Bacillus-Derived Manganese Superoxide Dismutase Relieves Ocular-Surface Inflammation and Damage by Reducing Oxidative Stress and Apoptosis in Dry Eye.

Purpose: We hypothesized that antioxidative enzymes supplementation could be a treatment option for dry eye. We investigated the efficacy of oral administration of Bacillus-derived superoxide dismutase (Bd-SOD) in a murine experimental dry eye (EDE). Methods: In part I, mice were randomly assigned to normal control, EDE, and mice groups that were treated with oral Bd-SOD after induction of EDE (EDE + Bd-SOD group; four mice in each group). Expression of SOD2, a major antioxidant enzyme with manganese as a cofactor, was assessed by immunofluorescence staining. In part II, mice were divided into seven groups (six mice in each group): normal control, EDE, vehicle-treated, topical 0.05% cyclosporin A (CsA)-treated, and oral Bd-SOD-treated (2.5, 5.0, and 10.0 mg/kg Bd-SOD) groups. Tear volume, tear-film break-up time (TBUT), and corneal fluorescein-staining scores (CFS) were measured at zero, five, and 10 days after treatment. Ten days after treatment, 2',7'-dichlorodihydrofluorescein diacetate for reactive oxygen species (ROS), enzyme-linked immunosorbent for malondialdehyde, and TUNEL assays for corneal apoptosis, flow cytometry inflammatory T cells, and histological assessment were performed. Results: Compared to the normal control group in part I, the EDE group showed significantly decreased SOD2 expression by immunofluorescence staining. However, the EDE + Bd-SOD group recovered similar to the normal control group. In part II, ROS, malondialdehyde, and corneal apoptosis were decreased in CsA and all Bd-SOD-treated groups. Corneal and conjunctival inflammatory T cells decreased, and conjunctival goblet cell density increased in CsA-treated and Bd-SOD-treated groups. Compared to the CsA-treated group, the 2.5 mg/kg Bd-SOD-treated group showed increased TBUT and decreased inflammatory T cells, and the 5.0 mg/kg Bd-SOD-treated group showed decreased CFS and increased conjunctival goblet cells. Conclusions: Oral Bd-SOD administration might increase autogenous SOD2 expression in ocular surface tissue in EDE and could be developed as a complementary treatment for DE in the future.

Assessment of Esophageal Reconstruction via Bioreactor Cultivation of a Synthetic Scaffold in a Canine Model.

OBJECTIVES: Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. METHODS: We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. RESULTS: Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. CONCLUSION: Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.

Melatonin Type 2 Receptor Activation Regulates Blue Light Exposure-Induced Mouse Corneal Epithelial Damage by Modulating Impaired Autophagy and Apoptosis.

The MT1/2 receptors, members of the melatonin receptor, belong to G protein-coupled receptors and mainly regulate circadian rhythms and sleep in the brain. Previous studies have shown that in many other cells and tissues, such as HEK293T cells and the retina, MT1/2 receptors can be involved in mitochondrial homeostasis, antioxidant, and anti-inflammatory responses. In our study, we aimed to investigate the effects of blue light (BL) exposure on the expression of melatonin and its receptors in the mouse cornea and to evaluate their functional role in corneal epithelial damage. After exposing 8-week-old C57BL/6 mice to BL at 25 and 100 J/cm2 twice a day for 14 days, a significant increase in the expression of 4-HNE and MT2 was observed in the cornea. MT2 antagonist-treated mice exposed to BL showed an increased expression of p62 and decreased expression of BAX and cleaved caspase 3 compared with mice exposed only to BL. In addition, MT2 antagonist-treated mice showed more enhanced MDA and corneal damage. In conclusion, BL exposure can induce MT2 expression in the mouse cornea. MT2 activation can modulate impaired autophagy and apoptosis by increasing the expression of BAX, an apoptosis activator, thereby regulating the progression of corneal epithelial damage induced by BL exposure.

Injection laryngoplasty of human adipose-derived stem cell spheroids with hyaluronic acid-based hydrogel improves the morphological and functional characteristics of geriatric larynx.

AIM: As the geriatric population increased, the need of treatment for laryngeal atrophy and dysfunction increased. This study was performed to evaluate the effects of injection of human adipose-derived stem cell (hASC) spheroid-loaded catechol-conjugated hyaluronic acid (HA-CA) hydrogel on therapeutic rejuvenation of the geriatric larynx. METHODS: Stem cell spheroids with hyaluronic acid-based hydrogel were injected into the laryngeal muscles of 18-month-old Sprague-Dawley rats. The effects of hASC spheroids were examined in the following four groups: SHAM, injected with PBS; GEL, injected with HA-CA hydrogel; MONO, injected with single hASCs in HA-CA hydrogel; and SP, injected with hASCs spheroids in HA-CA hydrogel. The rejuvenation efficacy in geriatric laryngeal muscle tissues at 12 weeks postinjection was evaluated and compared by histology, immunofluorescence staining, and functionality analysis. RESULTS: Total myofiber cross-sectional area and myofiber number/density, evaluated by detection of myosin heavy chain with antibodies against laminin and fast myosin heavy chain, were significantly higher in the SP group than in the other groups. The lamina propria of the larynx was evaluated by alcian blue staining, which showed that the HA was increased significantly in the SP group compared to the other groups. In functional analysis, the glottal gap area was significantly reduced in the SP group compared to the other groups. The phase difference in the vocal fold during vibration was also smaller in the SP group than in the other groups, but the difference did not reach statistical significance. CONCLUSION: Injection of hASC spheroids with hyaluronic acid-based hydrogel improves the morphological and functional characteristics of geriatric larynx.

Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.

Osteoporosis is one of the most common skeletal disorders caused by the imbalance between bone formation and resorption, resulting in quantitative loss of bone tissue. Since stem cell-derived extracellular vesicles (EVs) are growing attention as novel cell-free therapeutics that have advantages over parental stem cells, the therapeutic effects of EVs from adipose tissue-derived stem cells (ASC-EVs) on osteoporosis pathogenesis were investigated. ASC-EVs were isolated by a multi-filtration system based on the tangential flow filtration (TFF) system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme-linked immunosorbent assay. EVs are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor-κB ligand (RANKL), was highly enriched in ASC-EVs. We found that the intravenous administration of ASC-EVs attenuated bone loss in osteoporosis mice. Also, ASC-EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow-derived MSCs (BM-MSCs). However, OPG-depleted ASC-EVs did not show anti-osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC-EVs. Additionally, small RNA sequencing data were analysed to identify miRNA candidates related to anti-osteoporosis effects. miR-21-5p in ASC-EVs inhibited osteoclast differentiation through Acvr2a down-regulation. Also, let-7b-5p in ASC-EVs significantly reduced the expression of genes related to osteoclastogenesis. Finally, ASC-EVs reached the bone tissue after they were injected intravenously, and they remained longer. OPG, miR-21-5p, and let-7b-5p in ASC-EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC-EVs are highly promising as cell-free therapeutic agents for osteoporosis treatment.

The Antioxidant Effect of Small Extracellular Vesicles Derived from Aloe vera Peels for Wound Healing.

BACKGROUND: Extracellular vesicles (EVs) derived from plants have emerged as potential candidates for cosmetic and therapeutic applications. In this study, we isolated EVs from Aloe vera peels (A-EVs) and investigated the antioxidant and wound healing potential of A-EVs. METHODS: A-EVs were isolated by ultracentrifugation and tangential flow filtration and were characterized using transmission electron microscopy, nanoparticle tracking analysis. The cytotoxicity and cellular uptake of A-EVs were investigated by WST-1 assay and flow cytometry. The antioxidant effect of A-EVs was evaluated by superoxide dismutase (SOD) activity assay and cellular antioxidant activity (CAA) assay. The wound healing potential was assessed by in vitro scratch assay using human keratinocytes (HaCaT) and fibroblasts (HDF). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and their associated genes was analyzed by quantitative RT-PCR. RESULTS: A-EVs displayed a round shape and had diameters from 50 to 200 nm. A-EVs showed good cytocompatibility on human skin cells and were internalized into HaCaT cells via clathrin-, caveolae-mediated endocytosis, and membrane fusion. The SOD activity and CAA assays exhibited that A-EVs had antioxidant activity and reduced intracellular ROS levels in H2O2-treated HaCaT cells in a dose-dependent manner. A scratch assay showed that A-EVs enhanced the migration ability of HaCaT and HDF. Moreover, A-EVs significantly upregulated the mRNA expression of Nrf2, HO-1, CAT, and SOD genes in H2O2-treated HaCaT cells. Our findings reveal that A-EVs could activate the antioxidant defense mechanisms and wound healing process via the Nrf2 activation. CONCLUSION: Overall results suggest that the A-EVs are promising as a potential agent for skin regeneration.

Vitamin A-coupled stem cell-derived extracellular vesicles regulate the fibrotic cascade by targeting activated hepatic stellate cells in vivo.

Allogeneic transplantation of mesenchymal stem cell-derived extracellular vesicles (EVs) offers great potential for treating liver fibrosis. However, owing to their intrinsic surface characteristics, bare EVs are non-specifically distributed in the liver tissue after systemic administration, leading to limited therapeutic efficacy. To target activated hepatic stellate cells (HSCs), which are responsible for hepatic fibrogenesis, vitamin A-coupled small EVs (V-EVs) were prepared by incorporating vitamin A derivative into the membrane of bare EVs. No significant differences were found in the particle size and morphology between bare and V-EVs. In addition, surface engineering of EVs did not affect the expression of surface marker proteins (e.g., CD63 and CD9), as demonstrated by flow cytometry. Owing to the surface incorporation of vitamin A, V-EVs were selectively taken up by activated HSCs via receptor-mediated endocytosis. When systemically administered to mice with liver fibrosis, V-EVs effectively targeted activated HSCs in the liver tissue, resulting in reversal of the fibrotic cascade. Consequently, even at a 10-fold lower dose, V-EVs exhibited comparable anti-fibrotic effects to those of bare EVs, substantiating their therapeutic potential for liver fibrosis.

Hyaluronic Acid Coating on Hydrophobic Tracheal Scaffold Enhances Mesenchymal Stem Cell Adhesion and Tracheal Regeneration.

BACKGROUND: Long segmental tracheal repair is challenging in regenerative medicine due to low adhesion of stem cells to tracheal scaffolds. Optimal transplantation of stem cells for tracheal defects has not been established. We evaluated the role of hyaluronic acid (HA) coating of tracheal scaffolds in mesenchymal stem cell (MSC) adhesion and tracheal regeneration in a rabbit model. METHODS: A three-dimensionally printed tubular tracheal prosthesis was incubated with dopa-HA-fluorescein isothiocyanate in phosphate-buffered saline for 2 days. MSCs were incubated with an HA-coated scaffold, and their adhesion was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. HA coated scaffolds with or without MSC seeding were transplanted at the circumferential tracheal defect in rabbits, and survival, rigid bronchoscopy, radiologic findings, and histologic findings were compared between the two groups. RESULTS: HA-coated scaffolds showed better MSC adhesion than non-coated scaffolds. The HA-coated scaffolds with MSC group showed a wider airway and greater mucosal regeneration compared to the HA-coated scaffolds without MSC group. CONCLUSION: HA coating of scaffolds can promote MSC adhesion and tracheal regeneration.

Blue Light Induces Impaired Autophagy through Nucleotide-Binding Oligomerization Domain 2 Activation on the Mouse Ocular Surface.

In this study, we investigated the effects of blue light exposure on nucleotide-binding oligomerization domain 2 (NOD2) expression on the mouse ocular surface and evaluated the role of NOD2 activation in light-induced cell death. Mice were divided into wild-type (WT), NOD2-knock out (KO), WT + blue light (WT + BL), and NOD2-KO + blue light (NOD2-KO + BL) groups, and the mice in the WT+BL and NOD2-KO + BL groups were exposed to blue light for 10 days. After 10 days of blue light exposure, increased reactive oxygen species and malondialdehyde were observed in the WT + BL and NOD2-KO + BL groups, and the WT + BL group showed a higher expression of NOD2 and autophagy related 16 like 1. Although both WT+BL and NOD2-KO + BL groups showed an increase in the expression of light chain 3-II, NOD2-KO + BL mice had a significantly lower p62 expression than WT + BL mice. In addition, NOD2-KO+BL mice had significantly lower corneal epithelial damage and apoptosis than WT + BL mice. In conclusion, blue light exposure can induce impaired autophagy by activation of NOD2 on the ocular surface. In addition, the reactive oxygen species (ROS)-NOD2-autophagy related 16 like 1 (ATG16L) signaling pathway may be involved in the blue-light-induced autophagy responses, resulting in corneal epithelial apoptosis.

Endoscopically Applied Biodegradable Stent in a Rabbit Model of Pediatric Tracheomalacia.

OBJECTIVES: A polydioxanone (PDO) stent was developed to treat tracheomalacia in pediatric patients. However, its safety and efficacy need to be verified in animal studies before clinical trials in patients can be conducted. This study evaluated the safety and efficacy of a PDO stent in normal and tracheomalacia-model rabbits. METHODS: In total, 29 New Zealand white rabbits were used: 13 for evaluating the biocompatibility of the PDO stent in normal rabbits and 16 for the creation of a tracheomalacia model. The tracheomalacia model was successfully established in 12 rabbits, and PDO stents were placed in eight of those rabbits. RESULTS: The PDO stent was successfully positioned in the trachea of the normal rabbits using an endoscopic approach, and its degradation was observed 10 weeks later. The stent fragments did not induce distal airway obstruction or damage, and the mucosal changes that occurred after stent placement were reversed after degradation. The same procedure was performed on the tracheomalacia-model rabbits. The survival duration of the tracheomalacia rabbits with and without stents was 49.0±6.8 and 1.0±0.8 days, respectively. Thus, the PDO stent yielded a significant survival gain (P=0.001). In the tracheomalacia rabbits, stent degradation and granulation tissue were observed 7 weeks after placement, leading to airway collapse and death. CONCLUSION: We successfully developed a PDO stent and an endoscopic guide placement system. The degradation time of the stent was around 10 weeks in normal rabbits, and its degradation was accelerated in the tracheomalacia model. The mucosal changes associated with PDO stent placement were reversible. Placement of the PDO stent prolonged survival in tracheomalacia-model rabbits.

Prevascularized Tracheal Scaffolds Using the Platysma Flap for Enhanced Tracheal Regeneration.

OBJECTIVES: One of the greatest hurdles in tracheal tissue engineering is insufficient vascularization, which leads to delayed mucosal regeneration, inflammation, and restenosis. This study investigated whether a prevascularized segmental tracheal substitute using platysma can enhance tracheal mucosal regeneration. METHODS: Three-dimensional (3D) printed scaffolds with (group M) or without (group S) Matrigel coating were implanted under the feeding vessels of the platysma in New Zealand White rabbits (n = 3) to induce vascularization. After 1 or 2 weeks, tracheal defects were created and vascularized scaffolds with feeders of the platysma were transplanted as rotational flaps. As controls, scaffolds with or without Matrigel coating was transplanted into a tracheal defect without prevascularization. Airway patency and epithelization were examined using a rigid bronchoscope every 2 weeks. Surviving animals were euthanized at 24 weeks, and microcomputed tomography and histological evaluation were performed. RESULTS: Animals with 2 weeks of prevascularization showed longer survival than animals with 0 or 1 weeks of prevascularization regardless of the Matrigel coating. Wider airway patency was observed in group M than group S. Group M showed migration of epithelium over the scaffold from 4 weeks after transplantation and complete coverage with epithelium at 12 weeks, whereas group S showed migration of the epithelium from 14 weeks and incomplete coverage with epithelium even at 24 weeks. CONCLUSION: This two-step method, utilizing the platysma as an in vivo bioreactor, may be a promising approach to achieve long-term survival and enhanced luminal patency. Matrigel coating on the scaffold had a synergistic effect on epithelial regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1732-1740, 2021.

Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration.

Osteoarthritis (OA) is a chronic degenerative disease of articular cartilage that is the most common joint disease worldwide. Mesenchymal stem cells (MSCs) have been the most extensively explored for the treatment of OA. Recently, it has been demonstrated that MSC-derived extracellular vesicles (EVs) may contribute to the potential mechanisms of MSC-based therapies. In this study, we investigated the therapeutic potential of human adipose-derived stem cells EVs (hASC-EVs) in alleviating OA, along with the mechanism. EVs were isolated from the culture supernatants of hASCs by a multi-filtration system based on the tangential flow filtration (TFF) system. The isolated EVs were characterised using dynamic light scattering (DLS), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and flow cytometry analysis. The hASC-EVs not only promoted the proliferation and migration of human OA chondrocytes, but also maintained the chondrocyte matrix by increasing type Ⅱ collagen synthesis and decreasing MMP-1, MMP-3, MMP-13 and ADAMTS-5 expression in the presence of IL-1ß in vitro. Intra-articular injection of hASC-EVs significantly attenuated OA progression and protected cartilage from degeneration in both the monosodium iodoacetate (MIA) rat and the surgical destabilisation of the medial meniscus (DMM) mouse models. In addition, administration of hASC-EVs inhibited the infiltration of M1 macrophages into the synovium. Overall results suggest that the hASC-EVs should be considered as a potential therapeutic approach in the treatment of OA.

Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes.

Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming.

Transplantation of a 3D-printed tracheal graft combined with iPS cell-derived MSCs and chondrocytes.

For successful tracheal reconstruction, tissue-engineered artificial trachea should meet several requirements, such as biocompatible constructs comparable to natural trachea, coverage with ciliated respiratory mucosa, and adequate cartilage remodeling to support a cylindrical structure. Here, we designed an artificial trachea with mechanical properties similar to the native trachea that can enhance the regeneration of tracheal mucosa and cartilage through the optimal combination of a two-layered tubular scaffold and human induced pluripotent stem cell (iPSC)-derived cells. The framework of the artificial trachea was fabricated with electrospun polycaprolactone (PCL) nanofibers (inner) and 3D-printed PCL microfibers (outer). Also, human bronchial epithelial cells (hBECs), iPSC-derived mesenchymal stem cells (iPSC-MSCs), and iPSC-derived chondrocytes (iPSC-Chds) were used to maximize the regeneration of tracheal mucosa and cartilage in vivo. After 2 days of cultivation using a bioreactor system, tissue-engineered artificial tracheas were transplanted into a segmental trachea defect (1.5-cm length) rabbit model. Endoscopy did not reveal granulation ingrowth into tracheal lumen. Alcian blue staining clearly showed the formation of ciliated columnar epithelium in iPSC-MSC groups. In addition, micro-CT analysis showed that iPSC-Chd groups were effective in forming neocartilage at defect sites. Therefore, this study describes a promising approach for long-term functional reconstruction of a segmental tracheal defect.

Dexamethasone loaded bilayered 3D tubular scaffold reduces restenosis at the anastomotic site of tracheal replacement: in vitro and in vivo assessments.

Despite recent developments in the tracheal tissue engineering field, the creation of a patient specific substitute possessing both appropriate mechanical and biointerfacial properties remains challenging. Most tracheal replacement therapies fail due to restenosis at the anastomosis site. In this study, we designed a robust, biodegradable, 3D tubular scaffold by combining electrospinning (ELSP) and 3D (three-dimensional) printing techniques for use in transplantation therapy. After that, we loaded dexamethasone (DEX) onto the 3D tubular scaffold using mild surface modification reactions by using polydopamine (PDA), polyethyleneimine (PEI), and carboxymethyl-ß-cyclodextrin (ßCD). As a result, the fabricated 3D tubular scaffold had robust mechanical properties and the chemical modifications were confirmed to have proceeded successfully by physico-chemical analysis. The surface treatments allowed for a larger amount of DEX to be loaded onto the ßCD modified scaffold as compared to the bare group. In vitro and in vivo studies demonstrated that the DEX loaded 3D tubular scaffold exhibited significantly enhanced anti-inflammation activity, enhanced tracheal mucosal regeneration, and formation of a patent airway. From our results, we believe that our system may represent an innovative paradigm in tracheal tissue engineering by providing proper mechanical properties and successful formation of tracheal tissue as a means of remodeling and healing tracheal defects for use in transplantation therapy.

Human adipose stem cell-derived extracellular nanovesicles for treatment of chronic liver fibrosis.

Liver fibrosis is an excessive wound healing process that occurs in response to liver damage depending on underlying aetiologies. Currently, there are no effective therapies and FDA-approved therapeutics for the treatment of liver fibrosis except liver transplantation. Multipotent adipose-derived stem cells (ADSCs) have received significant attention as regenerative medicine for liver fibrosis owing to their advantages over stem cells with other origins. However, intrinsic limitations of stem cell therapies, such as cellular rejection and tumor formation, have impeded clinical applications of the ADSC-based liver therapeutics. To overcome these problems, the extracellular nanovesicles (ENVs) responsible for the therapeutic effect of ADSCs (A-ENVs) have shown considerable promise as cell-free therapeutics for liver diseases. However, A-ENVs have not been used for the treatment of intractable chronic liver diseases including liver fibrosis and cirrhosis. Therefore, in this study, we investigated the in vitro and in vivo antifibrotic efficacy of A-ENVs in thioacetamide-induced liver fibrosis models. A-ENVs significantly downregulated the expression of fibrogenic markers, such as matrix metalloproteinase-2, collagen-1, and alpha-smooth muscle actin. The systemic administration of A-ENVs led to high accumulation in fibrotic liver tissue and the restoration of liver functionality in liver fibrosis models through a marked reduction in α-SMA and collagen deposition. These results demonstrate the significant potential of A-ENVs for use as extracellular nanovesicles-based therapeutics in the treatment of liver fibrosis and possibly other intractable chronic liver diseases.

Chitosan oral patches inspired by mussel adhesion.

Oral mucosal drug delivery systems have been developed to expedite the regeneration of oral mucosa, there are still many challenges related to residence time for drugs because the ceaseless changes of saliva, mouth movement, and involuntary swallowing prevent robust adhesion of drugs and/or drug-loaded biomaterials. Thus, it is highly desirable to develop the delivery platforms exhibiting robust, stable adhesion within oral cavities. Herein, we have developed an adhesive polysaccharide oral patch called 'Chitoral' that utilizes chemical principles shown in wet-resistant mussel adhesion. Chitoral plays an important role as an adhesive layer in wet environments. We unexpectedly found that Chitoral instantly dissolves upon contact with saliva and a labial mucous layer, and then the dissolved Chitoral compounds forms an insoluble adhesion layer with mucins at Chitoral/mucous interface nearly immediate actions. Later, Chitoral gradually converts into adhesive hydrogels by the cooperative actions of covalent crosslinking and physical entanglement. The instant, robust muco-adhesion properties of Chitoral provides long-lasting therapeutic effects of drugs resulting enhanced healing of oral ulcer. Thus, mussel-inspired, mucous-resistant adhesive platforms, Chitoral, can be a platform for oral mucosal drug delivery systems.

Nanofiber-hydrogel composite-mediated angiogenesis for soft tissue reconstruction.

Soft tissue losses from tumor removal, trauma, aging, and congenital malformation affect millions of people each year. Existing options for soft tissue restoration have several drawbacks: Surgical options such as the use of autologous tissue flaps lead to donor site defects, prosthetic implants are prone to foreign body response leading to fibrosis, and fat grafting and dermal fillers are limited to small-volume defects and only provide transient volume restoration. In addition, large-volume fat grafting and other tissue-engineering attempts are hampered by poor vascular ingrowth. Currently, there are no off-the-shelf materials that can fill the volume lost in soft tissue defects while promoting early angiogenesis. Here, we report a nanofiber-hydrogel composite that addresses these issues. By incorporating interfacial bonding between electrospun poly(ε-caprolactone) fibers and a hyaluronic acid hydrogel network, we generated a composite that mimics the microarchitecture and mechanical properties of soft tissue extracellular matrix. Upon subcutaneous injection in a rat model, this composite permitted infiltration of host macrophages and conditioned them into the pro-regenerative phenotype. By secreting pro-angiogenic cytokines and growth factors, these polarized macrophages enabled gradual remodeling and replacement of the composite with vascularized soft tissue. Such host cell infiltration and angiogenesis were also observed in a rabbit model for repairing a soft tissue defect filled with the composite. This injectable nanofiber-hydrogel composite augments native tissue regenerative responses, thus enabling durable soft tissue restoration outcomes.

Reprogramming of cancer stem cells into non-tumorigenic cells using stem cell exosomes for cancer therapy.

Cancer stem cells (CSCs) are a small population of cells with stem cell-like properties found in tumors. CSCs are closely associated with tumor heterogeneity, which influences tumor progress, metastasis, and drug resistance. Here, we propose a concept to enhance efficacy of cancer therapy through CSC reprogramming into non-tumorigenic cells using stem cell-derived exosomes with osteoinductive potential. We hypothesized that exosomes derived from osteogenic differentiating human adipose-derived stem cells (OD-EXOs) contain specific cargos capable of inducing osteogenic differentiation of CSCs. Quantitative RT-PCR analysis revealed that OD-EXOs enhanced the expression of osteogenic-related genes, such as alkaline phosphatase (ALPL), osteocalcin (BGLAP), and runt-related transcription factor 2 (RUNX2). In addition, expression of drug-resistance genes such as ATP binding cassette (ABC) transporter, the breast cancer gene family (BCRA1 and BCRA2), and the ErbB gene family were significantly decreased in OD-EXO-treated CSCs. Our findings suggest that OD-EXOs function as a biochemical cue for CSC reprogramming and contribute to overcoming therapeutic resistance.

Functional recovery in photo-damaged human dermal fibroblasts by human adipose-derived stem cell extracellular vesicles.

Ultraviolet-B (UVB) irradiation causes imbalance between dermal matrix synthesis and degradation through aberrant upregulation of matrix metalloproteinases (MMPs), which leads to overall skin photoaging. We investigated the effects of extracellular vesicles (EVs) derived from human adipose-derived stem cells (HASCs) on photo-damaged human dermal fibroblasts (HDFs). EVs were isolated from conditioned media of HASCs with tangential flow filtration and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western blotting, micro RNA (miRNA) arrays, cytokine arrays and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The effects of EVs on the UVB-irradiated HDFs were evaluated using scratch assay, ELISA and real-time PCR. Microarrays exhibited that EVs are rich in various miRNAs and proteins, and that these EV contents are linked to a broad range of biological functions, including fibroblast proliferation, UV protection, collagen biosynthesis, DNA repair and cell ageing. A scratch assay showed that HASC-EVs enhanced the migration ability of UVB-irradiated HDFs. Real-time RT-PCR and ELISA analyses revealed that the HASC-derived EVs significantly suppressed the overexpression of MMP-1, -2, -3 and -9 induced by UVB irradiation and enhanced the expression of collagen types I, II, III and V and elastin. In particular, tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-ß1, which are important factors involved in MMP suppression and ECM synthesis, were upregulated in EV-treated HDFs after UVB irradiation. Overall results suggest that diverse components that are enriched in HASC-derived EVs could act as a biochemical cue for recovery from skin photoaging.