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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.

This article reports on the interactions of human bone cells, mesenchymal stem cells (hMSCs) from bone marrow and osteoblasts (hOBs), with a submicron-grooved Ti6Al4V alloy that promotes cell orientation in the direction of the anisotropy. Adhesion sites, actin and tubulin networks and fibronectin extracellular matrix of both cell types align with the direction of the grooves. hMSCs adhere at a higher rate on the patterned substrate than on the polished alloy, while no differences are found in hOBs attachment. Compared to the flat substrate, RhoA activity is higher in hMSCs and hOB cultured on the grooved alloy and treatment with C3 transferase leads to loss of organization of actin and tubulin cytoskeletons. Rho-associated kinase (ROCK) activity of hMSCs is upregulated on the anisotropic samples, but not affected in hOBs. Treatment with hydroxyfasudil disrupts the alignment of adhesion sites in hMSCs but not in hOBs. When cells are cultured in media that support osteogenic maturation, OPN secretion increases in hMSCs on the anisotropic alloy and it remains unaffected in hOBs. Cell layer calcification proceeds to a same extent in hMSCs cultured on the two metallic surfaces but decreases in hOBs cultured on the patterned samples. Taken together, these results indicate that hOBs are less sensitive than hMSCs to the patterned Ti6Al4V alloy. This effect can be attributed to their different stages of cell maturation and may be mediated, at least in part, through ROCK signaling because its activity increases on hMSCs cultured on the patterned alloy, while hOBs fail to upregulate it. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3154–3166, 2014.

The interactions between bone tissue and orthopedic implants are strongly affected by mechanical forces at the bone-implant interface, but the interplay between surface topographies, mechanical stimuli, and cell behavior is complex and not well understood yet. This study reports on the influence of mechanical stretch on human mesenchymal stem cells (hMSCs) attached to metallic substrates with different roughness. Controlled forces were applied to plasma membrane of hMSCs cultured on smooth and rough stainless steel surfaces using magnetic collagen-coated particles and an electromagnet system. Degree of phosphorylation of focal adhesion kinase (p-FAK) on the active form (Tyr-397), prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) levels increased on rough samples under static conditions. Cell viability and fibronectin production decreased on rough substrates, while hMSCs maturated to the osteoblastic lineage to a similar extent on both surfaces. PGE2 production and osteoprotegerin/receptor activator of nuclear factor kappa-B ligand ratio increased after force application on both surfaces, although to a greater extent on smooth substrates. p-FAK on Tyr-397 was induced fairly rapidly by mechanical stimulation on rough surfaces while cells cultured on smooth samples failed to activate this kinase in response to tensile forces. Mechanical forces enhanced VEGF secretion and reduced cell viability, fibronetin levels and osteoblastic maturation on smooth surfaces but not on rough samples. The magnetite beads model used in this study is well suited to characterize the response of hMSCs cultured on metallic surfaces to tensile forces and collected data suggest a mechanism whereby mechanotransduction driven by FAK is essential for stem cell growth and functioning on metallic substrates.

Titanium and its alloys are widely used as implant materials for dental and orthopaedic applications. To improve their wear and corrosion resistance, several surface modifications that give rise to an outer ceramic layer of rutile have been developed. It is expected that after a long period of functional loading, rutile debris will arise from these modified surfaces. We have compared the in vitro biocompatibility of subcytotoxic doses of rutile and titanium particles of phagocytosable size in primary cultures of human osteoblasts. Particles were visualized using a spectral confocal microscope by reflection. Both types of particles aggregated in the culture media and were efficiently internalized by osteoblasts as agglomerates. Treatment of isolated cultures of osteoblasts with rutile particles stimulated the release of IL-6, PGE2, and GM-CSF to a lesser extent than titanium. The influence of macrophages on the particle-induced stimulation of those local factors was analyzed by coculturing TPA-differentiated THP-1 cells with osteoblasts. Under these conditions, levels of IL-6 and PGE2 after treatment of cocultured osteoblasts with rutile particles were lower than after exposure to titanium. These results indicate that rutile debris shows a lower bioreactivity than titanium when tested in cultures of human osteoblasts and support the improved biocompatibility of titanium-based implants modified to create an outer layer of rutile on their surfaces.

We have evaluated the in-vitro biocompatibility of Ti6Al4V alloy coated by plasma spraying with an identical alloy. These surfaces are widely used in cementless prosthetic components, although osteoblasts behavior on this treated alloy has not been evaluated to date. Cross sectional examination revealed a thick and rough coating of identical composition without sign of delamination. Within the coating, small discontinuities and nonconnected pores were observed. Osteoblast response was evaluated by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared to the polished alloy, osteoblast adhesion measured as cell attachment and actin network reorganization was delayed on the plasma-sprayed surface. Cell proliferation and viability were also impaired on the rough surface. Several informative markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, osteoprotegerin, and mineralized nodule formation were evaluated and indicated that the plasma-sprayed alloy favored a more differentiated phenotype than polished alloy. Taken together, our in vitro results indicate that successful osseointegration of plasma spraying of Ti6Al4V with an identical alloy is mediated by modulation of osteoblastic differentiation and mineralization. © 2006 Wiley Periodicals, Inc.

We have recently reported that thermal oxidation treatments of Ti6Al4V at 500 degrees and 700 degrees C for 1 h result in the formation of an outer "ceramic" layer of rutile that do not decrease the high in vitro corrosion resistance of the alloy. In the present work, surface roughness was measured and found marginally increased as a consequence of oxidation of the alloy at 700 degrees C, but not at 500 degrees C. We have evaluated the biocompatibility of the oxidized surfaces, by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared with polished alloy, both thermal treatments increased osteoblast adhesion measured as cell attachment, beta1 integrin and FAK-Y397 expression, as well as cytoskeletal reorganization. Compared with treatment at 500 degrees C, thermal oxidation at 700 degrees C enhanced cell adhesion. Treatment at 700 degrees C transiently impaired cell proliferation and viability, which were not altered in alloys oxidized at 500 degrees C. Several markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, and mineralized nodule formation were found either unaffected or differentially increased by alloys treated either at 500 degrees or 700 degrees C. In addition, thermal oxidation at 700 degrees C also increased osteoprotegerin secretion. Taken together, our results indicate that thermal oxidation treatments at 500 degrees or 700 degrees C for 1 h improve the in vitro biocompatibility of Ti6Al4V.