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ln vitro assessment of immunomodulatory and osteogenic properties in 3D-Printed hydroxyapatite/barium titanate piezoelectric ceramic scaffolds
Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages. Abbreviations LPS Lipopolysaccharide ROS Reactive oxygen species ES Electrical stimulation BMDMs Bone marrow-derived macrophages NADPH Nicotinamide adenine dinucleotide phosphate G1P Glucose-1-phosphate G6P Glucose-6-phosphate F6P Fluctose-1-phosphate Ru5P Ribulose-5-phosphate OPEN
Piezoelectric, pyroelectric, and ferroelectric materials are considered unique biomedical materials due to their dielectric crystals and asymmetric centers that allow them to directly convert various primary forms of energy in the environment, such as sunlight, mechanical energy, and thermal energy, into secondary energy, such as electricity and chemical energy. These materials possess exceptional energy conversion ability and excellent catalytic properties, which have led to their widespread usage within biomedical fields. Numerous biomedical applications have demonstrated great potential with these materials, including disease treatment, biosensors, and tissue engineering. For example, piezoelectric materials have been used to stimulate cell growth in bone regeneration, while pyroelectric materials have been applied in skin cancer detection and imaging. Ferroelectric materials have even found use in neural implants that record and stimulate electrical activity in the brain. This paper reviews the relationship between ferroelectric, piezoelectric, and pyroelectric effects and the fundamental principles of different catalytic reactions. It also highlights the preparation methods of these three materials and the significant progress made in their biomedical applications. The review concludes by presenting key challenges and future prospects for efficient catalysts based on piezoelectric, pyroelectric, and ferroelectric nanomaterials for biomedical applications.
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Background
Most bone-related injuries to grassroots troops are caused by training or accidental injuries. To establish preventive measures to reduce all kinds of trauma and improve the combat effectiveness of grassroots troops, it is imperative to develop new strategies and scaffolds to promote bone regeneration.
Methods
In this study, a porous piezoelectric hydrogel bone scaffold was fabricated by incorporating polydopamine (PDA)-modified ceramic hydroxyapatite (PDA-hydroxyapatite, PHA) and PDA-modified barium titanate (PDA-BaTiO 3 , PBT) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physical and chemical properties of the Cs/Gel/PHA scaffold with 0–10 wt% PBT were analyzed. Cell and animal experiments were performed to characterize the immunomodulatory, angiogenic, and osteogenic capabilities of the piezoelectric hydrogel scaffold in vitro and in vivo.
Results
The incorporation of BaTiO 3 into the scaffold improved its mechanical properties and increased self-generated electricity. Due to their endogenous piezoelectric stimulation and bioactive constituents, the as-prepared Cs/Gel/PHA/PBT hydrogels exhibited cytocompatibility as well as immunomodulatory, angiogenic, and osteogenic capabilities; they not only effectively induced macrophage polarization to M2 phenotype but also promoted the migration, tube formation, and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) and facilitated the migration, osteo-differentiation, and extracellular matrix (ECM) mineralization of MC3T3-E1 cells. The in vivo evaluations showed that these piezoelectric hydrogels with versatile capabilities significantly facilitated new bone formation in a rat large-sized cranial injury model. The underlying molecular mechanism can be partly attributed to the immunomodulation of the Cs/Gel/PHA/PBT hydrogels as shown via transcriptome sequencing analysis, and the PI3K/Akt signaling axis plays an important role in regulating macrophage M2 polarization.
Conclusion
The piezoelectric Cs/Gel/PHA/PBT hydrogels developed here with favorable immunomodulation, angiogenesis, and osteogenesis functions may be used as a substitute in periosteum injuries, thereby offering the novel strategy of applying piezoelectric stimulation in bone tissue engineering for the enhancement of combat effectiveness in grassroots troops.
Reconstruction of bone defects and maintaining the continuity of the mandible is still a challenge in the maxillofacial surgery. Nowadays, the biomedical research within bone defect treatment is focussed on the therapy of using innovative biomaterials with specific characteristics consisting of the body’s own substances. Hydroxyapatite ceramic scaffolds have fully acceptable phase compositions, microstructures and compressive strengths for their use in regenerative medicine. The innovative hydroxyapatite ceramics used by us were prepared using the tape-casting method, which allows variation in the shape of samples after packing hydroxyapatite paste to 3D-printed plastic form. The purpose of our qualitative study was to evaluate the regenerative potential of the innovative ceramic biomaterial prepared using this method in the therapy of the cortical bone of the lower jaw in four mature pigs. The mandible bone defects were evaluated after different periods of time (after 3, 4, 5 and 6 months) and compared with the control sample (healthy cortical bone from the opposite side of the mandible). The results of the morphological, clinical and radiological investigation and hardness examination confirmed the positive regenerative potential of ceramic implants after treatment of the mandible bone defects in the porcine mandible model.
Bone implants fabricated using nanocomposites containing hydroxyapatite (HA) and barium titanate (BT) show osteoconductive, osteoinductive, osteointegration, and piezoelectricity properties for bone regeneration applications. In our present study, HA and BT nanopowders were synthesized using high-energy ball-milling-assisted solid-state reaction with precursors of calcium carbonate and ammonium dihydrogen phosphate, and barium carbonate and titanium oxide powder mixtures, respectively. Hexagonal HA and tetragonal BT phases were formed after calcination at 700 and 1000 °C, respectively. Subsequently, hydroxyapatite/barium titanate (HA/BT) nanocomposites with different weight percentages of HA and BT were prepared by ball-milling, then compacted and sintered at two different temperatures to endow these bioceramics with better mechanical, dielectric, and biological properties for bone regeneration. Microstructure, crystal phases, and molecular structure characterizations of these sintered HA/BT nanocomposite compacts (SHBNCs) were performed using field-emission scanning electron microscopy, x-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. Bulk density was evaluated using the Archimedes method. HA/BT nanocomposites with increased BT content showed enhanced dielectric properties, and the dielectric constant (ϵr ) value for 5HA/95BT was ∼182 at 100 Hz. Mechanical properties such as Vicker’s hardness, fracture toughness, yield strength, and diametral tensile strength were also investigated. The hemolysis assay of SHBNCs exhibited hemocompatibility. The effect of these SHBNCs as implants on the in vitro cytocompatibility and cell viability of MG-63 osteoblast-like cells was assessed by MTT assay and live/dead staining, respectively. 15HA/85BT showed increased metabolic activity with a higher number of live cells than BT after the culture period. Overall, the SHBNCs can be used as orthopedic implants for bone regeneration applications.
Three-dimensional (3D) bioprinting has emerged as a class of promising techniques in biomedical research for a wide range of related applications. Specifically, stereolithography apparatus (SLA) and digital light processing (DLP)-based vat-polymerization techniques are highly effective methods of bioprinting, which can be used to produce high-resolution and architecturally sophisticated structures. Our review aims to provide an overview of SLA- and DLP-based 3D bioprinting strategies, starting from factors that affect these bioprinting processes. In addition, we summarize the advances in bioinks used in SLA and DLP, including naturally derived and synthetic bioinks. Finally, the biomedical applications of both SLA- and DLP-based bioprinting are discussed, primarily centered on regenerative medicine and tissue modeling engineering.
Electrical stimulation has been suggested as a means for promoting the direct structural and functional bonding of bone tissue to an artificial implant, known as osseointegration. Previous work has investigated the impact of electrical stimulation in different models, both in vitro and in vivo, using various electrode configurations for inducing an electric field with a wide range of stimulation parameters. However, there is no consensus on optimal electrode configuration nor stimulation parameters. Here, we investigated a novel approach of delivering electrical stimulation to a titanium implant using parameters clinically tested in a different application, namely peripheral nerve stimulation. We propose an in vitro model comprising of Ti6Al4V implants precultured with MC3T3-E1 preosteoblasts, stimulated for 72 h at two different pulse amplitudes (10 µA and 20 µA) and at two different frequencies (50 Hz and 100 Hz). We found that asymmetric charge-balanced pulsed electrical stimulation improved cell survival and collagen production in a dose-dependent manner. Our findings suggest that pulsed electrical stimulation with characteristics similar to peripheral nerve stimulation has the potential to improve cell survival and may provide a promising approach to improve peri-implant bone healing, particularly to neuromusculoskeletal interfaces in which implanted electrodes are readily available.
The repair of critical bone defects remains challenging worldwide. Three canonical pillars (biomaterial scaffolds, bioactive molecules, and stem cells) of bone tissue engineering have been widely used for bone regeneration in separate or combined strategies, but the delivery of bioactive molecules has several obvious drawbacks. Biophysical stimuli have great potential to become the fourth pillar of bone tissue engineering, which can be categorized into three groups depending on their physical properties: internal structural stimuli, external mechanical stimuli, and electromagnetic stimuli. In this review, distinctive biophysical stimuli coupled with their osteoinductive windows or parameters are initially presented to induce the osteogenesis of mesenchymal stem cells (MSCs). Then, osteoinductive mechanisms of biophysical transduction (a combination of mechanotransduction and electrocoupling) are reviewed to direct the osteogenic differentiation of MSCs. These mechanisms include biophysical sensing, transmission, and regulation. Furthermore, distinctive application strategies of biophysical stimuli are presented for bone tissue engineering, including predesigned biomaterials, tissue-engineered bone grafts, and postoperative biophysical stimuli loading strategies. Finally, ongoing challenges and future perspectives are discussed.
In this study, a strategy to print hydroxyapatite (HA) ceramics based on falling‐down‐type digital light processing (DLP) 3D printing technology with high accuracy was proposed. The monolayer curing thickness and curing broadening of the samples were compared under different conditions, and the most suitable photoinitiator for the system and optimal amount of additive dye in the slurry were obtained. The light power density and single‐layer exposure time of the light source of the DLP printer were optimized, and a number of high‐resolution models of HA scaffolds with an exact square‐channel aperture width of 380 μm were successfully printed. The shrinkage, microstructural, and mechanical properties of the HA ceramic scaffolds were studied at different sintering temperatures. The microstructure and composition of the scaffolds were also characterized by scanning electron microscopy and X‐ray diffractometry. The compressive strength and compressive modulus of HA porous scaffold were 11.61 MPa and 1.26 GPa, respectively. And the porosity of scaffold was 31.67%.
Bone exhibits piezoelectric properties. Thus, electrical stimulations such as pulsed electromagnetic fields (PEMFs) and stimuli-responsive piezoelectric properties of scaffolds have been investigated separately to evaluate their efficacy in supporting osteogenesis. However, current understanding of cells responding under the combined influence of PEMF and piezoelectric properties in scaffolds is still lacking. Therefore, in this study, we fabricated piezoelectric scaffolds by functionalization of polycaprolactone-tricalcium phosphate (PCL-TCP) films with a polyvinylidene fluoride (PVDF) coating that is self-polarized by a modified breath-figure technique. The osteoinductive properties of these PVDF-coated PCL-TCP films on MC3T3-E1 cells were studied under the stimulation of PEMF. Piezoelectric and ferroelectric characterization demonstrated that scaffolds with piezoelectric coefficient d33 = −1.2 pC/N were obtained at a powder dissolution temperature of 100 °C and coating relative humidity (RH) of 56%. DNA quantification showed that cell proliferation was significantly enhanced by PEMF as low as 0.6 mT and 50 Hz. Hydroxyapatite staining showed that cell mineralization was significantly enhanced by incorporation of PVDF coating. Gene expression study showed that the combination of PEMF and PVDF coating promoted late osteogenic gene expression marker most significantly. Collectively, our results suggest that the synergistic effects of PEMF and piezoelectric scaffolds on osteogenesis provide a promising alternative strategy for electrically augmented osteoinduction. The piezoelectric response of PVDF by PEMF, which could provide mechanical strain, is particularly interesting as it could deliver local mechanical stimulation to osteogenic cells using PEMF.
During natural tissue regeneration, tissue microenvironment and stem cell niche including cell–cell interaction, soluble factors, and extracellular matrix (ECM) provide a train of biochemical and biophysical cues for modulation of cell behaviors and tissue functions. Design of functional biomaterials to mimic the tissue/cell microenvironment have great potentials for tissue regeneration applications. Recently, electroactive biomaterials have drawn increasing attentions not only as scaffolds for cell adhesion and structural support, but also as modulators to regulate cell/tissue behaviors and function, especially for electrically excitable cells and tissues. More importantly, electrostimulation can further modulate a myriad of biological processes, from cell cycle, migration, proliferation and differentiation to neural conduction, muscle contraction, embryogenesis, and tissue regeneration. In this review, endogenous bioelectricity and piezoelectricity are introduced. Then, design rationale of electroactive biomaterials is discussed for imitating dynamic cell microenvironment, as well as their mediated electrostimulation and the applying pathways. Recent advances in electroactive biomaterials are systematically overviewed for modulation of stem cell fate and tissue regeneration, mainly including nerve regeneration, bone tissue engineering, and cardiac tissue engineering. Finally, the significance for simulating the native tissue microenvironment is emphasized and the open challenges and future perspectives of electroactive biomaterials are concluded. This review provides a comprehensive understanding of the endogenous bioelectricity and piezoelectricity, as well as recent progress in the study of electroactive biomaterials and systems, and their mediated electrostimulation in tissue regeneration.
Piezoelectric ceramics, such as BaTiO3, have gained considerable attention in bone tissue engineering applications thanks to their biocompatibility, ability to sustain a charged surface as well as improve bone cells' adhesion and proliferation. However, the poor processability and brittleness of these materials hinder the fabrication of three-dimensional scaffolds for load bearing tissue engineering applications. For the first time, this study focused on the fabrication and characterisation of BaTiO3 composite scaffolds by using a multi-material 3D printing technology. Polycaprolactone (PCL) was selected and used as dispersion phase for its low melting point, easy processability and wide adoption in bone tissue engineering. The proposed single-step extrusion-based strategy enabled a faster and solvent-free process, where raw materials in powder forms were mechanically mixed and subsequently fed into the 3D printing system for further processing.
PCL, PCL/hydroxyapatite and PCL/BaTiO3 composite scaffolds were successfully produced with high level of consistency and an inner architecture made of seamlessly integrated layers. The inclusion of BaTiO3 ceramic particles (10% wt.) significantly improved the mechanical performance of the scaffolds (54 ± 0.5 MPa) compared to PCL/hydroxyapatite scaffolds (40.4 ± 0.1 MPa); moreover, the presence of BaTiO3 increased the dielectric permittivity over the entire frequency spectrum and tested temperatures. Human osteoblasts Saos-2 were seeded on scaffolds and cellular adhesion, proliferation, differentiation and deposition of bone-like extracellular matrix were evaluated. All tested scaffolds (PCL, PCL/hydroxyapatite and PCL/BaTiO3) supported cell growth and viability, preserving the characteristic cellular osteoblastic phenotype morphology, with PCL/BaTiO3 composite scaffolds exhibiting higher mineralisation (ALP activity) and deposited bone-like extracellular matrix (osteocalcin and collagen I).
The single-step multi-material additive manufacturing technology used for the fabrication of electroactive PCL/BaTiO3 composite scaffolds holds great promise for sustainability (reduced material waste and manufacturing costs) and it importantly suggests PCL/BaTiO3 scaffolds as promising candidates for load bearing bone tissue engineering applications to solve unmet clinical needs.
Proinflammatory (M1) macrophages play a vital role in antitumor immunity, and regulation of proinflammatory macrophage polarization is critical for immunotherapy. The polarization of macrophages can be regulated by biological or chemical stimulation, but investigations of the regulatory effect of physical stimulation are limited. Herein, regulating macrophage polarization with localized electrical signals derived from a piezoelectric β‐phase poly(vinylidene fluoride) (β‐PVDF) film in a wireless mode is proposed. Charges released on the surface of the β‐PVDF film driven by ultrasonic irradiation can significantly enhance the M1 polarization of macrophages. Mechanistic investigation confirms that electrical potentials rather than reactive oxygen species and mechanical forces enable Ca2+ influx through voltage‐gated channels and establishment of the Ca2+‐CAMK2A‐NF‐κB axis to promote the proinflammatory macrophage response during ultrasound treatment. Piezoelectric material‐mediated electrical signal‐activated proinflammatory macrophages significantly inhibit tumor cell proliferation. A method for electrogenetic regulation of immune cells as well as a powerful tool for engineering macrophages for immunotherapy is provided here. Regulating macrophage polarization with localized electrical signals derived from a piezoelectric β‐phase poly(vinylidene fluoride) (β‐PVDF) film in a wireless mode is proposed, providing a method for electrogenetic regulation of immune cells as well as a powerful tool for engineering macrophages for immunotherapy.
Developing bioactive materials for bone implants to enhance bone healing and bone growth has for years been the focus of clinical research. Barium titanate (BT) is an electroactive material that can generate electrical signals in response to applied mechanical forces. In this study, a BT piezoelectric ceramic coating was synthesized on the surface of a TC4 titanium alloy, forming a BT/TC4 material, and low-intensity pulsed ultrasound (LIPUS) was then applied as a mechanical stimulus. The combined effects on the biological responses of MC3T3-E1 cells were investigated. Results of scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction showed that an uniform nanospheres -shaped BT coating was formed on TC4 substrate. Piezoelectric behaviors were observed using piezoelectric force microscopy with the piezoelectric coefficient d33 of 0.42 pC/N. Electrochemical measures indicated that LIPUS-stimulated BT/TC4 materials could produce a microcurrent of approximately 10 μA/cm². In vitro, the greatest osteogenesis (cell adhesion, proliferation, and osteogenic differentiation) was found in MC3T3-E1 cells when BT/TC4 was stimulated using LIPUS. Furthermore, the intracellular calcium ion concentration increased in these cells, possibly because opening of the L-type calcium ion channels was promoted and expression of the CaV1.2 protein was increased. Therefore, the piezoelectric BT/TC4 material with LIPUS loading synergistically promoted osteogenesis, rending it a potential treatment for early stage formation of reliable bone-implant contact.
Electrical stimulation (ES) has potential to be an effective tool for bone injury treatment in clinics. However, the therapeutic mechanism associated with ES is still being discussed. This study aims to investigate the initial mechanism of action by characterising the physical and chemical changes in the extracellular environment during ES and correlate them with the responses of mesenchymal stem/stromal cells (MSCs). Computational modelling was used to estimate the electrical potentials relative to the cathode and the current density across the cell monolayer. We showed expression of phosphorylated ERK1/2, c-FOS, c-JUN, and SPP1 mRNAs, as well as the increased metabolic activities of MSCs at different time points. Moreover, the average of 2.5 μM of H 2 O 2 and 34 μg/L of dissolved Pt were measured from the electrically stimulated media (ES media), which also corresponded with the increases in SPP1 mRNA expression and cell metabolic activities. The addition of sodium pyruvate to the ES media as an antioxidant did not alter the SPP1 mRNA expression, but eliminated an increase in cell metabolic activities induced by ES media treatment. These findings suggest that H 2 O 2 was influencing cell metabolic activity, whereas SPP1 mRNA expression was regulated by other faradic by-products. This study reveals how different electrical stimulation regime alters cellular regenerative responses and the roles of faradic by-products, that might be used as a physical tool to guide and control cell behaviour.
Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation.
For large segmental bone defects, porous titanium scaffolds have some advantages, however, they lack electrical activity which hinders their further use. In this study, a barium titanate (BaTiO3) piezoelectric ceramic was used to modify the surface of a porous Ti6Al4V scaffold (pTi), which was characterized by scanning electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and roughness and water contact angle analyses. Low intensity pulsed ultrasound (LIPUS) was applied in vitro and in vivo study. The activity of bone marrow mesenchymal stem cells, including adhesion, proliferation, and gene expression, was significantly superior in the BaTiO3/pTi, pTi + LIPUS, and BaTiO3/pTi + LIPUS groups than in the pTi group. The activity was also higher in the BaTiO3/pTi + LIPUS group than in the BaTiO3/pTi and pTi + LIPUS groups. Additionally, micro-computed tomography, the mineral apposition rate, histomorphology, and the peak pull-out load showed that these scaffold conditions significantly enhanced osteogenesis and osseointegration 6 and 12 weeks after implantation in large segmental bone defects in the radius of rabbits compared with those resulting from the pTi condition. Consequently, the improved osteogenesis and osseointegration make the BaTiO3/pTi + LIPUS a promising method to promote bone regeneration in large segmental bone defects for clinical application.
Dental pulp stem cells (DPSCs) have great potential for use in tissue engineering (TE)-based dental treatments. Electrical stimulation (EStim) has been shown to influence cellular functions that could play an important role in the success of TE treatments. Despite many recent studies focused on DPSCs, few have investigated the effect EStim has on these cells. The aim of this research was to investigate the effects of direct current (DC) EStim on osteo-/odontogenic differentiation of DPSCs. To do so cells were isolated from male Sprague Dawley rats (7-8 weeks old), and phenotype characterization and multilineage differentiation analysis were conducted to verify their "stemness." Different voltages of DC EStim were administrated 1 h/day for 7 days, and the effect of EStim on DPSC osteo-/odontogenic differentiation was assessed by measuring calcium and collagen deposition, alkaline phosphatase (ALP) activity, and expression of osteo- and odontogenic marker genes at days 7 and 14 of culture. We found that while 10 and 50 mV/mm of EStim had no effect on cell number or metabolic activity, 100 mV/mm caused a significant reduction in cell number, and 150 mV/mm resulted in cell death. Despite increased gene expression of osteo-/odontogenic gene markers, Osteocalcin, RunX2, BSP, and DMP1, at day 7 in EStim treated cells, 50 mV/mm of EStim decreased collagen deposition and ALP activity at both time points, and calcium deposition was found to be lower at day 14. In conclusion, under the conditions tested, EStim appears to impair DPSC osteo-/odontogenic differentiation. Additional studies are needed to further characterize and understand the mechanisms involved in DPSC response to EStim, with an eye toward its potential use in TE-based dental treatments.
Human dental pulp-derived stem cells (hDPSCs) are promising cellular sources for bone healing. The acceleration of their differentiation should be beneficial to their clinical application. Therefore, a conductive polypyrrole (PPy)-made electrical stimulation (ES) device was fabricated to provide direct-current electric field (DCEF) treatment, and its effect on osteo-differentiation of hDPSCs was investigated in this study. To determine the optimal treating time, electrical field of 0.33 V/cm was applied to hDPSCs once for 4 h on different days after the osteo-induction. The alizarin red S staining results suggested that ES accelerated the mineralization rates of hDPSCs. The quantification analysis results revealed a nearly threefold enhancement in calcium deposition by ES at day 0, 2, and 4, whereas the promotion effect in later stages was in vain. To determine the ES-mediated signaling pathway, the expression of genes in the bone morphogenetic protein (BMP) family and related receptors were quantified using qPCR. In the early stages of osteo-differentiation, the mRNA levels of BMP2, BMP3, BMP4, and BMP5 were increased significantly in the ES groups, indicating that these genes were involved in the specific signaling routes induced by ES. We are the first using DCEF to improve the osteo-differentiation of hDPSCs, and our results promise the therapeutic applications of hDPSCs on cell-based bone tissue engineering.
Along with extensive research on the three-dimensional (3D) printing of polymers and metals, 3D printing of ceramics is now the latest trend to come under the spotlight. The ability to fabricate ceramic components of arbitrarily complex shapes has been extremely challenging without 3D printing. This review focuses on the latest advances in the 3D printing of ceramics and presents the historical origins and evolution of each related technique. The main technical aspects, including feedstock properties, process control, post-treatments and energy source–material interactions, are also discussed. The technical challenges and advice about how to address these are presented. Comparisons are made between the techniques to facilitate the selection of the best ones in practical use. In addition, representative applications of the 3D printing of various types of ceramics are surveyed. Future directions are pointed out on the advancement on materials and forming mechanism for the fabrication of high-performance ceramic components.
The piezoelectric effect of biological piezoelectric materials promotes bone growth. However, the material should be subjected to stress before it can produce an electric charge that promotes bone repair and reconstruction conducive to fracture healing. A novel method for in vitro experimentation of biological piezoelectric materials with physiological load is presented. A dynamic loading device that can simulate the force of human motion and provide periodic load to piezoelectric materials when co-cultured with cells was designed to obtain a realistic expression of piezoelectric effect on bone repair. Hydroxyapatite (HA)/barium titanate (BaTiO3) composite materials were fabricated by slip casting, and their piezoelectric properties were obtained by polarization. The d33 of HA/BaTiO3 piezoelectric ceramics after polarization was 1.3 pC/N to 6.8 pC/N with BaTiO3 content ranging from 80% to 100%. The in vitro biological properties of piezoelectric bioceramics with and without cycle loading were investigated. When HA/BaTiO3 piezoelectric bioceramics were affected by cycle loading, the piezoelectric effect of BaTiO3 promoted the growth of osteoblasts and interaction with HA, which was better than the effect of HA alone. The best biocompatibility and bone-inducing activity were demonstrated by the 10%HA/90%BaTiO3 piezoelectric ceramics.
Piezoelectric
ceramics are commonly used in various sensing applications. In this paper, the effect of poling and repoling conditions on thick-film piezoelectric
ceramics were investigated. The piezoelectric charge coefficient of the piezoelectric
ceramics were measured with varying poling conditions, where the effect of changing poling temperature and electrical field on the d
33
were analyzed. This was followed by investigating on the effect high applied electrical fields results in repolarization the alignment of the piezoelectric domain in the opposite direction. The temperature and electrical field dependence polarization of the thick-film piezoelectric
ceramics were varied near to its Curie temperature between 50°C to 250°C and at a range of electrical field from 20 V (400 kV/mm) up to 200 V (4 MV/mm). It was found that the piezoelectric properties increases with increasing the poling electric field and poling temperature significantly. The maximum values of piezoelectric coefficient were obtained for the piezoelectric
ceramics poled at the Curie temperature with high electric fields for 15 minutes. The aging behavior of the piezoelectric
ceramics shows that piezoelectric charge coefficient d
33
depends on the poling and repoling conditions.
The piezoelectric effect of bone has been observed similarly to the case of wood or ramie. The specimens were cut out from the femur of man and ox, and dried completely by heating. The piezoelectric constants were measured by three different experiments, that is, measurements of the static direct effect, the dynamic direct effect and the dynamic converse effect. The piezoelectric effect appears only when the shearing force is applied to the collagen fibres to make them slip past erch other. The magnitude of piezoelectric constant depends on the angle between the applied pressure and the axis of the bone. The maximum value of piezoelectric constant amounts to 6x 10-9c.g.s.e.s.u., which is about one-tenth of a piezoelectric constant du of quarts crystal. The specimens which were boiled in hot water and afterwards dried completely showed little change in the piezoelectric effect, the fact ascertaining that the effect is not of biological origin. The origin to piezoelectricity in bone may be ascribed to the piezoelectric effect of the crystalline micelle of collagen molecules. The consideration of the symmetry of the configulation of collagen fibres in the bone texture shows the existence of effects which are represented by only two piezoelectric constants d14 and d25, which are the same in magnitude but opposite in sign.
Hydroxyapatite (HA) and barium titanate (BT) powders were mixed and sintered to form hydroxyapatite - barium titanate (HABT) ceramics. These materials were then poled and their piezoelectric properties were measured. The microstructure of unpoled samples was examined using scanning electron microscopy (SEM).The piezoelectric constants (d 33 and d 31 ) of the ceramics were found to be dependent on the proportion of BT in the ceramic In materials containing less than 70% BT, no piezoelectric effect was found. Above this value, the piezoelectric constant increased with the addition of BT up to a value of 108pCN -1 for pure BT. Values of d 33 for ceramics containing more than 80% BT are above values previously shown to have a positive influence on bone growth in vivo . SEM analysis indicated that the grain size within the materials decreased as the proportion of BT in the material was reduced. Examination of the microstructure of the ceramics indicated the presence of electrical domains in the 100% BT and 95% BT ceramics. Domains were not visible below 95% BT. The reduction in grain size may influence the reduction in piezoelectric activity within the materials but cannot be considered to be the only cause.
Tissue engineering applications commonly encompass the use of three-dimensional (3D) scaffolds to provide a suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. These scaffolds serve to mimic the actual in vivo microenvironment where cells interact and behave according to the mechanical cues obtained from the surrounding 3D environment. Hence, the material properties of the scaffolds are vital in determining cellular response and fate. These 3D scaffolds are generally highly porous with interconnected pore networks to facilitate nutrient and oxygen diffusion as well as waste removal. This review focuses on the various fabrication techniques (e.g. conventional and rapid prototyping methods) that have been employed to fabricate 3D scaffolds of different pore sizes and porosity. The different pore size and porosity measurement methods will also be discussed. Scaffolds with graded porosity have also been studied for their ability to better represent the actual in vivo situation where cells are exposed to layers of different tissues with varying properties. In addition, the ability of pore size and porosity of scaffolds to direct cellular responses and alter the mechanical properties of scaffolds will be reviewed, followed by a look a nature's own scaffold, the extracellular matrix. Overall, the limitations of current scaffold fabrication approaches for tissue engineering applications as well as some novel and promising alternatives will be highlighted.
3D printing, also known as Additive manufacturing (AM), has emerged as a transformative technology with applications across various industries, including the medical sector. This review paper provides an overview of the current status of AM technology, its challenges, and its application in the medical industry. The paper covers the different types of AM technologies, such as fused deposition modeling, stereolithography, selective laser sintering, digital light processing, binder jetting, and electron beam melting, and their suitability for medical applications. The most commonly used biomedical materials in AM, such as plastic, metal, ceramic, composite, and bio-inks, are also viewed. The challenges of AM technology, such as material selection, accuracy, precision, regulatory compliance, cost and quality control, and standardization, are also discussed. The review also highlights the various applications of AM in the medical sector, including the production of patient-specific surgical guides, prosthetics, orthotics, and implants. Finally, the review highlights the Internet of Medical Things (IoMT) and artificial intelligence (AI) for regulatory frameworks and safety standards for 3D-printed biomedical devices. The review concludes that AM technology can transform the healthcare industry by enabling patients to access more personalized and reasonably priced treatment alternatives. Despite the challenges, integrating AI and IoMT with 3D printing technology is expected to play a vital role in the future of biomedical device applications, leading to further advancements and improvements in patient care. More research is needed to address the challenges and optimize its use for medical applications to utilize AM's potential in the medical industry fully.
Bone regeneration is a highly synchronized process that requires multiple biochemical, bioelectrical, mechanical, and other physiological cues. The restoration and delivery of electrical cues locally through piezoelectric materials have been demonstrated to facilitate osteogenesis in vitro and bone repair in vivo. However, the underlying mechanism by which piezoelectric stimulation promotes osteogenesis and bone repair remains unclear yet, limiting the design and clinical application of piezoelectric materials for bone repair. Herein, a piezoelectric BaTiO3/Ti6Al4V (BT/Ti) scaffold was prepared by hydrothermal synthesis of a uniform BaTiO3 layer on three dimensionally printed Ti6Al4V scaffold. The BT/Ti scaffolds exhibited piezoelectricity and favorable biocompatibility with RAW264.7 macrophages after polarization. In vitro results demonstrated that the piezoelectric effects of the poled BT/Ti scaffolds promoted M2 polarization of macrophages and immunoregulatory osteogenesis of MC-3T3 osteoblasts. In a subcutaneous implantation model, a higher proportion of CD68+ CD206+ M2 macrophages was observed in the tissues around the poled BT/Ti scaffolds under low intensity pulsed ultrasound (LIPUS) stimulation. Improvements in macrophage M2 polarization and bone regeneration were further identified in a sheep cervical corpectomy model. RNA sequencing and mechanistic investigation revealed that the piezoelectric BT/Ti (poled) scaffolds inhibited the inflammatory MAPK/JNK signaling cascade and activated oxidative phosphorylation (OXPHOS) and ATP synthesis in macrophages. Collectively, our study provides a promising method for regulating the immune microenvironment and enhancing bone regeneration using polarized piezoelectric BT/Ti scaffolds.
Inspired by the silkworm spinning process for production of tough cocoons, a gradient printing-assembly technique with silk fibroin (SF) and hydroxyapatite (HA) to achieve high strength scaffolds for bone regeneration is developed. A coaxial extrude-nozzle is employed to provide gathered thickening and shearing for aligned assembly. The aligned SF-HA assembles into the compacted nanostructure, which performs a maximum compressive strength of 166 MPa and bending strength of 40 MPa. Scaffolds with various morphologies could be arbitrarily constructed via extruded 3D printing for the regeneration of cortical bone or cancellous bone. The hemolysis quantification of red blood cells (RBCs), proliferation and flow cytometry of bone marrow stem cells (BMSCs) have proved the excellent biocompatibility of the printed scaffolds. Osteogenic induced differentiation assay in vitro and surgical intervention for rat femoral defect repairing have verified the successful osteogenesis with high mechanical strength and remarkable stability in the physiological environment. The silkworm spinning inspired 3D printing offers a facile approach for the fabrication of implantable scaffolds with high strength and excellent biocompatibility, which is highly desired for the applications of bone tissue engineering.
Advancements in polymer science and engineering have helped the scientific community to shift its attention towards the use of environmentally benign materials for reducing the environmental impact of conventional synthetic plastics. Biopolymers are environmentally benign, chemically versatile, sustainable, biocompatible, biodegradable, inherently functional, and ecofriendly materials that exhibit tremendous potential for a wide range of applications including food, electronics, agriculture, textile, biomedical, and cosmetics. This review also inspires the researchers toward more consumption of biopolymer-based composite materials as an alternative to synthetic composite materials. Herein, an overview of the latest knowledge of different natural- and synthetic-based biodegradable polymers and their fiber-reinforced composites is presented. The review discusses different degradation mechanisms of biopolymer-based composites as well as their sustainability aspects. This review also elucidates current challenges, future opportunities, and emerging applications of biopolymeric sustainable composites in numerous engineering fields. Finally, this review proposes biopolymeric sustainable materials as a propitious solution to the contemporary environmental crisis.
Bioelectrical phenomenon in natural bone has been well recognized for its role in bone development and fracture healing. For example, the piezoelectricity induced modulation in cellular functionality assists in the repair and regeneration of bone tissue. Against this backdrop, we review here the origin of dielectric and electrical responses (piezo-, pyro- and ferro-electricity) of natural bone along with their consequences in regulating the bone metabolic activities. The concept of piezoelectricity induced osteogenesis has driven the development of piezoimplants for bone regeneration applications. A number of recent studies have been critically analyzed to demonstrate as to how the surface charge polarization or electric field stimulation together with functional properties of piezoelectric biomaterials can synergistically modulate cell functionality, in vitro or tissue regeneration, in vivo. The examples are drawn from a range of piezoelectric bioceramics, (e.g. barium titanate, magnesium silicate etc.) and biopolymers (e.g. polyvinylidene fluoride (PVDF), collagen, etc). The challenging problem of processing the compositionally tailored bioceramics is emphasized in particular reference to (Na, K)NbO3, an implantable biomaterial with the most attractive combination of piezoresponsive properties. Taken together, this review comprehensively emphasizes the appealing relevance of piezo-bioceramics and piezo-biopolymers as next-generation orthopedic biomaterials.
In this research, hydroxyapatite (HA)-based ceramics were produced as suitable ceramic implants for orthopedic applications. To improve the physical, mechanical, electrical and biological properties of pure HA, we developed composite scaffolds of HA-barium titanate (BT) by cold isostatic pressing and sintering. Microstructure, crystal phases, and molecular structure were analyzed by using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), respectively. Bulk density values were measured using the Archimedes method. The effect of different percentages of BT on cell proliferation, viability, and ALP activity of dental pulp stem cells (DPSCs) was assessed by ProstoBlue assay, Live/Dead staining, and p-NPP assay. The obtained results indicate that the HA-BT scaffolds possess higher compressive strength, toughness, density, and hardness compared with pure HA scaffolds. After immersing the scaffold in SBF solution, more deposited apatite appeared on the HA-BT, which results in rougher surface on this scaffold thanpure HA. Electrical properties of HA in the presence of BT are improved. Based on the results of cell culture experiments, composites containing 40, 50 and 60 %wt of BT have excellent biocompatibility, with the best results occurring for the sample with 50 %wt BT.
Biomaterials can promote endogenous healing without delivering cells or therapeutics
Biomaterials with suitable surface modification strategies are contributing significantly to the rapid development of the field of bone tissue engineering. Despite these encouraging results, utilization of biomaterials is poorly translated to human clinical trials potentially due to lack of knowledge about the interaction between biomaterials and the body defense mechanism, the “immune system”. The highly complex immune system involves the coordinated action of many immune cells that can produce various inflammatory and anti‐inflammatory cytokines. Besides, bone fracture healing initiates with acute inflammation and may later transform to a regenerative or degenerative phase mainly due to the cross‐talk between immune cells and other cells in the bone regeneration process. Among various immune cells, macrophages possess a significant role in the immune defense, where their polarization state plays a key role in the wound healing process. Growing evidence shows that the macrophage polarization state is highly sensitive to the biomaterial's physiochemical properties, and advances in biomaterial research now allow well controlled surface properties. This review provides an overview of biomaterial‐mediated modulation of the immune response for regulating key bone regeneration events, such as osteogenesis, osteoclastogenesis, and inflammation, and it discusses how these strategies can be utilized for future bone tissue engineering applications. Recent approaches to biomaterial‐mediated immunomodulation for bone regeneration are summarized in this review. Engineered biomaterials with suitable properties can orchestrate the action of immune cells and their cross‐talk with key events in bone fracture healing such as osteogenesis, osteoclastogenesis, and inflammation. Such approaches can be a future tool for bridging the gap between laboratories to clinical trials for biomaterials.
It is well established that the piezoelectric effect plays an important physiological role in bone growth, remodeling and fracture healing. Barium titanate, as a well-known piezoelectric ceramic, is especially an attractive material as a scaffold for bone tissue engineering applications. In this regard, we tried to fabricate a highly porous barium titanate based scaffolds by foam replication method and polarize them by applying an external electric field. In order to enhance the mechanical and biological properties, polarized/non-polarized scaffolds were coated with gelatin and nanostructured HA and characterized for their morphologies, porosities, piezoelectric and mechanical properties. The results showed that the compressive strength and piezoelectric coefficient of porous scaffolds increased with the increase of sintering temperature. After being coated with Gel/HA nanocomposite, the interconnected porous structure and pore size of the scaffolds almost remain unchanged while the Gel/nHA-coated scaffolds exhibited enhanced compressive strength and elastic modulus compared with the uncoated samples. Also, the effect of polarizing and coating of optimal scaffolds on adhesion, viability, and proliferation of the MG63 osteoblast-like cell line was evaluated by scanning electron microscope (SEM) and MTT assay. The cell culture experiments revealed that developed scaffolds had good biocompatibility and cells were able to adhere, proliferate and migrate into pores of the scaffolds. Furthermore, cell density was significantly higher in the coated scaffolds at all tested time-points. These results indicated that highly porous barium titanate scaffolds coated with Gel/HA nanocomposite has great potential in tissue engineering applications for bone tissue repair and regeneration.
Bone defects are common and are associated with a significant burden of disease. The treatment of these injuries remains controversial, particularly those defects which are critical sized. Despite the need for decision making to be evidence based, a lack of consensus around definitions of critical-sized defects still exists, particularly around those defects in the 1-3 cm range. There is a need to define "critical" in bone defect size because noncritical defects may heal without planned reconstruction and secondary surgery. This article reviews the current evidence around the definition of a critical-sized bone defect and concludes that defects in the order of 2.5 cm or greater seem to have a poor natural history.
Objective:
Compare the musculoskeletal effects of low cadence cycling (LOW-CAD) with functional electrical stimulation (FES) to high cadence FES cycling (HIGH-CAD) for people with spinal cord injury (SCI). It was hypothesized that subjects using LOW-CAD would cycle with greater torque and thus show greater musculoskeletal improvements compared to subjects using HIGH-CAD.
Design:
Randomized pre-post design SETTING: Outpatient rehabilitation clinic PARTICIPANTS: Fourteen men and three women (22-67 years) with C4-T6 motor complete chronic SCI were randomized to LOW-CAD (n=9) or HIGH-CAD (n=8).
Interventions:
LOW-CAD cycled at 20 rpm and HIGH-CAD at 50 rpm three times/week for six months. Cycling torque (resistance per pedal rotation) increased if targeted cycling cadence was maintained.
Main outcome measures:
Dual-energy x-ray absorptiometry was used to assess distal femur aBMD, magnetic resonance imaging (MRI) to assess to assess trabecular and cortical bone architecture and thigh muscle volume, and biochemical markers to assess bone turnover.
Results:
15 subjects completed the study. LOW-CAD obtained maximal average torque of 2.9±2.8 Nm and HIGH-CAD of 0.8±0.2 Nm. LOW-CAD showed greater decreases in bone-specific-alkaline-phosphatase (BALP) indicating less bone formation for HIGH-CAD (15.5% decrease for LOW-CAD, 10.7% increase for HIGH-CAD). N-telopeptide (N-Tx) decreased 34% within LOW-CAD indicating decreased resorption. Both groups increased muscle volume (LOW-CAD by 19%; HIGH-CAD by 10%). LOW-CAD showed a non-significant 6-7% improvement in distal femur trabecular bone number (p=0.08) and separation (p=0.08) while HIGH-CAD showed a non-significant decline of 2-3%.
Conclusions:
This study suggests that the greater torque achieved with LOW-CAD cycling may result in improved bone health due to decreased bone turnover and improved trabecular bone. Longer term outcome studies are warranted to identify the effect on fracture risk.
The current understanding of electromechanical effects in fully hydrated bone is that they are electrokinetic (streaming potential) in nature. The presence of a second competing mechanism, piezoelectricity, which dominates in dry conditions, has been denied in conditions of full hydration based on the absence of a piezoelectric response from wet collagen. Since the mineralized collagenous matrix of bone can only absorb 26 wt% water (Relative Humidity (RH)=60%), there seems no reason to dismiss a piezoelectric component entirely and experimental evidence was sought using a new measuring technique. A comprehensive analysis has been developed to relate both mechanisms to bone structure at different levels of hydration. Our results indicate the presence of both mechanisms at full hydration, with the piezoelectric effect leading streaming potential in the time domain. The immediate implication of this finding is that it is the piezoelectric effect which determines the characteristics of the generated electrical signal and may subsequently influence bone generation and remodelling.
Autogenous cancellous bone graft provides an osteoconductive, osteoinductive, and osteogenic substrate for filling bone voids and augmenting fracture-healing.The iliac crest remains the most frequently used site for bone-graft harvest, but the proximal part of the tibia, distal end of the radius, distal aspect of the tibia, and greater trochanter are alternative donor sites that are particularly useful for bone-grafting in the ipsilateral extremity.The most common complication associated with the harvest of autogenous bone graft is pain at the donor site, with less frequent complications including nerve injury, hematoma, infection, and fracture at the donor site.Induced membranes is a method that uses a temporary polymethylmethacrylate cement spacer to create a bone-graft-friendly environment to facilitate graft incorporation, even in large segmental defects.
Bone stimulation represents a $500 million market in the United States. The use of electromagnetic stimulation in the treatment of fractures is common; however, the efficacy of this modality remains uncertain. We conducted a systematic review and meta-analysis of randomized controlled trials to evaluate the effect of electromagnetic stimulation on long-bone fracture-healing.
We searched four electronic databases (MEDLINE, EMBASE, CINAHL, and all Evidence-Based Medicine Reviews) for trials of electromagnetic stimulation and bone repair, in any language, published from the inception of the database to April 2008. In addition, we searched by hand seven relevant journals published between 1980 and April 2008 and the bibliographies of eligible trials. Eligible trials enrolled patients with long-bone lesions, randomly assigned them to electromagnetic stimulation or a control group, and reported on bone-healing. Information on the methodological quality, stimulation device, duration of treatment, patient demographics, and all clinical outcomes were independently extracted by two reviewers.
Of 2546 citations obtained in the literature search, eleven articles met the inclusion criteria. Evidence from four trials reporting on 106 delayed or ununited fractures demonstrated an overall nonsignificant pooled relative risk of 1.76 (95% confidence interval, 0.8 to 3.8; p = 0.15; I(2) = 60.4%) in favor of electromagnetic stimulation. Single studies found a positive benefit of electromagnetic stimulation on callus formation in femoral intertrochanteric osteotomies, a limited benefit for conservatively managed Colles fracture or for lower limb-lengthening, and no benefit on limb-length imbalance and need for reoperation in surgically managed pseudarthroses or on time to clinical healing in tibial stress fractures. Pain was reduced in one of the four trials assessing this outcome.
While our pooled analysis does not show a significant impact of electromagnetic stimulation on delayed unions or ununited long-bone fractures, methodological limitations and high between-study heterogeneity leave the impact of electromagnetic stimulation on fracture-healing uncertain.
The electromechanical properties of fluidfilled bone
- G W Hastings
- Medicine Fmjjomsmi
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- J Lee
- H Byun
- S K Madhurakkat Perikamana
- S Lee
- H Shin
J. Lee, H. Byun, S.K. Madhurakkat Perikamana, S. Lee, H. Shin, Current advances in
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Role and Mechanism of Mechanically Sensitive Ion Channel Trpm7 in Lipus Promoting Osteogenic Differentiation of MSCs
- H Yao
H. Yao, Role and Mechanism of Mechanically Sensitive Ion Channel Trpm7 in Lipus
Promoting Osteogenic Differentiation of MSCs, Chongqing Medical University,
Chongqing, 2018.