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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Two-Dimensional Black Phosphorus and Graphene Oxide Nanosheets Synergistically Enhance Cell Proliferation and Osteogenesis on 3D Printed Scaffolds Xifeng Liu,†,‡ A. Lee Miller, II,‡ Sungjo Park,§ Matthew N. George,† Brian E. Waletzki,‡ Haocheng Xu,† Andre Terzic,§ and Lichun Lu*,†,‡ Department of Physiology and Biomedical Engineering, ‡Department of Orthopedic Surgery, and §Department of Cardiovascular Diseases and Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota 55905, United States
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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 because of 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) three-dimensional (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 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, was 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
tissue engineering, the addition of 2D materials with useful chemical, material, or electrical properties to three-dimensional (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
Two-dimensional (2D) materials refer to ultrathin nanomaterials with high degrees of anisotropy and/or functionality.1,2 Currently, research on 2D materials is mainly focused on the characterization of properties 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, transitionmetal oxides, and transition-metal dichalcogenides.10 Within © 2019 American Chemical Society
Received: March 17, 2019 Accepted: June 3, 2019 Published: June 3, 2019 23558
DOI: 10.1021/acsami.9b04121 ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Research Article
ACS Applied Materials & Interfaces
Figure 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 material-functionalized 3D scaffolds for bone tissue engineering application.
broad applications in electronic or optoelectronic devices,28−32 while also retaining the biocompatibility of the materials, which is a highly desired trait of materials used in biomedical applications.33−35 In addition to biocompatibility, the 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 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
biomedical applications because of 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,17,18 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 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 because of 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 23559
DOI: 10.1021/acsami.9b04121 ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Research Article
ACS Applied Materials & Interfaces
polymerized to PPF via condensation under vacuum at 130 °C for another 4 h. 2.3. Printing of 3D Scaffolds. 2.3.1. 3D Printing Resin Preparation. To obtain a UV curable resin, the PPF polymer was dissolved in DEF (w/w 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. 2.3.2. 3D Scaffold Design in Computer-Aided Design. The 3D scaffold model was designed using a computer-aided design (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. 2.3.3. Scaffold Printing by 3D Stereolithography. The 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. The BFF files were read by the VIPER si2 STL system (3D Systems, Valencia, CA) to print 3D scaffolds layer by layer with a 365 nm UV laser through photo-cross-linking of PPF/DEF polymer resins. 2.3.4. Post-Printing Treatment. After printing, the supports were exfoliated gently and the scaffolds were washed with minimal amounts of acetone and absolute ethanol to remove excess resin. The 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. Afterward, Soxhlet scaffolds were rinsed in water for 2 days to remove excess THF. 2.4. Surface Functionalization of 3D Scaffolds. 2.4.1. Ammonolysis of 3D Scaffolds. The ammonolysis solution was prepared by dissolving 6.0 g of hexamethylenediamine (Sigma-Aldrich, Milwaukee, WI) in 100 mL of 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. 2.4.2. Surface Coating of 2D Materials. The ammonolyzed 3DPPF-Amine scaffolds were pretreated 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 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 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 3DPPF-Amine scaffolds were haphazardly grouped and then soaked for 1 h in BP solution, GO solution, and GO@BP solution. After coating, the scaffolds were dried under vacuum to generate 3D-PPF-AmineBP, 3D-PPF-Amine-GO, and 3D-PPF-Amine-GO@BP scaffolds. 2.5. Material Characterizations. 2.5.1. Protein Adsorption on 3D Scaffolds. To analyze protein adsorption, the scaffolds were soaked for 2 h at 37 °C in the 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) three times to remove any unabsorbed proteins. Proteins that are adsorbed on the 3D scaffolds were washed off with 1% sodium dodecyl sulfate (BioRad Laboratories, Hercules, CA) and the concentration of proteins was determined with Micro BCA protein assay kit (Pierce, Rockford, IL) according to the provided instructions. 2.5.2. Atomic Force Microscopy. The morphology and height of the GO and BP nanosheets were determined by atomic force microscopy (AFM) analysis using a Nanoscope IV PicoForce
implantable structures with sophisticated architectures that promote tissue growth and integration. Synthetic polymers currently in use in 3D printing today include poly(εcaprolactone),44−46 poly(L-lactic acid),47 poly(lactic-co-glycolic acid),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 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 In this study, we investigated the osteogenic capacity of 3D printed biodegradable scaffolds coated with 2D materials that alter their surface properties (Figure 1a). These functionalized 3D scaffolds consist of a cross-linked 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 (Figure 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 (Figure 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, was also investigated (Figure 1d). After implantation, the polymeric scaffolds were designed to enhance new bone formation in situ (Figure 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. GO 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) under vigorous stirring. The components were reacted at 50 °C under 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 3 days using a 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 of deionized (DI) H2O and sonicated for 1 h using a probe model sonicator (Qsonica Q500) to break the large BP nanosheets into small pieces. The mixture solution was then sonicated for another 12 h while using an ice bath sonicator (Elmasonic S10, Elma Schmidbauer GmbH, Germany). The obtained gray 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. PPF, used as the cross-linkable polymer for 3D printed scaffolds, was synthesized according to ref 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 obtain the intermediate dimer. The intermediate dimer was 23560
DOI: 10.1021/acsami.9b04121 ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Research Article
ACS Applied Materials & Interfaces MultiMode AFM (Bruker, Santa Barbara, CA) in an ambient environment.60 2.5.3. Scanning Electron Microscopy. 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 Xray spectrometry (EDXS) element mapping. 2.5.4. Transmission Electron Microscopy. The morphology of GO and BP nanosheets was observed using a transmission electron microscope (1200-EX II, JEOL Inc., Japan) at 80 kV voltage. 2.5.5. Attenuated Total Reflectance−Fourier Transform Infrared Spectroscopy. 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− 4000 cm−1. The functionalized scaffolds after mineralization in a simulated body fluid (SBF) were also characterized using attenuated total reflectance−Fourier transform infrared (ATR−FTIR) spectroscopy with the same parameters. 2.5.6. Coated Mass Determination. For mass determination, every 10 scaffolds were set into one group to increase the mass for easier determination. The weight of the scaffolds in each group was recorded as W1 before functionalization. Then, the independent groups underwent different functionalization processes 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. 2.5.7. 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, that is, room temperature and 37 °C. At each time point, 0.4 mL of the 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. 2.6.1. Live/Dead Cell Viability Staining. MC3T3 preosteoblast cells were expanded in α-MEM supplemented with 10% FBS and 0.5% streptomycin/penicillin. The functionalized 3D scaffolds were sterilized by UV irradiation for 2 h in a cell culture hood and then adhered to the bottom of 48-well tissue culture polystyrene (TCPS) plates. The 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. 2.6.2. Cytotoxicity of Leaching Medium. To further evaluate whether functionalized 3D scaffolds could release cytotoxic substances, the sterilized 3D scaffolds were placed into transwell chambers (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 a 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 a sterile silicone-based high vacuum grease (Dow Corning, Midland, MI). The MC3T3 cells were
seeded at a density of 30 000 cells per well. At 6 h 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 the MTS assay and absorbance at 490 nm was measured 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 an ascorbic acid (AA)-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 a UV−vis absorbance microplate reader. Immunofluorescence 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 nonspecific binding sites, scaffolds seeded with cells were soaked for 1 h in a PBS solution containing 1% bovine serum albumin. Permeabilized and blocked cells were then incubated with antivinculin−fluorescein isothiocyanate (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 (FA). After initial staining, secondary staining with rhodaminephalloidin (RP, Cytoskeleton Inc., Denver, CO, USA; 1:200 diluted in PBS) was conducted for an hour at 37 °C to stain cellular filaments. The immunofluorescence-stained cells on the functionalized 3D Scaffolds were immediately scoped on an inverted laser scanning confocal microscope (Carl Zeiss). 2.7.1. Total Collagen Production. The 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 h. The 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 a 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 a microscope. The relative collagen amount was calculated by normalizing the obtained optical density 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 SBF method, according to previous refs.63−65 The 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 1 M HCl (40 mL) in DI water, with a final volume of 1 L. The 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 scanning electron microscopy (SEM) and ATR−FTIR. To determine the cell proliferation on these biomineralized 3D scaffolds, the scaffolds were sterilized by UV irradiation for 2 h, 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 an AA-free nonosteogenic α-MEM, the medium was replaced with MTS solution and absorbance was measured by a UV− vis absorbance microplate reader at 490 nm. Immunofluorescence 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. The permeabilized cells were incubated with RP for an hour at 37 °C to stain cellular filaments, followed by incubation for 10 min in 4′,623561
DOI: 10.1021/acsami.9b04121 ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Research Article
ACS Applied Materials & Interfaces
Figure 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 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). 2.9.1. Alkaline Phosphatase Activity. Intracellular alkaline phosphatase (ALP) 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. The collected cells were repeatedly washed with sterilized DI H2O and centrifuged at least five times to remove potential ethylenediaminetetraacetic acid from trypsin solution and then counted and lysed in 0.2% Triton X-100 (0.5 mL of solution per 1 × 104 cells) at 4 °C overnight. The ALP
diamidino-2-phenylindole 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, that is, AA-free nonosteogenic α-MEM medium and osteogenic medium supplemented with 10 mM β-glycerophosphate (β-GP) and 50 μg/mL of AA. 23562
DOI: 10.1021/acsami.9b04121 ACS Appl. Mater. Interfaces 2019, 11, 23558−23572
Research Article
ACS Applied Materials & Interfaces
Figure 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-PPFAmine-GO@BP scaffold. (h) Schematic demonstration of the four types of 3D scaffolds. (i) AFM images and (j) averaged roughness (n = 5; *: p < 0.05) of the 3D scaffolds. (k) Protein adsorption and (l) total mass coated on the functionalized 3D scaffolds (n = 3; *: p < 0.05). 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, followed by Tukey’s posttest if necessary. Any two data groups with p-value calculated to be
