Impact of PLA/Mg films degradation on surface physical properties and biofilm survival.

New biocompatible and bioabsorbable materials are currently being developed for bone regeneration. These serve as scaffolding for controlled drug release and prevent bacterial infections. Films of polylactic acid (PLA) polymers that are Mg-reinforced have demonstrated they have suitable properties and bioactive behavior for promoting the osseointegration process. However little attention has been paid to studying whether the degradation process can alter the adhesive physical properties of the biodegradable film and whether this can modify the biofilm formation capacity of pathogens. Moreover, considering that the concentration of Mg and other corrosion products may not be constant during the degradation process, the question that arises is whether these changes can have negative consequences in terms of the bacterial colonization of surfaces. Bacteria are able to react differently to the same compound, depending on its concentration in the medium and can even become stronger when threatened. In this context, physical surface parameters such as hydrophobicity, surface tension and zeta potential of PLA films reinforced with 10% Mg have been determined before and after degradation, as well as the biofilm formation capacity of Staphylococcus epidermidis. The addition of Mg to the films makes them less hydrophobic and the degradation also reduces the hydrophobicity and increases the negative charge of the surface, especially over long periods of time. Early biofilm formation at 8 h is consistent with the physical properties of the films, where we can observe a reduction in the bacterial biofilm formation. However, after 24 h of incubation, the biofilm formation increases significantly on the PLA/Mg films with respect to PLA control. The explosive release of Mg ions and other corrosion products within the first hours were not enough to prevent a greater biofilm formation after this initial time. Consequently, the Mg addition to the polymer matrix had a bacteriostatic effect but not a bactericidal one. Future works should aim to optimize the design and biofunctionality of these promising bioabsorbable composites for a degradation period suitable for the intended application.

Bioactivity of dexamethasone-releasing coatings on polymer/magnesium composites.

We developed biodegradable polymeric coatings loaded with increasing amounts of dexamethasone on composites based on polylactic acid and Mg particles for bone repair. Incorporation of Mg particles into the polymeric matrix improves the compressive behaviour of the polymer. Mg-containing composites release Mg2+ ions into the culture medium and improve mesenchymal stem cell (MSC) viability, enhance their osteogenic potential and promote the release of angiogenic factors. Dexamethasone-loaded coatings deposited on composites delay Mg2+ ion dissolution while releasing controlled amounts of the drug, which are highly dependent on initial payload. Release kinetic of dexamethasone from the coatings exhibits a fast initial release of the drug followed by a slower secondary release. Bioactivity of the released dexamethasone was explored by monitoring dose-dependent responses of MSCs and macrophages. Biological effects exerted by the released drug are similar to those observed in cells treated with solutions of the glucocorticoid, indicating that the method employed for inclusion of dexamethasone into the coatings does not impair its bioactive behaviour. Culturing MSCs on dexamethasone-releasing coatings enhances extracellular matrix production and initial induction to osteogenic commitment as a function of drug payload. Dexamethasone incorporated into the coatings presents anti-inflammatory activity, as shown by the decrease in the production of cytokines and angiogenic factors by macrophages and MSCs. Deposition of dexamethasone-releasing coatings on polymer/Mg composites appears to be a promising approach to delay composite degradation at the early stage of implantation and may be useful to attenuate inflammation and adverse foreign body reactions.

Incorporation of Mg particles into PDLLA regulates mesenchymal stem cell and macrophage responses.

In this work, we investigated a new approach to incorporate Mg particles within a PDLLA matrix using a solvent-free commercially available process. PDLLA/Mg composites were manufactured by injection moulding and the effects of Mg incorporated into PDLLA on MSC and macrophage responses were evaluated. Small amounts of Mg particles (≤ 1 wt %) do not cause thermal degradation of PDLLA, which retains its mechanical properties. PDLLA/Mg composites release hydrogen, alkaline products and Mg(2+) ions without changing pH of culture media. Mg-containing materials provide a noncytotoxic environment that enhances MSC viability. Concentration of Mg(2+) ions in extracts of MSCs increases with the increment of Mg content in the composites. Incorporation of Mg particles into PDLLA stimulates FN production, ALP activity, and VEGF secretion in MSCs, an effect mediated by degradation products dissolved from the composites. Degradation products of PDLLA induce an increase in MCP-1, RANTES, and MIP-1α secretion in macrophages while products of composites have minimal effect on these chemokines. Regulation of MSC behavior at the biomaterial's interface and macrophage-mediated inflammatory response to the degradation products is related to the incorporation of Mg in the composites. These findings suggest that including small amounts of Mg particles into polymeric devices can be a valuable strategy to promote osseointegration and reduce host inflammatory response.

In vitro biocompatibility of an ultrafine grained zirconium.

We have investigated a novel ultrafine grained (UFG) Zr obtained by severe plastic deformation (SPD) which resulted in a refinement of the grain size by several orders of magnitude. Compared to conventional Zr, higher hardness values were measured on UFG Zr. Polished surfaces having similar topographical features from both materials were prepared, as assessed by atomic force microscopy (AFM). Surface hydrophobicity of Zr, evaluated by measuring water contact angles, was unaffected by grain size reduction. In vitro biocompatibility was addressed on conventional and UFG Zr surfaces and, for comparative purposes, a polished Ti6Al4V alloy was also investigated. Cell attachment and spreading, actin and beta-tubulin cytoskeleton reorganisation, fibronectin secretion and cellular distribution as well as cell viability were evaluated by culturing human osteoblastic Saos-2 cells on the surfaces. The osteoblastic response to conventional Zr was found to be essentially identical to Ti6Al4V and was not affected by grain size reduction. In order to evaluate the ability of the surfaces to promote osteogenic maturation and bone matrix mineralisation, human mesenchymal cells from bone marrow were switched to the osteoblastic phenotype by incubation in osteogenic induction media. Compared to undifferentiated mesenchymal cells, alkaline phosphatase activity and formation of mineralisation nodules were enhanced to the same extent on both Zr surfaces and Ti6Al4V alloy after induction of osteoblastic differentiation. In summary, improved mechanical properties together with excellent in vitro biocompatibility make UFG Zr a promising biomaterial for surgical implants.

Differential inflammatory macrophage response to rutile and titanium particles.

Titanium and its alloys are widely used as implant materials for dental and orthopaedic applications due to their advantageous bulk mechanical properties and biocompatibility, compared to other metallic biomaterials. In order to improve their wear and corrosion resistance, several surface modifications that give rise to an outer ceramic layer of rutile have been developed. The ability of rutile wear debris to stimulate the release of inflammatory cytokines from macrophages has not been addressed to date. We have compared the in vitro biocompatibility of sub-cytotoxic doses of rutile and titanium particles in THP-1 cells driven to the monocyte/macrophage differentiation pathway as well as in primary cultures of human macrophages. Confocal microscopy experiments indicated that differentiated THP-1 cells and primary macrophages efficiently internalised rutile and titanium particles. Treatment of THP-1 cells with rutile particles stimulated the release of TNF-alpha, IL-6 and IL-1beta to a lesser extent than titanium. The influence of osteoblasts on the particle-induced stimulation of TNF-alpha and IL-1beta was analysed by co-culturing differentiated THP-1 cells with human primary osteoblasts. Under these conditions, secretion levels of both cytokines after treatment of THP-1 cells with rutile particles were lower than after exposure to titanium. Finally, we observed that primary macrophages released higher amounts of TNF-alpha, IL-6 and IL-1beta after incubation with titanium particles than with rutile. Taken together, these data indicate that rutile particles are less bioreactive than titanium particles and, therefore, a higher biocompatibility of titanium-based implants modified with an outer surface layer of rutile is expected.