Polyvinyl alcohol associated with carbon nanotube scaffolds for osteogenic differentiation of rat bone mesenchymal stem cells

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Polyvinyl alcohol associated with carbon nanotube scaffolds for osteogenic differentiation of rat bone mesenchymal stem 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, Sa˜o Paulo, Brazil Department of Semiconductors, Instruments and Photonics, School of Electrical and Computer Engineering, University of Campinas, Sa˜o Paulo, Brazil c Centre of Natural and Human Sciences, Federal University of ABC, Santo Andre´, Sa˜o Paulo, Brazil b

A R T I C L E I N F O

A B S T R A C T

Article history:

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

Received 25 April 2011

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

Accepted 31 August 2011

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

Available online 6 September 2011

tion. The carbon material was characterised with a Renishaw Invia Raman microscope system 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 bromide and scanning electron microscopy, respectively. The materials did not interfere with the viability, metabolic activity, morphology and spreading of either of the cell types analysed. Nodules of mineralised organic matrix were identified with ARS and ALP, confirming osteogenic differentiation. These results indicated higher concentration of ALP and mineralised 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 orthopaedic surgery because the cartilage has a limited capacity for self-repair following trauma [1]. Biological treatment options are mainly based on bone marrow stimulation, tissue transplantation and tissue engineering techniques [2–8]. The

first option is based on joint debridement and the bone marrow stimulation principle [2,3]. The second option involves the transplantation of periosteum and autologous or allogeneic osteochondral plugs [4–6]. Autologous grafts are advantageous because they do not elicit immunological rejection [9]; however, their disadvantages include limited availability, reduced mechanical stability and morbidity of the donor area

* Corresponding author: Fax: +55 19 3521 7498. E-mail addresses: [email protected], [email protected] (A.A. Rodrigues). 0008-6223/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2011.08.071

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[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 advantage 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 threedimensional structure necessary to replace articular cartilage, and they can absorb large quantities of water and biological fluids [12]. Carbon nanomaterials offer potential as structural reinforcements 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 material whose mechanical performance can be changed and tailored to use in regenerative medicine [16,17]. Among several types of polymeric hydrogels, polyvinyl alcohol (PVAl) has received special attention because its physicochemical characteristics 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 order to assess its potential use to treat osteochondral defects.

2.

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Table 1 – Cytotoxicity of different samples studied for Vero or mesenchymal stem cells (MSC). Cell type/assay

SD

ER

0.10163 0.39575 0.54663 0.66175 0.65125

0.04402 0.30694 0.43755 0.54617 0.54038

0.01556 0.10852 0.1547 0.1931 0.19105

VC direct cytotoxicity PCT 0.0726 NCT 0.4858 PVAl 0.50954 PVAl-CNT 0.4144 PVAl-CNF 0424

0.02503 0.12979 0.03633 0.05562 0.01696

0.01119 0.05805 0.01625 0.02487 0.00758

MSC indirect cytotoxicity PCT 0099 NCT 0253 PVAl 0.35225 PVAl-CNT 0.29925 PVAl-CNF 0.3465

0.00693 0.04588 0.09895 0.07413 0.07781

0.00346 0.02294 0.04947 0.03706 0.03891

VC indirect cytotoxicity PCT NCT PVAl PVAl-CNT PVAl-CNF

Mean

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

Experiment

2.1. Preparation and characterisation of carbon nanoparticles The two types of carbon nanoparticles, carbon nanotubes (CNT) and carbon nanofibres (CNF), were produced by hot-filament chemical vapour deposition (HFCVD). CNT was produced on a copper substrate covered with a thin film of polyaniline. Two-tenths of a millilitre of a 2 mg/ml nickel nitrate-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 pressure 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 nanoparticles 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(vinyl alcohol) (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, Sa˜o 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 biomaterials [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, Sa˜o Paulo, Brazil. The cells were cultivated in Ham’s F12 medium (Nutricell) supplemented with 10% foetal calf serum (FCS, Nutricell) and 1% penicillin/streptomycin (PS, Hyclone) at 37 C in an atmosphere with 5% CO2.

2.4. Isolation and culture of rat bone marrow mesenchymal stem cells The femur, tibia and humerus bones were removed from Wistar–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

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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 centrifuged 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 centrifuging 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 concentration 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 obtained 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 suspensions (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 polystyrene 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 medium 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 paraphormaldehyde (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, criticalpoint 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 detect 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 replaced with the osteogenic induction medium (low-glucose DMEM supplemented with 15% FCS, 1% penicillin/streptomycin, 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: phosphatase and alizarin red staining

alkaline

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 contained 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 taken 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 transferred to a 96-well plate, and the absorbance reading was taken at k = 405 nm.

3.

Results

3.1. Characterisation of carbon nanoparticles, PVAl pure and 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

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

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 intense peaks of approximately 1350 cm 1 and 1580 cm 1, corresponding 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 (Spectrum 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 revealed 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 similar 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 statistically 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 observed. 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,

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

Fig. 3 – Direct cytotoxicity assay of PVAl, PVAl-CNT and PVAlCNF with Vero cells. There are no statistically significant differences (*p > 0.05) between the samples and the negative control of toxicity (NCT).

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).

along with signs of cellular division (Fig. 6A1). On the PVAlCNT samples, cells with a very irregular morphological pattern 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 without fibrils, could be observed (Fig. 6C1). A small number of MSCs were found adhered to the surface 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 morphology 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, suggesting cellular migration (Fig. 6A1, B1 and C1).

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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 biomaterial, the cells exhibited strongly staining basophilic nuclei 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.

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).

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

3.5.

Alkaline phosphatase and alizarin red staining

Fig. 8 shows the semi-quantitative analysis for ALP, a commonly used bone differentiation marker. This enzyme was present in all experimental conditions investigated. The mineralised bone matrix was proven by ARS’ incorporation 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

Fig. 6 – SEM of the material surfaces: (A) PVAl, (B) PVAl-CNTand (C) PVAl-CNF with Vero (A1, B1 and C1) and MSC cells (A2, B2 and C2) for 24 h. The asterisk indicate the cells adhered on the materials and the arrows the cellular extensions. Magnification of 2000·.

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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.)

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 preselection of these materials for in vivo assays [20–22]. The cytotoxicity 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

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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.

basophilia, with slightly metachromatic nuclei and evident nucleoli could be observed, indicating cell activity. The identification 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].

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 phenotypic 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 possible, 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.

Fig. 9 – Images of the MSC cells stained with ARS after 21 days of osteogenic differentiation. (A) Control; (B) PVAl; (C) PVAl-CNT and (D) PVAl-CNF. Magnification of 200·.

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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 Pen˜alver 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 proliferation 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 protein2. Facca et al. [48] evaluated CNT reinforced with hydroxyapatite 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 diseases 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 surgically implanted in defects artificially produced in articular cartilage of rats.

5.

Conclusion

The PVAl, PVAl-CNT and PVAl-CNF samples did not interfere with the growth and development of Vero cells and mesenchymal stem cells, or with their potential for osteogenic differentiation. The results obtained for the blends of PVAl and carbon nanoparticles (CNT and CNF) make these combinations more promising for treating osteochondral 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.

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