In vitro evaluation of osteoblast adhesion, proliferation and differentiation on chitosan-TiO2 nanotubes scaffolds with Ca2 + ions

Accepted Manuscript In vitro evaluation of osteoblast adhesion, proliferation and differentiation on chitosan-TiO2 nanotubes scaffolds with Ca2+ ions

Siew Shee Lim, Chun Ye Chai, Hwei-San Loh PII: DOI: Reference:

S0928-4931(16)30886-4 doi: 10.1016/j.msec.2017.03.075 MSC 7586

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

18 August 2016 6 January 2017 10 March 2017

Please cite this article as: Siew Shee Lim, Chun Ye Chai, Hwei-San Loh , In vitro evaluation of osteoblast adhesion, proliferation and differentiation on chitosan-TiO2 nanotubes scaffolds with Ca2+ ions. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.075

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ACCEPTED MANUSCRIPT In Vitro Evaluation of Osteoblast Adhesion, Proliferation and Differentiation on Chitosan-TiO2 nanotubes Scaffolds with Ca2+ Ions Siew Shee Lim1*, Chun Ye Chai2, Hwei-San Loh2,3* 1

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Department of Chemical with Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga 43500, Semenyih Selangor Darul Ehsan. 2 School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga 43500 Semenyih, Selangor Darul Ehsan 3 Biotechnology Research Centre, University of Nottingham Malaysia Campus, Jalan Broga 43500 Semenyih Selangor Darul Ehsan.

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Co-corresponding author emails: [email protected], [email protected]

ABSTRACT

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Hydrothermally synthesized TiO2 nanotubes (TNTs) were first used as a filler for chitosan scaffold for reinforcement purpose. Chitosan-TNTs (CTNTs) scaffolds prepared via direct blending and freeze drying retained cylindrical structure and showed enhanced compressive modulus and reduced degradation rate compared to chitosan membrane which experienced severe shrinkage after rehydration with ethanol. Macroporous interconnectivity with pore size of 70-230 μm and porosity of 88% were found in CTNTs scaffolds. Subsequently, the functionalization of CTNTs scaffolds with CaCl2 solutions (0.5 mM - 40.5 mM) was conducted at physiological pH. The adsorption isotherm of Ca2+ ions onto CTNTs scaffolds fitted well with Freundlich isotherm. CTNTs scaffolds with Ca2+ ions showed high biocompatibility by promoting adhesion, proliferation and early differentiation of MG63 in a non-dose dependent manner. CTNTs scaffolds with Ca2+ ions can be an alternative for bone regeneration.

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Keywords: Chitosan-titanium dioxide nanotubes (CTNTs), calcium ions, adsorption, osteoblast, biocompatibility.

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Introduction Bone tissue engineering seems to be a promising technology to overcome the limitations

of current bone grafts associated with limited source, immunological rejection and infection (Zhao et al., 2010). It is a technology that combines the principles of medical science and engineering to repair or recover damaged tissue by the substitution with scaffolds. Scaffold 1

ACCEPTED MANUSCRIPT serves as an artificial extracellular matrix (ECM) which mimics the extracellular environment by providing appropriate environmental conditions for intercellular contact and signaling. With high porosity and interconnected network, scaffolds allow cell penetration and adhesion and support structure (Navarro et al., 2006). They are always embedded with growth factors and have an appropriate degradation rate which matches with bone formation (Kim et al., 2011).

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With advanced technology, common materials such as synthetic polymeric materials,

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naturally derived polymers and composite materials have been under extensive investigation as

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artificial scaffolds (Kim et al., 2011). Chitosan is a semi-synthetic polymer derived from chitin which is widely distributed in exoskeleton of insects, fungi and crustaceans. It is a favorable material for tissue engineering, as it causes minimal foreign body reaction and would not trigger

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inflammation and of high biodegradability (Kim et al., 2008). However, it has to combine with other materials to form an ideal scaffold due to its low mechanical strength and

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osteoconductivity (Venkatesan & Kim 2010).

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Metallic titanium (Ti) is frequently used for orthopedic implantation because of its low toxicity, high mechanical strength, and good biocompatibility. Ti usually has a layer of titanium

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dioxide (TiO2). As Ti is bioinert, the presence of TiO2 layer confers the necessary bone-bonding ability (Das et al., 2008). Especially, TiO2 nanotubes (TNTs) washed with calcium acetate

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promoted a profound bone-forming ability (Kasuga 2006; Kubota et al. 2004). It was hypothesized that TNTs with adsorbed calcium (Ca) ions probably mediate cell adhesion and

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promote bone formation and hence this current study was conducted. Several previous studies also proved that Ca and phosphorous (P) ions involve in osteoblast differentiation (Ma et al.,

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2005, Maeno et al., 2005). In fact, the concentration of extracellular Ca2+ ions plays an important role in the regulation of osteoblast proliferation and differentiation through calcium/calmodulin signaling pathway (Zayzafoon 2006). In the present study, the incorporation of TNTs into chitosan matrix via direct blending was attempted and expected to improve the compressive modulus of chitosan scaffolds. The biocompatibility of such newly developed scaffolds was subsequently elevated via the liquidsolid adsorption of Ca precursor solution, as TNTs with higher specific surface area were hypothesized to provide adsorption sites for Ca2+ ions. The adsorption affinity of new chitosan composite scaffolds towards Ca2+ ions was determined and verified which would facilitate the 2

ACCEPTED MANUSCRIPT explanation on the profound bone-forming ability of TNTs as prepared by Kasuga (2006) and Kubota et al. (2004). With the adsorbed Ca2+ ions, these newly developed composite scaffolds were expected to exhibit more biologically inspired properties by promoting better osteoblastic functions on osteosarcoma cell lines (MG63). In particular, the dosage effect of Ca2+ ions on

Materials and Methods

2.1

Materials

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scaffolds in terms of adhesion, proliferation and differentiation was investigated.

Titanium dioxide (TiO2) powder, NaOH pellets, CaCl2 powder, chitosan powder (middle

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viscous, 80% deacetylated), sodium bicarbonate, sodium pyruvate, β-glycerophosphate, pnitrophenyl phosphate (pNPP), fluorescein diacetate (FDA), propidium iodide and dimethyl

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sulphoxide (DMSO) were purchased from Sigma Aldrich (Germany). The solvent used was analytical grade of acetic acid, hydrochloric acid and ethanol purchased from Merck (Germany),

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Sigma Aldrich (Germany) and R&M chemicals (UK), respectively. Ca/Mg hardness kit was purchased from HACH (USA). Phosphate buffered saline was prepared by dissolving sodium

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chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic, monopotassium and phosphate which were purchased from Sigma Aldrich (Germany) in distilled water. 24-well

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plates and 96-well plates were purchased from Orange Scientific (Belgium). Minimum essential media (MEM), Fetal bovine serum (FBS), penicillin/streptomycin, 0.25% trypsin and 3-(4, 5

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dimethylthiazol-2-yl)-2, 5-diphenyltetrazolim bromide (MTT) were purchased from Gibco (USA). Ascorbic acid was purchased from Merck (Germany), while Pro-Prep Protein Extraction

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Solution was purchased from iNtRON Biotechnology (South Korea). The deionised water was filtered by Mili-Q integral water purification unit. All chemicals were of analytical grade and used without further purification.

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Fabrication of chitosan and chitosan-TNTs (CTNTs) scaffolds TNTs were synthesized via hydrothermal synthesis. Two grams of TiO2 was mixed with

40ml of 10M NaOH homogenously on a hot plate (Heidolph, Germany) for 5 h before being subjected to hydrothermal reaction at 130oC for 72 h (Chen et al., 2002). The resultant powder was filtered and washed with deionized water and 0.01M HCl until neutral pH. Powder was then 3

ACCEPTED MANUSCRIPT dried at 55oC and heated to 400oC at 10oC/min for 5 h. One gram of chitosan flakes was stirred with 100ml of 0.2M acetic acid for 3 h. One mililitre of pure chitosan solution was transferred into each well of 24-well plate and frozen at -20°C for 24 hours. For the chitosan-TNTs (CTNTs) scaffold preparation, 16 weight percent (wt%) of TNTs was directly blended with chitosan solution for 5h. One mililitre of the colloidal mixture was also pipetted into each well of 24-well plate and stored at -20oC for 24h. Both solidified chitosan solution and the colloidal mixtures of

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chitosan and TNTs were then subjected to 24-h freeze drying at -40oC. Scaffolds were

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rehydrated in a series of ethanol by immersing them with 100% ethanol, 70% ethanol and 50%

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ethanol for 1h, 30 min and 30 min, respectively. The use of ethanol in the rehydration process served a purpose of sterilization which is required for cell-based evaluations. Scaffolds were

Microstructure characterization of TNTs, chitosan and chitosan-TNTs (CTNTs) scaffolds

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dried prior to characterization and functionalization with CaCl2 solution.

The particle size and internal structure of heated powder were verified by using

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Transmission Electron Microscope (Model No: Philips HMG 400 TEM, Netherlands) at an accelerating voltage of 100 kV. Surface morphology and pore size of chitosan scaffolds and

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chitosan-TNTs (CTNTs) scaffolds were studied by using Field Emission Scanning Electron Microscope (FESEM) at an accelerating voltage of 10-20kV. The estimation of pore diameter

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was obtained by analysis of digital FESEM images. Diameter and thickness of scaffolds were measured with a Digimatic micrometer to calculate the bulk volume of scaffolds. The skeletal

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volume of the scaffolds was determined by adopting the measurement protocol as developed by Tan et al. (2011) and measured by using a pycnometer (Laborglas, Germany). Subsequently, the

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porosity of each scaffold was determined using Equation (1):

 V  Porosity  1   skeletal  x100%   Vbulk 

(1)

where Vskeletal was the skeletal volume of scaffolds (cm3) and Vbulk was the bulk volume of scaffolds (cm3). Compressive modulus of scaffolds was determined using a mechanical tester (Model No. H25KS, USA). Scaffold was compressed by a load cell of 500N applied at crosshead speed of 1 mm/min until 60% of its original thickness was achieved (Takahashi et al., 2005). The 4

ACCEPTED MANUSCRIPT compressive modulus for scaffold was obtained as the slope of initial linear part of the stress against strain plot. Scaffolds were immersed in Phosphate Buffered Saline (PBS) solution for 14 days at pH 7.2 and 37 °C (Teng et al., 2008). The initial weight of scaffold was measured and recorded. After 14-day immersion, wet scaffold was taken out and rinsed with deionised water for three times. The rinsed scaffold was then subjected to 24-h freeze drying at -40°C followed by weight measurement. Any weight change in the scaffold was represented as the relative

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weight loss. Each test was performed in triplicate and the data reported were represented as

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means ± standard deviations.

Functionalization of scaffolds with CaCl2 solution via liquid-solid adsorption

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Scaffolds were immersed in 20ml of different concentrations of calcium chloride (CaCl2) solution: 0.5mM, 1.5mM, 4.5mM, 13.5mM and 40.5mM for functionalization. The pH of CaCl2

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solutions was adjusted to 7.2. Scaffolds were equilibrated in CaCl2 solutions at 100 rpm for 24h. The adsorption of Ca2+ ions onto scaffolds was determined by measuring the changes in

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concentration of solution before and after adsorption using Ca/Mg hardness kit. By using Equation (2), the amount of Ca2+ ions adsorbed onto scaffolds was calculated. After

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functionalization, scaffolds were washed with deionised water and dried prior to in vitro tests.

(Co - Ce) x V qe =

(2)

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M where qe is the amount of Ca2+ ions adsorbed by per gram scaffold (mg/g), Co and Ce are the

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concentrations of CaCl2 solutions before and after functionalization (mg/L), respectively, V is the volume of CaCl2 solution (L) and M is the dry mass of scaffold (g). The adsorption of Ca2+ ions onto scaffolds were analysed by using Langmuir, Freundlich and Temkin isotherm models. Langmuir isotherm assumes monolayer adsorption on homogenous surface with finite number of adsorption sites. The mathematical representation of Langmuir isotherm is shown by Equation (3):

Ce C 1   e qe bq m q m

(3)

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ACCEPTED MANUSCRIPT where qe represents equilibrium adsorption capacity per unit weight of scaffold (mg/g), qm is the maximum adsorption capacity of per unit weight of scaffold for Ca2+ ions (mg/g), Ce is the equilibrium liquid-phase concentration (mg/L) and b is the Langmuir isotherm constant (L/mg). Freundlich isotherm is used to represent multilayer adsorption on heterogeneous surface with the frequency of site associated with free energy of adsorption decreases exponentially with

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the increase of the free energy (Santoso et al., 2007). The Freundlich isotherm is represented in

1 ln Ce n

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ln qe  ln K F 

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Equation (4):

(4)

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where KF is the adsorption capacity of scaffolds (mg/g(L/mg)1/n) and 1/n represents the adsorption intensity or surface heterogeneity (Ho et al., 2000).

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Temkin isotherm takes into the account of the interaction between Ca2+ ions. The heat of adsorption of all molecules in the layer would decrease linearly with coverage due to

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adsorbate/adsorbate interactions (Hosseini et al., 2003). This model is also used for

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chemisorptions based on strong electrostatic interaction between positive and negative charges (Ma et al., 2008). The mathematical representation of Temkin model is shown in Equation (5). (5)

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qe  B ln K t  B ln Ce

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where Kt is an equilibrium binding constant (L/mg) corresponding to the maximum binding energy and B is related to the heat of adsorption.

In vitro cell culture

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Cell studies were conducted by using human MG63 cells (ATCC, USA), an osteosarcoma cell line. Cells were maintained using minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate and 1% Penicillin/Streptomycin, incubated at 37oC in a humidified incubator with 5% CO2. For passaging, MG63 cells were detached by using 0.25% trypsin and plated in culture flask. When 90-100% confluence was achieved, cells were ready for seeding. Cells were detached via similar trypsinization, centrifuged at 1800 rpm for 5 min followed by resuspension in medium. Cell 6

ACCEPTED MANUSCRIPT numbers were calculated by using a hemocytometer and then diluted to 6x104cells/ml. Before seeding, scaffolds were placed in 24-well plate and sterilized under UV irradiation for 15 min. Approximately 3x104cells were pipetted on each scaffold and polystyrene surface of the 24-well plate. Untreated (without Ca2+ adsorption) scaffolds with cells and cells on polystyrene (polystyrene) served as controls for the assessment of Ca2+-treated scaffolds. Each well was then fed with another 500µl culture medium followed by incubation in standard culture condition for

Cell adhesion assay

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pre-determined incubation periods. Culture medium was replaced every two-three days.

The adhesion of MG63 was investigated by using Fluorescein diacetate (FDA)/ Propidium iodide

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(PI) staining to determine the number of viable and dead cells on untreated scaffolds, Ca2+treated scaffolds and control (polystyrene). After seeding cells, samples were incubated for 4h to allow cell adhesion. Each sample was washed with PBS twice and then 1ml of staining solution

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containing 12.5µg/ml of FDA and 2µg/ml PI was added to each well. After 5-min incubation, staining solution was discarded and washed with PBS once. The scaffold was then observed

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under a fluorescence microscope (Nikon AZ100, Japan).

3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolim bromide (MTT) assay

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After 3, 5, and 7 days of incubation period, the culture medium was removed from each well. One mililitre of fresh culture medium and 100 µl of MTT solution (5mg/ml) were added to

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each well for further 4-h incubation. MTT was reduced by mitochondrial succinate dehydrogenase and formed an insoluble and dark purple formazan. Then, 1 ml of dimethyl

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sulphoxide (DMSO) was added to dissolve the formazan formed in each well. An aliquot resulting solution of 300 µl was transferred to a 96-well plate in triplicate, and the absorbance at 570 nm was measured using Varioskan™ Flash Multimode Reader. Three independent experiments for each prescribed time period were performed.

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Alkaline phosphatase (ALP) assay Osteoblast early differentiation marker, ALP was used to evaluate the osteoblast

differentiation property in the present study. Complete growth medium was supplemented with 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. Samples were collected after 7, 14, 21 7

ACCEPTED MANUSCRIPT and 28 days of incubation period. The culture medium was removed from each well and then washed with PBS twice. Subsequently, untreated scaffolds, Ca2+-treated scaffolds and polystyrene were scraped and incubated with 500µl of PRO-PREP Protein Extraction Solution on ice following manufacturer’s instructions to obtain protein lysates. ALP activity was calculated as the rate of p-nitrophenyl phosphate (pNPP) hydrolyzed by ALP into p-nitrophenol. In a 96-well plate, 100 µl of pNPP was added to 100 µl of cell lysis and incubated for 2 h at

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37oC. The absorbance was then measured at 405 nm by using Varioskan™ Flash Multimode

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Reader to detect for the p-nitrophenol. The experiments for each incubation time were

Statistical analysis

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performed in triplicate.

Results were presented as mean ± SD. The collected quantitative data of in vitro assays were analyzed by using one-way ANOVA. Least Significant Difference (LSD) and Duncan tests

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were done as post tests. P < 0.05 was considered as statistically significant.

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Results and Discussion

3.1

Microstructural characterization of chitosan and CTNTs scaffolds

The particle size and internal structure of TNTs were examined by using TEM as shown in Fig. 1 (a). Open-ended nanotubes with a diameter of 10-20 nm and length of few hundred nanometres

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to(a)1 micron were clearly seen. Chitosan (CTNTs) scaffold with 16 (b) scaffold and chitosan-TNTs(c) wt% of TNTs were fabricated and characterised in terms of their pore size, porosity, compressive

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modulus and degradation rate. Fig. 1 (b) shows a FESEM image of a chitosan membrane instead

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of three-dimensional (3D) scaffold. After rehydration with ethanol, chitosan scaffold lost its integrity and its whole structure experienced a severe shrinkage. Due to its loss of cylindrical structure, the pore size and porosity of chitosan scaffold were not possibly measured. Unlike the 400 μm

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pure chitosan membrane, CTNTs scaffold showed an interconnected 200 μm

Fig. 1. (a) TEM image of TNTs. (b) FESEM image of chitosan scaffold. (c) FESEM image of chitosanTNTs (CTNTs) scaffolds. (d) Compressive modulus of chitosan membrane and CTNTs scaffolds. (e) Degradation percent of chitosan membrane and CTNTs scaffolds after 14-day immersion in PBS.

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ACCEPTED MANUSCRIPT porous structure as seen in Fig. 1 (c). The pore size of CTNTs scaffold ranged from 70 μm to 230 μm. Evidently, the incorporation of TNTs into chitosan matrix aided to establish the macroporous structure and interconnectivity of scaffolds. Besides, CTNTs scaffold with approximately 88% total porosity exhibited compressive modulus of 2.1 MPa (Fig 1 (d)). The compressive modulus of CTNTs scaffold was much higher than that of chitosan membrane. After 14-day immersion in PBS, the degradation percent of chitosan membrane was

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about 10%. On the other hand, the degradation rate of CTNTs scaffold was only 4%. Again, the

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incorporation of TNTs in chitosan matrix greatly reduced the degradation rate of scaffolds by

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more than 2 folds. Based on these reinforced properties, CTNTs scaffold met the physical requirement as an ideal scaffold and was expected to facilitate cellular infiltration and allow the

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degradation rate of scaffolds to correspond with the formation of new bone (Karageorgiou and Kaplan 2005). This microstructural characterization section also concluded that 1% (v/v)

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chitosan solution was not sufficient to retain the cylindrical structure of scaffold. As a result, pure chitosan membrane was not further functionalized with Ca2+ ions and tested in any in vitro

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tests.

Adsorption isotherm

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CTNTs composite scaffolds were further functionalised with different concentrations of

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CaCl2 solution via 24-h batch adsorption at room temperature and pH 7.2. The amount of Ca2+

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ions adsorbed onto scaffolds (Qe) and the concentration of CaCl2 after adsorption (Ce) were

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Qe (mg/g)

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40.5 mM

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1.5 mM 0.5 mM 400

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Fig. 2. Adsorption capacity of Ca2+ ion by CTNTs scaffolds with different concentrations of CaCl2 solution. From left to right of the curve, the equilibrium concentration ranges of CaCl2 for initial concentration were 0.5mM, 1.5 mM, 4.5 mM, 13.5 mM and 40.5 mM, respectively.

measured. The adsorption isotherm of Ca2+ ions onto scaffolds (Fig. 2) showed a favorable trend.

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The amount of Ca2+ ions uptaken by scaffolds increased gradually from 2.21 mg/g to 17.85 mg/g corresponding to the initial concentration (Co) of CaCl2 starting from 50.05 mg/L (0.5 mM) to

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4053.84 mg/L (40.5 mM). Increase in initial concentration of CaCl2 facilitated sufficient driving force to overcome the resistance to the mass transfer of Ca2+ ions between aqueous and scaffolds

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(Srivastava et al., 2006). Type II isotherm was established between Ca2+ ions and chitosan-TNTs scaffolds. The multilayer adsorption in this study was initiated by monolayer of adsorbed Ca2+

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ions which is represented as the knee at the initial part of isotherm curve (Fig. 2).

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ACCEPTED MANUSCRIPT 3.2.1 Langmuir, Freundlich and Temkin isotherms The adsorption of Ca2+ ions onto the CTNTs scaffolds were analysed by using Langmuir, Freundlich and Temkin isotherm models. The correlation coefficient values (R2) derived from each isotherm model were compared to determine the applicability of isotherm models and the nature of adsorption. Graphs representing Langmuir, Freundlich and Temkin isotherms are

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shown in Fig. 3 (a), (b) and (c), respectively. The respective slope and intercept for each graph

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were also determined and are presented in Table 1. (b)

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(a)

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Fig. 3. (a) Langmuir (b) Freundlich and (c) Temkin isotherms for the adsorption of Ca2+ ions onto CTNTs scaffolds at room temperature and pH 7.2.

The adsorption of Ca2+ ions on CTNTs composite scaffolds in this study was novel and verified. An approximation of R2 (0.9709) close to unity in Freundlich isotherm represents the well correlation between the experimental data and this model. The values of K (1.3148) and n (3.006) were indicative of normal cooperative isotherm and heterogeneous in nature. The dissociation of water molecules conferred negative charges on TiO2 surface. The negatively charge Ti-OH groups then attracted Ca2+ ions via electrostatic interaction (Svetina et al., 2001). This was later followed by the multilayer formation of Ca2+ ions on CTNTs scaffolds. These 12

ACCEPTED MANUSCRIPT finding further verified the adsorption affinity of CTNTs scaffolds towards Ca2+ ions and explained the profound bone-forming ability of TNTs neutralized with calcium acetate in work by Kasuga et al. (2006) and Kubota et al. (2004).

Table 1.

Isotherm parameters for adsorption of Ca2+ ions onto CTNTs scaffolds. R2

Constants

Langmuir

qm (mg/g)=19.194 b (L/mg)=0.00455

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n= 3.006 KF (mg/g(L/mg)1/n)= 1.3148

0.9709

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Kt =351.0996 B = 0.3383

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0.8996

Cell adhesion

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Fig. 4 shows the results of FDA/PI-stained MG63 cells after 4-h incubation to investigate the osteoblast adhesion on scaffolds. The cell size of MG63 was measured at approximately 20

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µm. Fig. 5 shows the numbers of cell adhered on scaffolds per mm2 surface area. Cells grown on

polystyrene surface (no scaffold control) showed the highest viable and dead cell densities which

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were 49 ± 5.29 and 22 ± 3 cells per mm2, respectively. This was probably attributed to the poly-

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D-lysine coating of the culture plate which promoted the optimal cellular growth.

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FDA a

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4.5 mM CaCl2

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13.5 mM CaCl2

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Untreated scaffold

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Polystyrene

Fig. 4. Fluorescence images of viable and dead MG63 cells via staining of FDA (a – d) and PI (e – h) dyes on CTNTs scaffolds treated with 13.5 mM (a & e), 4.5 mM CaCl2 (b & f), polystyrene surface (c & g) and untreated scaffolds, (d & h), respectively after 4 h. The size of cell measured was 20 µm. Scale bars represent 1 mm.

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ACCEPTED MANUSCRIPT While cells on CTNTs scaffolds receiving 13.5 mM CaCl2 treatment had the highest viable cell and low dead cell densities amongst treated scaffolds which were 32.7 ± 3.51 and 8.3 ± 0.57 cells per mm2, respectively. The extracellular Ca2+ ions on scaffolds treated with 13.5 mM CaCl2 triggered the ligand binding with the integrin receptors that mediated cell adhesion (Leitinger et al., 1999). It is noteworthy to mention that viable cells on treated scaffolds could not be accurately quantified, as some cells had penetrated into such three-dimensional constructs

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instead of adhering on the top part of scaffolds.

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untreated polystyrene scaffold

CaCl2 concentration

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Cell number per mm²

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Fig. 5. The number of viable and dead cells on scaffolds per mm2 surface area after 4-h incubation. Comparisons of viable cells (a & b) and dead cells (aa & bb) between Ca2+-treated scaffolds and other groups. a: P < 0.05, significant difference of viable cells grown on polystyrene surface. b: P < 0.05, significant difference of viable cells grown on untreated scaffolds. aa: P < 0.05, significant difference of dead cells laid on polystyrene surface. bb: P < 0.05, significant difference of dead cells laid on untreated scaffolds.

The viable cell density on untreated scaffolds was approximately 15 times lower than that on treated scaffolds due to the acidic nature of untreated scaffolds after direct blending with chitosan solution. Primarily, the untreated CTNTs scaffolds are more acidic than treated

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ACCEPTED MANUSCRIPT scaffolds. The functionalization of CTNTs scaffolds with CaCl2 solution has changed the pH of the treated scaffolds to a neutral pH. They showed viable and dead cell densities of 2.67 ± 1.53 and 22 ± 3 cells per mm2, respectively. Scaffolds with difficulty in securing cell adhesion always fail to promote other osteoblastic behaviors. This finding further explains the low biocompatibility of untreated scaffolds in subsequent assays. Therefore, the acidic nature of untreated scaffolds necessitates the functionalization of scaffolds via liquid-solid adsorption

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which in turn increases the biocompatibility of scaffolds.

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Mechanism of Ca2+ ions on CTNT scaffolds for promoting adhesion is in fact not well understood. The initial adhesion of osteoblast is important for cell and scaffold interactions which in turn affect the ability of cells to proliferate and differentiate. Cell adhesion is mainly

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mediated by integrin binding to ECM proteins such as fibronectin, collagen and vitronectin. The surface characteristics of scaffold such as roughness, chemical composition and electrical charge

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determine how these ECM proteins would be adsorbed on surface (Barrère et al., 2006). Cationic surface enhances the adsorption of negatively charged ECM cell-adhesive proteins such as

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fibronectin and vitronectin. As an example, calcium-incorporated titanium enhanced osteoblast adhesion through promoting the adsorption of ECM proteins (Feng et al., 2004). In this study,

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CTNTs scaffolds with Ca2+ ions were deduced to attract these cell adhesive proteins when placed in culture medium. It was reported that transforming growth factor-beta 1 (TGF-beta1)

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stimulates intracellular Ca2+ signal in osteoblast leading to the induction of alpha5 integrin expression. Then, it enhances an interaction between fibronectin and alpha5 integrin resulting in

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cell adhesion (Nesti et al., 2002).

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Osteoblast proliferation Osteoblast proliferation was examined using MTT assay on the 3rd, 5th and 7th days for

the CTNTs scaffolds treated with five different concentrations of CaCl2 solution, untreated scaffolds and polystyrene surface (no scaffold control). Fig. 6 shows that there was significantly lower (P < 0.05) cellular proliferation rate in untreated scaffolds as compared to polystyrene

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control across all the time points tested. Similarly, cells grew much better on scaffolds treated

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with various concentrations of CaCl2 solution than the untreated counterparts. On the 3rd day of culture, the optical density value observed in scaffolds treated with 40.5 mM CaCl2 was

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significantly higher (P < 0.05) than that of the control cells on polystyrene surface and untreated scaffolds. Besides, it was found that only scaffolds treated with 4.5 mM CaCl2 possessed a slight

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higher rate than that of the polystyrene control on the 5th day of culture, whereas, all the other treated scaffolds showed a lower proliferation rate as compared to this control. While on the 7th

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day of culture, a lower proliferation rate was noticed in 1.5 mM, 4.5 mM and 13.5 mM CaCl2

2.00 bbb

1.80 1.60 1.20 1.00 0.60 0.40 0.20 0.00

aaa

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treated scaffolds as compared to that of treated with 40.5 mM CaCl2.

0.5mM

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Fig. 6. Proliferation rates of MG63 measured by MTT assay on the 3rd (a & b), 5th (aa & bb) and 7th (aaa & bbb) days of culture grown on scaffolds treated with different concentrations of CaCl2. a, aa, aaa: P < 0.05, significant difference of cells grown on polystyrene surface and treated scaffolds on the 3rd, 5th and 7th days, respectively. b, bb, bbb: P < 0.05, significant difference of cells grown on untreated scaffolds and treated scaffolds on the 3rd, 5th and 7th days, respectively. 17

ACCEPTED MANUSCRIPT From the MTT finding, we observed that there was a lower cellular proliferation rate for the untreated scaffolds as compared to that of control cells grown on polystyrene surface. Cells grew better on the smoother polystyrene surface than rough surface. In comparison to scaffolds without functionalization, cells grown on scaffolds with adsorbed Ca2+ ions exhibited a significant higher proliferation rate. These results suggested that Ca2+ ions have promoted osteoblast proliferation. Maeno et al. (2005) investigated the effect of Ca2+ ions on osteoblast

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proliferation and differentiation at monolayer and 3D culture by using mouse primary osteoblast.

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By incubating the cells embedded in collagen gel with various concentrations of Ca2+ ions

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containing media, they found that 2-6 mM of Ca2+ ion was suitable for osteoblast proliferation; 6-8 mM was good for osteoblast differentiation and higher concentration, i.e. > 10mM was

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however becoming cytotoxic. Chattopadhyay and coworkers (2004) also studied the effect of extracellular Ca2+ ions in term of promoting proliferation. They proved that the increase in

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extracellular Ca2+ ions lifted up the intracellular Ca2+ ions via calcium sensing receptors (CaSR). CaSR induced cyclin D expression in response to 5 mM Ca2+ ions and enhanced osteoblast proliferation (Chattopadhyay et al., 2004). Contrasting to these previous studies, our experiment

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design was using CTNTs scaffolds to immobilize Ca2+ ions before seeding the cells and scaffolds

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acted as reservoirs for Ca2+ ions. In the functionalization of 40.5mM, 13.5mM and 4.5mM CaCl2, only a small portion of Ca2+ ions was adsorbed which did not cause any significant

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cytotoxic effect to cells. CTNTs scaffolds with adsorbed Ca2+ ions in this study promoted cell

Osteoblast differentiation

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proliferation, but they did not show a prominent dose-dependent manner.

ALP is an early marker of osteoblast differentiation which is important in bone formation and mineralization. Fig. 7 shows the cellular ALP activity on CTNTs scaffolds treated with five different concentrations of CaCl2 solution, untreated scaffolds and polystyrene surface on the 7th, 14th, 21st and 28th days. On 7th day of culture, 4.5 mM Ca2+-treated scaffold and control cells grown on polystyrene showed the highest ALP activity. When progressing to 14th day of culture, ALP activities of cells grown on scaffolds were dramatically raised to 3 folds over the 7th-day ALP activity. This indicates bone cells grown on CTNTs scaffolds have initiated the expression of mature osteoblast properties. The 40.5 mM CaCl2 treated scaffolds showed the highest ALP 18

ACCEPTED MANUSCRIPT activity. Yet, 13.5 mM and 4.5 mM CaCl2 treated scaffolds had also exhibited similar results. These three scaffolds that were treated with higher CaCl2 concentration indicate that Ca2+ ions play a role in mediating osteoblast differentiation. Throughout the ALP course of study, control cells grown on polystyrene surface did not show a raise in ALP activity indicating that these cells

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Fig. 7. Alkaline phosphatase (ALP) activity of osteoblast cells during 28 days of culture on polystyrene surface, untreated scaffolds and scaffolds treated with different concentrations of CaCl2. Each bar represents the mean value of ALP activity ± SD. a: P < 0.05, significant difference of cellular ALP activity in comparison between polystyrene control and treated scaffolds. b: P < 0.05, significant difference of cellular ALP activity in comparison between untreated and treated scaffolds after 14 days of culture. The scaffolds receiving 40.5mM CaCl2 treatment had the highest differentiation rate.

Steadily, ALP activity of cells grown on treated scaffolds declined on 21st and 28th days. It is suggested that there was a development of more differentiated osteoblast to mineralize ECM. ALP synthesis was downregulated, while osteopontin (OPN) and osteocalcin (OCN) were induced to mineralize ECM (Owen et al., 1990). Due to the lack of understanding on culture conditions that trigger the mineralization of MG63, terminal differentiation and mineralization cannot be studied by using MG63 cells conforming to a previous suggestion (Lincks et al., 1998). 19

ACCEPTED MANUSCRIPT During bone formation, it can be divided into four periods of development (adhesion, proliferation, differentiation and mineralization). There are two transition points of gene expression for cell proliferation and differentiation. First phase is after the completion of proliferation and initiation of ECM maturation, when ALP is upregulated. The second phase initiates with the ECM mineralization (Aronow et al., 1990). The role of Ca2+ ions in osteoblast differentiation under the effects of other hormones are not clearly understood. From the 7th to 14th

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days, it is suggested that osteoblast proliferation was ceased and differentiation started. ALP

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activity reached a peak level on 14th day indicates that MG63 cells on scaffolds have

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differentiated and started to express the encoded proteins for ECM. As compared to untreated scaffold, higher CaCl2 concentrations treated scaffolds, namely 4.5 mM, 13.5 mM and 40.5 mM

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had significant increase in promoting differentiation while the other two concentrations of 1.5 mM and 0.5 mM also had slight improvement in promoting differentiation. This result

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corresponds with other studies that Ca2+ ions induced differentiation (Maeno et al., 2005). Ma et al. (2005) also revealed how dissolved Ca2+ ions from hydroxyapatite regulated osteoblast response. They found that osteoblast precursor cell lines, human embryonic palatal mesenchyme

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cells grown in media containing high Ca2+ ions showed a significantly higher ALP activity and

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OCN. While Zayzafoon et al. (2005) showed an increase in extracellular Ca2+ ions activated the Ca2+/calmodulin-dependent protein kinase IIα, this induced expression of c-fos, AP-1 activation

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and promoted differentiation of resting osteoblast. In addition, Kubota et al. (2004) showed that after the 7th day of insertion of pelletized TNTs with Ca2+ ions into rats, the formation of new

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osteoblasts.

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bone was observed. Most importantly, cells adjacent to the new bone were identified as

Conclusion The results of this study demonstrated the potential of CTNTs scaffolds functionalized

with Ca2+ ions in bone tissue engineering. Newly developed scaffolds with macroporosity and improved mechanical properties met the physical requirement of scaffolds and outperformed the pure chitosan membrane. Besides, CTNTs scaffolds exhibited heterogeneous nature and adsorption affinity towards Ca2+ ions by showing a close approximation with Freundlich isotherm. CTNTs scaffolds functionalized with Ca2+ ions promoted adhesion, proliferation and 20

ACCEPTED MANUSCRIPT differentiation of osteoblast-like cells, MG63 but not in a dose-dependent manner. In conclusion, CTNTs scaffolds with Ca2+ ions can be an effective method in enhancing bone tissue regeneration by accelerating the proliferation and differentiation of osteoblast cells. Yet, some fundamental studies in future elucidating the adhesion, proliferation and differentiation

Acknowledgements

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mechanisms are still required in order to obtain a definite answer.

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Authors would like to extend our gratitude towards funding bodies. This work was supported by Ministry of Science Technology and Innovation (03-02-12-SF0002) and University

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of Nottingham Malaysia Campus.

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Vitae of authors

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Siew Shee Lim

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Siew Shee Lim received her Bachelor and Master degrees in Chemical Engineering from State University of New York at Buffalo (SUNY, Buffalo). She completed her PhD (Chemical Engineering) at the University of Nottingham Malaysia Campus in 2015. She is now an assistant professor in University of Nottingham Malaysia Campus and her current research work focuses on the fabrication of nanocomposite scaffolds for bone regeneration.

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Chun Ye Chai

Chun Ye Chai obtained a first class in Bachelor’s honours degree in Biotechnology from The University of Nottingham in 2014. During his final year of first degree, he conducted his research project on nanotechnology field. Some parts of his study have been described in this research article. Other than his interest in nanotechnology, he also mastered in various molecular biological techniques.

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Hwei-San Loh

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Sandy Hwei-San Loh is a Professor of The University of Nottingham Malaysia Campus, a Fellow of the Higher Education Academy, UK and has been lecturing in the molecular biology and biotechnology areas for ten years. Her current research interests include bioproduction of high value pharmaceuticals and industrial proteins via plant molecular pharming strategies; drugs discovery from natural products; combinatorial treatments for cancers; applications of nanomaterials for the developments of new therapeutics, scaffolds and biosensors.

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ACCEPTED MANUSCRIPT Highlights

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CTNTs scaffolds showed improved compressive modulus and reduced degradation rate. CTNTs scaffolds showed adsorption affinity towards Ca2+ ions. Adsorption isotherm of Ca2+ ions on CTNTs scaffolds fit with Freundlich isotherm. CTNTs scaffolds with Ca2+ ions promoted adhesion, growth and differentiation.

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