CTNNAL1 deficiency suppresses CFTR expression in HDM-induced asthma mouse model through ROCK1-CAL signaling pathway.

The downregulation of adhesion molecule catenin alpha-like 1 (CTNNAL1) in airway epithelial cells of asthma patients and house dust mite (HDM)-induced asthma animal models was illustrated in our previous study. It is assumed to contribute to airway inflammation and mucus hypersecretion. In this work, we further explore the underlying mechanism of CTNNAL1 in asthma. CTNNAL1-silenced female mice exhibit a decreased level of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated and ATP-gated Cl - channel that correlates with mucus hypersecretion. Our previous study demonstrated that ROCK1 expression decreases but ROCK2 expression increases in the lungs of a CTNNAL1-silenced mouse model. Inhibition of ROCK1 leads to a reduction in CFTR expression in CTNNAL1-overexpressing and CTNNAL1-silenced human bronchial epithelial (HBE) cells. It has been reported that ROCK1 is a downstream target of RhoA and that activation of RhoA increases CFTR expression after CTNNAL1 deficiency in vitro and in vivo. The above results indicate that CTNNAL1 regulates CFTR expression through the ROCK1 pathway. In addition, the expression of CFTR-associated ligand (CAL) is increased after CTNNAL1 silencing, and immunoprecipitation results confirm the interaction between ROCK1 and CAL. Inhibition of CAL does not influence ROCK1 expression but increases CFTR expression in CTNNAL1-silenced HBE cells. These data suggest that CTNNAL1 deficiency decreases CFTR expression in the HDM-induced asthma mouse model through the ROCK1-CAL signaling pathway.

Mst1 attenuates myocardial ischemia/reperfusion injury following heterotopic heart transplantation in mice through regulating Keap1/Nrf2 axis.

Ischemia reperfusion (I/R) injury remains a frequent adverse event that accompanies heart transplantation. Oxidative stress and aberrant production of free radicals were regarded as the culprit of cell death and tissue damage in post-transplant IR injury. Mst1 has been identified as a mediator of oxidative stress and Nrf2 regulates anti-oxidative enzymes, however, the interaction between Mst1 and Nrf2 anti-oxidative stress pathway remains to be clarified in the event of cardiac IR injury. Herein, the model of ischemia-reperfusion injury in heterotopic heart transplantation mice was firstly established.. We observed that cardiac IR induced upregulation of Mst1 and activation of Nrf2/HO-1pathway in mice receiving heterotopic heart transplantation. Further Cobalt dichloride-induced oxidative stress model of RAW264.7 macrophage cells were then established to mimic cardiac I/R injury, results showed that exposure to CoCl2 induced the upregulation of Mst1 and activation of Keap1/Nrf2 pathway, and genetic ablation of Mst-1 and inhibition of Keap1/Nrf2 pathway aggravated oxidative damage in those cells. Additional in vivo study showed that transfection of Mst1 shRNA spurred ROS generation and worsened cardiac damage in IR mice. Meanwhile, Mst1-KD mice receiving heart transplantation showed markedly downregulation of Nrf2, HO-1 yet upregulation of Keap1, indicating diminished protective effect against tissue damage caused by IR probably owing to the frustration of Keap1/Nrf2 pathway. Taken together, our findings demonstrated the protective effect of Mst1 from cardiac IR injury via triggering Keap1/Nrf2 axis and suppressing ROS generation, which shed light on the promising role of Mst1 in transitional management of IR injury resulted from cardiac transplantation.

Preparation and evaluation of stingray skin collagen/oyster osteoinductive composite scaffolds.

The regeneration of bone defects is a major challenge for clinical orthopaedics. Herein, we designed and prepared a new type of bioactive material, using stingray skin collagen and oyster shell powder (OSP) as raw materials. A stingray skin collagen/oyster osteoinductive composite scaffold (Col-OSP) was prepared for the first time by genipin cross-linking, pore-forming and freeze-drying methods. These scaffolds were characterized by ATR-FTIR, SEM, compression, swelling, cell proliferation, cell adhesion, alkaline phosphatase activity, alizarin red staining and RT-PCR etc. The Col-OSP scaffold had an interconnected three-dimensional porous structure, and the mechanical properties of the Col-OSP composite scaffold were enhanced compared with Col, combining with the appropriate swelling rate and degradation rate, the scaffold was more in line with the requirements of bone tissue engineering scaffolds. The Col-OSP scaffold was non-toxic, promoted the proliferation, adhesion, and differentiation of MC3T3-E1 cells, and stimulated the osteogenesis-related genes expressions of osteocalcin (OCN), collagen type I (COL-I) and RUNX2 of MC3T3-E1 cells.

Temperature- and pH-responsive injectable chitosan hydrogels loaded with doxorubicin and curcumin as long-lasting release platforms for the treatment of solid tumors.

The efficacy of treating solid tumors with chemotherapy is primarily hindered by dose-limiting toxicity due to off-target effects and the heterogeneous drug distribution caused by the dense extracellular matrix. The enhanced permeability and retention (EPR) effect within tumors restricts the circulation and diffusion of drugs. To overcome these obstacles, hydrogels formed in situ at the tumor site have been proposed to promote drug accumulation, retention, and long-lasting release. We developed a thiolated chitosan (CSSH) hydrogel with a gelation point of 37°C. Due to the pH-sensitive characteristics of disulfides, the prepared hydrogel facilitated drug release in the acidic tumor environment. A drug release system composed of hydrophilic doxorubicin (Dox) and hydrophobic liposome-encapsulated curcumin (Cur-Lip) was designed to enhance the long-lasting therapeutic impacts and reduce adverse side effects. These composite gels possess a suitable gelation time of approximately 8-12 min under physiological conditions. The cumulative release ratio was higher at pH = 5.5 than at pH = 7.4 over the first 24 h, during which approximately 10% of the Dox was released, and Cur was released slowly over the following 24-120 h. Cell assays indicated that the Cur-Lip/Dox/CSSH gels effectively inhibited the growth of cancer cells. These in situ-formed Cur-Lip/Dox gels with long-term drug release capabilities have potential applications for tumor suppression and tissue regeneration after surgical tumor resection.

VEGFA-Enriched Exosomes from Tendon-Derived Stem Cells Facilitate Tenocyte Differentiation, Migration, and Transition to a Fibroblastic Phenotype.

Tendon-derived stem cells (TDSCs) play a vital role in repair of rotator cuff tear injuries by secreting paracrine proteins that regulate resident cell functions. Secreted exosomes may play a role in tendon injury repair by mediating intercellular communication; however, the detailed mechanisms by which TDSC-derived exosomes affect tenocyte development remain unknown. Here, we examined the effects of exosomes isolated from conditioned medium of TDSCs on tenocyte differentiation, migration, and transition to a fibroblastic phenotype in vitro. Successful isolation of exosomes from TDSCs was confirmed by high expression levels of CD81, CD63, CD9, and TSG101. Treatment with TDSC-derived exosomes promoted the growth and migration of cultured rat tenocytes, and increased the levels of the fibrosis markers collagen I, collagen III, scleraxis, tenascin C, and α-smooth muscle actin. Furthermore, vascular endothelial growth factor A (VEGFA) expression was higher in TDSC-derived exosomes than in TDSCs, and genetic knockdown of VEGFA suppressed the stimulatory effect of TDSC-derived exosomes on tenocyte development. Overall, these results demonstrate that VEGFA-enriched exosomes isolated from TDSCs promote differentiation and migration of cultured tenocytes and their transition to a fibroblastic phenotype. These data provide a new potential clinical treatment strategy for tendon injury.

In-situ formable dextran/chitosan-based hydrogels functionalized with collagen and EGF for diabetic wounds healing.

Delayed or non-healing skin wounds causing gangrene or even amputation, greatly threats diabetic patients lives. Herein, a bioactive, in-situ formable hydrogel based wound dressing was designed through simple Schiff base reaction. Oxidized dextran (OD) and carboxyethyl chitosan (CEC) were crosslinked together and applied as the main porous framework of hydrogel. To improve the mechanical strength and biocompatibility, collagen (Col) and EGF (Epidermal Growth Factor) were introduced into OD-CEC precursors: (1) after addition of only Col, the mechanical strength of hydrogels was improved by participating the functional -NH2 group of Col into the crosslinking process. Moreover, swelling ratio was as high as 750% on 3%OD-3%CEC-Col (water retention rate was 65 wt% after 7 days). (2) Once we introduced both Col and EGF into the OD-CEC hydrogel, the proliferation of mouse embryonic fibroblast (NIH 3T3) cells was promoted using 3%OD-3%CEC-Col/EGF, an accelerated wound healing was observed with 86% wound closure after only 14 operative days. Hematoxylin and eosin (H&E) staining and Masson staining indicated the synergy of Col and EGF might promote new tissue's formation, well collagen distributions and thus accelerate skin regeneration, presenting great potentials in wound healing of diabetic patients.

Long noncoding RNA H19 accelerates tenogenic differentiation by modulating miR-140-5p/VEGFA signaling.

Rotator cuff tear (RCT) is a common tendon injury, but the mechanisms of tendon healing remain incompletely understood. Elucidating the molecular mechanisms of tenogenic differentiation is essential to develop novel therapeutic strategies in clinical treatment of RCT. The long noncoding RNA H19 plays a regulatory role in tenogenic differentiation and tendon healing, but its detailed mechanism of action remains unknown. To elucidate the role of H19 in tenogenic differentiation and tendon healing, tendon-derived stem cells were harvested from the Achilles tendons of Sprague Dawley rats and a rat model of cuff tear was established for the exploration of the function of H19 in promoting tenogenic differentiation. The results showed that H19 overexpression promoted, while H19 silencing suppressed, tenogenic differentiation of tendon-derived stem cells (TDSCs). Furthermore, bioinformatic analyses and a luciferase reporter gene assay showed that H19 directly targeted and inhibited miR-140-5p to promote tenogenic differentiation. Further, inhibiting miR-140-5p directly increased VEGFA expression, revealing a novel regulatory axis between H19, miR-140-5p, and VEGFA in modulating tenogenic differentiation. In rats with RTC, implantation of H19-overexpressing TDSCs at the lesion promoted tendon healing and functional recovery. In general, the data suggest that H19 promotes tenogenic differentiation and tendon-bone healing by targeting miR-140-5p and increasing VEGFA levels. Modulation of the H19/miR-140-5p/VEGFA axis in TDSCs is a new potential strategy for clinical treatment of tendon injury.

A synergy between dopamine and electrostatically bound bactericide in a poly (vinyl alcohol) hybrid hydrogel for treating infected wounds.

Antibacterial hydrogels have emerged as viable options for battling infections associated with impaired wound healing. It is challenging in developing antibacterial hydrogels that have sustained and stable bactericidal activity while avoiding the use of any agents that may adversely affect safety. In view of this concern, a multi-functional polyvinyl alcohol (PVA)/sodium alginate-dopamine (SA-DA) hydrogel matrix-based wound dressing embedding with bis-quaternary triphenyl-phosphonium salt (BTPP+), that would present long-term intrinsic antimicrobial properties was developed using freeze-thawing (F-T) method herein. DA endows the hydrogel with efficient bacteria capture ability and subsequently the captured bacterial pathogens were in situ killed by electrostatically bound BTPP+, and hence significantly augmented the antibacterial efficacy. Furthermore, DA, co-operating with BTPP+ could promote erythrocyte and platelet aggregation on hydrogels, which ensures hydrogels with improved hemostasis capacity. Thus, this investigation provides a feasible simple avenue for development of long-term intrinsic antimicrobial hydrogel dressings with efficient hemostasis efficacy for infected wounds.

Fabrication of a multi-level drug release platform with liposomes, chitooligosaccharides, phospholipids and injectable chitosan hydrogel to enhance anti-tumor effectiveness.

Some anti-cancer drugs have poor solubility and availability, and are easily eliminated by rapid metabolism in vivo. To fix the drugs at the administration site and delay their release, a release platform with multi-level and multi-function was designed. The results showed that the curcumin (Cur) loaded liposomes (Cur@Lip) were coated sequentially with positive Chitooligosaccharides (Cur@Lip-Cos) and negative phospholipids (Cur@Lip-Cos-PC), to enhance water solubility, encapsulation efficiency, and delayed the release of the Cur, stability and cell intake of the liposomes, and the bioactivity of the system. The Cur@Lip-Cos could significantly enhance the inhibitory effect of MCF-7, better than the Cur@Lip-Cos-PC. The Lips were then fixed in an injectable thiolated chitosan hydrogel for local immobilization and sustained release which can effectively delay the release of Cur to inhibit MCF-7 growth. In summary, the innovative and biomimetic liposomal hydrogels are expected to provide more ideas for the design of drug carriers.

Injectable and In Situ-Formable Thiolated Chitosan-Coated Liposomal Hydrogels as Curcumin Carriers for Prevention of In Vivo Breast Cancer Recurrence.

To improve water solubility and bioavailability, curcumin (Cur) was encapsulated by liposomes (Cur-Lip), which was further coated with thiolated chitosan (CSSH) to form liposomal hydrogels (CSSH/Cur-Lip gel). The hydrogels were thermosensitive with in situ injectable performance, which were fluidic at room temperature and gelled quickly at 37 °C. The cumulative release ratio of the 200 µM CSSH/Cur-Lip gel was 31.57 ± 1.34% at 12 h, which could effectively delay the release of curcumin. Worthily, the resilient hydrogels were compressive even after five cycles of compression. The cytotoxicity test indicated that the liposomal hydrogels had good cytocompatibility, but after encapsulation of curcumin, MCF-7 cells were suppressed and killed dramatically after 72 h. The in vivo breast cancer recurrence experiment showed that the CSSH/Cur-Lip gel inhibited breast cancer recurrence after tumors were resected, and the tissue of defect in the CSSH/Cur-Lip gel group was repaired. The results showed that the drug-loaded liposomal hydrogels can deliver curcumin continuously and exerted an excellent tumoricidal effect in vitro and in vivo. The injectable, in situ-formable, and thermosensitive CSSH/Cur-Lip gel can be designed as a promising novel drug delivery vehicle to be used as carriers for local accurate and sustained drug delivery to minimize burst release and as tissue engineering scaffolds for tissue regeneration after tumor resection.

Histatin1-modified thiolated chitosan hydrogels enhance wound healing by accelerating cell adhesion, migration and angiogenesis.

It is urgently needed for effective treatments of extensive skin loss, wherein lack of angiogenesis is a major obstacle. In this study, we present a thermosensitive thiolated chitosan (CSSH) hydrogel conjugated with Histatin1 (Hst1) as a wound dressing to study its efficacy in enhancing the cell adhesion, spreading, migration, and angiogenesis. The composite hydrogels with gelation time of 5-7 min, showed a prolonged release of Hst1. Cell culture indicated that the adhesion, spreading, migration and tubule formation of HUVECs were promoted, especially for the Hst1-H group. The in vivo healing evaluation showed that the rate of recovery in Hst1-H group was increased to 84% at day 7, and the CD31 positive cells, vascular endothelial growth factor (VEGF) positive cells and aligned collagen fibers were significantly more than the controlled groups. Therefore, CSSH/Hst1 hydrogel is a promising candidate for wound healing by accelerating cell adhesion, migration and angiogenesis.

PEGylated Bis-Quaternary Triphenyl-Phosphonium Tosylate Allows for Balanced Antibacterial Activity and Cytotoxicity.

Quaternary triphenylphosphonium compounds (TPP+) have been widely recognized as an important antimicrobial because of their fast antimicrobial speed and broad antimicrobial spectrum. However, small-molecule TPP+ compounds have the defects of toxicity, which is the key factor that limits their practical applications. Here, two mono- and one bis-quaternary phosphonium tosylate compounds with different lengths of oligo(ethylene glycol) (OEG) chains and TPP+ as the active moiety were synthesized. Bis-TPP+ have a short OEG chain coupling two TPP+ at both ends, while mono-TPP+ attaches the OEG chain at one end in one molecule. In vitro antibacterial activities were evaluated against both Gram-positive as well as Gram-negative bacteria in terms of the inhibition zone (ZOI) and minimum inhibitory concentration (MIC). To investigate the antibacterial mechanism, ß-galactosidase activity was monitored for measuring the degree of membrane permeability correlated to the abilities to disrupt the membranes of bacteria. Moreover, their structure-antibacterial activity and structure-cytotoxicity relationships were further analyzed. The results indicated that bis-TPP+ synthesized can reach the sterilization rate 90% or more against Escherichia coli and Staphylococcus aureus at MICs of 3.1 and 1.5 mg/mL, respectively, and meanwhile, the cell proliferation can reach more than 80%. This paper represents an excellent approach for development of bis-TPP+ bactericidal molecules that would achieve an optimal balance between antimicrobial activity and cytotoxicity.

Liposomes coated with thiolated chitosan as drug carriers of curcumin.

Liposome is one of a promising delivery system to improve water solubility, stability, and bioavailability of curcumin. But its instability is not favorable for long-circulating treatment, controlled release or conservation. To overcome the disadvantages, thiol derivatised chitosan (CSSH) were synthesized and utilized to coat liposomes. The CSSH coated curcumin liposomes (Cur-Lip-CSSH) had an encapsulation efficiency (EE) of 93.95%, a drug loading (DL) of 7.95%, an average particle size of 406.0nm, and a positive zeta-potential of 36.6mV, which were all higher than that of Cur-Lip. Cur-Lip-CSSH showed slower in vitro release than Cur-Lip at pH5.5 and pH7.4, and the higher retention of curcumin would be remained for the following uptake of cells. The stability of the both liposomes at 4°C was almost the same, but Cur-Lip-CSSH displayed a higher stability at room temperature and higher temperature by DSC characterization. Curcumin can inhibit the growth of cancer cells under certain conditions. MCF-7 cell line was used to study its inhibition and proliferation after treating with curcumin and Cur-Lip-CSSH. Treatment of MCF-7 with curcumin and Cur-Lip-CSSH showed dose and time dependent cytotoxicity, with growth suppression at 200µM, 72h, obviously. These results indicate that the proper coating of liposomes is able to improve the stability of liposomes and the Lip-CSSH can function as potential drug delivery system.

Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition.

To mimic the natural extracellular matrix (ECM) and to facilitate tissue regeneration, in-situ formable collagen/hyaluronan composite hydrogels were prepared by a facile approach via Michael addition reaction, with maleilated collagen (Col-MA) and thiol derivatized hyaluronan (HA-SH). The hydrogels were denoted as CHG-1, CHG-2, CHG-3 and CHG-4 by vinyl/free thiol molar ratio (f) of 1:1, 1:2, 1:3 and 1:4, respectively. Results showed that with decrease of the f values, the gelation time decreased from 43s to 15s, Young's modulus increased from 1671.65Pa to 9105.86Pa, and the swelling ratio increased from 1.5 to 12.7. SEM images of the air-dried samples revealed that the chemical modification process did not denature the collagen, and the interwoven collagen fibrils were indeed observed. Cell culture confirmed that the CHG facilitated the growth and proliferation of MC3T3-E1. Cells displayed spreading morphology and formed nearly aligned cell layers with the extension of culture time. Therefore, it is suggested that CHG can be used as injectable materials for tissue regeneration.

A novel biomimetic scaffold with hUCMSCs for lumbar fusion.

Discectomy and lumbar fusion are common clinical approaches to treating intervertebral disc (IVD) degeneration with the aid of autologous bone and/or biomaterials. Biomaterials are considered suitable if they are biodegradable and can guide tissue regeneration. These features are necessary for total IVD removal, when it degenerates, as lumbar fusion treatment, avoiding secondary damage caused by the use of autogenous bone. In this work, a novel biomimetic porous chitosan/poly(l-lactic acid) scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) was applied in lumbar fusion. Hierarchically porous chitosan scaffolds were prepared using molds, porogens and freeze drying, and then poly(l-lactic acid) networks were distributed throughout the interior scaffold to enhance mechanical strength. We conducted cell culture in vitro and animal experiments in vivo. The scaffolds were incubated in hUCMSCs medium and co-cultured for 8 days, and then implanted into half-destroyed IVDs in New Zealand rabbits. The results show that scaffolds with hUCMSCs had greater ability to guide disc regeneration than the blank control and autologous bone, as determined using X-rays, computed tomography, and histologic analyses. Findings confirmed the role of hUCMSCs stimulation in bone-tissue healing and IVD regeneration. This hMUMSCs-based approach, together with the strategy proposed for incorporating osteoblastic cells into scaffolds that promote endogenous or synthetic repair mechanisms, may then be used to develop strategies for the stem-cell-based healing of other acute injuries and chronic diseases.