Accelular nanofibrous bilayer scaffold intrapenetrated with polydopamine network and implemented into a full-thickness wound of a white-pig model affects inflammation and healing process.

Treatment of complete loss of skin thickness requires expensive cellular materials and limited skin grafts used as temporary coverage. This paper presents an acellular bilayer scaffold modified with polydopamine (PDA), which is designed to mimic a missing dermis and a basement membrane (BM). The alternate dermis is made from freeze-dried collagen and chitosan (Coll/Chit) or collagen and a calcium salt of oxidized cellulose (Coll/CaOC). Alternate BM is made from electrospun gelatin (Gel), polycaprolactone (PCL), and CaOC. Morphological and mechanical analyzes have shown that PDA significantly improved the elasticity and strength of collagen microfibrils, which favorably affected swelling capacity and porosity. PDA significantly supported and maintained metabolic activity, proliferation, and viability of the murine fibroblast cell lines. The in vivo experiment carried out in a domestic Large white pig model resulted in the expression of pro-inflammatory cytokines in the first 1-2 weeks, giving the idea that PDA and/or CaOC trigger the early stages of inflammation. Otherwise, in later stages, PDA caused a reduction in inflammation with the expression of the anti-inflammatory molecule IL10 and the transforming growth factor ß (TGFß1), which could support the formation of fibroblasts. Similarities in treatment with native porcine skin suggested that the bilayer can be used as an implant for full-thickness skin wounds and thus eliminate the use of skin grafts.

The Role of ARHGAP1 in Rho GTPase Inactivation during Metastasizing of Breast Cancer Cell Line MCF-7 after Treatment with Doxorubicin.

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 µM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

Calcined Hydroxyapatite with Collagen I Foam Promotes Human MSC Osteogenic Differentiation.

Collagen I-based foams were modified with calcined or noncalcined hydroxyapatite or calcium phosphates with various particle sizes and pores to monitor their effect on cell interactions. The resulting scaffolds thus differed in grain size, changing from nanoscale to microscopic, and possessed diverse morphological characteristics and resorbability. The materials' biological action was shown on human bone marrow MSCs. Scaffold morphology was identified by SEM. Using viability test, qPCR, and immunohistochemical staining, we evaluated the biological activity of all of the materials. This study revealed that the most suitable scaffold composition for osteogenesis induction is collagen I foam with calcined hydroxyapatite with a pore size of 360 ± 130 µm and mean particle size of 0.130 µm. The expression of osteogenic markers RunX2 and ColI mRNA was promoted, and a strong synthesis of extracellular protein osteocalcin was observed. ColI/calcined HAP scaffold showed significant osteogenic potential, and can be easily manipulated and tailored to the defect size, which gives it great potential for bone tissue engineering applications.

The Effect of Osteoblast Isolation Methods from Adult Rats on Osteoclastogenesis in Co-Cultures.

Co-cultures of osteoblasts and osteoclasts are on the rise because they enable a more complex study. Diseases such as osteoporosis are related to a higher age. Thus, cell isolation from adult individuals is necessary. Osteoblasts can be isolated from the rat femur by three methods: explant culture, explant culture with enzymatic pre-treatment, or enzymatic treatment. The isolation methods yield different populations of osteoblasts which, in a co-culture with peripheral blood mononuclear cells, might result in differences in osteoclastogenesis. Therefore, we examined the differences in osteogenic markers, cell proliferation, and the metabolic activity of isolated osteoblast-like cells in a growth and differentiation medium. We then evaluated the effect of the isolated populations of osteoblast-like cells on osteoclastogenesis in a subsequent co-culture by evaluating osteoclast markers, counting formed osteoclast-like cells, and analyzing their area and number of nuclei. Co-cultures were performed in the presence or absence of osteoclastogenic growth factors, M-CSF and RANKL. It was discovered that enzymatic isolation is not feasible in adult rats, but explant culture and explant culture with enzymatic pre-treatment were both successful. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation than explant culture in a growth medium. The differentiation medium reduced differences in proliferation during the culture. Some differences in metabolic activity and ALP activity were also found between the osteoblast-like cells isolated by explant culture or by explant culture with enzymatic pre-treatment, but only on some days of cultivation. According to microscopy, the presence of exogenous growth factors supporting osteoclastogenesis in co-cultures was necessary for the formation of osteoclast-like cells. In this case, the formation of a higher number of osteoclast-like cells with a larger area was observed in the co-culture with osteoblast-like cells isolated by explant culture compared to the explant culture with enzymatic pre-treatment. Apart from this observation, no differences in osteoclast markers were noted between the co-cultures with osteoblast-like cells isolated by explant culture and the explant culture with enzymatic pre-treatment. The TRAP and CA II activity was higher in the co-cultures with exogenous growth than that in the co-cultures without exogenous growth factors on day 7, but the opposite was true on day 14. To conclude, explant culture and explant culture with enzymatic pre-treatment are both suitable methods to yield osteoblast-like cells from adult rats capable of promoting osteoclastogenesis in a direct co-culture with peripheral blood mononuclear cells. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation. The explant culture yielded osteoblast-like cells which induced the formation of a higher number of osteoclast-like cells with a larger area compared to the explant culture with enzymatic pre-treatment when cultured with exogenous M-CSF and RANKL.

Cellular Response to Individual Components of the Platelet Concentrate.

Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Calcaneal Tendon Collagen Fiber Morphometry and Aging.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.

[Histological evaluation of biomaterials administration in vivo on the cartilage, bone and skin healing]. / Histologické hodnocení vlivu in vivo aplikace biomateriálu na hojení chrupavky, kosti a kuze.

Our aim was to show the benefits and limitations of histological assessment of healing supported by implantable biomaterials. We reviewed and showed photographs of the histological and immunohistochemical methods applicable for the assessment of desirable and undesirable effects of biomaterials on the healing of hard and soft tissues. Currently used methods for evaluating the microscopic effects of bioengineered materials on the recipient tissue are reviewed. For histopathological analysis, semiquantitative scoring systems can be used. Alternatively, the main tissue constituents may be quantified using continuous variables giving the numerical densities of cells, lengths of microvessels or connective tissue fibres, area surfaces, area and volumes fractions, or clustering and colocalization of microscopic objects. Using systematic uniform random sampling strategies at the level of tissue blocks, sections, and image fields leads to a reasonable low variability of the quantitative results.

Modelling the role of membrane mechanics in cell adhesion on titanium oxide nanotubes.

Titanium surface treated with titanium oxide nanotubes was used in many studies to quantify the effect of surface topography on cell fate. However, the predicted optimal diameter of nanotubes considerably differs among studies. We propose a model that explains cell adhesion to a nanostructured surface by considering the deformation energy of cell protrusions into titanium nanotubes and the adhesion to the surface. The optimal surface topology is defined as a geometry that gives the membrane a minimum energy shape. A dimensionless parameter, the cell interaction index, was proposed to describe the interplay between the cell membrane bending, the intrinsic curvature, and the strength of cell adhesion. Model simulation shows that an optimal nanotube diameter ranging from 20 nm to 100 nm (cell interaction index between 0.2 and 1, respectively) is feasible within a certain range of parameters describing cell membrane adhesion and bending. The results indicate a possibility to tune the topology of a nanostructural surface in order to enhance the proliferation and differentiation of cells mechanically compatible with the given surface geometry while suppressing the growth of other mechanically incompatible cells.

The Gradual Release of Alendronate for the Treatment of Critical Bone Defects in Osteoporotic and Control Rats.

Purpose: Osteoporosis is a severe health problem with social and economic impacts on society. The standard treatment consists of the systemic administration of drugs such as bisphosphonates, with alendronate (ALN) being one of the most common. Nevertheless, complications of systemic administration occur with this drug. Therefore, it is necessary to develop new strategies, such as local administration. Methods: In this study, emulsion/dispersion scaffolds based on W/O emulsion of PCL and PF68 with ALN, containing hydroxyapatite (HA) nanoparticles as the dispersion phase were prepared using electrospinning. Scaffolds with different release kinetics were tested in vitro on the co-cultures of osteoblasts and osteoclast-like cells, isolated from adult osteoporotic and control rats. Cell viability, proliferation, ALP, TRAP and CA II activity were examined. A scaffold with a gradual release of ALN was tested in vivo in the bone defects of osteoporotic and control rats. Results: The release kinetics were dependent on the scaffold composition and the used system of the poloxamers. The ALN was released from the scaffolds for more than 22 days. The behavior of cells cultured in vitro on scaffolds with different release kinetics was comparable. The difference was evident between cell co-cultures isolated from osteoporotic and control animals. The PCL/HA scaffold show slow degradation in vivo and residual scaffold limited new bone formation inside the defects. Nevertheless, the released ALN supported bone formation in the areas surrounding the residual scaffold. Interestingly, a positive effect of systemic administration of ALN was not proved. Conclusion: The prepared scaffolds enabled tunable control release of ALN. The effect of ALN was proved in vitro and in in vivo study supported peri-implant bone formation.

Core/shell nanofibers with embedded liposomes as a drug delivery system.

The broader application of liposomes in regenerative medicine is hampered by their short half-life and inefficient retention at the site of application. These disadvantages could be significantly reduced by their combination with nanofibers. We produced 2 different nanofiber-liposome systems in the present study, that is, liposomes blended within nanofibers and core/shell nanofibers with embedded liposomes. Herein, we demonstrate that blend electrospinning does not conserve intact liposomes. In contrast, coaxial electrospinning enables the incorporation of liposomes into nanofibers. We report polyvinyl alcohol-core/poly-ε-caprolactone-shell nanofibers with embedded liposomes and show that they preserve the enzymatic activity of encapsulated horseradish peroxidase. The potential of this system was also demonstrated by the enhancement of mesenchymal stem cell proliferation. In conclusion, intact liposomes incorporated into nanofibers by coaxial electrospinning are very promising as a drug delivery system.

A simple drug anchoring microfiber scaffold for chondrocyte seeding and proliferation.

The structural properties of microfiber meshes made from poly(2-hydroxyethyl methacrylate) (PHEMA) were found to significantly depend on the chemical composition and subsequent cross-linking and nebulization processes. PHEMA microfibres showed promise as scaffolds for chondrocyte seeding and proliferation. Moreover, the peak liposome adhesion to PHEMA microfiber scaffolds observed in our study resulted in the development of a simple drug anchoring system. Attached foetal bovine serum-loaded liposomes significantly improved both chondrocyte adhesion and proliferation. In conclusion, fibrous scaffolds from PHEMA are promising materials for tissue engineering and, in combination with liposomes, can serve as a simple drug delivery tool.

Advances in Electrospun Hybrid Nanofibers for Biomedical Applications.

Electrospun hybrid nanofibers, based on functional agents immobilized in polymeric matrix, possess a unique combination of collective properties. These are beneficial for a wide range of applications, which include theranostics, filtration, catalysis, and tissue engineering, among others. The combination of functional agents in a nanofiber matrix offer accessibility to multifunctional nanocompartments with significantly improved mechanical, electrical, and chemical properties, along with better biocompatibility and biodegradability. This review summarizes recent work performed for the fabrication, characterization, and optimization of different hybrid nanofibers containing varieties of functional agents, such as laser ablated inorganic nanoparticles (NPs), which include, for instance, gold nanoparticles (Au NPs) and titanium nitride nanoparticles (TiNPs), perovskites, drugs, growth factors, and smart, inorganic polymers. Biocompatible and biodegradable polymers such as chitosan, cellulose, and polycaprolactone are very promising macromolecules as a nanofiber matrix for immobilizing such functional agents. The assimilation of such polymeric matrices with functional agents that possess wide varieties of characteristics require a modified approach towards electrospinning techniques such as coelectrospinning and template spinning. Additional focus within this review is devoted to the state of the art for the implementations of these approaches as viable options for the achievement of multifunctional hybrid nanofibers. Finally, recent advances and challenges, in particular, mass fabrication and prospects of hybrid nanofibers for tissue engineering and biomedical applications have been summarized.

The Preparation and Biological Testing of Novel Wound Dressings with an Encapsulated Antibacterial and Antioxidant Substance.

Chronic wounds represent a significant socio-economic problem, and the improvement of their healing is therefore an essential issue. This paper describes the preparation and biological properties of a novel functionalized nanofiber wound dressing consisting of a polycaprolactone nanofiber carrier modified by a drug delivery system, based on the lipid particles formed by 1-tetradecanol and encapsulated gentamicin and tocopherol acetate. The cytotoxicity of extracts was tested using a metabolic activity assay, and the antibacterial properties of the extracts were tested in vitro on the bacterial strains Staphylococcus aureus and Pseudomonas aeruginosa. The effect of the wound dressing on chronic wound healing was subsequently tested using a mouse model. Fourteen days after surgery, the groups treated by the examined wound cover showed a lower granulation, reepithelization, and inflammation score compared to both the uninfected groups, a lower dermis organization compared to the control, a higher scar thickness compared to the other groups, and a higher thickness of hypodermis and bacteria score compared to both the uninfected groups. This work demonstrates the basic parameters of the safety (biocompatibility) and performance (effect on healing) of the dressing as a medical device and indicates the feasibility of the concept of its preparation in outpatient conditions using a suitable functionalization device.

The Effect of Alendronate on Osteoclastogenesis in Different Combinations of M-CSF and RANKL Growth Factors.

Bisphosphonates (BPs) are compounds resembling the pyrophosphate structure. BPs bind the mineral component of bones. During the bone resorption by osteoclasts, nitrogen-containing BPs are released and internalized, causing an inhibition of the mevalonate pathway. As a consequence, osteoclasts are unable to execute their function. Alendronate (ALN) is a bisphosphonate used to treat osteoporosis. Its administration could be associated with adverse effects. The purpose of this study is to evaluate four different ALN concentrations, ranging from 10-6 to 10-10 M, in the presence of different combinations of M-CSF and RANKL, to find out the effect of low ALN concentrations on osteoclastogenesis using rat and human peripheral blood mononuclear cells. The cytotoxic effect of ALN was evaluated based on metabolic activity and DNA concentration measurement. The alteration in osteoclastogenesis was assessed by the activity of carbonic anhydrase II (CA II), tartrate-resistant acid phosphatase staining, and actin ring formation. The ALN concentration of 10-6 M was cytotoxic. Low ALN concentrations of 10-8 and 10-10 M promoted proliferation, osteoclast-like cell formation, and CA II activity. The results indicated the induction of osteoclastogenesis with low ALN concentrations. However, when high doses of ALN were administered, their cytotoxic effect was demonstrated.

Smart Electrospun Hybrid Nanofibers Functionalized with Ligand-Free Titanium Nitride (TiN) Nanoparticles for Tissue Engineering.

Herein, we report the fabrication and characterization of novel polycaprolactone (PCL)-based nanofibers functionalized with bare (ligand-free) titanium nitride (TiN) nanoparticles (NPs) for tissue engineering applications. Nanofibers were prepared by a newly developed protocol based on the electrospinning of PCL solutions together with TiN NPs synthesized by femtosecond laser ablation in acetone. The generated hybrid nanofibers were characterised using spectroscopy, microscopy, and thermal analysis techniques. As shown by scanning electron microscopy measurements, the fabricated electrospun nanofibers had uniform morphology, while their diameter varied between 0.403 ± 0.230 µm and 1.1 ± 0.15 µm by optimising electrospinning solutions and parameters. Thermal analysis measurements demonstrated that the inclusion of TiN NPs in nanofibers led to slight variation in mass degradation initiation and phase change behaviour (Tm). In vitro viability tests using the incubation of 3T3 fibroblast cells in a nanofiber-based matrix did not reveal any adverse effects, confirming the biocompatibility of hybrid nanofiber structures. The generated hybrid nanofibers functionalized with plasmonic TiN NPs are promising for the development of smart scaffold for tissue engineering platforms and open up new avenues for theranostic applications.

Healing and Angiogenic Properties of Collagen/Chitosan Scaffolds Enriched with Hyperstable FGF2-STAB® Protein: In Vitro, Ex Ovo and In Vivo Comprehensive Evaluation.

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

Combinations of growth factors for human mesenchymal stem cell proliferation and osteogenic differentiation.

AIMS: Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides - growth factors, influencing cell fate through surface cellular receptors. METHODS: In our study transforming growth factor ß (TGF-ß), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. RESULTS: TGF-ß and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. CONCLUSION: A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades.Cite this article: Bone Joint Res 2020;9(7):412-420.

In vitro evaluation of a novel nanostructured Ti-36Nb-6Ta alloy for orthopedic applications.

Aim: To evaluate the impact of a nanostructured surface created on ß-titanium alloy, Ti-36Nb-6Ta, on the growth and differentiation of human mesenchymal stem cells. Materials & methods: The nanotubes, with average diameters 18, 36 and 46 nm, were prepared by anodic oxidation. Morphology, hydrophilicity and mechanical properties of the nanotube layers were characterized. The biocompatibility and osteogenic potential of the nanostructured surfaces were established using various in vitro assays, scanning electron microscopy and confocal microscopy. Results: The nanotubes lowered elastic modulus close to that of bone, positively influenced cell adhesion, improved ALP activity, synthesis of type I collagen and osteocalcin expression, but diminished early cell proliferation. Conclusion: Nanostructured Ti-36Nb-6Ta with nanotube diameters 36 nm was the most promising material for bone implantation.

Mesenchymal stem cell interaction with Ti6Al4V alloy pre-exposed to simulated body fluid.

Titanium and its alloys are widely used for substitution of hard tissues, especially in orthopaedic and dental surgery. Despite the benefit of the use of titanium for such applications, there are still questions which must be sorted out. Surface properties are crucial for cell adhesion, proliferation and differentiation. Mainly, micro/nanostructured surfaces positively influence osteogenic differentiation of human mesenchymal stem cells. Ti6Al4V is a biocompatible α + ß alloy which is widely used in orthopaedics. The aim of this study was to investigate the interaction of the nanostructured and ground Ti6Al4V titanium alloys with simulated body fluid complemented by the defined precipitation of hydroxyapatite-like coating and to study the cytotoxicity and differentiation capacity of cells with such a modified titanium alloy. Nanostructures were fabricated using electrochemical oxidation. Human mesenchymal stem cells (hMSC) were used to evaluate cell adhesion, metabolic activity and proliferation on the specimens. The differentiation potential of the samples was investigated using PCR and specific staining of osteogenic markers collagen type I and osteocalcin. Our results demonstrate that both pure Ti6Al4V, nanostructured samples, and hydroxyapatite-like coating supported hMSC growth and metabolic activity. Nanostructured samples improved collagen type I synthesis after 14 days, while both nanostructured and hydroxyapatite-like coated samples enhanced collagen synthesis on day 21. Osteocalcin synthesis was the most enhanced by hydroxyapatite-like coating on the nanostructured surfaces. Our results indicate that hydroxyapatite-like coating is a useful tool guiding hMSC osteogenic differentiation.