 28 ACS Paragon Plus Environment

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cell adhesion, taking advantage of the large surface area provided by the 2D GO layers. On the other hand, the inclusion of BP nanosheets allowed for continuous phosphate release from scaffolds as the material degraded, resulting in a stretched cell shape and the development of cellular filaments around their edges. Moreover, 3D confocal scanning and the measurement of cellular markers produced during osteogenesis confirmed that the most robust cell attachment, proliferation, and differentiation were achieved on scaffolds functionalized with both BP and GO nanosheets. These results clearly demonstrated that the incorporation of two-dimensional BP and GO could synergistically stimulate cell proliferation and osteogenesis, making this method a promising route for tissue engineering applications.

ASSOCIATED CONTENT Supporting Information: Additional SEM and immunofluorescence images. The Supporting Information is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION *Corresponding Author E-mail: [email protected]. Tel.: 507-284-2267. Fax: 507-284-5075. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grant R01 AR56212.

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