1
0
S. Sartori et al. / Reactive & Functional Polymers xxx (2013) xxx–xxx
The unsuitability of NS-BC2000 was also confirmed by Western
results reported in this work suggest a better biological response
for polymers having surface with less ordered structure, as those
observed in C-BC2000 and K-BC2000 by AFM studies. The poor cell
adhesion of NS-BC2000 may be ascribed to its higher degree of or-
der, compared to the other analysed polymers. However, a more
detailed study should be conducted to confirm this hypothesis
and comprehensive experiments concerning this aspect will be
the subject of future studies.
In the series of polyurethanes studied in this work, the most
promising polymer for the repair of muscle tissues was
K-BC2000, since it better matched the elastomeric behaviour of
muscle tissues (such as skeletal muscle and cardiac tissue) and,
in the meantime, cell tests performed with myoblasts on this
substrate showed high viability, adequate cell adhesion, spreading
and proliferation. Additional experiments are needed to evaluate
the contractile cell ability and preservation of the contractile
phenotype over time.
Blot (WB) analysis (Fig. 10) reporting a down-regulated expression
of vinculin, a membrane-cytoskeletal protein involved in cell
spreading and focal adhesion formation. On the contrary,
K-BC2000 and C-BC2000 showed a vinculin expression comparable
to the control, while on A-BC2000 this parameter resulted slighly
down-regulated even if still indicating an appropriate cell adhesion
on the substrate. Vinculin expression assessed with WB is in accor-
dance with the results of MTS assay and morphology observations
(
Fig. 11). The analysis of the expression of the proliferating cell nu-
clear antigen (PCNA) protein at 3 days of myoblast seeding pro-
vided information about cell proliferation. The samples K-BC2000
and C-BC2000 showed PCNA protein expression approximately
comparable to the positive control, while a weak decrease was ob-
served, as expected, on NS-BC2000 and A-BC2000. The ability of
cells to proliferate should be due to the synthesis of extracellular
matrix proteins in the first hours of cell adhesion, promoting an
adequate environment for cell proliferation.
Fluorescence staining for the f-actin component of the cytoskel-
Acknowledgment
2
eton showed in Fig. 11 illustrates how, C C12 myoblast cells
have adhered and proliferated on all surfaces after 7 days. On
K-BC2000, C-BC2000 and A-BC2000 substrates, actin stress fibres
can be observed; cells formed a defined cytoskeletal arrangement
and adopted an adequate spindle-shape morphology. NS-BC2000
does not promote myoblast spreading, since cells maintained an al-
most round shape. These observations confirmed the data obtained
by western blot analysis on vinculin expression.
This study was financially supported by POR Piattaforme Inno-
vative (F.E:S.R. 2007-2013) ‘‘Active-Advanced Cardiovascular Ther-
apies’’ and the Italian MUR PRIN2008.
Appendix A. Supplementary material
at
4
. Conclusion
References
In this work the influence of the chain extender in the physico-
chemical properties of a series of polyester-urethanes was studied.
The chemical structure of the chain extenders affects the crystal-
linity and the mechanical properties of the resulting polymers. In
detail, crystallinity estimated by DSC analysis correlates with the
morphologies of polymer thin films observed by AFM: PURs that
possess the highest crystallinity (A-BC2000 and NS-BC2000)
showed high order spherulitic morphologies; on the opposite, the
PUR with the lowest crystallinity (K-BC2000) showed a clear phase
separation but no spherulites and lamellar structures were
detected.
Thermal characterisation hightlighted that all the synthesised
PURs can be suitable substrates for biomedical applications.
Mechanical tests showed that K-BC2000 and NS-BC2000 are good
candidates as scaffold materials for the regeneration of contractile
tissues because of their elastomeric behaviour.
[
1] K.A. Gross, L.M. Rodr ´ı guez-Lorenzo, Biomaterials 25 (2004) 4955–4962.
[2] G.F. Muschler, C. Nakamoto, L.G. Griffith, J. Bone Joint Surg. (2004) 1541–1558.
[
[
[
3] M. Sabir, X. Xu, L. Li, J. Mater. Sci. 44 (2009) 5713–5724.
4] A.R. Webb, J. Yang, G.A. Ameer, Expert Opin. Biol. Therm. 4 (2004) 801–812.
5] M.M. Aygen, E. Braunwald, Circulation 26 (1962) 516–524.
[6] A. Biewener, R. Baudinette, J. Exp. Biol. 198 (1995) 1829–1841.
[
7] A. Silvestri, P.M. Serafini, S. Sartori, P. Ferrando, F. Boccafoschi, S. Milione, L.
Conzatti, G. Ciardelli, J. Appl. Polym. Sci. 122 (2011) 3661–3671.
8] S. Sartori, A. Rechichi, G. Vozzi, M. D’Acunto, E. Heine, P. Giusti, G. Ciardelli,
React. Funct. Polym. 68 (2008) 809–821.
[
[9] P.A. Gunatillake, G.F. Meijs, S.J. McCarthy, R. Adhikari, N. Sherriff, J. Appl.
Polym. Sci. 69 (1998) 1621–1633.
10] G. Ciardelli, A. Rechichi, S. Sartori, M. D’Acunto, A. Caporale, E. Peggion, G.
Vozzi, P. Giusti, Polym. Adv. Technol. 17 (2006) 786–789.
[11] P. Lu, K. Takai, V.M. Weaver, Z. Werb, Extracellular matrix degradation and
remodeling in development and disease, Cold Spring Harb. Perspect. Biol 3
[
(
2011) 1–2.
[
12] J. Guan, W.R. Wagner, Biomacromolecules 6 (2005) 2833–2842.
[13] G. Ciardelli, A. Rechichi, P. Cerrai, M. Tricoli, N. Barbani, P. Giusti, Macromol.
Symp. 218 (2004) 261–272.
14] H. Kweon, M.K. Yoo, I.K. Park, T.H. Kim, H.C. Lee, H.-S. Lee, J.-S. Oh, T. Akaike, C.-
S. Cho, Biomaterials 24 (2003) 801–808.
[15] M. Cooke, N. Leeves, C. White, Arch. Oral. Biol. 48 (2003) 323–327.
16] K.L. Fujimoto, K. Tobita, W.D. Merryman, J. Guan, N. Momoi, D.B. Stolz, M.S.
Sacks, B.B. Keller, W.R. Wagner, J. Am. Coll. Cardiol. 49 (2007) 2292–2300.
17] N.M. Bless, D. Smith, J. Charlton, B.J. Czermak, H. Schmal, H.P. Friedl, P.A. Ward,
Curr. Biol. 7 (1997) 877–880.
Myoblast adhesion on the synthesised PURs was lower than
that observed for the tissue culture polystyrene control; approxi-
mately 40/50% of the seeded cells adhered to the analysed sub-
strates. Moreover, our studies on cell growth over time (3 and
[
[
[
[
[
[
[
7
days) demonstrated the ability of the adhered cells to spread
and proliferate on the synthesised PURs, with the exception of
NS-BC2000.
It is apparent from all the biocompatibility tests that C-BC2000
and K-BC2000 exhibited the best performances.
18] R.N. Zuckermann, J.M. Kerr, S.B.H. Kent, W.H. Moos, J. Am. Chem. Soc. 114
(
1992) 10646–10647.
19] A. Rechichi, G. Ciardelli, M. D’Acunto, G. Vozzi, P. Giusti, J. Biomed. Mater. Res.
A 84 (2008) 847–855.
20] M. Penco, L. Sartore, F. Bignotti, S. D’Antone, L. Di Landro, Eur. Polym. J. 36
Despite some authors relate polymer biocompatibility to hydro-
philicity [30,31] or to mechanical properties [32,33], we could not
observe a trend in biological response related to these properties.
A relationship between phase separation and biological re-
sponse was observed by Hsu [34] and Rodriguez [35]. Kohr et al.
reported that heterogeneous morphologies developed by different
monomer compositions of block copolymers showed different cell
adhesion morphologies with regard to serum proteins [36]. Never-
theless, the detailed relationship between microphase separation
and biocompatibility has not been extensively studied yet. The
(
2000) 901–908.
21] J. Guan, M.S. Sacks, E.J. Beckman, W.R. Wagner, J. Biomed. Mater. Res. 61
(2002) 493–503.
[22] K.D. Kavlock, T.W. Pechar, J.O. Hollinger, S.A. Guelcher, A.S. Goldstein, Acta
Biomater. 3 (2007) 475–484.
23] S.L. Cooper, A.V. Tobolsky, J. Appl. Polym. Sci. 10 (1966) 1837–1844.
24] L. Averous, L. Moro, P. Dole, C. Fringant, Polymer 41 (2000) 4157–4167.
[
[
[25] D. Sarkar, J.-C. Yang, A.S. Gupta, S.T. Lopina, J. Biomed. Mater. Res. A 90A (2009)
63–271.
2
[
[
26] N. Maffulli, J. Bone Joint Surg. 81 (1999) 1019–1036.
27] Z. Ge, F. Yang, J.C. Goh, S. Ramakrishna, E.H. Lee, J. Biomed. Mater. Res. A 77
(2006) 639–652.