Chitosan Based Scaffolds, but

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Homologous Sodium Alginate/Chitosan Based Scaffolds, but Contrasting Effect on Stem Cell Shape and Osteogenesis Lili Zhang, Haowei Fang, Kunxi Zhang, and Jingbo Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18859 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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ACS Applied Materials & Interfaces

Homologous Sodium Alginate/Chitosan Based Scaffolds, but Contrasting Effect on Stem Cell Shape and Osteogenesis Lili Zhang, Haowei Fang, Kunxi Zhang* and Jingbo Yin*

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Department of Polymer Materials, Shanghai University, 99 Shangda Road, Shanghai 200444, PR China. *Corresponding Author E-mail: [email protected] (Kunxi Zhang) E-mail: [email protected] (Jingbo Yin)

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Abstract. Stem cell shape appears to be involved in the regulation of osteogenesis, which has been confirmed in two dimensional surfaces and three dimensional hydrogels. The present study was to evaluate the effect of matrix-controlled cellular shape on osteogenesis in three dimensional porous scaffolds based on the sodium alginate (ALG) and chitosan (CS). Three ALG/CS scaffolds, especially including a stiff one, were fabricated from different precursor matrices. Soft scaffold A was fabricated from ALG/CS polyelectrolyte, and further crosslinked by Ca2+ and glutaraldehyde to achieve soft scaffold B with alterative hydrophilicity. Stiff scaffold C with “hard-to-deform” feature was fabricated from “ALG/CS preformed gel”, which was an ALG gel network expanded by swelling force of the dissolving CS, and fixed using Ca2+ and glutaraldehyde. SEM and F-actins staining showed mesenchymal stem cell (MSCs) on the inner surfaces inside scaffold A with high swelling behavior exhibited rounded shape, but spindle-like shape in scaffold B. Stiff scaffold C forced MSCs to adhere to 1 - Environment ACS Paragon -Plus

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polygonal shape. Fibronectin (Fn) adsorption was found to be weakened in scaffold A. Integrin α5β1 expression, as well as osteogenesis-related genes (ALP, OCN) expression were detected to be higher in the stiff scaffold C. Thus the present study illustrated that the stiff scaffold C responded to cells with “hard-to-deform” information, leading to amplification of focal adhesions and inducing high tension of cells, consequently enhancing osteogenesis.

Keywords: sodium alginate; chitosan; cell shape; stiffness; osteogenesis

1. Introduction Towards stem cell based bone tissue engineering, it has been already demonstrated that mesenchymal stem cells (MSCs) allowed to adhere, flatten and spread underwent osteogenesis

1,2

. According to comparison between globally isotropic circular, square,

triangular, and star cells, Ding J. and his colleagues found that optimal osteogenic differentiation happened in star cells 3. Thus, cellular shape appear to be integral to the commitment of stem cell fate. Properties of substrates, such as mechanical property, hydrophilicity & hydrophobicity and surface topography, show significant effect on cell adhesion, cell shape and spread

4-6

.

Researches have indicated that varying physical and chemical properties of substrates would modulates fibronectin (Fn) conformation and directs integrin binding and specificity, thus result in changes of cellular shape so to realize the regulation of cell differentiation 1,2. Matrix interfacial hydrophobicity showed the enhanced effect on osteogenesis of stem cell by harnessing the cytoskeletal organization of MSCs on substrate 7. Besides, the elasticity and rigidity of three-dimensional microenvironments regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem cell populations 8. Thus, bone scaffolds should be designed not only to act as the temporary residence and delivery system for stem cells, but also to regulate stem cell differentiation of certain lineages. 2 - Environment ACS Paragon -Plus

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However, although the effect of matrix-controlled cellular shape on osteogenesis has been confirmed in two dimensional surfaces and three dimensional hydrogels, whether the three dimensional porous scaffolds perform the similar effect of matrix-controlled cellular shape on osteogenesis with two dimensional surfaces and three dimensional hydrogels, remains elusive. Sodium alginate (ALG) and chitosan (CS) are two kinds of natural macromolecules that have been widely used in bone tissue engineering 9. There were various methods to fabricate scaffolds based on ALG and CS to act as scaffolds. However, due to the hydrophilic nature of ALG and CS, the ALG/CS based scaffolds generally exhibited soft character when wetted in water and culture medium. These sponge-like scaffolds performed well in absorbing water and deformation. It was a challenge to fabricate an ALG/CS based scaffold that was stiff and hard to deform, so to reinforce the traction forces of stem cells, promoting osteogenesis. Towards bone tissue engineering, it is worth to construct a stiffer ALG/CS based scaffold for bone tissue engineering, and to evaluate the effect of the stiff feature of ALG/CS based scaffolds on regulation of cellular shape, as well as the effect of shape regulation on promoting osteogenesis of stem cells in three dimensional porous scaffold. Here in the present study, soft and stiff scaffolds were all fabricated based on ALG and CS. The soft scaffolds were prepared from ALG/CS polyelectrolyte matrices. At the same time, cross-linking components were employed to alter the hydrophilicity of the scaffold matrix. A new strategy that using swelling pressure of CS during dissolution to expend the ALG gel network, was proposed to prepare the stiff scaffold with “hard-to-deform” property. These ALG/CS scaffolds were created for the purpose of evaluating the effect of scaffold on promoting osteogenic differentiation of stem cells through forcing the adhesive stem cells to present different cellular shape on the inner surfaces inside the scaffolds. The structure, swelling property and mechanical property of ALG/CS scaffolds were studied. MSCs were seeded into these scaffolds. The shape and proliferation of adherent MSCs was observed.

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Furthermore, the osteogenic differentiation of MSCs in different ALG/CS scaffolds was monitored to evaluate the effect of scaffolds-controlled cellular shape on osteogenesis in three dimensional porous scaffold.

2. Experimental Section 2.1. Preparation of ALG/CS based scaffolds All scaffolds in this paper were obtained by mixing chitosan(Mv = 4×104, DA≤5%, purchased from Jinan Haidebei Marine Bioengineering Corp) and sodium alginate(Mv: 7.8 × 105, purchased from Sinopharm Chemical Reagent Co., Ltd.). The mole ratio of –COOH in ALG and –NH2 of CS was 1 : 1. The solid content (the sum of mass fraction of ALG and CS) was 3%. The fabrication of three types of ALG/CS based scaffolds named scaffold A, scaffold B and scaffold C was listed blew. To study the viscoelastic behavior of the ALG/CS precursor matrices during the preparation of different types of scaffolds, rheological experiments were performed on an AR2000 rheometer from TA Instruments with 25 mm diameter parallel plates at 37 °C in the oscillatory mode.

The frequency sweep test was performed, which covered a range of

frequencies from 1 to 100 rad/s at the regular strain of γ = 0.01. The storage modulus G′, loss modulus G″ and dynamic viscosity η were obtained with respect to frequency. FT-IR measurements were performed with an AVATAR 370 FTIR spectrophotometer from Nicolet. The KBr used for pellet preparation, each matrix were detected after drying. 2.1.1. Preparation of soft scaffold A Sodium alginate was dissolved in deionized water to form a homogeneous solution. CS powder (3 wt%) that evenly dispersed in ALG solution was dissolved by slowly adding dilute acids until the pH value reached 4-5. Then it was sped up the emulsion with FA25 High Shear 4 - Environment ACS Paragon -Plus

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Emulsifier (FA25, FLUKO, Shanghai) and kept stirring for 12 h to form a homogeneous polyelectrolyte suspension. After lyophilization at -20 °C, scaffold A was obained. 2.1.2. Preparation of soft scaffold B Scaffold A was firstly soaked in 3% calcium chloride (CaCl2) solution for 15 min and then in 1% glutaraldehyde aqueous solution for 15 min. After being lyophilized, scaffold B was achieved. 2.1.3. Preparation of stiff scaffold C After dissolving ALG in deionized water, CS powder (3 wt%) that evenly dispersed in ALG solution was dissolved by adding dilute acids to immediately reach the pH value lower than 3. Then colorless transparent gel-like mixture was obtained. After lyophilization at -20 o

C, the matrix was firstly soaked in 3% calcium chloride (CaCl2) solution for 15 min,

followed by being soaked in 1% glutaraldehyde aqueous solution for 15 min. After lyophilization at -20°C, scaffold C was obained. 2.2. Characterization of ALG/CS based scaffolds 2.2.1. Porous structure of scaffolds The microstructures of scaffolds were observed by scanning electron microscopy (SEM) (JXA-840, JEOL, Japan). Pore diameters of scaffolds were obtained by analyzing the SEM images from sectioned microspheres. 2.2.2. Swelling behavior of scaffolds Each sample was measured immediately after vacuum dried as the dry weights (Wd) ; next, these samples were immersed in phosphate buffered saline (PBS) solution at a constant 37 oC and weighed at given time notes to conform the swollen weights (Ws); the completely swollen samples were centrifuged at 500 rpm for 3 min and then weighed straightway as Ws’. The swelling ratio of each simple was calculated by the following equation: swelling ratio = 5 - Environment ACS Paragon -Plus


(Ws -Wd)/Wd ×100%; The water retention was calculated by the following equation: ratio of water retention = (Ws’ -Wd)/Wd ×100%. 2.2.3. Mechanical properties of scaffolds The compressive performance of scaffolds was evaluated using a Dynamic Mechanical Analyzer (TA Q800, instrument, TA Instruments Waters LLC, USA) in 0.1 M PBS at 37 oC, fitted with a custom compression clamp. A cubic sample (4mm ⅹ 4mm ⅹ 4mm) was compressed with a constant strain rate of 10% min-1. 2.3. Evaluation of cellular shape and proliferation 2.3.1. Cell seeding and in vitro culture MSCs, derived from femurs of neonatal SD rats, were purchased from Servicebio (China). MSCs were cultured in low glucose DMEM supplemented with 10% FBS, 50 µg ml-1 ascorbic acid, 100 U ml-1 penicillin, and 100 µg ml-1 streptomycin in 100 mm diameter culture dishes at 37 oC in a 5% CO2 atmosphere. MSCs prior to passage 3 were collected and resuspended in culture medium at a concentration of 1×105 cells/ml, followed by being seeded into ALG/CS based scaffolds. After incubation for 3 h to allow cell attachment, osteogenic induced medium, which was composed of high-glucose DMEM, 10% FBS, 50 µM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate and 100 nM dexamethasone, was added for in vitro induction. 2.3.2. Observation of cellular shape SEM was used to observe the cellular shape inside the scaffolds. At 3 h and 24 h postseeding, the scaffolds/MSCs were collected and washed with PBS for 3 times, followed by being immersed in 4% paraformaldehyde. After gradually replacing H2O by ethanol, hexamethyldisilazane was used to replace ethanol. The samples, collected after the


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evaporation of hexamethyldisilazane, were treated with gold sputtering and observed with SEM. Rhodamine phalloidin was also used to stain F-actins to further identify the cellular shape. The scaffolds/MSCs samples were collected washed with PBS for 3 times, followed by being immersed in 4% paraformaldehyde. 0.5% Triton was used to treat the samples and then washed by PBS for 20 min. After being immersed in staining solution for 30 min, samples were washed by PBS and observed with confocal laser microscope (FV-1000, Japan, Instrumental Analysis and Research Center, Shanghai University). 2.3.3. Adsorption of Fn on scaffolds The scaffolds immersed in culture medium for 4 h were washed with PBS for 2 h, followed by being determined by immunofluorescent staining. Briefly, samples were blocked with BSA and reacted with polyclonal to Fn antibody at 4 oC, followed by being immersed in Alexa Fluor 568 labeled secondary antibody at 37 oC and observed using confocal laser scanning microscopy. 2.3.4. Evaluation of cellular proliferation in scaffolds Cell numbers in the scaffold at 1, 7 and 14 days post-seeding were quantified by DNA assay using Hoechst 33258 dye (Sigma-Aldrich). Briefly, the scaffolds/MSCs samples were collected and washed with PBS for 3 times, and crushed, followed by being treated with 0.5 mg/ml proteinase K at 56 oC for 12 h. After centrifugation, DNA content in supernatant was quantified spectrophotometrically using Hoechst 33258 dye by correlating with a standard curve (SpectraMax M2, Molecular Devices, USA). To visualize the growth and distribution of MSCs in scaffolds, MSCs were pre-labeled with fluorescent 3,3’-dioc tadecyloxacarbocyanine perchlorate (DiO) dye (Molecular Probes, USA) for 15 min at 37 oC before seeding. The cells grown in scaffolds were observed by confocal 7 - Environment ACS Paragon -Plus


laser microscope. The relative cytotoxicity was evaluated by Cell Counting Kit-8 (CCK-8). The scaffolds extracts were collected by immersing 200 mg scaffolds in 1 ml DMEM & 10% FBS for 1, 2, 4, 6 and 8 weeks. DMEM & 10% FBS and DMSO were employed as control groups. MSCs were seeded in 96-well plates(10000 cells per well) for 24 h, followed by changing growth medium with 110 µL extract to culture for another 48 h. Then, 10 µL of CCK-8 solution was added to each well of the plate to incubate for 4 h, followed by measuring the absorbance (OD value) at 450 nm using a microplate reader (SpectraMax M2, Molecular Devices, USA). Cell viability%=(ODsample-ODDMEM)/ODDMEM×100%. 2.4. Evaluation of in vitro osteogenic differentiation Scaffolds/MSCs samples were fixed with 4% paraformaldehyde and embedded in paraffin, sectioned (5 µm thick). Von Kossa and alkaline phosphatase (ALP) staining were carried out to show the osteogenesis of MSCs in the three scaffolds. For von Kossa staining, the sections were incubated in 5% silver nitrate solution for 1 h, and washed. ALP staining was performed according to the manufacturer’s protocol of Leukocyte Alkaline Phosphatase Kit (Sigma; MO, USA). Total RNA was isolated using Trizol and determined from the optical absorbance at 260nm. Then, cDNA was synthesized from RNA using PrimeScript 1st Stand cDNA synthesis kit (TaKaRa). Real-time PCR was performed using SybrGreen PCR MasterMix in each reaction. Expression of osteogenesis-related genes, including ALP, OCN and OPG were detected. Relative expression levels for each gene were calculated by normalizing the quantified cDNA transcript level to that of the GAPDH. And the relative gene amount of non-induced group was set as 1. GAPDH: 5’-tgaacaggaagctcactgg-3’ & 5’-tccaccaccctgttgctgta-3’; OCN: 5’tctctctgctcactctgctg-3’ & 5’-attttggagcagctgtgccg-3’; ALP: 5’-acgtggctaagaatgtcatc-3’ & 5’acgtggctaagaatgtcatc-3’; OPG: 5’-caggagagtgaggcaggctatt-3’ & 5’-cttcaggggttggagatttagc-3’. 8 - Environment ACS Paragon -Plus

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Besides, expression of cell adhesion molecules, integrin α5 and β1 was also monitored by RTPCR. Relative expression levels for these two genes were calculated by normalizing the quantified

cDNA

transcript

acttagggcaagtcgggaatt-3’

&

level

to

that

of

the

GAPDH.

5’-gggcagtgagtggggtttat-3’;

Integrin

Integrin

α5: β1:

5’5’-

tctgtggaggaaatggtgtgtg-3’ & 5’-tttagtgaggttgaaatgggagc-3’. 2.5. Statistical analysis All date were reported as mean ± standard deviation (SD). Single-factor analysis of variance (ANOVA) was used to assess the statistical significance of the results. Values of p < 0.05 were indicative of significant differences.

3. Results and Discussion 3.1. Construction of ALG/CS scaffolds using different methods Thanks to numerous carboxyl groups of ALG and amino groups of CS, various methods were reported to fabricate scaffolds based on ALG and CS

9-12

. ALG is a water soluble

polymer, the network of which has to be fixed by other components such as Ca2+ to yield a 3D structure. CS is well known to be a semi-crystalline polymer. The stable, crystalline structure make CS a normally insoluble in aqueous solutions above pH 7 but fully soluble in dilute acids, thus providing a convenient mechanism for processing under mild conditions. The viscous CS solutions can be gelled in high pH solutions or baths of solvents. Thus, CS possessed excellent ability to be processed into porous scaffold 13. Here in the present study, three types of scaffold based on ALG and CS were fabricated from different ALG/CS precursor matrices. 3.1.1 Preparation of ALG/CS precursor matrices for soft scaffolds



Theoretically, ALG is a polyelectrolyte carrying negative charges, which could interact with CS, a polyelectrolyte carrying positive charges. In the present study, the electronic interaction between ALG and CS was found to significantly depend on pH value. ALG aqueous solution (3 wt%) showed the pH of 7. CS power (3 wt%) that evenly dispersed in ALG solution was dissolved by slowly adding dilute acids until the pH value reached 4-5. Along with the protonation of amino groups on CS, the electronic interaction between CS and ALG was formed strongly enough to overcome their solvation effect, leading to the precipitation of ALG/CS polyelectrolyte (Fig. 1). With the effect of emulsification and longtime continuous agitation, the ALG/CS polyelectrolyte suspension, which was used to fabricate porous scaffold by freeze-drying, was formed (Fig. 2a). As FT-IR spectra showed (Fig. 2b), the characteristic peaks of CS was at 1635 cm-1 and 1600 cm-1 corresponding to C=O stretching band (amide I) and N-H bending band (amide II). Besides, the peak at 1076 cm-1 corresponded to C-N stretching vibration. The peak at 1029 cm-1 corresponded to C-O-C stretching vibration. The ALG spectrum showed characteristic bands of C=O stretching vibration (1793 cm-1), O-H bending vibration (1417 cm-1), C-O stretching vibration (1259 cm1

), as well as C-O-C stretching vibration (1029 cm-1) which was same to CS. The ALG/CS

polyelectrolyte formation was recognized as the new peak observed at 1627 cm-1, together with a shoulder peak at 1570 cm-1, which were respectively assigned to the carboxylic groups of ALG associated with CS and to the amino groups of CS associated with ALG. Besides, with the protonation of amino groups at pH of 4-5, the peak at 1076 cm-1 that corresponded to C-N stretching vibration of CS was weakened. At the same time, rheological test was used to analyze the pH depended interaction between ALG and CS. As shown in Fig. 2c, ALG aqueous solution (3 wt%) showed the η* of 40 Pa.s. After adding CS, the η* of ALG solution/CS powder was 170 Pa.s. And the η* of ALG/CS polyelectrolyte suspension was 2400 Pa.s. As shown in Fig. 2d, Gꞌꞌ was higher than Gꞌ, indicating that ALG/CS polyelectrolyte suspension possessed typical flow behavior. 10 -Environment ACS Paragon-Plus

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3.1.2 Preparation of ALG/CS precursor matrices for stiff scaffold A new strategy, based on the gelation characteristics of ALG and dissolution characteristics of CS, was introduced to create the stiff ALG/CS scaffold. Briefly, CS dissolution was designed to take place inside the ALG gel, so to provide swelling pressure to expand the ALG network to an expansion state. After freeze-drying, the networks were fixed by Ca2+ and glutaraldehyde to yield the stiff scaffold (Fig. 1 and Fig. 2a). Along with the protonation of carboxyl groups in ALG, ALG can be gelled through hydrogen bonds and hydrophobic effects. As shown in Fig. 2c, the η* of ALG solution was 43 Pa.s. When pH value was lower than 3, the η* dramatically reached at 4900 Pa.s. And the Gꞌ was higher than Gꞌꞌ, indicating the gel formation (Fig. 2e). At the same time, CS dissolved along with the pH value decreased from 7 to < 3. Thus, CS power (3 wt%) was evenly dispersed in ALG solution to reach the combination of ALG and CS. Then, dilute acids was added immediately to reach the pH value of 3, the ALG gelled, at the same time, CS began to dissolve in side of the ALG gel. As shown in Fig. 2c, the η* of ALG/CS mixture increased from 170 Pa.s to 280000 Pa.s. According to Fig. 2d, unlike ALG/CS polyelectrolyte suspension, the Gꞌ of ALG/CS preformed gel was 27000 Pa, significantly higher than Gꞌꞌ and the Gꞌ value of ALG/CS polyelectrolyte suspension. Besides, due to the protonation of carboxyl groups and restricted effect of gelation, electronic interaction between ALG and CS was limited. Thus, the absence of peaks at 1627 cm-1and 1570 cm-1 in FT-IR spectra illustrated that there was no significant polyelectrolyte formation in ALG/CS preformed gel (Fig. 2b). Then, the ALG/CS polyelectrolyte suspension and ALG/CS preformed gel were freezedried at -20 oC to achieve the porous structure. Due to the uniform and extensive electronic interaction in ALG/CS polyelectrolyte suspension, the porous scaffold kept stably after neutralization. Then, a soft and sponge-like scaffold was achieved, which was named as 11 -Environment ACS Paragon-Plus


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scaffold A. As shown in Fig. 3a, as characterized by SEM, the apertures of scaffold A were 150-200 µm after freezing at -20 oC. The existence of small pores on the walls of pores impacted the matrix continuity, indicated a loose matrix of scaffold A. Moreover, scaffold A was further cross-linked by Ca2+ and glutaraldehyde to shield carboxyl and amino groups on the surfaces of walls of pores to evaluate the cellular adhesion. This scaffold was named as scaffold B. According to Fig. 3b, the porous structure of scaffold B was similar as scaffold A, showing no significant change. In contrast, the porous matrix dried form ALG/CS preformed gel was fixed by Ca2+ and glutaraldehyde. Then, scaffold C was achieved. Unlike the sponge-like feature of scaffold A and scaffold B, scaffold C exhibited a stiff characteristic. Besides, as the SEM images illustrated (Fig. 3c), scaffold C possessed a quite continuous porous structure. The apertures of scaffold C were 200-300 µm after freezing at -20 oC, significant larger than those of scaffold A and scaffold B. 3.2. Swelling behavior of different ALG/CS scaffolds Hydrophilicity is an important factor that affect cellular adhesion

14,15

. To define the

hydrophilicity of porous scaffolds, swelling behavior including water uptake and water retention ability was put into account. Both ALG and CS are hydrophilic macromolecule, exhibiting strong interaction with H2O via hydroxyl and ether bond, as well as carboxyl and amino groups. However, although scaffold A, scaffold B and scaffold C were all based on ALG and CS, the swelling behavior, including water uptake and water retention, was different. As shown in Fig. 3d, the sponge-like scaffold A showed a quick swelling after being immersed in ddH2O. The water uptake ratio (swelling ratio) reached over 1000% within 10 min. Unlike scaffold A, the swelling ratio curves of both scaffold B and scaffold C showed a mild rise of water uptake ratio, indicating an obvious slower swelling in ddH2O. The water uptake ratio of scaffold B and scaffold C reached about 800% and 700% within 10 min, 12 -Environment ACS Paragon-Plus

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respectively, significant lower than that of scaffold A. After 24h, the equilibrium swelling ratio of scaffold A was significantly higher than that of scaffold B and scaffold C. The above result illustrated that scaffold A possessed a well-performed water uptake capacity. Furthermore, water retention was detected to evaluate the H2O bonding ability of the three scaffolds, which was directly associated with the cell adhesion behavior. During water retention detection, water that remaining in scaffolds after centrifugation was considered to combine to material matrix, which may form the hydration to affect cell-matrix interaction. According to Fig. 3e, after centrifugation, the water retention ratio of scaffold A was about 800%. Both the water retention ratios of scaffold B and scaffold C were 500%, significant lower than that of scaffold A. Compared to scaffold A, both scaffold B and scaffold C underwent the reaction with Ca2+ and glutaraldehyde, consuming lots of carboxyl and amino groups, which could efficiently combine H2O by more strongly ionic solvation. Thus, the H2O bonding ability of scaffold B and scaffold C was different from scaffold A. 3.3. Cytotoxicity of different ALG/CS scaffolds ALG and CS have been recognized to possess biocompatibility. However, glutaraldehyde is a cross-linking agent with cytotoxicity. Although scaffold B and scaffold C were dialyzed with H2O for a really long time, cytotoxicity of the scaffolds still needed to be evaluated to make sure that cell viability did not influence the study of cellular shape and differentiation. Thus, the relative cytotoxicity of the scaffolds extracts collected by immersing 200 mg scaffolds in 1 ml DMEM for 1, 2, 4, 6 and 8 weeks was evaluated. As shown in Fig. 3f, the leaching liquor was evaluated to possess no significant cytotoxicity when compared to DMEM control group. Viability of MSCs in extracts of scaffold B and scaffold C was as high as that in extracts of scaffold A, indicating that the introduction of glutaraldehyde did not cause significant cell death during in vitro culture and evaluation. Furthermore, the three scaffolds were implanted subcutaneously as shown in Fig. S1. After 7 d post-implantation, 13 -Environment ACS Paragon-Plus


fibrous capsules formed to encapsulate implanted scaffolds in all groups. Host tissues were observed invaded into ambient pore spaces. However, along with the degradation of scaffolds, the long term release of glutaraldehyde may make the present scaffolds not suitable for in vivo tissue regeneration. Other cross-linking agents have to be developed for further in vivo tissue regeneration. 3.4. Mechanical properties of different ALG/CS scaffolds Besides hydrophilicity, mechanical features of matrix also affect cellular adhesion 16. What is more, for osteogenesis, mechanical features of matrix also guide stem cell differentiation fate. Cells that attach to the substrate were found to exert contractile forces, causing tensile stresses in the cytoskeleton

1,2,17,18

. The relationship between these forces and mechanical

stiffness of the substrate have a major influence on cell migration, proliferation and differentiation. Relatively stiff substrates with bone-like properties were found to be osteogenic

19

. However, ALG/CS based scaffolds usually exhibited soft feature, due to the

hydrophilic nature of the two macromolecules, even though these scaffolds were evaluated to be effective in bone tissue engineering as the temporary residence. Thus, it is worth, as well as challenged to contract a stiff scaffold based on ALG and CS. In the present study, mechanical properties of scaffold A, scaffold B and scaffold C were evaluated. Like most currently practiced scaffolds based on ALG and CS, both scaffold A and scaffold B exhibited the soft feature when wetted in water. They were easily deformed under the loading (Fig. 3g). However, different from the soft nature of scaffold A and scaffold B, scaffold C exhibited significantly stiff and “hard-to-deform” feature even at wetted state (Fig. 3h). As the stress-strain curves shown, when strain was below 20%, the stress of wetted scaffold C was significant higher than that of scaffold A and scaffold B. The compressive modulus of scaffold C was 1.45 MPa (strain was 10%), much higher than that of scaffold A and scaffold B, both of which were lower than 0.05 MPa (Fig. 3i). Nevertheless, the 14 -Environment ACS Paragon-Plus

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toughness of scaffold C was poor, which was illustrated by the low damage point at a strain of 20%. Both scaffold A and scaffold B showed well performed deformability, which were not destroyed even when strain reached over 80%. For one thing, the strength and stiffness, especially the “hard-to-deform” feature of scaffold C were came from the expended networks. During processing, ALG network formed quickly. CS dissolved and fulfilled the space inside of ALG network. At the same time, the dissolved CS exerted swelling force to drive ALG network to an expansion state, which was strong enough to resist deformation. For another, the expanded networks were kept, fixed, especially enhanced by the cross-linking effect of Ca2+ and glutaraldehyde. Actually it was a double cross-linked networks, which further contributed to the stiff nature. However, the double cross-linking had to base on the ALG/CS preformed gel to develop the mechanical enhancement effect. Otherwise, although scaffold B underwent the same cross-linking management, it was still as soft as scaffold A. Because during the formation of ALG/CS polyelectrolyte, the ALG and CS molecule bonded together to cause the phase separation. Further cross-linking effect may destroy the electrostatic interaction but could not built a strong matrix structure. 3.5. Adhesive cell shape inside the different ALG/CS scaffolds The swelling behavior and mechanical properties of different ALG/CS scaffolds were evaluated as factors that affect the cellular adhesion. Then, mesenchymal stem cells (MSCs) were employed as the model cell to evaluate the effect of swelling behavior and mechanical properties on cellular shape after adhesion on the inner surfaces inside the scaffolds. As Fig. 4 shown, most MSCs with rounded profile attached on the surfaces of pores of all the scaffolds after 3 h post-seeding. However, pseudopodium was observed clearly in scaffold C (Fig. 4g). At 24 h post-seeding, MSCs in scaffold A still showed rounded shape. And

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individual cell could be observed adhering on the inner surfaces of scaffold. Unlike scaffold A, MSCs adhered on inner surfaces of scaffold B were dragged along the cellular long axis, showing a spindle-like shape. In scaffold C, most MSCs changed from rounded and spheroidal at 3 h post-seeding to flattened and polygonal morphologies, presenting lots of pseudopodium. When compared to MSCs in scaffold A and scaffold B, adhered cells in scaffold C showed the largest adhesive area, indicating that the MSC-matrix interaction in scaffold C was significantly up-regulated. To further confirm and clearly observe the cellular shape inside the three scaffolds, fluorescent staining of F-actins was carried out to show the cytoskeleton (Fig. 4j,k,l). Representative fluorescence images of F-actins clearly confirmed that MSCs with rounded shape and spindle-like shape on the surfaces of pores of scaffold A and scaffold B, respectively, while with flattened and polygonal morphologies in scaffold C at 24 post-seeding. Moreover, due to the larger adhesive area (Fig. 5a), fluorescence of cytoskeleton in scaffold C took wider area than that in scaffold A and scaffold B in the same size of visual field, indicating that there were more chances for cell-to-cell contacts in scaffold C. Accordingly, a sequence of physicochemical reactions happened after cell adhering to matrix surface. Proteins, such as Fn adsorption to matrix surface occurs firstly to mediate the cell adhesion and also provide signals to the cell through the cell adhesion receptors. Cells do not interact directly with the matrix but via proteins that absorbed on matrix surface 14,15,20,21. The matrix physicochemical parameters were known to influence the adhesion of proteins and cells. For one thing, hydration was recognized as a barrier to affect proteins absorption, thus cellular adhesion

14,15

. ALG and CS are polyhydrophilic materials, which share the

hydrophilic nature and electrical neutrality. Water that bonded to matrix through hydrogen bond and even more strongly ionic solvation weakened the cell-matrix interaction, resulting in the limited cellular adhesion. As shown in Fig. 5b, Fn, which was visualized as red


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fluorescence lights, was adsorbed significantly fewer in scaffold A than in the other two scaffolds. MSCs in scaffold A showed low tractions and low degrees of cell spreading, with rounded and spheroidal morphologies. Nevertheless, the water binding ability through strongly ionic solvation was restricted after Ca2+ and glutaraldehyde cross-linking. More significantly adsorbed Fn was observed well distributed on the surfaces of innerpores of scaffold B and scaffold C. The interactions between cells and matrices in scaffold B and scaffold C were thus significantly stronger. MSCs in scaffold B and scaffold C exhibited high tractions and high degrees of cell spreading. For another, MSCs adhered on scaffold B and scaffold C exhibited different morphologies, which might have relationship with the stiffness of matrices. Accordingly, matrices stiffness have great impact on cell adhesion probably through a traction-force-mediated inside-outsidein mechanotransduction pathway 16-18,22-26. Generally, increasing matrices stiffness promotes cellular adhesion. Briefly, adherent cells exert traction forces, which generated by actomyosin contractility and transmitted to extracellular space via integrin-mediated anchoring junction, to sense mechanical responses of matrices. According to the applied tension forces and the corresponding matrix deformation, adhesions reinforce or disassemble. On stiffer matrices of scaffold C, which was hard to deform, the adhesion reinforced, leading to the amplification of peripheral focal adhesions and reorganization of the actin cytoskeleton. Integrin was reported to provide the anchoring force for cell attachment to substrate α5β1 by Fn adsorption is essential for initial cell adhesion

27

. The activation of integrin

28,29

. As shown in Fig. 5c, the

intracellular protein expression of integrin α5 and β1 in scaffold C were significantly higher than those in scaffold B and scaffold A. The difference of integrin α5β1 expression in scaffold A and scaffold B may be attributed to the weak Fn adsorption of scaffold A induced by hydration effect. Since the Fn absorption was weakened, the Fn activated integrin α5β1 expression in scaffold A was thus down-regulated.



After adhesion, cells began to proliferate. The proliferation of MSCs was observed and quantified in Fig. 5d,e. As shown in Fig. 5d, cell membrane was stained fluorescently by DiO. At 7 d post-seeding, the fluorescence range of MSCs in scaffold A was the lowest. In scaffold B and scaffold C, Dio labeled cells spread along with the inner surfaces, exhibiting more extensive fluorescence of MSCs. According to DNA assay (Fig. 5e), cell number in scaffold A at 7 d was significantly lower than that in scaffold B and scaffold C. And there was no difference in cell number between scaffold B and scaffold C. However, the fluorescence range of MSCs in scaffold C was wider than that in scaffold B, which may be attributed to the larger adhesive area. At 14 d post-seeding, MSCs with rounded profile dispersed inside the scaffold A, showing insignificant proliferation. Cell number in scaffold A was still the lowest. However, in scaffold B and scaffold C, due to the significant proliferation of MSCs, the fluorescence ranges of MSCs were wider than those at 7 d. Cell number in scaffold B was the same as that in scaffold C. Unlike 7d, the fluorescence ranges at 14 d in scaffold B and scaffold C showed no significant difference. Moreover, cell-to-cell contacts in scaffold B and scaffold C were further evaluated according to the fluorescent staining of F-actins (Fig. 5d). Unlike the significant difference of the fluorescence images of F-actins between scaffold B and scaffold C at 24 h post-seeding (Fig. 5a), abundantly overlapped F-actins were found both in scaffold B and scaffold C at 7 d and 14 d post-seeding, showing no significant difference. 3.5. Osteogenesis of MSCs in different ALG/CS scaffolds Cell fate has been known to be related to matrix-regulated cell spreading shape. Cells with large adhesion areas preferred to the osteogenic differentiation. Here, after the 7 days in vitro osteogenic induction, representative images of Van Kossa and ALP staining were used to identify the osteogenic characteristics. As shown in Fig. 6a, Van Kossa staining of MSCs area of all histological sections gave rise to the brown color at 7 d, indicating calcium deposition. 18 -Environment ACS Paragon-Plus

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In accordance with the previous studies, the staining range in the stiff scaffold C was the widest among the three scaffolds, revealing that MSCs in scaffold C exhibited promoted osteogenesis when comparing to MSCs in soft scaffold B and scaffold A. ALP staining showed the similar results with Von Kossa staining in Fig. 6b. After 14 d, the staining became darkened and more widely distributed in all sections, revealing the up-regulation of osteogenesis. However, according to the histological staining, MSCs in scaffold A showed the lowest degree of calcium deposition. Besides, although calcium deposition in scaffold B was lower than that in scaffold C at 7 d, the difference reduced after 14 d. Furthermore, ALP and OCN gene expressions were evaluated to confirm the osteogenic differentiation. According to Fig. 6c,d, either at 7 d or 14 d, MSCs in scaffold C showed significantly higher gene expression of ALP and OCN than MSCs in scaffold A and scaffold B. At 7 d, ALP and OCN gene expression in scaffold B showed no significant difference when comparing with scaffold A, but significantly lower than that in scaffold C. ALP and OCN gene expression was up-regulated after 14 days’ induction, indicating the osteogenic differentiation of MSCs in scaffold A, scaffold B and scaffold C was enhanced. However, osteogenic gene expression of MSCs in scaffold A was still lower than that in scaffold B and scaffold C. Remarkably, the gap of osteogenic gene expression between scaffold B and scaffold C significantly narrowed. At the same time, the expression of OPG was monitored. OPG secreted by MSCs competitively inhibits RANKL, interrupting the maturation of osteoclasts and bone resorption. In the present study, MSCs in all the three scaffolds showed significant but similar OPG gene expression, which did not significantly change during 7 d and 14 d osteoblastic differentiation of MSC. The underlying mechanism of the shape-related osteogenesis in the present study may mainly involve the Fn activated integrin α5β1 and the mechanical features of matrix. Integrin α5β1 activated by Fn adsorption is essential for not only initial cell adhesion, but also the 19 -Environment ACS Paragon-Plus


subsequent osteogenesis gene expression 28,29. Stem cells interrogate matrix stiffness through traction forces that transmitted via integrin-mediated anchoring junction, making decisions on their lineage specifications after gauging the mechanical resistance of matrix to the forces 1,2,46

. Thus, the promotion of osteogenesis by stiff matrix should own to the high cell tension that

mediated by integrin α5β1. During mechanical coupling of MSCs to substrate mediated by the binding of Fn with integrin α5β1, the stiff scaffold C responded to cells with “hard-to-deform” information, leading to amplification of peripheral focal adhesions (large cell spreading area) thus inducing high tension of cells, consequently enhancing osteogenesis. At the same time, effect of Ca2+, which presented in scaffold B and scaffold C but in scaffold A, on osteogenesis of MSCs also should be taken into account. Thus, another scaffold showed in Fig. S2, named scaffold A/Ca2+ was fabricated by soaking scaffold A in 3% calcium chloride (CaCl2) solution for 15 min. The scaffold A/Ca2+ showed similar porous structure with scaffold A and similar rounded shape of attached MSC. After undergoing osteogenic induction at same condition, osteogenesis-related genes expression of MSCs in scaffold A/Ca2+, including ALP and OCN was detected to show no significant difference with that in scaffold A. But in scaffold B, scaffold C, as well as scaffold A/Ca2+ that consist of Ca2+, the significantly different osteogenesis-related genes expression illustrated the obvious cell shape-related effect on osteogenesis in the present study. The unapparent effect of Ca2+ here might be attributed to the few amount loaded in scaffolds and the presence of Ca2+ in culture medium. At last, towards tissue regeneration application, three dimensional scaffolds with porous structure were different from two dimensional substrates. Along with the in vitro proliferation for 14 d, cell density in scaffold B and scaffold C increased remarkably, taking over the inner spaces of the scaffold, leading to the increase of cell density, thus the promotion of cell-to-cell contacts, which contributed to intercellular interactions. Extensive intercellular interactions 20 -Environment ACS Paragon-Plus

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was reported to exhibit great effect on cell differentiation 30. However, the effects of high density resulted cell-to-cell contact were complicated 31. As illustrated by Ding J., there was a competition between size effect and contact effect along with the increase of cell density. In the case of high cell density in three dimensions, the cell-to-cell contact extent might be higher than that on two dimensional cultures due to more contact directions. The effect of cell-to-cell contact thus could be more significant in three dimensional in vitro cultures, partly making up for size effect. This might explain that at 14 d, the osteogenesis of MSCs in scaffold B was significantly up-regulated. In brief, the stiff and “hard-to-deform” matrix of scaffold C induced polygonal cellular adhesion with high degrees of cell spreading, leading to high tractions. The high traction then resulted in the promotion of osteogenic differentiation. Thus, the stiff-performed scaffold C based on ALG/CS exhibited the advantage to control the lineage commitment of MSCs into osteoblastic phenotype by regulating cell shape.

4. Conclusions In summary, based on ALG/CS polyelectrolyte suspension and ALG/CS preformed gel, scaffold A and scaffold B with soft feature, as well as stiffer scaffold C were fabricated to exhibit different swelling behavior and mechanical properties. Scaffold A, freeze-dried from ALG/CS polyelectrolyte suspension, possessed high swelling ratio and water retention ratio, weakened cellular adhesion, leading to the rounded shape of adhesive MSCs. Scaffold B, fabricated from scaffold A through being cross-linked using Ca2+ and glutaraldehyde, supported cellular adhesion with spindle-like shape. Scaffold C, freeze-dried from ALG/CS preformed gel, which was an ALG gel network expanded by swelling CS, and cross-linked using Ca2+ and glutaraldehyde, exhibited stiff and “hard-to-deform” nature, and forced MSCs to adhere to polygonal shape, thus promoting the osteogenesis of MSCs. Supporting Information 21 -Environment ACS Paragon-Plus


Subcutaneous implantation of scaffolds in vivo, cell shape and osteogenesis in scaffold A/Ca2+ (Document) Acknowledgements: The work was supported by the National Natural Science Foundation of China (Nos. 51503119, 51373094, 51473090), the Science and Technology Commission of Shanghai Municipality (No. 15JC1490400). Also, we acknowledge the Instrumental Analysis Research Centre (Shanghai University) for the use and direction of SEM. References [1] Guilak, F.; Cohen, D.M.; Estes, B.T.; Gimble, J.M.; Liedtke, W.; Chen, C.S. Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix. Cell Stem Cell 2009, 5, 17-26 [2] McBeath, R.; Pirone, D.M.; Nelson, C.M.; Bhadriraju, K.; Chen, C.S. Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment. Dev. Cell 2004, 6, 483-495. [3] Peng, R.; Yao, X.; Ding, J. Effect of Cell Anisotropy on Differentiation of Stem Cells on Micropatterned Surfaces through the Controlled Single Cell Adhesion. Biomaterials 2011, 32, 8048-8057. [4] Engler, A.J.; Sen, S.; Sweeney, H.L.; Discher, D.E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126, 677-689. [5] Miranda Coelho, N.; Gonzalez-Garcia, C.; Planell, J.A.; Salmeron-Sanchez, M.; Altankov, G. Different Assembly of Type IV Collagen on Hydrophilic and Hydrophobic Substrata Alters Endothelial Cells Interaction. Eur. Cells Mater. 2010, 19, 262-272; [6] Gittens, R.A.; McLachlan, T.; Olivares-Navarrete, R.; Cai, Y.; Berner, S.; Tannenbaum, R.; Schwartz, Z.; Sandhage, K.H.; Boyan, B.D. The Effects of Combined Micron/Submicron-scale Surface Roughness and Nanoscale Features on Cell Proliferation and Differentiation. Biomaterials 2011, 32, 3395-3403.


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[7] Ayala, R.; Zhang, C.; Yang, D.; Hwang, Y.; Aung, A.; Shroff, S.S.; Arce, F.T.; Lal, R.; Arya, G.; Varghese, S. Engineering the Cell-Material Interface for Controlling Stem Cell Adhesion, Migration, and Differentiation. Biomaterials 2011, 32, 3700-3711. [8] Huebsch, N.; Arany, P.R.; Mao, A.S.; Shvartsman, D.; Ali, O.A.; Bencherif, S.A.; RiveraFeliciano, J.; Mooney, D.J. Harnessing Traction-Mediated Manipulation of the Cell/Matrix Interface to Control Stem-Cell Fate. Nat. Mater. 2010, 9, 518-526. [9] Li, Z.; Ramay, H.R.; Hauc,h K.D.; Xiao, D.; Zhang, M. Chitosan-Alginate Hybrid Scaffolds for Bone Tissue Engineering. Biomaterials 2005, 26, 3919-3928. [10] Florczyk, S.J.; Kim, D.J.; Wood, D.L.; Zhang, M. Influence of Processing Parameters on Pore Structure of 3D Porous Chitosan-Alginate Polyelectrolyte Complex Scaffolds. J. Biomed. Mater. Res. A 2011, 98, 614-620. [11] Florczyk, S.J.; Leung, M.; Li, Z.; Huang, J.I.; Hopper, R.A.; Zhang, M. Evaluation of Three-dimensional Porous Chitosan-Alginate Scaffolds in Rat Calvarial Defects for Bone Regeneration Applications. J. Biomed. Mater. Res. A 2013, 101, 2974-2983. [12] Amir Afshar, H.; Ghaee, A. Preparation of Aminated Chitosan/Alginate Scaffold Containing Halloysite Nanotubes with Improved Cell Attachment. Carbohydr. Polym. 2016, 151, 1120-1131. [13] Francis Suh, J.K.; Matthew, H.W.T. Application of Chitosan-based Polysaccharide Biomaterials in Cartilage Tissue negineering: A Review. Biomaterials 2000, 21, 2589-2598. [14] Lih, E.; Oh, S.H.; Joung, Y.K.; Lee, J.H.; Han, D.K. Polymers for Cell/Tissue AntiAdhesion. Prog. Polym. Sci. 2015, 44, 28-61. [15] Chen, S.; Li, L.; Zhao, C.; Zheng, J. Surface Hydration: Principles and Applications toward Low-fouling/Nonfouling Biomaterials. Polymer 2010, 51, 5283-5293. [16] Young, D.A.; Choi, Y.S.; Engler, A.J.; Christman, K.L. Stimulation of Adipogenesis of Adult Adipose-derived Stem Cells Using Substrates that Mimic the Stiffness of Adipose Tissue. Biomaterials 2013, 34, 8581-8588. 23 -Environment ACS Paragon-Plus


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[17] Missirlis, D.; Spatz, J.P. Combined Effects of PEG HydrogelElasticity and CellAdhesive Coating on Fibroblast Adhesion and Persistent Migration. Biomacromolecules 2014, 15, 195-205. [18] Ye, K.; Wang, X.; Cao, L.; Li, S.; Li, Z.; Yu, L.; Ding, J. Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate. Nano Lett. 2015, 15, 4720-4729. [19] Huebsch, N.; Lippens, E.; Lee, K.; Mehta, M.; Koshy, S.T.; Darnell, M.C.; Desai, R.; Madl, C.M.; Xu, M.; Zhao, X.; Chaudhuri, O.; Verbeke, C.; Kim, W.S.; Alim, K.; Mammoto, A.; Ingber, D.E.; Duda, G.N.; Mooney, D.J. Matrix Elasticity of Void-forming Hydrogels Controls Transplanted-Stem-Cell-Mediated Bone Formation. Nat. Mater. 2015, 14, 12691277. [20] Grieshaber, S.E.; Farran, A.J.E.; Bai, S.; Kiick, K.L.; Jia, X. Tuning the Properties of Elastin

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Figure 1. Schematic representation for the preparation of ALG/CS precursor matrices for the soft scaffolds and stiff scaffold. For soft scaffold, pH was adjusted slowly to 4-5 to support the ALG/CS polyelectrolyte suspension formation. For stiff scaffold, a new preparation strategy was created. CS dissolution was designed to take place inside the ALG gel, so to provide swelling pressure to expand the ALG network to an expansion state. After freezedrying, the networks were fixed by Ca2+ and glutaraldehyde to yield the stiff scaffold.


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Figure 2. Evaluation of the different ALG/CS precursor matrices during the preparation of different types of scaffolds. (a) general view of the ALG/CS polyelectrolyte, ALG gel and ALG/CS preformed gel; (b) FT-IR spectra of CS, ALG, ALG/CS polyelectrolyte and ALG/CS preformed gel; (c) Frequency dependence of complex viscosity |η*| of ALG solution, ALG gel, ALG/CS polyelectrolyte suspension, ALG solution/CS power and ALG/CS 27 -Environment ACS Paragon-Plus


preformed gel; (d) storage modulus/loss modulus (Gꞌ / Gꞌꞌ) of ALG/CS preformed gel and ALG/CS polyelectrolyte suspension; (e) storage modulus/loss modulus (Gꞌ / Gꞌꞌ) of ALG solution and ALG gel.

Figure 3. Features of the three scaffolds. SEM images of scaffold A (a), scaffold B (b), scaffold C (c); the swelling ratio (d), water retention ratio (e) of the three scaffolds; (f) CCK-8 assay for evaluation of the relative cytotoxicity of scaffolds extracts; (g) scaffolds bearing 0.5 kg weight; Compression tests for the wetted scaffolds at 25 °C: (h) the stress-strain curves and (i) the compressive modulus. (*p < 0.05, **p < 0.01)


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Figure 4. Observation of cell shape and cytoskeleton. SEM image of MSCs adhered on the inner surfaces of scaffold A (a-c), scaffold B (d-f) and scaffold C (g-i). a, d, g at 3 h postseeding; b, e, h at 24 h post-seeding; c, f and i were higher-magnification images selected from b, e and h, respectively; fluorescent staining of F-actins of MSCs adhered on the inner surfaces of scaffold A (j), scaffold B (k) and scaffold C (l). (Bar scales: 100 µm for a, b, d, e, g, h; 30 µm for c, f, i, j, k, l)



Figure 5. Cytoskeleton and proliferation of MSCs in scaffolds. (a) representative fluorescence images of F-actins in scaffolds at 24 h post-seeding; (b) fluorescence images of Fn adsorbed in scaffolds; (c) Expression of integrin α5 and β1 genes; (d) Dio prelabeled MSCs adhered and spread on the pore surfaces of scaffold A, scaffold B and scaffold C at 7 and 14 d postseeding; (e) DNA assay to show the proliferation of MSCs; (f) representative images of Factins in scaffold B and scaffold C at 7 and 14 d, respectively. (Bar scales: 50 µm for a, f; 30 -Environment ACS Paragon-Plus

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100 µm for b,d) (* means the data was significantly different with the others at the same time point; All the data in c showed significant difference with the others among the three scaffolds)

Figure 6. Osteogenic differentiation of MSCs in the three scaffolds. (a) Von Kossa staining and (b) ALP staining of histological sections selected from samples underwent 7 d and 14 d osteogenic induction; ALP, OCN and OPG gene expression of MSCs in scaffold A, scaffold B and scaffold C at 7 d (c) and 14 d (d). (Bar scales: 100 µm for all) (*p < 0.05) (All the data in d showed significant difference with the others except OPG)



Graphical Abstract


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Chitosan Based Scaffolds, but

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