Dilation-Responsive Microshape Programing Prevents Vascular Graft Stenosis.

Shape memory materials have been successfully applied to minimally invasive implantation of medical devices. However, organ-movement-specific shape programing at a microscale level has never been demonstrated despite significant unmet needs. As vein-to-artery grafting induces vein dilation and stenosis, a polymeric self-enclosable external support (SES) is designed to wrap the vascular out-wall. Its micropores are programmed to increase sizes and interconnections upon dilation. Vessel dilation promotes venous maturation, but overdilation induces stenosis by disturbed blood flow. Therefore, the unique elastic shape-fixity of SES provides a foundation to enable a stable microscale shape transition by maintaining the vein dilation. The shape transition of micropore architecture upon dilation induces beneficial inflammation, thereby regenerating vasa vasorum and directing smooth muscle cell migration toward adventitia with the consequent muscle reinforcement of veins. This game-changer approach prevents the stenosis of vein-to-artery grafting by rescuing ischemic disorders and promoting arterial properties of veins.

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.

Potential roles of aquaporin 9 in the pathogenesis of endometriosis.

Aquaporins (AQPs) are involved in cell migration, proliferation and carcinogenesis in tumor development and physiologic inflammatory processes, but their associations with endometriosis have not been fully evaluated. In this study, tissue samples were obtained from women undergoing laparoscopic surgery for endometriosis and other benign conditions. Analysis of expressions of AQP subtypes in eutopic and ectopic endometrium of patients with endometriosis (Eu-EMS and Ect-EMS, respectively) and eutopic endometrium of control patients without endometriosis (Eu-CTL) were performed using the NanoString nCounter System and western blotting. Human endometrial stromal cells (HESCs) were cultured and transfected with the siRNA of the AQP of interest. Among the AQP1-9 subtypes, endometrial expression of AQP2 and AQP8 was significantly increased, whereas AQP9 expression was significantly decreased in the Eu-EMS group compared to the Eu-CTL group. Comparison of expression of AQP2, AQP8 and AQP9 among Eu-EMS, Ect-EMS and Eu-CTL groups revealed significant differences for only AQP9. Expression of AQP9 in the Eu-EMS group was decreased compared with that in Eu-CTL. After transfection of AQP9 siRNA in HESCs, expressions of MMP2 and MMP9 were significantly elevated. Increased expression of phosphorylated ERK 1/2 and phosphorylated p38 MAPK proteins after transfection was also confirmed using western blot analysis. Increased migration and invasion potentials of HESCs after transfection were determined by migration and wound healing assays. These findings suggest that AQP9 may be involved in the pathogenesis of endometriosis and warrant further investigation as a potential therapeutic target for treating endometriosis.

Therapeutic Efficacy-Potentiated and Diseased Organ-Targeting Nanovesicles Derived from Mesenchymal Stem Cells for Spinal Cord Injury Treatment.

Human mesenchymal stem cell (hMSC)-derived exosomes have been spotlighted as a promising therapeutic agent for cell-free regenerative medicine. However, poor organ-targeting ability and insufficient therapeutic efficacy of systemically injected hMSC-exosomes were identified as critical limitations for their further applications. Therefore, in this study we fabricated iron oxide nanoparticle (IONP)-incorporated exosome-mimetic nanovesicles (NV-IONP) from IONP-treated hMSCs and evaluated their therapeutic efficacy in a clinically relevant model for spinal cord injury. Compared to exosome-mimetic nanovesicles (NV) prepared from untreated hMSCs, NV-IONP not only contained IONPs which act as a magnet-guided navigation tool but also carried greater amounts of therapeutic growth factors that can be delivered to the target cells. The increased amounts of therapeutic growth factors inside NV-IONP were attributed to IONPs that are slowly ionized to iron ions which activate the JNK and c-Jun signaling cascades in hMSCs. In vivo systemic injection of NV-IONP with magnetic guidance significantly increased the amount of NV-IONP accumulating in the injured spinal cord. Accumulated NV-IONP enhanced blood vessel formation, attenuated inflammation and apoptosis in the injured spinal cord, and consequently improved spinal cord function. Taken together, these findings highlight the development of therapeutic efficacy-potentiated extracellular nanovesicles and demonstrate their feasibility for repairing injured spinal cord.

A Disposable Photovoltaic Patch Controlling Cellular Microenvironment for Wound Healing.

Electrical stimulation (ES) is known to affect the wound healing process by modulating skin cell behaviors. However, the conventional clinical devices that can generate ES for promoting wound healing require patient hospitalization due to large-scale of the extracorporeal devices. Herein, we introduce a disposable photovoltaic patch that can be applied to skin wound sites to control cellular microenvironment for promoting wound healing by generating ES. In vitro experiment results show that exogenous ES could enhance cell migration, proliferation, expression of extracellular matrix proteins, and myoblast differentiation of fibroblasts which are critical for wound healing. Our disposable photovoltaic patches were attached to the back of skin wound induced mice. Our patch successfully provided ES, generated by photovoltaic energy harvested from the organic solar cell under visible light illumination. In vivo experiment results show that the patch promoted cutaneous wound healing via enhanced host-inductive cell proliferation, cytokine secretion, and protein synthesis which is critical for wound healing process. Unlike the current treatments for wound healing that engage passive healing processes and often are unsuccessful, our wearable photovoltaic patch can stimulate regenerative activities of endogenous cells and actively contribute to the wound healing processes.

Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells.

Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs.

The Role of Vasculature and Angiogenic Strategies in Bone Regeneration.

Bone regeneration is a complex process that involves various growth factors, cell types, and extracellular matrix components. A crucial aspect of this process is the formation of a vascular network, which provides essential nutrients and oxygen and promotes osteogenesis by interacting with bone tissue. This review provides a comprehensive discussion of the critical role of vasculature in bone regeneration and the applications of angiogenic strategies, from conventional to cutting-edge methodologies. Recent research has shifted towards innovative bone tissue engineering strategies that integrate vascularized bone complexes, recognizing the significant role of vasculature in bone regeneration. The article begins by examining the role of angiogenesis in bone regeneration. It then introduces various in vitro and in vivo applications that have achieved accelerated bone regeneration through angiogenesis to highlight recent advances in bone tissue engineering. This review also identifies remaining challenges and outlines future directions for research in vascularized bone regeneration.

3D printing modality effect: Distinct printing outcomes dependent on selective laser sintering (SLS) and melt extrusion.

A direct and comprehensive comparative study on different 3D printing modalities was performed. We employed two representative 3D printing modalities, laser- and extrusion-based, which are currently used to produce patient-specific medical implants for clinical translation, to assess how these two different 3D printing modalities affect printing outcomes. The same solid and porous constructs were created from the same biomaterial, a blend of 96% poly-ε-caprolactone (PCL) and 4% hydroxyapatite (HA), using two different 3D printing modalities. Constructs were analyzed to assess their printing characteristics, including morphological, mechanical, and biological properties. We also performed an in vitro accelerated degradation study to compare their degradation behaviors. Despite the same input material, the 3D constructs created from different 3D printing modalities showed distinct differences in morphology, surface roughness and internal void fraction, which resulted in different mechanical properties and cell responses. In addition, the constructs exhibited different degradation rates depending on the 3D printing modalities. Given that each 3D printing modality has inherent characteristics that impact printing outcomes and ultimately implant performance, understanding the characteristics is crucial in selecting the 3D printing modality to create reliable biomedical implants.

Engineering of Cell Derived-Nanovesicle as an Alternative to Exosome Therapy.

BACKGROUND: Exosomes, nano-sized vesicles ranging between 30 and 150 nm secreted by human cells, play a pivotal role in long-range intercellular communication and have attracted significant attention in the field of regenerative medicine. Nevertheless, their limited productivity and cost-effectiveness pose challenges for clinical applications. These issues have recently been addressed by cell-derived nanovesicles (CDNs), which are physically synthesized exosome-mimetic nanovesicles from parent cells, as a promising alternative to exosomes. CDNs exhibit structural, physical, and biological properties similar to exosomes, containing intracellular protein and genetic components encapsulated by the cell plasma membrane. These characteristics allow CDNs to be used as regenerative medicine and therapeutics on their own, or as a drug delivery system. METHODS: The paper reviews diverse methods for CDN synthesis, current analysis techniques, and presents engineering strategies to improve lesion targeting efficiency and/or therapeutic efficacy. RESULTS: CDNs, with their properties similar to those of exosomes, offer a cost-effective and highly productive alternative due to their non-living biomaterial nature, nano-size, and readiness for use, allowing them to overcome several limitations of conventional cell therapy methods. CONCLUSION: Ongoing research and enhancement of CDNs engineering, along with comprehensive safety assessments and stability analysis, exhibit vast potential to advance regenerative medicine by enabling the development of efficient therapeutic interventions.

Fibroblast function recovery through rejuvenation effect of nanovesicles extracted from human adipose-derived stem cells irradiated with red light.

Fibroblasts (hDFs) are widely employed for skin regeneration and the treatment of various skin disorders, yet research were rarely investigated about restoration of diminished therapeutic efficacy due to cell senescence. The application of stem cell and stem cell-derived materials, exosomes, were drawn attention for the restoration functionality of fibroblasts, but still have limitation for unintended side effect or low yield. To advance, stem cell-derived nanovesicle (NV) have developed for effective therapeutic reagents with high yield and low risk. In this study, we have developed a method using red light irradiated human adipose-derived stem cells (hADSCs) derived NV (R-NVs) for enhancing the therapeutic efficacy and rejuvenating hDFs. Through red light irradiation, we were able to significantly increase the content of stemness factors and angiogenic biomolecules in R-NVs. Treatment with these R-NVs was found to enhance the migration ability and leading to rejuvenation of old hDFs to levels similar to those of young hDFs. In subsequent in vivo experiments, the treatment of old hDFs with R-NVs demonstrated a superior skin wound healing effect, surpassing that of young hDFs. In summary, this study successfully induced rejuvenation and leading to increased therapeutic efficacy to R-NVs treated old hDFs previously considered as biowaste.

Advanced In Vitro Three-Dimensional Skin Models of Atopic Dermatitis.

Atopic dermatitis (AD) is one of the most prevalent inflammatory skin diseases that is characterized by eczematous rashes, intense itching, dry skin, and sensitive skin. Although AD significantly impacts the quality of life and the number of patients keeps increasing, its pathological mechanism is still unknown because of its complexity. The importance of developing new in vitro three-dimensional (3D) models has been underlined in order to understand the mechanisms for the development of therapeutics since the limitations of 2D models or animal models have been repeatedly reported. Thus, the new in vitro AD models should not only be created in 3D structure, but also reflect the pathological characteristics of AD, which are known to be associated with Th2-mediated inflammatory responses, epidermal barrier disruption, increased dermal T-cell infiltration, filaggrin down-regulation, or microbial imbalance. In this review, we introduce various types of in vitro skin models including 3D culture methods, skin-on-a-chips, and skin organoids, as well as their applications to AD modeling for drug screening and mechanistic studies.

Piezoelectric and Triboelectric Nanogenerators for Enhanced Wound Healing.

Wound healing is a highly orchestrated biological process characterized by sequential phases involving inflammation, proliferation, and tissue remodeling, and the role of endogenous electrical signals in regulating these phases has been highlighted. Recently, external electrostimulation has been shown to enhance these processes by promoting cell migration, extracellular matrix formation, and growth factor release while suppressing pro-inflammatory signals and reducing the risk of infection. Among the innovative approaches, piezoelectric and triboelectric nanogenerators have emerged as the next generation of flexible and wireless electronics designed for energy harvesting and efficiently converting mechanical energy into electrical power. In this review, we discuss recent advances in the emerging field of nanogenerators for harnessing electrical stimulation to accelerate wound healing. We elucidate the fundamental mechanisms of wound healing and relevant bioelectric physiology, as well as the principles underlying each nanogenerator technology, and review their preclinical applications. In addition, we address the prominent challenges and outline the future prospects for this emerging era of electrical wound-healing devices.

Enhancing viability and angiogenic efficacy of mesenchymal stem cells via HSP90α and HSP27 regulation based on ROS stimulation for wound healing.

Light-based therapy has been reported as a potential preconditioning strategy to induce intracellular reactive oxygen species (ROS) signaling and improve the angiogenic properties of various types of cells. However, bio-stimulation mechanisms of light therapy in terms of ROS-heat shock proteins (HSPs) mediated anti-apoptotic and angiogenic pathways in human adult stem cells have not been fully delineated yet. Commonly used light sources such as light-emitting diode (LED) and laser are accompanied by drawbacks, such as phototoxicity, thermal damage, and excessive ROS induction, so the role and clinical implications of light-induced HSPs need to be investigated using a heat-independent light source. Here, we introduced organic LED (OLED) at 610 nm wavelength as a new light source to prevent thermal effects from interfering with the expression of HSPs. Our results showed that light therapy using OLED significantly upregulated anti-apoptotic and angiogenic factors in human bone marrow mesenchymal stem cells (hMSCs) at both gene and protein levels via the activation of HSP90α and HSP27, which were stimulated by ROS. In a mouse wound-closing model, rapid recovery and improved re-epithelization were observed in the light-treated hMSCs transplant group. This study demonstrates that the upregulation of Akt (protein kinase B)-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, caused by HSP90α and HSP27 expression, is the mechanism behind the anti-apoptotic and angiogenic effects of OLED treatment on stem cells.

Subaqueous 3D stem cell spheroid levitation culture using anti-gravity bioreactor based on sound wave superposition.

BACKGROUND: Recently, various studies have revealed that 3D cell spheroids have several advantages over 2D cells in stem cell culture. However, conventional 3D spheroid culture methods have some disadvantages and limitations such as time required for spheroid formation and complexity of the experimental process. Here, we used acoustic levitation as cell culture platform to overcome the limitation of conventional 3D culture methods. METHODS: In our anti-gravity bioreactor, continuous standing sonic waves created pressure field for 3D culture of human mesenchymal stem cells (hMSCs). hMSCs were trapped and aggerated in pressure field and consequently formed spheroids. The structure, viability, gene and protein expression of spheroids formed in the anti-gravity bioreactor were analyzed by electron microscope, immunostaining, polymerase chain reaction, and western blot. We injected hMSC spheroids fabricated by anti-gravity bioreactor into the mouse hindlimb ischemia model. Limb salvage was quantified to evaluate therapeutic efficacy of hMSC spheroids. RESULTS: The acoustic levitation in anti-gravity bioreactor made spheroids faster and more compact compared to the conventional hanging drop method, which resulted in the upregulation of angiogenic paracrine factors of hMSCs, such as vascular endothelial growth factor and angiopoietin 2. Injected hMSCs spheroids cultured in the anti-gravity bioreactor exhibited improved therapeutic efficacy, including the degree of limb salvage, capillary formation, and attenuation of fibrosis and inflammation, for mouse hindlimb ischemia model compared to spheroids formed by the conventional hanging drop method. CONCLUSION: Our stem cell culture system using acoustic levitation will be proposed as a new platform for the future 3D cell culture system.

Advanced Human BBB-on-a-Chip: A New Platform for Alzheimer's Disease Studies.

The blood-brain barrier (BBB) is a unique vascular structure that serves as a molecular transport gateway for the maintenance of brain homeostasis. Chronic disruption or breakdown of the BBB reportedly leads to neurodegenerative diseases. Nonetheless, research on human BBB pathophysiology and drug development remains highly dependent on studies using inherently different animals. Moreover, more studies have shown that animal models are not appropriate in modeling Alzheimer's disease (AD), underlining the importance of in vitro models of the human BBB with physiological relevance. In this review, recent advances in human BBB-on-a-chip technologies are highlighted and their potential for pathogenesis studies and drug prescreening for AD treatment are discussed.

Microvascularized tumor organoids-on-chips: advancing preclinical drug screening with pathophysiological relevance.

Recent developments of organoids engineering and organ-on-a-chip microfluidic technologies have enabled the recapitulation of the major functions and architectures of microscale human tissue, including tumor pathophysiology. Nevertheless, there remain challenges in recapitulating the complexity and heterogeneity of tumor microenvironment. The integration of these engineering technologies suggests a potential strategy to overcome the limitations in reconstituting the perfusable microvascular system of large-scale tumors conserving their key functional features. Here, we review the recent progress of in vitro tumor-on-a-chip microfluidic technologies, focusing on the reconstruction of microvascularized organoid models to suggest a better platform for personalized cancer medicine.

Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs.

Lipid nanoparticles (LNPs) for the efficient delivery of drugs need to be designed for the particular administration route and type of drug. Here we report the design of LNPs for the efficient delivery of therapeutic RNAs to the lung via nebulization. We optimized the composition, molar ratios and structure of LNPs made of lipids, neutral or cationic helper lipids and poly(ethylene glycol) (PEG) by evaluating the performance of LNPs belonging to six clusters occupying extremes in chemical space, and then pooling the lead clusters and expanding their diversity. We found that a low (high) molar ratio of PEG improves the performance of LNPs with neutral (cationic) helper lipids, an identified and optimal LNP for low-dose messenger RNA delivery. Nebulized delivery of an mRNA encoding a broadly neutralizing antibody targeting haemagglutinin via the optimized LNP protected mice from a lethal challenge of the H1N1 subtype of influenza A virus, and delivered mRNA more efficiently than LNPs previously optimized for systemic delivery. A cluster approach to LNP design may facilitate the optimization of LNPs for other administration routes and therapeutics.

Advanced Fabrication Techniques of Microengineered Physiological Systems.

The field of organs-on-chips (OOCs) has experienced tremendous growth over the last decade. However, the current main limiting factor for further growth lies in the fabrication techniques utilized to reproducibly create multiscale and multifunctional devices. Conventional methods of photolithography and etching remain less useful to complex geometric conditions with high precision needed to manufacture the devices, while laser-induced methods have become an alternative for higher precision engineering yet remain costly. Meanwhile, soft lithography has become the foundation upon which OOCs are fabricated and newer methods including 3D printing and injection molding show great promise to innovate the way OOCs are fabricated. This review is focused on the advantages and disadvantages associated with the commonly used fabrication techniques applied to these microengineered physiological systems (MPS) and the obstacles that remain in the way of further innovation in the field.

Microchannel network hydrogel induced ischemic blood perfusion connection.

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases.

Interleukin-4 Gene Transfection and Spheroid Formation Potentiate Therapeutic Efficacy of Mesenchymal Stem Cells for Osteoarthritis.

Osteoarthritis (OA) is a painful intractable disease that significantly affects patients' quality of life. However, current therapies, such as pain killers and joint replacement surgery, do not lead to cartilage protection. Mesenchymal stem cells (MSCs) have been proposed as an alternative strategy for OA therapy because MSCs can secrete chondroprotective and anti-inflammatory factors. However, interleukin-4 (IL-4), a potent anti-inflammatory cytokine, is barely produced by MSCs, and MSC therapy suffers from rapid MSC death following intra-articular implantation. MSCs in spheroids survive better than naïve MSCs in vitro and in vivo. IL-4-transfected MSCs in spheroids (IL-4 MSC spheroid) show increased chondroprotective and anti-inflammatory effects in an OA chondrocyte model in vitro. Following intra-articular implantation in OA rats, IL-4 MSC spheroids show better cartilage protection and pain relief than naïve MSCs. Thus, IL-4 MSC spheroid may potentiate the therapeutic efficacy of MSCs for OA.