Mesenchymal Stem Cell Aggregation-Released Extracellular Vesicles Induce CD31+ EMCN+ Vessels in Skin Regeneration and Improve Diabetic Wound Healing.

The blood vessel system is essential for skin homeostasis and regeneration. While the heterogeneity of vascular endothelial cells has been emergingly revealed, whether a regeneration-relevant vessel subtype exists in skin remains unknown. Herein, a specialized vasculature in skin featured by simultaneous CD31 and EMCN expression contributing to the regeneration process is identified, the decline of which functionally underlies the impaired angiogenesis of diabetic nonhealing wounds. Moreover, enlightened by the developmental process that mesenchymal condensation induces angiogenesis, it is demonstrated that mesenchymal stem/stromal cell aggregates (CAs) provide an efficacious therapy to enhance regrowth of CD31+ EMCN+ vessels in diabetic wounds, which is surprisingly suppressed by pharmacological inhibition of extracellular vesicle (EV) release. It is further shown that CAs promote secretion of angiogenic protein-enriched EVs by proteomic analysis, which directly exert high efficacy in boosting CD31+ EMCN+ vessels and treating nonhealing diabetic wounds. These results add to the current knowledge on skin vasculature and help establish feasible strategies to benefit wound healing under diabetic condition.

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

Targeted inhibition of osteoclastogenesis reveals the pathogenesis and therapeutics of bone loss under sympathetic neurostress.

Sympathetic cues via the adrenergic signaling critically regulate bone homeostasis and contribute to neurostress-induced bone loss, but the mechanisms and therapeutics remain incompletely elucidated. Here, we reveal an osteoclastogenesis-centered functionally important osteopenic pathogenesis under sympatho-adrenergic activation with characterized microRNA response and efficient therapeutics. We discovered that osteoclastic miR-21 was tightly regulated by sympatho-adrenergic cues downstream the ß2-adrenergic receptor (ß2AR) signaling, critically modulated osteoclastogenesis in vivo by inhibiting programmed cell death 4 (Pdcd4), and mediated detrimental effects of both isoproterenol (ISO) and chronic variable stress (CVS) on bone. Intriguingly, without affecting osteoblastic bone formation, bone protection against ISO and CVS was sufficiently achieved by a (D-Asp8)-lipid nanoparticle-mediated targeted inhibition of osteoclastic miR-21 or by clinically relevant drugs to suppress osteoclastogenesis. Collectively, these results unravel a previously underdetermined molecular and functional paradigm that osteoclastogenesis crucially contributes to sympatho-adrenergic regulation of bone and establish multiple targeted therapeutic strategies to counteract osteopenias under stresses.

Extracellular Vesicles for Immunomodulation in Tissue Regeneration.

A large number of people suffer from tissue injury and defect worldwide, which constitutes a critical challenge for regenerative medicine. During the complicated process of tissue repair and regeneration, immune response that involves many kinds of immune cells often concurrently exists and plays a significant role, thus providing a promising target for the development of therapeutic strategies. As a critical player in cell-cell communication, extracellular vesicles (EVs) are a cluster of nano-sized vesicles of different categories, which have been reported to possess favorable immunoregulatory potential, and participate in the process of tissue repair and regeneration. Furthermore, EVs can be engineered with genetic or chemical strategies for optimized performance as therapeutic mediators. Here, we provide an outline on the biology of EVs as well as the role of EVs in immune regulation, focusing on exosomes, microvesicles, and apoptotic vesicles. We further summarize the applications of EV-based therapies for tissue regeneration, with particular emphasis on the modulation of immune system. Also, we have discussed the construction strategies of engineered EVs and the immunomodulatory capability of engineered EVs as well as their therapeutic potential in tissue repair. This review will highlight the outstanding potential of EV-based therapeutic strategies for tissue repair and regeneration. Impact statement Extracellular vesicles (EVs) have been shown to possess potent immunomodulatory abilities and thus hold great therapeutic potential for regenerative medicine via immune modulation. Also, the development of engineering technology has provided a feasible tool for optimizing the performance of natural EVs. We hereby outline the current knowledge regarding the features of EVs and the construction strategies of engineered EVs as well as the immunomodulation effects of natural and engineered EVs. We also highlight the applications of EV-based therapies for tissue regeneration, with particular emphasis on the modulation of immune system.

Dynamic Modification of Palladium Catalysts with Chain Alkylamines for the Selective Hydrogenation of Alkynes.

Selective hydrogenation of alkynes plays a pivotal role in the field of chemical production but still suffers from restrained catalytic activity and low alkene selectivity. Herein, a dynamic modification strategy was utilized by preferentially attaching diethylenetriamine (DETA) to the surface of the support to modify the Pd catalyst. The DETA-modified Pd catalyst demonstrates unprecedented reactivity (14,412 h-1) and selectivity as high as 94% for the semihydrogenation of 2-methyl-3-butyn-2-ol at 35 °C, presenting a 36-fold higher reactivity than the Lindlar catalyst. Moreover, the yield exceeds 98.2% at full conversion under no solvent and organic adsorbate conditions, indicating the potential applications for industrial production. Systematic studies reveal that flexible DETA serves in a reversible "breathing pattern" for the molecular discrimination by constructing dynamic metal-support interaction (DMSI), enabling selective exclusion of alkenes from the Pd surface. DETA is competent to dynamically adjust the adsorption behaviors of reactants and effectively boost the intrinsic activity of the modified catalyst. Impressively, the DETA-modified Pd catalyst exhibits exceptional stability even after being recycled 20 times. This work sheds light on a novel and applicable method for the rational design of heterogeneous catalysts via DMSI.

A composite sponge based on alkylated chitosan and diatom-biosilica for rapid hemostasis.

Rapid control of bleeding is of great significance in military trauma and traffic accidents. In this study, alkylated chitosan (AC) and diatom biosilica (DB) were combined to develop a safe and effective hemostatic composite sponge (AC-DB sponge) for hemorrhage control. Due to the procoagulant chemical structure of AC-DB sponge, it exhibited rapid hemostatic ability in vitro (clotting time was shortened by 78% than that of control group), with favorable biocompatibility (hemolysis ratio < 5%, no cytotoxicity). The strong interface effect between AC-DB sponge and blood induced the erythrocyte and platelets activation, deformation and aggregation, intrinsic coagulation pathway activation, resulting in significant coagulation acceleration. AC-DB sponge had excellent performance in in vivo assessments with shortest clotting time (106.2 s) and minimal blood loss (328.5 mg). All above results proved that AC-DB sponge had great potential to be a safe and rapid hemostatic material.

Chitosan/Diatom-Biosilica Aerogel with Controlled Porous Structure for Rapid Hemostasis.

Uncontrolled hemorrhage is the main reason of possible preventable death after accidental injury. It is necessary to develop a hemostatic agent with rapid hemostatic performance and good biocompatibility. In this study, a chitosan/diatom-biosilica-based aerogel is developed using dopamine as cross-linker by simple alkaline precipitation and tert-butyl alcohol replacement. The chitosan/diatom-biosilica aerogel exhibits favorable biocompatibility and multiscale hierarchical porous structure (from nanometer to micrometer), which can be controlled by the concentration of tert-butyl alcohol. The displacement of tert-butyl alcohol can keep the porosity of diatom-biosilica in aerogel and give it large surface with efficient water absorption ratio. 30% tert-butyl alcohol replacement of aerogel possesses the largest surface area (74.441 m2 g-1 ), water absorption capacity (316.83 ± 2.04%), and excellent hemostatic performance in vitro blood coagulation (≈70 s). Furthermore, this aerogel exhibits the shortest clotting time and lowest blood loss in rat hemorrhage model. The strong interface effect between aerogel and blood is able to promote erythrocytes aggregation, platelets adhesion, and activation, as well as, activate the intrinsic coagulation pathway to accelerate blood coagulation. All the above results demonstrate that chitosan/diatom-biosilica aerogel has great potential to be a safe and rapid hemostatic material.

Hydroxybutyl chitosan/diatom-biosilica composite sponge for hemorrhage control.

Effective bleeding control is critical first step in current civilian and military trauma treatment, however commercially available hemostatic materials are difficult to achieve expected effects. In this study, a composite sponge (H-D) based on hydroxybutyl chitosan (HBC) and diatom-biosilica (DB) was designed for hemorrhage control. H-D exhibited hierarchical porous structure, favorable biocompatibility (hemolysis ratio < 5 %, no cytotoxicity), along with high and fast fluid absorbability (11-16 times than that of weight), given effective hemostasis effect (clotting time shortened by 70 % than that of control). In vitro coagulation tests demonstrated that H-D could provide strong interface effect to induce erythrocyte absorption and aggregation, as well as activating the intrinsic coagulation pathway and thus accelerated blood coagulation. These results proved that H-D composite sponge has great potential for hemorrhage control.

pH-sensitive amphiphilic chitosan-quercetin conjugate for intracellular delivery of doxorubicin enhancement.

A novel pH-responsive nanomicelle (QT-CA-CS) based on Chitosan, Quercetin and Citraconic anhydride was reported in this study. The QT-CA-CS could self-assemble into nanomicelles for encapsulating anticancer drug doxorubicin (DOX) by ultrasound. The novel nanomicelles had P-gp inhibition and pH responsiveness, which was capable of inhibiting drug efflux and responding to an endo/lysosomal acidic environment. The drug loaded nanomicelles had high encapsulation rate (more than 80%), small particle size (133.52 ± 4.13 nm) and positive zeta potential (+13.5 mV). The release rate of doxorubicin and quercetin in pH 4.5 was faster than that in pH 7.4. QT-CA-CS-DOX nanomicelles could promote cellular uptake of doxorubicin by drug resistance cell line (MCF-7/ADR), which was 8.62 folds higher than that of free doxorubicin. Most importantly, QT-CA-CS-DOX nanomicelles could escape from lysosomes and rapidly release doxorubicin and quercetin in the cytoplasm, which had an enhanced inhibitory effect on tumor cells, especially for MCF-7/ADR. The above results proved that the high potential of QT-CA-CS-DOX nanomicelles for multidrug resistance related tumor therapy.

Multifunctional quercetin conjugated chitosan nano-micelles with P-gp inhibition and permeation enhancement of anticancer drug.

In this study, quercetin-chitosan conjugate (QT-CS) was synthesized for oral delivery of doxorubicin (DOX) to improve its oral bioavailability by increasing its water solubility, opening tight junction and bypassing the P-glycoprotein (P-gp). The prepared QT-CS self-assembled into micelles which could encapsulate DOX with high encapsulation rate, small particle size (136.9 nm) and strong zeta potential (+16.2 mV). QT-CS-DOX micelles displayed sustained-release profile in gastrointestinal simulation fluid (pH 1.2/pH 7.4). QT-CS micelles could promote cellular uptake of doxorubicin, which was 2.2 folds higher than that of free doxorubicin. The trans epithelial electrical resistance (TEER) value of Caco-2 monolayer cells was significantly reduced (about 57%) by drug loaded QT-CS micelles, leading to a high apparent permeability coefficient (Papp) of doxorubicin, which was 10.17 folds higher than that of free doxorubicin. Above results indicate that QT-CS micelles are promising vehicles for the oral delivery of insoluble anticancer drugs.

Multifunctional chitosan/dopamine/diatom-biosilica composite beads for rapid blood coagulation.

Uncontrollable bleeding is the main cause of death in wars and accidents. The development of emergency material for rapid hemostatic can effectively reduce bleeding-related death. The commercial hemostatic materials available in the market are difficult to meet requirements of rapid hemostasis, good biocompatibility, low cost and ease of use. In this study, we developed chitosan/dopamine/diatom-biosilica composite beads (CDDs) for rapid hemostasis with good biocompatibility. CDDs were prepared by combining chitosan with diatom-biosilica (DB) using dopamine as bio-glue. The porous internal structure of CDDs led to rapid and large amount of water absorption, which contributed to the rapid hemostasis (83 s, 22% of the control group). The hemolytic rate of CDDs was less than 5% and cell viability was above 80%, confirming its good biocompatibility. All the above results indicated that CDDs had potential to develop into safe and non-toxic hemostatic material.

Biosynthetic calcium-doped biosilica with multiple hemostatic properties for hemorrhage control.

In this study, we reported calcium-doped biosilica (Ca-biosilica) with multiple hemostatic properties derived from Coscinodiscus sp. frustule. The incorporation of calcium into the diatom frustule was achieved by a simple biosynthetic route through feeding the diatoms with calcium chloride, which was confirmed by calcein staining and EDXS. Ca-Biosilica exhibited an efficient water absorption ratio (36.36 ± 1.44 times its own weight of liquid), superior compatibility (hemolysis ratio <5%, no cytotoxicity against MEFs) and excellent hemostatic effect (203.67 ± 15.63 s at 5 mg mL-1; 145.01 ± 20.41 s at 10 mg mL-1). The intrinsic blood coagulation pathway was clearly strengthened by the unique interface of Ca-biosilica, which was rich in silanol groups and calcium, leading to fast hemorrhage control in rat-tail amputation model. The clotting time of Ca-biosilica was 88.34 ± 28.54 s, which was similar to that of Quickclot® zeolite, whereas only one-third blood loss by weight (0.21 ± 0.16 g) was found in Ca-biosilica-treated group compared with that of the Quickclot® zeolite group (0.63 ± 0.09 g). The results prove that Ca-biosilica is promising as a quick hemostatic agent due to its effectiveness, excellent biocompatibility and simple and environmentally friendly preparation process.