Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue

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Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue Engineering Abiy Woldetsadik, Sudhir Sharma, Sachin Khapli, Ramesh Jagannathan, and Mazin Magzoub ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00301 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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ACS Biomaterials Science & Engineering

Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue Engineering Abiy D. Woldetsadika, Sudhir K. Sharmab, Sachin Khaplib, Ramesh Jagannathanb,*, Mazin Magzouba,*

a

Biology Program, Division of Science, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. b Nano and Bio Materials Laboratory, Engineering Division, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.

Short Title: Hierarchically Porous CaCO3 scaffolds

A.D.W. and S.K.S. contributed equally to this work. Correspondence: * M.M., Biology Program, Division of Science, New York University Abu Dhabi, P.O. Box 129188, Saadiyat Island Campus, Abu Dhabi, United Arab Emirates. E-mail: [email protected]. * R.J., Engineering Division, New York University Abu Dhabi, P.O. Box 129188, Saadiyat Island Campus, Abu Dhabi, United Arab Emirates. E-mail: [email protected].

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Hierarchically Porous CaCO3 scaffolds

Abbreviations ARS, Alizarin Red S; AFM, atomic force microscope; APTES, 3-aminopropyl triethoxysilane; BSA, bovine serum albumin; CaCO3, calcium carbonate; CL, type I collagen; CLSM, confocal laser scanning microscopy; CPC, cetylpyridinium chloride; DIC, Differential interference contrast; EDS, energy dispersive X-ray spectroscopy; ELISA, enzyme-linked immunosorbent assay; ECM, extracellular matrix; FN, fibronectin; FITC, fluorescein isothiocyanate; FTIR, fourier transform infrared spectroscopy; HA, hydroxyapatite; MTS, CellTiter 96 AQueous One Solution cell proliferation assay; KBr, potassium bromide; OD, optical density; PBS, Phosphate-buffered saline; PFA, paraformaldehyde; PMA, phorbol 12-myristate 13-acetate; PGA, polyglycolic acid; PLGA, polyd-l-lactic-co-glycolic acid,; pNPP, p-nitrophenyl phosphate; polystyrene, poly-l-lactic acid, PLLA; polyglycolic acid, PGA; Ra, average surface roughness; RGD, Arg-Gly-Asp motifs in ECM proteins; SEM, scanning electron microscope; Si, silicon; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; sc-CO2, supercritical carbon dioxide; TGF-β, transforming growth factor beta; TNF-α; tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; VN, vitronectin; XRD, X-ray diffraction.

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Abstract Hierarchically porous CaCO3 scaffolds comprised of micron- (diameter = 2 ± 0.3 µm) and nano-sized (diameter = 50.42 ± 14.38 nm) pores were fabricated on silicon substrates using a supercritical CO2 process. Differentiated human THP-1 monocytes exposed to the CaCO3 scaffolds produced negligible levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α), confirming the lack of immunogenicity of the scaffolds. Extracellular matrix (ECM) proteins, vitronectin (VN) and fibronectin (FN), displayed enhanced adsorption to the scaffolds compared to the silicon controls. ECM protein-coated CaCO3 scaffolds promoted adhesion, growth and proliferation of osteoblast MC3T3 cells. MC3T3 cells grown on the CaCO3 scaffolds produced substantially higher levels of transforming growth factor-beta (TGF-β) and vascular endothelial growth factor-A (VEGFA), which regulate osteoblast differentiation, and exhibited markedly increased alkaline phosphatase (ALP) activity, a marker of early osteoblast differentiation, compared to controls. Moreover, the CaCO3 scaffolds stimulated matrix mineralization (calcium deposition), an endpoint of advanced osteoblast differentiation and an important biomarker for bone tissue formation. Taken together, these results demonstrate the significant potential of the hierarchically porous CaCO3 scaffolds for bone tissue engineering applications. Keywords: supercritical CO2; hierarchically porous CaCO3 scaffolds; osteoblast MC3T3 cells; osteoblast adhesion, proliferation and differentiation; matrix mineralization




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1. Introduction Bone grafting is performed in cases of trauma, deformity, cancer, and ageing related bone damage (1). While autografts, which are harvested from a remote site within the patient’s body (2,3), and allografts, which are harvested from a deceased donor or a cadaver (2,4), are standard clinical procedures, they have limited utility (5) making it necessary to develop synthetic bone substitutes. However, development of biomaterials in bone regenerative medicine remains a major challenge because of the need for the substitute material to simultaneously meet multiple biological, mechanical, and manufacturing constraints (1). The biological requirements for biomaterials used for bone tissue engineering applications include: biocompatibility, in order to avoid any local or systemic toxic or inflammatory responses; bioactivity, which is defined as the ability to develop direct and strong bonding with bone tissue; osteoconductivity, that is the biomaterial must serve as a scaffold to guide formation of newly forming bone along their surfaces by providing appropriate binding sites to promote osteoblast adhesion, differentiation and subsequent extracellular matrix (ECM) formation; and bioresorbability, as the scaffold must degrade at a controlled rate to make way for the growth of new bone tissue (6) . A critical consideration for bone substitutes is porosity, which is important for the osteoconductivity and bioresorbability of the biomaterials (7). Similar to natural bone tissue, artificial scaffolds must be porous on various length scales. Micropores (< 20 µm diameter) promote adhesion of osteoblasts and growth factors, while macropores (200–350 µm diameter) are necessary for capillary in-growth and vascularization of bone tissue (8,9). Enhanced porosity, however, modulates the elastic response of the scaffold and needs to be optimized for achieving both goals simultaneously. In addition to porosity, scaffold surface properties such as chemical composition, topography, roughness and wettability have been shown to strongly influence osteoblast adhesion, growth and differentiation (10). Further to the aforementioned constraints, it necessary to fabricate the bone scaffolds material in the desired three-dimensional shape under Good Manufacturing Practice (GMP) conditions in a reproducible and quality controlled fashion, and the material and manufacturing costs should be reasonable. Various attempts have been made to develop scaffolds for tissue engineering from natural (collagen, silk and hyaluronic acid) or synthetic polymers, such as polystyrene, poly-l-lactic acid (PLLA), polyglycolic acid (PGA) and poly-d-l-lactic-co-glycolic acid (PLGA) (11). Composite scaffolds have been used to overcome some of the problems associated with using natural or synthetic polymers alone, such as poor mechanical properties (related to natural polymers) or toxicity (associated with rapid degradation of synthetic polymers, which produces CO2 and leached ions that lower the local pH and cause cell or tissue necrosis (11-15). However, the use of composite scaffolds are often characterized by structural complexity which hinders technological manipulation (16). Bone mineral is composed largely of calcium salts, phosphate and carbonate (17), and calcium plays a significant role in enhancing osteogenesis of bone cells (18). One of the most commonly used


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calcium-based bone substitutes is hydroxyapatite (HA), a naturally occurring mineral form of calcium phosphate (19). Although similar in composition to bone mineral, HA is brittle and has little tensile strength, and therefore provides limited biomechanical support (19). As an alternative to HA and CaPO4based biomaterials, research efforts have focused on CaCO3 (20,21). CaCO3-based biomaterials possess several excellent properties that make them ideally suited for bone implantation and regeneration, including biocompatibility, bioactivity, and high osteoconductivity leading to formation of a uniquely strong bone-biomaterial interface (22). Additionally, CaCO3 offers superior biodegradation properties compared to calcium phosphate (20). However, scaling-up of fabrication methods for the production of porous CaCO3 scaffolds has remained a major challenge (21,23). Most commonly developed scaffold techniques are solution-based processes utilizing sacrificial templates, e.g. colloidal crystals (24-26), resulting in porous crystal structures. True scaffold coatings are generally limited to polymers using techniques such as electro spinning (27). Recently, rapid prototyping processes have been used to create hierarchically porous structures of polymer/ceramic systems (16,2831) . We have developed a template-free, scalable technology using supercritical carbon dioxide (scCO2) as a green platform (Figure 1) to prepare CaCO3 scaffolds (22). Our method, which is applicable to a broad range of ceramic materials, can generate hierarchical porosity on the macro and micro scale. An attractive feature of the sc-CO2 based process is that it introduces nanoscale features in the hierarchically porous scaffolds. Nanoscale features are important because mammalian cells respond to mechano-transductive stimuli from nanotopographic structures within the extracellular matrix, and the presence of such features in a synthetic scaffold influences osteoblast adhesion, morphology, proliferation and differentiation. In addition, the sc-CO2 based process can be carried out under GMP conditions and potentially integrated with the SFF (Solid Freeform Fabrication) methods of bone tissue engineering (32). In preliminary investigations, we demonstrated the utility of our technique for coating planar Si substrates and screw shaped Ti-implants with porous scaffolds of CaCO3 bioceramic (33). The sc-CO2 process, which generates a fine, sub-micron size aerosol of calcium acetate (34) is impinged on a hot substrate, such as silicon, thereby subjecting the aerosol droplets to the so called “coffee-ring effect”, resulting in the creation of a unique scaffold containing a distribution of micro and nano sized pores. It has been previously established that the rapid de-pressurization of sc-CO2 is able to produce submicron sized droplets, unlike standard nebulization techniques, which generate much larger sized droplets (34,35). The aim of the current study is to characterize the mechanical properties of the CaCO3 scaffolds and extensively investigate the in vitro biological response (adhesion, proliferation, differentiation, and matrix mineralization) of osteoblasts grown on the scaffolds. Our results show that the CaCO3 scaffolds, with a well-defined inter-connected pore structure, represent highly promising biomaterials for bone tissue engineering applications.




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2. Materials and methods

2.1. Reagents Alizarin Red S (ARS), ascorbic acid, bovine serum albumin (BSA), β-Glycerophosphate, calcium acetate, cetylpyridinium chloride (CPC), Coomassie Brilliant Blue R250, ethanol, fibronectin (FN), fluorescein isothiocyanate labeled phalloidin (FITC-phalloidin), glacial acetic acid, L-glutamine, methanol, Minimum Essential Medium (MEM), paraformaldehyde (PFA), penicillin/streptomycin, phorbol 12-myristate 13-acetate (PMA), phosphate buffered saline (PBS), lipopolysaccharide (LPS), rat tail type I collagen (CL), RPMI 1640, sodium dodecyl sulfate (SDS), Triton X-100 and vitronectin (VN) were purchased from all purchased Sigma (St. Louis, MO). Hoechst 33342, pre-stained protein ladder (40-300kDa) and ELISA kits (for detection of transforming growth factor-beta 1 (TGF-β1), tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor-A (VEGF-A)) were all from Life Science Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from GE Healthcare Life Sciences (Logan, UT). Alkaline Phosphatase (ALP) Assay kit was from Abcam (Cambridge, MA). Bradford Protein Assay was procured from Bio-Rad Laboratories (Hercules, CA). CellTiter 96 AQueous One Solution (MTS) Cell Proliferation Assay kit was purchased from Promega (Madison, WI).

2.2. Scaffold deposition and characterization Hierarchically porous CaCO3 scaffolds were deposited by a supercritical carbon dioxide (scCO2) assisted nebulization process (Figure 1) reported previously (22,36). In brief, an aqueous solution of calcium acetate was mixed with a continuous stream of sc-CO2 in a static mixer. Mixing of these two streams in a small volume mixer enhances the dissolution of CO2 in the aqueous phase (36). In order to generate the aerosol, the mixture was first passed through a heat exchanger (temperature = 90 °C) and allowed to expand to atmospheric pressure via a capillary tubing. This depressurization process produces a fine and dense stream of aerosols. The aerosol stream is then directed to impinge on a heated silicon substrate, resulting in the formation of the porous scaffolds due to the “coffee ring” effect. In the present work, we used an aqueous solution of calcium acetate (0.1 wt %). The temperature of the mixing chamber was maintained at 110 °C. Continuous flow of superciticalCO2 was used to maintain the pressure of the mixing chamber at 120 bar. We used square shaped silicon wafers mounted on a heated aluminum substrate heater whose temperature was optimized and maintained constant at 130 °C throughout the deposition process. The coating duration was systematically varied from 5 to 30 min to investigate the influence of deposition time on pore size. We selected a coating duration of 20 min for these studies. These scaffolds were sintered at a temperature of 450 °C for 2 h. The scaffolds and substrates were 10 mm ×10 mm in size.


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The morphology of the CaCO3 scaffolds was analyzed using scanning electron microscopy (SEM) (Quanta FEG 450; FEI, Hillsboro, Oregon). The composition of the scaffolds was determined by energy dispersive X-ray spectroscopy (EDS) (Quanta FEG 450). A Panalytical Empyrean X-ray diffractometer (Empyrean X-Ray Diffractometer; PANalytical, The Netherlands) equipped with a Cu anode was used to measure X-ray diffraction (XRD) data from the CaCO3 scaffolds. The X-ray tube was operated typically at 45 kV and 40 mA for these measurements. The diffraction pattern was collected at a scanning rate of 0.5° per minute, and the diffraction angle 2θ was varied from 10° to 50° in Bragg–Brentano geometry, as reported previously (22). Atomic force microscopy (AFM) (5500 Atomic Force Microscope; Keysight Technologies Inc., Santa Rosa, CA) was used to determine surface roughness of the scaffolds, and the mechanical properties of the scaffolds and substrates were characterized using an Agilent G200 Nano Indentor.

2.3. Analysis of ECM protein adsorption The hierarchically porous CaCO3 scaffolds, and silicon and cell culture glass (Nunc Thermanox Coverslips; Thermofisher) control substrates, were soaked in 70% (v/v) ethanol for 30 min, then washed with phosphate buffered saline (PBS) and placed in a vacuum desiccator to remove air bubbles. The pre-wetted scaffolds and substrates were incubated in extracellular matrix (ECM) protein – type I collagen (CL), vitronectin (VN) or fibronectin (FN) – solutions (0-20 µg/mL) for 24 h at 4 °C. Thereafter, the protein solutions were removed, and the scaffolds and substrates were washed thoroughly with PBS and incubated in a 1% (w/v) sodium dodecyl sulfate (SDS) solution to recover adsorbed proteins. Recovered protein concentrations were determined by the colorimetric Bradford Protein assay. For qualitative analysis, the recovered ECM protein solutions were electrophoresed on a 3-10% SDS polyacrylamide gel, followed by staining with Coomassie Blue solution (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid) for 30 min at room temperature. For densitometry analysis, an image of the stained gel was captured and post-processed using Fiji image processing software (USA) (37). Regions of interest were defined for each band and intensities were compared with a bovine serum albumin (BSA) standard.

2.4. Cell lines Mouse calvarial bone osteoblast MC3T3 cells (ATCC no. CRL-2593) were cultured in standard medium (MEM supplemented with 10% FBS, 4 mM L-glutamine and 1% penicillin/streptomycin), or differentiation medium (MEM supplemented with 10% FBS, 4 mM L-glutamine, 50 µg/ml ascorbic acid, 10 mM β-Glycerophosphate and 1% penicillin/streptomycin), in 5% CO2 at 37 oC. Human monocytic leukemia THP-1 cells (ATCC no. TIB-202) were cultured in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 4 mM L-glutamine, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 1% penicillin/streptomycin, in 5% CO2 at 37 °C. THP-1 cells were differentiated into a




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macrophage-like phenotype by incubating them with 10 ng/mL PMA in complete medium for 72 h in 5% CO2 at 37 oC.

2.5. Inflammatory cytokine assay Differentiated THP-1 cells were seeded at a density of 5 × 104 cells/mL on uncoated hierarchically porous CaCO3 scaffolds and silicon substrates in 24-well plates. Cells treated with lipopolysaccharide (LPS) (38) were used as a positive control, while cells cultured on cell culture glass surface served as a negative control. After culturing for 48 h, the cell culture medium was assayed for secretion of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) using a commercial ELISA kit. The total TNF-α level was determined from the absorbance (λ = 490 nm) measured on a Synergy H1MF Multi-Mode microplate-reader (BioTek, Winooski, VT) using a standard TNF-α concentration calibration curve.

2.6. Osteoblast adhesion and proliferation Adhesion and proliferation of osteoblasts was evaluated using the CellTiter 96 AQueous One Solution (MTS) assay, which measures reduction of the tetrazolium compound MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) to soluble formazan, by dehydrogenase enzymes, in living cells (39,40). Osteoblast MC3T3 cells were seeded at a density of 5 × 104 cells/mL in standard medium on hierarchically porous CaCO3 scaffolds (uncoated and ECM protein-coated) and silicon substrates in 24-well plates, and cultured for the indicated durations. Thereafter, the medium was replaced with fresh medium, and 20 µL MTS reagent was added to each well. The MTS reagent was incubated for 4 h at 37 °C, and absorbance of the soluble formazan product (λ = 490 nm) of MTS reduction was measured on a Synergy H1MF MultiMode microplate-reader, with a reference wavelength of 650 nm to subtract background. Cells grown on cell culture glass were used as control, and wells with medium alone served as a blank. The total cellular protein content was determining using the colorimetric Bradford Protein assay. After culturing for the indicated durations, the cells were harvested and lysed with PBS containing 1% Triton X-100. 10 µL aliquots of the cell lysates were transferred to 96-well plates. 200 µL dye reagent (diluted 1:4 with deionized water) was then added to each well, and the plate was incubated for 15 min at room temperature. Finally, the absorbance (λ = 595 nm) was measured on a Synergy H1MF Multi-Mode microplate-reader, and the total protein concentration was determined using a standard BSA calibration curve.

2.7. Confocal fluorescence microscopy Osteoblast MC3T3 cells were seeded at a density of 5 × 104 cells/mL in standard medium on hierarchically porous CaCO3 scaffolds (uncoated and ECM protein-coated) and silicon substrates in 4chambered 35 mm glass bottom Cellview cell culture dishes (Greiner Bio-One, Monroe, NC). After


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culturing for 3 days, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, washed three times with PBS, permeabilized with 0.2% Triton-X-100 for 5 min, and blocked with 1% bovine serum albumin (BSA) for 30 min. The cells were then stained with 10 µg/mL fluorescein isothiocyanate labeled phalloidin (FITC-phalloidin) for 30 min at 37 oC to visualize filamentous actin (F-actin), washed three times with PBS (10 min per wash), followed by staining with 5 µg/mL Hoechst 33342 for 10 min at 37 oC to visualize the nuclei. Imaging was done on an Olympus Fluoview FV-1000 confocal laser scanning microscope, using a 63× Plan-Apo/1.3 NA oil immersion objective with differential interference contrast (DIC) capability. Image processing was done using the Fiji image processing software (37).

2.8. Quantification of growth factor production Osteoblast MC3T3 cells were seeded at a density of 5 × 104 cells/mL in differentiation medium on uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds and control substrates (silicon and cell culture glass) in 24-well plates, and cultured for 14 days. Culture medium collected from uncoated samples (on days 3, 7 and 14) and ECM protein-coated samples (on day 14) were assayed for VEGF-A and TGF-β levels using commercial ELISA kits. The total VEGF-A and TGF-β levels were determined from the absorbance (λ = 490 nm) measured on a Synergy H1MF Multi-Mode microplate-reader using standard VEGF-A and TGF-β concentration calibration curves.

2.9. Measurement of alkaline phosphatase (ALP) activity Alkaline phosphatase (ALP) activity was measured using a highly sensitive colorometric assay. The assay uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate, which turns yellow when dephosphorylated by ALP (41). Osteoblast MC3T3 cells were seeded at a density of 5 × 104 cells/mL in differentiation medium on uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds and control substrates (silicon and cell culture glass) in 24-well plates. After culturing for the indicated durations, the cells were harvested and lysed with PBS containing 1% Triton X-100. 50 µL aliquots of the cell lysates were added to 96-well plates, along with 50 µL assay buffer and 50 µL pNPP. The samples were incubated for 1 h in the dark for at room temperature. Thereafter, 20 µL stop solution was added to the wells, and absorbance (λ = 490 nm) was measured on a Synergy H1MF Multi-Mode microplate-reader. The results were normalized to the total cellular protein content determined using the Bradford Protein assay.

2.10. Detection of matrix mineralization Calcium deposition was detected using Alizarin Red S (ARS) staining (42). Calcium forms a complex with ARS, an anthraquinone derivative, in a chelation process, and the end product is an orange-red stain. Osteoblast MC3T3 cells were seeded at a density of 5 × 104 cells/mL in differentiation medium on uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds




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and control substrates (silicon and cell culture glass) in 24-well plates, and cultured for the indicated durations. Thereafter, the medium was aspirated gently and the cells were fixed with 70% ethanol for 10 min. The cells were then washed with PBS, stained with ARS solution (1%  w/v, pH 4.1) for 10 min, and subsequently washed thoroughly with PBS. This staining duration was chosen to ensure that no dissolution of the scaffolds occurred due to exposure to the acidic ARS solution. Imaging was done a Nikon Eclipse LV100ND microscope equipped with a Nikon DS-Fi2 camera. Calcium deposition was quantified using cetylpyridinium chloride (CPC) extraction. ARS-stained cells were incubated in 0.5 mL CPC solution (10% w/v) for 1 h at room temperature. The CPC incubation solution was then transferred to 96-well plates (200  µL/well), and the absorbance (λ = 570 nm) was measured on a Synergy H1MF Multi-Mode microplate-reader. Mineral composition was analyzed by Fourier transform infrared spectroscopy (FTIR) on an Agilent Cary 600 Series FTIR spectrometer using ATR mode. Potassium bromide (KBr) was mixed with the cell lysates collected from scaffolds and control substrates to prepare the pellets for FTIR measurements. Spectra were acquired in the 400-4000 cm-1 range, at a resolution of 0.5  cm−1, with an average of 64 scans.

2.11. Statistical Analysis Confidence intervals in this work represent the standard error of the mean (S.E.M.) across at least three independent trials. Statistical analysis was performed using the Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance between test groups and controls, or among three or more groups, was assessed by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s post hoc test. p < 0.05 was considered to be statistically significant.


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3. Results and discussion We investigated fully the suitability of our hierarchically porous CaCO3 scaffolds as bone implant materials. The immunogenicity potential was tested by evaluating the inflammatory response of differentiated human THP-1 monocytes, a widely used model of monocyte/macrophage activation (43) , exposed to the scaffolds. Multiple complementary assays were then used to evaluate the adhesion, proliferation and differentiation of MC3T3, an osteoblast precursor cell line derived from mouse calvaria (44), seeded onto the CaCO3 scaffolds. The results from the CaCO3 scaffolds were compared to two commonly used control substrates with the same dimensions (10 mm ×10 mm): silicon, which is the substrate on which the CaCO3 scaffold is formed, and cell culture glass (Nunc Thermanox Coverslips; Thermofisher) coated with a proprietary polyester film surface to enhance cell adhesion, growth and differentiation (45,46).

3.1. Hierarchically porous CaCO3 scaffold deposition and characterization Hierarchical porous CaCO3 scaffolds were deposited onto silicon substrates under optimal process conditions (P = 120 bar, T = 130 oC and t = 20 min) and yielded high quality scaffolds. Immediately following deposition, the scaffolds were sintered at 450 oC for 2 h in air. The scaffolds were then subjected to microstructural, compositional and mechanical characterization (Figure 2). Scanning electron microscopy (SEM) micrographs of the CaCO3 scaffolds, in the 250 µm range (left panel), 60 µm range (middle panel), and 3 µm range (right panel), revealed the presence of micron- and nano-sized pores within a uniform three-dimensional structure (Figure 2a). X-ray diffraction (XRD) analysis confirmed the presence of calcite phase in the CaCO3 scaffolds (Figure 2b, left panel). We observed a dominant diffraction peak located at 2θ = 29.4°, corresponding to (104) texture, along with evidence of (012), (110), (113), (202), (018), and (116) calcite diffraction peaks. We did not observe any impurity or secondary phase formation in these scaffolds. The presence of sharp diffraction peaks also indicates that these CaCO3 scaffolds are well aligned. Immersion of the scaffolds in cell culture medium for seven days resulted in the re-crystallization of small CaCO3 crystallites. These crystallites were analysed using energy-dispersive X-ray spectroscopy (EDS) through placement of the probe directly on the fine crystals (Figure 2b, right panel). The EDS data clearly showed the presence of Ca, C, O and silicon substrate, confirming the presence of CaCO3 in the crystallites (47-49). We suspect that immersion in medium facilitates some level of Ostwald ripening that results in the re-crystallization of these fine crystals. The pore size distribution of the CaCO3 scaffolds as a function of deposition time was characterized by AFM (> 10 measurements per sample) (Figure 2c). The typical pore size distributions measured for deposition times ranging from 5 to 20 min showed that pore sizes are independent of deposition time. The pore size of the scaffolds was typically 2 ± 0.3 µm. The mechanical properties of the scaffolds, such as micro-hardness and Young’s modulus, were determined using a nano-indentation technique (Figure 2d). For a typical penetration depth, the




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maximum load values for the scaffolds were lower than that for glass and silicon, indicating that the scaffold structure was softer (left panel). The relative softness of the scaffold structure compared to silicon and glass is also reflected in the hardness (middle panel) and Young’s modulus (right panel) data obtained using the constant stiffness measurements (CSM). However, the hardness and Young’s modulus of the scaffolds are within the range of values reported for natural bone (50,51). Atomic force microscopy (AFM) was used to characterize the micro (∼ 1-3 µm) and nano structures (50.42 ± 14.38 nm) (Figure 2e). The CaCO3 scaffolds demonstrated a much higher average surface roughness (Ra = 1.95 nm, top middle panel; Ra = 20 nm, lower middle panel) compared to the cell culture glass substrate (Ra = 1.12 nm, top right panel; Ra = 2.34 nm, lower right panel) and silicon (Ra = 1.12 nm, top right panel; Ra = 2.34 nm, lower right panel). The higher surface roughness for the scaffolds is consistent with the presence of the micro- and nano-porous structures. Remarkably, despite their highly porous structure, the elastic modulus of the CaCO3 scaffolds is comparable to that of natural bone (16.6–38.5 GPa) (50-52). Scaffold porosity facilitates transport of nutrients and metabolites, and promotes adhesion of growth factors, osteoblast invasion and tissue growth, as well as vascularization (7). Moreover, porosity improves mechanical interlocking between the scaffold and the surrounding natural bone, thereby providing greater mechanical stability at this critical interface (9). However, there appears to be no consensus regarding the optimum pore size for bone formation and growth (53). Notably, the porosity of our scaffolds could, in principle, be tuned as a function of the deposition process parameters, as described previously (22). Thus, the hierarchically porous CaCO3 scaffolds generated by our sc-CO2 process appear to have the desired biomechanical properties to facilitate optimal adhesion, growth and differentiation of osteoblasts for bone tissue regeneration.

3.2. Scaffold immunogenicity A critical consideration for bone substitutes is their potential to trigger significant inflammation. Although low levels of inflammation aid the healing process, significant inflammation can result in failure of implants (54,55). To assess the inflammatory potential of the hierarchically porous CaCO3 scaffolds, we quantified the production of tumor necrosis factor-alpha (TNF-α) by differentiated human THP-1 monocytes exposed to the scaffolds (Figure 3a). TNF-α is an inflammatory cytokine primarily produced by macrophages/monocytes during acute inflammation (56). The macrophage activator lipopolysaccharide (LPS) (38) was used as a positive control, while THP-1 cells cultured on cell culture glass surface (Nunc Thermanox Coverslips) served as a negative control. Culturing THP-1 cells on the cell culture glass surface or silicon substrate resulted in ∼13 pg/mL TNF-α (Figure 3a). Likewise, THP-1 cells grown on the CaCO3 scaffolds produced negligible levels of TNF-α (∼10 pg/mL). In contrast, THP-1 cells treated with LPS produced ∼135 pg/ml TNF-α,


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which is in the range of values reported for differentiated THP-1 cells treated with LPS (38,57). These results clearly demonstrate the lack of immunogenicity of the hierarchically porous CaCO3 scaffolds.

3.3. ECM protein adsorption In bone tissue, osteoblasts are surrounded by an extracellular matrix (ECM) – a complex and dynamic network of proteins, proteoglycans, glycosaminoglycans and growth actors – which provides structural support and the biochemical cues that modulate bone remodeling (58,59). Biomimetic scaffolds are therefore commonly coated with ECM proteins, such as type I collagen (CL), fibronectin (FN) or vitronectin (VN), in order to mimic the in vivo ECM microenvironment and promote osteoblast adhesion, growth and proliferation (60,61). Here, we measured adsorption of ECM proteins to the hierarchically porous CaCO3 scaffolds using the Bradford protein assay (62) (Figure 3b). Adsorption of VN to the scaffolds increased steadily in a dose dependent manner up to a protein concentration of 20 µg/mL (middle panel). Significantly, VN adsorption to the scaffolds was substantially higher compared to the silicon substrate controls (at the 20 µg/mL dose, scaffold adsorption was ~ 4-fold higher than that of the controls). Likewise, we observed dose-dependent increase in FN adsorption to the scaffolds, which was considerably higher than the adsorption to the silicon controls at all protein concentrations (right panel). On the other hand, CL did not significantly adhere to the scaffolds (left panel). The strong adsorption to the scaffolds of VN/FN relative to CL was confirmed by SDS-PAGE analysis (Figure 3c). ECM protein adsorption is highly dependent on the physicochemical properties of the biomaterial, including surface chemistry, topography and roughness (63-66). The greater adsorption to the glass substrate (Nunc Thermanox Coverslips) is likely due to their hydrophilic polyester film surface. It should be noted that although the CaCO3 scaffolds and control substrates have the same dimensions, the presence of micron- and nano-sized pores in the scaffolds means that the surface areas of the samples are not necessarily perfectly matched. At present we are unable to accurately determine the contribution of the pores to the surface area of the scaffolds. However, it has been reported that with a high degree of nanoscale roughness the topography of scaffolds appears smooth to ECM proteins and has little effect on the adsorption process (67-70). Our results show that the properties of the hierarchically porous CaCO3 scaffolds are favorable for strong adsorption of ECM FN and VN. FN and VN have been shown to enhance osteoblast adhesion to biomaterials surfaces due to presence of the Arg-Gly-Asp (RGD) motifs in the proteins, which are recognized and bound by adhesion receptors, namely integrins, on osteoblast surfaces (71,72). Conversely, CL adsorbs poorly to the scaffolds, which can be attributed to the relatively weak interaction of CL with synthetic scaffold surfaces (65,66,73,74). Furthermore, due to the inaccessibility of its RGD sequences for cell surface adhesion receptors, CL is generally not recommended for surface modification of scaffolds (71,72,75). Thus, we subsequently examined adhesion, proliferation and differentiation of osteoblasts on FN- and VN-coated CaCO3 scaffolds.




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3.4. Osteoblast adhesion, morphology and proliferation Attachment to bone substitute surfaces and subsequent proliferation are prerequisites for osteoblast differentiation and ECM mineralization (76). We therefore assessed the ability of the hierarchically porous CaCO3 scaffolds to promote adhesion, growth and proliferation of osteoblast MC3T3 cells (Figure 4). First, we quantified the initial adhesion of MC3T3 cells to the hierarchically porous CaCO3 scaffolds and control substrates at 24 h following seeding (Figure 4a). We observed ∼ 3-fold higher adhesion of MC3T3 cells to the uncoated CaCO3 scaffolds compared to the silicon substrate controls. Importantly, adhesion efficiency of the uncoated scaffolds was comparable to that of the cell culture glass surface (Nunc Thermanox Coverslips). Coating the CaCO3 scaffolds with ECM proteins, VN or FN, increased cell adhesion compared to the uncoated scaffolds (∼ 89 vs 66% for ECM protein-coated vs uncoated scaffolds, respectively). Next, the morphology of MC3T3 cells adhered to uncoated or ECM protein-coated hierarchically porous CaCO3 scaffolds for 3 days was examined by confocal fluorescence microscopy (Figure 4b). Substantially higher F-actin staining was observed in MC3T3 cells cultured on uncoated CaCO3 scaffolds compared to cells grown on the silicon substrate controls. MC3T3 cells grown on the uncoated scaffolds also showed greater F-actin organization throughout the cells, with distinct bundles of fibers clearly visible, compared to controls (top and lower left panels). Cells grown on the FN- or VN-coated scaffolds exhibited even higher levels of F-actin, and increased F-actin organization, compared to uncoated scaffolds (top and lower right panels). Finally, we measured the proliferation of osteoblast MC3T3 cells cultured on uncoated and ECM protein-coated CaCO3 scaffolds over a period of 21 days (Figure 4c). Culturing the cells on uncoated scaffolds increased the proliferation rate relative to the silicon substrate controls (left panel). Specifically, there were ∼ 3- and 13-fold more cells adhered to the scaffolds than the silicon substrate on days 7 and 14, respectively. Moreover, coating the CaCO3 scaffolds with VN or FN increased the proliferation rate even further, with the number of cells on the ECM protein-coated scaffolds 2–3-fold higher than on the uncoated scaffolds on day 14 (middle and right panels). These results were supported by quantitative analysis of total protein synthesis by MC3T3 cells cultured on uncoated or ECM protein-coated CaCO3 scaffolds for 28 days (Figure 4d). Protein content of MC3T3 cells cultured on uncoated CaCO3 scaffolds was substantially higher than cells grown on the silicon substrate controls (35 vs 5 mg/mL total protein for uncoated scaffolds vs controls, respectively, after 28 days; left panel). Likewise, culturing cells on ECM protein-coated scaffolds increased the protein content ∼ 5-fold relative to the uncoated scaffolds (right panel). Taken together, our results show that the chemical and physical properties of the hierarchically porous CaCO3 scaffolds stimulate osteoblast attachment, growth and proliferation. Coating the scaffolds with ECM proteins further enhances the biological response of osteoblasts. In the following


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sections, we evaluate the ability of ECM protein-coated scaffolds to promote osteoblast differentiation and matrix mineralization.

3.5. Growth factor production Vascular endothelial growth factor-A (VEGF-A), one of the most potent pro-angiogenic growth factors, directly stimulates osteoblast migration, proliferation and differentiation (77). Likewise, the multifunctional cytokine transforming growth factor-beta (TGF-β) plays a critical role in bone remodeling and repair by regulating osteoblast proliferation and differentiation (77,78). Here, we quantified production of VEGF-A and TGF-β by osteoblast cells cultured on hierarchically porous CaCO3 scaffolds using ELISA (Figure 5). MC3T3 cells cultured on uncoated CaCO3 scaffolds showed substantially higher production of growth factors compared to cells grown on the silicon substrate controls (Figure 5a). On days 7 and 14, VEGF-A levels on the CaCO3 scaffolds were 103 ± 11 and 151 ± 18 pg/mL, respectively, which correspond to ∼ 10- and 15-fold increases, respectively, compared to controls (left panel). Similarly, TGF-β production on the CaCO3 scaffolds was 52 ± 4 and 110 ± 6 ng/mL on days 7 and 14, respectively, which represent ∼ 5- and 11-fold higher levels, respectively, than observed on controls (right panel). Thus, the CaCO3 scaffolds stimulate production of growth factors by osteoblasts. Interestingly, growth factor production by osteoblast MC3T3 cells cultured on ECM proteincoated CaCO3 scaffolds was significantly higher compared to cells cultured on uncoated scaffolds (Figure 5b). VEGF-A levels on VN- and FN-coated CaCO3 scaffolds was ∼ 3- and 4 -fold higher, respectively, than on uncoated scaffolds (left panel). Likewise, TGF-β production on the VN and FNcoated scaffolds was ∼ 2- and 3-fold, respectively, that observed on uncoated scaffolds (right panel). Importantly, the levels of VEGF-A and TGF-β produced by MC3T3 cells cultured on our ECM protein-coated scaffolds were greater than to those reported for osteoblasts cultured on other CaCO3 scaffolds (49). These results demonstrate the potential of the ECM protein-coated hierarchically porous CaCO3 scaffolds to enhance osteoblast differentiation (61).

3.6. Alkaline phosphatase (ALP) activity Proliferating osteoblasts produce alkaline phosphatase (ALP), and the activity of the enzyme is greatly enhanced during in vitro bone formation (79,80). ALP activity is therefore commonly used as a biochemical marker of early osteoblast differentiation (79,81). We measured the ALP activity of osteoblast MC3T3 cells cultured for 3–14 days on cell culture glass (Nunc Thermanox Coverslips), silicon substrate and uncoated hierarchically porous CaCO3 scaffolds (Figure 6a). ALP activity of MC3T3 cells grown on the uncoated CaCO3 scaffolds steadily increased over 14 days, whereas for the same duration the ALP activity of the cells grown on the silicon substrate remained largely unchanged (0.47 vs 0. 64 nmol/mg protein at days 3 vs 14, respectively). Significantly, ALP activity of the cells grown on the CaCO3 scaffolds was significantly




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higher than that of cells grown on the silicon substrate controls (at days 7 and 14, ALP activity on scaffolds was ∼ 7- and 9-fold higher, respectively, than on the silicon substrates). Therefore, the CaCO3 scaffolds promote osteoblast adherence, proliferation, and differentiation. Next, we examined the effect of coating the CaCO3 scaffolds with ECM proteins on osteoblast differentiation (Figure 6b). MC3T3 cells cultured for 14 days on FN- and VN-coated CaCO3 scaffolds showed comparable ALP activity. However, ALP activity of cells grown on the ECM protein-coated CaCO3 scaffolds was ∼ 3–fold higher than that of cells grown on uncoated scaffolds. These results confirm that ECM proteins significantly enhance adhesion, proliferation and differentiation of osteoblasts on the hierarchically porous CaCO3 scaffolds.

3.7. Matrix mineralization Matrix mineralization (calcium deposition) is one of the most important biomarkers of bone formation, and is considered an endpoint of advanced osteoblast differentiation (82,83). Here, we assessed the matrix mineralization by differentiated osteoblast M3CT3 cells cultured on uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds using three independent assays (Figure 7). Calcium deposition by M3CT3 cells cultured on silicon substrate, uncoated CaCO3 scaffolds and glass substrate (Nunc Thermanox Coverslips) for 14 days was detected using Alizarin Red S (ARS) staining (42,84-87) (Figure 7a). The CaCO3 scaffold samples displayed intensive ARS staining, indicative of extensive calcium deposition, compared to the silicon substrate samples. Moreover, formation of mineralized nodules was observed in the CaCO3 scaffold samples, which was largely absent from the silicon control samples. Interestingly, mineralized nodule formation was also significantly higher in the scaffold samples compared to the glass substrate samples. The amount of calcium deposited in the different samples was quantified by cetylpyridinium chloride (CPC) extraction (Figure 7b). Calcium deposition by M3CT3 cells cultured on uncoated CaCO3 scaffolds for 14 and 21 days was ∼ 7 - and 9-fold higher, respectively, compared to cells grown on the silicon substrate (left panel). Significantly, even higher calcium deposition was detected in ECM-protein coated CaCO3 scaffold samples (right panel). The levels of calcium deposition on FN- and VN-coated CaCO3 scaffolds were ∼ 10- and 6-fold, respectively, those on the uncoated scaffolds. Thus, the hierarchically porous CaCO3 the hierarchically porous CaCO3 scaffolds promote matrix mineralization, and the effect is enhanced by coating the scaffolds with ECM proteins. The composition of minerals from the different samples was analysed by Fourier transform infrared spectroscopy (FTIR) (88-91) (Figure 7c). The FTIR spectra revealed that the minerals formed by MC3T3 cells cultured on the CaCO3 scaffolds for 14 days are phosphate and carbonate, which are the main components of bone mineral (left panel). Specifically, the spectrum showed the characteristic absorption bands of the phosphate groups: a strong band at 1021-1158 cm-1 and a weak band at 595599 cm-1. The absorption bands at 1452 and 1415 cm-1, together with the weak band at 855 cm-1, are characteristic of hydroxyl carbonate (92). These results strongly support the calcium deposition


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measurements (Figure 7a, b), confirming matrix formation and mineralization by the osteoblasts grown on the CaCO3 scaffolds. The mineral-to-matrix ratio, calculated as the ratio of the integrated area under the phosphate band to that of the amide I band (93), was significantly higher for the CaCO3 scaffold samples compared to the silicon substrate control samples (1.06 for CaCO3 vs 0.17 for silicon; right panel). When compared with intact bone (90), the value for the CaCO3 scaffold samples was lower, suggesting that the in vitro cell culture environment requires further optimization. However, the mineral-to-matrix ratio observed with our CaCO3 scaffolds is comparable to that reported for other artificial bone scaffolds (94). Taken together, our results show that the hierarchically porous CaCO3 scaffolds promote osteoblast differentiation and matrix mineralization.




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4. Conclusion In this work, we have presented a systematic and thorough investigation of the in vitro biological response of osteoblasts grown on CaCO3 scaffolds fabricated using an efficient, tunable, scalable and ecofriendly supercritical carbon dioxide (sc-CO2) process. The resulting scaffolds are highly porous, with inter-connected micro- and nano-pores, yet have mechanical properties (such as hardness and Young’s modulus) that are comparable to natural bone. The scaffolds possess the physicochemical properties, including topography and roughness, to promote osteoblast adhesion and proliferation, stimulate osteoblast differentiation and induce matrix mineralization. Moreover, the scaffolds can readily be coated with ECM proteins, which further enhances the biological response of osteoblasts. These findings underline the potential of the hierarchically porous CaCO3 scaffolds as biomaterials in bone regenerative medicine.


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Acknowledgements The authors thank Drs. Ina Rianasari and Vineeth Mukundan for help with the early phase of the project. The authors also thank Dr. James Weston for help with the XRD characterization of the scaffolds. The authors acknowledge NYU Abu Dhabi Core Technology Platform resources for essential equipment and technical support. This work was supported by New York University Abu Dhabi research grants for M.M. and R.J.




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Author contributions M.M. and R.J. conceived the research. A.D.W. designed and performed the experiments. S.K. and R.J. developed the CaCO3 scaffold fabrication process. S.K.S. fabricated and characterized the CaCO3 scaffolds. A.D.W. and M.M. analyzed the data and wrote the manuscript. All authors commented on the manuscript.


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Competing financial interests The authors declare that they have no competing financial interests.




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(70) Leong, M. F.; Chian, K. S.; Mhaisalkar, P. S.; Ong, W. F.; Ratner, B. D. Effect of electrospun poly(D,L-lactide) fibrous scaffold with nanoporous surface on attachment of porcine esophageal epithelial cells and protein adsorption. Journal of biomedical materials research. Part A 2009, 89, 10401048. (71) Rezania, A.; Healy, K. E. Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 1999, 17, 615-623. (72) Takada, Y.; Ye, X.; Simon, S. The integrins. Genome biology 2007, 8, 215. (73) Rabe, M.; Verdes, D.; Seeger, S. Understanding protein adsorption phenomena at solid surfaces. Advances in colloid and interface science 2011, 162, 87-106. (74) Wahlgren, M.; Arnebrant, T. Protein adsorption to solid surfaces. Trends in biotechnology 1991, 9, 201-208. (75) Ruoslahti, E. RGD and other recognition sequences for integrins. Annual review of cell and developmental biology 1996, 12, 697-715. (76) Vandrovcova, M.; Bacakova, L. Adhesion, growth and differentiation of osteoblasts on surfacemodified materials developed for bone implants. Physiological research 2011, 60, 403-417. (77) Liu, Y. O., B.R. Distinct VEGF Functions During Bone Development and Homeostasis. Arch. Immunol. Ther. Exp 2014. (78) Erlebacher, A.; Filvaroff, E. H.; Ye, J. Q.; Derynck, R. Osteoblastic responses to TGF-beta during bone remodeling. Molecular biology of the cell 1998, 9, 1903-1918. (79) Bellows, C. G.; Aubin, J. E.; Heersche, J. N. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone and mineral 1991, 14, 27-40. (80) Golub, E. E.; Harrison, G.; Taylor, A. G.; Camper, S.; Shapiro, I. M. The role of alkaline phosphatase in cartilage mineralization. Bone and mineral 1992, 17, 273-278. (81) Shafiu Kamba, A.; Zakaria, Z. A. Osteoblasts growth behaviour on bio-based calcium carbonate aragonite nanocrystal. BioMed research international 2014, 2014, 215097. (82) Owen, T. A.; Aronow, M.; Shalhoub, V.; Barone, L. M.; Wilming, L.; Tassinari, M. S.; Kennedy, M. B.; Pockwinse, S.; Lian, J. B.; Stein, G. S. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. Journal of cellular physiology 1990, 143, 420-430. (83) Degasne I, B. M., Demais V, Huré G, Lesourd M, Grolleau B, et al. . Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces. Calcif.Tissue Int. 1999, 499-507. (84) Bizios, R. Mini-review: Osteoblasts: An in vitro model of bone-implant interactions. Biotechnology and bioengineering 1994, 43, 582-585. (85) Schiller PC, D. I. G., Balkan W, Roos BA, Howard GA. Gap-junctional communication mediates parathyroid hormone stimulation of mineralisation in osteoblastic cultures. Bone 2001, 28, 38–44. (86) Ohgushi, H.; Dohi, Y.; Katuda, T.; Tamai, S.; Tabata, S.; Suwa, Y. In vitro bone formation by rat marrow cell culture. Journal of biomedical materials research 1996, 32, 333-340. (87) Coelho, M. J.; Fernandes, M. H. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000, 21, 1095-1102. (88) Prabhakaran, M. P.; Venugopal, J.; Ramakrishna, S. Electrospun nanostructured scaffolds for bone tissue engineering. Acta biomaterialia 2009, 5, 2884-2893. (89) D. Depan, a. T. C. P. a. R. D. K. M. The synergistic effect of a hybrid graphene oxide–chitosan system and biomimetic mineralization on osteoblast functions. Biomaterials Science 2014, 2, 264. 26 ACS Paragon Plus Environment

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(90) Bonewald, L. F.; Harris, S. E.; Rosser, J.; Dallas, M. R.; Dallas, S. L.; Camacho, N. P.; Boyan, B.; Boskey, A. von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcified tissue international 2003, 72, 537-547. (91) Nauman, E. A.; Ebenstein, D. M.; Hughes, K. F.; Pruitt, L.; Halloran, B. P.; Bikle, D. D.; Keaveny, T. M. Mechanical and chemical characteristics of mineral produced by basic fibroblast growth factor-treated bone marrow stromal cells in vitro. Tissue engineering 2002, 8, 931-939. (92) Renno, A. C.; Bossini, P. S.; Crovace, M. C.; Rodrigues, A. C.; Zanotto, E. D.; Parizotto, N. A. Characterization and in vivo biological performance of biosilicate. BioMed research international 2013, 2013, 141427. (93) Takata, S.; Yonezu, H.; Shibata, A.; Enishi, T.; Sato, N.; Takahashi, M.; Nakao, S.; Komatsu, K.; Yasui, N. Mineral to matrix ratio determines biomaterial and biomechanical properties of rat femur-application of Fourier transform infrared spectroscopy. The journal of medical investigation : JMI 2011, 58, 197-202. (94) Raynaud, S.; Champion, E.; Bernache-Assollant, D.; Thomas, P. Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders. Biomaterials 2002, 23, 1065-1072.




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Figure legends Figure 1. Schematic diagram illustrating the supercritical carbon dioxide (Sc-CO2) process used for fabrication of hierarchically porous CaCO3 scaffolds. An aqueous solution of calcium acetate was mixed in a small volume with a continuous stream of carbon dioxide (sc-CO2) under supercritical conditions. The mixture was passed through a heat exchanger and then allowed to expand at atmospheric pressure through capillary tubing. The continuous collection of the aerosol onto heated silicon substrates resulted in the simultaneous release of dissolved CO2 and evaporation of water. The described method does not require any sacrificial templates to create three-dimensional scaffolds. Figure 2. Characterization of the hierarchically porous CaCO3 scaffolds. (a) Scanning electron microscopy (SEM) images of the CaCO3 scaffolds over scan areas in the 250 µm (scale bar = 50 µm) (left panel), 60 µm (scale bar = 20 µm) (middle panel), and 3 µm (scale bar = 1 µm) (right panel), ranges. (b) Powder XRD pattern of the CaCO3 scaffolds (left panel). Reflections at values of 2θ = 23, 29.5, 31.8, 36.1, 39.5, 43.2, 47.2, 47.6, 48.6, 56.6, 57.5, 60.7, 64.7 and 65.7° correspond to the (012), (104), (006), (110), (113), (202), (024), (018) and (116) lattice planes of the calcium carbonate polymorph, calcite, respectively. Energy dispersive X-ray spectroscopy (EDS) analysis of the CaCO3 scaffolds (right panel). (c) Typical pore size distribution of the CaCO3 scaffolds as a function of deposition time (t = 5, 10, 15 and 20 min). (d) Mechanical properties of the CaCO3 scaffolds. Typical load-displacement curve (left panel), hardness (middle panel), and Young's modulus (right panel), of the CaCO3 scaffolds. (e) Topographical AFM images and average roughness (Ra) values of the silicon substrate (Ra = 0.107 nm, top left panel; Ra = 1.023 nm, lower left panel), CaCO3 scaffolds (Ra = 1.95 nm, top middle panel; Ra = 20 nm, lower middle panel), and glass substrate (Ra = 1.12 nm, top right panel; Ra = 2.34 nm, lower right panel). Scan area = 5 µm × 5 µm (top panels) and 20 µm × 20 µm (lower panels); scale bar = 2 nm (top panels) and 5 µm (lower panels). Figure 3. Immunogenicity and ECM protein adsorption capacity of the hierarchically porous CaCO3 scaffolds. (a) Levels of inflammatory cytokine tumor necrosis factor-alpha (TNF-α) produced by differentiated human monocytic leukemia THP-1 cells exposed to uncoated CaCO3 scaffolds or silicon substrates for 48 h. Cells treated with lipopolysaccharide (LPS) were used as a positive control, while cells cultured on cell culture glass surface (Nunc Thermanox Coverslips) served as a negative control. TNF-α levels in the culture medium were assayed using a commercial ELISA kit. (b,c) Adsorption of extracellular matrix (ECM) proteins to the CaCO3 scaffolds. (b) Pre-wetted scaffolds and substrates were incubated in 0-20 µg/mL solutions of type I collagen (CL, left panel), vitronectin (VN, middle panel) or fibronectin (FN, right panel) for 24 h at 4 °C. Adsorbed ECM proteins were recovered using a 1% (w/v) sodium dodecyl sulfate (SDS) solution, and protein concentrations were determined by the colorimetric Bradford Protein assay. (c) Representative SDS-PAGE of the ECM proteins


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recovered in (b) stained with Coomassie Blue. Lanes: Mr, protein ladder; I, silicon substrate; II, CaCO3 scaffold; III, cell culture glass substrate. *, p < 0.01; ***, p < 0.0001; non-significant (ns), p > 0.05 compared with silicon substrate controls. Figure 4. Osteoblast adhesion efficiency, morphology and proliferation. (a) Adhesion of osteoblast MC3T3 cells to the uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds and silicon substrates at 24 h following seeding. Adhesion efficiency was quantified using the CellTiter 96 AQueous One Solution (MTS) assay. Cells grown on cell culture glass (Nunc Thermanox Coverslips) were used as control, and wells with medium alone served as a blank. (b) Confocal fluorescence microscopy images of MC3T3 cells grown on uncoated and ECM protein-coated hierarchically porous CaCO3 scaffolds and control substrates (silicon and cell culture glass). Cells were cultured for 3 days, then fixed, permeabilized and stained with 10 µg/mL fluorescein isothiocyanate labeled phalloidin (FITC-phalloidin) and 5 µg/mL Hoechst 33342. Scale bar = 50 µM. (c) Proliferation of MC3T3 cells cultured on uncoated (left panel) and ECM protein-coated (middle and right panels) hierarchically porous CaCO3 scaffolds and silicon substrates over 21 days. Adhesion efficiency was quantified using the CellTiter 96 AQueous One Solution (MTS) assay. Cells grown on cell culture glass were used as control, and wells with medium alone served as a blank. (d) Total cellular protein content of MC3T3 cells grown on uncoated (left panel) and ECM protein-coated (right panel) scaffolds and control substrates. At the indicated time-points, the cells were harvested, lysed, and the total protein concentration was determined using the colorimetric Bradford Protein assay. *, p < 0.01; ***, p < 0.0001; non-significant (ns), p > 0.05 compared with silicon substrate controls. Figure 5. Growth factor production by osteoblasts cultured on the hierarchically porous CaCO3 scaffolds. Osteoblast MC3T3 cells were grown on (a) uncoated, or (b) ECM protein-coated, CaCO3 scaffolds and control substrates (silicon and Nunc Thermanox Coverslips). The culture medium was collected from uncoated samples (on days 3, 7 and 14) and ECM protein-coated samples (on day 14) and assayed for vascular endothelial growth factor-A (VEGF-A, left panels) and transforming growth factor-beta (TGF-β, right panels) levels using commercial ELISA kits. ***, p < 0.0001; non-significant (ns), p > 0.05 compared with silicon substrate controls. Figure 6. Alkaline phosphatase (ALP) activity of osteoblasts cultured on the hierarchically porous CaCO3 scaffolds. Osteoblast MC3T3 cells were grown on (a) uncoated, or (b) ECM proteincoated, CaCO3 scaffolds and control substrates (silicon and Nunc Thermanox Coverslips). Cell lysates were harvested from uncoated samples (on days 3, 7 and 14) and ECM protein-coated samples (on day 14) and lysed. ALP activity in the lysates was measured with a colorometric assay that uses pnitrophenyl phosphate (pNPP) as a phosphatase substrate, and normalized to the total cellular protein




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determined using the Bradford Protein assay. ***, p < 0.0001; non-significant (ns), p > 0.05 compared with silicon substrate controls. Figure 7. Matrix mineralization by osteoblasts cells cultured on the hierarchically porous CaCO3 scaffolds. (a) Alizarin Red S (ARS) staining of calcium deposition by osteoblast MC3T3 cells grown on silicon substrate, uncoated CaCO3 scaffolds and glass substrate (Nunc Thermanox Coverslips) for 14 days (left panels). Scale bar = 200 µm. The number of mineralized nodules (examples indicated by yellow arrows) in the scaffold and substrate samples was quantified on day 14 (right panel). (b) Quantification of calcium deposition by cetylpyridinium chloride (CPC) extraction from ARS-stained cells grown for the indicated durations on uncoated (left panel), or ECM protein-coated (right panel), CaCO3 scaffolds and control substrates (silicon and cell culture glass). (c) Fourier transform infrared spectroscopy (FTIR) analysis of lysates of MC3T3 cells cultured on uncoated scaffolds and control substrates (silicon and cell culture glass) for 14 days (left panel). Arrows at 900–1200 cm−1 represent phosphate and mineralization peaks, while arrowheads at 1590–1700 cm−1 represent amide I, II and III peaks. Mineral-to-matrix ratio, calculated as the ratio of the integrated area under the phosphate band to that of the amide I band, of the scaffolds and control substrates at day 14 (right panel). ***, p < 0.0001; non-significant (ns), p > 0.05 compared with silicon substrate controls.


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Silicon

CaCO3

Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue

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