Enhanced Osteogenesis of Injectable Calcium Phosphate Bone

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Bio-interactions and Biocompatibility

Enhanced Osteogenesis of Injectable Calcium Phosphate Bone Cement Mediated by Loading Chondroitin Sulfate Haishan Shi, Xiaoling Ye, Jing Zhang, and Jiandong Ye ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00871 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 22, 2018

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ACS Biomaterials Science & Engineering

Enhanced Osteogenesis of Injectable Calcium Phosphate Bone Cement Mediated by Loading Chondroitin Sulfate Haishan Shi,1,2,3‡ Xiaoling Ye,1,2‡ Jing Zhang1,2 and Jiandong Ye1,2,4* 1School

of Materials Science and Engineering, South China University of Technology,

Guangzhou 510640, China. 2National

Engineering Research Center for Tissue Restoration and Reconstruction, South China

University of Technology, Guangzhou 510006, China. 3College

4Key

of Chemistry and Materials Science, Jinan University, Guangzhou 510632, China.

Laboratory of Biomedical Materials of Ministry of Education, South China University of

Technology, Guangzhou 510006, China. ‡The

first two authors contribute equally to this work.

*Corresponding

author: Jiandong Ye, [email protected].

KEYWORDS: calcium phosphate cement, chondroitin sulfate, injectability, osteogenesis

ACS Paragon Plus Environment

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ABSTRACT: Towards repairing critical-sized bone defects, calcium phosphate cement (CPC) has been well recognized as a fairly promising bone graft owing to its properties of injectability, selfsetting, biocompatibility and osteoconductivity. However, poor osteogenic capacity of CPC still limits its applications for meeting the demands of bone healing. In this work, chondroitin sulfate (CS), as an important component of the extracellular matrix network, was introduced into CPC to enhance its osteogenesis ability. Incorporation of CS had no evident effect on the phase, morphology, apparent porosity and compressive strength of hydrated cement products, but notably enhanced the injectability and improved the anti-washout property of the cement pastes. CS was able to be sustainedly released from CS-CPCs in a CS-dose-dependent manner and supposed to have a long-term release potential for constant biological stimulation. CS-CPCs markedly accelerated the preferential adsorption of fibronectin. Furthermore, CS-CPCs significantly improved the adhesion, proliferation and osteogenic differentiation of bone mesenchymal stem cells, which was synergistically mediated by the adhesion events of cells on the hydrated cements and the stimulation effects of CS molecules. Herein, utilization of CS is supposed to endow injectable calcium phosphate bone cements with enhanced osteogenic capacity and suitable physicochemical properties for numerous promising orthopedic applications.

1. INTRODUCTION Critical-sized bone defects caused by sports, pathological injury or trauma have raised significant clinical challenges around the world.1-2 There have been numerous major advances in the development of calcium phosphate based materials which are utilized to augment the bone repair.3-5 For instance, calcium phosphate ceramics in the form of granules or porous scaffolds show the capacity of stimulating the osteogenic differentiation of bone mesenchymal stem cells


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(BMSCs) in vitro and bone regeneration of critical-sized defects in vivo.3-5 However, the ceramic granules can easily migrate into the surrounding tissue after surgery, while the porous scaffolds cannot completely fill in the defects.6,7 This leads to limited bone-implant bonding and poor osseointegration. Moreover, shape nonconformity of the fabricated implants to the defect sites can increase bone loss, weaken bone regeneration, cause trauma to the surrounding tissue, and prolong surgery time. In response to these issues, with the emerging of minimally invasive surgery techniques, biomaterials with self-setting characteristics have been studied for clinical applications.7-9 Due to a moldable paste-like consistency, calcium phosphate cement (CPC), which can harden and fit into the defect site in situ, is an alternative to other bone filling materials.8-10 CPC has good liquidity and can be used as a binder to fill the peri-implant defects.11,12 CPC attracts much interest also due to its potential tissue compatibility and excellent osteoconductivity.13-15 Utilization of CPC can address the issues such as interfacial loosening commonly seen in the application of polymethylmethacrylate (PMMA) based bone cement to some extent.16 Nonetheless, CPC still lacks osteoinductivity and osseointegration ability without a good balance of tissue ingrowth and biodegradation/resorption, which will easily decrease the implant stability, bone-implant contact and bone remodeling.17-18 In some cases, CPC materials did not allow repair of the critical-size defects which were generally filled with connective fibrous tissue, whilst the materials were almost totally resorbed.17-19 At present, the trend in developing a new generation of biodegradable materials demands good osteogenesis and integration capacity.20 CPC-based minimally invasive systems can be beneficial for the delivery of drugs or factors when used in specific conditions that best serve the purpose of bone repair.2123

The aforementioned limitations of CPC can be overcame by loading growth factors or other

factor-like substances into the cement matrix to enhance new bone formation and substitution.23-


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25 However,

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attempts to exploit growth factors have always failed in vivo because of the short

half-life time and the high diffusibility of these factors in situ.26 Inspirations of endochondral ossification perhaps can provide a new perspective for the development of alternative options to growth factors.27,28 Chondroitin sulfate (CS), a sulfated glycosaminoglycan, is mainly found in cartilage and bone as a structural component of the extracellular matrix (ECM) network.29-31 CS can mediate cellular adhesion, migration, proliferation and differentiation mainly by incorporation with fibronectin, cytokines and growth factors.32-34 CS, as one of the non-collagenous proteoglycans, also plays a crucial role in the regulation of biological mineralization of cartilage and bone tissues through binding calcium ions and promoting apatite formation.35-37 Numerous promising results which employed CS as part of bone substitutes had been demonstrated in various animal models.38-43 Incorporation of CS into bone cements within critical-sized bone defects had led to enhancements of bone remodeling and increases of new bone ingrowth.40-42 However, for potential surgical operations, the effects of incorporation of CS on the physicochemical properties of injectable bone cements are not fully understood. The further cytological molecular mechanisms leading to the in vivo stimulation effects of CS-CPC also remain unclear. Furthermore, the release behavior of CS from CS-CPC, regarded as a good CS carrier, are not yet determined to verify a promising long-term effect of CS-CPC in bone regeneration. In this work, CS was introduced into CPC as an alternative bioactive factor-like substance to improve the osteogenic capacity along with appropriate physicochemical properties. With the addition of CS, the phase, component, morphology, apparent porosity and compressive strength of the hydrated cements, as well as the setting time, injectability and anti-washout performance of the cement pastes were systematically investigated. The behaviors of the in vitro CS release,


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specific proteins adsorption and cellular response of CS-CPC were also unraveled. Herein, an appropriate content of CS is supposed to have a predicted important effect on the physicochemical properties and osteogenic activity of CPC, which will provide a reference for developing novel bone cements with great performances. 2. MATERIALS AND METHODS 2.1 Preparation procedure Calcium phosphate cement raw materials comprised of solid phase and setting liquid. The solid powder consisted of α-Ca3(PO4)2 (α-TCP), CaHPO4 (DCPA, monetite), CaCO3 (CC, calcite) and Ca10(PO4)6(OH)2 (HA) at a mass ratio of 60:25:10:5. The setting liquid contained 250 mM Na2HPO4 and 250 mM NaH2PO4. The α-TCP powder was prepared by calcining a mixture of CaHPO4•2H2O (DCPD, brushite) and CC with a molar ratio of 2:1 at 1350ºC for 2 h. The DCPA powder was prepared from DCPD through ball-milling for 2 h and then air drying at 120ºC for 12 h. The HA powder was prepared by a chemical precipitation method. In brief, Ca(NO3)2 solution was added into (NH4)2HPO4 solution dropwise at a Ca/P molar ratio of 1.67:1 and stirred at 25ºC for 12 h with adjusting pH 10-12. The precipitate was then obtained, centrifuged, rinsed, air dried and calcinated at 1000ºC for 2 h. Afterwards, the as-calcinated HA powder was ball-milled for 8 h and air dried for 24 h. All the used chemicals were analytical reagents (Guangzhou Chemical Reagent Factory, China). CS-loaded CPC samples (CS-CPCs) were prepared by mixing solid powders with the setting liquids containing different contents of CS (Mw: ~5 × 104 g/mol, ≥98%, Qiyun Biotechnology, China) at a liquid-to-powder ratio of 0.6 mL/g. Afterward, cement pastes were obtained and then injected into cylinder molds (φ6 mm × 12 mm) and disk molds (φ6 mm × 1.5 mm; φ10 mm × 1.5


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mm). Sequentially, the cement columns and disks were demolded, incubated at 37ºC and 98% relative humidity for 3 days, and air dried at 37ºC for 24 h. These products were obtained and used as the hydrated cements for the subsequent tests unless otherwise specified. These CS-CPCs were labelled as CPC-con, CPC-0.5CS, CPC-1.0CS, CPC-1.5CS and CPC-2.0CS with CS contents of 0, 0.5, 1.0, 1.5 and 2.0 wt.% of the hydrated cements, respectively. 2.2 Materials characterization Phases of the hydrated cement products were analyzed by an X-ray diffractometer (XRD; Empyrean, PANalytical, Netherlands) using CuKα radiation (λ = 1.5418 Å). Data were collected for 2θ (10º-60º) at a step size of 0.013º. Fourier-transform infrared spectra (FTIR) were recorded by an infrared spectroscope (Nicolet iS10, Thermo Scientific, USA) using samples in the form of pellets formed with spectroscopic grade potassium bromide (Sigma Aldrich, USA). Micromorphology of the cross-sections of the hydrated CS-CPCs was analyzed by a fieldemission scanning electron microscope (FESEM; NOVA NanoSEM 430, FEI, USA). Compressive strength of the hydrated cement columns (Φ6 mm × 10 mm) was tested using a universal mechanical testing machine (Instron 5967, INSTRON, USA) pursuant to ISO13779-1. Each test was repeated six times. Apparent porosity of the hydrated cement columns (Φ6 mm × 10 mm) was measured using the Archimedes method. In brief, the cement columns were initially weighted as W0, then put into pycnometers that were weighed as W1 when full of ethanol, and vacuumized for 2 h. Afterwards, the pycnometers with cement columns were weighed as W2 with ethanol filling up again. The columns were then taken out, wiped by filter papers steeping with ethanol, and weighed as W3. The apparent porosity was calculated by the Equation 1. Each test was repeated six times.


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Porosity  %  

W3 - W0 100 W1  W3 - W2

(Equation 1)

Setting time of the cement pastes was determined by a Gilmore apparatus pursuant to ASTM C266-13. The Gilmore apparatus included an initial setting indenter (113.4 ± 0.5 g, Φ2.12 ± 0.05 mm) and a final setting indenter (453.6 ± 0.5 g, Φ1.06 ± 0.05 mm). After mixing the solid powder with setting liquid, the paste was transferred into a mold (φ15 × 8 mm) and placed at 37ºC and 98% relative humidity. The indenters were lowered vertically on the cement surface for 5 s to determine the setting time at intervals of 60 s until the paste was hardened. Initial and final setting occurred when there was no a complete cyclic penetration using corresponding indenters. Each test was repeated six times. Injectability of the cement pastes was measured using a syringe (φ12.5 mm) which was fitted with a round needle (φ2 mm). After mixing the solid powder with setting liquid, the paste was transferred into the syringe and loaded vertically with 5 kg force for 120 s. The injectability of the cement was calculated as the mass percentage of the paste extruded from the syringe. Each test was repeated thrice. Anti-washout property of the cement pastes was measured by a shaking method. After mixing the solid powder with setting liquid, the paste was immediately injected into the deionized water, and then placed in a shaker (120 rpm) at 37ºC until the paste was hardened. The anti-washout property of the paste was determined if the paste did not visibly disintegrate during shaking. Optical appearance of the cements was photographed by a digital camera (D5100, Nikon, Japan). 2.3 In vitro CS release test of CS-CPCs


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Hydrated cement columns (Φ6 mm × 10 mm) were immersed in PBS (pH 7.4, Toscience, China) at a column’s superficial area to PBS’s volume ratio of 0.5 cm2/mL and transferred in a shaker (120 rpm) at 37ºC for 14 days. The immersion liquids were collected and replenished every other day. The CS concentration of collected liquids was tested by the dimethylmethylene blue (DMMB) colorimetric assay. In brief, the DMMB working solution was prepared by dissolving sodium chloride (40 mM), glycine (40 mM) and DMMB (46 µM) in aqueous solution at pH 3.0. The immersion liquids were mixed with the DMMB working solution with a certain ratio at 25ºC for 5 min according to the instructions. The absorbance at 525 nm was read using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific, USA) referenced to a standard curve of CS. Each experiment was repeated thrice. 2.4 In vitro specific adsorption of fibronectin (FN) on CS-CPCs Hydrated cement disks (Φ6 mm × 1.5 mm) were immersed in PBS containing 10 vol.% fetal bovine serum (FBS; Gibco, USA) at 37ºC for 2 h. Then, the samples were rinsed thrice to remove residual proteins, and blocked by a PBS solution containing 5 wt.% bovine serum albumin (BSA; Sigma Aldrich, USA) at 4ºC for 2 h. Subsequently, these samples were incubated with rabbit monoclonal to fibronectin antibody (Abcam, USA) at 4ºC overnight, and stained with Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech Group, China) for 1 h. Samples were rinsed with PBS thrice and observed by a confocal laser scanning microscope (CLSM; TCS SP5, Leica Microsystems, Germany). 2.5 Cell culture BMSCs (CRL-12424, ATCC, USA) were cultured with culture media in a incubator at 37ºC with 95% relative humidity and 5% CO2 atmosphere. The culture medium containing 10 vol.% FBS


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(Gibco, USA) and 90 vol.% high-glucose DMEM (Gibco, USA), was replenished every two days. Cells were digested with 0.25 wt.% trypsin solution (Gibco, USA) and collected for the subsequent cellular experiments. 2.6 Cellular seeding, attachment, viability and proliferation Hydrated cement disks were sterilized by Co60 gamma radiation. Cells were seeded onto the cement disks (Φ6 mm × 1.5 mm) at an initial density of 2 × 104 cells per well. After being cultured for 24 h, cells were fixed with 2.5 vol.% glutaraldehyde solution, dehydrated with gradient alcohol (30, 50, 70, 80, 90, 95 and 100 vol.%, respectively), and air dried at 25ºC. Morphology of these cells were observed using the FESEM. After 3 days of culture, cells were slowly rinsed with PBS, and double-stained with Calcein-AM (Biotium, USA) and propidium iodide (Biotium, USA). The stained cells were observed by an inverted fluorescence microscope (Eclipsc Ti-U, Nikon, Japan). Cellular viability was assessed by a CCK-8 assay (Dojindo, Japan). After 1, 3 and 5 days, the samples were sucked out, transferred into a new plate, slowly added with CCK-8 working solution and placed at 37ºC for 1 h. The absorbance was recorded at 450 nm by the microplate reader. 2.7 Immunofluorescence microscopy Cells were seeded onto the cement disks (Φ6 mm × 1.5 mm) at an initial density of 2 × 104 cells per well. After 24 h, cells were rinsed with PBS and fixed with 4.0 vol.% formalin. The fixed cells were successively incubated with 5 wt.% BSA solution at 4ºC for 2 h and 0.1 vol.% Triton X-100 (Sigma Aldrich, USA) at room temperature for 5 min. Subsequently, focal adhesion (FA) contact was identified by vinculin staining by anti-vinculin antibody (Abcam, USA) and Cy3conjugated Affinipure Goat Anti-Rabbit IgG (H+L), while integrin α5 was identified using anti-


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integrin α5 antibody (Abcam, USA) and the Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (H+L). Actin microfilaments and nuclei were stained by Alexa-Fluor® 488 phalloidin (AAT Bioquest, USA) and DAPI (Beyotime, China), respectively. Immunofluorescence images of cell nuclei (blue), cytoskeleton arrangement (green), FA (red) and integrin α5 distribution (red) of BMSCs were captured by the CLSM. Cellular area and FA area on the CS-CPCs disks were statistically analyzed with software ImageJ 1.50g (National Institutes of Health, NIH, USA). More than 200 viable cells were used for the statistical analyses. 2.8 Real-time quantitative PCR (RT-qPCR) Gene expressions of cellular adhesion and osteogenic differentiation were quantitatively analyzed using the RT-qPCR method. For cellular adhesion-related gene expression, cells were seeded onto cement disks (Φ10 mm × 1.5 mm) at an initial density of 1 × 105 cells per well. After being cultured for 24 h, the RNA was extracted with HiPure Total RNA Micro Kit (Magen, China) and then reverse-transcribed into cDNA with iScript cDNA Synthesis Kit (Bio-Rad, USA). PCR analysis was performed using SYBR Green detection reagent (iQTM SYBR Green Supermix, Bio-Rad, USA) to examine the gene expression levels of integrin α5, integrin β1 and β-actin. The relative gene expression was normalized against that of the housekeeper gene (βactin). Data was finally calculated using the formula 2−(normalized average Ct). For osteogenic differentiation-related gene expression, cells were seeded onto the cement disks (Φ10 mm × 1.5 mm) in the osteogenic media at an initial density of 5 × 104 cells per well. The osteogenic medium contained cell culture medium supplemented with 50 mg/L ascorbic acid (Sigma Aldrich, USA), 10 mM β-glycerol phosphate (Merck Millipore, USA) and 100 nM dexamethasone (Sigma Aldrich, USA). The medium was refreshed every two days. After 7 and


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14 days, the PCR was conducted to examine the gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), collagen I (Col1), osteocalcin (OCN), osteopontin (OPN) through the aforementioned procedure. GAPDH was employed as the housekeeper gene. The primer sequences are summarized in Table S1. 2.9 Statistical analysis Cellular experiments were repeated four times unless specified otherwise. The data were expressed as "means ± SD" for all the above experiments. Statistical analyses were performed by one-way analysis of variance. Differences of the data were compared with Tukey's test, and P value of < 0.05 was considered to be significant. 3. RESULTS AND DISCUSSION 3.1 Phase, morphology, physicochemical properties and CS release of CS-CPCs XRD patterns and FTIR spectra and SEM images of the cross-sections of the hydrated cements are shown in Figure 1. Phases of the hydrated CS-CPCs were similar to that of CPC-con. Main diffraction peaks of all the hydrated cement products belonged to typical HA (PDF#09-0432). Small amounts of the unhydrated raw materials, α-TCP (PDF#29-0359) and CC (calcite, PDF#83-0578), were also found, whilst freely soluble DCPA was hardly detected. Herein, DCPA was supposed to completely convert into the final products during the hydration process. A bit of impurity, β-tricalcium phosphate (β-TCP; PDF#09-0169), was derived from impure synthetic α-TCP powder (Figure S1). With the increment of CS content, phases of the hydrated cement products had no evident changes. Characteristic vibration bands of phosphate groups (PO43-) were found for all the hydrated cement products. Three vibration bands at around 875,


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1415 and 1460 cm-1 were attributed to carbonate groups (CO32-). It was of indicative that the main phase component of the hydrated products was type B carbonated hydroxyapatite (B-CHA) which contained carbonate species replacing the phosphate groups.44 In addition, incorporation of CS did not significantly affect the components of the hydrated cement products. Lamellar crystals were clearly observed in all the cement matrix, indicating that addition of CS also did not notably affect the morphology of the hydrated cement products. Besides, great amounts of nanopores were observed in the cement matrix, providing good carriers for the controlled delivery systems of drugs or other molecules. Chains of CS also could combine with calcium ions.36 In this regard, CS molecules could be left in the confined space of the hydrated cement products and filled the micro-holes left by the liquid phase after hydration. As a result, a relatively denser matrix of CS-CPCs formed compared with that of CPC-con. Compressive strength, apparent porosity, setting time, injectability and CS kinetic release curves of CS-CPCs are presented in Figure 2. The compressive strength of the hydrated CPCs was comparable with each other without any effect of CS content. With the increasing CS content, the apparent porosity of the hydrated cements slightly increased ranging from ~55% to ~60%, whereas the setting time of the pastes was notably prolonged. Initial and final setting times were even extended from 5 ± 1 and 35 ± 5 min to 7 ± 2 and 53 ± 3 min as CS content increased, respectively. Incorporation of CS also markedly improved the injectability of the cement pastes from 38.67 ± 3.45% (CPC-con) to 90.22 ± 2.74% (CPC-2.0CS) in a contentdependent manner, which facilitated the clinical applications of CPCs in a surgical procedure. As mentioned above, CS molecules could attach on the surface of raw powders, binding with calcium/phosphate ions, which inhibited the setting behaviors of the cement pastes to some extent. In addition, the presence of CS molecules in the intergranular interface reduced the


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frictional resistance among the particles derived from the rough surfaces, and thus improved the flowability of cement pastes. In some perspective, CS played a role of water reducer in the setting process of the cement pastes. Owing to the high porosity, CPC was suitable to be employed as a good CS carrier. As immersed in PBS, CS was released from the hydrated CSCPCs products in a CS-content-dependent manner. Besides, according to the sustained release content of CS of all the hydrated cement products, CS would continue to be steadily released with the degradation of the cements in the subsequent immersion time, which provided a longterm potential of CS release for the constant stimulation of biological response. Anti-washout property of the cement pastes was determined by their appearance integrity after injected into the water as given in Figure 3. Thanks to the good injectability, CS-CPCs had good continuous shapes in contrast to the intermittent paste of CPC-con when injected in the water. With the time prolonged, all the cement pastes disintegrated to a variable extent and parts of them were stripped after the shaking cycles. Compared with the obvious disintegration of CPCcon paste, CS-CPC pastes showed more complete appearances. With the increase of CS content, the cement pastes became more stable in the liquids. It might well be contributed by the chemical chelation of a large number of carboxyl groups of CS molecules with Ca2+ of cement matrix. Nevertheless, CPC-2.0CS paste collapsed more distinctly compared with other CS-CPC pastes. It was supposed that the delayed setting behavior induced by CS addition was harmful to the shape preserving in the face of washing out. Besides, a great amount of CS also broke the combination of solid powders when the paste contacted with immersion liquids, which was similar to the capillarity phenomenon. Thus, the addition of CS with a certain content enhanced the antiwashout property of the cement pastes. 3.2 Fibronectin adsorption behaviors and cellular response of CS-CPCs


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Protein adsorption is the first step reaction between cells and biomaterials, which manipulate subsequent cellular responses including adhesion, spreading, migration, growth, proliferation and differentiation.45,46 In this regard, fibronectin plays a major role in initial cellular adhesion and subsequent biological events. SEM images and fluorescence photographs of BMSCs adhered on the hydrated cement products as well as immunofluorescence photographs of FN adsorbed on the cements are shown in Figure 4. Cells adhered tightly with good spreading shape on the surface of all the hydrated cement products, indicating good cellular affinity of these hydrated cement products. In addition, more viable BMSCs adhered onto CS-CPCs and exhibited affluent pseudopods, compared to those on CPC-con. Specific marker of FN adsorbed onto the hydrated cements typically revealed the adsorption of specific proteins closely associated with cellular adhesion.47,48 As CS content increased, the adsorption amount of FN significantly increased. It was indicated that addition of CS notably enhanced the preferential adsorption of specific proteins in favor of cellular adhesion on the hydrated cements. In particular, as CS content increased to 2.0 wt.%, a lower FN adsorption amount on the products was detected compared to that on CPC-1.5CS. As discussed above, high CS content could result in the poor integrity of cement pastes, which left delicate cement surfaces and was not helpful for the proteins adsorption. For the further illumination of initial cellular adhesion and subsequent biological events, we also characterized the vinculin secretion (FA formation) and integrin α5β1 gene expression of BMSCs on the hydrated cements. Figure 5 gives immunofluorescence images of FA formation (vinculin) and integrin α5 expression of BMSCs on hydrated CS-CPCs, respectively. Meanwhile, nuclei and cytoskeleton filaments of cells were raveled by DAPI and Alexa-Fluor 488 phalloidin, respectively. Quantitative analysis of areas of cells and FA contacts, as well as qPCR evaluation


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of cellular adhesion related gene expression (integrin α5β1) of BMSCs were further performed. Cells spread well and exhibited spindle on the cements products. Moreover, cells expressed more cytoplasmic extensions accompanied with more attachment points and pseudopodia on CS-CPCs compared with CPC-con. With CS content increasing, areas of cells on CS-CPCs gradually and significantly increased, but slightly decreased for CPC-2.0CS. It might well be positively correlated with the specific adsorption of FN on the hydrated cements. FA contact areas of cells on CS-CPCs were also distinctly greater compared with those on CPC-con, showing a good cellular adhesion state. From the fluorescence observation, relatively higher secretion of integrin α5 of cells was found on CS-CPCs than that on CPC-con. Relative to an indistinctive gene expression of integrin α5 of cells on all the hydrated cements, markedly higher integrin β1 mRNA expression was detected for CS-CPCs compared to CPC-con. Results indicated that the cells exhibited a better spreading morphology and adhesion capacity on the hydrated cements with the incorporation of CS. Proliferation and osteogenic differentiation-related gene expression of BMSCs on the hydrated cements are given in Figure 6. Cells presented stable proliferation ability on all the hydrated cements as culture time prolonged. In addition, CS-CPCs stimulated notably a greater proliferation capability of BMSCs than CPC-con and showed dependence with the CS content. It was suggested that incorporation of CS into the cements was beneficial to the proliferation of BMSCs. BMSCs can differentiate into osteoblast in vitro and in vivo. In the bone formation process, osteoblast is the essential functional cell that is accountable for the synthesis, secretion and biomineralization of bone matrix.49,50 Hence, osteogenic differentiation of BMSCs is one of the primary processes for bone regeneration.51,52 Compared with CPC-con, CS-incorporated products contributed slightly to gene expressions of Runx2, Col1 and OCN in BMSCs, but


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significantly up-regulated the mRNA expressions of ALP and OPN in a CS-content-dependent manner. ALP mRNA expression was directly related to ALP activity, which is well known to be the obvious osteogenic mark of osteogenic differentiation ability of BMSCs. In addition, OPN expression were correlated with cellular adhesion behaviors.53 As one of the primary components of ECM network, CS could affect the immobilization of soluble molecules (e.g. growth factors and other cytokines) on the surface of the implants, and interact with bone cells (e.g. osteoblast and osteoclast) via integrins or other specific receptors, which directly/indirectly influenced the adhesion, migration, growth, proliferation and differentiation of these cells.54-56 Herein, the adhesion capacity of BMSCs enhanced by incorporation of CS provided evidence for the upregulation of OPN expression in BMSCs to some extent. It was of indicative that CS addition improved the osteogenic differentiation ability of BMSCs through mediating the cellular adhesion events. In consideration of sustained CS release during cellular culture, the effect of CS itself on cellular response was discussed. In this regard, a certain amount of CS was directly added in the culture media and co-cultured with BMSCs. CS could significantly improve the proliferation and osteogenic differentiation of BMSCs as directly co-cultured with the cells (Figure S2). Results showed that the addition of CS into CPC could enhance osteogenic differentiation of BMSCs, which was synergistically attributed to the enhanced cellular adhesion on the hydrated cement and the stimulation effects of CS molecules. This study concentrated on exploring the cytological response and osteogenic activity of CSCPC. Further investigations are needed to be carried out in vivo. Specifically, the release of CS would be of great importance because there are several ways with regard to the in vivo delivery of CS during new bone formation. To be best of the authors’ knowledge, at present, clinical applications of non-protein factor-like compounds in injectable bone cement are yet to be


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reported. The future work will incorporate animal models to validate the positive responses of BMSCs, which would be of help for clinical trials. 4. CONCLUSIONS Chondroitin sulfate, an important component of the ECM network, was introduced into CPC to improve the osteogenesis activity. Incorporation of CS had no evident effect on the phase, morphology, apparent porosity and mechanical strength of the hydrated cements, whilst notably prolonged the setting time of the cement pastes and enhanced corresponding injectability. Addition of CS with a certain amount also enhanced the anti-washout property of the cement pastes. Because of its high porosity and good self-setting characteristics, CPC was suggested as a suitable CS carrier. CS could be sustainedly released from CS-CPCs products in a contentdependent manner as immersion and supposed to have a long-term release potential for constant biological stimulation. CS-CPCs notably accelerated the preferential adsorption of specific proteins, such as fibronectin, which was in favor of cellular adhesion behaviors. CS-CPCs significantly improved the proliferation and osteogenic differentiation of BMSCs, which was synergistically mediated by the adhesion events of cells on the cements and the stimulation effects of CS molecules. Hence, incorporation of CS is supposed to obtain enhanced osteogenic capacity and suitable physicochemical properties of injectable calcium phosphate bone cements, providing references for the developments of bone repair biomaterials meeting numerous orthopedic applications. FIGURES:


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Figure 1. Phase and morphology of CS-CPCs. XRD patterns (a), FTIR spectra (b) and SEM images (c) of the cross sections of hydrated CS-CPCs.


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Figure 2. Physiochemical properties and CS release behaviors of CS-CPCs. Compressive strength (a), apparent porosity (a), setting time (b), injectability (b) and CS kinetic release curves (c) of CS-CPCs.


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Figure 3. Anti-washout property of CS-CPC pastes. Optical digital images of cement pastes after injected into deionized water and shaken at a speed of 120 rpm.


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Figure 4. Proteins adsorption and cellular adhesion on the hydrated CS-CPCs. SEM images and live/dead staining fluorescence photographs of BMSCs adhered on the hydrated CS-CPCs, as well as immunofluorescence images of FN (red) adsorbed on the cements. Red arrows show the adhered cells on the hydrated cements.


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Figure 5. Cellular adhesion on the hydrated CS-CPCs. Immunofluorescence images show focal adhesion (FA) formation (maturation) through vinculin (red), nuclei (blue), actin cytoskeleton


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filaments (green) and merged images of BMSCs on hydrated CS-CPCs after being cultured for 24 h (a). Quantitative analysis data for cell area (c) and FA area (d). Real-time PCR results of cellular adhesion related gene expression (Integrin α5 and β1) of BMSCs after being cultured on hydrated CS-CPCs for 24 h (e and f). Immunofluorescence analysis of integrin α5 expression (red) in BMSCs on the hydrated CS-CPCs for 24 h (b). Nuclei were stained by DAPI (blue) and cytoskeleton filaments were stained with Alexa-Fluor 488 phalloidin (green). * CS-CPCs groups compared with CPC-con group. * P < 0.05, ** P < 0.005, *** P < 0.001.

Figure 6. Cellular behaviors on CS-CPCs. Proliferation (a) of BMSCs after being cultured on CS-CPCs for 1, 3 and 5 days, respectively. Osteogenic differentiation related gene expression (b) of BMSCs after being cultured on CS-CPCs for 7 days. * CS-CPCs groups compared with CPCcon group. * P < 0.05, ** P < 0.005, *** P < 0.001. ASSOCIATED CONTENT


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Supporting Information. The following file is available free of charge. Details of the experimental procedures and characterization results of the XRD patterns of raw materials and the behaviors of bone mesenchymal stem cells co-cultured with chondroitinsulfate-containing media (PDF) AUTHOR INFORMATION ORCID Haishan Shi: 0000-0003-3831-6898 Jiandong Ye: 0000-0002-5366-2054 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡The first two authors contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (Grant 2016YFB0700803), the National Natural Science Foundation of China (Grant 51672087), the Science and Technology Program of Guangzhou City of China (Grant 201508020017), and the China Postdoctoral Science Foundation (Grants 2016M602601 and 2018T110926). REFERENCES


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Page 25 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1) Cuthbert, R. J.; Churchman, S. M.; Tan, H. B.; McGonagle, D.; Jones, E.; Giannoudis, P. V. Induced periosteum a complex cellular scaffold for the treatment of large bone defects. Bone 2013, 57 (2), 484-492. DOI: 10.1016/j.bone.2013.08.009. (2) Tang, W.; Lin, D.; Yu, Y.; Niu, H.; Guo, H.; Yuan, Y.; Liu, C. Bioinspired trimodal macro/micro/nano-porous scaffolds loading rhBMP-2 for complete regeneration of critical size bone defect. Acta Biomater. 2016, 32, 309-323. DOI: 10.1016/j.actbio.2015.12.006. (3) Yuan, H.; Fernandes, H.; Habibovic, P.; de Boer, J.; Barradas, A. M. C.; de Ruiter, A.; Walsh, W. R.; van Blitterswijk, C. A.; de Bruijn, J. D. Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. P. Natl. Acad. Sci. USA 2010, 107 (31), 13614-13619. DOI: 10.1073/pnas.1003600107. (4) Zhang, W. J.; Chang, Q.; Xu, L.; Li, G. L.; Yang, G. Z.; Ding, X.; Wang, X. S.; Cui, D. X.; Jiang, X. Q. Graphene oxide-copper nanocomposite-coated porous CaP scaffold for vascularized bone regeneration via activation of Hif-1. Adv. Healthc. Mater. 2016, 5 (11), 1299-1309. DOI: 10.1002/adhm.201500824. (5) Santos, P. S.; Cestari, T. M.; Paulin, J. B.; Martins, R.; Rocha, C. A.; Arantes, R. V. N.; Costa, B. C.; dos Santos, C. M.; Assis, G. F.; Taga, R. Osteoinductive porous biphasic calcium phosphate ceramic as an alternative to autogenous bone grafting in the treatment of mandibular bone critical-size defects. J. Biomed. Mater. Res. B 2018, 106 (4), 1546-1557. DOI: 10.1002/jbm.b.33963.


25


Page 26 of 40

(6) Hotz, G. Alveolar ridge augmentation with hydroxylapatite using fibrin sealant for fixation: Part II: Clinical application. Int. J. Oral Max. Surg. 1991, 20, 208-213. DOI: 10.1016/S09015027(05)80176-6. (7) Zhang, Y. D.; Cui, X.; Zhao, S. C.; Wang, H.; Rahaman, M. N.; Liu, Z. T.; Huang, W. H.; Zhang, C. Q. Evaluation of Injectable Strontium-Containing Borate Bioactive Glass Cement with Enhanced Osteogenic Capacity in a Critical-Sized Rabbit Femoral Condyle Defect Model. ACS Appl. Mater. Inter. 2015, 7 (4), 2393-2403. DOI: 10.1021/am507008z. (8) Liu, Z. G.; Chen, J. M.; Zhang, G. W.; Zhao, J. S.; Fu, R.; Tang, K. Y.; Zhi, W.; Duan, K.; Weng, J.; Li, W.; Qu, S. X. Enhanced Repairing of Critical-Sized Calvarial Bone Defects by Mussel-Inspired Calcium Phosphate Cement. ACS Biomater. Sci. Eng. 2018, 4 (5), 1852-1861. DOI: 10.1021/acsbiomaterials.8b00243. (9) Paul, K.; Lee, B. Y.; Abueva, C.; Kim, B.; Choi, H. J.; Bae, S. H.; Lee, B. T., In vivo evaluation of injectable calcium phosphate cement composed of Zn- and Si-incorporated betatricalcium phosphate and monocalcium phosphate monohydrate for a critical sized defect of the rabbit femoral condyle. J. Biomed. Mater. Res. B 2017, 105 (2), 260-271. DOI: 10.1002/jbm.b.33537. (10) Aghyarian, S.; Bentley, E.; Hoang, T. N.; Gindri, I. M.; Kosrnopoulos, V.; Kim, H. K. W.; Rodrigues, D. C. In Vitro and In Vivo Characterization of Premixed PMMA-CaP Composite Bone Cements. ACS Biomater. Sci. Eng. 2017, 3 (10), 2267-2277. DOI: 10.1021/acsbiomaterials.7600276.


26

Page 27 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11) Arısan, V.; Anıl, A.; Wolke, J. G.; Özer, K. The effect of injectable calcium phosphate cement on bone anchorage of titanium implants: an experimental feasibility study in dogs, Int. J. Oral Max. Surg. 2010, 39 (5), 463-468. DOI: 10.1016/j.ijom.2010.01.004. (12) Zou, D.; Guo, L.; Lu, J.; Zhang, X.; Wei, J.; Liu, C.; Zhang, Z.; Jiang, X. Engineering of bone using porous calcium phosphate cement and bone marrow stromal cells for maxillary sinus augmentation with simultaneous implant placement in goats. Tissue Eng. A. 2012, 18 (13-14), 1464-1478. DOI: 10.1089/ten.TEA.2011.0501. (13) Zhang, J.; Ma, X.; Lin, D.; Shi, H.; Yuan, Y.; Tang, W.; Zhou, H.; Guo, H.; Qian, J.; Liu, C. Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell behavior via an integrin-mediated mechanism. Biomaterials 2015, 53, 251-264. DOI: 10.1016/j.biomaterials.2015.02.097. (14) Schumacher, M.; Gelinsky, M. Strontium modified calcium phosphate cements - approaches towards targeted stimulation of bone turnover. J. Mater. Chem. B 2015, 3 (23), 4626-4640. DOI: 10.1039/c5tb00654f. (15) Liu, W. J.; Zhai, D.; Huan, Z. G.; Wu, C. T.; Chang, J., Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive strength, in vitro bioactivity and cytocompatibility. Acta Biomater. 2015, 21, 217-227. DOI: 10.1016/j.actbio.2015.04.012. (16) Vaishya, R.; Chauhan, M.; Vaish, A. Bone cement. J. Clin. Orthop. Trauma 2013, 4 (4), 157-163. DOI: 10.1016/j.jcot.2013.11.005.


27


Page 28 of 40

(17) Cancian, D. C.; Hochuli-Vieira, E.; Marcantonio, R. A.; Garcia Júnior, I. R. Utilization of autogenous bone, bioactive glasses, and calcium phosphate cement in surgical mandibular bone defects in Cebus apella monkeys. Int J Oral Max Impl. 2004, 19 (1), 73-79. DOI: null. (18) Schneider, G.; Blechschmidt, K.; Linde, D.; Litschko, P.; Körbs, T.; Beleites, E. Bone regeneration with glass ceramic implants and calcium phosphate cements in a rabbit cranial defect model. J. Mater. Sci Mater. Med. 2010, 21 (10), 2853-2859. DOI: 10.1007/s10856-0104143-0. (19) Thormann, U.; Ray, S.; Sommer, U.; Elkhassawna, T.; Rehling, T.; Hundgeburth, M.; Henß, A.; Rohnke, M.; Janek, J.; Lips, K. S.; Heiss, C.; Schlewitz, G.; Szalay, G.; Schumacher, M.; Gelinsky, M.; Schnettler, R.; Alt, V. Bone formation induced by strontium modified calcium phosphate cement in critical-size metaphyseal fracture defects in ovariectomized rats. Biomaterials 2013,34 (34), 8589-8598. DOI: 10.1016/j.biomaterials.2013.07.036. (20) Yu, X.; Tang, X.; Gohil, S. V.; Laurencin, C. T. Biomaterials for bone regenerative engineering. Adv. Healthc. Mater. 2015, 4 (9), 1268-1285. DOI: 10.1002/adhm.201400760. (21) Ginebra, M.-P.; Canal, C.; Espanol, M.; Pastorino, D.; Montufar, E. B. Calcium phosphate cements as drug delivery materials. Adv. Drug Deliver. Rev. 2012, 64 (12), 1090-1110. DOI: 10.1016/j.addr.2012.01.008. (22) Ghosh, S.; Wu, V.; Pernal, S.; Uskokovic, V., Self-setting calcium phosphate cements with tunable antibiotic release rates for advanced antimicrobial applications. ACS Appl. Mater. Inter. 2016, 8 (12), 7691-7708. DOI: 10.1021/acsami.6b01160.


28

Page 29 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Akkineni, A. R.; Luo, Y. X.; Schumacher, M.; Nies, B.; Lode, A.; Gelinsky, M. 3D plotting of growth factor loaded calcium phosphate cement scaffolds. Acta Biomater. 2015, 27, 264-274. DOI: 10.1016/j.actbio.2015.08.036. (24) Schumacher, M.; Reither, L.; Thomas, J.; Kampschulte, M.; Gbureck, U.; Lode, A.; Gelinsky, M. Calcium phosphate bone cement/mesoporous bioactive glass composites for controlled growth factor delivery. Biomater. Sci. 2017, 5 (3), 578-588. DOI: 10.1039/c6bm00903d. (25) Huang, B. L.; Wu, Z. H.; Ding, S.; Yuan, Y.; Liu, C. S. Localization and promotion of recombinant human bone morphogenetic protein-2 bioactivity on extracellular matrix mimetic chondroitin sulfate-functionalized calcium phosphate cement scaffolds. Acta Biomater. 2018, 71, 184-199. DOI: 10.1016/j.actbio.2018.01.004. (26) Tabata, Y.; Ikada, Y. Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities. Biomaterials. 1999, 20 (22): 2169-2175. DOI: 10.1016/S0142-9612(99)00121-0. (27) Berendsen, A. D.; Olsen, B. R. Bone development. Bone 2015, 80, 14-18. DOI: 10.1016/j.bone.2015.04.035. (28) Freeman, F. E.; McNamara, L. M. Endochondral priming: a developmental engineering strategy for bone tissue regeneration. Tissue Eng. B 2017, 23 (2), 128-141. DOI: 10.1089/ten.teb.2016.0197.


29


Page 30 of 40

(29) Baeurle, S. A.; Kiselev, M. G.; Makarova, E. S.; Nogovitsin, E. A. Effect of the counterion behavior on the frictional–compressive properties of chondroitin sulfate solutions. Polymer 2009, 50 (7), 1805-1813. DOI: 10.1016/j.polymer.2009.01.066. (30) Ryan, C. N. M.; Sorushanova, A.; Lomas, A. J.; Mullen, A. M.; Pandit, A.; Zeugolis, D. I. Glycosaminoglycans in tendon physiology, pathophysiology, and therapy. Bioconjugate Chem. 2015, 26 (7), 1237-1251. DOI: 10.1021/acs.bioconjchem.5b00091. (31) Wang, T. Y.; Lai, J. H.; Han, L. H.; Tong, X. M.; Yang, F. Modulating stem cellchondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels. J. Mater. Chem. B 2016, 4 (47), 7641-7650. DOI: 10.1039/c6tb01583b. (32) Kowitsch, A.; Zhou, G. Y.; Groth, T. Medical application of glycosaminoglycans: a review. J. Tissue Eng. Regen. M. 2018, 12 (1), E23-E41. DOI: 10.1002/term.2398. (33) Hayes, A.; Sugahara, K.; Farrugia, B.; Whitelock, J. M.; Caterson, B.; Melrose, J. Biodiversity of CS-proteoglycan sulphation motifs: chemical messenger recognition modules with roles in information transfer, control of cellular behaviour and tissue morphogenesis. Biochem. J. 2018, 475, 587-620. DOI: 10.1042/bcj20170820. (34) Canning, D. R.; Brelsford, N. R.; Lovett, N. W. Chondroitin sulfate effects on neural stem cell differentiation. In Vitro Cell Dev.-An. 2016, 52 (1), 35-44. DOI: 10.1007/s11626-015-99418. (35) Rhee, S. H.; Tanaka, J. Effect of chondroitin sulfate on the crystal growth of hydroxyapatite. J. Am. Ceram. Soc. 2010, 83 (8), 2100-2102. DOI: 10.1111/j.1151-2916.2000.tb01522.x.


30

Page 31 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Uchisawa, H.; Okuzaki, B. I.; Ichita, J.; Matsue, H. Binding between calcium ions and chondroitin sulfate chains of salmon nasal cartilage glycosaminoglycan. International Congress Series 2001, 1223 (01), 205-220. DOI: 10.1016/s0531-5131(01)00458-7. (37) Hoshi, K.; Ejiri, S.; Ozawa, H. Localizational alterations of calcium, phosphorus, and calcification-related organics such as proteoglycans and alkaline phosphatase during bone calcification. J. Bone Miner. Res. 2010, 16 (2), 289-298. DOI: 10.1359/jbmr.2001.16.2.289. (38) Schneiders, W.; Ruhnow, R. M. Effect of chondroitin sulphate on material properties and bone romodelling around hydroxyapatite/collagen. J. Biomed. Mater. Res. A 2010, 85A (3), 638645. DOI: 10.1002/jbm.a.31611. (39) Yang, S.; Guo, Q.; Shores, L. S.; Aly, A.; Ramakrishnan, M.; Kim, G. H.; Lu, Q.; Su, L.; Elisseeff, J. H. Use of a chondroitin sulfate bioadhesive to enhance integration of bioglass particles for repairing critical-size bone defects. J. Biomed. Mater. Res. A 2015, 103 (1), 235242. DOI: 10.1002/jbm.a.35143. (40) Yoshikawa, M.; Toda, T. In vivo estimation of periapical bone reconstruction by chondroitin sulfate in calcium phosphate cement. J. Eur. Ceram. Soc. 2004, 24 (2), 521-531. DOI: 10.1016/s0955-2219(03)00196-1. (41) Yoshikawa, M.; Hayami, S.; Toda, T. In vivo estimation of calcium phosphate cements containing chondroitin sulfate in subcutis. Mater. Sci. Eng. C 2002, 20 (1), 135-141. DOI: 10.1016/s0928-4931(02)00023-1. (42) Huang, B.; Wu, Z.; Ding, S.; Yuan, Y.; Liu, C. Localization and promotion of recombinant human bone morphogenetic protein-2 bioactivity on extracellular matrix mimetic chondroitin


31


Page 32 of 40

sulfate-functionalized calcium phosphate cement scaffolds. Acta Biomater. 2018, 71, 184-199. DOI: 10.1016/j.actbio.2018.01.004. (43) Schneiders, W.; Reinstorf, A.; Biewener, A.; Serra, A.; Grass, R.; Kinscher, M.; Heineck, J.; Rehberg, S.; Zwipp, H.; Rammelt, S. In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia. J. Ortho. Res. 2009, 27 (1), 15-21. DOI: 10.1002/jor.20719. (44) Wang, M.; Qian, R.; Bao, M.; Gu, C.; Zhu, P.; Wang, M.; Qian, R.; Bao, M.; Gu, C.; Zhu, P. Raman, FT-IR and XRD study of bovine bone mineral and carbonated apatites with different carbonate levels. Mater. Lett. 2018, 210, 203-206. DOI: 10.1016/j.matlet.2017.09.023. (45) Gittens, R. A.; Scheideler, L.; Rupp, F.; Hyzy, S. L.; Geis-Gerstorfer, J.; Schwartz, Z.; Boyan, B. D. A review on the wettability of dental implant surfaces II: Biological and clinical aspects. Acta Biomater. 2014, 10 (7), 2907-2918. DOI: 10.1016/j.actbio.2014.03.032. (46) Falde, E. J.; Yohe, S. T.; Colson, Y. L.; Grinstaff, M. W. Superhydrophobic materials for biomedical applications. Biomaterials 2016, 104, 87-103. DOI: 10.1016/j.biomaterials.2016.06.050. (47) Zollinger, A. J.; Smith, M. L. Fibronectin, the extracellular glue. Matrix Bio. 2017, 60-61, (27-37). DOI: 10.1016/j.matbio.2016.07.011. (48) Foster, T. J.; Geoghegan, J. A.; Ganesh, V. K.; Hook, M. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat. Rev. Microbiol. 2014, 12 (1), 49-62. DOI: 10.1038/nrmicro3161.


32

Page 33 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49) Karner, C. M.; Long, F. X. Wnt signaling and cellular metabolism in osteoblasts. Cell. Mol. Life Sci. 2017, 74 (9), 1649-1657. DOI: 10.1007/s00018-016-2425-5. (50) Capulli, M.; Paone, R.; Rucci, N., Osteoblast and osteocyte: games without frontiers. Arch. Biochem. Biophys. 2014, 561, 3-12. DOI: 10.1016/j.abb.2014.05.003. (51) Grayson, W. L.; Bunnell, B. A.; Martin, E.; Frazier, T.; Hung, B. P.; Gimble, J. M. Stromal cells and stem cells in clinical bone regeneration. Nat. Rev. Endocrinol. 2015, 11 (3), 140-150. DOI: 10.1038/nrendo.2014.234. (52) Tang, D.; Tare, R. S.; Yang, L.-Y.; Williams, D. F.; Ou, K.-L.; Oreffo, R. O. C. Biofabrication of bone tissue: approaches, challenges and translation for bone regeneration. Biomaterials 2016, 83, 363-382. DOI: 10.1016/j.biomaterials.2016.01.024. (53) Johnson, G. A.; Burghardt, R. C.; Bazer, F. W., Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep. J. Anim. Sci. Biotechnol. 2014, 5, 14. DOI: 10.1186/2049-1891-5-56. (54) Ruoslahti, E.; Yamaguchi, Y. Proteoglycans as modulators of growth factor activities. Cell 1991, 64 (5), 867-869. DOI: 10.1016/0092-8674(91)90308-L. (55) Lee, J. W.; Juliano, R. Mitogenic signal transduction by integrin- and growth factor receptor-mediated pathways. Mol. Cells 2004, 17 (2), 188-202. DOI: null. (56) Rhee, S. H.; Tanaka, J. Self-assembly phenomenon of hydroxyapatite nanocrystals on chondroitin sulfate. J. Mater. Sci. Mater. M. 2002, 13 (6), 597-600. DOI: 10.1023/A:1015135112302.


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For Table of Contents Use Only Enhanced Osteogenesis of Injectable Calcium Phosphate Bone Cement Mediated by Loading Chondroitin Sulfate Haishan Shi,1,2,3‡ Xiaoling Ye,1,2‡ Jing Zhang1,2 and Jiandong Ye1,2,4* Table of Content


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Figure 1. Phase and morphology of CS-CPCs. XRD patterns (a), FTIR spectra (b) and SEM images (c) of the cross sections of hydrated CS-CPCs.



Figure 2. Physiochemical properties and CS release behaviors of CS-CPCs. Compressive strength (a), apparent porosity (a), setting time (b), injectability (b) and CS kinetic release curves (c) of CS-CPCs. 85x167mm (300 x 300 DPI)


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Figure 3. Anti-washout property of CS-CPC pastes. Optical digital images of cement pastes after injected into deionized water and shaken at a speed of 120 rpm.



Figure 4. Proteins adsorption and cellular adhesion on the hydrated CS-CPCs. SEM images and live/dead staining fluorescence photographs of BMSCs adhered on the hydrated CS-CPCs, as well as immunofluorescence images of FN (red) adsorbed on the cements. Red arrows show the adhered cells on the hydrated cements.


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Figure 5. Cellular adhesion on the hydrated CS-CPCs. Immunofluorescence images show focal adhesion (FA) formation (maturation) through vinculin (red), nuclei (blue), actin cytoskeleton filaments (green) and merged images of BMSCs on hydrated CS-CPCs after being cultured for 24 h (a). Quantitative analysis data for cell area (c) and FA area (d). Real-time PCR results of cellular adhesion related gene expression (Integrin α5 and β1) of BMSCs after being cultured on hydrated CS-CPCs for 24 h (e and f). Immunofluorescence analysis of integrin α5 expression (red) in BMSCs on the hydrated CS-CPCs for 24 h (b). Nuclei were stained by DAPI (blue) and cytoskeleton filaments were stained with Alexa-Fluor 488 phalloidin (green). * CS-CPCs groups compared with CPC-con group. * P < 0.05, ** P < 0.005, *** P < 0.001.



Figure 6. Cellular behaviors on CS-CPCs. Proliferation (a) of BMSCs after being cultured on CS-CPCs for 1, 3 and 5 days, respectively. Osteogenic differentiation related gene expression (b) of BMSCs after being cultured on CS-CPCs for 7 days. * CS-CPCs groups compared with CPC-con group. * P < 0.05, ** P < 0.005, *** P < 0.001. 170x89mm (300 x 300 DPI)


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Enhanced Osteogenesis of Injectable Calcium Phosphate Bone

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