C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9

.sc iencedi rect .com

Avai lab le at www

journal homepage: www.elsev ier .com/ locate /carbon

Polyvinyl alcohol associated with carbon nanotube scaffoldsfor osteogenic differentiation of rat bone mesenchymalstem cells

Ana A. Rodrigues a,*, Nilza A. Batista a, Vanessa P. Bavaresco a, Vitor Baranauskas b,Helder J. Ceragioli b, Alfredo C. Peterlevitz b, Arnaldo R. Santos Jr. c,William D. Belangero a

a Orthopaedic Biomaterials Laboratory, School of Medical Sciences, University of Campinas, Sao Paulo, Brazilb Department of Semiconductors, Instruments and Photonics, School of Electrical and Computer Engineering, University of Campinas,

Sao Paulo, Brazilc Centre of Natural and Human Sciences, Federal University of ABC, Santo Andre, Sao Paulo, Brazil

A R T I C L E I N F O

Article history:

Received 25 April 2011

Accepted 31 August 2011

Available online 6 September 2011

0008-6223/$ - see front matter � 2011 Elsevidoi:10.1016/j.carbon.2011.08.071

* Corresponding author: Fax: +55 19 3521 749E-mail addresses: labimo@fcm.unicamp.b

A B S T R A C T

Polyvinyl alcohol (PVAl) hydrogel, alone and reinforced with two types of carbon nanopar-

ticles, was studied in cultured cells to assess its potential use in treating osteochondral

defects. The carbon nanoparticles were produced by hot-filament chemical vapour deposi-

tion. The carbon material was characterised with a Renishaw Invia Raman microscope sys-

tem and the morphological particles were characterised with field emission scanning

electron microscopy and high-resolution transmission electron microscopy. Cytotoxicity

was evaluated by measuring the Vero fibroblast-type cells’ metabolic activity and studying

their morphology. The osteogenic differentiation of mesenchymal stem cells obtained from

rat bone marrow was evaluated by alkaline phosphatase (ALP) and alizarin red S (ARS)

staining. Cell viability and morphology were assessed with thiazolyl blue tetrazolium bro-

mide and scanning electron microscopy, respectively. The materials did not interfere with

the viability, metabolic activity, morphology and spreading of either of the cell types ana-

lysed. Nodules of mineralised organic matrix were identified with ARS and ALP, confirming

osteogenic differentiation. These results indicated higher concentration of ALP and min-

eralised matrix for PVAl with carbon nanoparticles. The results of this study indicate the

potential use of carbon nanoparticles with PVAl hydrogels as orthopaedic biomaterials to

treat osteochondral defects, but further in vivo investigations are still necessary.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Articular cartilage lesions are an unsolved problem in ortho-

paedic surgery because the cartilage has a limited capacity

for self-repair following trauma [1]. Biological treatment op-

tions are mainly based on bone marrow stimulation, tissue

transplantation and tissue engineering techniques [2–8]. The

er Ltd. All rights reserved

8.r, anaarodriguess@gmail

first option is based on joint debridement and the bone mar-

row stimulation principle [2,3]. The second option involves

the transplantation of periosteum and autologous or alloge-

neic osteochondral plugs [4–6]. Autologous grafts are advanta-

geous because they do not elicit immunological rejection [9];

however, their disadvantages include limited availability, re-

duced mechanical stability and morbidity of the donor area

..com (A.A. Rodrigues).

Table 1 – Cytotoxicity of different samples studied for Veroor mesenchymal stem cells (MSC).

Cell type/assay Mean SD ER

VC indirect cytotoxicityPCT 0.10163 0.04402 0.01556NCT 0.39575 0.30694 0.10852PVAl 0.54663 0.43755 0.1547PVAl-CNT 0.66175 0.54617 0.1931PVAl-CNF 0.65125 0.54038 0.19105

VC direct cytotoxicityPCT 0.0726 0.02503 0.01119NCT 0.4858 0.12979 0.05805PVAl 0.50954 0.03633 0.01625PVAl-CNT 0.4144 0.05562 0.02487PVAl-CNF 0424 0.01696 0.00758

MSC indirect cytotoxicityPCT 0099 0.00693 0.00346NCT 0253 0.04588 0.02294PVAl 0.35225 0.09895 0.04947PVAl-CNT 0.29925 0.07413 0.03706PVAl-CNF 0.3465 0.07781 0.03891

Cytotoxicity results for PVAl, PVAl-CNT and PVAl-CNF showed

statistically significant differences (*p < 0.05) when compared to

positive control of toxicity (PCT) but not when compared to nega-

tive control of toxicity (NCT) (*p > 0.05).

C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9 451

[4,5,10,11]. The third option, tissue engineering techniques,

involves using a bioartificial implant to transplant cells onto

a three-dimensional scaffold. This procedure has the advan-

tage of a reduced risk of disease transmission [7,8]. Synthetic

polymers, such as hydrogels, are used for cartilage tissue

engineering scaffolds because they can provide the three-

dimensional structure necessary to replace articular cartilage,

and they can absorb large quantities of water and biological

fluids [12].

Carbon nanomaterials offer potential as structural rein-

forcements because they have high mechanical strength

and good biocompatibility [13–16]. The carbon nanoparticles

associated with hydrogels allow changes in the hydrogels’

mechanic and structural properties, resulting in a new mate-

rial whose mechanical performance can be changed and tai-

lored to use in regenerative medicine [16,17]. Among several

types of polymeric hydrogels, polyvinyl alcohol (PVAl) has re-

ceived special attention because its physicochemical charac-

teristics and viscoelastic properties are similar to articular

cartilage [18,19].

The aim of this study is to investigate the cytotoxicity of

PVAl hydrogel, both alone and reinforced with two types of

carbon nanoparticles, used as scaffold for cell culture, in or-

der to assess its potential use to treat osteochondral defects.

2. Experiment

2.1. Preparation and characterisation of carbon nano-particles

The two types of carbon nanoparticles, carbon nanotubes

(CNT) and carbon nanofibres (CNF), were produced by hot-fil-

ament chemical vapour deposition (HFCVD). CNT was pro-

duced on a copper substrate covered with a thin film of

polyaniline. Two-tenths of a millilitre of a 2 mg/ml nickel ni-

trate-acetone solution (nickel being the catalyst for CNT

growth) was dropped on the dry polyaniline film. After drying

at room temperature, the polyaniline film was placed in the

HFCVD reactor in a nitrogen atmosphere at 450 �C and a pres-

sure of 26 mbar for 30 min. An acetone solution of camphor

bubbled in hydrogen gas was used as a carbon source. The

same procedure was used to synthesise CNF, except for the

absence of the Ni catalyst.

Raman spectra were recorded at room temperature using a

Renishaw Invia Raman microscope system with a laser for

excitation (k = 785 nm) at a laser power of approximately

6 mW.

The morphological characterisation of the carbon nano-

particles was obtained with field emission scanning electron

microscopy (FESEM) using a JEOL JSM 6330F microscope and

high-resolution transmission electron microscopy (HRTEM)

using a JEOL 3010 microscope.

2.2. Preparation and characterisation of poly(vinylalcohol) (PVAl) pure and associated with carbon nanoparticles

A 10% aqueous solution of PVAl (Sigma–Aldrich mW 89,000–

98,000 g/mol, 99% hydrolysed) was prepared. CNT and CNF

were diluted at a ratio of 2.5 mg per 10 ml of PVAl solution

and homogenised at 60 �C for 1 h in magnetic stirrer to obtain

the PVAl membranes associated with CNT (PVAl-CNT) and

CNF (PVAl-CNF). The solution was transferred to a Petri dish

and kept at room temperature for 7 days. The membranes

were acetalised by a chemical treatment and crosslinked by

electron beam irradiation at 25 kGy produced by a Radiation

Dynamiton electron beam accelerator (Institute of Energy

and Nuclear Research, Sao Paulo, Brazil).

The PVAl, PVAl-CNT and PVAl-CNF were characterised by

Raman spectroscopy using a laser for excitation (k = 785 nm)

at a laser power of approximately 6 mW.

2.3. Cell culture of fibroblastic cells

Fibroblastic cells are recommended as standard for studies of

cytotoxicity and cell-substratum interactions with biomateri-

als [20–22]. These cells were chosen because they are present

in the initial phase of healing and implant integration. The

Vero fibroblast-type cells used in this study were obtained

from Adolfo Lutz Institute, Sao Paulo, Brazil. The cells were

cultivated in Ham’s F12 medium (Nutricell) supplemented

with 10% foetal calf serum (FCS, Nutricell) and 1% penicil-

lin/streptomycin (PS, Hyclone) at 37 �C in an atmosphere with

5% CO2.

2.4. Isolation and culture of rat bone marrow mesen-chymal stem cells

The femur, tibia and humerus bones were removed from Wis-

tar–Kyoto rats to obtain bone mesenchymal stem cells (MSC).

The epiphyses were removed, and the bone shafts were placed

in 5-ml blood collection tubes and centrifuged at 400g for

10 min. The precipitated bone marrow was homogenised with

a phosphate buffer saline/ethylenediamine tetraacetic acid

452 C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9

solution (PBS/EDTA) at a concentration of 2 ml PBS/EDTA per

bone, filtered with an ultra-thin (20 lm) filter, placed on

15 ml of Ficoll-Hypaque� and centrifuged at 300g for 25 min.

The fraction of mononucleal cells was collected and centri-

fuged for 10 min at 200g, the supernatant was discarded and

the precipitate was homogenised in 10 ml of PBS/EDTA. The

obtained suspension was washed in three cycles of centrifug-

ing at 200g for 10 min. A count in a Neubauer chamber was

conducted after the last cycle, and the cells were placed in

Dulbecco’s Modified Eagle’s Medium (DMEM) with a low con-

centration of glucose, supplemented with 10% of FCS.

2.5. Cytotoxicity assay

The modified Mosmann method was used [23]. Extracts of the

tested materials (PVAl, PVAl-CNT and PVAl-CNF) were ob-

tained by incubating them in Ham’s F-12 medium containing

10% FCS at a proportion of 0.2 g/ml medium for 48 h at 5% CO2

and 37 �C. This method is in agreement with the standards for

evaluation of biomedical devices [20–23].

For the indirect cytotoxicity assay, Vero and MSC cell sus-

pensions (3 · 106 cells/ml) were inoculated into a 96-well cell

culture plate (n = 5) and incubated at 37 �C for 24 h. After this,

the culture medium was replaced by the extract obtained from

the tested materials, and the cells were maintained under

these conditions for 24 h. For the direct cytotoxicity assay, a

suspension of 3 · 106 cells/ml (n = 5) was directly cultivated

on the materials for 24 h. Ham’s F-12 medium with phenol

0.5% was used as the positive control toxicity (PCT) and poly-

styrene extract as the negative control toxicity (NCT) in both

tests.

After incubation, the medium was removed and the wells

were washed with 200 ll PBS. Next, 200 ll of Ham’s F12 med-

ium with 10 mM of Hepes buffer and 50 ll of thiazolyl blue

tetrazolium bromide solution (MTT, Sigma) were added, and

the plate was incubated in the dark for 4 h at 37 �C. After that,

the medium with MTT was removed, and 200 ll of dimethyl

sulphoxide (DMSO) was added. The absorbance curve was

determined in a microplate reader (Bio-Rad 550 microplate

spectrophotometer) at k = 540 nm.

Commercial software (Microcal TM Origin� version 6.0) was

used for statistical calculation. Student’s t-test was employed

for assessing statistical differences between each sample and

the NCT and PCT, while one-way analysis of variance (ANOVA)

was employed for assessing statistical differences between all

samples. P < 0.05 was considered statistically significant.

2.6. Scanning electron microscopy of cell morphology

The Vero and MSCs cells (3 · 106 cell/ml) were inoculated in

100 lL of Ham’s F12 medium supplemented with 10% FCS in

a 96-well plate containing the three different materials and

incubated at 37 �C in 5% of CO2 for 24 h. After fixation in par-

aphormaldehyde (Sigma) 2.5% and glutaraldehyde (Sigma)

2.5%, the samples were post-fixed in a 1% solution of osmium

tetroxide (Sigma) for 1 h at room temperature in the dark,

washed in distilled water, dehydrated in ethanol, critical-

point dried in CO2 (Balzers, CDT 030), coated with gold in a

sputter coater (Balzers CTD 050) and viewed with electron

microscopy (JEOL 5800).

2.7. Cytochemical analysis

The Vero cells and MSCs were cultivated in contact with PVAl,

PVAl-CNT and PVAl-CNF. A 3 · 106 cell/ml in low-glucose

DMEM medium with 10% FCS cell suspension was inoculated

on the materials, and the plate was cultivated for 24 h at

37 �C. After this, the cells were fixed in formaldehyde 10%

for 24 h and stained with toluidine blue (TB) at pH 4.0 to de-

tect nucleic and glycosaminoglycan acids [24,25].

2.8. Induction of osteogenic differentiation

The MSCs were inoculated in 24-well plates and induced to

osteogenic differentiation, as described by Neuhuber et al.

[26]. After 48 h, the PVAl, PVAl-CNT and PVAl-CNF samples

were added to the wells, and the culture medium was re-

placed with the osteogenic induction medium (low-glucose

DMEM supplemented with 15% FCS, 1% penicillin/streptomy-

cin, 100 nM dexamethasone, 50 lM ascorbate-2-phosphate

and 10 mM glycerol-phosphate). A control culture (CC) was

prepared similarly, but without any PVAl. The medium was

changed every three days over a 21-day period.

2.9. Analyses of osteogenic differentiation: alkalinephosphatase and alizarin red staining

ALP was assessed with a specific kit (Sigma Fast p-nitrophenyl

phosphatase tablets N1891), according to the manufacturer’s

recommendations: 50 ll of the differentiation medium con-

tained in the 24-well plate was transferred to a 96-well plate

and incubated for 2 h at 37 �C. Next, 200 ll of ALP substrate

solution was added, and the plate was kept in the dark for

30 min at room temperature. An absorbance reading was ta-

ken at k = 405 nm.

For ARS, the cells were fixed in 4% paraformaldehyde for

10 min, washed with distilled water and stained for 2 min,

then washed again in distilled water and differentiated in

95% ethanol and hydrochloric acid 100% for 15 s. The cells

were kept in distilled water while images were obtained with

a Leica inverted microscope. ARS activity was evaluated with

semiquantification colorimetric method described by Gregory

et al. [27]. After the water was drained, the plate was kept at

room temperature until it was completely dry. Next, 280 ll

of acetic acid 10% was added, and the plate was shaken for

30 min. The contents of each well were then transferred to

Eppendorf tubes, which were warmed to 85 �C for 10 min

and then cooled down in ice for 5 min. After that, the tubes

were centrifuged at 16,000g for 20 min, and 100 ll of the

supernatant were transferred to a new tube containing 40 ll

of ammonium hydroxide 10%. The final solution was trans-

ferred to a 96-well plate, and the absorbance reading was ta-

ken at k = 405 nm.

3. Results

3.1. Characterisation of carbon nanoparticles, PVAl pureand associated with carbon nanoparticles

Fig. 1 shows the typical Raman spectrum obtained from the

samples of CNT (Fig. 1A), CNF (Fig. 1B), and membranes of

1000 2000 3000 4000

0

1000

2000

3000

4000

5000

6000

Inte

nsity

(a.

u)

Raman shift (cm-1) Raman shift (cm-1)

1341

1598

2679 2937

32201145

1000 2000 3000 40000

200

400

600

800

1000

1200

1400

1600

1800

Inte

nsity

(a.

u.)

13481582

2690 2910

1000 2000 3000 4000

0

2000

4000

6000

8000

10000

Inte

nsity

(a.

u.)

Raman shift (cm-1) Raman shift (cm-1)

3280

29272878

27922700

1484

14461364

1310

1251

1174

1150

1106921705

859

835

801501

345

A B

DC

1000 2000 3000 4000

0

2000

4000

6000

8000

10000

12000

Inte

nsity

(a.

u)

3270

2927

2889

27922700

1594

1483

14461364

1310

1251

1174

1150

1100921

859

835

801

705

501

356

Fig. 1 – Raman spectra obtained from: (A) CNT, (B) CNF, (C) pure PVAl and (D) PVA1-CNT.

C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9 453

pure PVA1 (Fig. 1C) and PVA1 doped with CNF (Fig. 1D). The

spectrum corresponding to CNT samples showed peaks at

1341 cm�1, 1598 cm�1, 1145 cm�1, 2679 cm�1, 2937 cm�1 and

3220 cm�1, while the spectrum corresponding to the CNF

showed peaks at 1348 cm�1, 1582 cm�1, 2690 cm�1 and

2910 cm�1.

The first-order region of both samples presented two in-

tense peaks of approximately 1350 cm�1 and 1580 cm�1, cor-

responding to the disorder-induced sp2 peak (D-line) and

the highly oriented graphite E2g mode sp2 peak (G-line). In

the second order region, there were peaks at 2679 cm�1 (Spec-

trum 1A) and at 2690 cm�1 (Spectrum 1B), which correspond

to the D-line second harmonic (2 · D). The peaks at 2937 cm�1

(Spectrum 1A) and at 2910 cm�1 (Spectrum 1B) correspond to

the sum of D- and G-line frequencies (D + G). Finally, a small

peak at 3220 cm�1 (Spectrum 1A) corresponds to the G-line

second harmonic (2 · G).

The Raman spectra of pure PVAl (Spectrum 1C), of CNT

doped PVAl (Spectrum 1D) and of CNF doped PVAl (not shown)

have very complex Raman scattering peaks and is difficult to

identify any scattering provoked by the CNT or CNF.

Fig. 2 shows the typical CNT and CNF morphologies re-

vealed by the FESEM (Fig. 2A and B) and HRTEM (Fig. 2C and

D). Both CNTs (Fig. 2C) and nanorods, i.e., CNFs (Fig. 2D), are

visible.

3.2. Cytotoxicity assay

The results of the cytotoxicity of Vero cells by MTT assay

against samples of PVAl, PVAl-CNT and PVAl-CNF were simi-

lar to the results obtained for NCT and completely different

from those obtained for PCT (Figs. 3 and 4). According to the

Student’s t-test, there were statistically significant differences

(*p < 0.05) between the readings obtained for all samples and

those obtained for PCT. The same could be observed with

MSCs (Fig. 5). In this case, the cell proliferation was even sta-

tistically higher than those obtained for the NCT, as shown in

Fig. 5 (see Table 1).

3.3. Scanning electron microscopy of cell morphology

The spread of Vero cells over the surface of PVAl, PVAl-CNT

and PVAl-CNF samples ( Fig. 6A1, B1 and C1) could be ob-

served. Cells with irregular morphology (retracted, elongated

and always well-flattened) were found on the PVAl samples.

Vesicles and microcavities were also found in a few cells,

Fig. 2 – Typical sample morphologies revealed by FESEM: (A) CNT and (B) CNF. Images obtained with HRTEM: (C) CNT and (D)

CNF.

Fig. 4 – Indirect cytotoxicity assay of PVAl, PVAl-CNT and

PVAl-CNF with Vero cells. There are no statistically

significant differences (*p > 0.05) between the samples and

the negative control of toxicity (NCT).

Fig. 3 – Direct cytotoxicity assay of PVAl, PVAl-CNTand PVAl-

CNF with Vero cells. There are no statistically significant

differences (*p > 0.05) between the samples and the negative

control of toxicity (NCT).

454 C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9

along with signs of cellular division (Fig. 6A1). On the PVAl-

CNT samples, cells with a very irregular morphological pat-

tern and with cytoplasmic bridges and filamentous material

between them could be found (Fig. 6B1). On the PVAl-CNF

samples, flattened cells with irregular morphology, but with-

out fibrils, could be observed (Fig. 6C1).

A small number of MSCs were found adhered to the sur-

face of the studied materials. On the PVAl and PVAl-CNT

spread cells with bipolar morphology and star-shaped

(Fig. 6A2 and B2). On the PVAl-CNF, cells with irregular mor-

phology and well-flattened could be observed (Fig. 6C2).

In the two experimental conditions, with Vero and MSC

cells, thin cellular extensions could be observed from the

edges in several regions of the cells (Fig. 6A1, B1, C1, A2, B2

and C2). In a few cases, larger extensions were noticed, sug-

gesting cellular migration (Fig. 6A1, B1 and C1).

Fig. 5 – Indirect cytotoxicity assay of PVAl, PVAl-CNT and

PVAl-CNF with MSC cells. There are no statistically

significant differences (*p > 0.05) between the samples and

the negative control of toxicity (NCT).

C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9 455

3.4. Cytochemical analysis

TB staining showed growth and confluence between in Vero

cells in PVAl, PVAl-CNT and PVAl- CNF, similar to the pattern

Fig. 6 – SEM of the material surfaces: (A) PVAl, (B) PVAl-CNTand (C)

C2) for 24 h. The asterisk indicate the cells adhered on the mater

2000·.

of growth observed in NCT (Fig. 7A1, B1, C1 and D1). In PVAl

samples, a semi-confluent layer of cells and possibly dividing

cells were observed. The same could be seen in PVAl-CNT and

PVAl-CNF, but with larger cells. Regardless of the type of bio-

material, the cells exhibited strongly staining basophilic nu-

clei with prominent nucleoli and slight metachromasia,

which are signs of substantial cellular activity. After 24 h,

the MSC culture showed typical behaviour, i.e., it was in a

stage of semiconfluence, with cells having an oblong shape

and bipolar morphology (Fig. 7A2, B2, C2 and D2) and regions

of clusters with a polygonal cell aspect. The same pattern of

cytoplasmic basophilia with metachromasia and prominent

nucleoli could be noticed.

3.5. Alkaline phosphatase and alizarin red staining

Fig. 8 shows the semi-quantitative analysis for ALP, a com-

monly used bone differentiation marker. This enzyme was

present in all experimental conditions investigated.

The mineralised bone matrix was proven by ARS’ incorpora-

tion into the MSCs after 21 days of osteogenic differentiation. In

Fig. 9, it is possible to observe the development of mineralised

nodules characteristic of osteogenic differentiation after

PVAl-CNF with Vero (A1, B1 and C1) and MSC cells (A2, B2 and

ials and the arrows the cellular extensions. Magnification of

Fig. 7 – Cytochemical analysis of cells cultured in contact with: (A) culture control; (B) PVAl; (C) PVAl-CNT; (D) PVAl-CNF. From

A1 to D1: Vero cells. From A2 to D2: MSCs. Toluidine blue (TB) stain with a magnification of 200·. (For interpretation of the

references in colour in this figure legend, the reader is referred to the web version of this article.)

456 C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9

contact with PVAl, PVAl-CNT and PVAl-CNF. The quantification

of the osteogenic matrix production is presented in Fig. 10,

where it can be noticed that, for all conditions studied, the

counts were similar to the CC.

4. Discussion

The CNT and CNF samples were characterised with Raman

spectroscopy, FESEM and HRTEM [28,29]. The HRTEM images

showed longitudinal and parallel lines corresponding to mul-

tiwalled structures that may be identified as CNTs. However,

some tubes appeared to have their inner space almost filled.

We called these CNFs. The Raman results identified both

CNTs and CNFs as multiwalled structures [28,29].

In vitro tests represent the initial phase of the assessment

process for biocompatibility study, which is part of the prese-

lection of these materials for in vivo assays [20–22]. The cyto-

toxicity assays showed that the tested materials materials

had no toxicity against cells. No cytochemical variations were

observed in the Vero cells or the MSCs, which grew for 24 h

on the PVAl, PVAl-CNT and PVAl-CNF. Cells with intense

Fig. 8 – Quantification of ALP in MSC cells after 21 days of

osteogenic differentiation in contact with culture control

and samples of PVAl, PVAl-CNT and PVAl-CNF.

Fig. 10 – Quantification of the mineralised matrix of MSC

cells after 21 days of osteogenic differentiation, using the

colorimetric method of ARS extraction.

C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9 457

basophilia, with slightly metachromatic nuclei and evident

nucleoli could be observed, indicating cell activity. The identi-

fication of metabolically active cells, made clear by the TB

staining, is in line with previously published data on different

polymers [30–32].

SEM is an effective technique for evaluating cell growth

and spreading on the materials under study. Changes in cell

morphology, as well as the cells’ ability to aggregate and

scatter, may indicate different behaviours toward these

materials [33]. In the present study, the Vero cells did not

show morphological alterations, indicating that the materials

did not interfere with the cellular spreading and adhesion.

According to the literature, the surface of the materials can

impact the initial phases of adhesion and spreading, thus

causing a delay in cell growth and consequently morphological

alterations [34–37].

Fig. 9 – Images of the MSC cells stained with ARS after 21 days of

and (D) PVAl-CNF. Magnification of 200·.

Small quantities of MSCs were observed on the materials.

This result was expected because these cells show kinetic

adhesion and a slower division capacity compared with Vero

cells [38,39]. In accordance with international standards, MSCs

are identified based not only on their morphological and phe-

notypic characteristics, but also on their ability to differentiate

into osteoblasts, chondrocytes and adipocytes [40–42].

The MSCs were isolated and differentiated into osteogenic

cells, according to a specific protocol [26]. After 21 days of

induction, the cells started producing ALP, and it was possi-

ble, by means of ARS, to confirm the presence of mineralised

nodules, thus attesting to the MSCs’ differentiation potential.

The PVAl, PVAl-CNT and PVAl-CNF cultivated in contact with

the MSCs did not interfere with this differentiation, which

was confirmed by the fact that their ALP quantification and

ARS staining were similar to those of the control cells.

osteogenic differentiation. (A) Control; (B) PVAl; (C) PVAl-CNT

458 C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9

Our results are in accordance with the literature. Chen

et al. [43] analysed the development of newborn rat tooth

germ cells cultured in contact with PVAl, and they observed

ALP produced by cells and mineralisation nodules with mRNA

expression for osteocalcin and osteopontin production. Tay

et al. [44] found that CNT had a positive influence on the

adhesion and differentiation of human mesenchymal stem

cells (hMSCs). According to those authors, CNT modulated

the growth and differentiation of the hMSCs, providing a

higher adhesion rate and cellular spreading. These findings

were confirmed by Penalver et al. [45], who found that CNT

films promoted greater adhesion of MSC cells and did not

interfere with the osteogenic differentiation process.

Some studies described in the literature will be important

to guide the use of CNTs in biomaterials to be used to repair

or replace defects bone and osteochondral [46–48]. Akasaka

et al. [46] prepared culture dishes with homogeneous thin

or thick films of non-modified CNTs and examined the effect

on human osteoblastic cells. The authors showed that the

CNTs films were the most effective substrate for the prolifer-

ation of osteoblastic cells, indicating that these materials

can be used as an effective biomaterial for osteoblastic cells

culturing and proliferation. Usui et al. [47] evaluated the

CNTs implanted subperiosteally in skull and tibial defects

created in mouse to examine the bone–tissue compatibility.

After 4 weeks implantation, the results demonstrate that

CNTs permitting bone repair and accelerate bone formation

in response to recombinant human morphogenetic protein-

2. Facca et al. [48] evaluated CNT reinforced with hydroxyap-

atite coating on titanium implants in bone mouse. The

authors observed higher osseointegration around the

implants with CNT.

Although there are few studies using hydrogels associated

with carbon nanotubes, the results presented here encourage

further investigation of this combination’s use in treating

osteochondral lesions and possible applications to other dis-

eases that involve the locomotor apparatus. Since the

in vitro results alone are not sufficient to indicate the most

suitable material for treatment of osteochondral defects, the

next step of this study is to investigate the in vivo behaviour

of PVAl hydrogels reinforced with carbon nanoparticles surgi-

cally implanted in defects artificially produced in articular

cartilage of rats.

5. Conclusion

The PVAl, PVAl-CNT and PVAl-CNF samples did not inter-

fere with the growth and development of Vero cells and

mesenchymal stem cells, or with their potential for osteo-

genic differentiation. The results obtained for the blends

of PVAl and carbon nanoparticles (CNT and CNF) make

these combinations more promising for treating osteochon-

dral defects than PVAl alone.

Acknowledgements

We would like to thank CAPES, CNPq, FAPESP, INCT-BIOFABRIS

and National Laboratory of Synchrotron Light – LNLS, Brazil.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.carbon.2011.08.071.

R E F E R E N C E S

[1] Hunziker EB. Articular cartilage repair: basic science andclinical progress. A review of the current status andprospects. Osteoarthr Cartil 2002;10:432–63.

[2] Chen F, Frenkel S, Di CP. Rapair of articular cartilage defects:part II. Treatment options. Am J Orthop 1999;28:88–96.

[3] Baumgaertner MR, Cannon WD, Vittori JM, Schmidt ES,Maurer RC. Arthrocospic debridement of the arthritic knee.Clin Orthop 1990;253:197–202.

[4] Brown MD, Mailinint I, David PB. A roentgenographicevaluation of frozen allografts versus autografts in anteriorcervical spine fusions. Clin Orthop 1976;119:231–6.

[5] Beaver RJ, Mahomed M, Backstein D, Davis A, Zukor DJ, GrossAE. Fresh osteochondral allografts for post-traumatic defectsin the knee. A survivorship analysis. J Bone Joint Surg1992;74B:105–10.

[6] Rish BL, Mcfadden JT, Penix JO. Anterior cervical fusion usinghomologous bone grafts. A comparative study. Surg Neurol1976;5:119–21.

[7] Ishaug-Riley SL, Crane GM, Gurlek A, Miller MJ, Yasko AW,Yaszemski MJ, et al. Ectopic bone formation by marrowstromal osteoblast transplantation using poly(L-lactic-co-glycolic acid) foams implanted into the rat mesentery. JBiomed Mater Res 1997;36:1–8.

[8] Guillot P, Cui W, Fisk NM. Stem cell differentiation andexpansion for clinical applications and tissue engineering. JCell Mol Med 2007;11:935–44.

[9] Younger EM, Chapman MN. Morbidity at bone graft donorsites. J Orthop Trauma 1989;3:192–5.

[10] Finkemeier CG. Bone-grafting and bone-graft substitutes. JBone Joint Surg 2002;84:454–64.

[11] Bayne O, Langer F, Pritzker KP, Gross AE. Osteochondralallografts in the treatment of osteoarthritis of the knee.Orthop Clin North Am 1985;16:727–40.

[12] Hutmacher DW. Scaffolds in tissue engineering bone andcartilage. Biomaterials 2000;21:2529–43.

[13] Iijima S. Helical microtubes of graphitic carbon. Nature1991;354:56–8.

[14] Li WZ, Xie SS, Qian LX, Chang BH, Zou BS, Wang G. Large-scale synthesis of carbon nanotubos. Science1996;274:1701–3.

[15] Sinnott SB, Andrews R. Carbon nanotubes: synthesis,properties and applications. Crit Rev Solid State Mater Sci2001;26:145–249.

[16] Chen Y, Bahar Bilgen BS, Pareta RA, Myles AJ, Fenniri, AaronRK, et al. Self-assembled rosette nanotube/hydrogelcomposites for cartilage tissue engineering. Tissue Eng Part C2010;16:1233–44.

[17] Sangram KS, Fernandes EG, Chiellini F, Chiellini E. Thermalanalysis of PVA/CNTs 2D membrane. J Therm Anal Calorim2009;97:859–64.

[18] Peppas NA, Korsmeyer RW. Hydrogels in medicine andpharmacology. Boca Raton: CRC Press; 1987.

[19] Krumova M, Lopes D, Benavente R, Mijangos C, Perena JM.Effect of crosslinking on the mechanical and thermalproperties of poly (viny alcohol). Polymer 2000;41:9265–72.

[20] ISO 10993-5 I – (E). Biological evaluation of medical devices.Part 5: Tests for cytotoxicity: in vitro methods; 1992.

C A R B O N 5 0 ( 2 0 1 2 ) 4 5 0 – 4 5 9 459

[21] Kirkpatrick CJ. Biological testing of materials and medicaldevices – a critical view of current and proposedmethodologies for biocompatibility testing: cytotoxicityin vitro. Reg Affairs 1992;4:13–32.

[22] ASTM F813-83. Standard practice for direct contact cellculture evaluation of materials for medical devices.

[23] Mossmam TJ. A rapid colorimetric assay of cellular growthand survival: application to proliferation and cytotoxicityassays. J Immunol Methods 1983;65:55–63.

[24] Lison, L. Histochemie et Cytochemie Animales – Principles etMethodes. Paris, France: Gauthier Villars; 1960.

[25] Mello MLS. Cytochemistry of DNA, RNA and nuclear proteins.Braz J Genetics 1997;20:257–64.

[26] Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I.Effects of plating density and culture time on bone marrowstromal cell characteristics. Exp Hematol 2008;36:1176–85.

[27] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture:comparison with cetylpyridinium chloride extraction. AnalBiochem 2004;329:77–84.

[28] Thomsem C, Reich S. Double resonant Raman scattering ingraphite. Phys Rev Lett 2000;85:5214–7.

[29] Dresselhaus M, Dresselhaus G, Jorio A, Filho AGS, PimentoMA, Saito R. Single nanotube raman spectroscopy. Acc ChemRes 2002;35:1070–8.

[30] Santos Jr AR, Ferreira BMP, Duek EAR, Dolder H, Wada MLF.Use of blend of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate-co-hydroxyvalerate) as surface forVero cell cultured. Braz J Med Biol Res 2005;38:1623–32.

[31] Santos Jr AR, Barbanti SH, Duek EAR, Wada MLF. Analysis ofthe growth pattern of Vero cells cultured on dense andporous poly(L-lactic acid) scaffolds. Mater Res 2009;12:257–63.

[32] Lombello CB, Malmonge SM, Wada MLF. PolyHEMA and Poly-HEMA-poly(MMA-co-AA) as substrates for culturing Verocells. J Mater Sci Mater Med 2000;11:541–6.

[33] Buser D, Shenk RK, Steinemman S, Fiorellini JP, Fox CH, SitichH. Influence of surface characteristics on bone integration oftitanium implants. J Biomed Mater Res 1991;25:889–902.

[34] Eisenbarth E, Meyle J, Breme Nachtigall W. Influence of thesurface structure of titanium materials on the adhesion offibroblasts. Biomaterials 1996;17:1399–403.

[35] Deligianni DD, Katasla N, Sotiropolou D. Effects of surfaceroughness of the titanium alloy Ti–6Al–4V on bone marrowcell response and on protein adsorption. Biomaterials2001;22:1241–51.

[36] Anselme K, Bigerelle M, Noel B. Effect of grooved titaniumsubstratum on human osteoblastic cell growth. J BiomedMater Res 2002;60:529–40.

[37] Curtis A, Wilknson C. Topographical control of cells.Biomaterials 1997;18:1573–83.

[38] Chamberlain G, Fox J, Aston B. Concise review: mesenchymalstem cells: their phenotype, differentiation capacity,immunological features, and potential for homing. StemCells 2007;25:2739–49.

[39] Chiu RC. Bone marrow stem cells as a source for cell therapy.Heart Fail Rev 2003;8:247–51.

[40] Abdallah BM, Kassem M. Human mesenchymal stem cells:from basic biology to clinical applications. Gene Ther2008;15:109–16.

[41] Bydlowski SP, Debes AA, Maselli LMF, Janz FL. Caracterısticasbiologicas das celulas-tronco mesenquimais. Rev Bras HematHemot 2009;31:25–35.

[42] Anghileri E, Marconi S, Pignatelli A. Neuronal differentiationpotential of human adipose-derived mesenchymal stemcells. Stem Cell Dev 2008;17:909–16.

[43] Chen R-S, Chen M-H, Young T-H. Induction of differentiationand mineralization in rat tooth germ cells on PVA throughinhibition of ERK1/2. Biomaterials 2009;30:541–7.

[44] Tay CY, Gu H, Leong WS, Yu H, Li HQ, Heng BC, et al. Cellularbehavior of human mesenchymal stem cells cultured onsingle-walled carbon nanotube film. Carbon2010;48:1095–104.

[45] Penalver JL, Linares-Fernandez JL, Farıas VA, Lopez-RamonMV, Tassi M, Oliver FJ, et al. Activated carbon cloth assupport for mesenchymal stem cell growth anddifferentiation to osteocytes. Carbon 2009;47:3574–84.

[46] Akasaka T, Yokoyama A, Matsuoka M, Hashimoto T, Watari F.Thin films of single-walled carbon nanotubes promotehuman osteoblastic cells (Saos-2) proliferation in low serumconcentrations. Mater Sci Eng C 2010;30:391–9.

[47] Usui Y, Aoki K, Narita N, Murakami N, Nakamura I, NakamuraK, et al. Carbon nanotubes with high bone–tissuecompatibility and bone-formation acceleration effects. Small2008;2:240–6.

[48] Facca S, Lahiri D, Fioretti F, Messadeq N, Mainard D,Benkirane-Jessel N, et al. In vivo osseointegration of nano-designed composite coatings on titanium implants. ACSNano 2011;5:4790–9.