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. 2013 Jan 9;10(80):20120833.
doi: 10.1098/rsif.2012.0833. Print 2013 Mar 6.

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

A Gloria  1 , T Russo, U D'Amora, S Zeppetelli, T D'Alessandro, M Sandri, M Bañobre-López, Y Piñeiro-Redondo, M Uhlarz, A Tampieri, J Rivas, T Herrmannsdörfer, V A Dediu, L Ambrosio, R De Santis
Affiliations

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

A Gloria et al. J R Soc Interface. .

Abstract

In biomedicine, magnetic nanoparticles provide some attractive possibilities because they possess peculiar physical properties that permit their use in a wide range of applications. The concept of magnetic guidance basically spans from drug delivery and hyperthermia treatment of tumours, to tissue engineering, such as magneto-mechanical stimulation/activation of cell constructs and mechanosensitive ion channels, magnetic cell-seeding procedures, and controlled cell proliferation and differentiation. Accordingly, the aim of this study was to develop fully biodegradable and magnetic nanocomposite substrates for bone tissue engineering by embedding iron-doped hydroxyapatite (FeHA) nanoparticles in a poly(ε-caprolactone) (PCL) matrix. X-ray diffraction analyses enabled the demonstration that the phase composition and crystallinity of the magnetic FeHA were not affected by the process used to develop the nanocomposite substrates. The mechanical characterization performed through small punch tests has evidenced that inclusion of 10 per cent by weight of FeHA would represent an effective reinforcement. The inclusion of nanoparticles also improves the hydrophilicity of the substrates as evidenced by the lower values of water contact angle in comparison with those of neat PCL. The results from magnetic measurements confirmed the superparamagnetic character of the nanocomposite substrates, indicated by a very low coercive field, a saturation magnetization strictly proportional to the FeHA content and a strong history dependence in temperature sweeps. Regarding the biological performances, confocal laser scanning microscopy and AlamarBlue assay have provided qualitative and quantitative information on human mesenchymal stem cell adhesion and viability/proliferation, respectively, whereas the obtained ALP/DNA values have shown the ability of the nanocomposite substrates to support osteogenic differentiation.

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Figures

Figure 1.
Figure 1.
Schematic of moulding and solvent-casting techniques used to obtain polymeric and nanocomposite disc-shaped specimens. (Online version in colour.)
Figure 2.
Figure 2.
XRD spectra relative to PCL, FeHA and the composite containing the biggest amount of magnetic phase (PCL/FeHA 70/30).
Figure 3.
Figure 3.
SEM image of PCL/FeHA (80/20).
Figure 4.
Figure 4.
EDS image of PCL/FeHA (80/20). (Online version in colour.)
Figure 5.
Figure 5.
SEM–EDS P-, Ca- and Fe-mapping photographs of PCL/FeHA (80/20).
Figure 6.
Figure 6.
Load–displacement curves obtained from small punch tests performed on neat PCL and PCL reinforced with FeHA nanoparticles.
Figure 7.
Figure 7.
Results obtained from micro-CT analysis: reconstruction of (a) PCL/FeHA (90/10 w/w) and (b) PCL/FeHA (70/30 w/w) obtained by integrating Skyscan's software package, ImageJ software, Materialise Mimics and Rapidform 2006. (Online version in colour.)
Figure 8.
Figure 8.
Typical image qualitatively representing the water contact angle. (Online version in colour.)
Figure 9.
Figure 9.
Field-dependent magnetization of the three PCL/FeHA compositions investigated in the article. Each curve was taken at T = 310 K (human body temperature). The coercive field is approximately 15 Oe, independent of the composition. Details are given in the text.
Figure 10.
Figure 10.
Temperature dependence of the magnetization of the PCL/FeHA (90/10) sample. The sample was cooled in zero-field, then heated in a small field (50 Oe—fh branch) and again cooled in field (fc branch). Details are given in the text.
Figure 11.
Figure 11.
Frequency dependence of the (a) real (b) and imaginary parts of the magnetic susceptibility of PCL/FeHA (90/10). Details are given in the text.
Figure 12.
Figure 12.
Hyperthermia curves of the PCL/FeHA magnetic scaffolds under application of a RF magnetic field of f = 260 kHz and H = 27 mT, suitable for in vivo applications. (Online version in colour.)
Figure 13.
Figure 13.
Cell adhesion study: CLSM images at different times after cell seeding. (i) From top to bottom, PCL, PCL/FeHA 90/10, PCL/FeHA 80/20 and PCL/FeHA 70/30 at 7 days after cell seeding. (ii) From top to bottom, PCL, PCL/FeHA 90/10, PCL/FeHA 80/20 and PCL/FeHA 70/30 at 14 days after cell seeding. (iii) From top to bottom, PCL, PCL/FeHA 90/10, PCL/FeHA 80/20 and PCL/FeHA 70/30 at 21 days after cell seeding. Scale bar, 100 µm.
Figure 14.
Figure 14.
Results obtained from AlamarBlue assay at 7, 14 and 21 days after seeding. Error bar represents the s.d. *p < 0.05; **p < 0.01; ***p < 0.001, indicate statistically significant differences between nanocomposite and PCL substrates, at the same time from cell seeding (one-way ANOVA followed by Tukey's post hoc test).
Figure 15.
Figure 15.
Results obtained from ALP/DNA assay at 7, 14 and 21 days after cell seeding. Error bar represents the s.d.
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