Design and Characterization of a Composite Material Based on Sr(II)-Loaded Clay Nanotubes Included within a Biopolymer Matrix

INTRODUCTION Materials for bone tissue engineering should guide and promote the regeneration of bone tissue. Various biocompatible and biodegradable porous scaffolds have been recently designed, with the aim to provide a temporary substitute which mimics bone extracellular matrix structure. Several materials have been recently suggested in the literature as promising candidates, including bioceramics, bioglasses, and hydroxyapatite particles embedded in polymeric scaffolds. Nevertheless, synthetic scaffolds still display many limitations, boosting the search for materials integrating both osteoconductive and osteoinductive properties1,2. In this study we formulated and evaluated the in vitro cytotoxicity of a nanocomposite biomaterial for bone regeneration that integrates the structural properties of Halloysite nanotubes (HNT) and the bioactivity of strontium (Sr(II)) included into a biocompatible and biodegradable 3-polyhydroxybutyrate-co-3-hydroxy-valerate (PHBV) matrix. EXPERIMENTAL METHODS HNT powder (Imerys Minerals Ltd; Auckland, New Zealand) was functionalized with a solution of APTES in ethanol (2.5 %wt). The APTES amount was chosen to cover about 20% of the HNT external surface. To upload Sr(II), 100 mg of HNTs were mixed with 10 ml of a 100 mg/ml SrCl2o6H2O solution. The dispersion was vigorously mixed for 24 h and then centrifuged. The solid was recollected, dried, and then used for the subsequent preparation of the nanocomposites. HNT dispersions were spin coated by means of a P6700 spin coater (Speciality Coating Systems Inc.) onto the glass previously functionalized with APTES, and then a PHBV chloroform solution (2% wt) was spin coated on top of it. The coating process was carried out at r. t. in two stages at constant rotating speed (1500 and 2500 rpm), separated by an acceleration step. The composite morphology was evaluated after degradation in PBS at 37°C for 28 days by SEM microscopy. The retained amount of loaded Sr(II) in HNT-Sr and HNT-Sr-PHBV was evaluated by inductively coupled plasma-atomic emission spectroscopy. For cytotoxicity evaluation, nanocomposite samples were grinded and suspended in distilled water by a Ultra-Turrax® T25 Basic mechanical homogenize. L929 fibroblasts (4x103 cell/well) were incubated with HNT, HNT-Sr, HNT-PHBV and HNT-Sr-PHBV sample solutions (25, 50 and 100 µg/mL) and cell viability was evaluated by MTT and Trypan blue assay after 72 h of incubation, while cell proliferation by BrdU assay at 48 h. RESULTS AND DISCUSSION SEM image of the degraded composite evidenced a random bulk erosion process with a increase of exposed HNT respect the control sample, where HNT was almost completely embedded in PHBV matrix. The value of retained amount of loaded Sr(II) was similar in HNT-Sr and HNT-Sr-PHBV samples (~ 60% Sr(II) after 28 days) indicating that PHBV did not affect the release of Sr(II) and simply modulated the exposure of HNT to the surrounding medium. The cytotoxicity and proliferation assays showed a cell viability (>80%) and proliferation (similar to control) of the cells incubated with HNT, HNT-PHBV and HNT-Sr-PHBV at all concentrations, indicating high biocompatibility of HNT, HNT-PHBV and HNT-Sr-PHBV up to 100 ?g/mL. However, the HNT-Sr induces a significant viability reduction and proliferation already at 25 ?g/mL, suggesting the need for a barrier to modulate its interaction with cells. CONCLUSION In this study we demonstrated the possibility to use a combination of Halloysite nanotubes and PHBV biopolymer for drug delivery of Sr(II). Furthermore, we have prepared for the first time a HNT-PHBV composite capable of effectively uploading and carrying high doses of Sr(II) with a low cytotoxicity modulated by PHBV coating.