Enterotoxin-related genes PPFIA4 and SCN3B promote colorectal cancer development and progression.

To identify the role of enterotoxin-related genes in colorectal cancer (CRC) development and progression. Upregulated differentially expressed genes shared by three out of five Gene Expression Omnibus (GEO) data sets were included to screen the key enterotoxin-induced oncogenes (EIOGs) according to criteria oncogene definition, enrichment, and protein-protein interaction (PPI) network analysis, followed by prognosis survival, immune infiltration, and protential drugs analyses was performed via integration of RNA-sequencing data and The Cancer Genome Atlas-derived clinical profiles. We screened nine common key EIOGs from at least three GEO data sets. A Cox proportional hazards regression models verified that more alive cases, decreased overall survival, and highest 4-year survival prediction in CRC patients with high-risk score. Protein tyrosine phosphatase receptor type F polypeptide-interacting protein alpha-4 (PPFIA4), STY11, SCN3B, and SPTBN5 were shared in the same PPI network. Immune infiltration results showed that SCN3B and synaptotagmin 11 expression were obviously associated with B cell, macrophage, myeloid dendritic cell, neutrophils, and T cell CD4+ and CD8+ in both colon adenocarcinoma and rectal adenocarcinoma. CHIR-99021, MLN4924, and YK4-279 were identified as the potential drugs for treatment. Finally, upregulated EIOGs genes PPFIA4 and SCN3B were found in colon adenocarcinoma and PPFIA4 and SCN3B were proved to promote cell proliferation and migration in vitro. We demonstrated here that EIOGs promoting a malignancy phenotype was related with poor survival and prognosis in CRC, which might be served as novel therapeutic targets in CRC management.

Xuebijing improves inflammation and pyroptosis of acute lung injury by up-regulating miR-181d-5p-mediated SPP1 inactivation.

BACKGROUND: Xuebijing (XBJ) is widely applied in the treatment of Acute Lung Injury (ALI). This study focused on the potential mechanism of XBJ in Lipopolysaccharide (LPS)-induced ALI. METHODS: The rat ALI model was established by injection of LPS (10 mg/kg) and pretreated with XBJ (4 mL/kg) three days before LPS injection. BEAS-2B cell line was stimulated with LPS (1 µg/mL) and ATP (5 mM) to induce pyroptosis, and XBJ (2 g/L) was pretreated 24h before induction. The improvement effects of XBJ on pulmonary edema, morphological changes, and apoptosis in ALI lung tissue were evaluated by lung wet/dry weight ratio, HE-staining, and TUNEL staining. Inflammatory cytokines in lung tissue and cell supernatant were determined by ELISA. pyroptosis was detected by flow cytometry. Meanwhile, the expressions of miR-181d-5p, SPP1, p-p65, NLRP3, ASC, caspase-1, p20, and GSDMD-N in tissues and cells were assessed by RT-qPCR and immunoblotting. The relationship between miR-181d-5p and SPP1 in experimental inflammation was reported by dual luciferase assay. RESULTS: XBJ could improve inflammation and pyroptosis of ALI by inhibiting contents of inflammatory cytokines, and levels of inflammation- and pyroptosis-related proteins. Mechanistically, XBJ could up-regulate miR-181d-5p and inhibit SPP1 in ALI. miR-181d-5p can target the regulation of SPP1. Depressing miR-181d-5p compensated for the ameliorative effect of XBJ on ALI, and overexpressing SPP1 suppressed the attenuating effect of XBJ on LPS-induced inflammation and pyroptosis. CONCLUSION: XBJ can regulate the miR-181d-5p/SPP1 axis to improve inflammatory response and pyroptosis in ALI.

Long-term drinking of green tea combined with exercise improves hepatic steatosis and obesity in male mice induced by high-fat diet.

Dietary habits and exercise play an important role in the well-being of human health. Currently, how long of drinking tea combined with exercise could efficiently ameliorate hepatic steatosis and obesity still needs to be investigated. Here, short-term and long-term green tea drinking combined with exercise were studied to improve hepatic steatosis and obesity in high-fat diet-induced (HF) mice. Our results showed that Yunkang 10 green tea (GT) combined with exercise (Ex) exhibited synergistic prevention effects on ameliorating hepatic steatosis and obesity. Especially, 22-week intervention with GT or Ex improved all symptoms of obesity, which indicated that long-term intervention exhibited profound preventive effects than the short term. Moreover, the combined intervention of 22 weeks inhibited the activation of NF-κB pathway and the expression of proinflammatory cytokines, which suggests that tea combined exercise may improve liver steatosis mainly by inhibiting inflammation. The key molecules for regulating lipid and glucose metabolism SCD1 were obviously downregulated, and GLU2 and PPARγ were significantly upregulated by GT and exercise in the liver of high-fat diet-induced mice. This study demonstrated that long-term intervention with GT and exercise effectively relieved hepatic steatosis and obesity complications by ameliorating hepatic inflammation, reducing lipid synthesis, and accelerating glucose transport.

Pro-vasculogenic Fibers by PDA-Mediated Surface Functionalization Using Cell-Free Fat Extract (CEFFE).

Formation of adequate vascular network within engineered three-dimensional (3D) tissue substitutes postimplantation remains a major challenge for the success of biomaterials-based tissue regeneration. To better mimic the in vivo angiogenic and vasculogenic processes, nowadays increasing attention is given to the strategy of functionalizing biomaterial scaffolds with multiple bioactive agents. Aimed at engineering electrospun biomimicking fibers with pro-vasculogenic capability, this study was proposed to functionalize electrospun fibers of polycaprolactone/gelatin (PCL/GT) by cell-free fat extract (CEFFE or FE), a newly emerging natural "cocktail" of cytokines and growth factors extracted from human adipose tissue. This was achieved by having the electrospun PCL/GT fiber surface coated with polydopamine (PDA) followed by PDA-mediated immobilization of FE to generate the pro-vasculogenic fibers of FE-PDA@PCL/GT. It was found that the PDA-coated fibrous mat of PCL/GT exhibited a high FE-loading efficiency (∼90%) and enabled the FE to be released in a highly sustained manner. The engineered FE-PDA@PCL/GT fibers possess improved cytocompatibility, as evidenced by the enhanced cellular proliferation, migration, and RNA and protein expressions (e.g., CD31, vWF, VE-cadherin) in the human umbilical vein endothelial cells (huvECs) used. Most importantly, the FE-PDA@PCL/GT fibrous scaffolds were found to enormously stimulate tube formation in vitro, microvascular development in the in ovo chick chorioallantoic membrane (CAM) assay, and vascularization of 3D construct in a rat subcutaneous embedding model. This study highlights the potential of currently engineered pro-vasculogenic fibers as a versatile platform for engineering vascularized biomaterial constructs for functional tissue regeneration.

Enterotoxigenic Bacteroides fragilis (ETBF) Enhances Colorectal Cancer Cell Proliferation and Metastasis Through HDAC3/miR-139-3p Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) is believed to promote the malignant process of colorectal cancer (CRC), but the underlying molecular mechanism still needs to be revealed. CRC cells (SW480 and HCT-116) were treated with ETBF strain. Cell proliferation, invasion and, migration were evaluated by cell counting kit 8 assay, EdU assay, colony formation assay, transwell assay, and wound healing assay. Protein expression was analyzed by western blot. MicroRNA (miR)-139-3p and histone deacetylase 3 (HDAC3) expression levels in tissues and cells were determined by qRT-PCR. Xenograft tumor model was conducted to evaluate the effect of miR-139-3p on CRC tumor growth. ETBF treatment could promote CRC cell proliferation, invasion and migration. MiR-139-3p expression was decreased by ETBF, and its overexpression reversed the effect of ETBF on CRC cell progression. HDAC3 negatively regulated miR-139-3p expression, and its overexpression facilitated CRC cell behaviors via reducing miR-139-3p expression. Moreover, HDAC3 expression was increased by ETBF, and its knockdown also abolished ETBF-mediated CRC cell progression. Additionally, miR-139-3p overexpression could reduce CRC tumor growth in vivo. ETBF aggravated CRC proliferation and metastasis via the regulation of HDAC3/miR-139-3p axis. The discovery of ETBF/HDAC3/miR-139-3p axis may provide a new direction for CRC treatment.

Physiological cyclic stretching potentiates the cell-cell junctions in vascular endothelial layer formed on aligned fiber substrate.

In vascular tissue engineering, formation of stable endothelial cell-cell and cell-substrate adhesions is essential for maintaining long-term patency of the tissue-engineered vascular grafts (TEVGs). In this study, sheet-like aligned fibrous substrates of poly(l-lactide-co-caprolactone) (PLCL) were prepared by electrospinning to provide basement membrane-resembling structural support to endothelial cells (ECs). Cyclic stretching at physiological and pathological levels was then applied to human umbilical vein endothelial cells (HUVECs) cultured on chosen fibrous substrate using a force-loading device, from which effects of the cyclic stretching on cell-cell and cell-substrate adhesions were examined. It was found that applying uniaxial 1 Hz cyclic stretch at physiological levels (5 % and 10 % elongation) strengthened the cell-cell junctions, thus leading to improved structural integrity, functional expression and resistance to thrombin-induced damaging impacts in the formed endothelial layer. The cell-cell junctions were disrupted at pathological level (15 % elongation) cyclic stretching, which however facilitated the formation of focal adhesions (FAs) at cell-substrate interface. Mechanistically, the effects of cyclic stretching on endothelial cell-cell and cell-substrate adhesions were identified to be correlated with the RhoA/ROCK signaling pathway. Results from this study highlight the relevance between applying dynamic mechanical stimulation and maintaining the structural integrity of the formed endothelial layer, and implicate a necessity to implement appropriate dynamic mechanical training (i.e., preconditioning) to obtain tissue-engineered blood vessels with long-term patency post-implantation.

Xuebijing improves inflammation and pyroptosis of acute lung injury by up-regulating miR-181d-5p-mediated SPP1 inactivation

Abstract Background Xuebijing (XBJ) is widely applied in the treatment of Acute Lung Injury (ALI). This study focused on the potential mechanism of XBJ in Lipopolysaccharide (LPS)-induced ALI. Methods The rat ALI model was established by injection of LPS (10 mg/kg) and pretreated with XBJ (4 mL/kg) three days before LPS injection. BEAS-2B cell line was stimulated with LPS (1 μg/mL) and ATP (5 mM) to induce pyroptosis, and XBJ (2 g/L) was pretreated 24h before induction. The improvement effects of XBJ on pulmonary edema, morphological changes, and apoptosis in ALI lung tissue were evaluated by lung wet/dry weight ratio, HE-staining, and TUNEL staining. Inflammatory cytokines in lung tissue and cell supernatant were determined by ELISA. pyroptosis was detected by flow cytometry. Meanwhile, the expressions of miR-181d-5p, SPP1, p-p65, NLRP3, ASC, caspase-1, p20, and GSDMD-N in tissues and cells were assessed by RT-qPCR and immunoblotting. The relationship between miR-181d-5p and SPP1 in experimental inflammation was reported by dual luciferase assay. Results XBJ could improve inflammation and pyroptosis of ALI by inhibiting contents of inflammatory cytokines, and levels of inflammation- and pyroptosis-related proteins. Mechanistically, XBJ could up-regulate miR-181d-5p and inhibit SPP1 in ALI. miR-181d-5p can target the regulation of SPP1. Depressing miR-181d-5p compensated for the ameliorative effect of XBJ on ALI, and overexpressing SPP1 suppressed the attenuating effect of XBJ on LPS-induced inflammation and pyroptosis. Conclusion XBJ can regulate the miR-181d-5p/SPP1 axis to improve inflammatory response and pyroptosis in ALI.

Shape-memory responses compared between random and aligned electrospun fibrous mats.

Significant progress has been made in the design of smart fibers toward achieving improved efficacy in tissue regeneration. While electrospun fibers can be engineered with shape memory capability, both the fiber structure and applied shape-programming parameters are the determinants of final performance in applications. Herein, we report a comparison study on the shape memory responses compared between electrospun random and aligned fibers by varying the programming temperature T prog and the deforming strain ε deform . A PLLA-PHBV (6:4 mass ratio) polymer blend was first electrospun into random and aligned fibrous mat forms; thereafter, the effects of applying specific T prog (37°C and 46°C) and ε deform (30%, 50%, and 100%) on the morphological change, shape recovery efficiency, and switching temperature T sw of the two types of fibrous structures were examined under stress-free condition, while the maximum recovery stress σ max was determined under constrained recovery condition. It was identified that the applied T prog had less impact on fiber morphology, but increasing ε deform gave rise to attenuation in fiber diameters and bettering in fiber orientation, especially for random fibers. The efficiency of shape recovery was found to correlate with both the applied T prog and ε deform , with the aligned fibers exhibiting relatively higher recovery ability than the random counterpart. Moreover, T sw was found to be close to T prog , thereby revealing a temperature memory effect in the PLLA-PHBV fibers, with the aligned fibers showing more proximity, while the σ max generated was ε deform -dependent and 2.1-3.4 folds stronger for the aligned one in comparison with the random counterpart. Overall, the aligned fibers generally demonstrated better shape memory properties, which can be attributed to the macroscopic structural orderliness and increased molecular orientation and crystallinity imparted during the shape-programming process. Finally, the feasibility of using the shape memory effect to enable a mechanoactive fibrous substrate for regulating osteogenic differentiation of stem cells was demonstrated with the use of aligned fibers.

Formation of low-density electrospun fibrous network integrated mesenchymal stem cell sheet.

Cell sheets combined with electrospun fibrous mats represent an attractive approach for the repair and regeneration of injured tissues. However, the conventional dense electrospun mats as supportive substrates in forming "cell sheet on fiber mat" complexes suffer from problems of limiting the cellular function and eliciting a host response upon implantation. To give full play to the role of electrospun biomimicking fibers in forming quality cell sheets, this study proposed to develop a cell-fiber integrated sheet (CFIS) featuring a spatially homogeneous distribution of cells within the fiber structure by using a low-density fibrous network for cell sheet formation. A low-density electrospun polycaprolactone (PCL) fibrous network at a density of 103.8 ± 16.3 µg cm-2 was produced by controlling the fiber deposition for a short period of 1 min and subsequently transferred onto polydimethylsiloxane rings for facilitating cell sheet formation, in which rat bone marrow-derived mesenchymal cells were used. Using a dense electrospun PCL fibrous mat (481.5 ± 7.5 µg cm-2) as the control, it was found that cells on the low-density fibrous network (L-G) exhibited improved capacities in spreading, proliferation, stemness maintenance and matrix-remodeling during the process of CFIS formation. Structurally, the CFIS constructs revealed strong integration between the cells and the fibrous network, thus providing excellent cohesion and physical integrity to enable strengthening of the formed cell sheet. By contrast, the cell sheet formed on the dense fibrous mat (D-G) showed a two-layer (biphasic) structure due to the limitation of cellular invasion. Moreover, such engineered CFIS was identified with enhanced immunomodulatory effects by promoting LPS-stimulated macrophages towards an M2 phenotype in vitro. Our results suggest that the CFIS may be used as a native tissue equivalent "cell sheet" for improving the efficacy of the tissue engineering approach for the repair and regeneration of impaired tissues.

[Chondrogenic and ameliorated inflammatory effects of chitosan-based biomimetic scaffold loaded with icariin].

Icariin (ICA) is a small molecule drug capable of promoting cartilage repair and ameliorating inflammation. Loading ICA into a biomaterial scaffold for cartilage tissue engineering will thus potentially enhance the biological functionality of the engineered scaffold. In this study, short fibers processed from electrospun poly(l-lactide-co-caprolactone) (PLCL) fibers which were prior coated with polydopamine (PDA), were mixed with citric acid doped chitosan solution (CC) for preparing short fibers reinforced chitosan hydrogel (PDA@PLCL/CC) by a freeze-thawing combined freeze-drying method. Thereafter, ICA was loaded into the PDA@PLCL/CC scaffold through physical adsorption to generate a newly engineered biomimetic cartilage scaffold (ICA-PDA@PLCL/CC). Finally, ICA-mediated chondrogenic and ameliorated inflammatory effects of the ICA-PDA@PLCL/CC scaffold were examined in vitro using rabbit chondrocytes. The results showed that the ICA-free PDA@PLCL/CC scaffold possessed appropriate pore size and porosity (> 80%), high water absorbance capacity and improved mechanical performance, and also promoted chondrocyte proliferation and adhesion. The ICA-laden ICA-PDA@PLCL/CC scaffold was evidenced to maintain cytomorphology, upregulate the expression of chondrogenic gene (sox-9), glycosaminoglycan gene (gag), and type Ⅱ collagen gene (col Ⅱ) as well as the synthesis of the cartilage matrix. In the presence of a simulated inflammation, the ICA-PDA@PLCL/CC scaffold was found to reduce chondrocyte fibrosis, effectively downregulate the expression of proinflammatory factors interleukin-6 (il-6), interleukin-1 (il-1), and inducible nitric oxide synthase (inos) in chondrocytes. It can also reduce matrix metalloproteinase-3 (mmp-3) expression and promote the synthesis of the extracellular matrix glycosaminoglycan (GAG) and type II collagen (Col II). The newly developed ICA-PDA@PLCL/CC scaffold may find applications in the regeneration and repair of cartilage defects.

Engineering a Mechanoactive Fibrous Substrate with Enhanced Efficiency in Regulating Stem Cell Tenodifferentiation.

Electrospun-aligned fibers in ultrathin fineness have previously demonstrated a limited capacity in driving stem cells to differentiate into tendon-like cells. In view of the tendon's mechanoactive nature, endowing such aligned fibrous structure with mechanoactivity to exert in situ mechanical stimulus by itself, namely, without any forces externally applied, is likely to potentiate its efficiency of tenogenic induction. To test this hypothesis, in this study, a shape-memory-capable poly(l-lactide-co-caprolactone) (PLCL) copolymer was electrospun into aligned fibrous form followed by a "stretching-recovery" shape-programming procedure to impart shape memory capability. Thereafter, in the absence of tenogenic supplements, human adipose-derived stem cells (ADSCs) were cultured on the programmed fibrous substrates for a duration of 7 days, and the effects of constrained recovery resultant stress-stiffening on cell morphology, proliferation, and tenogenic differentiation were examined. The results indicate that the in situ enacted mechanical stimulus due to shape memory effect (SME) did not have adverse influence on cell viability and proliferation, but significantly promoted cellular elongation along the direction of fiber alignment. Moreover, it revealed that tendon-specific protein markers such as tenomodulin (TNMD) and tenascin-C (TNC) and gene expression of scleraxis (SCX), TNMD, TNC, and collagen I (COL I) were significantly upregulated on the mechanoactive fibrous substrate with higher recovery stress compared to the counterparts. Mechanistically, the Rho/ROCK signaling pathway was identified to be involved in the substrate self-actuation-induced enhancement in tenodifferentiation. Together, these results suggest that constrained shape recovery stress may be employed as an innovative loading modality to regulate the stem cell tenodifferentiation by presenting the fibrous substrate with an aligned tendon-like topographical cue and an additional mechanoactivity. This newly demonstrated paradigm in modulating stem cell tenodifferentiation may improve the efficacy of tendon tissue engineering strategy for tendon healing and regeneration.

Engineering a Highly Biomimetic Chitosan-Based Cartilage Scaffold by Using Short Fibers and a Cartilage-Decellularized Matrix.

Engineering scaffolds with structurally and biochemically biomimicking cues is essential for the success of tissue-engineered cartilage. Chitosan (CS)-based scaffolds have been widely used for cartilage regeneration due to its chemostructural similarity to the glycosaminoglycans (GAGs) found in the extracellular matrix of cartilage. However, the weak mechanical properties and inadequate chondroinduction capacity of CS give rise to compromised efficacy of cartilage regeneration. In this study, we incorporated short fiber segments, processed from electrospun aligned poly(lactic-co-glycolic acid) (PLGA) fiber arrays, into a citric acid-modified chitosan (CC) hydrogel scaffold for mechanical strengthening and structural biomimicking and meanwhile introduced cartilage-decellularized matrix (CDM) for biochemical signaling to promote the chondroinduction activity. We found that the incorporation of PLGA short fibers and CDM remarkably strengthened the mechanical properties of the CC hydrogel (+349% in compressive strength and +153% in Young's modulus), which also exhibited a large pore size, appropriate porosity, and fast water absorption ability. Biologically, the engineered CDM-Fib/CC scaffold significantly promoted the adhesion and proliferation of chondrocytes and supported the formation of matured cartilage tissue with a cartilagelike structure and deposition of abundant cartilage ECM-specific GAGs and type II collagen (+42% in GAGs content and +295% in type II collagen content). The enhanced mechanical competency and chondroinduction capacity with the engineered CDM-Fib/CC scaffold eventually fulfilled successful in situ osteochondral regeneration in a rabbit model. This study thereby demonstrated a great potential of the engineered highly biomimetic chitosan-based scaffold in cartilage tissue repair and regeneration.

Collagen and chondroitin sulfate functionalized bioinspired fibers for tendon tissue engineering application.

Functional tendon tissue engineering depends on harnessing the biochemical and biophysical cues of the native tendon extracellular matrix. In this study, we fabricated highly-aligned poly(L-lactic acid) (PLLA) fibers with surfaces decorated by two of the crucial tendon ECM components, type 1 collagen (COL1) and chondroitin sulfate (CS), through a coaxial stable jet electrospinning approach. Effects of the biomimetic COL1-CS (shell)/PLLA (core) fibers on the tenogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro were investigated. Higher rates of cell spreading and proliferation are observed on the aligned COL1-CS/PLLA fibers compared to that on the plain PLLA fibers. Expression of the tendon-associated genes scleraxis (SCX) and COL1 as well as protein tenomodulin (TNMD) are significantly increased. Introduction of mechanical stimulation gives rise to synergistic effect on tenogenic differentiation of hMSCs. Higher expression of TGF-ß2, TGFßR-II, and Smad3 by the cells on the COL1-CS/PLLA fiber substrates are observed, which indicates that COL1-CS/PLLA ultrafine fibers dictate the hMSC tenogenic differentiation through activating the TGF-ß signaling pathway. Animal study in rat Achilles tendon repair model corroborated the promoting role of COL1-CS/PLLA in regenerating a tendon-like tissue. Thus, our highly aligned biomimicking fibers may serve as an efficient scaffolding system for functional tendon regeneration.

Shape Memory and Osteogenesis Capabilities of the Electrospun Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Modified Poly(l-Lactide) Fibrous Mats.

Poly(l-lactide) (PLLA) as one of the most well-known biodegradable polyesters has been studied extensively for bone tissue engineering. If being properly programmed, scaffolds from PLLA can also be endowed with the capability of shape memory. However, several noted issues, for example, mechanical brittleness, high glass transition temperature Tg, and relatively poor shape retention and recovery properties, necessitate modification of the PLLA to improve its application efficacy in physiological conditions. This study is proposed to modify PLLA by having the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) incorporated to form ultrafine composite fibers (i.e., PLLA-PHBV) through electrospinning. Different pairs of PLLA-PHBV at the varying mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 0:10 can be successfully electrospun into fibrous form with the fineness of 2-3 µm. Incorporation of PHBV enables to give rise to desired Tg decreases and also, interestingly, increases in the Young's modulus of the PLLA-PHBV blends, while gradually increasing the PHBV mass ratios up to 30%. The PLLA-PHBV (7:3) formulation is identified to present excellent shape memory properties with high shape fixing ratio (>98%) and shape recovery ratio (>96%) compared to the unmodified PLLA fiber counterpart. Moreover, the PLLA-PHBV (7:3) fibers also show enhanced osteogenesis-inducing ability in the mouse bone mesenchymal stem cells, even under nonosteoinductive conditions. Collectively, for the first time this study demonstrates the enhanced shape memory and osteogenesis capabilities of the electrospun PLLA-PHBV composite fibers, and the researched PLLA-PHBV (7:3) fiber system could be potentially applied as a multifunctional scaffolding material for applications in bone tissue repair and regeneration. Impact statement By first converting the poly(l-lactide) (PLLA)-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hybrids into fibrous form at varied mass ratios followed by a thorough characterization, we reasonably demonstrated that incorporation of an appropriate amount of PHBV (i.e., 30%) into the PLLA fibers could give rise to significant improvement on the shape memory capability of the PLLA, along with the desired decreases in the transition temperature (Tg). Moreover, the fibrous PLLA-PHBV (7:3) scaffold was also found to significantly promote the osteogenic commitment in bone mesenchymal stem cells with osteoinductive factors in a synergistic manner. Our biomimicking and shape memory enabled fibrous scaffold of PLLA-PHBV could be used to construct multifunctional three-dimensional scaffold with shape memory effect for bone regeneration.

Understanding the cellular responses based on low-density electrospun fiber networks.

Fibers produced from electrospinning are well-known to be extremely fine with diameters ranging from tens of nanometers to a few microns. Such ultrafine fibers not only allow for engineering scaffolds resembling the ultrastructure of the native extracellular matrix, but also offer possibility to explore the remodeling behavior of cells in vitro, due to their mechanically 'adequate' softness endowed by their ultrafine fineness. However, the remodeling effect of cells on the biomimicking fibrous substrates remains to be understood, because the crisscrossing and entangling among nanofibers in those tightly packed fibrous mats ultimately lead to merely a topological phenomenon, similar to that of the nanofiber-like topography embossed on the surface of a solid matter. In this study, the effect of nanofiber density on cellular response behavior was investigated by reducing the density of electrospun fiber networks. Using polycaprolactone (PCL) as a model polymer, randomly oriented fiber networks with various densities, namely, 37.7 ± 16.3 µg/cm2 (D1), 103.8 ± 16.3 µg/cm2 (D2), 198.2 ± 40.0 µg/cm2 (D3), and 471.8 ± 32.7 µg/cm2 (D4), were prepared by electrospinning for varied collection durations (10 s, 50 s, 100 s, and 10 min, respectively). By examining the responsive behavior of the human induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSCs) cultured on these nanofibrous networks, we showed that the fiber network with a moderate density (D2) is beneficial to the cell attachment, spreading, actin polymerization, contractility and migration. There also showed an increased tendency in nuclear localization of the Yes-associated protein (YAP) and subsequent activation of YAP responsive gene transcription, and cell proliferation and collagen synthesis were also enhanced on the D2. However, further increasing the fiber density (D3, D4) gave rise to weakened induction effect of fibers on the cellular responses. These results enrich our understanding on the effect of fiber density on cell behavior, and disclose the dependence of cellular responses on fiber density. This study paves the way to precisely design biomimetic fibrous scaffolds for achieving enhanced cell-scaffold interactions and tissue regeneration.

Stiffness of the aligned fibers affects structural and functional integrity of the oriented endothelial cells.

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, effects of the stiffness of scaffolds with anisotropic topography on ECs within vivo like oriented morphology has received little attention. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68-2141.72 MPa), similar to the range of stiffness of the healthy and diseased subendothelial matrix, were used to investigate the effects of fiber stiffness on ECs' attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs, capable of providing topographical cues, is a crucial endothelium-protective microenvironmental factor by maintaining stable and quiescent endothelium with in vivo like orientation and strong cell-cell junctions. Stiffer AFSs exacerbated the disruption of endothelium integrity, the occurrence of endothelial-to-mesenchymal transition (EndMT), and the inflammation-induced activation in the endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels. STATEMENT OF SIGNIFICANCE: In vascular tissue engineering, promoting endothelialization on scaffold surface has been considered as a paramount strategy to reduce post-implantation complications. Electrospun aligned fibers have been known to provide contact guidance effect in directing endothelial cells' oriented growth, however, whether the formed EC monolayer in 'correct' orientation shape is of 'correct' function hasn't been explored yet. Given the recognized important role of substrate stiffness in endothelial function, AFSs across physiologically relevant range of moduli (14.68-2141.72 MPa) while maintaining consistent surface chemistry and topographical features were employed to investigate the fiber stiffness effects on ECs function in anisotropic morphology. This study will provide more insightful perspectives in the physiologically remodeling progression of vascular endothelium and design of vascular scaffolds.

Dietary supplement of Yunkang 10 green tea and treadmill exercise ameliorate high fat diet induced metabolic syndrome of C57BL/6 J mice.

BACKGROUND: Diet and exercise play important roles in ameliorating metabolic syndrome. Yunkang 10 (Camellia sinensis var. assamica) is a most cultivated tea variety for making tea in the Southwestern China. Currently, there is no report of healthy effects of Yunkang 10 green tea (YKGT) and treadmill exercise (Ex) on high fat diet induced metabolic syndrome (MetS). We aimed to investigate the beneficial effects and molecular mechanism of YKGT and Ex using high fat diet induced MetS of C57BL/6 mice. METHODS: Catechins and caffeine in water extract of YKGT were measured via high performance liquid chromatography (HPLC). 10-week old mice were fed with high fat diet (HFD) for 10 weeks to induce obese mice. Then the obese mice were fed with continuous high fat diet (HFD), HFD with YKGT, HFD with Ex, and HFD with both YKGT and Ex for 8 weeks, respectively. The another group of 10-week old mice fed with low fat diet (LFD) were used as control. RESULTS: HPLC data revealed that YKGT has abundantly high concentration of epigallocatechin gallate (EGCG) and caffeine compared to Longjing 43 (Camellia sinensis var. sinensis) green tea. YKGT and Ex significantly decreased the level of blood glucose, serum total cholesterol (TC), triglyceride (TG), insulin, and alanine aminotransferase activity (ALT) when compared to HFD group. The fatty liver and hepatic pro-inflammatory gene expression in the YKGT, Ex and YKGT+Ex groups was mitigated significantly compared with HFD group, respectively. The phosphorylation of inhibitor of nuclear factor kappa-B kinase α/ß (IKKα/ß) and inhibitor of nuclear factor kappa-B α (IkBα) protein in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway was also decreased in YKGT or YKGT+Ex groups. The combination of YKGT and Ex prevented gene expression for lipid synthesis in the liver tissue, and significantly upregulated mRNA level of glucose transport genes in the skeletal muscles, when compared to the HFD group. CONCLUSIONS: This study demonstrated that YKGT supplement or exercise appeared to reverse preexisting metabolic syndrome, and effectively relieved the fatty liver and hepatic inflammatory response induced by high fat diet. YKGT supplement and treadmill exercise together had better beneficial effects than only one intervention.

Asiaticoside loading into polylactic-co-glycolic acid electrospun nanofibers attenuates host inflammatory response and promotes M2 macrophage polarization.

Polylactic-co-glycolic acid (PLGA) is one of the most promising synthetic materials for tissue engineering due to its excellent biocompatibility, good mechanical properties, and tunable biodegradation time. However, the accumulation of PLGA degradative products could cause significant host inflammatory response, a microenvironment favoring tissue fibrosis that is mainly mediated by M1 subtype macrophage. Drug loading is an emerging technology to modify electrospun nanofibers, and asiaticoside (AS) was demonstrated as an anti-inflammatory drug. This study investigated the potential effect of AS incorporating into PLGA electrospun nanofibers on modulating host inflammatory response. The results showed that AS co-electrospun with PLGA nanofibers could significantly reduce the infiltration of inflammatory cells at the implantation site as opposed to the site of regular PLGA nanofibers. In particular, immunohistochemistry demonstrated decreased M1 macrophage infiltration whereas increased M2 macrophage infiltration in the implantation site of AS-PLGA nanofibers when compared to the PLGA implantation site. In vitro study also revealed that culture of human fibroblasts on PLGA nanofibers resulted in significantly enhanced gene expression of inflammatory cytokines when compared to non-seeded fibroblasts, but these genes were significantly downregulated when seeded on AS-PLGA. Furthermore, culture of macrophage on AS-PLGA led to upregulated M2 marker gene expression and downregulated M1 marker gene expression. Collectively, these results indicate that, AS might be an ideal drug for loading into electrospun polymer nanofibers and thus favoring for tissue regeneration via mediating macrophage polarization.

Electrospun acid-neutralizing fibers for the amelioration of inflammatory response.

Biodegradable aliphatic polyesters, especially polylactide (PLA), polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), are the most representative and widely used synthetic polymers in the field of tissue engineering and regenerative medicine. However, these polyesters often give rise to aseptic inflammation because of their acidic degradation products after implantation. Here, unidirectional shell-core structured fibers of chitosan/poly(lactide-co-glycolide) (i.e., CTS/PLGA) with acid-neutralizing capability were developed for addressing the noted issue by coating the PLGA fiber surfaces with a layer of the alkaline chitosan by coaxial electrospinning. Our results showed that during a period of 8-week degradation, the shell-layer of chitosan with its unique alkaline nature for acid-neutralization obviously hindered the pH decrease as a result of the degradation of PLGA-core. In a mocked acidic environment testing of the human dermal fibroblasts, chitosan-enabled acidity neutralization could significantly reduce in vitro the secretion of inflammatory factors and downregulate the expression of related inflammatory genes. Thereafter, biocompatibility assessment in vitro showed that the CTS/PLGA fibers had poorer cell adhesion capacity than the PLGA fibers but were cytocompatible and promoted cell migration and secretion of collagen. Moreover, subcutaneous embedding for two and four weeks in vivo revealed that the CTS/PLGA fibers significantly reduced the recruitment of inflammatory cells and the formation of foreign body giant cells (FBGCs). This study thereby demonstrated the evident acid-neutralizing effect of the chitosan-coating layer on alleviating the inflammatory responses caused by the acidic degradation products of the PLGA-core. Our highly aligned CTS/PLGA fibers, as a kind of quasi "pH-neutral fibers" with the acid-neutralizing capability, could be potentially applied for engineering those architecturally anisotropic tissues (e.g., tendon/ligament) toward improved efficacy of regeneration. STATEMENT OF SIGNIFICANCE: It is well known that acidic degradation products from representative aliphatic polyesters (e.g., PLA, PGA, and PLGA) give rise to the problem of aseptic inflammation. Various alkaline components acting as neutralizing agents have been used to address the noted issue. However, rather less attention has been paid to engineer these polyesters into a fibrous form with acid-neutralizing functionality. The present study proposes the concept of "pH-neutral fibers" and develops shell-core structured unidirectional fibers of chitosan/poly(lactide-co-glycolide) with acid-neutralizing capability for ameliorating inflammatory responses caused by the acidic degradation products of PLGA. It provides a comprehensive study encompassing fiber characterization and in vitro and in vivo evaluation, which would pave the way for developing sophisticated pH-neutral fibers for functional tissue regeneration.