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.

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.

Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns, respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues.

Transversus abdominis plane block using a short-acting local anesthetic for postoperative pain after laparoscopic colorectal surgery: a systematic review and meta-analysis.

BACKGROUND: Transversus abdominis plane (TAP) block using a short-acting local anesthetic as part of multimodal analgesia is efficient in various abdominal surgeries, including laparoscopic surgery. However, information regarding its use in laparoscopic colorectal surgery is still limited and sometimes controversial. Therefore, we conducted a systematic review and meta-analysis to determine whether TAP block using a short-acting anesthetic has a positive postoperative analgesic outcome in patients who have undergone laparoscopic colorectal surgery. METHODS: We searched for studies comparing the postoperative pain outcome after laparoscopic colorectal surgery between patients who received TAP block and a control group (placebo or no treatment). Outcome measures were early pain at rest (numeric rating scale [NRS] score at 0-2 h postoperatively), late pain at movement (NRS score at 24 h postoperatively), late pain at rest (NRS score at 24 h postoperatively), and postoperative opioid consumption (up to 24 h postoperatively). We used a random-effects model for the meta-analysis and Egger's regression test to detect publication bias. RESULTS: We included six studies involving 452 patients (224 in the TAP block group, 228 in the control group). Early and late pain scores at movement were significantly different between the TAP block and control groups (standardized mean difference: - 0.695, P < 0.0001 for early pain and - 0.242, P = 0.029 for late pain). There was no significant difference between the TAP block and control groups in early pain at rest (P = 0.475), late pain at rest (P = 0.826), and postoperative opioid consumption (P = 0.257). CONCLUSIONS: The TAP block using a short-acting anesthetic had a significant effect on the postoperative pain outcome in the early (0-2 h) and late (24 h) period at movement. However, it did not have a significant effect on the postoperative pain outcome in the early (0-2 h) and late (24 h) periods at rest after laparoscopic surgery.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Directly Induced Neural Differentiation of Human Adipose-Derived Stem Cells Using Three-Dimensional Culture System of Conductive Microwell with Electrical Stimulation.

Adipose-derived stem cells (ADSCs) have the capacity to differentiate into neural precursor cells which can be used for nerve regeneration. However, their inherently low neurogenic differentiation efficiency limits further clinical applications. This study was designed to promote neurogenic differentiation efficacy of ADSCs by integrating conductive hydrogel-based microwells with electrical stimulation (ES). We hypothesize that ADSCs will differentiate more efficiently into neural precursor cells when electrically stimulated in conductive hydrogel microwells. To make the conductive hydrogel-based microwell, polyethylene glycol (PEG) diacrylate aqueous solution mixed with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was patterned with the polydimethylsiloxane mold and exposed to UV light to induce photo-cross-linking of the conductive hydrogel. After seeding the ADSCs in the microwells, the cells formed distinct cell spheres in PEG microwells and wide disks in the PEG/PEDOT:PSS microwells. Although the microwells yielded varying three-dimensional (3D) cell aggregate structure, cell viability was not affected. After neurogenic differentiation with ES, the ADSC aggregates in PEG/PEDOT:PSS microwells with ES expressed greater positive neuronal differentiation markers compared to nonstimulated PEG/PEDOT:PSS microwells. Although all neuronal gene expression levels were greater in PEG microwells with ES, the increased rates of gene expression levels between treated and untreated PEG/PEDOT:PSS microwells were much higher compared to PEG microwells. This would mean that electrically stimulating ADSC aggregates in conductive microwells is an effective method in increasing neurogenic differentiation. Therefore, we propose a most effective strategy taking advantage of a 3D conductive culture system which can be useful in a wide variety of electrical application.