Implantable system for chronotherapy.

Diurnal variation in enzymes, hormones, and other biological mediators has long been recognized in mammalian physiology. Developments in pharmacobiology over the past few decades have shown that timing drug delivery can enhance drug efficacy. Here, we report the development of a battery-free, refillable, subcutaneous, and trocar-compatible implantable system that facilitates chronotherapy by enabling tight control over the timing of drug administration in response to external mechanical actuation. The external wearable system is coupled to a mobile app to facilitate control over dosing time. Using this system, we show the efficacy of bromocriptine on glycemic control in a diabetic rat model. We also demonstrate that antihypertensives can be delivered through this device, which could have clinical applications given the recognized diurnal variation of hypertension-related complications. We anticipate that implants capable of chronotherapy will have a substantial impact on our capacity to enhance treatment effectiveness for a broad range of chronic conditions.

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 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.

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.

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.

Enhanced neuronal differentiation of neural stem cells with mechanically enhanced touch-spun nanofibrous scaffolds.

We studied NE-4C neural cells differentiation on 2D polycaprolactone (PCL) nanofibrous scaffolds with systematically varied mechanical characteristics of nanofibers while retaining an unchanged fiber alignment, diameter, and chemical composition. Our experiments demonstrated that the nanofibers with enhanced mechanical properties are beneficial for the preferential development of neuronal cells vs. glial cells. Electrospun (ES) and touch-spun (TS) nanofibers were fabricated with Young's modulus in the range of 10 MPa to 230 MPa and a fraction of crystallinity from 30% to 80%. The TS fibers undergo a greater drawing ratio and thus approach a greater polymer chain stretching and alignment that resulted in an increased crystallinity. The TS scaffolds demonstrated improved stability in the aqueous cell culture environment, resisting misalignment and entanglement after a period of 2 weeks of swelling followed by 14 days of neural differentiation. The results confirmed that the neurites on the TS fibers have a preferred orientation even after swelling.

Touch-Spun Nanofibers for Nerve Regeneration.

In the current study, we examined the potential for neural stem cell (NSCs) proliferation on novel aligned touch-spun polycaprolactone (PCL) nanofibers. Electrospun PCL nanofibers with similar diameter and alignment were used as a control. Confocal microscopy images showed that NSCs grew and differentiated all over the scaffolds up to 8 days. Neurite quantification analysis revealed that the NSCs cultured on the touch-spun fibers with incorporated bovine serum albumin promoted the expression of neuron-specific class III ß-tubulin after 8 days. More importantly, NSCs grown on the aligned touch-spun PCL fibers exhibited a bipolar elongation along the direction of the fiber, while NSCs cultured on the aligned electrospun PCL fibers expressed a multipolar elongation. The structural characteristics of the PCL nanofibers analyzed by X-ray diffraction indicated that the degree of crystallinity and elastic modulus of the touch-spun fiber are significantly higher than those of electrospun fibers. These findings indicate that the aligned and stiff touch-spun nanofibrous scaffolds show considerable potential for nerve injury repair.

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.

Development of 3D printable conductive hydrogel with crystallized PEDOT:PSS for neural tissue engineering.

Bioelectronic devices enable efficient and effective communication between medical devices and human tissue in order to directly treat patients with various neurological disorders. Due to the mechanical similarity to human tissue, hydrogel-based electronic devices are considered to be promising for biological signal recording and stimulation of living tissues. Here, we report the first three-dimensionally (3D) printable conductive hydrogel that can be photocrosslinked while retaining high electrical conductivity. In addition, we prepared dorsal root ganglion (DRG) cell-encapsulated gelatin methacryloyl (GelMA) hydrogels which were integrated with the 3D printed conductive structure and evaluated for efficiency neural differentiation under electrical stimulation (ES). For enhanced electrical conductivity, a poly(3,4-ethylenedioxythiophene) (PEDOT): polystyrene sulfonate (PSS) aqueous solution was freeze-dried and mixed with polyethylene glycol diacrylate (PEGDA) as the photocurable polymer base. Next, the conductive hydrogel was patterned on the substrate by using a table-top stereolithography (SLA) 3D printer. The fabricated hydrogel was characterized for electrochemical conductivity. After printing with the PEDOT:PSS conductive solution, the patterned hydrogel exhibited decreased printing diameters with increasing of PEDOT:PSS concentration. Also, the resultant conductive hydrogel had significantly increased electrochemical properties with increasing PEDOT:PSS concentration. The 3D printed conductive hydrogel provides excellent structural support to systematically transfer the ES toward encapsulated DRG cells for enhanced neuronal differentiation. The results from this study indicate that the conductive hydrogel can be useful as a 3D printing material for electrical 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.

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.

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.

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.

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.