Autor(es): Mendes, Maria C. ; Martins, Elisa A.G. ; Chernozem, Roman V. ; Chernozem, Polina V. ; Custódio, Catarina C. ; Surmenev, Roman A. ; Kholkin, Andrei L. ; Silva, Ana S. ; Mano, João F.
Data: 2025
Identificador Persistente: http://hdl.handle.net/10773/46076
Origem: RIA - Repositório Institucional da Universidade de Aveiro
Assunto(s): Core-shell nanoparticles; Magnetoelectric stimulation; Static and cyclic magnetic fields; 3D spheroids; Osteogenic differentiation
Magnetoelectric nanoparticles (MENPs), combining a magnetostrictive core with a piezoelectric shell, offer a promising route for remote-controlled biomedical applications by converting external magnetic fields into electric cues. However, the clinical translation of these materials remains limited due to the toxicity of high-performance piezoelectric materials, which typically contain lead. Previously, we developed lead-free MENPs comprising manganese ferrite oxide (MFO) core nanoparticles (NPs) coated with Ba0.85Ca0.15Zr0.1Ti0.9O3 (BCZT) piezoelectric shell (MFO@BCZT). While these nanotransducers exhibit robust magnetic responsiveness and piezoelectric performance comparable to lead-based ceramics, their role in producing in situ electrical cues to accelerate bone repair remains unexplored. Given the established role of electrical stimulation in bone remodeling, this study explores the potential of MFO@BCZT MENPs to promote the osteogenic differentiation of human adipose-derived stem cells (hASCs) after internalization, assembly into magnetized 3D spheroids, and subsequent embedding in gelatin methacryloyl hydrogels, to better recapitulate physiologically relevant microenvironments. Differentiation was assessed under static and cyclic magnetic fields (CMF) conditions and compared to spheroids containing bare MFO NPs and spheroids without NPs. Results revealed that MFO and MFO@BCZT NPs were cytocompatible; however, MFO@BCZT MENPs significantly enhanced osteogenic marker expression and mineral deposition compared to both controls, with CMF further amplifying these effects. Under CMF stimulation, MFO@BCZT MENPs produced a mineralized matrix with a calcium-to-phosphorus molar ratio of 1.67, aligning precisely with native bone apatite. Overall, by restoring the bioelectric properties of bone at the target region, this study position MFO@BCZT MENPs as a compelling platform for future smart bone therapies. Bone tissue is an essential part of the human body, providing structural support, protecting internal organs, and serving as a reservoir for essential minerals. Beyond its mechanical role, bone possesses intrinsic bioelectrical properties including dielectric, piezoelectric, pyroelectric, and ferroelectric properties, which all play a central role in its development, remodeling, and fracture healing.1 While these properties endow bone with a capacity for self-repair, critical-sized defects exceed this regenerative potential, requiring clinical intervention for proper repair.2–4 Common treatment strategies include bone fixation implants, autographs, allografts, and electrical stimulating therapies.2–6 However, each approach presents limitations: fixation devices may necessitate secondary surgeries; autografts can lead to donor-site morbidity; and allografts risk immune rejection and disease transmission.2 Electrical stimulation therapies, which involve the implantation of electrodes percutaneously or transcutaneously at the defect site and rely on an external power source or electromagnetic coil to deliver the appropriate electrical signal over time, introduce additional risks such as infection, skin irritation and thermal injury, electrical shock, and concerns regarding device biocompatibility and battery disposal.3,7 Understanding bone physiology has been relevant to develop solutions for bone repair, in particular for bone regeneration.8 Inspired by the electrophysiological properties of native bone, piezoelectric materials have attracted growing interest in biomedical applications due to their ability to deliver electrical stimuli to cells in response to applied mechanical stress (direct piezoelectricity).9,10 This unique property enables the creation of self-powered electrical systems favorable for clinical use, without the need for electrode implantation and any external electrical power source.11,12 Despite these advantages, the development of functional piezoelectric materials for biomedical applications faces two main challenges, namely the mechanical stimuli required to activate the piezoelectric effect, and the materials composition required to ensure biocompatibility, biodegradability, and a robust piezoelectric response.9,11,13 Although body motion can activate piezoelectric materials, physical activity is often limited postoperatively, failing to generate stress for piezoelectric responses.11–13 As an alternative, low-powered ultrasounds can be used to stimulate the piezoelectric materials and generate electrical signals.9 While ultrasounds offer a noninvasive approach with adjustable frequencies to minimize cell damage, ultrasound waves are subjected to refraction across tissues of varying densities, scattering at tissue interfaces, reflection, and absorption, ultimately leading to heat generation, which may be harmful to tissues with prolonged exposure.11,12,14 To overcome these challenges, magnetoelectric (ME) composite nanomaterials have emerged as a promising strategy for electrical stimulation, as they generate electrical charges in response to external magnetic fields (direct magnetoelectric effect), which, unlike ultrasound, penetrate deeply into the body without attenuation.7,15–18 ME materials are typically composed of a ferromagnetic and a piezoelectric phase coupled elastically.18–20 Under a low-frequency magnetic field, the ferromagnetic phase undergoes magnetostrictive strain, which is transferred to the piezoelectric phase, producing a localized electric field.21 The concept of ME nanoparticles (NPs) (MENPs) was first introduced by Yue et al in 2012, as a noninvasive brain stimulation method capable of crossing the blood–brain barrier (BBB) and restoring neuronal communication in Parkinson’s disease.22 Subsequent in vitro, ex vivo, and in vivo studies validated their safety, BBB penetration, magnetic targeting, and ability to trigger cellular biophysical and biochemical processes.15,16,19,23–25 Importantly, MENPs, typically smaller than 30 nm, have shown no cytotoxic effects in the brain or major organs (kidneys, lungs, liver, spleen), nor do they impair hepatic, renal, or neurobehavioral functions.26,27 Their small size also enables rapid clearance, further supporting their potential as safe and effective nanotherapeutics.27 Core–shell nanostructures represent one of the most extensively studied MENP configurations to date. Many reported systems use Fe3O4 NPs as the magnetostrictive core, as it is the only metal iron NPs approved by the FDA, and explore doping with Co²⁺, Ni²⁺, or Mn²⁺ to improve stability and performance, yielding CoFe₂O₄, NiFe₂O₄, or MnFe₂O₄ NPs, respectively.18,28 For the piezoelectric shell, a key design criterion is the d33 coefficient, which quantifies the amount of induced charge per unit of mechanical stress.29 While lead-based ceramics such as PZT (d₃₃ = 300–1000 pC/N) and PMN-PT (d₃₃ ≥800 pC/N) exhibit exceptional performance, their toxicity precludes biomedical use.3,18 Biocompatible candidates include barium titanate (BaTiO3, BT, d3 3= 190 pC/N) and its derivatives like sodium bismuth titanate (NBT, d33 = 100–400 pC/N), as well as polyvinylidene fluoride (PVDF, d33 = 22 pC/N) derivatives, though they exhibit lower d33 values which results in an reduced piezoelectric response.30,31 To overcome this challenge, we have previously developed a lead-free MENPs composed of MnFe₂O₄ (MFO) cores coated with a Ba0.85Ca0.15Zr0.1Ti0.9O3 (BCZT) piezoelectric shell (MFO@BCZT).32 MFO is a soft magnetic material characterized by high magnetic permeability, low coercivity, and high chemical stability. Their high permeability enables rapid magnetization even under low-intensity magnetic fields, while low coercivity ensures easy and reversible demagnetization. Mn2+ doping enhances chemical stability and biocompatibility due to the body's natural manganese metabolism.33–35 Meanwhile, the inclusion of BCZT provides a biocompatible shell with a d33 value of 620 pC/N, which is one of the highest coupling coefficients for lead-free piezoelectric materials available today, offering strong piezoelectric performance with excellent cytocompatibility.29,31 Leveraging on the synergy between MFO and BCZT, we hypothesized that MFO@BCZT MENPs can significantly enhance mechanical sensitivity and ME conversion under low intensity magnetic fields, supporting bone regeneration via in situ electrical stimulation. To closely replicate the in vivo environment, MFO@BCZT MENPs were internalized into human adipose-derived stem cells (hASCs) and subsequently assembled into 3D spheroids. Outcomes were assessed under static and cyclic magnetic field (CMF) exposure and compared to spheroids containing bare MFO NPs lacking the piezoelectric shell and spheroids devoid of magnetic functionality, demonstrating the superior performance of MFO@BCZT MENPs in promoting osteogenic differentiation via intracellular ME stimulation.
