Learning models for classifying Raman spectra of genomic DNA from tumor subtypes.

An early and accurate detection of different subtypes of tumors is crucial for an effective guidance to personalized therapy and in predicting the ability of tumor to metastasize. Here we exploit the Surface Enhanced Raman Scattering (SERS) platform, based on disordered silver coated silicon nanowires (Ag/SiNWs), to efficiently discriminate genomic DNA of different subtypes of melanoma and colon tumors. The diagnostic information is obtained by performing label free Raman maps of the dried drops of DNA solutions onto the Ag/NWs mat and leveraging the classification ability of learning models to reveal the specific and distinct physico-chemical interaction of tumor DNA molecules with the Ag/NW, here supposed to be partly caused by a different DNA methylation degree.

Revealing Low Amplitude Signals of Neuroendocrine Cells through Disordered Silicon Nanowires-Based Microelectrode Array.

Today, the key methodology to study in vitro or in vivo electrical activity in a population of electrogenic cells, under physiological or pathological conditions, is by using microelectrode array (MEA). While significant efforts have been devoted to develop nanostructured MEAs for improving the electrophysiological investigation in neurons and cardiomyocytes, data on the recording of the electrical activity from neuroendocrine cells with MEA technology are scarce owing to their weaker electrical signals. Disordered silicon nanowires (SiNWs) for developing a MEA that, combined with a customized acquisition board, successfully capture the electrical signals generated by the corticotrope AtT-20 cells as a function of the extracellular calcium (Ca2+ ) concentration are reported. The recorded signals show a shape that clearly resembles the action potential waveform by suggesting a natural membrane penetration of the SiNWs. Additionally, the generation of synchronous signals observed under high Ca2+ content indicates the occurrence of a collective behavior in the AtT-20 cell population. This study extends the usefulness of MEA technology to the investigation of the electrical communication in cells of the pituitary gland, crucial in controlling several essential human functions, and provides new perspectives in recording with MEA the electrical activity of excitable cells.

Tailoring the Microarchitectures of 3D Printed Bone-like Scaffolds for Tissue Engineering Applications.

Material extrusion (MEX), commonly referred to as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a versatile and cost-effective technique to fabricate suitable scaffolds for tissue engineering. Driven by a computer-aided design input, specific patterns can be easily collected in an extremely reproducible and repeatable process. Referring to possible skeletal affections, 3D-printed scaffolds can support tissue regeneration of large bone defects with complex geometries, an open major clinical challenge. In this study, polylactic acid scaffolds were printed resembling trabecular bone microarchitecture in order to deal with morphologically biomimetic features to potentially enhance the biological outcome. Three models with different pore sizes (i.e., 500, 600, and 700 µm) were prepared and evaluated by means of micro-computed tomography. The biological assessment was carried out seeding SAOS-2 cells, a bone-like cell model, on the scaffolds, which showed excellent biocompatibility, bioactivity, and osteoinductivity. The model with larger pores, characterized by improved osteoconductive properties and protein adsorption rate, was further investigated as a potential platform for bone-tissue engineering, evaluating the paracrine activity of human mesenchymal stem cells. The reported findings demonstrate that the designed microarchitecture, better mimicking the natural bone extracellular matrix, favors a greater bioactivity and can be thus regarded as an interesting option for bone-tissue engineering.

Severely Damaged Freeze-Injured Skeletal Muscle Reveals Functional Impairment, Inadequate Repair, and Opportunity for Human Stem Cell Application.

BACKGROUND: The regeneration of severe traumatic muscle injuries is an unsolved medical need that is relevant for civilian and military medicine. In this work, we produced a critically sized nonhealing muscle defect in a mouse model to investigate muscle degeneration/healing phases. MATERIALS AND METHODS: We caused a freeze injury (FI) in the biceps femoris of C57BL/6N mice. From day 1 to day 25 post-injury, we conducted histological/morphometric examinations, an analysis of the expression of genes involved in inflammation/regeneration, and an in vivo functional evaluation. RESULTS: We found that FI activates cytosolic DNA sensing and inflammatory responses. Persistent macrophage infiltration, the prolonged expression of eMHC, the presence of centrally nucleated myofibers, and the presence of PAX7+ satellite cells at late time points and with chronic physical impairment indicated inadequate repair. By looking at stem-cell-based therapeutic protocols of muscle repair, we investigated the crosstalk between M1-biased macrophages and human amniotic mesenchymal stem cells (hAMSCs) in vitro. We demonstrated their reciprocal paracrine effects where hAMSCs induced a shift of M1 macrophages into an anti-inflammatory phenotype, and M1 macrophages promoted an increase in the expression of hAMSC immunomodulatory factors. CONCLUSIONS: Our findings support the rationale for the future use of our injury model to exploit the full potential of in vivo hAMSC transplantation following severe traumatic injuries.

Editorial for the Special Issue on Nanostructured Surfaces and Devices for Biomedical Applications.

The ability to control and modify the surface topography of materials at the nanoscale, which produces features with a comparable size to that of biological entities, so as to effectively probe and influence processes at both the cellular and the molecular level, has facilitated incredible possibilities in the fields of biomedicine, biosensing, and diagnostics [...].

Statistical Classification for Raman Spectra of Tumoral Genomic DNA.

We exploit Surface-Enhanced Raman Scattering (SERS) to investigate aqueous droplets of genomic DNA deposited onto silver-coated silicon nanowires, and we show that it is possible to efficiently discriminate between spectra of tumoral and healthy cells. To assess the robustness of the proposed technique, we develop two different statistical approaches, one based on the Principal Components Analysis of spectral data and one based on the computation of the ℓ2 distance between spectra. Both methods prove to be highly efficient, and we test their accuracy via the Cohen's κ statistics. We show that the synergistic combination of the SERS spectroscopy and the statistical analysis methods leads to efficient and fast cancer diagnostic applications allowing rapid and unexpansive discrimination between healthy and tumoral genomic DNA alternative to the more complex and expensive DNA sequencing.

Biological Response to Bioinspired Microporous 3D-Printed Scaffolds for Bone Tissue Engineering.

The scaffold is a key element in the field of tissue engineering, especially when large defects or substitutions of pathological tissues or organs need to be clinically addressed. The expected outcome is strongly dependent on the cell-scaffold interaction and the integration with the surrounding biological tissue. Indeed, mimicking the natural extracellular matrix (ECM) of the tissue to be healed represents a further optimization that can limit a possible morphological mismatch between the scaffold and the tissue itself. For this aim, and referring to bone tissue engineering, polylactic acid (PLA) scaffolds were 3D printed with a microstructure inspired by the trabecular architecture and biologically evaluated by means of human osteosarcoma SAOS-2 cells. The cells were seeded on two types of scaffolds differing for the designed pore size (i.e., 400 and 600 µm), showing the same growth exponential trend found in the control and no significant alterations in the actin distribution. The microporous structure of the two tested samples enhanced the protein adsorption capability and mRNA expression of markers related to protein synthesis, proliferation, and osteoblast differentiation. Our findings demonstrate that 3D-printed scaffolds support the adhesion, growth, and differentiation of osteoblast-like cells and the microporous architecture, mimicking the natural bone hierarchical structure, and favoring greater bioactivity. These bioinspired scaffolds represent an interesting new tool for bone tissue engineering and regenerative medicine applications.

Raman Mapping of Biological Systems Interacting with a Disordered Nanostructured Surface: A Simple and Powerful Approach to the Label-Free Analysis of Single DNA Bases.

This article demonstrates the possibility to use a novel powerful approach based on Raman mapping of analyte solutions drop casted on a disordered array of Ag covered silicon nanowires (Ag/SiNWs), to identify the characteristic spectral signal of the four DNA bases, adenine (A), thymine (T), cytosine (C), and guanine (G), at concentration as low as 10 ng/µL, and to study their specific way of interacting with the nanostructured substrate. The results show a distinctive and amplified interaction of guanine, the base that is most susceptible to oxidation, with the nanostructured surface. Our findings explain the recently revealed diverse behaviour of cancer and normal DNA deposited on the same Ag/SiNWs, which is ascribed to mechanical deformation and base lesions present on the oxidised DNA molecule backbone and causes detectable variation in the Raman signal, usable for diagnostic purposes. The notable bio-analytical capability of the presented platform, and its sensitivity to the molecule mechanical conformation at the single-base level, thus provides a new reliable, rapid, label-free DNA diagnostic methodology alternative to more sophisticated and expensive sequencing ones.

Silver-coated silicon nanowire platform discriminates genomic DNA from normal and malignant human epithelial cells using label-free Raman spectroscopy.

Genomic deoxyribonucleic acid (DNA) stores and carries the information required to maintain and replicate cellular life. While much efforts have been devoted in decoding the sequence of DNA basis to detect the genetic mutations related to cancer disease, it is becoming clear that physical properties, like structural conformation, stiffness and shape, can play an important role to recognize DNA modifications. Here, silver-coated silicon nanowires (Ag/SiNWs) are exploited as Raman spectroscopic platform to easily discriminate healthy and cancer genomic DNA, extracted from human normal skin and malignant melanoma cells, respectively. In particular, aqueous DNA droplets are directly deposited onto a forest of Ag/SiNWs and Raman maps are acquired after sample dehydration. By applying principal component analysis (PCA) to the Raman spectra collected within the droplets, healthy and cancer cell DNA can be distinguished without false negative identifications and with few false positive results (< 2%). The discrimination occurs regardless the analysis of specific DNA sequencing, but through Raman bands strictly related to the interfacing of the DNA and the NWs. The observed phenomenon can be ascribed to conformational differences and/or diverse charge properties between healthy and cancer cell DNA determining a different arrangement of the molecules adsorbed onto the NWs upon water evaporation. The unique interaction with DNA and facile fabrication technology make Ag/SiNWs an effective platform for a robust, rapid and label-free cancer diagnosis, as well as a potential tool to investigate physical properties of DNA.

Biocompatibility assessment of sub-5 nm silica-coated superparamagnetic iron oxide nanoparticles in human stem cells and in mice for potential application in nanomedicine.

Ultrasmall superparamagnetic iron oxide nanoparticles with a size <5 nm are emerging nanomaterials for their excellent biocompatibility, chemical stability, and tunable surface modifications. The applications explored include dual-modal or multi-modal imaging, drug delivery, theranostics and, more recently, magnetic resonance angiography. Good biocompatibility and biosafety are regarded as the preliminary requirements for their biomedical applications and further exploration in this field is still required. We previously synthesized and characterized ultrafine (average core size of 3 nm) silica-coated superparamagnetic iron oxide fluorescent nanoparticles, named sub-5 SIO-Fl, uniform in size, shape, chemical properties and composition. The cellular uptake and in vitro biocompatibility of the as-synthesized nanoparticles were demonstrated in a human colon cancer cellular model. Here, we investigated the biocompatibility of sub-5 SIO-Fl nanoparticles in human Amniotic Mesenchymal Stromal/Stem Cells (hAMSCs). Kinetic analysis of cellular uptake showed a quick nanoparticle internalization in the first hour, increasing over time and after long exposure (48 h), the uptake rate gradually slowed down. We demonstrated that after internalization, sub-5 SIO-Fl nanoparticles neither affect hAMSC growth, viability, morphology, cytoskeletal organization, cell cycle progression, immunophenotype, and the expression of pro-angiogenic and immunoregulatory paracrine factors nor the osteogenic and myogenic differentiation markers. Furthermore, sub-5 SIO-Fl nanoparticles were intravenously injected into mice to investigate the in vivo biodistribution and toxicity profile for a time period of 7 weeks. Our findings showed an immediate transient accumulation of nanoparticles in the kidney, followed by the liver and lungs, where iron contents increased over a 7-week period. Histopathology, hematology, serum pro-inflammatory response, body weight and mortality studies demonstrated a short- and long-term biocompatibility and biosafety profile with no apparent acute and chronic toxicity caused by these nanoparticles in mice. Overall, these results suggest the feasibility of using sub-5 SIO-Fl nanoparticles as a promising agent for stem cell magnetic targeting as well as for diagnostic and therapeutic applications in oncology.

Combination of cord blood-derived human hepatic progenitors and hepatogenic factors strongly improves recovery after acute liver injury in mice through modulation of the Wnt/ß-catenin signaling.

Cell therapy represents a promising alternative strategy for end-stage liver disease, and hepatic progenitors are the best candidates. The possibility to maximize the paracrine effects of transplanted cells represents a great potential benefit for cell therapy success. We studied how cell type and microenvironment modulate the Wnt/ß-catenin signaling in vitro and in vivo. In vitro, the onset of hepatocyte commitment was characterized by the presence of nuclear truncated ß-catenin. In vivo, we analyzed the effect of human hepatic progenitors on damage recovery and functional regeneration in a mouse model of acute liver injury, either in combination or in absence of a selected mix of hepatogenic factors. Animals injected with human hepatic progenitors and hepatogenic factors showed improved engraftment triggering the Wnt/ß-catenin signaling cascade. Human hepatic progenitors expressing the human oval cell marker OV6 displayed a consistent colocalization with ß-catenin and colocalized with Wnt1 main ligand of the canonical pathway. Wnt5a, on the contrary, was expressed in distinct liver cell populations. Epithelial mesenchymal transition-related markers showed enhanced expression and wider distribution, and the hepato-mesenchymal population Thy1 + CK19- was also present. Control animals injected with hepatogenic factors alone exhibited higher ß-catenin, decreased Wnt5a levels, and persistent proliferation of the hepato-mesenchymal population. In conclusion, the combination of human hepatic progenitors with selected hepatogenic factors creates a positive synergy with local microenvironment, ameliorates cell engraftment, stimulates and accelerates regenerative process, and improves the rescue of hepatic function by modulating the Wnt/ßcatenin signaling and activating hepato-mesenchymal population.

Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine.

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for "in vitro" stem cell commitment, preparatory to the "in vivo" stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

Array of disordered silicon nanowires coated by a gold film for combined NIR photothermal treatment of cancer cells and Raman monitoring of the process evolution.

Photothermal therapy (PTT) assisted by nanomaterials is a promising minimally invasive technique for cancer treatment. Here, we explore the PTT properties of a silicon- and gold-based nanostructured platform suitable for being directly integrated in fibre laser systems rather than injected into the human body, which occurs for the most commonly unreported PTT nanoagents. In particular, the photothermal properties of an array of disordered silicon nanowires coated by a thin gold film (Au/SiNWs) were tested on a monolayer of human colon adenocarcinoma cells (Caco-2) irradiated with a 785 nm laser. Au/SiNWs allowed an efficient photothermal action and simultaneous monitoring of the process evolution through the Raman signal coming from the irradiated cellular zone. Strong near infra-red (NIR) absorption, overlapping three biological windows, cell-friendly properties and effective fabrication technology make Au/SiNWs suitable both to be integrated in surgical laser tools and as an in vitro platform to develop novel PTT protocols using different cancer types and NIR sources.

Electromagnetic information transfer through aqueous system.

Several beneficial effects of the electromagnetic information transfer through aqueous system (EMITTAS) procedure have previously been reported in vitro. The clinical potential of this procedure has also started to be evaluated. Information flow in biological systems can be investigated through chemical and molecular approaches or by a biophysical approach focused on endogenous electrodynamic activities. Electromagnetic signals are endogenously generated at different levels of the biological organization and, likely, play an active role in synchronizing internal cell function or local/systemic adaptive response. Consequently, each adaptive response can be described by its specific electromagnetic pattern and, therefore, correlates with a unique and specific electromagnetic signature. A biophysical procedure synchronously integrating the EMITTAS procedure has already been applied for the treatment of articular pain, low-back pain, neck pain and mobility, fluctuating asymmetry, early-stage chronic kidney disease, refractory gynecological infections, minor anxiety and depression disorders. This clinical strategy involves a single treatment, since the EMITTAS procedure allows the patient to continue his/her own personal treatment at home by means of self-administration of the recorded aqueous system. A significant and long-lasting improvement has been reported, showing a potential beneficial use of this biophysical procedure in the management of common illnesses in an efficient, effective and personalized way. Data from recent studies suggest that aqueous systems may play a key role in providing the basis for recording, storing, transferring and retrieving clinically effective quanta of biological information. These features likely enable to trigger local and systemic self-regulation and self-regeneration potential of the organism.

In vitro biocompatibility study of sub-5 nm silica-coated magnetic iron oxide fluorescent nanoparticles for potential biomedical application.

Magnetic iron oxide nanoparticles (IONPs), for their intriguing properties, have attracted a great interest as they can be employed in many different biomedical applications. In this multidisciplinary study, we synthetized and characterized ultrafine 3 nm superparamagnetic water-dispersible nanoparticles. By a facile and inexpensive one-pot approach, nanoparticles were coated with a shell of silica and contemporarily functionalized with fluorescein isothiocyanate (FITC) dye. The obtained sub-5 nm silica-coated magnetic iron oxide fluorescent (sub-5 SIO-Fl) nanoparticles were assayed for cellular uptake, biocompatibility and cytotoxicity in a human colon cancer cellular model. By confocal microscopy analysis we demonstrated that nanoparticles as-synthesized are internalized and do not interfere with the CaCo-2 cell cytoskeletal organization nor with their cellular adhesion. We assessed that they do not exhibit cytotoxicity, providing evidence that they do not affect shape, proliferation, cellular viability, cell cycle distribution and progression. We further demonstrated at molecular level that these nanoparticles do not interfere with the expression of key differentiation markers and do not affect pro-inflammatory cytokines response in Caco-2 cells. Overall, these results showed the in vitro biocompatibility of the sub-5 SIO-Fl nanoparticles promising their safe employ for diagnostic and therapeutic biomedical applications.

Placenta Derived Mesenchymal Stem Cells Hosted on RKKP Glass-Ceramic: A Tissue Engineering Strategy for Bone Regenerative Medicine Applications.

In tissue engineering protocols, the survival of transplanted stem cells is a limiting factor that could be overcome using a cell delivery matrix able to support cell proliferation and differentiation. With this aim, we studied the cell-friendly and biocompatible behavior of RKKP glass-ceramic coated Titanium (Ti) surface seeded with human amniotic mesenchymal stromal cells (hAMSCs) from placenta. The sol-gel synthesis procedure was used to prepare the RKKP glass-ceramic material, which was then deposited onto the Ti surface by Pulsed Laser Deposition method. The cell metabolic activity and proliferation rate, the cytoskeletal actin organization, and the cell cycle phase distribution in hAMSCs seeded on the RKKP coated Ti surface revealed no significant differences when compared to the cells grown on the treated plastic Petri dish. The health of of hAMSCs was also analysed studying the mRNA expressions of MSC key genes and the osteogenic commitment capability using qRT-PCR analysis which resulted in being unchanged in both substrates. In this study, the combination of the hAMSCs' properties together with the bioactive characteristics of RKKP glass-ceramics was investigated and the results obtained indicate its possible use as a new and interesting cell delivery system for bone tissue engineering and regenerative medicine applications.

The trail from quantum electro dynamics to informative medicine.

Several years ago just before Christmas, in a small meeting room at the Institute of Pharmacology at the University of Rome, we had the opportunity to attend a meeting on "The role of QED in medicine" by Emilio Del Giudice and Giuliano Preparata. Before that meeting, we were more oriented towards a mechanistic view of Biochemistry and Medicine, believing that chemical reactions could only take place when a random collision between molecules with a gain in energy takes place. We envisioned water as just a solvent in which was possible to dissolve a solute. After we listened to Giuliano's and Emilio's speech on the "New physics of water", and on "The possible origin of coherence in cell, tissues and the interaction of very weak and low frequency magnetic fields with the ions, systems of the cell", we realized that living organisms are complex electrochemical systems which evolved in a relatively narrow range of well-defined environmental parameters. Environmental natural electro-magnetic fields are an ubiquitous factor in nature. If nature gave certain organisms the ability to receive information about the environment via invisible electromagnetic signals, then there must also the capability to discriminate between significant and meaningless ones. Bearing in mind that electromagnetic fields can be perceived by living organisms by means a resonance effect, we should not be amazed if they can be able to induce different biological effects. The work that we will present in memory of Emilio is based on the hypotheses that an aqueous system a chemical differentiation agent such as retinoic acid (RA) were electronically captured and transferred to the culture medium of Neuroblastoma Cell Line (LAN-5) and the proliferation rate was assessed to assess cell responses to the electromagnetic information transfer through the aqueous system. Like those enfolded in living organisms could play a synergic role in modulating biological functions, generating dissipative structures under appropriate patterns of electromagnetic signals providing basis for storing and retrieving biological activities. An external electro-magnetic stimulus from a source molecule can be stored, translated, and transferred by the aqueous systems to the biological target, selectively driving their endogenous activity and mimicking the effect of a source molecule.

Copper ion fluxes through the floating water bridge under strong electric potential.

We have performed a series of experiments applying high voltage between two electrodes, immersed in two beakers containing bidistilled water in a way similar to experiments conducted by Fuchs and collaborators, which showed that a water bridge can be formed between the two containers. We also observed the formation of water bridge. Moreover, choosing different pairs of electrodes depending on the material they are made up of, we observed that copper ions flow can pass along the bridge if the negative electrode is made up of copper. We show that the direction of the flux not only depends on the applied electrostatic field but on the relative electronegativity of the electrodes too. These results open new perspectives in understanding the properties of water. We suggest a possible explanation of the obtained results.

Interdisciplinary approach to cell-biomaterial interactions: biocompatibility and cell friendly characteristics of RKKP glass-ceramic coatings on titanium.

In this work, titanium (Ti) supports have been coated with glass-ceramic films for possible applications as biomedical implant materials in regenerative medicine. For the film preparation, a pulsed laser deposition (PLD) technique has been applied. The RKKP glass-ceramic material, used for coating deposition, was a sol-gel derived target of the following composition: Ca-19.4, P-4.6, Si-17.2, O-43.5, Na-1.7, Mg-1.3, F-7.2, K-0.2, La-0.8, Ta-4.1 (all in wt%). The prepared coatings were compact and uniform, characterised by a nanometric average surface roughness. The biocompatibility and cell-friendly properties of the RKKP glass-ceramic material have been tested. Cell metabolic activity and proliferation of human colon carcinoma CaCo-2 cells seeded on RKKP films showed the same exponential trend found in the control plastic substrates. By the phalloidin fluorescence analysis, no significant modifications in the actin distribution were revealed in cells grown on RKKP films. Moreover, in these cells a high mRNA expression of markers involved in protein synthesis, proliferation and differentiation, such as villin (VIL1), alkaline phosphatase (ALP1), ß-actin (ß-ACT), Ki67 and RPL34, was recorded. In conclusion, the findings, for the first time, demonstrated that the RKKP glass-ceramic material allows the adhesion, growth and differentiation of the CaCo-2 cell line.

Nonpulsed sinusoidal electromagnetic fields as a noninvasive strategy in bone repair: the effect on human mesenchymal stem cell osteogenic differentiation.

In vivo control of osteoblast differentiation is an important process needed to maintain the continuous supply of mature osteoblast cells for growth, repair, and remodeling of bones. The regulation of this process has also an important and significant impact on the clinical strategies and future applications of cell therapy. In this article, we studied the effect of nonpulsed sinusoidal electromagnetic field radiation tuned at calcium-ion cyclotron frequency of 50 Hz exposure treatment for bone differentiation of human mesenchymal stem cells (hMSCs) alone or in synergy with dexamethasone, their canonical chemical differentiation agent. Five days of continuous exposure to calcium-ion cyclotron resonance affect hMSC proliferation, morphology, and cytoskeletal actin reorganization. By quantitative real-time polymerase chain reaction, we also observed an increase of osteoblast differentiation marker expression such as Runx2, alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN) together with the osteoprotegerin mRNA modulation. Moreover, in these cells, the increase of the protein expression of OPN and ALP was also demonstrated. These results demonstrate bone commitment of hMSCs through a noninvasive and biocompatible differentiating physical agent treatment and highlight possible applications in new regenerative medicine protocols.