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.

4D Self-Morphing Culture Substrate for Modulating Cell Differentiation.

As the most versatile and promising cell source, stem cells have been studied in regenerative medicine for two decades. Currently available culturing techniques utilize a 2D or 3D microenvironment for supporting the growth and proliferation of stem cells. However, these culture systems fail to fully reflect the supportive biological environment in which stem cells reside in vivo, which contain dynamic biophysical growth cues. Herein, a 4D programmable culture substrate with a self-morphing capability is presented as a means to enhance dynamic cell growth and induce differentiation of stem cells. To function as a model system, a 4D neural culture substrate is fabricated using a combination of printing and imprinting techniques keyed to the different biological features of neural stem cells (NSCs) at different differentiation stages. Results show the 4D culture substrate demonstrates a time-dependent self-morphing process that plays an essential role in regulating NSC behaviors in a spatiotemporal manner and enhances neural differentiation of NSCs along with significant axonal alignment. This study of a customized, dynamic substrate revolutionizes current stem cell therapies, and can further have a far-reaching impact on improving tissue regeneration and mimicking specific disease progression, as well as other impacts on materials and life science research.

In vitro and in vivo evaluation of 3D bioprinted small-diameter vasculature with smooth muscle and endothelium.

The ability to fabricate perfusable, small-diameter vasculature is a foundational step toward generating human tissues/organs for clinical applications. Currently, it is highly challenging to generate vasculature integrated with smooth muscle and endothelium that replicates the complexity and functionality of natural vessels. Here, a novel method for directly printing self-standing, small-diameter vasculature with smooth muscle and endothelium is presented through combining tailored mussel-inspired bioink and unique 'fugitive-migration' tactics, and its effectiveness and advantages over other methods (i.e. traditional alginate/calcium hydrogel, post-perfusion of endothelial cells) are demonstrated. The biologically inspired, catechol-functionalized, gelatin methacrylate (GelMA/C) undergoes rapid oxidative crosslinking in situ to form an elastic hydrogel, which can be engineered with controllable mechanical strength, high cell/tissue adhesion, and excellent bio-functionalization. The results demonstrate the bioprinted vascular construct possessed numerous favorable, biomimetic characteristics such as proper biomechanics, higher tissue affinity, vascularized tissue manufacturing ability, beneficial perfusability and permeability, excellent vasculoactivity, and in vivo autonomous connection (∼2 weeks) as well as vascular remodeling (∼6 weeks). The advanced achievements in creating biomimetic, functional vasculature illustrate significant potential toward generating a complicated vascularized tissue/organ for clinical transplantation.

4D anisotropic skeletal muscle tissue constructs fabricated by staircase effect strategy.

Like the morphology of native tissue fiber arrangement (such as skeletal muscle), unidirectional anisotropic scaffolds are highly desired as a means to guide cell behavior in anisotropic tissue engineering. In contrast, contour-like staircases exhibit directional topographical cues and are judged as an inevitable defect of fused deposition modeling (FDM). In this study, we will translate this staircase defect into an effective bioengineering strategy by integrating FDM with surface coating technique (FCT) to investigate the effect of topographical cues on regulating behaviors of human mesenchymal stem cells (hMSCs) toward skeletal muscle tissues. This integrated approach serves to fabricate shape-specific, multiple dimensional, anisotropic scaffolds using different biomaterials. 2D anisotropic scaffolds, first demonstrated with different polycaprolactone concentrations herein, efficiently direct hMSC alignment, especially when the scaffold is immobilized on a support ring. By surface coating the polymer solution inside FDM-printed sacrificial structures, 3D anisotropic scaffolds with thin wall features are developed and used to regulate seeded hMSCs through a self-established rotating bioreactor. Using layer-by-layer coating, along with a shape memory polymer, smart constructs exhibiting shape fix and recovery processes are prepared, bringing this study into the realm of 4D printing. Immunofluorescence staining and real-time quantitative polymerase chain reaction analysis confirm that the topographical cues created via FCT significantly enhance the expression of myogenic genes, including myoblast differentiation protein-1, desmin, and myosin heavy chain-2. We conclude that there are broad application potentials for this FCT strategy in tissue engineering as many tissues and organs, including skeletal muscle, possess highly organized and anisotropic extracellular matrix components.

Recent advances in 3D printing: vascular network for tissue and organ regeneration.

Over the past years, the fabrication of adequate vascular networks has remained the main challenge in engineering tissues due to technical difficulties, while the ultimate objective of tissue engineering is to create fully functional and sustainable organs and tissues to transplant in the human body. There have been a number of studies performed to overcome this limitation, and as a result, 3D printing has become an emerging technique to serve in a variety of applications in constructing vascular networks within tissues and organs. 3D printing incorporated technical approaches allow researchers to fabricate complex and systematic architecture of vascular networks and offer various selections for fabrication materials and printing techniques. In this review, we will discuss materials and strategies for 3D printed vascular networks as well as specific applications for certain vascularized tissue and organ regeneration. We will also address the current limitations of vascular tissue engineering and make suggestions for future directions research may take.

3D Printed scaffolds with hierarchical biomimetic structure for osteochondral regeneration.

Osteochondral defects resulting from trauma and/or pathologic disorders are critical clinical problems. The current approaches still do not yield satisfactory due to insufficient donor sources and potential immunological rejection of implanted tissues. 3D printing technology has shown great promise for fabricating customizable, biomimetic tissue matrices. The purpose of the present study is to investigate 3D printed scaffolds with biomimetic, biphasic structure for osteochondral regeneration. For this purpose, nano-hydroxyapatite and transforming growth factor beta 1 nanoparticles were synthesized and distributed separately into the lower and upper layers of the biphasic scaffold, which was fabricated using 3D stereolithography printer. Our results showed that this scaffold design successfully promoted osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells, as well as enhanced gene expression associated with both osteogenesis and chondrogenesis alike. The finding demonstrated that 3D printed osteochondral scaffolds with biomimetic, biphasic structure are excellent candidates for osteochondral repair and regeneration.

A novel near-infrared light responsive 4D printed nanoarchitecture with dynamically and remotely controllable transformation.

Four-dimensional (4D) printing is an emerging and highly innovative additive manufacturing process by which to fabricate pre-designed, self-assembly structures with the ability to transform over time. However, one of the critical challenges of 4D printing is the lack of advanced 4D printing systems that not only meet all the essential requirements of shape change but also possess smart, dynamic capabilities to spatiotemporally and instantly control the shape-transformation process. Here, we present a facile 4D printing platform which incorporates nanomaterials into the conventional stimuli-responsive polymer, allowing the 4D printed object to achieve a dynamic and remote controlled, on-time and position shape transformation. A proof-of-concept 4D printed brain model was created using near-infrared light (NIR) responsive nanocomposite to evaluate the capacity for controllable 4D transformation, and the feasibility of photothermal stimulation for modulating neural stem cell behaviors. This novel 4D printing strategy can not only be used to create dynamic 3D patterned biological structures that can spatiotemporally control their shapes or behaviors of transformation under a human benign stimulus (NIR), but can also provide a potential method for building complex self-morphing objects for widespread applications.

Advanced 4D Bioprinting Technologies for Brain Tissue Modeling and Study.

Although the process by which the cortical tissues of the brain fold has been the subject of considerable study and debate over the past few decades, a single mechanistic description of the phenomenon has yet to be fully accepted. Rather, two competing explanations of cortical folding have arisen in recent years; known as the axonal tension and the differential tangential expansion models. In the present review, these two models are introduced by analyzing the computational, theoretical, materials-based, and cell studies which have yielded them. Then Four-dimensional bioprinting is presented as a powerful technology which can not only be used to test both models of cortical folding de novo, but can also be used to explore the reciprocal effects that folding associated mechanical stresses may have on neural development. Therein, the fabrication of "smart" tissue models which can accurately simulate the in vivo folding process and recapitulate physiologically relevant stresses are introduced. We also provide a general description of both cortical neurobiology as well as the cellular basis of cortical folding. Our discussion also entails an overview of both 3D and 4D bioprinting technologies, as well as a brief commentary on recent advancements in printed central nervous system tissue engineering.

3D bioprinting for cardiovascular regeneration and pharmacology.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide. Compared to traditional therapeutic strategies, three-dimensional (3D) bioprinting is one of the most advanced techniques for creating complicated cardiovascular implants with biomimetic features, which are capable of recapitulating both the native physiochemical and biomechanical characteristics of the cardiovascular system. The present review provides an overview of the cardiovascular system, as well as describes the principles of, and recent advances in, 3D bioprinting cardiovascular tissues and models. Moreover, this review will focus on the applications of 3D bioprinting technology in cardiovascular repair/regeneration and pharmacological modeling, further discussing current challenges and perspectives.

Photolithographic-stereolithographic-tandem fabrication of 4D smart scaffolds for improved stem cell cardiomyogenic differentiation.

4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

Three-Dimensional-Bioprinted Dopamine-Based Matrix for Promoting Neural Regeneration.

Central nerve repair and regeneration remain challenging problems worldwide, largely because of the extremely weak inherent regenerative capacity and accompanying fibrosis of native nerves. Inadequate solutions to the unmet needs for clinical therapeutics encourage the development of novel strategies to promote nerve regeneration. Recently, 3D bioprinting techniques, as one of a set of valuable tissue engineering technologies, have shown great promise toward fabricating complex and customizable artificial tissue scaffolds. Gelatin methacrylate (GelMA) possesses excellent biocompatible and biodegradable properties because it contains many arginine-glycine-aspartic acids (RGD) and matrix metalloproteinase sequences. Dopamine (DA), as an essential neurotransmitter, has proven effective in regulating neuronal development and enhancing neurite outgrowth. In this study, GelMA-DA neural scaffolds with hierarchical structures were 3D-fabricated using our custom-designed stereolithography-based printer. DA was functionalized on GelMA to synthesize a biocompatible printable ink (GelMA-DA) for improving neural differentiation. Additionally, neural stem cells (NSCs) were employed as the primary cell source for these scaffolds because of their ability to terminally differentiate into a variety of cell types including neurons, astrocytes, and oligodendrocytes. The resultant GelMA-DA scaffolds exhibited a highly porous and interconnected 3D environment, which is favorable for supporting NSC growth. Confocal microscopy analysis of neural differentiation demonstrated that a distinct neural network was formed on the GelMA-DA scaffolds. In particular, the most significant improvements were the enhanced neuron gene expression of TUJ1 and MAP2. Overall, our results demonstrated that 3D-printed customizable GelMA-DA scaffolds have a positive role in promoting neural differentiation, which is promising for advancing nerve repair and regeneration in the future.

Stereolithographic 4D Bioprinting of Multiresponsive Architectures for Neural Engineering.

4D printing represents one of the most advanced fabrication techniques for prospective applications in tissue engineering, biomedical devices, and soft robotics, among others. In this study, a novel multiresponsive architecture is developed through stereolithography-based 4D printing, where a universal concept of stress-induced shape transformation is applied to achieve the 4D reprogramming. The light-induced graded internal stress followed by a subsequent solvent-induced relaxation, driving an autonomous and reversible change of the programmed configuration after printing, is employed and investigated in depth and details. Moreover, the fabricated construct possesses shape memory property, offering a characteristic of multiple shape change. Using this novel multiple responsive 4D technique, a proof-of-concept smart nerve guidance conduit is demonstrated on a graphene hybrid 4D construct providing outstanding multifunctional characteristics for nerve regeneration including physical guidance, chemical cues, dynamic self-entubulation, and seamless integration. By employing this fabrication technique, creating multiresponsive smart architectures, as well as demonstrating application potential, this work paves the way for truly initiation of 4D printing in various high-value research fields.

Bio-Based Polymers for 3D Printing of Bioscaffolds.

Three-dimensional (3D) printing technologies enable not only faster bioconstructs development but also on-demand and customized manufacturing, offering patients a personalized biomedical solution. This emerging technique has a great potential for fabricating bioscaffolds with complex architectures and geometries and specifically tailored for use in regenerative medicine. The next major innovation in this area will be the development of biocompatible and histiogenic 3D printing materials with bio-based printable polymers. This review will briefly discuss 3D printing techniques and their current limitations, with a focus on novel bio-based polymers as 3D printing feedstock for clinical medicine and tissue regeneration.

Enhanced neural stem cell functions in conductive annealed carbon nanofibrous scaffolds with electrical stimulation.

Carbon-based nanomaterials have shown great promise in regenerative medicine because of their unique electrical, mechanical, and biological properties; however, it is still difficult to engineer 2D pure carbon nanomaterials into a 3D scaffold while maintaining its structural integrity. In the present study, we developed novel carbon nanofibrous scaffolds by annealing electrospun mats at elevated temperature. The resultant scaffold showed a cohesive structure and excellent mechanical flexibility. The graphitic structure generated by annealing renders superior electrical conductivity to the carbon nanofibrous scaffold. By integrating the conductive scaffold with biphasic electrical stimulation, neural stem cell proliferation was promoted associating with upregulated neuronal gene expression level and increased microtubule-associated protein 2 immunofluorescence, demonstrating an improved neuronal differentiation and maturation. The findings suggest that the integration of the conducting carbon nanofibrous scaffold and electrical stimulation may pave a new avenue for neural tissue regeneration.

4D printing of polymeric materials for tissue and organ regeneration.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex, stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applications. Although the 4D concept was first highlighted in 2013, extensive research has rapidly developed, along with more-in-depth understanding and assertions regarding the definition of 4D. In this review, we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in the field. Both transformation-preprogrammed 4D printing and 4D printing of shape memory polymers are intensively surveyed. Afterwards we will explore and discuss the applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissues and implantable scaffolds, as well as future perspectives and conclusions.

3D Bioprinting a Cell-Laden Bone Matrix for Breast Cancer Metastasis Study.

Metastasis is one of the deadliest consequences of breast cancer, with bone being one of the primary sites of occurrence. Insufficient 3D biomimetic models currently exist to replicate this process in vitro. In this study, we developed a biomimetic bone matrix using 3D bioprinting technology to investigate the interaction between breast cancer (BrCa) cells and bone stromal cells (fetal osteoblasts and human bone marrow mesenchymal stem cells (MSCs)). A tabletop stereolithography 3D bioprinter was employed to fabricate a series of bone matrices consisting of osteoblasts or MSCs encapsulated in gelatin methacrylate (GelMA) hydrogel with nanocrystalline hydroxyapatite (nHA). When BrCa cells were introduced into the stromal cell-laden bioprinted matrices, we found that the growth of BrCa cells was enhanced by the presence of osteoblasts or MSCs, whereas the proliferation of the osteoblasts or MSCs was inhibited by the BrCa cells. The BrCa cells co-cultured with MSCs or osteoblasts presented increased vascular endothelial growth factor (VEGF) secretion in comparison to that of monocultured BrCa cells. Additionally, the alkaline phosphatase activity of MSCs or osteoblasts was reduced after BrCa cell co-culture. These results demonstrate that the 3D bioprinted matrix, with BrCa cells and bone stromal cells, provides a suitable model with which to study the interactive effects of cells in the context of an artificial bone microenvironment and thus may serve as a valuable tool for the investigation of postmetastatic breast cancer progression in bone.

4D printing smart biomedical scaffolds with novel soybean oil epoxidized acrylate.

Photocurable, biocompatible liquid resins are highly desired for 3D stereolithography based bioprinting. Here we solidified a novel renewable soybean oil epoxidized acrylate, using a 3D laser printing technique, into smart and highly biocompatible scaffolds capable of supporting growth of multipotent human bone marrow mesenchymal stem cells (hMSCs). Porous scaffolds were readily fabricated by simply adjusting the printer infill density; superficial structures of the polymerized soybean oil epoxidized acrylate were significantly affected by laser frequency and printing speed. Shape memory tests confirmed that the scaffold fixed a temporary shape at -18 °C and fully recovered its original shape at human body temperature (37 °C), which indicated the great potential for 4D printing applications. Cytotoxicity analysis proved that the printed scaffolds had significant higher hMSC adhesion and proliferation than traditional polyethylene glycol diacrylate (PEGDA), and had no statistical difference from poly lactic acid (PLA) and polycaprolactone (PCL). This research is believed to significantly advance the development of biomedical scaffolds with renewable plant oils and advanced 3D fabrication techniques.

High-Intensity Sweeteners in Alternative Tobacco Products.

INTRODUCTION: Sweeteners in tobacco products may influence use initiation and reinforcement, with special appeal to adolescents. Recent analytical studies of smokeless tobacco products (snuff, snus, dissolvables) detected flavorants identical to those added to confectionary products such as hard candy and chewing gum. However, these studies did not determine the levels of sweeteners. The objective of the present study was to quantify added sweeteners in smokeless tobacco products, a dissolvable product, electronic cigarette liquids and to compare with sweetener levels in confectionary products. METHODS: Sweetener content of US-sourced smokeless tobacco, electronic cigarette liquid, and confectionary product samples was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). RESULTS: All smokeless products contained synthetic high intensity sweeteners, with snus and dissolvables exceeding levels in confectionary products (as much as 25-fold). All snus samples contained sucralose and most also aspartame, but no saccharin. In contrast, all moist snuff samples contained saccharin. The dissolvable sample contained sucralose and sorbitol. Ethyl maltol was the most common sweet-associated component in electronic cigarette liquids. DISCUSSION: Sweetener content was dependent on product category, with saccharin in moist snuff, an older category, sucralose added at high levels to more recently introduced products (snus, dissolvable) and ethyl maltol in electronic cigarette liquid. The very high sweetener concentrations may be necessary for the consumer to tolerate the otherwise aversive flavors of tobacco ingredients. Regulation of sweetener levels in smokeless tobacco products may be an effective measure to modify product attractiveness, initiation and use patterns. IMPLICATIONS: Dissolvables, snus and electronic cigarettes have been promoted as risk-mitigation products due to their relatively low content of nitrosamines and other tobacco toxicants. This study is the first to quantify high intensity sweeteners in snus and dissolvable products. Snus and dissolvables contain the high intensity sweetener, sucralose, at levels higher than in confectionary products. The high sweetness of alternative tobacco products makes these products attractive to adolescents. Regulation of sweetener content in non-cigarette products is suggested as an efficient means to control product palatability and to reduce initiation in adolescents.

Four-Dimensional Printing Hierarchy Scaffolds with Highly Biocompatible Smart Polymers for Tissue Engineering Applications.

The objective of this study was to four-dimensional (4D) print novel biomimetic gradient tissue scaffolds with highly biocompatible naturally derived smart polymers. The term "4D printing" refers to the inherent smart shape transformation of fabricated constructs when implanted minimally invasively for seamless and dynamic integration. For this purpose, a series of novel shape memory polymers with excellent biocompatibility and tunable shape changing effects were synthesized and cured in the presence of three-dimensional printed sacrificial molds, which were subsequently dissolved to create controllable and graded porosity within the scaffold. Surface morphology, thermal, mechanical, and biocompatible properties as well as shape memory effects of the synthesized smart polymers and resultant porous scaffolds were characterized. Fourier transform infrared spectroscopy and gel content analysis confirmed the formation of chemical crosslinking by reacting polycaprolactone triol and castor oil with multi-isocyanate groups. Differential scanning calorimetry revealed an adjustable glass transition temperature in a range from -8°C to 35°C. Uniaxial compression testing indicated that the obtained polymers, possessing a highly crosslinked interpenetrating polymeric networks, have similar compressive modulus to polycaprolactone. Shape memory tests revealed that the smart polymers display finely tunable recovery speed and exhibit greater than 92% shape fixing at -18°C or 0°C and full shape recovery at physiological temperature. Scanning electron microscopy analysis of fabricated scaffolds revealed a graded microporous structure, which mimics the nonuniform distribution of porosity found within natural tissues. With polycaprolactone serving as a control, human bone marrow-derived mesenchymal stem cell adhesion, proliferation, and differentiation greatly increased on our novel smart polymers. The current work will significantly advance the future design and development of novel and functional biomedical scaffolds with advanced 4D printing technology and highly biocompatible smart biomaterials.

Rhamnolipids as platform molecules for production of potential anti-zoospore agrochemicals.

Rhamnolipid biosurfactants have potential applications in the control of zoosporic plant pathogens. However, rhamnolipids have not been closely investigated for the anti-zoospore mechanism or for developing new anti-zoospore chemicals. In this study, RhL-1 and RhL-3 groups of rhamnolipids were used to generate the corresponding RhL-2 and RhL-4 groups and the free diacids. Conversion of RhL-3 to RhL-1 was also accomplished in vitro with cellobiase as the catalyst. The anti-zoospore effects of RhL-1-RhL-4 and the diacids were investigated with zoospores of Phytophthora sojae. For RhL-1-RhL-4, approximately 20, 30, 40, and 40 mg/L, respectively, were found to be the lowest concentrations required to stop movement of all zoospores, which indicates that the anti-zoospore effect remains strong even after RhL-1 and RhL-3 are hydrolyzed into RhL-2 and RhL-4. The free diacids required a significantly higher critical concentration of about 125 mg/L. Rhamnose can be obtained as a co-product.