Nanomaterials in 4D Printing: Expanding the Frontiers of Advanced Manufacturing.

As an innovative technology, four-dimentional (4D) printing is built upon the principles of three-dimentional (3D) printing with an additional dimension: time. While traditional 3D printing creates static objects, 4D printing generates "responsive 3D printed structures", enabling them to transform or self-assemble in response to external stimuli. Due to the dynamic nature, 4D printing has demonstrated tremendous potential in a range of industries, encompassing aerospace, healthcare, and intelligent devices. Nanotechnology has gained considerable attention owing to the exceptional properties and functions of nanomaterials. Incorporating nanomaterials into an intelligent matrix enhances the physiochemical properties of 4D printed constructs, introducing novel functions. This review provides a comprehensive overview of current applications of nanomaterials in 4D printing, exploring their synergistic potential to create dynamic and responsive structures. Nanomaterials play diverse roles as rheology modifiers, mechanical enhancers, function introducers, and more. The overarching goal of this review is to inspire researchers to delve into the vast potential of nanomaterial-enabled 4D printing, propelling advancements in this rapidly evolving field.

4D Printed Nerve Conduit with In Situ Neurogenic Guidance for Nerve Regeneration.

Nerve repair poses a significant challenge in the field of tissue regeneration. As a bioengineered therapeutic method, nerve conduits have been developed to address damaged nerve repair. However, despite their remarkable potential, it is still challenging to encompass complex physiologically microenvironmental cues (both biophysical and biochemical factors) to synergistically regulate stem cell differentiation within the implanted nerve conduits, especially in a facile manner. In this study, a neurogenic nerve conduit with self-actuated ability has been developed by in situ immobilization of neurogenic factors onto printed architectures with aligned microgrooves. One objective was to facilitate self-entubulation, ultimately enhancing nerve repairs. Our results demonstrated that the integration of topographical and in situ biological cues could accurately mimic native microenvironments, leading to a significant improvement in neural alignment and enhanced neural differentiation within the conduit. This innovative approach offers a revolutionary method for fabricating multifunctional nerve conduits, capable of modulating neural regeneration efficiently. It has the potential to accelerate the functional recovery of injured neural tissues, providing a promising avenue for advancing nerve repair therapies.

3D printing of thick myocardial tissue constructs with anisotropic myofibers and perfusable vascular channels.

Engineering of myocardial tissues has become a promising therapeutic strategy for treating myocardial infarction (MI). However, a significant challenge remains in generating clinically relevant myocardial tissues that possess native microstructural characteristics and fulfill the requirements for implantation within the human body. In this study, a thick 3D myocardial construct with anisotropic myofibers and perfusable branched vascular channels is created with clinically relevant dimensions using a customized beam-scanning stereolithography printing technique. To obtain tissue-specific matrix niches, a decellularized extracellular matrix microfiber-reinforced gelatin-based bioink is developed. The bioink plays a crucial role in facilitating the precise manufacturing of a hierarchical microstructure, enabling us to better replicate the physiological characteristics of the native myocardial tissue matrix in terms of structure, biomechanics, and bioactivity. Through the integration of the tailored bioink with our printing method, we demonstrate a biomimetic architecture, appropriate biomechanical properties, vascularization, and improved functionality of induced pluripotent stem cell-derived cardiomyocytes in the thick tissue construct in vitro. This work not only offers a novel and effective means to generate biomimetic heart tissue in vitro for the treatment of MI, but also introduces a potential methodology for creating clinically relevant tissue products to aid in other complex tissue/organ regeneration and disease model applications.

Chitosan and its derivatives in 3D/4D (bio) printing for tissue engineering and drug delivery applications.

Tissue engineering research has undergone to a revolutionary improvement, thanks to technological advancements, such as the introduction of bioprinting technologies. The ability to develop suitable customized biomaterial inks/bioinks, with excellent printability and ability to promote cell proliferation and function, has a deep impact on such improvements. In this context, printing inks based on chitosan and its derivatives have been instrumental. Thus, the current review aims at providing a comprehensive overview on chitosan-based materials as suitable inks for 3D/4D (bio)printing and their applicability in creating advanced drug delivery platforms and tissue engineered constructs. Furthermore, relevant strategies to improve the mechanical and biological performances of this biomaterial are also highlighted.

4D Thermo-Responsive Smart hiPSC-CM Cardiac Construct for Myocardial Cell Therapy.

Purpose: 4D fabrication techniques have been utilized for advanced biomedical therapeutics due to their ability to create dynamic constructs that can transform into desired shapes on demand. The internal structure of the human cardiovascular system is complex, where the contracting heart has a highly curved surface that changes shape with the heart's dynamic beating motion. Hence, 4D architectures that adjust their shapes as required are a good candidate to readily deliver cardiac cells into the damaged heart and/or to serve as self-morphing tissue scaffolds/patches for healing cardiac diseases. In this proof-of-concept in vitro study, a two-in-one 4D smart cardiac construct that integrates the functions of minimally invasive cell vehicles and in situ tissue patches was developed for repairing damaged myocardial tissue. Methods: For this purpose, a series of thermo-responsive 4D structures with different shapes and sizes were fabricated via the combination of fused deposition modeling (FDM)-printing and stamping molding. The thermo-responsive 4D constructs were firstly optimized to exhibit their shape transformation behavior at the designated temperature for convenient control. After which, the mechanical properties, shape recovery rate, and shape recovery speed of the 4D constructs at different temperatures were thoroughly evaluated. Also, the proliferation and functional prototype of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the 4D constructs were quantified and evaluated using F-actin staining and immunostaining. Results: Our results showed that the 4D constructs possessed the desirable capability of shape-changing from spherical carriers to unfolded patches at human body temperature and exhibited excellent biocompatibility. Moreover, myocardial maturation in vitro with a uniform and printing pattern-specific cell distribution was observed on the surface of the unfolded 4D constructs. Conclusion: We successfully developed a 4D smart cardiac construct that integrates the functions of minimally invasive cell vehicles and in situ tissue patches for repairing damaged myocardial tissue.

3D printed biomimetic flexible blood vessels with iPS cell-laden hierarchical multilayers.

Successful recovery from vascular diseases has typically relied on the surgical repair of damaged blood vessels (BVs), with the majority of current approaches involving the implantation of autologous BVs, which is plagued by donor site tissue damage. Researchers have attempted to develop artificial vessels as an alternative solution to traditional approaches to BV repair. However, the manufacturing of small-diameter (< 6 mm) BVs is still considered one of the biggest challenges due to its difficulty in the precise fabrication and the replication of biomimetic architectures. In this study, we successfully developed 3D printed flexible small-diameter BVs that consist of smooth muscle cells and a vascularized endothelium. In the developed artificial BV, a rubber-like elastomer was printed as the outermost layer of the vessel, which demonstrated enhanced mechanical properties, while and human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iSMCs) and endothelial cells (iECs) embedded fibrinogen solutions were coaxially extruded with thrombin solution to form cell-laden fibrin gel inner layers. Our results showed that the 3D BVs possessed proper mechanical properties, and the cells in the fibrin layers substantially proliferated over time to form a stable BV construct. Our study demonstrated that the 3D printed flexible small-diameter BV using iPSCs could be a promising platform for the treatment of vascular diseases.

An in vitro analysis of the effect of geometry-induced flows on endothelial cell behavior in 3D printed small-diameter blood vessels.

Clinical recovery from vascular diseases has increasingly become reliant upon the successful fabrication of artificial blood vessels (BVs) or vascular prostheses due to the shortage of autologous vessels and the high incidence of vessel graft diseases. Even though many attempts at the clinical implementation of large artificial BVs have been reported to be successful, the development of small-diameter BVs remains one of the significant challenges due to the limitation of micro-manufacturing capacity in complexity and reproducibility, as well as the development of thrombosis. The present study aims to develop 3D printed small-diameter artificial BVs that recapitulate the longitudinal geometric elements in the native BVs using biocompatible polylactic acid (PLA). As their intrinsic physical properties are crystallinity dependent, we used two PLA filaments with different crystallinity to investigate the suitability of their physical properties in the micro-manufacturing of BVs. To explore the mechanism of venous thrombosis, our study provided a preliminarily comparative analysis of the effect of geometry-induced flows on the behavior of human endothelial cells (ECs). Our results showed that the adhered healthy ECs in the 3D printed BV exhibited regulated patterns, such as elongated and aligned parallel to the flow direction, as well as geometry-induced EC response mechanisms that are associated with hemodynamic shear stresses. Furthermore, the computational fluid dynamics simulation results provided insightful information to predict velocity profile and wall shear stress distribution in the geometries of BVs in accordance with their spatiotemporally-dependent cell behaviors. Our study demonstrated that 3D printed small-diameter BVs could serve as suitable candidates for fundamental BV studies and hold great potential for clinical applications.

Emerging 4D Printing Strategies for Next-Generation Tissue Regeneration and Medical Devices.

The rapid development of 3D printing has led to considerable progress in the field of biomedical engineering. Notably, 4D printing provides a potential strategy to achieve a time-dependent physical change within tissue scaffolds or replicate the dynamic biological behaviors of native tissues for smart tissue regeneration and the fabrication of medical devices. The fabricated stimulus-responsive structures can offer dynamic, reprogrammable deformation or actuation to mimic complex physical, biochemical, and mechanical processes of native tissues. Although there is notable progress made in the development of the 4D printing approach for various biomedical applications, its more broad-scale adoption for clinical use and tissue engineering purposes is complicated by a notable limitation of printable smart materials and the simplistic nature of achievable responses possible with current sources of stimulation. In this review, the recent progress made in the field of 4D printing by discussing the various printing mechanisms that are achieved with great emphasis on smart ink mechanisms of 4D actuation, construct structural design, and printing technologies, is highlighted. Recent 4D printing studies which focus on the applications of tissue/organ regeneration and medical devices are then summarized. Finally, the current challenges and future perspectives of 4D printing are also discussed.

4D printing in biomedical applications: emerging trends and technologies.

Nature's material systems during evolution have developed the ability to respond and adapt to environmental stimuli through the generation of complex structures capable of varying their functions across direction, distances and time. 3D printing technologies can recapitulate structural motifs present in natural materials, and efforts are currently being made on the technological side to improve printing resolution, shape fidelity, and printing speed. However, an intrinsic limitation of this technology is that printed objects are static and thus inadequate to dynamically reshape when subjected to external stimuli. In recent years, this issue has been addressed with the design and precise deployment of smart materials that can undergo a programmed morphing in response to a stimulus. The term 4D printing was coined to indicate the combined use of additive manufacturing, smart materials, and careful design of appropriate geometries. In this review, we report the recent progress in the design and development of smart materials that are actuated by different stimuli and their exploitation within additive manufacturing to produce biomimetic structures with important repercussions in different but interrelated biomedical areas.

Acoustic Droplet Vaporization of Perfluorocarbon Droplets in 3D-Printable Gelatin Methacrylate Scaffolds.

Scientists face a significant challenge in creating effective biomimetic constructs in tissue engineering with sustained and controlled delivery of growth factors. Recently, the addition of phase-shift droplets inside the scaffolds is being explored for temporal and spatial control of biologic delivery through vaporization using external ultrasound stimulation. Here, we explore acoustic droplet vaporization (ADV) in gelatin methacrylate (GelMA), a popular hydrogel used for tissue engineering applications because of its biocompatibility, tunable mechanical properties and rapid reproducibility. We embedded phase-shift perfluorocarbon droplets within the GelMA resin before crosslinking and characterized ADV and inertial cavitation (IC) thresholds of the embedded droplets. We were successful in vaporizing two different perfluorocarbon---perfluoropentane (PFP) and perfluorohexane (PFH)--cores at 2.25- and 5-MHz frequencies and inside hydrogels with varying mechanical properties. The ADV and IC thresholds for PFP droplets in GelMA scaffolds increased with frequency and in stiffer scaffolds. The PFH droplets exhibited ADV and IC activity only at 5 MHz for the range of excitations below 3MPa investigated here and at threshold values higher than those of PFP droplets. The results provide a proof of concept for the possible use of ADV in hydrogel scaffolds for tissue engineering.

Recent advances in bioprinting technologies for engineering cardiac tissue.

Annually increasing incidence of cardiac-related disorders and cardiac tissue's minimal regenerative capacity have motivated the researchers to explore effective therapeutic strategies. In the recent years, bioprinting technologies have witnessed a great wave of enthusiasm and have undergone steady advancements over a short period, opening the possibilities for recreating engineered functional cardiac tissue models for regenerative and diagnostic applications. With this perspective, the current review delineates recent developments in the sphere of engineered cardiac tissue fabrication, using traditional and advanced bioprinting strategies. The review also highlights different printing ink formulations, available cellular opportunities, and aspects of personalized medicines in the context of cardiac tissue engineering and bioprinting. On a concluding note, current challenges and prospects for further advancements are also discussed.

Recent advances in bioprinting technologies for engineering hepatic tissue.

In the sphere of liver tissue engineering (LTE), 3D bioprinting has emerged as an effective technology to mimic the complex in vivo hepatic microenvironment, enabling the development of functional 3D constructs with potential application in the healthcare and diagnostic sector. This review gears off with a note on the liver's microscopic 3D architecture and pathologies linked to liver injury. The write-up is then directed towards unmasking recent advancements and prospects of bioprinting for recapitulating 3D hepatic structure and function. The article further introduces available stem cell opportunities and different strategies for their directed differentiation towards various hepatic stem cell types, including hepatocytes, hepatic sinusoidal endothelial cells, stellate cells, and Kupffer cells. Another thrust of the article is on understanding the dynamic interplay of different hepatic cells with various microenvironmental cues, which is crucial for controlling differentiation, maturation, and maintenance of functional hepatic cell phenotype. On a concluding note, various critical issues and future research direction towards clinical translation of bioprinted hepatic constructs are discussed.

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study.

Two-dimensional (2D) in vitro cell cultures and laboratory animals have been used traditionally as the gold-standard preclinical cancer model systems. However, for cancer stem cell (CSC) studies, they exhibit notable limitations on simulating native environment, which depreciate their translatability for clinical development purposes. In this study, different three-dimensional (3D) printing platforms were used to establish novel 3D cell cultures enriched in CSCs from non-small cell lung cancer (NSCLC) patients and cell lines. Rigid scaffolds with an elevated compressive modulus and uniform pores and channels were produced using different filaments. Hydrogel-based scaffolds were printed with a more irregular distribution of pores and a lower compressive modulus. As a 3D model of reference, suspension spheroid cultures were established. Therein, cancer cell lines exhibited enhanced proliferation profiles on rigid scaffolds compared to the same cells grown on either hydrogel scaffolds or tumor spheres. Meanwhile, primary cancer cells grew considerably better on hydrogel scaffolds or in tumor sphere culture, compared to cells grown on rigid scaffolds. Gene expression analysis confirmed that tumor spheres and cells seeded on hydrogel scaffolds significantly overexpress most of stemness and invasion promoters tested compared to control cells grown in 2D culture. A different phenomenon was observed within cells growing on the rigid scaffolds, where fewer significant variations in gene expression were detected. Our findings provide strong evidence for the advantageous usage of 3D printed models, especially those which use GelMA-PEGDA hydrogels as the primary scaffold material, for studying lung CSCs. The results demonstrated that the 3D printed scaffolds were better to mimic tumor complexity and regulate cancer cell behavior than in vivo 2D culture models.

Dual 3D printing for vascularized bone tissue regeneration.

The development of sufficient vascular networks is crucial for the successful fabrication of tissue constructs for regenerative medicine, as vascularization is essential to perform the metabolic functions of tissues, such as nutrient transportation and waste removal. In recent years, efforts to 3D print vascularized bone have gained substantial attention, as bone disorders and defects have a marked impact on the older generations of society. However, conventional and previous 3D printed bone studies have been plagued by the difficulty in obtaining the nanoscale geometrical precision necessary to recapitulate the distinct characteristics of natural bone. Additionally, the process of developing truly biomimetic vascularized bone tissue has been historically complex. In this study, a biomimetic nano-bone tissue construct with a perfusable, endothelialized vessel channel was developed using a combination of simple stereolithography (SLA) and fused deposition modeling (FDM) 3D printing systems. The perfusable vessel channel was created within the SLA printed bone scaffold using an FDM printed polyvinyl alcohol (PVA) sacrificial template. Within the fabricated constructs, bone tissue was formed through the osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), and distinct capillaries sprouted through the angiogenesis of the endothelialized vessel channel after human umbilical vein endothelial cells (HUVECs) had been perfused throughout. Furthermore, the fabricated constructs were evaluated in physiologically relevant culture conditions to predict tissue development after implantation in the human body. The experimental results revealed that the custom-designed bioreactor with an hMSC-HUVEC co-culture system enhanced the formation of vascular networks and the osteogenic maturation of the constructs for up to 20 days of observation.

4D Printed Cardiac Construct with Aligned Myofibers and Adjustable Curvature for Myocardial Regeneration.

As an innovative additive manufacturing process, 4D printing can be utilized to generate predesigned, self-assembly structures which can actuate time-dependent, and dynamic shape-changes. Compared to other manufacturing techniques used for tissue engineering purposes, 4D printing has the advantage of being able to fabricate reprogrammable dynamic tissue constructs that can promote uniform cellular growth and distribution. For this study, a digital light processing (DLP)-based printing technique was developed to fabricate 4D near-infrared (NIR) light-sensitive cardiac constructs with highly aligned microstructure and adjustable curvature. As the curvature of the heart is varied across its surface, the 4D cardiac constructs can change their shape on-demand to mimic and recreate the curved topology of myocardial tissue for seamless integration. To mimic the aligned structure of the human myocardium and to achieve the 4D shape change, a NIR light-sensitive 4D ink material, consisting of a shape memory polymer and graphene, was created to fabricate microgroove arrays with different widths. The results of our study illustrate that our innovative NIR-responsive 4D constructs exhibit the capacity to actuate a dynamic and remotely controllable spatiotemporal transformation. Furthermore, the optimal microgroove width was discovered via culturing human induced pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells onto the constructs' surface and analyzing both their cellular morphology and alignment. The cell proliferation profiles and differentiation of tricultured human-induced pluripotent stem cell-derived cardiomyocytes, mesenchymal stem cells, and endothelial cells, on the printed constructs, were also studied using a Cell Counting Kit-8 and immunostaining. Our results demonstrate a uniform distribution of aligned cells and excellent myocardial maturation on our 4D curved cardiac constructs. This study not only provides an efficient method for manufacturing curved tissue architectures with uniform cell distributions, but also extends the potential applications of 4D printing for tissue regeneration.

3D Bioprinting-Tunable Small-Diameter Blood Vessels with Biomimetic Biphasic Cell Layers.

Blood vessel damage resulting from trauma or diseases presents a serious risk of morbidity and mortality. Although synthetic vascular grafts have been successfully commercialized for clinical use, they are currently only readily available for large-diameter vessels (>6 mm). Small-diameter vessel (<6 mm) replacements, however, still present significant clinical challenges worldwide. The primary objective of this study is to create novel, tunable, small-diameter blood vessels with biomimetic two distinct cell layers [vascular endothelial cell (VEC) and vascular smooth muscle cell (VSMC)] using an advanced coaxial 3D-bioplotter platform. Specifically, the VSMCs were laden in the vessel wall and VECs grew in the lumen to mimic the natural composition of the blood vessel. First, a novel bioink consisting of VSMCs laden in gelatin methacryloyl (GelMA)/polyethylene(glycol)diacrylate/alginate and lyase was designed. This specific design is favorable for nutrient exchange in an ambient environment and simultaneously improves laden cell proliferation in the matrix pore without the space restriction inherent with substance encapsulation. In the vessel wall, the laden VSMCs steadily grew as the alginate was gradually degraded by lyase leaving more space for cell proliferation in matrices. Through computational fluid dynamics simulation, the vessel demonstrated significantly perfusable and mechanical properties under various flow velocities, flow viscosities, and temperature conditions. Moreover, both VSMCs in the scaffold matrix and VECs in the lumen steadily proliferated over time creating a significant two-cell-layered structure. Cell proliferation was confirmed visually through staining the markers of alpha-smooth muscle actin and cluster of differentiation 31, commonly tied to angiogenesis phenomena, in the vessel matrices and lumen, respectively. Furthermore, the results were confirmed quantitatively through gene analysis which suggested good angiogenesis expression in the blood vessels. This study demonstrated that the printed blood vessels with two distinct cell layers of VECs and VSMCs could be potential candidates for clinical small-diameter blood vessel replacement applications.

Three-Dimensional Printing Biologically Inspired DNA-Based Gradient Scaffolds for Cartilage Tissue Regeneration.

Cartilage damage caused by aging, repeated overloading, trauma, and diseases can result in chronic pain, inflammation, stiffness, and even disability. Unlike other types of tissues (bone, skin, muscle, etc.), cartilage tissue has an extremely weak regenerative capacity. Currently, the gold standard surgical treatment for repairing cartilage damage includes autografts and allografts. However, these procedures are limited by insufficient donor sources and the potential for immunological rejection. After years of development, engineered tissue now provides a valuable artificial replacement for tissue regeneration purposes. Three-dimensional (3D) bioprinting technologies can print customizable hierarchical structures with cells. The objective of the current work was to prepare a 3D-printed three-layer gradient scaffold with lysine-functionalized rosette nanotubes (RNTK) for improving the chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). Specifically, biologically inspired RNTKs were utilized in our work because they have unique surface chemistry and biomimetic nanostructure to improve cell adhesion and growth. Different ratios of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) were printed into a three-layer GelMA-PEGDA gradient scaffold using a stereolithography-based printer, followed by coating with RNTKs. The pores and channels (∼500 µm) were observed in the scaffold. It was found that the population of ADSCs on the GelMA-PEGDA-RNTK scaffold increased by 34% compared to the GelMA-PEGDA scaffold (control). Moreover, after 3 weeks of chondrogenic differentiation, collagen II, glycosaminoglycan, and total collagen synthesis on the GelMA-PEGDA-RNTK scaffold significantly respectively increased by 59%, 71%, and 60%, as compared to the control scaffold. Gene expression of collagen II α1, SOX 9, and aggrecan in the ADSCs growing on the GelMA-PEGDA-RNTK scaffold increased by 79%, 52%, and 47% after 3 weeks, compared to the controls, respectively. These results indicated that RNTKs are a promising type of nanotubes for promoting chondrogenic differentiation, and the present 3D-printed three-layer gradient GelMA-PEGDA-RNTK scaffold shows considerable promise for future cartilage repair and regeneration.

4D physiologically adaptable cardiac patch: A 4-month in vivo study for the treatment of myocardial infarction.

There has been considerable progress in engineering cardiac scaffolds for the treatment of myocardial infarction (MI). However, it is still challenging to replicate the structural specificity and variability of cardiac tissues using traditional bioengineering approaches. In this study, a four-dimensional (4D) cardiac patch with physiological adaptability has been printed by beam-scanning stereolithography. By combining a unique 4D self-morphing capacity with expandable microstructure, the specific design has been shown to improve both the biomechanical properties of the patches themselves and the dynamic integration of the patch with the beating heart. Our results demonstrate improved vascularization and cardiomyocyte maturation in vitro under physiologically relevant mechanical stimulation, as well as increased cell engraftment and vascular supply in a murine chronic MI model. This work not only potentially provides an effective treatment method for MI but also contributes a cutting-edge methodology to enhance the structural design of complex tissues for organ regeneration.

Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation.

OBJECTIVES: Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures. METHODS: A customized ultrasound (US) exposure setup was used for the variation of key US parameters: intensity, excitation duration, and duty cycle. Cell proliferation was quantified by 5-bromo-2'-deoxyuridine and alamarBlue assays after LIPUS excitation. RESULTS: At a 20% duty cycle and 10-minute excitation period, we varied LIPUS intensity from to 100 mW/cm2 (spatial-average temporal-average) to find a gradual decrease in T47D cell proliferation, the decrease being strongest at 100 mW/cm2 . In contrast, healthy MCF-12A breast cells showed an increase in proliferation when exposed to the same conditions. Above a 60% duty cycle, T47D cell proliferation decreased drastically. Effects of continuous wave US stimulation were further explored by varying the intensity and excitation period. CONCLUSIONS: These experiments concluded that, irrespective of the waveform (pulsed or continuous), LIPUS stimulation could inhibit the proliferation of T47D breast cancer cells, whereas the same behavior was not observed in healthy cells. The study demonstrates the beneficial bioeffects of LIPUS on breast cancer cells and offers the possibility of developing novel US-mediated cancer therapy.

Integrating cold atmospheric plasma with 3D printed bioactive nanocomposite scaffold for cartilage regeneration.

The progressive degeneration of articular cartilage or osteoarthritis of the knee is a serious clinical problem affecting patient quality of life. In recent years, artificially engineered cartilage scaffolds have been widely studied as a promising method to stimulate cartilage regeneration. In this study, a novel biomimetic cartilage scaffold was developed by integrating a cold atmospheric plasma (CAP) treatment with prolonged release of bioactive factors. Specifically, a surface of 3D printed hydrogel scaffold with drug-loaded nanoparticles was treated with CAP. Our results showed that the scaffolds with CAP treatment can improve hydrophilicity as well as surface nano-roughness and can thus facilitate stem cell adhesion. More importantly, this study demonstrated that integrating CAP treatment with drug-loaded nanoparticles can synergistically enhance chondrogenesis of human bone marrow mesenchymal stem cells when compared to control scaffolds. The results in this study indicate the great potential of applying CAP and drug-loaded nanoparticles into 3D printed tissue scaffolds for promoting cartilage regeneration.