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PURPOSE Breast cancer care requires a multimodal approach and a multidisciplinary team who must work together to obtain good clinical results. The fragmentation of care can affect the breast cancer care; however, it has not been measured in a low-resource setting. The aim of this study was to identify fragmentation of care, the geographic variation of this and its association with 4-year overall survival (OS), and costs of care for patients with breast cancer enrolled in Colombia's contributory health care system. MATERIALS AND METHODS A retrospective cohort study was conducted using administrative databases. Women with breast cancer who were treated from January 1, 2013, to December 31, 2015, were included. Fragmentation of care was the exposure, which was measured by the number of different health care provider institutions (HCPIs) that treated a patient during the first year after diagnosis. Crude mortality rates were estimated, survival functions were calculated using the nonparametric Kaplan-Meier approach, and adjusted hazard ratios (HRs) were estimated using multivariate Cox regression model to identify the association of fragmentation with 4-year OS. The association between fragmentation and costs of care was assessed using a multivariate linear regression model. RESULTS A total of 10,999 patients with breast cancer were identified, and 1,332 deaths were observed. The 4-year crude mortality rate was 31.97 (95% CI, 30.25 to 33.69) per 1,000 person-years for the whole cohort, and the highest rate was in the cohort defined for the fourth quartile of the fragmentation measurement (eight or more HCPIs), 40.94 (95% CI, 36.49 to 45.39). The adjusted HR for 4-year OS was 1.04 (95% CI, 1.01 to 1.07) for each HCPI additional. The cost of care is increased for each additional HCPIs (cost ratio, 1.25; 95% CI, 1.23 to 1.26). CONCLUSION Fragmentation of care decreases overall 4-year OS and increases the costs of care in women with breast cancer for Colombia.

Aims A significant reduction in wear at five and ten years was previously reported when comparing Durasul highly cross-linked polyethylene with nitrogen-sterilized Sulene polyethylene in total hip arthroplasty (THA). We investigated whether the improvement observed at the earlier follow-up continued, resulting in decreased osteolysis and revision surgery rates over the second decade. Methods Between January 1999 and December 2001, 90 patients underwent surgery using the same acetabular and femoral components with a 28 mm metallic femoral head and either a Durasul or Sulene liner. A total of 66 hips of this prospective randomized study were available for a minimum follow-up of 20 years. The linear femoral head penetration rate was measured at six weeks, one year, and annually thereafter, using the Dorr method on digitized radiographs with a software package. Results In the Durasul group, no patients underwent revision due to loosening or showed radiological evidence of osteolysis. In the Sulene group, four patients (four hips) were revised due to femoral component loosening. The 20-year cumulative failure incidence in the presence of the competing event of death for revision surgery was 4.5% (95% confidence interval (CI) 0.8 to 13.6) in the Durasul group, and 8.9% (95% CI 2.8 to 19.5) in the Sulene group. The mean wear one year after surgery was 0.09 mm (SD 0.007) in the Durasul group and 0.24 (SD 0.015) in the Sulene group (p < 0.001). From one to 20 years after surgery, the mean total penetration was 0.32 mm (SD 0.045) in the Durasul group and 1.07 mm (SD 0.13) in the Sulene group (p < 0.001). Mean femoral head penetration at 20 years was approximately 70.0% less in the Durasul group than the Sulene group. Conclusion The significant reduction in femoral head penetration obtained with the Durasul compared with Sulene in uncemented THA resulted in lower osteolysis and revision rates after 20 years. Cite this article: Bone Joint J 2022;104-B(9):1032–1038.

Aims Bone stock restoration of acetabular bone defects using impaction bone grafting (IBG) in total hip arthroplasty may facilitate future re-revision in the event of failure of the reconstruction. We hypothesized that the acetabular bone defect during re-revision surgery after IBG was smaller than during the previous revision surgery. The clinical and radiological results of re-revisions with repeated use of IBG were also analyzed. Methods In a series of 382 acetabular revisions using IBG and a cemented component, 45 hips (45 patients) that had failed due to aseptic loosening were re-revised between 1992 and 2016. Acetabular bone defects graded according to Paprosky during the first and the re-revision surgery were compared. Clinical and radiological findings were analyzed over time. Survival analysis was performed using a competing risk analysis. Results Intraoperative bone defect during the initial revision included 19 Paprosky type IIIA and 29 Paprosky type IIIB hips; at re-revision, seven hips were Paprosky type II, 27 type IIIA and 11 were type IIIB (p = 0.020). The mean preoperative Harris Hip Score was 45.4 (SD 6.4), becoming 80.7 (SD 12.7) at the final follow-up. In all, 12 hips showed radiological migration of the acetabular component, and three required further revision surgery. The nine-year cumulative failure incidence (nine patients at risk) of the acetabular component for further revision surgery was 9.6% (95% confidence interval (CI) 2.9 to 21.0) for any cause, and 7.5% (95% CI 1.9 to 18.5) for aseptic loosening. Hips with a greater hip height had a higher risk for radiological migration (odds ratio 1.09, 95% CI 1.02 to 1.17; p = 0.008). Conclusion Bone stock restoration can be obtained using IBG in revision hip surgery. This technique is also useful in re-revision surgery; however, a better surgical technique including a closer distance to hip rotation centre could decrease the risk of radiological migration of the acetabular component. A longer follow-up is required to assess potential fixation deterioration. Cite this article: Bone Joint J 2021;103-B(3):492–499.

Background Healthcare fragmentation is a phenomenon in which patients experience a discontinuity in healthcare. Patients with colorectal cancer are more likely to experience fragmented care because they require specialized treatment by multidisciplinary teams. The objective of this study was to identify the association between healthcare fragmentation and 3-year survival for patients with colorectal cancer in Colombia. Methods A retrospective cohort study was performed using administrative databases, with an electronic algorithm to identify patients with colorectal cancer based on codes. The patients were recruited between January 1, 2013 and December 31, 2016. The exposure variable was fragmentation, which was measured based on the number of different providers that treated a patient during the first year after diagnosis. Three-year mortality rates and incidence rate ratios (IRRs) were estimated for quartiles of fragmentation. A cut-off point was determined that divided the population between exposure to higher fragmentation versus lower fragmentation. Matching was performed using propensity scores to control for confounding, and the hazard ratio for exposure to higher fragmentation was calculated for the matched sample. Results A total of 5,036 patients with colorectal cancer was identified, 2,525 (49.88%) of whom were women. The mean number of network providers for the total sample was 5.71 (SD 1.98). The patients in the quartile with higher fragmentation had the highest mortality rate, 35.67 (95%CI; 33.63–38.06) per 100 patients. The comparison of higher and lower quartiles of fragmentation resulted in an IRR of 1.23 (95%CI; 1.04–1.45; p 0.02). Of the 5,036 patients, 422 (8.38%) were classified as the exposed cohort (higher fragmentation). The total matched sample consisted of 844 subjects, and an HR of 1.35 (95%CI; 1.14–1.61) was estimated. Conclusions Exposure to more highly fragmented healthcare networks decreases overall 3-year survival for patients with colorectal cancer in Colombia.

Bone stock restoration of acetabular bone defects using impaction bone grafting (IBG) in total hip arthroplasty may facilitate future re-revision in the event of failure of the reconstruction. We hypothesized that the acetabular bone defect during re-revision surgery after IBG was smaller than during the previous revision surgery. The clinical and radiological results of re-revisions with repeated use of IBG were also analyzed.In a series of 382 acetabular revisions using IBG and a cemented component, 45 hips (45 patients) that had failed due to aseptic loosening were re-revised between 1992 and 2016. Acetabular bone defects graded according to Paprosky during the first and the re-revision surgery were compared. Clinical and radiological findings were analyzed over time. Survival analysis was performed using a competing risk analysis.Intraoperative bone defect during the initial revision included 19 Paprosky type IIIA and 29 Paprosky type IIIB hips; at re-revision, seven hips were Paprosky type II, 27 type IIIA and 11 were type IIIB (p = 0.020). The mean preoperative Harris Hip Score was 45.4 (SD 6.4), becoming 80.7 (SD 12.7) at the final follow-up. In all, 12 hips showed radiological migration of the acetabular component, and three required further revision surgery. The nine-year cumulative failure incidence (nine patients at risk) of the acetabular component for further revision surgery was 9.6% (95% confidence interval (CI) 2.9 to 21.0) for any cause, and 7.5% (95% CI 1.9 to 18.5) for aseptic loosening. Hips with a greater hip height had a higher risk for radiological migration (odds ratio 1.09, 95% CI 1.02 to 1.17; p = 0.008).Bone stock restoration can be obtained using IBG in revision hip surgery. This technique is also useful in re-revision surgery; however, a better surgical technique including a closer distance to hip rotation centre could decrease the risk of radiological migration of the acetabular component. A longer follow-up is required to assess potential fixation deterioration. Cite this article

The development of new drugs for musculoskeletal regeneration purposes has attracted much attention in the last decades. In this work, we present three novel vitamin B9 (folic acid)-derivatives bearing divalent cations (ZnFO, MgFO and MnFO), providing their synthesis mechanism and physicochemical characterization. In addition, a strong emphasis has been placed on evaluating their biological properties (along with our previously reported SrFO) using human mesenchymal stem cells (hMSC). In all the cases, pure folate derivatives (MFOs) with a bidentate coordination mode between the metal and the folate anion, and a 1:1 stoichiometry, were obtained in high yields. A non-cytotoxic dose of all the MFOs (50 ¿g/mL) was demonstrated to modulate by their own the mRNA profiles towards osteogenic-like or fibrocartilaginous-like phenotypes in basal conditions. Moreover, ZnFO increased the alkaline phosphatase activity in basal conditions, while both ZnFO and MnFO increased the matrix mineralization degree in osteoinductive conditions. Thus, we have demonstrated the bioactivity of these novel compounds and the suitability to further studied them in vivo for musculoskeletal regeneration applications., This work has been funded by the Spanish MICINN(MAT201573656-JIN, RTI2018-095159-B-I00), ISCIII-FEDER-MINECO-AES (PI15/00752, PI15/01118, PI18/00643) CAM (IND2018/BMD-9485) and ERASMUSþTraineeship Programmes. D.F.-V. isfinancially supported by a predoctoral program MICINN FPU18/04683. L.S is financially supported by a Miguel Servet contract(CPII16/00038) from ISCIII- FEDER-MINECO-AES- FSE. N.V is financially supported by Program I2 from CAM. B. V. and L. R. are members of the SusPlast platform from the Spanish National Research Council (CSIC)

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied., The authors thank financial support to Ministry of Science, Innovation and Universities (Spain) (MAT2017- 2017-84277-R), Instituto Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER), MINECOAES (PI18/00643 and PI15/01118). Ana Mora-Boza is supported by “La Caixa” foundation (Scholarship of, code LCF/BQ/ES16/11570018), Laura Saldaña is supported by a Miguel Servet contract from ISCIII-MINECO-AESFEDER- FSE. Nuria Vilaboa is supported by Program I2 from Comunidad Autónoma de Madrid. The authors are indebted to Rosana Ramirez (ICTP-CSIC) and Fatima Bensiamar (IdiPAZ and CIBER-BBN) for excellent technical assistance with cell culture experiments and gene expression analysis, respectively.

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.

Mesenchymal stem cells (MSC) modulate the macrophage-mediated inflammatory response through the secretion of soluble factors. In addition to its classical effects on calcium homeostasis, 1,25-dihydroxyvitamin D3 (1,25D3) has emerged as an important regulator of the immune system. The present study investigates whether 1,25D3 modulates the paracrine interactions between MSC and macrophages. 1,25D3 stimulated MSC to produce PGE2 and VEGF and regulated the interplay between macrophages and MSC toward reduced pro-inflammatory cytokine production. Conditioned media (CM) from co-cultures of macrophages and MSC impaired MSC osteogenesis. However, MSC cultured in CM from 1,25D3-treated co-cultures showed increased matrix maturation and mineralization. Co-culturing MSC with macrophages prevented the 1,25D3-induced increase in RANKL levels, which correlated with up-regulation of OPG secretion. MSC seeding in three-dimensional (3D) substrates potentiated their immunomodulatory effects on macrophages. Exposure of 3D co-cultures to 1,25D3 further reduced the levels of soluble factors related to inflammation and chemotaxis. As a consequence of 1,25D3 treatment, the recruitment of monocytes toward CM of 3D co-cultures decreased, while the osteogenic maturation of MSC increased. These data add new insights into the pleiotropic effects of 1,25D3 on the crosstalk between MSC and macrophages and highlight the role of the hormone in bone regeneration.

A deeper knowledge on the degradation pattern of a biodegradable material is required before considering it for a specific application as a bioabsorbable implant. In the last few years, composites formed by polylactic-acid reinforced with Mg-based particles have been proposed for biodegradable implants. The aim of the present work is to study the effect of the type and crystalline degree of the polymeric matrix on in vitro degradation kinetics of PLA/Mg composites. The effect of the nature of the matrix has been studied by comparing a composite with a poly-L-lactic (PLLA) matrix with another with a poly-L,D-lactic (PLDA) matrix. The influence of the crystalline degree has been studied by comparing a near amorphous with a high crystalline composite. The degradation behaviour has been determined through a joint analysis of the microstructure, the changes in mass and water intake, the pH evolution in buffered and non-buffered media, and the amount of H released up to 28 days of immersion. The viability of human mesenchymal stem cells (MSCs) on the composites was also evaluated. Results show that a PLDA matrix is more effective lowering the degradation rate of Mg particles than PLLA. The crystalline degree plays a major role in PLA/Mg composites degradation. The composite with the near amorphous matrix exhibited the lowest degradation rate and the highest cell viability. Whereas, cell viability decreases markedly on the high-crystalline composites, which is likely related to the accelerated release of degradation products. These data highlight the importance of matrix crystallinity on the degradation kinetics and cytocompatibility of PLA/Mg composites., The authors thank funding from MAT2015-63974-C4-1-R , MAT2016-79869-C2-1-P, PCIN-2017-036, S2013/MIT-2862 and PI15/00752 ( ISCIII -MINECO-AES) projects. LS is supported by a Miguel Servet contract from ISCIII-MINECO-AES-FEDER-FSE. SCC is supported by a Juan de la Cierva contract FJCI-2016-29004 .

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.

One of the most important complications of total joint arthroplasty is failure associated with periprosthetic osteolysis, a process mainly initiated by the biological response to wear-derived products from the biomaterials in service. The inflammatory mediator interleukin-6 (IL-6) plays a key role in the establishment and progression of aseptic loosening. Metal particles specifically up-regulate IL-6 production in bone-forming cells and implant-bone interfacial tissues. The use of statins has been recently associated with a significantly reduced risk of revision in patients that undergo total hip arthroplasty. We hypothesized that simvastatin (Simv) could modulate the osteoblastic response to titanium particles (Ti) by attenuating the production of IL-6. Pre-treatment of human osteoblastic cells with Simv down-regulated Ti particle-induced IL-6 gene expression at mRNA and protein levels. The effect of Simv on Ti-induced IL-6 production in osteoblastic cells could not be explained by inhibition of the internalization of metal particles. The mechanism involved in this down-regulation is based in the inhibition of the HMG-CoA/GGPP/RhoA/ROCK pathway, independently of Simv effects in the cholesterol synthesis. The cytokine-lowering property of Simv has been observed in Saos-2 cells and human primary osteoblasts (hOBs) exposed to Ti particles, and was further enhanced when hOBs were co-cultured with macrophages.

The colonization of an implant surface by bacteria is an extremely important medical problem, which often leads to the failure of medical devices. Modern surface modification techniques, such as ion implantation, can confer to the surfaces very different properties from those of the bulk underlying material. In this work, austenitic stainless steel 316 LVM has been superficially modified by Si + ion implantation. The effect of surface modification on the biocompatibility and bacterial adhesion to 316 LVM stainless steel has been investigated. To this aim, human mesenchymal stem cells (hMSCs), as precursor of osteoblastic cells, and bacterial strains relevant in infections related to orthopedic implants, i.e., Staphylococcus aureus and Staphylococcus epidermidis , have been assayed. For the understanding of changes in the biological response associated to ion implantation, variations in the chemical surface composition, topography, surface Gibbs energy, isoelectric point and in vitro corrosion behavior have been evaluated. hMSCs adhesion, viability and differentiation to the osteoblastic lineage were unaffected by Si + ion implantation. On the other hand, Si + ion implantation diminished the number of attached bacteria in static conditions and led to smaller adhesion rates and retention strength. The ability of implanted surfaces to reduce the bacterial adhesion was higher for Staphylococcus epidermidis than for Staphylococcus aureus. This study proposes Si + ion implantation as an effective way of reducing bacterial adhesion on 316 LVM stainless steel surfaces without compromising its good biocompatibility.

The purpose of the current study was to investigate whether the cryopolymerization technique is capable of producing suitable scaffolds for bone tissue engineering. Cryopolymers made of 2-hydroxyethyl methacrylate and acrylic acid with (W1 and W20) and without (W0) wollastonite particles were prepared. The elastic modulus of the specimens rose one order of magnitude from W1 to W20. Total porosity reached 56% for W0, 72% for W1 and 36% for W20, with pore sizes of up to 2 mm, large interconnection sizes of up to 1 mm and small interconnection sizes of 50-80 µm on dry specimens. Cryogels swell up to 224 ± 17% for W0, 315 ± 18% for W1 and 231 ± 27% for W20 specimens, while maintaining the integrity of the bodies. Pore sizes > 5 mm can be observed for swollen specimens. The biocompatibility of the samples was tested using human mesenchymal stem cells isolated from bone marrow and adipose tissues. Both types of cells attached and grew on the three tested substrates, colonized their inner regions and organized an extracellular cell matrix. Fibronectin and osteopontin levels decreased in the media from cells cultured on W20 samples, likely due to increased binding on the ECM deposited by cells. The osteoprotegerin-to-receptor activator of nuclear factor-κB ligand secretion ratios increased with increasing wollastonite content. Altogether, these results indicate that an appropriate balance of surface properties and structure that favours stromal cell colonization in the porous cryogels can be achieved by modulating the amount of wollastonite.

Commercial 316 LVM austenitic stainless steel samples have been coated by immersion in a bath of molten Al-12.6%Si alloy for 120 s. The coating consists of the Al12(Fe,Cr)3Si2 intermetallic. In vitro corrosion behaviour has been evaluated in the Ringer's solution by means of potentiodynamic curves and electrochemical impedance spectroscopy. The results reveal that the coated specimens exhibit lower susceptibility to localised corrosion with respect to the substrate. XPS analysis suggests that the ennoblement of the pitting potential is due to the formation of a chromium oxyhydroxide containing passive layer. The intermetallic coating shows a good biocompatibility, as demonstrated by culturing human mesenchymal stem cells isolated from bone marrow which attached, grew and differentiated to the osteoblastic lineage to a similar extent on coated and bare steels. In summary, this study proposes a method that generates Ni-free coatings of the stainless steel with useful properties for biomedical applications. © Springer Science+Business Media, LLC 2011.

Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and β-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell–particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca2+ uptake, allowing the development of a functional osteoblast phenotype.

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 Mg 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 Mg 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.

We investigated a Ti6Al4V alloy modified by means of laser peening in the absence of sacrificial coatings. As a consequence of the temperature rise during laser focusing, melting and ablation generated an undulated surface that exhibits an important increase in the content of titanium oxides and OH- ions. Human mesenchymal stem cells and osteoblasts cultured on the oxidized alloy develop noticeable filopodia and lamellipodia. Their paxillin-stained focal adhesions are smaller than in cells attached to the untreated alloy and exhibit a marked loss of colocalization with the ends of actin stress fibers. An important imbalance of phosphorylation and/or dephosphorylation of the focal adhesion kinase is detected in cells grown on the oxidized alloy. Although these mechanisms of adhesion are deeply altered, the surface treatment does not affect cell attachment or proliferation rates on the alloy. Human mesenchymal stem cells cultured on the treated alloy in media containing osteogenic inducers differentiate towards the osteoblastic phenotype to a higher extent than those on the untreated surface. Also, the specific functions of human osteoblasts cultured on these media are enhanced on the treated alloy. In summary, laser peening tailors the Ti6Al4V surface to yield an oxidized layer with increased roughness that allows the colonization and activities of bone-lineage cells., This work was supported by grants PI12/01698 from Fondo de Investigaciones Sanitarias (FIS, Spanish Ministry of Economy and Competitiveness, MINECO, Spain), S2013/MIT-2862 from Comunidad de Madrid, MAT2009-14695-C04-01-02-04 from the former Spanish Ministry of Science and Innovation (MICINN), MAT2012-37736-C05-03-05 and MAT2014-52905- REDT (MINECO) and GR10149 (Junta de Extremadura, Spain). LC was the recipient of predoctoral fellowship BES-2010-034989 from MICINN. LS is supported by grant award CP11/00022 (FIS). NV is supported by Program I2 from Comunidad de Madrid (Spain).

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 Ti6Al4 V alloy was also investigated. Cell attachment and spreading, actin and β-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. © 2007 Elsevier Ltd. All rights reserved.

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.

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.

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.

One of the problems associated with the modern biomaterials used in prostheses is osteolysis, which, although its exact origin is unknown, has been associated with wear particles. Osteoblasts seem to participate directly in this phenomenon. This paper investigates in vitro cellular response to the wear particles from the metal substrate and ceramic covering (alpha-alumina) of a new titanium yttrium aluminum alloy, MA 956, that has been proposed as a biomaterial because of its exceptional mechanical and electrochemical properties. The effect of different sizes (10 and 80 microm) of MA 956 and alpha-alumina particles on osteoblast function was studied in primary human bone cell cultures. Cells were harvested from trabecular bone fragments obtained during knee arthroplasty. Osteoblastic cell response to the particles was measured by assaying C-terminal type I procollagen (PICP), alkaline phosphatase, and osteocalcin secretion, with and without 1.25(OH)(2)D(3) stimulation, in the cell-conditioned medium. Both sizes of MA 956 and alpha-alumina particles decreased PICP secretion in nonstimulated osteoblastic cells, but this secretion was not affected in the cultures stimulated with 1.25(OH)(2)D(3). Only the 10 microm alpha-alumina particles inhibited alkaline phosphatase activity in 1.25(OH)(2)D(3)-stimulated and nonstimulated cultures. The rise in osteocalcin levels after 1.25(OH)(2)D(3) stimulation was lower in the presence of the 10 microm MA 956 particles than in the presence of alpha-alumina particles. Although both materials seem to have directly affected in vitro osteoblastic cell function, the increase in osteocalcin levels after 1.25(OH)(2)D(3) stimulation was lower after exposure to MA 956 particles than the increase observed after exposure to alpha-alumina particles. Therefore, it does not seem that osteocalcin stimulated bone resorption, suggesting that MA 956 would be less likely to provoke osteolysis.

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.

[EN] Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300 μm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80% of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups., We are indebted to MINECO (BFU2010-17450), AGAUR (2009SGR-0108 and SGR2009-516), DGI (CTQ2010-19501) and CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN, Spain) for funding our research on Inclusion bodies. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, and Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. EGF is supported by the Programa Personal de Tecnico de Apoyo (Modalidad Infraestructuras cientifico-tecnologicas, MICINN). We also thank technical assistance from the Servei de Cultius Celulars, Produccio d'Anticossos i Citometria (SCAC) Laboratori de Luminescencia i Espectroscopia de Biomolecules (LLEB) and Servei de Microscopia, all at the Universitat Autonoma de Barcelona (UAB). We are also indebted to the Protein Production Platform (CIBER-BBN) for helpful technical assistance and for protein production and purification services (http://bbn.ciber-bbn.es/programas/plataformas/equipamiento). AV received an ICREA ACADEMIA award.

Polylactic acid/Mg composites have been recently proposed for biodegradable osteosynthesis devices because, with regards to the neat polymer, they combine an enhanced biocompatibility and bioactivity with better mechanical properties, particularly creep strength. A question still arises about their bacterial behavior. For this purpose, composites of poly-L-D-lactic acid (PLDA) loaded with 1 and 10 wt.% of Mg microparticles were evaluated using Staphylococcus epidermidis, with special emphasis on the study of bacterial adhesion and biofilm formation. During biofilm formation the bacteria viability of the composites decreased up to 65.3% with respect to PLDA. These antibacterial properties do not compromise the cytocompatibility of the material as the composites enhanced the viability of mesenchymal stem cells and their osteogenic commitment. These findings provide an important added value to the biodegradable and biocompatible PLDA/Mg composites for the manufacture of osteosynthesis devices., This work was supported by Grants MAT2012‐37736‐C05‐01-03-04-05 from the ‘Ministerio de Economía y Competitividad’ (MINECO, Spain); PI15/00752 and PI15/01118 from Instituto Salud Carlos III (ISCIII)-Fondos FEDER (MINECO-AES, Spain); GR15025 from the ‘Consejería de Economía, Ciencia e Innovación’ (Junta de Extremadura, Spain); S2013/MIT-2862 from Comunidad Autónoma de Madrid (CAM) and FEDER and CIBER‐BBN funds. LS is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00022). NV is supported by Program I2 from CAM.

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.

Formation of thin films on titanium alloys incorporating bioactive small molecules or macromolecules is a route to improve their biocompatibility. Aminoalkylsilanes are commonly employed as interface reagents that combine good adhesion properties with an amino tail group susceptible of further functionalization. This article introduces a reproducible methodology to obtain a cross-linked polymer-type brush structure of covalently-bonded aminoalkylsiloxane chains on Ti6Al4V. The experimental protocol can be fine-tuned to provide a high density of surface-coated amino groups (threshold value: 2.1±0.1×10(-8) mol cm(-2)) as proven by chemical and spectrophotometric analyses. Using a model reaction involving the condensation of 3-aminopropyltrimethoxysilane (APTMS) on Ti6Al4V alloy, we herein show the effects of reaction temperature, reaction time and solvent humidity on the composition and structure of the film. The stability of the resulting coating under physiological-like conditions as well as the possibility of surface re-silanization has also been evaluated. To verify if detrimental effects on the biological performance of the Ti6Al4V alloy were induced by this coverage, human primary osteoblasts behavior, Staphylococci adhesion and biofilm formation have been tested and compared to the Ti6Al4V oxidized surface. Reaction with trans-cinnamaldehyde has used in order to determine useful amino groups at aminosilanized surface, XPS and UV analyses of imino derivatives generated reveal that almost a 50% of these groups are actually available at the siloxane chains.

This study reports on the biocompatibility of 316 LVM steel blasted with small and rounded ZrO2 particles or larger and angular shaped Al2O3 particles. The effect of blasting on the in vitro corrosion behavior and the associated ion release is also considered. Surface of Al2O3 blasted samples was rougher than that of ZrO2 blasted samples, which was also manifested by a higher surface area. Compared to the polished alloy, blasted steels exhibited a lower corrosion resistance at the earlier stages of immersion, particularly when using Al2O3 particles. With increasing immersion time, blasted samples experienced an improvement of the corrosion resistance, achieving impedance values typical of passive alloys. Blasting of the alloy led to an increase in Fe release and the leaching of Ni, Mn, Cr and Mo. On all surfaces, ion release is higher during the first 24 h exposure and tends to decrease during the subsequent exposure time. Despite the lower corrosion resistance and higher amount of ions released, blasted alloys exhibit a good biocompatibility, as demonstrated by culturing osteoblastic cells that attached and grew on the surfaces.

Osteosarcoma-derived cells have been routinely used for studying osteoblastic functions, but it remains unclear to what extent they mimic the behavior of primary osteoblasts in the study of cells and materials interactions. This study reports comparatively on the responses of three human osteosarcoma cell lines, MG-63, Saos-2 and U-2 OS, and human primary osteoblasts cultured on Ti6Al4V surfaces or exposed to Ti particles. Phenotypic characterization of the cell lines revealed that Saos-2 cells and primary osteoblasts displayed similar expression patterns of Cbfa1, SP7 and osteocalcin. Unlike primary cells, the cell lines expressed markers of undifferentiated cells, had high proliferative rates and poor fibronectin matrix assembly. None of the three cell lines faithfully reproduced the adhesive behavior of primary osteoblasts when cultured on Ti6Al4V surfaces or exposed to Ti particles. Differences in cell growth between the cell lines and primary osteoblasts cultured on Ti6Al4V surfaces were also observed. Ti particles inhibited the growth of Saos-2 cells and primary osteoblasts to a similar extent, while no such effect was observed in U-2 OS and MG-63 cells. Saos-2 cells reproduced the alkaline phosphatase (ALP) activity profile of primary osteoblasts cultured on metallic surfaces or exposed to particles. Altogether, these results show that none of the osteoblast-like cells studied perfectly mimic the behavior of human osteoblast cells (hOB) on Ti6Al4V surfaces or exposed to Ti particles. Saos-2 cells reproduce some of the hOB responses such as the profile of enzymatic ALP activity when cultured on the surfaces or treated with particles as well as cell growth inhibition when exposed to Ti particles. Although in vitro cytocompatibility studies involve the evaluation of multiple parameters, Saos-2 cells may be used as representative of human osteoblasts when these standard tests are evaluated.

Patterned surfaces direct cell spatial dynamics, yielding cells oriented along the surface geometry, in a process known as contact guidance. The Rho family of GTPases controls the assembly of focal adhesions and cytoskeleton dynamics, but its role in modulating bone-cell alignment on patterned surfaces remains unknown. This article describes the interactions of two human cell types involved in osseointegration, specifically mesenchymal stem cells and osteoblasts, with submicron- or nano-scale Ti6Al4V grooved surfaces generated by mechanical abrasion. The surface chemistry of the alloy was not affected by grinding, ensuring that the differences found in cellular responses were exclusively due to changes in topography. Patterned surfaces supported cell growth and stimulated mesenchymal stem cell viability. Anisotropic surfaces promoted cell orientation and elongation along the grates. Both cell types oriented on nanometric surfaces with grooves of 150 nm depth and 2 μm width. The number of aligned cells increased by approximately 30% on submicrometric grooves with sizes of about 1 μm depth and 10 μm width. Cells were treated with drugs that attenuate the activities of the GTPase RhoA and one of its downstream effectors, Rho-associated kinase (ROCK), and contact guidance of treated cells on the grooved surfaces was investigated. The data indicate that the RhoA/ROCK pathway is a key modulator of both mesenchymal stem cell and osteoblast orientation on nanometric surface features. RhoA and its effector participate in the alignment of mesenchymal stem cells on submicrometric grooves, but not of osteoblasts. These findings show that RhoA/ROCK signaling is involved in contact guidance of bone-related cells on metallic substrates, although to a varying extent depending on the specific cell type and the dimensions of the pattern. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC–polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC–polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin–TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.

Titanium (Ti) and its alloys are widely used in biomedical devices as bone tissue replacements due to their advantageous bulk mechanical properties and biocompatibility. It is known that particles released from Ti-based implants impair essential functions of osteoblasts, which for survival require attachment to specific extracellular matrix proteins at the bone surface. This study investigates whether Ti particles of micrometric sizes affect the osteoblast attachment machinery. Exposure of human osteoblastic Saos-2 cells to Ti particles impaired their adhesion strength, migration and proliferation. Attenuation of these functions was associated with reduced cell spreading, cell membrane disruptions and loss of cell shape. Cell exposure to Ti particles led to changes in cytoskeletal structures, including reduced ventral stress fibers combined with a disorderly arrangement of β-tubulin and acetylated α-tubulin fibers. Cytoskeleton disassembly was associated with a reduction in overall cell adhesion area, characterized by fewer centrally localized focal adhesions and shorter focal contacts at the periphery. Paxillin adaptor protein redistributed to peripheral corner regions, colocalizing with poorly organized actin fibers at attachment sites. Total focal adhesion kinase (FAK) protein amounts, as well as its degree of phosphorylation on the active form p-FAK (Tyr-397), decreased, which was accompanied by a lesser extent of co-localization with paxillin in focal contacts. On the other hand, p-FAK (Tyr-407), an inhibitory form of FAK, accumulated in the focal contacts of Ti-treated cells. Pyk2 phosphorylated on Tyr-402 colocalized with paxillin in focal contacts of untreated cells, while it was barely detected upon exposure to particles. In summary, changes in the phosphorylation states of both FAK and Pyk2 tyrosine kinases at focal contacts underlie impaired bone-forming cell attachment after exposure to Ti particles of micrometric sizes.

The purpose of the current study was to evaluate the effects of alumina particles on secretion of several cytokines involved in bone resorption in cocultures of macrophages and osteoblasts. To distinguish the contribution of each individual cell type, we have established a heterologous in vitro system that makes use of mouse J774 cells and primary cultured human osteoblasts. J744 cells decreased the production of TNF-α when they were cocultured with osteoblasts. Treatment of J744 cells with alumina particles increased TNF-α secretion, but the induction was lower when cells were cocultured with osteoblasts. Secretion of IL-6 by J744 cells was very low, and increased in the presence of osteoblasts. Alumina particles were only able to stimulate the release of IL-6 by J744 cells when cells were cocultured with osteoblasts. On the other hand, incubation of osteoblasts with alumina particles enhanced the release of IL-6 and GM-CSF. Coculturing osteoblasts with J744 cells induced them to release IL-6 and GM-CSF, and treatment with alumina further increased the secretion of both mediators by osteoblasts. According to these in vitro results, it seems rather plausible that alumina particles are able to initiate an inflammatory response in vivo. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Thermal oxidation treatments of Ti6Al4V, at 500 and 700°C, for 1 h result in the formation of an outer "ceramic" layer of rutile, which enhances osteoblast response. In the present study, we have measured in vitro Ti and Al ion release from Ti64 alloy in the as-received state and after thermal oxidation treatments at 500 or 700°C, to culture medium under standard cell-culture conditions. Concentrations of both Ti and Al released from both thermal oxidation treatments were lower than from polished alloy. Al was released from the treated or untreated surfaces in substantially lower extent than Ti. Titanium and aluminium ions affected primary human osteoblast proliferation, metabolic activity, and differentiation in a dose-dependent manner. Treatments with individual Ti or Al metal ions in similar concentration ranges than released from the surfaces did not alter osteoblast response, which also remained unaffected after treatments with combinations of Ti plus Al applied in the proportional relations than detected in ion-release experiments. We then selected higher concentrations of Ti that impaired osteoblast proliferation and differentiation, while the proportional lower concentrations of Al did not alter osteoblast behavior. In spite of its inert character, it was found that Al significantly enhanced the deleterious effect of Ti on osteoblast differentiation. Therefore, thermal oxidation treatments of Ti6Al4V alloy may improve the biocompatibility of the alloy by reducing both Ti and Al release, and thus attenuating ion-mediated interference with osteoblast differentiation. © 2005 Wiley Periodicals, Inc.

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-α, IL-6 and IL-1β to a lesser extent than titanium. The influence of osteoblasts on the particle-induced stimulation of TNF-α and IL-1β 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-α, IL-6 and IL-1β 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. © 2006 Elsevier Ltd. All rights reserved.

The effect of two biomaterials, polyethylene and α-alumina, on interleukin-6 (IL-6) secretion and expression has been studied in human osteoblasts in primary culture. Human osteoblastic cells were derived from fresh trabecular bone explants removed during total knee arthroplasty. On reaching confluence, cells were subcultured in 6 well plates; the resulting subcultures were incubated until confluence and polyethylene or α-alumina particles were added to some while the rest were left as controls. The IL-6 mRNA levels were assessed by reverse transcription (RT) followed by polymerase chain reaction (PCR). IL-6 secretion was measured in the conditioned medium. The IL-6 expression was higher in the presence of both biomaterials. Maximum expression occurred in response to a dose of 50mg particles/well with both biomaterials and was greater after polyethylene particle addition than after α-alumina particle addition at this dose. The maximum IL-6 secretion elicited by α-alumina was produced at 10mg particles/well while maximum response with polyethylene required 50mg/well. At a dose of 10mg/well, α-alumina particles induced more secretion than 10mg of polyethylene particles. Nevertheless, at a dose of 50mg/well maximum secretion was produced with polyethylene particles. In conclusion and in our experimental conditions, polyethylene as well as α-alumina increased both the expression and the secretion of IL-6 in human osteoblastic cells in primary culture and stimulation from polyethylene appears stronger than that from α-alumina at the same dose. © 2001 Elsevier Science Ltd. All rights reserved.

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