Conjugated linoleic acid (CLA) is a bioactive compound known for its anti-inflammatory, anti-carcinogenic, and metabolic effects, with growing interest in its role in supporting bone health. Preclinical studies, particularly those involving the t10c12 isomer, have shown that CLA can enhance bone mineral density (BMD) by enhancing bone formation and reducing bone resorption, indicating its potential as a therapeutic agent to improve bone health. However, clinical trials have yielded inconsistent results, underscoring the difficulty in translating animal model successes to human applications. A major challenge is CLA's low water solubility, poor absorption, and limited bioavailability, which restrict its therapeutic effectiveness. To address these issues, nanoparticle-based delivery systems have been proposed to improve its solubility, stability, and resistance to oxidative damage, thereby enhancing its bioactivity. Recent studies also suggest that electrical stimulation can stimulate bone regeneration by promoting bone cell proliferation, differentiation, and adherence to scaffolds. This review explores the combined use of CLA supplementation and electrical stimulation as a novel approach to improving bone health, particularly in osteoporosis management. By integrating CLA's biological effects with the regenerative potential of electrical stimulation, this multimodal strategy offers a promising method for enhancing bone restoration, with significant implications for clinical applications in bone health.

Biophysical stimuli such as alternating electrical fields can mimic endogenous electrical potentials and currents in natural bone. This can help to improve the healing and reconstruction of bone tissue. However, little is known about the combined influence of biomaterials and alternating electric fields on bone cells. Therefore, this study aimed to investigate the impact of both, biomaterials and alternating electric fields, on osteoblast as well as osteoclast differentiation. Initially, either RAW 264.7 or MC3T3-E1 cells were seeded on Ti6Al4V substrates as a load-bearing implant material, modified with biomimetic calcium phosphate (BCP), or uncoated as a reference. The cells were stimulated towards osteoclastic and osteoblastic differentiation via respective growth factors. The effects of BCP substrate modification on cell differentiation were examined after 7 days for RAW 264.7 and after 14 days for MC3T3-E1 cells. In a further series of tests, either RAW 264.7 or MC3T3-E1 cells were seeded on BCP-modified Ti6Al4V substrates, stimulated towards differentiation using growth factors, and further electrically stimulated via alternating electric fields of different voltages and frequencies. In parallel to the first test series RAW 264.7 and MC3T3-E1 cells were stimulated for 7 and 14 days, respectively. Cell morphology was examined via scanning electron microscopy. Cell viabilities were assessed via WST-8 assay. Electrically stimulated MC3T3-E1 cell orientation was evaluated based on fluorescence microscopy images. Marker genes were examined via qPCR. While BCP increased osteoclast-specific gene expression, it had the opposite effect on osteoblast-related genes compared to respective cells seeded on uncoated Ti6Al4V substrates. ES with different parameters showed a broad cellular response due to electrocoupling. While cell viability assessments and gene expression analyses showed clear differences between ES samples and unstimulated controls, only minor cell morphology and orientation differences were observed. Furthermore, there was no clear trend towards a dominant influence of either voltage or frequency as control parameters. Further studies were initiated to investigate the underlying intracellular mechanisms targeted by ES. This work provides an introduction to the targeted control of cellular processes using defined electric fields. The optimization of voltage and frequency could provide therapeutic windows to control specific cellular functions and potentially improve bone regeneration and remodeling processes.

In the repair of large bone defects, loss of the periosteum can result in diminished osteoinductive activity, nonunion, and incomplete regeneration of the bone structure, ultimately compromising the efficiency of bone regeneration. Therefore, the research and development of tissue-engineered periosteum which can replace the periosteum function has become the focus of current research. The functionalized electrospinning periosteum is expected to mimic the natural periosteum and enhance bone repair processes more effectively. This review explores the construction strategies for functionalized electrospun periosteum from the following perspectives: ⅰ) bioactive factor modification (bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) etc.), ⅱ) inorganic compound modification, ⅲ) drug modification, ⅳ) artificial periosteum in response to physical stimuli. Furthermore, the construction of artificial periosteum through electrospinning, in conjunction with other strategies, is also analyzed. Finally, the current challenges and prospects for the development of electrospinning periosteum are also discussed.

Electrical stimulation has been used with tissue engineering-based models to develop three-dimensional (3D), dynamic, research models that are more physiologically relevant than static two-dimensional (2D) cultures. For bone tissue, the effect of electrical stimulation has focused on promoting healing and regeneration of tissue to prevent bone loss. However, electrical stimulation can also potentially affect mature bone parenchymal cells such as osteoblasts to guide bone formation and the secretion of paracrine or endocrine factors. Due to a lack of physiologically relevant models, these phenomena have not been studied in detail. In vitro electrical stimulation models can be useful for gaining an understanding of bone physiology and its effects on paracrine tissues under different physiological and pathological conditions. Here, we use a 3D, dynamic, in vitro model of bone to study the effects of electrical stimulation conditions on protein and gene expression of SaOS-2 human osteosarcoma osteoblast-like cells. We show that different stimulation regimens, including different frequencies, exposure times, and stimulation patterns, can have different effects on the expression and secretion of the osteoblastic markers alkaline phosphatase and osteocalcin. These results reveal that electrical stimulation can potentially be used to guide osteoblast gene and protein expression.

Bone defects repair continues to be a significant challenge facing the world. Biological scaffolds, bioactive molecules, and cells are the three major elements of bone tissue engineering, which have been widely used in bone regeneration therapy, especially with the rise of bioactive molecules in recent years. According to their physical properties, they can be divided into force, magnetic field (MF), electric field (EF), ultrasonic wave, light, heat, etc. However, the transmission of bioactive molecules has obvious shortcomings that hinder the development of the tissue-rearing process. This paper reviews the mechanism of physical signal induction in bone tissue engineering in recent years. It summarizes the application strategies of physical signal in bone tissue engineering, including biomaterial designs, physical signal loading strategies and related pathways. Finally, the ongoing challenges and prospects for the future are discussed.

Neuronal differentiation and maturation are crucial for developing research models and therapeutic applications. Brain-derived neurotrophic factor (BDNF) is a widely used biochemical stimulus for promoting neuronal maturation. However, the broad effects of biochemical stimuli on multiple cellular functions limit their applicability in both in vitro models and clinical settings. Electrical stimulation (ES) offers a promising physical method to control cell fate and function, but it is hampered by lack of standard and optimised protocols. In this study, we demonstrate that ES outperforms BDNF in promoting neuronal maturation in human neuroblastoma SH-SY5Y. Additionally, we address the question regarding which ES parameters regulate biological responses. The neuronal differentiation and maturation of SH-SY5Y cells were tested under several pulsed ES regimes. We identified accumulated charge and effective electric field time as novel criteria for determining optimal ES regimes. ES parameters were obtained using electrochemical characterisation and equivalent circuit modelling. Our findings show that neuronal maturation in SH-SY5Y cells correlates with the amount of accumulated charge during ES. Higher charge accumulation (~ 50 mC/h) significantly promotes extensive neurite outgrowth and ramification, and enhances the expression of synaptophysin, yielding effects exceeding those of BDNF. In contrast, fewer charge injection to the culture (~ 0.1 mC/h) minimally induces maturation but significantly increases cell proliferation. Moreover, ES altered the concentration and protein cargo of secreted extracellular vesicles (EV). ES with large enough accumulated charge significantly enriched EV proteome associated with neural development and function. These results demonstrate that each ES regime induces distinct cellular responses. Increased accumulated charge facilitates the development of complex neuronal morphologies and axonal ramification, outperforming exogenous neurotrophic factors. Controlled ES methods are immediately applicable in creating mature neuronal cultures in vitro with minimal chemical intervention.

Bone injuries and diseases are associated with profound changes in the biophysical properties of living bone tissues, particularly their electrical and mechanical properties. The biophysical properties of healthy bone are attributed to the complex network of interactions between its various cell types (i.e., osteocytes, osteoclast, immune cells and vascular endothelial cells) with the surrounding extracellular matrix (ECM) against the backdrop of a myriad of biomechanical and bioelectrical stimuli arising from daily physical activities. Understanding the pathophysiological changes in bone biophysical properties is critical to developing new therapeutic strategies and novel scaffold biomaterials for orthopedic surgery and tissue engineering, as well as provides a basis for the application of various biophysical stimuli as therapeutic agents to restore the physiological microenvironment of injured/diseased bone tissue, to facilitate its repair and regeneration. These include mechanical, electrical, magnetic, thermal and ultrasound stimuli, which will be critically examined in this review. A significant advantage of utilizing such biophysical stimuli to facilitate bone healing is that these may be applied non-invasively with minimal damage to surrounding tissues, unlike conventional orthopedic surgical procedures. Furthermore, the effects of such biophysical stimuli can be localized specifically at the bone defect site, unlike drugs or growth factors that tend to diffuse away after delivery, which may result in detrimental side effects at ectopic sites.

The repair of critical-sized bone defects represents significant clinical challenge. An alternative approach is the use of 3D composite scaffolds to support bone regeneration. Hydroxyapatite (HA) and tri-calcium phosphate (β-TCP), combined with polycaprolactone (PCL), offer promising mechanical resistance and biocompatibility. Bioelectrical stimulation (ES) at physiological levels is proposed to reestablishes tissue bioeletrocity and modulates cell signaling communication, such as the BMP/TGF-β and the RANK/RANK-L/OPG pathways. This study aimed to evaluate the use HA/TCP scaffolds and ES therapy for bone regeneration and their impact on the TGF-β/BMP pathway, alongside their relationship with the RANK/RANKL/OPG pathway in critical bone defects. The scaffolds were implanted at the bone defect in animal model (calvarial bone) and the area was subjected to ES application twice a week at 10 µA intensity of current for 5 min each session. Samples were collected for histomorphometry, immunohistochemistry, and molecular analysis. The TGF-β/BMP pathway study showed the HA/TCP+ES group increased BMP-7 gene expression at 30 and 60 days, and also greater endothelial vascular formation. Moreover, the HA/TCP and HA/TCP+ES groups exhibited a bone remodeling profile, indicated by RANKL/OPG ratio. HA/TCP scaffolds with ES enhanced vascular formation and mineralization initially, while modulation of the BMP/TGF pathway maintained bone homeostasis, controlling resorption via ES with HA/TCP.

Traditional bioreactor systems involve the use of three-dimensional (3D) scaffolds or stem cell aggregates, limiting the accessibility to the production of cell-secreted biomolecules. Herein, we present the use a pulse electromagnetic fields (pEMFs)-assisted wave-motion bioreactor system for the dynamic and scalable culture of human bone marrow-derived mesenchymal stem cells (hBMSCs) with enhanced the secretion of various soluble factors with massive therapeutic potential. The present study investigated the influence of dynamic pEMF (D-pEMF) on the kinetic of hBMSCs. A 30-min exposure of pEMF (10V-1Hz, 5.82 G) with 35 oscillations per minute (OPM) rocking speed can induce the proliferation (1 × 105 → 4.5 × 105) of hBMSCs than static culture. Furthermore, the culture of hBMSCs in osteo-induction media revealed a greater enhancement of osteogenic transcription factors under the D-pEMF condition, suggesting that D-pEMF addition significantly boosted hBMSCs osteogenesis. Additionally, the RNA sequencing data revealed a significant shift in various osteogenic and signaling genes in the D-pEMF group, further suggesting their osteogenic capabilities. In this research, we demonstrated that the combined effect of wave and pEMF stimulation on hBMSCs allows rapid proliferation and induces osteogenic properties in the cells. Moreover, our study revealed that D-pEMF stimuli also induce ROS-scavenging properties in the cultured cells. This study also revealed a bioactive and cost-effective approach that enables the use of cells without using any expensive materials and avoids the possible risks associated with them post-implantation.

Tissue repair and reconstruction are a clinical difficulty. Bioelectricity has been identified as a critical factor in supporting tissue and cell viability during the repair process, presenting substantial potential for clinical application. This review delves into various sources of electrical stimulation and identifies appropriate electrode materials for clinical use. It also highlights the biological mechanisms of electrical stimulation at both the subcellular and cellular levels, elucidating how these interactions facilitate the repair and regeneration processes across different organs. Moreover, specific electrode materials and stimulation sources are outlined, detailing their impact on cellular activity. The future development trends are projected from two perspectives: the optimization of equipment performance and the fulfillment of clinical demands, focusing on the feasibility, safety, and cost-effectiveness of technologies.

Bacterial infection and insufficient osteogenic activity are the main causes of orthopedic implant failure. Conventional surface modification methods are difficult to meet the requirements for long-term implant placement. In order to better regulate the function of implant surfaces, especially to improve both the antibacterial and osteogenic activity, external stimuli-responsive (ESR) strategies have been employed for the surface modification of orthopedic implants. External stimuli act as "smart switches" to regulate the surface interactions with bacteria and cells. The balance between antibacterial and osteogenic capabilities of implant surfaces can be achieved through these specific ESR manifestations, including temperature changes, reactive oxygen species production, controlled release of bioactive molecules, controlled release of functional ions, etc. This Review summarizes the recent progress on different ESR strategies (based on light, ultrasound, electric, and magnetic fields) that can effectively balance antibacterial performance and osteogenic capability of orthopedic implants. Furthermore, the current limitations and challenges of ESR strategies for surface modification of orthopedic implants as well as future development direction are also discussed.

Infertility was reported in approximately 15% of all heterozygous couples, with the male factor accounting for nearly half of the cases. This typically occurs due to low sperm production, sperm dysfunction, and sperm delivery obstruction. In this randomized controlled single-blind clinical trial, 90 infertile male subjects diagnosed with oligospermia, hypospermia, asthenozoospermia, or necrozoospermia were recruited. Semen samples were obtained with the masturbation method and an assessment of semen volume, sperm count, and motility was performed. Five milliamps of electrical shock was delivered to the participants through the fertility improvement device. Semen analysis was collected 4 months post-intervention from all subjects. Data were collected and an analysis of pre- and post-intervention results was performed. There was an improvement in the count, volume, and motility of the patient's sperm after electrical shock treatment compared with the control group. By using the analysis of variance (ANOVA) test, there were statistically significant differences between the first and the second seminal analysis results (<.05). All other results were found to be independently correlated. This study demonstrated that using a painless, convenient at-home device, which is designed to contain all the testis tissue as a cup and then extend to include the scrotal roots reaching the penile root to include the epididymis, could significantly improve sperm motility and count. This device can be utilized to tackle the significant issue of infertility in a cost-effective, safe, and efficacious manner. An ultrasound was done before and after using the device as well as years after with no changes noted.Clinical Trial's Registration Number: NCT04173052.

Recent interest in tissue engineering (TE) has focused on electrically conductive biomaterials. This has been inspired by the characteristics of the cells' microenvironment where signalling is supported by electrical stimulation. Numerous studies have demonstrated the positive influence of electrical stimulation on cell excitation to proliferate, differentiate, and deposit extracellular matrix. Even without external electrical stimulation, research shows that electrically active scaffolds can improve tissue regeneration capacity. Tissues like bone, muscle, and neural contain electrically excitable cells that respond to electrical cues provided by implanted biomaterials. To introduce an electrical pathway, TE scaffolds can incorporate conductive polymers, metallic nanoparticles, and ceramic nanostructures. However, these materials often do not meet implantation criteria, such as maintaining mechanical durability and degradation characteristics, making them unsuitable as scaffold matrices. Instead, depositing conductive layers on TE scaffolds has shown promise as an efficient alternative to creating electrically conductive structures. A stratified scaffold with an electroactive surface synergistically excites the cells through active top-pathway, with/without electrical stimulation, providing an ideal matrix for cell growth, proliferation, and tissue deposition. Additionally, these conductive coatings can be enriched with bioactive or pharmaceutical components to enhance the scaffold's biomedical performance. This review covers recent developments in electrically active biomedical coatings for TE. The physicochemical and biological properties of conductive coating materials, including polymers (polypyrrole, polyaniline and PEDOT:PSS), metallic nanoparticles (gold, silver) and inorganic (ceramic) particles (carbon nanotubes, graphene-based materials and Mxenes) are examined. Each section explores the conductive coatings' deposition techniques, deposition parameters, conductivity ranges, deposit morphology, cell responses, and toxicity levels in detail. Furthermore, the applications of these conductive layers, primarily in bone, muscle, and neural TE are considered, and findings from in vitro and in vivo investigations are presented. STATEMENT OF SIGNIFICANCE: Tissue engineering (TE) scaffolds are crucial for human tissue replacement and acceleration of healing. Neural, muscle, bone, and skin tissues have electrically excitable cells, and their regeneration can be enhanced by electrically conductive scaffolds. However, standalone conductive materials often fall short for TE applications. An effective approach involves coating scaffolds with a conductive layer, finely tuning surface properties while leveraging the scaffold's innate biological and physical support. Further enhancement is achieved by modifying the conductive layer with pharmaceutical components. This review explores the under-reviewed topic of conductive coatings in tissue engineering, introducing conductive biomaterial coatings and analyzing their biological interactions. It provides insights into enhancing scaffold functionality for tissue regeneration, bridging a critical gap in current literature.

Microcarrier applications have made great advances in tissue engineering in recent years, which can load cells, drugs, and bioactive factors. These microcarriers can be minimally injected into the defect to help reconstruct a good microenvironment for tissue repair. In order to achieve more ideal performance and face more complex tissue damage, an increasing amount of effort has been focused on microcarriers that can actively respond to external stimuli. These microcarriers have the functions of directional movement, targeted enrichment, material release control, and providing signals conducive to tissue repair. Given the high controllability and designability of magnetic and electroactive microcarriers, the research progress of these microcarriers is highlighted in this review. Their structure, function and applications, potential tissue repair mechanisms, and challenges are discussed. In summary, through the design with clinical translation ability, meaningful and comprehensive experimental characterization, and in-depth study and application of tissue repair mechanisms, stimuli-responsive microcarriers have great potential in tissue repair.

Piezoelectric effect produces an electrical signal when stress is applied to the bone. When the integrity of the bone is destroyed, the biopotential within the defect site is reduced and several physiological responses are initiated to facilitate healing. During the healing of the bone defect, the bioelectric potential returns to normal levels. Treatment of fractures that exceed innate regenerative capacity or exhibit delayed healing requires surgical intervention for bone reconstruction. For bone defects that cannot heal on their own, exogenous electric fields are used to assist in treatment. This paper reviews the effects of exogenous electrical stimulation on bone healing, including osteogenesis, angiogenesis, reduction in inflammation and effects on the peripheral nervous system. This paper also reviews novel electrical stimulation methods, such as small power supplies and nanogenerators, that have emerged in recent years. Finally, the challenges and future trends of using electrical stimulation therapy for accelerating bone healing are discussed.

Transforming growth factor beta-1 (TGFβ-1) and connective tissue growth factor (CTGF) are upregulated in the implanted human cochlea.

Cochlear implantation can lead to insertion trauma and intracochlear new tissue formation, which can detrimentally affect implant performance. TGFβ-1 and CTGF are profibrotic proteins implicated in various pathologic conditions, but little is known about their role in the cochlea. The present study aimed to characterize the expression of these proteins in the human implanted cochlea.

Archival human temporal bones (HTB) acquired from 12 patients with previous CI and histopathological evidence of new tissue formation as well as surgical samples of human intracochlear scar tissue surrounding the explanted CI were used in this study. Histopathologic analysis of fibrosis and osteoneogenesis was conducted using H&E. Protein expression was characterized using immunofluorescence. RNA expression from surgical specimens of fibrotic tissue surrounding the CI was quantified using qRT-PCR.

TGFβ-1 and CTGF protein expressions were upregulated in the areas of fibrosis and osteoneogenesis surrounding the CI HTB. Similarly, surgical samples demonstrated upregulation of protein and mRNA expression of TGFβ-1 and mild upregulation of CTGF compared with control. TGFβ-1 was expressed diffusely within the fibrous capsule, whereas CTGF was expressed in the thickened portion toward the modiolus and the fibrosis-osteoneogensis junction.

To our knowledge, this is the first study to demonstrate increased expression of TGFβ-1 and CTGF in the human implanted cochlea and may provide better understanding of the mechanism behind this pathogenic process to better develop future mitigating interventions.

Gelatin methacryloyl (GelMA) hydrogels is a widely used bioink because of its good biological properties and tunable physicochemical properties, which has been widely used in a variety of tissue engineering and tissue regeneration. However, pure GelMA is limited by the weak mechanical strength and the lack of continuous osteogenic induction environment, which is difficult to meet the needs of bone repair. Moreover, GelMA hydrogels are unable to respond to complex stimuli and therefore are unable to adapt to physiological and pathological microenvironments. This review focused on the functionalization strategies of GelMA hydrogel based bioinks for bone regeneration. The synthesis process of GelMA hydrogel was described in details, and various functional methods to meet the requirements of bone regeneration, including mechanical strength, porosity, vascularization, osteogenic differentiation, and immunoregulation for patient specific repair, etc. In addition, the response strategies of smart GelMA-based bioinks to external physical stimulation and internal pathological microenvironment stimulation, as well as the functionalization strategies of GelMA hydrogel to achieve both disease treatment and bone regeneration in the presence of various common diseases (such as inflammation, infection, tumor) are also briefly reviewed. Finally, we emphasized the current challenges and possible exploration directions of GelMA-based bioinks for bone regeneration.

Introduction: Electrical stimulation has been used as a promising approach in bone repair for several decades. However, the therapeutic use is hampered by inconsistent results due to a lack of standardized application protocols. Recently, electrical stimulation has been considered for the improvement of the osseointegration of dental and endoprosthetic implants. Methods: In a pilot study, the suitability of a specifically developed device for electrical stimulation in situ was assessed. Here, the impact of alternating electric fields on implant osseointegration was tested in a gap model using New Zealand White Rabbits. Stimulation parameters were transmitted to the device via a radio transceiver, thus allowing for real-time monitoring and, if required, variations of stimulation parameters. The effect of electrical stimulation on implant osseointegration was quantified by the bone-implant contact (BIC) assessed by histomorphometric (2D) and µCT (3D) analysis. Results: Direct stimulation with an alternating electric potential of 150 mV and 20 Hz for three times a day (45 min per unit) resulted in improved osseointegration of the triangular titanium implants in the tibiae of the rabbits. The ratio of bone area in histomorphometry (2D analysis) and bone volume (3D analysis) around the implant were significantly increased after stimulation compared to the untreated controls at sacrifice 84 days after implantation. Conclusion: The developed experimental design of an electrical stimulation system, which was directly located in the defect zone of rabbit tibiae, provided feedback regarding the integrity of the stimulation device throughout an experiment and would allow variations in the stimulation parameters in future studies. Within this study, electrical stimulation resulted in enhanced implant osseointegration. However, direct electrical stimulation of bone tissue requires the definition of dose-response curves and optimal duration of treatment, which should be the subject of subsequent studies.

Bone is remodeled through a dynamic process facilitated by biophysical cues that support cellular signaling. In healthy bone, signaling pathways are regulated by cells and the extracellular matrix and transmitted via electrical synapses. To this end, combining electrical stimulation (ES) with conductive scaffolding is a promising approach for repairing damaged bone tissue. Therefore, "smart" biomaterials that can provide multifunctionality and facilitate the transfer of electrical cues directly to cells have become increasingly more studied in bone tissue engineering. Herein, 3D-printed electrically conductive composite scaffolds consisting of demineralized bone matrix (DBM) and polycaprolactone (PCL), in combination with ES, for bone regeneration were evaluated for the first time. The conductive composite scaffolds were fabricated and characterized by evaluating mechanical, surface, and electrical properties. The DBM/PCL composites exhibited a higher compressive modulus (107.2 MPa) than that of pristine PCL (62.02 MPa), as well as improved surface properties (i.e., roughness). Scaffold electrical properties were also tuned, with sheet resistance values as low as 4.77 × 105 Ω/sq for our experimental coating of the highest dilution (i.e., 20%). Furthermore, the biocompatibility and osteogenic potential of the conductive composite scaffolds were tested using human mesenchymal stromal cells (hMSCs) both with and without exogenous ES (100 mV/mm for 5 min/day four times/week). In conjunction with ES, the osteogenic differentiation of hMSCs grown on conductive DBM/PCL composite scaffolds was significantly enhanced when compared to those cultured on PCL-only and nonconductive DBM/PCL control scaffolds, as determined through xylenol orange mineral staining and osteogenic protein analysis. Overall, these promising results suggest the potential of this approach for the development of biomimetic hybrid scaffolds for bone tissue engineering applications.

Electrical Stimulation (ES) is an excellent technique to promote the differentiation and proliferation of precursor cells towards new linages. We studied the effects of alternated current (AC) electrical stimulation on mouse neuroblastoma cell line N2A differentiation towards neuronal tissue. We developed an ES system with electronics, culture-ware with electrodes and encapsulation, designing a setup and protocols for stimulation. The applied ES were biphasic voltage pulses, with programmable amplitudes and frequencies. After ES, N2A cells were analyzed using microscope images. Differentiated and non-differentiated cells were counted. Results show that ES facilitates the differentiation of N2A cells in N2A. The best values for the applied electric field (pulse biphasic signals) in terms of amplitude and frequency, are around 250-500 mV/mm and 100 Hz, these conditions are proposed as the most suitable for future ES of N2A.

The regenerative capacity of bone is indispensable for growth, given that accidental injury is almost inevitable. Bone regenerative capacity is relevant for the aging population globally and for the repair of large bone defects after osteotomy (e.g., following removal of malignant bone tumours). Among the many therapeutic modalities proposed to bone regeneration, electrical stimulation has attracted significant attention owing to its economic convenience and exceptional curative effects, and various electroactive biomaterials have emerged. This review summarizes the current knowledge and progress regarding electrical stimulation strategies for improving bone repair. Such strategies range from traditional methods of delivering electrical stimulation via electroconductive materials using external power sources to self-powered biomaterials, such as piezoelectric materials and nanogenerators. Electrical stimulation and osteogenesis are related via bone piezoelectricity. This review examines cell behaviour and the potential mechanisms of electrostimulation via electroactive biomaterials in bone healing, aiming to provide new insights regarding the mechanisms of bone regeneration using electroactive biomaterials.

This review examines the roles of electroactive biomaterials in rehabilitating the electrical microenvironment to facilitate bone regeneration, addressing current progress in electrical biomaterials and the mechanisms whereby electrical cues mediate bone regeneration. Interactions between osteogenesis-related cells and electroactive biomaterials are summarized, leading to proposals regarding the use of electrical stimulation-based therapies to accelerate bone healing.

Understanding the molecular and physical complexity of the tissue microenvironment (TiME) in the context of its spatiotemporal organization has remained an enduring challenge. Recent advances in engineering and data science are now promising the ability to study the structure, functions, and dynamics of the TiME in unprecedented detail; however, many advances still occur in silos that rarely integrate information to study the TiME in its full detail. This review provides an integrative overview of the engineering principles underlying chemical, optical, electrical, mechanical, and computational science to probe, sense, model, and fabricate the TiME. In individual sections, we first summarize the underlying principles, capabilities, and scope of emerging technologies, the breakthrough discoveries enabled by each technology and recent, promising innovations. We provide perspectives on the potential of these advances in answering critical questions about the TiME and its role in various disease and developmental processes. Finally, we present an integrative view that appreciates the major scientific and educational aspects in the study of the TiME.

In this work, we propose a simple, reliable, and versatile strategy to create 3D electroconductive scaffolds suitable for bone tissue engineering (TE) applications with electrical stimulation (ES). The proposed scaffolds are made of 3D-extruded poly(ε-caprolactone) (PCL), subjected to alkaline treatment, and of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), anchored to PCL with one of two different crosslinkers: (3-glycidyloxypropyl)trimethoxysilane (GOPS) and divinyl sulfone (DVS). Both cross-linkers allowed the formation of a homogenous and continuous coating of PEDOT:PSS to PCL. We show that these PEDOT:PSS coatings are electroconductive (11.3-20.1 S cm-1), stable (up to 21 days in saline solution), and allow the immobilization of gelatin (Gel) to further improve bioactivity. In vitro mineralization of the corresponding 3D conductive scaffolds was greatly enhanced (GOPS(NaOH)-Gel - 3.1 fold, DVS(NaOH)-Gel - 2.0 fold) and cell colonization and proliferation were the highest for the DVS(NaOH)-Gel scaffold. In silico modelling of ES application in DVS(NaOH)-Gel scaffolds indicates that the electrical field distribution is homogeneous, which reduces the probability of formation of faradaic products. Osteogenic differentiation of human bone marrow derived mesenchymal stem/stromal cells (hBM-MSCs) was performed under ES. Importantly, our results clearly demonstrated a synergistic effect of scaffold electroconductivity and ES on the enhancement of MSC osteogenic differentiation, particularly on cell-secreted calcium deposition and the upregulation of osteogenic gene markers such as COL I, OC and CACNA1C. These scaffolds hold promise for future clinical applications, including manufacturing of personalized bone TE grafts for transplantation with enhanced maturation/functionality or bioelectronic devices.

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Piezoelectric scaffolds have been recently developed to explore their potential to enhance the bone regeneration process using the concept of piezoelectricity, which also inherently occurs in bone. In addition to providing mechanical support during bone healing, with a suitable design, they are supposed to produce electrical signals that ought to favor the cell responses. In this study, using finite element analysis (FEA), a piezoelectric scaffold was designed with the aim of providing favorable ranges of mechanical and electrical signals when implanted in a large bone defect in a large animal model, so that it could inform future pre-clinical studies. A parametric analysis was then performed to evaluate the effect of the scaffold design parameters with regard to the piezoelectric behavior of the scaffold. The designed scaffold consisted of a porous strut-like structure with piezoelectric patches covering its free surfaces within the scaffold pores. The results showed that titanium or PCL for the scaffold and barium titanate (BT) for the piezoelectric patches are a promising material combination to generate favorable ranges of voltage, as reported in experimental studies. Furthermore, the analysis of variance showed the thickness of the piezoelectric patches to be the most influential geometrical parameter on the generation of electrical signals in the scaffold. This study shows the potential of computer tools for the optimization of scaffold designs and suggests that patches of piezoelectric material, attached to the scaffold surfaces, can deliver favorable ranges of electrical stimuli to the cells that might promote bone regeneration.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Bone defect repair remains a critical challenge in current orthopedic clinical practice, as the available therapeutic strategies only offer suboptimal outcomes. Therefore, bone tissue engineering (BTE) approaches, involving the development of biomimetic implantable scaffolds combined with osteoprogenitor cells and native-like physical stimuli, are gaining widespread interest. Electrical stimulation (ES)-based therapies have been found to actively promote bone growth and osteogenesis in both in vivo and in vitro settings. Thus, the combination of electroactive scaffolds comprising conductive biomaterials and ES holds significant promise in improving the effectiveness of BTE for clinical applications. The aim of this study was to develop electroconductive polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning, which are capable of emulating the native tissue's fibrous extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The resulting nanofibers were successfully functionalized with apatite-like structures to mimic the inorganic phase of the bone ECM. The conductive electrospun scaffolds presented nanoscale fiber diameters akin to those of collagen fibrils and displayed bone-like conductivity. PEDOT:PSS incorporation was shown to significantly promote scaffold mineralization in vitro. The mineralized electroconductive nanofibers demonstrated improved biological performance as observed by the significantly enhanced proliferation of both human osteoblast-like MG-63 cells and human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs). Moreover, mineralized PAN/PEDOT:PSS nanofibers up-regulated bone marker genes expression levels of hBM-MSCs undergoing osteogenic differentiation, highlighting their potential as electroactive biomimetic BTE scaffolds for innovative bone defect repair strategies.

Osteoporotic-related fractures are among the leading causes of chronic disease morbidity in Europe and in the US. While a significant percentage of fractures can be repaired naturally, in delayed-union and non-union fractures surgical intervention is necessary for proper bone regeneration. Given the current lack of optimized clinical techniques to adequately address this issue, bone tissue engineering (BTE) strategies focusing on the development of scaffolds for temporarily replacing damaged bone and supporting its regeneration process have been gaining interest. The piezoelectric properties of bone, which have an important role in tissue homeostasis and regeneration, have been frequently neglected in the design of BTE scaffolds. Therefore, in this study, we developed novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) nanofibers via electrospinning capable of replicating the tissue's fibrous extracellular matrix (ECM) composition and native piezoelectric properties. The developed PVDF-TrFE/HAp nanofibers had biomimetic collagen fibril-like diameters, as well as enhanced piezoelectric and surface properties, which translated into a better capacity to assist the mineralization process and cell proliferation. The biological cues provided by the HAp nanoparticles enhanced the osteogenic differentiation of seeded human mesenchymal stem/stromal cells (MSCs) as observed by the increased ALP activity, cell-secreted calcium deposition and osteogenic gene expression levels observed for the HAp-containing fibers. Overall, our findings describe the potential of combining PVDF-TrFE and HAp for developing electroactive and osteoinductive nanofibers capable of supporting bone tissue regeneration.

Morphoceuticals are a new class of interventions that target the setpoints of anatomical homeostasis for efficient, modular control of growth and form. Here, we focus on a subclass: electroceuticals, which specifically target the cellular bioelectrical interface. Cellular collectives in all tissues form bioelectrical networks via ion channels and gap junctions that process morphogenetic information, controlling gene expression and allowing cell networks to adaptively and dynamically control growth and pattern formation. Recent progress in understanding this physiological control system, including predictive computational models, suggests that targeting bioelectrical interfaces can control embryogenesis and maintain shape against injury, senescence and tumorigenesis. We propose a roadmap for drug discovery focused on manipulating endogenous bioelectric signaling for regenerative medicine, cancer suppression and antiaging therapeutics. Teaser: By taking advantage of the native problem-solving competencies of cells and tissues, a new kind of top-down approach to biomedicine becomes possible. Bioelectricity offers an especially tractable interface for interventions targeting the software of life for regenerative medicine applications.

Various methods have been used to treat bone defects caused by genetic disorders, injury, or disease. Yet, there is still great need to develop alternative approaches to repair damaged bone tissue. Bones naturally exhibit piezoelectric potential, or the ability to convert mechanical stresses into electrical impulses. This phenomenon has been utilized clinically to enhance bone regeneration in conjunction with electrical stimulation (ES) therapies; however, oftentimes with critical-sized bone defects, the bioelectric potential at the site of injury is compromised, resulting in less desirable outcomes. In the present study, the potential of a 3D-printed conductive polymer blend to enhance bone formation through restoration of the bioelectrical microenvironment was evaluated. A commercially available 3D printer was used to create circular, thin-film scaffolds consisting of either polylactide (PLA) or a conductive PLA (CPLA) composite. Preosteoblast cells were seeded onto the scaffolds and subjected to direct current ES via a purpose-built cell culture chamber. It was found that CPLA scaffolds had no adverse effects on cell viability, proliferation or differentiation when compared with control scaffolds. The addition of ES, however, resulted in a significant increase in the expression of osteocalcin, a protein indicative of osteoblast maturation, after 14 days of culture. Furthermore, xylenol orange staining also showed the presence of increased mineralized calcium nodules in cultures undergoing stimulation. This study demonstrates the potential for low-cost, conductive scaffolding materials to support cell viability and enhance in vitro mineralization in conjunction with ES.

Biomaterial properties, such as surface roughness, morphology, stiffness, conductivity, and chemistry, significantly influence a cell's ability to sense and adhere to its surface and regulate cell functioning. Understanding how biomaterial properties govern changes in cellular function is one of the fundamental goals of tissue engineering. Still, no generalized rule is established to predict cellular processes (adhesion, spreading, growth and differentiation) on biomaterial surfaces. A few studies have highlighted that cells sense biomaterial properties at multiple length scales and regulate various intracellular biochemical processes like cytoskeleton organization, gene regulation, and receptor expression to influence cell function. However, recent studies have found cellular metabolism as another critical aspect of cellular processes that regulate cell behavior, co-relating metabolism to cellular functions like adhesion, proliferation, and differentiation. Now researchers have started to uncover previously overlooked factors on how biomaterial properties govern changes in cellular functions mediated through metabolism. This review highlights how different physiochemical properties of scaffolds designed from different biomaterials influence cell metabolism. The review also discusses the role of metabolism change in cellular functions and cell behavior in the context of bone tissue engineering. It also emphasizes the importance of cell metabolism as a missing link between the cellular behavior and physicochemical properties of scaffolds and serves as a guiding principle for designing scaffolds for tissue engineering.

Developing scalable electrical stimulating platforms for cell and tissue engineering applications is limited by external power source dependency, wetting resistance, microscale size requirements, and suitable flexibility. Here, a versatile and scalable platform is developed to enable tunable electrical stimulation for biological applications by harnessing the giant magnetoelastic effect in soft systems, converting gentle air pressure (100-400 kPa) to yield a current of up to 10.5 mA and a voltage of 9.5 mV. The platform can be easily manufactured and scaled up for integration in multiwell magnetoelastic plates via 3D printing. The authors demonstrate that the electrical stimulation generated by this platform enhances the conversion of fibroblasts into neurons up to 2-fold (104%) and subsequent neuronal maturation up to 3-fold (251%). This easily configurable electrical stimulation device has broad applications in high throughput organ-on-a-chip systems, and paves the way for future development of neural engineering, including cellular therapy via implantable self-powered electrical stimulation devices.

Direct current electrical stimulation may serve as a promising nonpharmacological adjunct promoting osteogenesis and fusion. The aim of this study was to evaluate the utility of electroactive spine instrumentation in the focal delivery of therapeutic electrical stimulation to enhance lumbar bone formation and interbody fusion.

A finite element model of adult human lumbar spine (L4-L5) instrumented with single-level electroactive pedicle screws was simulated. Direct current electrical stimulation was routed through anodized electroactive pedicle screws to target regions of fusion. The electrical fields generated by electroactive pedicle screws were evaluated in various tissue compartments including isotropic tissue volumes, cortical, and trabecular bone. Electrical field distributions at various stimulation amplitudes (20-100 µA) and pedicle screw anodization patterns were analyzed in target regions of fusion (eg, intervertebral disc space, vertebral body, and pedicles).

Electrical stimulation with electroactive pedicle screws at various stimulation amplitudes and anodization patterns enabled modulation of spatial distribution and intensity of electric fields within the target regions of lumbar spine. Anodized screws (50%) vs unanodized screws (0%) induced high-amplitude electric fields within the intervertebral disc space and vertebral body but negligible electric fields in spinal canal. Direct current electrical stimulation via anodized screws induced electrical fields, at therapeutic threshold of >1 mV/cm, sufficient for osteoinduction within the target interbody region.

Selective anodization of electroactive pedicle screws may enable focal delivery of therapeutic electrical stimulation in the target regions in human lumbar spine. This study warrants preclinical and clinical testing of integrated electroactive system in inducing target lumbar fusion in vivo.

The findings of this study provide a foundation for clinically investigating electroactive intrumentation to enhance spine fusion.

Critical bone defects are the most difficult challenges in the area of tissue repair. Polycaprolactone (PCL) scaffolds, associated with hydroxyapatite (HA) and tricalcium phosphate (TCP), are reported to have an enhanced bioactivity. Moreover, the use of electrical stimulation (ES) has overcome the lack of bioelectricity at the bone defect site and compensated the endogenous electrical signals. Such treatments could modulate cells and tissue signaling pathways. However, there is no study investigating the effects of ES and bioceramic composite scaffolds on bone tissue formation, particularly in the view of cell signaling pathway. This study aims to investigate the application of HA/TCP composite scaffolds and ES and their effects on the Wingless-related integration site (Wnt) pathway in critical bone repair. Critical bone defects (25 mm²) were performed in rats, which were divided into four groups: PCL, PCL + ES, HA/TCP and HA/TCP + ES. The scaffolds were grafted at the defect site and applied with the ES application twice a week using 10 µA of current for 5 min. Bone samples were collected for histomorphometry, immunohistochemistry and molecular analysis. At the Wnt canonical pathway, HA/TCP and HA/TCP + ES groups showed higher Wnt1 and β-catenin gene expression levels, especially HA/TCP. Moreover, HA/TCP + ES presented higher Runx2, Osterix and Bmp-2 levels. At the Wnt non-canonical pathway, HA/TCP group showed higher voltage-gated calcium channel (Vgcc), calmodulin-dependent protein kinase II, and Wnt5a genes expression, while HA/TCP + ES presented higher protein expression of VGCC and calmodulin (CaM) at the same period. The decrease in sclerostin and osteopontin genes expressions and the lower bone sialoprotein II in the HA/TCP + ES group may be related to the early bone remodeling. This study shows that the use of ES modulated the Wnt pathways and accelerated the osteogenesis with improved tissue maturation.

The combination of electrical stimulation (ES) and bone tissue engineering (BTE) has been successful in treatments of bone regeneration. This study evaluated the effects of ES combined with PCL + β-TCP 5% scaffolds obtained by rotary jet spinning (RJS) in the regeneration of bone defects in the calvaria of Wistar rats. We used 120 animals with induced bone defects divided into 4 groups (n = 30): (C) without treatment; (S) with PCL+ β-TCP 5% scaffold; (ES) treated with ES (10 μA/5 min); (ES + S) with PCL + β-TCP 5% scaffold. The ES occurred twice a week during the entire experimental period. Cell viability (in vitro: Days 3 and 7) and histomorphometric, histochemical, and immunohistochemical (in vivo; Days 30, 60, and 90) analysis were performed. In vitro, ES + S increased cell viability after Day 7 of incubation. In vivo, it was observed modulation of inflammatory cells in ES therapy, which also promoted blood vessels proliferation, and increase of collagen. Moreover, ES therapy played a role in osteogenesis by decreasing ligand kappa B nuclear factor-TNFSF11 (RANKL), increasing alkaline phosphatase (ALP), and decreasing the tartarate-resistant acid phosphatase. The combination of ES with RJS scaffolds may be a promising strategy for bone defects regeneration, since the therapy controlled inflammation, favored blood vessels proliferation, and osteogenesis, which are important processes in bone remodeling.

Aim: The purpose of the study was to identify the morphological features of reparative osteogenesis in the rats lower jaw under the conditions of using electrical stimulation.

Materials and Methods: An experiment was conducted on 24 mature male rats of the WAG population. Two groups were formed. Group 1 included 12 rats that were modeled with a perforated defect of the lower jaw body. Group 2 included 12 animals that were modeled with a perforated defect similar to group 1. In animals, a microdevice for electrical action was implanted subcutaneously in the neck area on the side of the simulated bone defect (a temporary Videx AG 4 battery; a constant sinusoidal electric current of an unchanging nature 1 milliampere, frequency 30 W). The negative electrode connected to the negative pole of the battery was in contact with the bone defect. The battery and electrode were insulated with plastic heat shrink material. Morphological and statistical methods were used.

Results: The positive effect of electrical stimulation on reparative osteogenesis was due to a decrease in the severity of hemodynamic disorders, activation of angiogenesis in granulation tissue, which was one of the components of the regenerate that filled the bone defect, matured and turned into connective tissue; stimulation of the proliferative potential of fibroblastic cells and cells with osteoblastic activity in granulation tissue; increasing the proliferative potential of osteoblastic elements of bone tissue bordering the cavity; stimulation of macrophage cells and processes of cleansing the bone cavity from fragments of a blood clot and alteratively changed tissues; formation of clusters of adipocytes in the loci of connective and granulation tissue of the regenerate; the process of metaplasia of connective tissue into bone tissue; an increase of the foci of hematopoiesis in the intertrabecular spaces of lamellar bone tissue.

Conclusions: A comprehensive clinical and experimental study conducted by the authors proved that electrical stimulation activates the reparative osteogenesis in the lower jaw, which occurs through direct osteogenesis and does not finish on the 28th day of the experiment.

Noninvasive electronic bone growth stimulators (EBGSs) have been in clinical use for decades. However, systematic reviews show inconsistent and limited clinical efficacy. Further, noninvasive EBGS studies in small animals, where the stimulation electrode is closer to the fracture site, have shown promising efficacy, which has not translated to large animals or humans. We propose that this is due to the weaker electric fields reaching the fracture site when scaling from small animals to large animals and humans. To address this gap, we measured the electric field strength reaching the bone during noninvasive EBGS therapy in human and sheep cadaver legs and in finite element method (FEM) models of human and sheep legs. During application of 1100 V/m with an external EBGS, only 21 V/m reached the fracture site in humans. Substantially weaker electric fields reached the fracture site during the later stages of healing and at increased bone depths. To augment the electric field strength reaching the fracture site during noninvasive EBGS therapy, we introduced the Injectrode, an injectable electrode that spans the distance between the bone and subcutaneous tissue. Our study lays the groundwork to improve the efficacy of noninvasive EBGSs by increasing the electric field strength reaching the fracture site.

Bone regeneration is a well-orchestrated process involving electrical, biochemical, and mechanical multiple physiological cues. Electrical signals play a vital role in the process of bone repair. The endogenous potential will spontaneously form on defect sites, guide the cell behaviors, and mediate bone healing when the bone fracture occurs. However, the mechanism on how the surface charges of implant potentially guides osteogenesis and osteoimmunology has not been clearly revealed yet. In this study, piezoelectric BaTiO₃/β-TCP (BTCP) ceramics are prepared by two-step sintering, and different surface charges are established by polarization. In addition, the cell osteogenesis and osteoimmunology of BMSCs and RAW264.7 on different surface charges were explored. The results showed that the piezoelectric constant d₃₃ of BTCP was controllable by adjusting the sintering temperature and rate. The polarized BTCP with a negative surface charge (BTCP-) promoted protein adsorption and BMSC extracellular Ca²⁺ influx. The attachment, spreading, migration, and osteogenic differentiation of BMSCs were enhanced on BTCP-. Additionally, the polarized BTCP ceramics with a positive surface charge (BTCP+) significantly inhibited M1 polarization of macrophages, affecting the expression of the M1 marker in macrophages and changing secretion of proinflammatory cytokines. It in turn enhanced osteogenic differentiation of BMSCs, suggesting that positive surface charges could modulate the bone immunoregulatory properties and shift the immune microenvironment to one that favored osteogenesis. The result provides an alternative method of synergistically modulating cellular immunity and the osteogenesis function and enhancing the bone regeneration by fabricating piezoelectric biomaterials with electrical signals.