Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

Biomaterials 34 (2013) 7073e7085 Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomateri...

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Biomaterials 34 (2013) 7073e7085

Contents lists available at SciVerse ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro Greeshma Thrivikraman a, b, Prafulla K. Mallik a, Bikramjit Basu a, * a b

Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore 560012, India Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore 560012, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2013 Accepted 29 May 2013 Available online 21 June 2013

In designing and developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of the substrate conductivity on cell fate processes is rather limited. In order to address this issue, spark plasma sintered HA-CaTiO3 (Hydroxyapatite-Calcium titanate) has been used as a model material system to showcase the effect of varying conductivity on cell functionality. Being electroactive in nature, mouse myoblast cells (C2C12) were selected as a model cell line in this study. It was inferred that myoblast adhesion/growth systematically increases with substrate conductivity due to CaTiO3 addition to HA. Importantly, parallel arrangement of myoblast cells on higher CaTiO3 containing substrates indicate that self-adjustable cell patterning can be achieved on conductive biomaterials. Furthermore, enhanced myoblast assembly and myotube formation were recorded after 5 days of serum starvation. Overall, the present study conclusively establishes the positive impact of the substrate conductivity towards cell proliferation and differentiation as well as confirms the efficacy of HA-CaTiO3 biocomposites as conductive platforms to facilitate the growth, orientation and fusion of myoblasts, even when cultured in the absence of external electric field. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Substrate conductivity Cell proliferation and differentiation Myoblast and myotube alignment Biomaterial Cell and myotube density

1. Introduction In the recent years, research in the field of biomaterials for orthopedic applications has been focused to develop new strategies for better and effective osseointegration of implant materials into the host. In its narrowest sense, the cellesubstrate interaction has been proven to regulate cellular fate processes in the vicinity of the implants [1,2]. Some biological tissues exhibit a wide range of electrical activities for maintaining cellular homeostasis and modulating molecular events, engaged in development, adaptation, repair and regeneration of tissues [3e5]. Specifically, cardiac, neural, bone and muscle tissues use the mechanism of electrical conductivity i.e. accumulation and flow of charges (bioelectricity) to regulate its physiological behavior and to propagate electrical potentials through their cellular components [6e8]. For example, in the process of bone regeneration, piezoelectric property of the collagen is hypothesized to generate electric field involved in bone remodeling. This, in turn, establishes the fact that electrical

* Corresponding author. E-mail address: [email protected] (B. Basu). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.05.076

stimulation offers repair and healing of tissues in the clinical situation, where normal healing is impaired [5]. Limited studies came up showing the efficacy of electrical stimulation in inducing cellular response in a variety of cell types, including fibroblast, osteoblast, myoblast, neuronal cells etc. [9]. Therefore, designing electrically conductive biomaterials is of major interest amongst researchers due to its outstanding ability to interface with bioelectric fields in cells and tissues to simulate normal electrophysiology of the body. A few studies have reported the use of electrically active bioceramics and polymers as substrates for implantation to bring about enhanced biological response [10,11]. Jun et al. [12] demonstrated that electrically conductive PLCL/PANi nanofibrous construct stimulated myoblast proliferation and differentiation in the absence of external electric field, highlighting the suitability of these substrates as target cues to control cellular functions [12]. Even though earlier studies suggest the non-suitability of stiffer substrates for myoblast growth and differentiation [13,14], the current study was designed to use HA-CaTiO3 bioceramics and C2C12 myoblast cell line as a simplistic model for examining the effect of substrate conductivity on functionality of smooth muscle cells. This is because of the fact that orthopedic implants not only have access to surrounding bony tissues, but also are exposed to the

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adjacent skeletal muscle cells, which can be adversely affected by implant-related factors (physical, chemical and electrical) acting over a period of healing time [15]. As a result, the effect of implantation on neighboring biological environment continues to be an intensive area of clinical research. Sun et al. made an attempt to evaluate the effect of calcium phosphate bioceramics on adjacent skeletal muscles, when used as bone substitutes [15]. They inferred the inhibitory effect of HA and b-TCP on the cell attachment and growth rate of myoblast, indicating the possibility of the substrates to interfere with repair and regeneration of skeletal muscles injured during surgery at the time of implantation. Till date, in spite of the progress in the development of various synthetic and natural biomaterials, ceramic material such as hydroxyapatite has been receiving wide recognition in clinical research, due to its excellent bioactive and osteoconductive nature, but it has low electrical conductivity property [16,17]. In contrast, natural bone has significant electrical properties and develops a potential under mechanical stress to induce osseous formation and healing [18]. In this study, we used a combination of bioactive HA and electroactive CaTiO3 (CT) to develop cytocompatible composites with enhanced electrical conductivity. Moreover, we adopted multistage spark plasma sintering technique that employs lower temperature and shorter duration to make HA-CT composite substrates. Given the potential of electroconductive biomaterials for orthopedic applications, the major issue being addressed in this paper is the effect of electrical conductivity on the behavior of myoblast cells seeded on HA-CT composites. In vitro studies such as cellular adhesion, spreading, proliferation and differentiation of C2C12 myoblast were carried out on HA-CT composites using myoblast cells. In a nutshell, the present study will demonstrate how myoblast cells behave on hydrophobic stiff bioceramic substrates in a conductivity dependent manner.

2. Materials and methods 2.1. Powder synthesis Hydroxyapatite Ca10(PO4)6(OH)2 was synthesized using suspensionprecipitation route, one of the most commonly accepted method [16]. The powders used were CaO (SD Fine-Chem) and H3PO4 (Merck). Initially, CaO was dispersed in distilled water at a concentration of 18.6 g/l. The dispersed solution was kept on a hot plate with electromagnetic stirrer for continuous stirring of the dispersed medium. By keeping the Ca/P ratio as desired (1.67), an equivalent amount of 0.17 M H3PO4 solution was added drop wise in the dispersed CaO solution. The above set up was held at 80  C and stirred for 3e4 h to allow the reaction to occur completely. Thereafter, concentrated NH4OH (Qualikems) was added drop wise to bring the pH of the solution to 10. For synthesizing 25 g of HA, about 5e10 ml of NH4OH is required. After the completion of the reaction, the product was allowed to precipitate by keeping it at room temperature for 24 h which was collected by filtration. When the precipitate was dried, the lump was powdered in an agate mortar and subsequently calcined at 800  C for 2 h. The XRD analysis of the sample was taken which ensured the presence of all characteristic peaks corresponding to hydroxyapatite (not shown). Crystalline calcium titanate (CaTiO3) was synthesized by mechanical activation of a mixture of CaO and TiO2 (anatase). Stoichiometric proportions of the powders were mixed in a planetary ball mill (Fritsch, Puloverisette 1583, Germany) for 6 h. The powder to ball ratio was 1:4 and 50% of the jar volume was occupied by the wet mixing medium, acetone. The powder was collected by filter paper, dried in hot air oven at 70  C and calcined at 850  C for 2 h, followed by sintering at 950  C for 4 h to obtain pure CaTiO3 phase. The effect of milling on the solid-state reaction and the purity of the prepared powder were confirmed by X-ray diffraction studies (not shown). 2.2. SPS sintering and composite fabrication In order to prepare composite powders of varying compositions, appropriate amounts of CaTiO3 (40e80 wt%) were wet ball milled with HA for 16 h. Two purposes were achieved by ball milling, one is the proper mixing of the powders and the other is the reduction in particle size. The sintering of the milled powder was performed in the SPS (Dr. Sinter, Model 515S, SPS syntax Inc, Japan) under vacuum in the temperature range of 950e1200  C. High DC pulse current was passed between the graphite electrodes and simultaneously uniaxial pressure in range of 40e60 MPa was applied from the beginning of the sintering cycle. The sintering behavior was

Fig. 1. SEM-BSE images showing the polished microstructure to illustrate CaTiO3 distribution in HA matrix in two investigated composites: (a) HA40CT and (b) HA80CT. Also, the EDS spectrum taken from the region marked on each BSE image is provided below the respective image.

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monitored by measuring the change in the axial dimension of the compact body. After final stage of holding at sintering temperature, the sample was allowed to cool in the vacuum chamber to less than 500  C at a cooling rate of 100  C/min. Various HA-CT compositions were sintered via multi-stage sintering schedule by holding the powder compacts at two intermediate temperatures of 950  C and 1100  C, followed by holding at final sintering temperature of 1200  C with hold time of 5 min at each temperature. The rate of heating was kept constant at 100  C/min for each cycle. It is to be noted that the net heating time was kept constant in all the sintering experiments. The relative densities of the resulting samples were measured by Archimedes’ method. “Relative density” refers to the density of the sintered ceramic sample, which is calculated based on the formula,

rs ¼ rbulk =rtheo  100% where rs, rbulk and rtheo are relative density, bulk density and X-ray theoretical density respectively. The optimized sintering conditions and the relative densities of the sintered pellets are summarized in Table 1. After sintering, the samples with higher sinter densities (>98%) were chosen for cytocompatibility assessment, to leave out the possibility of the influence of porosity on cellematerial interaction. The use of porous HA-CT substrates in the present study would otherwise have brought two parameters: porosity and conductivity. In order to investigate the sole influence of substrate conductivity alone, almost fully dense HA-CT substrates were used, since the porosity is known to degrade the conductivity property. It was otherwise difficult to study the beneficial role of CaTiO3 addition to conductivity enhancement and consequently its influence on cell functionality and differentiation. 2.3. Bioceramic substrate characterization The surfaces of the SPSed samples were ground and polished using standard metallographic techniques. The surface roughness and topography of the HA-CT composites were analyzed by Atomic force microscopy (AFM) (A100 SGS, APE Research nanotechnology, Italy). The observations were performed in non-contact mode using silicon-nitride (Si3N4) cantilevers, scanned over 10  10 mm2 area. The polished SPS sintered HA-CT samples were etched under argon ion beam to visualize the microstructural features. The etched samples were then imaged using Scanning Electron Microscope (SEM, FEI Inspect F20) equipped with an energy dispersive spectrometer (EDS) system to study the distribution of CaTiO3 in HACaTiO3 composites. The surface chemistry of the composites were analyzed using Kratos X-ray photoelectron spectrometer using standard Al Ka excitation source (hn ¼ 1486.6 eV). The binding energy (BE) scale was calibrated by measuring reference peak of C 1s (BE ¼ 284.8 eV) from the surface contamination, with a precision of about 0.1 eV. XRD analysis of the samples were carried out using CuKa radiation operated at 30 kV and 40 mA (Brucker Xpert diffractometer) at a scan rate of 0.5 /min for 2q varying from 20 to 80 with a step size of 0.02 to characterize the phases present in the samples. The surface wettability was studied by measuring contact angles using a sessile drop method with both distilled water and complete culture media (DMEMþ10% FBS) deposited on a HA-CT sample surface. The wettability inspection was performed optically using goniometer (Dataphysis, Germany) and an inspection microscope that combine together charge-coupled device (CCD) camera and digital imaging techniques. The measured contact angle not only provides information about the wettability of a HA-CT composites surface but also reveals the contribution of surface roughness and chemical heterogeneity to the observed wettability state. During the experiment, volume of the liquid was kept constant (9 ml) all over the specimen surface. The wettability investigations were carried out with an accuracy of 1 at room temperature and a humidity of 35  5%. For AC conductivity measurement, the polished samples of 1e2 mm thickness were coated with silver paste, which acts as electrodes for the sample. The capacitance and dielectric loss were measured as a function of frequency (100 Hze1 MHz) at room temperature by precision impedance analyzer (Wayne Kerr 6500B, UK). The samples were heated at a heating rate of 2  C/min. AC conductivity (sac) was calculated using the following relationship,

sac

 G  d ¼ Ohm: cm1 A

where, conductance G ¼ uCD, d is the thickness, C is the capacitance, A is the area of the sample and D is the dielectric loss. 2.4. Cell culture All the in vitro cell culture experiments were performed using C2C12 mouse myoblast cell line, procured from National Centre for Biological Science (NCBS), Bangalore. Cells were revived from cryopreserved stock and expanded in Complete media containing Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 20% fetal bovine serum (FBS; Invitrogen), 1% antibiotic antimycotic solution (Sigma) and 2 mM L-glutamine (Invitrogen). Cells were maintained in a humidified CO2 incubator at 37  C (Sanyo, MCO-18AC, USA) and media change is

Fig. 2. (a) XRD plot showing the phase assemblage in SPS processed materials and (b) the frequency dependent conductivity property of the investigated materials at a temperature close to cell incubation temperature.

done every two days. Once when the cells reached 70e80% confluency, they were detached from the culture flask using 0.05% trypsin-EDTA (Invitrogen) and subcultured for further use. All the cell culture experiments were conducted with cells at passage of 2e7. The differentiation of myoblast cells was induced by replacing complete media with DMEM containing 1% FBS, after it reaches almost 90% confluency. It was then cultured under serum starvation condition for 5 days, to attain complete fusion of myoblast to myotubes. The formation of myotubes on the ceramic samples was analyzed by observing the morphological features under scanning electron microscope. The number of myotubes present were counted and averaged from 5 representative images of each samples. Myotubes of length greater than 200 mm only were considered for counting, to avoid false positives. Similarly, the

Table 1 Sintering conditions and relative density of multi-stage spark plasma sintered (MSSPS) of HA, CT, HA-40%wtCT and HA-80%wt CT ceramics. Samples

Sintering temperature ( C) and holding time (min)

Sintering pressure (MPa)

Relative density (% rth)

Ca10(PO4)6(OH)2 (HA) CaTiO3 (CT)

850  C (5 min)/950  C (5 min)/1100  C (5 min) 950  C (5 min)/1100  C (5 min)/1200  C (5 min) 950  C (5 min)/1100  C (5 min)/1200  C (5 min) 950  C (5 min)/1100  C (5 min)/1200  C (5 min)

30

99.4

50

98.2

50

99.0

50

98.6

HA-40 wt%CaTiO3 (HA-40CT) HA-80 wt%CaTiO3 (HA-80CT)

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dimensions of myotubes such as length and width were quantified using Image J software and averaged from 5 representative SEM images per biomaterial substrate. 2.5. Cell adhesion studies Cylindrical pellets of about 1 cm diameter and 1.5 mm thickness were used for the cell culture experiments. All the samples were sterilized in steam autoclave,

followed by washing with 70% ethanol and PBS respectively before cell seeding. For the cell adhesion test, about 5  104 cells were seeded onto each substrate placed in 24 well plate. After the stipulated time of culture, the samples were washed with PBS and fixed with 1.5% glutaraldehyde (Loba Chemie, India) in PBS for 30 min at 4  C. A series of ethanol wash (30, 50, 70, 90, and 100%) was done subsequently to dehydrate the samples completely. The dried samples were then sputter-coated (Vacuum Tech, Bangalore, India) with gold and were observed under scanning electron microscope (Philips, Quanta).

Fig. 3. XPS analysis showing (a) Wide scan spectra of HA, HA 40CT, HA 80CT and CT samples (b) deconvoluted high resolution peaks obtained for Ca2p, O1s and Ti2p.

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Table 2 Binding energy (eV) values obtained from XPS spectra of various HA-CT samples with C1s (284.8 eV) as reference binding energy for calibration. Samples

Binding energy (eV) Ca2p Ca2p3/2

Atomic ratio P2p

O1s O2

Ca2p1/2

Ti2p OH

Ads.H2O

Ti2p3/2 Ti

HA HA 40CT

347.1 347.1

350.7 350.7

133.0 133.2

HA 80CT

346.8

350.4

133.0

CT

346.7

350.3

e

530.9 531.1(HA) 529.8(CT) 531.2(HA) 530.3(CT) 530.1



Ti2p1/2 Ti



Ti



Ti3þ

Ti4þ



Ti

531.8 531.6

532.7 532.8

e 457.7

e 458.4

e 463.0

e 464.1

e 0.28

e 0.72

532.1

532.7

457.9

458.5

463.7

464.4

0.22

0.78

531.8

532.8

457.7

458.7

463.2

464.3

0.21

0.79

Fig. 4. AFM analysis showing a) the three dimensional overview of surface topography of HA-CT composites (b) surface roughness (Rms) of 10  10 mm2 area on HA-CT surfaces.

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2.6. MTT assay Cell viability test was performed using MTT (3(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Sigma) assay on pure HA, Pure CT and HA-CT composites. C2C12 myoblast cells of approximately 5  l04 cells/ml were seeded on the sterilized samples placed in 24 well plates and incubated for 1, 2 and 3 days in CO2 incubator at 37  C. After the desired incubation period, the medium in the well plate was removed and washed twice with PBS, followed by addition of 15% MTT reagent (Sigma) prepared in DMEM (without phenol red) for 5 h. The purple colored formazan crystals developed on the cells were solubilized using DMSO (Merck) and the optical density was measured at 595 nm in a microplate reader (iMark, Bio-rad laboratories, India). The amount of the formazan crystals measured by the absorbance at 595 nm is directly proportional to the number of metabolically active C2C12 mouse myoblast cells on the substrate. The cell viability was calculated using the following equation, Mean absorbance of the sample  100 % Viability ¼ Mean absorbance of control

Table 3 Table showing the list of contact angle values and image of water and DMEM droplets on the surface of HA, CT and HA-CT composites. Contact angle ( )

Image of drops

Water

DMEM

Water

HA

77.4  1.8

53.3  1.4

CT

82.8  3.8

51.6  2.0

HA40CT

56.7  1.4

49.4  0.6

HA80CT

69.5  0.9

57.5  0.8

Samples

DMEM

2.7. Cell morphological analysis Cell proliferation and orientation of the C2C12 mouse myoblast cells were studied using fluorescence microscopy (Nikon Eclipse, model LV100D, Japan). C2C12 cells, grown on ceramic samples and control were fixed with freshly prepared 4% paraformaldehyde (PFA). After 20 min, PFA was removed and washed with PBS. In order to permeabilize the cells, the samples were treated with 0.1% Triton X solution for 8e10 min. Again after thorough washing with PBS, the samples were incubated in 1% BSA to prevent non-specific binding of fluorescent dyes. To detect the expression of myogenin, the constructs were incubated at room temperature for 3 h in Mouse Anti-Human Myogenin Monoclonal Antibody (Santa Cruz Biotechnology, Inc.) after 1:100 dilution. The secondary antibody used was FITC conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.) for 1 h at a dilution of 1:500. In order to visualize the actin filaments, the cells were stained with Alexa Fluor 488 (Invitrogen) for 20 min and counterstained with Hoechst stain 33342 (Invitrogen) to visualize the cell nuclei. Cell density was calculated manually by taking the average cell count (from 5 images) and dividing it by the total area (per cm2), using Image J software. 2.8. Statistical analysis Statistical analysis was performed by using SPSS-16.0 (IBM, USA) software. At least 3 samples of each composition were used (n ¼ 3) for all the experiments and it was repeated for at least 3 times. The analyzed data were plotted as mean  standard deviation. Comparison of cell number and cell viability over time, on different composition were carried out by mixed model two-way ANOVA, followed by Bonferroni’s post-hoc test, whereas univariate ANOVA was used for rest of the statistical analysis. Values of p < 0.05 were considered as statistically significant.

3. Results

any regular patterns. In other words, the spark plasma sintered materials, as investigated in the present study, do not show any oriented morphology or texture in terms of CaTiO3 distribution. The phase assemblage of SPSed HA-CT composite samples was analyzed using XRD. The majority of the XRD peaks matched with the main peaks for HA and CaTiO3, as shown in Fig. 2a. Two additional peaks were also noticed in the sintered HA40CT and HA80CT, which did not correspond to either HA or CaTiO3. An absence of a and b-tricalcium phosphate (Ca3(PO4)2) and CaO peaks were observed along with the presence of only few weak peaks of unreacted TiO2 in the XRD spectrum of CaTiO3. X-ray diffraction patterns, as shown in Fig. 2a, clearly reveal the characteristic peaks for HA and CaTiO3, thereby confirming the formation of HA-CaTiO3 composites without any phase dissociation or new phase. AC conductivity values for various HA-CaTiO3 composites, measured at room temperature as a function of frequency, is shown in Fig. 2b. With the exception of HA, a systematic increase in conductivity with frequency is recorded for all the investigated compositions. However, such increase is most significant in case of HA80CT and CT. For HA, frequency dependent conductivity increase is rather weak and only recorded at above 1 kHz frequency. Also, a maximum increase in conductivity of HA by one order of magnitude is recorded with increase in frequency from 1 kHz to 1 MHz. In sharp

3.1. HA-CaTiO3 biomaterial substrate properties Substrate compositional properties of the investigated HA-CT substrates were studied using an array of characterization tools like XRD, AFM, SEM, Impedance analyzer and XPS. As mentioned in subsection 2.2, the investigated materials have less than 2% open porosity (see Table 1). Fig. 1 shows the backscattered electron images of the typical microstructures of HA 40CT and HA 80CT composites along with their corresponding EDS spectra. The bright contrast depicts the CaTiO3 phases (with higher mean atomic number) that are distributed in a darker matrix, which is detected as HA, having comparatively lesser mean atomic number. Elemental analysis of the darker region in Fig. 1a (enclosed in white) revealed the presence of large amount of calcium and phosphorous, validating HA as the major phase in HA 40CT composites. Similarly, the EDS spectrum collected from selected regions of HA 80CT (Fig. 1b), allowed detection of both calcium and titanium as the major constituents, with lesser amount of phosphorous, thereby confirming the phase with bright contrast as CaTiO3. It is apparent from both the images (Fig. 1a and b) that the fine microstructures of CaTiO3 having grain size in the range of 0.5e2.0 mm are homogenously dispersed, without forming

Fig. 5. Contact angles of investigated samples measured with deionised (DI) water and DMEM complete culture media.

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contrast, almost five to six orders of magnitude increase in conductivity is measured with CT and HA80CT in the frequency space of 0.1 kHze1 MHz. For HA40CT, a sharp increase in conductivity is observed above 100 kHz. Overall, the addition of 40% CaTiO3 to HA does not increase conductivity to a significant extent and the conductivity of HA80CT closely follows the frequency dependent increase like CT. Around two orders of magnitude difference is grossly maintained between HA80CT and CT over the investigated frequency region. Such a frequency dependent increase in AC

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conductivity at room temperature is mainly caused by the ionic polarization of CaTiO3. These results imply that higher amount of CaTiO3 loading enhances the electrical properties of HA-CaTiO3 systems to a significant extent. Besides, the lower conductivity value of HA 40CT correlate well with the conductivity of natural bone which is in the range of 109 U1 cm1 [19] and the highest conductivity value of CT approximates that of muscle tissue, which is in the order of 103 U1 cm1 [20]. Such a matching conductivity of HA-CT substrates with that of the bone and muscles finds great

Fig. 6. Fluorescence image of myoblast cells proliferated on (a) HA (b) HA 40CT (c) HA 80CT (d) CT, for a time period of 24 h. (e), (f), (g) and (h) represents the fluorescent images captured after 72 h of culture on HA, HA 40CT, HA 80CT and CT, respectively.

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application of these materials as potential orthopedic implants. In addition, CaTiO3 is reported to be involved in apatite growth and in enabling osseointegration and osteoconductivity [21]. The wide scan XPS spectra of various HA-CT specimens (Fig. 3a), detected the presence of elements such as Ca, P, O and Ti that are characteristics of HA and CT, with a low concentration of surface contaminants like adventitious carbon and nitrogen. A slow scan of Ca2p showed a doublet which was deconvoluted to Ca2p1/2 and Ca2p3/2 whose respective binding values are listed in Table 2. The symmetric P2p peak referenced at 133 eV was identified for all composites except CT. The typical O1s spectrum of HA sample was deconvoluted into three main peaks, namely O2(PO3 4 ) at 531, OH at 531.6e531.8 and adsorbed H2O at 532.5e 533.2 eV, respectively. The extra peak at 529.9e530.3 eV in HA 40CT and HA 80CT can be attributed to the signals from lattice oxygen (O2) of CT. The shape (Fig. 3b) and binding energy values (Table 2) of the Ti2p spectra of HA 40CT and HA 80CT were nearly identical to those of CT, showing the existence of two oxidation states namely Ti4þ and Ti3þ. From the calculated atomic ratios, it can be deciphered that around 25% of Ti3þ exists along with Ti4þ. Such a reduction of Ti4þ in all the CT samples would have happened during the spark plasma sintering process. Even so, the ratio of Ti3þ to Ti4þ remains the same in various CT containing samples, illustrating identical surface chemistry on all composite surfaces (see Table 2). The topographical image and average roughness of the HA-CT sample surfaces were captured using AFM. The three dimensional representation of the surface morphology, shown in Fig. 4a, depicts almost similar features for all the four examined samples. The measured roughness values for HA, HA 40CT, HA 80CT and CT were found to be 33.2  8 nm, 31.2  7.9 nm, 29.8  4.8 nm and 39.2  8.8 nm respectively. It is also clear from the plot (Fig. 4b) that, the surface roughness values are comparable to each other and does not indicate any statistically significant variation with increase in CT content. Overall, the surface of HA-CT samples were extremely smooth, with a roughness value of <50 nm. As far as the elastic stiffness property of HA-CaTiO3 substrates is concerned, the elastic modulus, measured using nanoindentation varies in the range of 80e120 GPa. This indicates that the substrates are relatively stiffer and it is therefore of interest to assess how myoblast cells grow and differentiate on such elastically stiffer substrates. The average contact angles of HA-CaTiO3 composite and monolithic ceramic surfaces are shown in Table 3. Sessile drop method was generally adopted to determine the contact angle of SPSed ceramic surfaces. It involves the deposition of a drop of liquid on a flat surface and observing the wetting of such sessile drop on the surface. The contact angle is formed between the solid/liquid interface and the vapour/liquid interface, which is determined optically. For HA with 40% and 80% CaTiO3, the contact angles obtained were 56.7  1.4 and 69.5  0.9 in water and 49.4  0.6 and 57.5  0.8 in DMEM, respectively (see Table 3). It can be observed from Table 3 that the addition of CT decreased the contact angle of the substrate, making it more hydrophilic, whereas pure HA and CaTiO3 are hydrophobic in nature. More pronounced decrease in hydrophobicity was recorded for contact angle measurements in DMEM culture media. In summary, the HA-CaTiO3 substrates are characterized to be elastically stiff, relatively hydrophobic and moderately electroconductive. Also, both HA and CaTiO3 phases are predominantly retained. Especially, AFM analysis revealed insignificant difference in terms of surface roughness and XPS analysis confirmed similar surface chemistry in terms of Ti3þ/Ti4þ ratio. Therefore, the influence of substrate conductivity on cell functionality can be judiciously studied in the present work.

Fig. 7. MTT assay results showing number of viable cells on HA, HA 40CT, HA 80CT and CT samples cultured for a time period of 1, 2 and 3 days respectively. * indicates statistically significant difference in the cell number with respect to day 1 (p < 0.05).

3.2. Myoblast cell viability The viability of cells grown on material surface is commonly determined using MTT assay, which is regarded as one of the quantitative assays to determine the metabolic activity of the cells. MTT is a chemical reagent that interacts directly with the mitochondria of live cells which are in contact with the materials surface and the resulting change in optical density (OD) is correlated with the number of viable cells adhered on the samples surface. Fig. 7 shows the number of myoblasts cells proliferated on each sample (including control) after 24, 48 and 72 h. There was no significant difference in the cell count amongst HA-CaTiO3 samples on all days and showed almost identical values like that of control. Also, the number of cells on all samples increased markedly from day 1 to day 3, indicating that the addition of CaTiO3 can induce cell proliferation as good as HA. Similarly, percentage viability of cells with respect to control on all samples remained the same, which is

Fig. 8. Plot of the percentage cell viability of myoblasts cells grown on HA-CT samples, measured with respect to control. No statistically significant difference was observed for p < 0.05.

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shown in Fig. 8. None of the HA-CaTiO3 substrate showed statistically significant difference in cell viability for p value less than 0.05. These data demonstrate that HA-CaTiO3 substrates are equally compatible to myoblast cells as that of the control glass cover slip and monolithic HA. 3.3. Myoblast cell functionality The initial attachment of C2C12 mouse myoblast cells on the surface of HA, CaTiO3 as well as the HA-CaTiO3 composites were observed under SEM, after 12 h of culture (not shown). There was not much difference in terms of the morphology and number of cells adhered on various ceramic sample, during the initial period of culture. The cultured C2C12 cells were well spread and appeared to have strong adhesion on all the substrates. In order to visualize the details of cytoskeletal structures and the shape of the nuclei, myoblasts grown for 3 days were immunostained for F-actin and counterstained for nuclei. Neither of the HA-CaTiO3 samples showed difference in cellular morphology and cytoskeletal organization on day 1, when examined under fluorescence microscope (Fig. 6a, b, c, d). But after 3 days, myoblasts reached complete confluency in all the samples. Moreover, the cells appeared more organized and strongly oriented in samples with higher CaTiO3 content (Fig. 6g, h), whereas cells appeared to be perturbed and nonaligned on HA and HA 40CT substrates (Fig. 6e, f). 3.4. Myotube formation Upon reaching confluency, the cells kept in serum starvation condition, started fusing into myotubes and the SEM images in Fig. 10 allow clear visualization of long myotubes formed on HA-CT samples. HA samples, with lower conductivity showed shorter, less prominent myotubes with irregular morphology, compared to that all other CaTiO3 containing samples. As the time of culture progressed to 5 days, the differentiated cells displayed more defined

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and neatly aligned myotubes, with maximum number of myotubes on CT substrates (Fig. 10d). Furthermore, to confirm the commitment of myoblast to form myotubes, immunofluorescent labeling of myogenin was performed on all samples (including control), after 3 days of serum starvation. Regardless of the detection of myogenin on all samples (Fig. 9), well defined assembly of myoblasts was observed only on HA 80CT and CT substrates (Fig. 9c, d), demonstrating the capability of conductivity in inducing the expression of myogenic regulatory factors. In Fig. 12, the myotube density (numbers averaged over substrate area) is plotted for various substrates. Clearly, the results indicate that HA-CT composites support myotube formation to a significantly higher extent than HA or tissue culture plastic control. A marked increase is recorded on HA-80CT and CT substrates and the increase in myotube density with CaTiO3 addition to HA is systematic and almost linear. More quantification on myotube formation was made by measuring lengths and widths and the results are plotted in Fig. 11a and b, respectively. Again, both the myotube width and length increased systematically with an increase in CaTiO3 content. The widths of the myotubes on HA was around 7 mm, whereas it was around 10e12 mm on HA-40CT and HA-80CT substrates. However, a much higher myotube width of 16 mm was measured on CT. Comparing myotube width with size of cultured myoblasts, it appears that myoblasts contract in the process of differentiation into myotubes. With regard to the myotube length, almost three times longer myotubes, on an average were formed on HA-40CT and around six times longer on pure CT. Again a number of myoblasts clearly fused together to form myotubes as can be realized from the results plotted in Fig. 11. Taken together, the results of the differentiation study once again confirm the positive impact of substrate conductivity on myoblast cell fate. The key result of variation in myoblast and myotube density with increasing conductivity is illustrated in Fig. 12.

Fig. 9. Immunostaining of myogenin expressed on (a) HA (b) HA 40CT (c) HA 80CT (d) CT samples.

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Fig. 10. Scanning electron microscopy images showing myotube organization and alignment on (a) HA (b) HA 40CT (c) HA 80CT (d) CT, after 5 days of differentiation (serum starvation). (e) and (f) shows the magnified image of myotube formed on HA 80CT and CT respectively.

4. Discussion The surgical procedure during orthopedic implantation results in injury of skeletal muscles, in and around the defect site. Hence, it is necessary to ensure that the implant material that we employ as bone substitute should also provide a favorable platform for the muscle cells to grow and proliferate. With this fact in mind, the present study is mainly focused on assessing the behavior of myoblast cells on stiff conductive ceramic substrates. The major emphasis of the current study is therefore to illustrate how the conductivity property of substrate influences the muscle cell functionality. For this purpose, we designed HA-CaTiO3 substrates with different conductivities by varying the CaTiO3 content (0, 40, 80, and 100 wt%) in the biocomposites to address the following questions:

a) How does the substrate conductivity influence the cell growth? b) How would the time in culture influence cell growth on electroconductive substrates? c) Can inherent substrate conductivity influence cell functionality in vitro, even in the absence of electric field stimulated conditions? d) Can the differentiation of myoblast to myotubes be regulated on electroconductive substrates? In addressing all the above issues in an integrated manner, a planned set of in vitro culture experiments were conducted using C2C12 mouse myoblast cells. Muscle tissue uses electrical conductivity for maintaining homeostasis, development and regeneration [22] and hence myoblasts were chosen as a model cell line to assess the effect of conductivity on cell proliferation and differentiation in vitro.

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Fig. 12. Graph showing the variation of both myoblast and myotube density as a function of A.C conductivity.

Fig. 11. Quantification of myotube formation on control and HA-CT substrates. (a) Average width of the myotube formed, (b) Length of the myotube. All measurements were carried out on samples after 3 days of differentiation. * denotes statistically significant difference with respect to control, and # indicates statistical significance with respect to HA.

On the investigated ceramic substrates, the characteristic shape of C2C12 was maintained, that is viewed immediately after one day of culture. The cultured myoblast cells appeared either star-shaped or fusiform (Fig. 6a, b, c, d), with most of the cells trying to bridge or overlap with each other [23]. Likewise, the viability of myoblast cells on the HA-CaTiO3 composites was not affected by the addition of CaTiO3 to HA even after 72 h of incubation (Fig. 6). However, the density of myoblast cells, obtained by counting the cell number from fluorescence images were significantly higher in HA-CaTiO3 composites than HA. Moreover, a further investigation was carried out on these composites in order to correlate the effect of inherent electrical conductivity on the attachment and proliferation of myoblast cells. It was observed that cell density followed the same trend as that of the electrical conductivity with increasing amount of CaTiO3 (Fig. 12). It is inferred from these results that substrate conductivity positively influences the proliferation of myoblast cells. It has been reported in literature that myoblasts, being very contractile in nature, can grow and spread substantially well on

both softer and stiffer substrates; but for the fusion of myotubes and for the formation of striations, it prefers substrates with tissuelike stiffness [14]. With increase in time of culture, well developed myotubes have a greater tendency to detach readily from the rigid glass substrates due to higher degree of actinomyosin contraction [13]. We also experienced a similar kind of detachment of cell layer from control cover slip after 5 days of differentiation (images not shown). Hence, in general, substrates with intermediate (tissue like) stiffness is always suggested for the culture of myoblast. In spite of this, our attempt is not to establish the suitability of stiffer ceramic materials as potential substrate for myoblast culture, but to indicate the fact that the electroconductive nature of bone substitutes can aid better muscle regeneration as well. The assembly of myoblasts to form multinucleated myotubes is one of the prerequisite to promote muscle regeneration and function [12,24]. In order to demonstrate the effect of substrate conductivity on myogenesis, myoblasts grown to almost 90% confluency were subjected to serum starvation (1% FBS). Immunofluorescent staining against myogenin was performed on all samples after 3 days of serum starvation. The presence of myogenin was poorly detected on cells grown on lower conducting substrates (Fig. 9a and b), whereas mature myotubes with higher levels of myogenin expression was detected on substrates with higher conductivity (Fig. 9c and d). The up-regulation of myogenin induces expression of myotube specific genes, causing myofibril formation [25e27]. Such a prominent rise in the expression of myogenin on CaTiO3 substrates illustrates the dependency of substrate conductivity on the entry of muscle progenitors to terminal differentiation. To provide further confirmatory evidence of myotube formation, imaging of the substrates under SEM was performed. Although distinct myotubes started appearing within 3 days of differentiation on substrates with increasing CT content (not shown), after 5 days, CT substrate showed neatly organized myotubes aligned predominantly along one direction (Fig. 10). The orientation of myotubes in this parallel manner helps in the generation of large forces to enable muscle contraction during movement [28]. In addition, such preferential organization and well-defined linear geometries of myotubes defines its physiological shape and governs the formation of myofibrils, which are the structural unit of skeletal muscles [29]. While in most cases of conventional cultures, differentiation of myoblasts on glass coverslips results in the development of complex and branched network of myotubes, that has no physiological relevance to native muscle tissue [30].

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Fig. 13. Schematic diagram represents the overview of myoblast behavior on conductive ceramic substrates.

Moreover, the number of myotubes formed was significantly higher on CT substrates than that on HA and control. Even though the cultured myoblast cells were crowded, the myotubes were sparsely oriented on sintered HA. Also, they were fewer in number and were not well distinguished on HA samples (Fig. 10). A nearly linear trend was noticed for the width (Fig. 11b) and length (Fig. 11c) of myotubes, with CT displaying the highest aspect ratio. Herein the opposing effect played by stiffer substrates on myoblast growth seems to be masked by the positive role played by the conductive substrates. SEM images of sintered HA-CaTiO3 substrates (Fig. 1) exhibit randomly dispersed CaTiO3 phases on the surface of HA matrix indicating that the myotube alignment is independent of surface distribution of CT grains. Surface analysis by XPS leaves out any difference in surface chemistry among CT containing samples (Fig. 3). A further investigation by AFM, demonstrated similarity in the surface topology, with comparable roughness values among all the four samples (Fig. 4). As a result, the influence of surface parameters like topology and roughness on cell adhesion and orientation [31,32] can be discretely ruled out in the current study. Comparing the cell growth and cell viability results with surface wettability (see Figs. 5 and 7), it is evident that, myoblast cells prefer to proliferate on hydrophobic surfaces, as the contact angle of greater than 50 was measured with all the investigated substrates, independent of their compositions. Furthermore, the differentiation of myoblast to myotube is also facilitated on the hydrophobic substrates (Figs. 5 and 11). However, we do not find any clear correlation between the results of myoblast proliferation/ growth/differentiation with surface wettability in terms of contact angle, because substrates with lowest (HA) and highest (CT) conductivity values exhibited almost similar wettability behavior. Therefore, substrate conductivity emerges as an important factor determining the myoblast cell functionality in vitro in HA-CT system. It is expected that the myoblast cells, grown on substrates with increasing conductivity are exposed to Ca2þ ions that triggers the activation of terminal differentiation. A net influx of calcium ions and generation of hyperpolarization are required to promote the fusion of myoblast to multinucleated myotubes by membrane reorganization [33,34]. It can also be assumed that the electrically conducting environment can easily favor the transmittance of electric potential generated to maintain muscle functionality. Overall, the proliferation and fusion of myoblast on CaTiO3 containing electroconductive substrates clearly demonstrate the influence of inherent substrate conductivity on the growth and organization pattern of C2C12 cells. In summary, we attempted to understand the suitability of moderately conducting ceramic substrates for short term muscle growth and differentiation (see Fig. 13). Our results indicate that CaTiO3 based substrates enhance initial stage of myotube orientation and assembly to a similar extent like that of skeletal muscle organization. An increase in conductivity of the substrates

correlated well with increase in cell density and aspect ratio of myotubes. The benefit of studying such effects clarifies the ambiguity of inhibitory role played by stiffer bone substrates in the regeneration and restoration of neighboring damaged skeletal muscles. 5. Conclusions Based on the experimental results reported and analyzed in the present paper, the following conclusions emerge. a) The SPS sintered HA-CaTiO3 bioceramics have similar surface chemistry (Ti3þ/Ti4þ ratio) and topography as analyzed from the XPS and AFM. The surface roughness was determined to be <50 nm in all the samples. b) The myoblast cell viability and growth on HA-CaTiO3 platforms increases in a conductivity dependent manner, in culture up to 5 days. c) Importantly, all the cultured myoblasts exhibit oriented cell growth on both CaTiO3 and HA-80 CaTiO3 substrate after 3 days of incubation. Such self-organized cellular patterns were not observed for incubation time of 1 or 2 days. d) When cultured in serum starvation media, myoblast cells differentiated to well aligned parallel myotubes on HA-CaTiO3 substrates. Both, the myotube density, myotube length/diameter, as well as myogenin expression systematically increase with increase in substrate conductivity. Overall, the present results unambiguously demonstrate that substrate conductivity plays an important role in guiding myoblast proliferation and differentiation on elastically stiff and hydrophobic bioceramic substrates. Acknowledgments The authors would like to thank Department of Science & Technology (DST) and Department of Biotechnology (DBT), Government of India for the financial support. The authors are also grateful to Dr. Girish Kunte, Suma, Venkatesh (MNCF facility, CeNSE, IISc) and Srinivas (AFM facility, MRC, IISc) for their assistance in using the research facilities. References [1] Basu B, Katti DS, Kumar A. Advanced biomaterials: fundamentals, processing, and applications. John Wiley & Sons Ltd; 2009. [2] Magnani A, Priamo A, Pasqui D, Barbucci R. Cell behaviour on chemically microstructured surfaces. Mater Sci Eng C 2003;23(3):315e28. [3] Kitchen S, Bazin S. Electrotherapy: evidence-based practice. Churchill Livingstone; 2002. [4] Martino S, D’Angelo F, Armentano I, Kenny JM, Orlacchio A. Stem cellbiomaterial interactions for regenerative medicine. Biotechnol Adv 2012;30(1):338e51.

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