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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)

Conventional tissue engineering approaches rely on scaffold-based delivery of exogenous proteins, genes, and/or cells to stimulate regeneration via growth factor signaling. However, scaffold-based approaches do not allow active control of dose, timing, or spatial localization of a delivered growth factor once the scaffold is implanted, yet these are all crucial parameters in promoting tissue regeneration. To address this limitation, we developed a stable cell line containing a heat-activated and rapamycin-dependent gene expression system. In this study, we investigate how high intensity focused ultrasound (HIFU) can spatiotemporally control firefly luciferase (fLuc) transgene activity both in vitro and in vivo by the tightly controlled generation of hyperthermia. Cells were incorporated into composite scaffolds containing fibrin and hydroxyapatite particles, which yielded significant increases in acoustic attenuation and heating in response to HIFU compared to fibrin alone. Using 2.5 MHz HIFU, transgene activation was observed at acoustic intensities of 201 W/cm2 and higher. Transgene activation was spatially patterned in the scaffolds by rastering HIFU at speeds up to 0.15 mm/s. In an in vivo study, a 67-fold increase in fLuc activity was observed in scaffolds exposed to HIFU and rapamycin versus rapamycin only at 2 days post implantation. Repeated activation of transgene expression was also demonstrated 8 days after implantation. No differences in in vivo scaffold degradation or compaction were observed between +HIFU and -HIFU groups. These results highlight the potential utility of using this heat-activated and rapamycin-dependent gene expression system in combination with HIFU for the controlled stimulation of tissue regeneration.

We examined the hypothesis that substrate microarchitecture regulates the crosstalk between human mesenchymal stem cells (hMSC) and cell types involved in bone regeneration. Compared with polyester flat substrates having uniformly distributed homogenous pores (2D), three-dimensional polystyrene substrates with randomly oriented and interconnected pores of heterogeneous size (3D) stimulated the stromal secretion of IGF-1 while lessened the production of VEGFR-1, MCP-1 and IL-6. The medium conditioned by hMSC cultured in 3D substrates stimulated tube formation by human endothelial cells (hEC) to a higher extent than medium from 2D cultures. 3D co-cultures of hMSC and hEC contained higher secreted levels of IGF-1, EGF and FGF-2 than 2D co-cultures, resulting in increased hEC proliferation and migration. Substrate microarchitecture influenced the secretion of factors related to bone remodeling as the ratio RANKL to OPG, and the levels of M-CSF and IL-6 were higher in 3D co-cultures of hMSC and human osteoblasts (hOB) than in 2D co-cultures. Cytokine microenvironment in 3D co-cultures stimulated osteoblast matrix reorganization while demoted the late steps of osteoblastic maturation. Altogether, data in this study may unveil a new role of scaffold microarchitecture during bone regeneration, as modulator of the paracrine relationships that hMSC establish with hEC and hOB.

The aim of this paper is to achieve in situ photochemical synthesis of silver nanoclusters (AgNCs) stabilized by the multiple-amine groups of chitosan (Ch@AgNCs) with luminescent and photothermal properties. Ch@AgNCs were obtained by applying a fast and simple methodology previously described by our group. Direct functionalization of AgNCs with chitosan template provided new nanohybrids directly in water solution, both in the presence or absence of oxygen. The formation of hybrid AgNCs could be monitored by the rapid increase of the absorption and emission maximum band with light irradiation time. New Ch@AgNCs not only present photoluminescent properties but also photothermal properties when irradiated with near infrared light (NIR), transducing efficiently NIR into heat and increasing the temperature of the medium up to 23 °C. The chitosan polymeric shell associated to AgNCs works as a protective support stabilizing the metal cores, facilitating the storage of nanohybrids and preserving luminescent, photothermal and bactericide properties., The authors are thankful for the financial support from Ministerio de Economía y Competitividad (MINECO) through the project MAT2017- 84277-R and MAT2014-57429-R and Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigaciónn (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, UE) through the projects: PGC2018-095364-B-I00 and RTI2018-095159-B-10

Achievement of spatiotemporal control of growth factors production remains a main goal in tissue engineering. In the present work, we combined inducible transgene expression and near infrared (NIR)-responsive hydrogels technologies to develop a therapeutic platform for bone regeneration. A heat-activated and dimerizer-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in hydrogels based on fibrin and plasmonic gold nanoparticles that transduced incident energy of an NIR laser into heat. In the presence of dimerizer, photoinduced mild hyperthermia induced the release of bioactive BMP-2 from NIR-responsive cell constructs. A critical size bone defect, created in calvaria of immunocompetent mice, was filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the heat-activated and dimerizer-dependent gene circuit. In animals that were treated with dimerizer, NIR irradiation of implants induced BMP-2 production in the bone lesion. Induction of NIR-responsive cell constructs conditionally expressing BMP-2 in bone defects resulted in the formation of new mineralized tissue, thus indicating the therapeutic potential of the technological platform., This work was supported by grant PI15/01118 from ISCIII-Fondos FEDER, Ministerio de Economía y Competitividad (MINECO), Spain, grant RTI2018-095159-B-I00 from Ministerio de Ciencia, Innovación y Universidades (MICINN), Spain, grant Roche-IdiPAZ from the intramural funding program of Foundation for Biomedical Research of Hospital Universitario La Paz-IdiPAZ, grant ERC-2013-CoG-614715 (NANOHEDONISM) from ERC Consolidator Grant program, grant R21AR072336 from NIH, United States, and by HSF Pharmaceuticals S.A. C.E-D. was the recipient of predoctoral grant FI14/00447 from ISCIII-Fondos FEDER, Ministerio de Economía y Competitividad (MINECO). N.V. is supported by Program I2 from Comunidad Autónoma de Madrid.

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-L-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo., This work was supported by grant PI15/01118 from Instituto de Salud Carlos III (ISCIII)-Fondos FEDER, Ministry of Economy and Competitiveness (MINECO), Spain, grants RTI2018- 095159-B-I00 and SAF2013-50364-EXP from MINECO, grant Roche- IdiPAZ from the intramural funding program of Foundation for Biomedical Research of La Paz University Hospital-IdiPAZ, grant ERC-2013-CoG-614715 (NANOHEDONISM) from ERC Consolidator Grant program and by HSF Pharmaceuticals S.A. C.E-D. was the re- cipient of predoctoral grant FI14/00447 from ISCIII-Fondos FEDER, MINECO. N.V. is supported by Program I2 from CAM

Background: Immunoregulatory capacity of mesenchymal stem cells (MSC) is triggered by the inflammatory environment, which changes during tissue repair. Macrophages are essential in mediating the inflammatory response after injury and can adopt a range of functional phenotypes, exhibiting pro-inflammatory and anti-inflammatory activities. An accurate characterization of MSC activation by the inflammatory milieu is needed for improving the efficacy of regenerative therapies. In this work, we investigated the immunomodulatory functions of MSC primed with factors secreted from macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype. We focused on the role of TNF-α and IL-10, prototypic pro-inflammatory and anti-inflammatory cytokines, respectively, as priming factors for MSC. Methods: Secretion of immunoregulatory mediators from human MSC primed with media conditioned by human macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype was determined. Immunomodulatory potential of primed MSC on polarized macrophages was studied using indirect co-cultures. Involvement of TNF-α and IL-10 in priming MSC and of PGE2 in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Collagen hydrogels were used to study MSC and macrophages interactions in a more physiological environment. Results: Priming MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages enhanced their immunomodulatory potential through increased PGE2 secretion. We identified the pro-inflammatory cytokine TNF-α as a priming factor for MSC. Notably, the anti-inflammatory IL-10, mainly produced by pro-resolving macrophages, potentiated the priming effect of TNF-α. Collagen hydrogels acted as instructive microenvironments for MSC and macrophages functions and their crosstalk. Culturing macrophages on hydrogels stimulated anti-inflammatory versus pro-inflammatory cytokine secretion. Encapsulation of MSC within hydrogels increased PGE2 secretion and potentiated immunomodulation on macrophages, attenuating macrophage pro-inflammatory state and sustaining antiinflammatory activation. Priming with inflammatory factors conferred to MSC loaded in hydrogels greater immunomodulatory potential, promoting anti-inflammatory activity of macrophages Conclusions: Factors secreted by pro-inflammatory and anti-inflammatory macrophages activated the immunomodulatory potential of MSC. This was partially attributed to the priming effect of TNF-α and IL-10. Immunoregulatory functions of primed MSC were enhanced after encapsulation in hydrogels. These findings may provide insight into novel strategies to enhance MSC immunoregulatory potency., This work was supported by grants from Instituto Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER), MINECO-AES (PI15/00752 and PI15/ 01118) and Comunidad Autónoma de Madrid (CAM) (S2013/MIT-2862). LS is supported by a Miguel Servet contract from ISCIII-MINECO-AES-FEDER-FSE. NV is supported by Program I2 from CAM.

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.

Total hip replacement has become one of the most successful interventions achieved in modern medicine, with excellent technical outcomes and benefits for patients suffering end-stage joint diseases. In-service degradation of prosthetic materials results in the production and accumulation of wear debris particles with devastating effects for the peri-implant tissues. Exposure to wear debris induces a localized inflammatory response and periprosthetic osteolysis, leading to aseptic loosening and failure of the implant. In order to solve this clinical problem, efforts have been focused on improving the tribological properties of biomaterials and elucidating the biological response to wear debris. Unravelling the cellular and molecular mechanisms involved in this pathology will aid to identify diagnostic biomarkers and therapeutic targets.

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 present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety.

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 PGE

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.

Herpes simplex virus (HSV) causes significant morbidity on the human population through such clinical syndromes as cold sores, genital herpes, herpes stromal keratitis, and encephalitis. Attempts to generate efficacious vaccines to date have failed. We have recently described the use of a conditionally replication-competent HSV-1 vector to immunize mice against a lethal challenge of HSV-1. The unique feature of this vaccine vector is that its replication is tightly controlled and can only occur in the presence of local heat and the presence of a small molecule inducer (an antiprogestin). This gives it the safety advantage of a replication-defective vaccine vector as well as the advantage of a replication-competent vector in that it is able to stimulate innate and adaptive aspects of the immune response in a natural context that a replication-defective vector cannot. In this chapter we provide a brief overview of HSV vaccines followed by the methodology used to propagate and utilize replication-conditional HSV vectors as vaccines.

This article focuses on the electrochemical study of two titanium alloys employed in the manufacture of orthopedic implants – Ti-6Al-4V and Ti-6Al-7Nb – both of them obtained through powder metallurgy (PM). For comparative purposes, Ti-6Al-4V fabricated conventionally has also been investigated. Samples were immersed in a simulated body fluid (SBF) and incubated at 37 °C for different immersion time. Under these experimental conditions, we compared the influence of the processing method of alloys (PM or conventional) and their composition in the corrosion resistance. The corrosion resistance of these alloys in contact with SBF was evaluated with electrochemical impedance spectroscopy (EIS). The resulting impedance plots of all of them showed good reproducibility. For the lowest frequency tested (10 mHz) all of the samples showed high impedance modulus value approximately on the order of 106 Ω. This behavior is usually ascribed to a high corrosion protection performance. Although no significant differences in the evolution of the corrosion behavior for different immersion times has been found; the Ti-6Al-7Nb alloy processed by PM delivers a steady growth of corrosion resistance from day one until twelve weeks immersion. This sample showed the best performance between the two studied compositions. The resulting impedance plots show how powder metallurgy allows obtaining materials with similar or superior corrosion resistance in physiological conditions, than alloys obtained conventionally. Alloys characterization by scanning electron microscopy revealed no evidence of pitting corrosion phenomenon. Authors acknowledge funds provided by Spanish Government (programme MINECO, Ref. MAT2012-38650-C02-01) and regional government of Madrid (programme MULTIMAT-CHALLENGE, Ref. S2013/MIT-2862).

Temporal and spatial control of growth factor gradients is critical for tissue patterning and differentiation. Reinitiation of this developmental program is also required for regeneration of tissues during wound healing and tissue regeneration. Devising methods for reconstituting growth factor gradients remains a central challenge in regenerative medicine. In the current study we develop a novel gene therapy approach for temporal and spatial control of two important growth factors in bone regeneration, vascular endothelial growth factor, and bone morphogenetic protein 2, which involves application of high intensity focused ultrasound to cells engineered with a heat-activated- and ligand-inducible gene switch. Induction of transgene expression was tightly localized within cell-scaffold constructs to subvolumes of ∼30 mm³, and the amplitude and projected area of transgene expression was tuned by the intensity and duration of ultrasound exposure. Conditions for ultrasound-activated transgene expression resulted in minimal cytotoxicity and scaffold damage. Localized regions of growth factor expression also established gradients in signaling activity, suggesting that patterns of growth factor expression generated by this method will have utility in basic and applied studies on tissue development and regeneration.

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.

Suspensions in phosphate buffered saline (PBS) of gold nanorods stabilized with cetyltrimethyl ammonium chloride (CTABr), polystyrenesulfonate (PSS) and methyl-polyethyleneglycol-thiol (m-PEG-SH) have been prepared and the evolution of their colloidal stability and plasmonic response over time has been evaluated. Their performance after lyophilization, alcoholic sterilization and resuspension has also been characterized. Sub-cytotoxic doses on HeLa cells were calculated for the three surface functionalizations used. Their heating efficiency at different exposure times was also evaluated after being irradiated with near infrared light. The best results were obtained for m-PEG-SH stabilized rods, which were not only stable, sterilizable and lyophilizable, but also biocompatible at all doses tested, showing potential as a stable, re-suspendible and biocompatible hyperthermic agent.

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.

Spatial and temporal patterns of bone morphogenetic protein (BMP) signaling are crucial to the assembly of appropriately positioned and shaped bones of the face and head. This review advances the hypothesis that reconstitution of such patterns with cutting-edge gene therapies will transform the clinical management of craniofacial bone defects attributed to trauma, disease, or surgical resection. Gradients in BMP signaling within developing limbs and orofacial primordia regulate proliferation and differentiation of mesenchymal progenitors. Similarly, vascular and mesenchymal cells express BMPs in various places and at various times during normal fracture healing. In non-healing fractures of long bones, BMP signaling is severely attenuated. Devices that release recombinant BMPs promote healing of bone in spinal fusions and, in some cases, of open fractures, but cannot control the timing and localization of BMP release. Gene therapies with regulated expression systems may provide substantial improvements in efficacy and safety compared with protein-based therapies. Synthetic gene switches, activated by pharmacologics or light or hyperthermic stimuli, provide several avenues for the non-invasive regulation of the expression of BMP transgenes in both time and space. Through new gene therapy platforms such as these, active control over BMP signaling can be achieved to accelerate bone regeneration.

Comparative modeling of the DNA-binding domain of human HSF1 facilitated the prediction of possible binding pockets for small molecules and definition of corresponding pharmacophores. In silico screening of a large library of lead-like compounds identified a set of compounds that satisfied the pharmacophoric criteria, a selection of which compounds was purchased to populate a biased sublibrary. A discriminating cell-based screening assay identified compound 001, which was subjected to systematic analysis of structure–activity relationships, resulting in the development of compound 115 (IHSF115). IHSF115 bound to an isolated HSF1 DNA-binding domain fragment. The compound did not affect heat-induced oligomerization, nuclear localization and specific DNA binding but inhibited the transcriptional activity of human HSF1, interfering with the assembly of ATF1-containing transcription complexes. IHSF115 was employed to probe the human heat shock response at the transcriptome level. In contrast to earlier studies of differential regulation in HSF1-naïve and -depleted cells, our results suggest that a large majority of heat-induced genes is positively regulated by HSF1. That IHSF115 effectively countermanded repression in a significant fraction of heat-repressed genes suggests that repression of these genes is mediated by transcriptionally active HSF1. IHSF115 is cytotoxic for a variety of human cancer cell lines, multiple myeloma lines consistently exhibiting high sensitivity.

The aim of this work was the generation of a multifunctional nanopolymeric system that incorporates IR-780 dye, a near-infrared (NIR) imaging probe that exhibits photothermal and photodynamic properties; and a derivate of α-tocopheryl succinate (α-TOS), a mitochondria-targeted anticancer compound. IR-780 was conjugated to the hydrophilic segment of copolymer PEG-b-polyMTOS, based on poly(ethylene glycol) (PEG) and a methacrylic derivative of α-tocopheryl succinate (MTOS), to generate IR-NP, self-assembled nanoparticles (NPs) in aqueous media which exhibit a hydrophilic shell and a hydrophobic core. During assembly, the hydrophobic core of IR-NP could encapsulate additional IR-780 to generate derived subspecies carrying different amount of probe (IR-NP-eIR). Evaluation of photo-inducible properties of IR-NP and IR-NP-eIR were thoroughly assessed in vitro. Developed nanotheranostic particles showed distinct fluorescence and photothermal behavior after excitation by a laser light emitting at 808 nm. Treatment of MDA-MB-453 cells with IR-NP or IR-NP-eIR resulted in an efficient internalization of the IR-780 dye, while subsequent NIR-laser irradiation led to a severe decrease in cell viability. Photocytoxicity conducted by IR-NP, which could not be attributed to the generation of lethal hyperthermia, responded to an increase in the levels of intracellular reactive oxygen species (ROS). Therefore, the fluorescence imaging and inducible phototoxicity capabilities of NPs derived from IR-780-PEG-b-polyMTOS copolymer confer high value to these nanotheranostics tools in clinical cancer research. Statement of Significance Multifunctional polymeric nanoparticles (NPs) that combine imaging and therapeutic properties are highly valuable in cancer treatment. In this paper we describe the development of NPs that are fluorescent in the near-infrared (NIR). This is important for their visualization in living tissues that present low absorption and low autofluorescence in this wavelength region (between 700 and 1000 nm). Moreover, NPs present photothermal and photodynamic properties when NIR irradiated: the NPs produce an efficient increment of temperature and increase the intracellular reactive oxygen species (ROS) when laser irradiated at 808 nm. These tuneable photoinduced properties make the NPs highly cytotoxic after NIR irradiation and provide a new tool for highly precise cancer treatment.

Herpes simplex virus (HSV) causes significant morbidity on the human population through such clinical syndromes as cold sores, genital herpes, herpes stromal keratitis, and encephalitis. Attempts to generate efficacious vaccines to date have failed. We have recently described the use of a conditionally replication-competent HSV-1 vector to immunize mice against a lethal challenge of HSV-1. The unique feature of this vaccine vector is that its replication is tightly controlled and can only occur in the presence of local heat and the presence of a small molecule inducer (an antiprogestin). This gives it the safety advantage of a replication-defective vaccine vector as well as the advantage of a replication-competent vector in that it is able to stimulate innate and adaptive aspects of the immune response in a natural context that a replication-defective vector cannot. In this chapter we provide a brief overview of HSV vaccines followed by the methodology used to propagate and utilize replication-conditional HSV vectors as vaccines.

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.

Implantation of scaffolds may elicit a host foreign body response triggered by monocyte/macrophage lineage cells. Growing evidence suggests that topographical cues of scaffolds play an important role in MSC functionality. In this work, we examined whether surface topographical features can regulate paracrine interactions that MSCs establish with macrophages. Three-dimensional (3D) topography sensing drives MSCs into a spatial arrangement that stimulates the production of the anti-inflammatory proteins PGE2 and TSG-6. Compared to two-dimensional (2D) settings, 3D arrangement of MSCs co-cultured with macrophages leads to an important decrease in the secretion of soluble factors related with inflammation and chemotaxis including IL-6 and MCP-1. Attenuation of MCP-1 secretion in 3D co-cultures correlates with a decrease in the accumulation of its mRNA levels in MSCs and macrophages. Using neutralizing antibodies, we identified that the interplay between PGE2, IL-6, TSG-6 and MCP-1 in the co-cultures is strongly influenced by the micro-architecture that supports MSCs. Local inflammatory milieu provided by 3D-arranged MSCs in co-cultures induces a decrease in monocyte migration as compared to monolayer cells. This effect is partially mediated by reduced levels of IL-6 and MCP-1, proteins that up-regulate each other's secretion. Our findings highlight the importance of topographical cues in the soluble factor-guided communication between MSCs and macrophages.

The development of noninvasive technologies for remote control of gene expression has received increased attention for their therapeutic potential in clinical scenarios, including cancer, neurological disorders, immunology, tissue engineering, as well as developmental biology research. Near-infrared (NIR) light is a suitable source of energy that can be employed to pattern transgene expression in plasmonic cell constructs. Gold nanoparticles tailored to exhibit a plasmon surface band absorption peaking at NIR wavelengths within the so called tissue optical window (TOW) can be used as fillers in fibrin-based hydrogels. These biocompatible composites can be loaded with cells harboring heat-inducible gene switches. NIR laser irradiation of the resulting plasmonic cell constructs causes the local conversion of NIR photon energy into heat, achieving spatially restricted patterns of transgene expression that faithfully match the illuminated areas of the hydrogels. In combination with cells genetically engineered to harbor gene switches activated by heat and dependent on a small-molecule regulator (SMR), NIR-responsive hydrogels allow reliable and safe control of the spatiotemporal availability of therapeutic biomolecules in target tissues.

Gold nanoparticles (Au NPs) are promising vectors for gene delivery applications. In order to gain insight on the influence of particle size on cell transfection, Au NPs were combined with poly(ethylenimine) (PEI) to prepare two sets of PEI-coated Au NPs having particle-size distributions centered at about 6 nm (

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.

Cancer therapy has been characterized throughout history by ups and downs, not only due to the ineffectiveness of treatments and side effects, but also by hope and the reality of complete remission and cure in many cases. Within the therapeutic arsenal, alongside surgery in the case of solid tumors, are the antitumor drugs and radiation that have been the treatment of choice in some instances. In recent years, immunotherapy has become an important therapeutic alternative, and is now the first choice in many cases. Nanotechnology has recently arrived on the scene, offering nanostructures as new therapeutic alternatives for controlled drug delivery, for combining imaging and treatment, applying hyperthermia, and providing directed target therapy, among others. These therapies can be applied either alone or in combination with other components (antibodies, peptides, folic acid, etc.). In addition, gene therapy is also offering promising new methods for treatment. Here, we present a review of the evolution of cancer treatments, starting with chemotherapy, surgery, radiation and immunotherapy, and moving on to the most promising cutting-edge therapies (gene therapy and nanomedicine). We offer an historical point of view that covers the arrival of these therapies to clinical practice and the market, and the promises and challenges they present.

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.

Mesoporous silica nanoparticles have been synthesized and functionalized with four different types of molecules containing amino groups, i.e., with primary amines only, with quaternary amines, with quaternized cyclic amines, or with polyethylenimine (PEI), which is formed by primary, secondary, and tertiary amines. These nanoparticles were then incubated with reporter plasmids and the ability of the resulting complexes to transfect human cells was studied. Only nanoparticles functionalized with PEI were efficient for transfection. The agglomeration behavior and the electrokinetic potential of the nanoparticle–plasmid complexes have been studied, as well as their cell internalization behavior using a fluorescent-labeled plasmid that allows its monitorization by confocal microscopy. The results indicate that the efficiency of PEI-functionalized nanoparticles for transfection resides to some extent in the different characteristics imparted to the nanoparticles regarding agglomeration and surface charge behavior.

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.

Millions of people receive annually an internal device aimed to repair or reconstruct damaged bone, and different therapeutic moieties are administered during or after surgery generally using systemic routes. The local administration of those moieties using in situ drug-eluting devices emerges as an alternative to minimize undesired side effects in healthy tissues, optimize the amount of drug needed, and reduce the costs.In vitro and in vivo published evidence regarding the performance of internally implantable drug-loaded biomedical devices for traumatology and orthopedic surgery are reviewed in this article, the problems that are encountered, and the main challenges in the development of a new generation of devices.An insight of past and current efforts to control the rate of drug release from devices, as well as the requirements that future developments may fulfill, such as responding 'on demand' after biological signaling or communicating the device with the exterior.The main drawback for proper design of devices having improved capabilities mainly remains in the lack of understanding with regards to the complex regulation of physiopathological mechanisms and the thermodynamics at the interface between the implant surface and the surrounding tissue. The potential toxicity and risk versus benefits of any new drug-eluting device need to be evaluated carefully.

SummaryObjectiveOsteoarthritis (OA) is clinically characterized by degeneration of the joints and has been traditionally considered a primary disorder of articular cartilage, with secondary changes in the subchondral bone. The increased bone mass and generalized changes in bone quality observed in osteoarthritic patients suggest that OA may be a primary systemic bone disorder with secondary articular cartilage damage. The iliac crest is a skeletal site distant from the affected joint, with a minimal load-bearing function. To provide evidence that OA is a systemic disorder, we searched for differentially expressed genes in the iliac crest bone of patients suffering from hip OA.Material and methodsGene expression levels between bone samples collected at surgery from the iliac crest of patients undergoing total hip arthroplasty for primary OA and younger donors, who were undergoing spinal arthrodesis, were investigated by means of oligonucleotide microarrays. To verify data detected by microarrays technology, Real Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays were performed with specimens from osteoarthritic patients and donors, as well as from elderly donors who were undergoing arthroplasty for subcapital femoral neck fracture.ResultsThe microarray analysis surveyed 8327 genes and identified 83 whose expression levels differed at least 1.5-fold in the OA group (P

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.

The development of noninvasive technologies for remote control of gene expression has received increased attention for their therapeutic potential in clinical scenarios, including cancer, neurological disorders, immunology, tissue engineering, as well as developmental biology research. Near-infrared (NIR) light is a suitable source of energy that can be employed to pattern transgene expression in plasmonic cell constructs. Gold nanoparticles tailored to exhibit a plasmon surface band absorption peaking at NIR wavelengths within the so called tissue optical window (TOW) can be used as fillers in fibrin-based hydrogels. These biocompatible composites can be loaded with cells harboring heat-inducible gene switches. NIR laser irradiation of the resulting plasmonic cell constructs causes the local conversion of NIR photon energy into heat, achieving spatially restricted patterns of transgene expression that faithfully match the illuminated areas of the hydrogels. In combination with cells genetically engineered to harbor gene switches activated by heat and dependent on a small-molecule regulator (SMR), NIR-responsive hydrogels allow reliable and safe control of the spatiotemporal availability of therapeutic biomolecules in target tissues.

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.