Toward morphologically relevant extracellular matrix: nanofiber-hydrogel composites for tumor cell culture.

The natural extracellular matrix (ECM) consists of a continuous integrated fibrin network and a negatively charged proteoglycan-based matrix. In this work, we report a novel three-dimensional nanofiber hydrogel composite that mimics the natural ECM structure, exhibiting both degradability and mechanical characteristics comparable to that of tumor tissue. The embedded nanofiber improves the hydrogel mechanical properties, and varying the fiber density can match the elastic modulus of different tumor tissues (1.51-10.77 kPa). The degradability of the scaffold gives sufficient space for tumor cells to secrete and remodel the ECM. The expression levels of cancer stem cell markers confirmed the development of aggressive and metastatic phenotypes of prostate cancer cells in the 3D scaffold. Similar results were obtained in terms of anticancer resistance of prostate cancer cells in 3D scaffolds showing stem cell-like properties, suggesting that the current bionic 3D scaffold tumor model has broad potential in the development of effective targeted agents.

Spider-Silk-like Fiber Mat-Covered Polypropylene Warp-Knitted Hernia Mesh for Inhibition of Fibrosis under Dynamic Environment.

Hernia surgery is a widely performed procedure, and the use of a polypropylene mesh is considered the standard approach. However, the mesh often leads to complications, including the development of scar tissue that wraps around the mesh and causes it to shrink. Consequently, there is a need to investigate the relationship between the mesh and scar formation as well as to develop a hernia mesh that can prevent fibrosis. In this study, three different commercial polypropylene hernia meshes were examined to explore the connection between the fabric structure and mechanical properties. In vitro dynamic culture was used to investigate the mechanism by which the mechanical properties of the mesh in a dynamic environment affect cell differentiation. Additionally, electrospinning was employed to create polycaprolactone spider-silk-like fiber mats to achieve mechanical energy dissipation in dynamic conditions. These fiber mats were then combined with the preferred hernia mesh. The results demonstrated that the composite mesh could reduce the activation of fibroblast mechanical signaling pathways and inhibit its differentiation into myofibroblasts in dynamic environments.

Micro-nano hierarchical scaffold providing temporal-matched biological constraints for tendon reconstruction.

Due to the limitations of tendon biology, high-quality tendon repair remains a clinical and scientific challenge. Here, a micro-nano hierarchical scaffold is developed to promote orderly tendon regeneration by providing temporal-matched biological constraints. In short, fibrin (Fb), which provides biological constraints, is loaded into poly (DL-lactide-co-glycolide) nanoyarns with suitable degradation cycles (Fb-loaded nanofiber yarns (Fb-NY)). Then further combined with braiding technology, temporary chemotactic Fb scaffolds with tendon extracellular matrix-like structures are obtained to initiate the regeneration process. At the early stage of healing (2 w), the regeneration microenvironment is regulated (inducing M2 macrophages and restoring the early blood supply necessary for healing) by Fb, and the alignment of cells and collagen is induced by nanoyarn. At the late healing stage (8 w), with the degradation of Fb-NY, non-functional vascular regression occurs, and the newborn tissues gradually undergo load-bearing remodeling, restoring the anvascularous and ordered structure of the tendon. In summary, the proposed repair strategy provides temporal-matched biological constraints, offering a potential pathway to reconstruct the ordered structure and function of tendons.

Shish-kebab structure fiber with nano and micro diameter regulate macrophage polarization for anti-inflammatory and bone differentiation.

Biopolymer grafts often have limited biocompatibility, triggering excessive inflammatory responses similar to foreign bodies. Macrophage phenotype shifts are pivotal in the inflammatory response and graft success. The effects of the morphology and physical attributes of the material itself on macrophage polarization should be the focus. In this study, we prepared electrospun fibers with diverse diameters and formed a shish-kebab (SK) structure on the material surface by solution-induced crystallization, forming electrospun fiber scaffolds with diverse pore sizes and roughness. In vitro cell culture experiments demonstrated that SK structure fibers could regulate macrophage differentiation toward M2 phenotype, and the results of in vitro simulation of in vivo tissue reconstruction by the microenvironment demonstrated that the paracrine role of M2 phenotype macrophages could promote bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. In rats implanted with a subcutaneous SK-structured fiber scaffold, the large-pore size and low-stiffness SK fiber scaffolds demonstrated superior immune performance, less macrophage aggregation, and easier differentiation to the anti-inflammatory M2 phenotype. Large pore sizes and low-stiffness SK fiber scaffolds guide the morphological design of biological scaffolds implanted in vivo, which is expected to be an effective strategy for reducing inflammation when applied to graft materials in clinical settings.

Cobweb-Inspired Micro/Nanostructured Scaffolds for Soft Tissue Regeneration with Inhibition Effect of Fibrosis under Dynamic Environment.

In soft tissue repair, fibrosis can lead to repair failure and long-term chronic pain in patients. Excessive mechanical stimulation of fibroblasts is one of the causes of fibrosis during abdominal wall regeneration. Inspired by the cobweb, a polycaprolactone beaded fiber is prepared by electrospinning. The cobweb-inspired structure attenuates the mechanical stimulation of cells under a dynamic environment. Nano-protrusions are introduced into the scaffold for further inhibition of fibrosis by self-induced crystallization. A machine is built for in vitro dynamic culture and rat abdominal subcutaneous embedding experiments are performed to verify the inhibiting effect of fibrosis in a dynamic environment in vivo. Results show that the expression of integrin ß1 and α-smooth muscle actin is inhibited by the cobweb-inspired structure under dynamic culture. The results of hematoxylin and eosin and Masson's trichrome indicate that the cobweb-inspired structure has a good inhibitory effect on fibrosis in a dynamic environment in vivo. In general, the cobweb-inspired scaffold with nano-protrusions has a good ability to inhibit fibrosis under both static and dynamic environments. It is believed that the scaffold has promising applications in the field of inhibiting fibrosis caused by mechanical stimulation.

Pathway elucidation of bioactive rhamnosylated ginsenosides in Panax ginseng and their de novo high-level production by engineered Saccharomyces cerevisiae.

Rg2 and Re are both rhamnose-containing ginsenosides isolated exclusively from Panax plants, which exhibit broad spectrum of pharmacological activities. However, limitations of current plant-relied manufacturing methods have largely hampered their medical applications. Here, we report elucidation of the complete biosynthetic pathway of these two ginsenosides by the identification of a rhamnosyltransferase PgURT94 from Panax ginseng. We then achieve de novo bio-production of Rg2 and Re from glucose by reconstituting their biosynthetic pathways in yeast. Through stepwise strain engineering and fed-batch fermentation, the maximum yield of Rg2 and Re reach 1.3 and 3.6 g/L, respectively. Our work completes the identification of the last missing enzyme for Rg2 and Re biosynthesis and achieves their high-level production by engineered yeasts. Once scaled, this microbial biosynthesis platform will enable a robust and stable supply of Rg2 and Re and facilitate their food and medical applications.

A loofah-inspired scaffold with enhanced mimicking mechanics and tumor cells distribution for in vitro tumor cell culture platform.

Tumor cells cultured in a physiologically related three-dimensional (3D) matrix can replicate many basic characteristics of tumor tissue. Tumor tissues are harder than normal, so when using hydrogels for 3D tumor cell culture, attempts have been made to prepare hydrogel scaffolds that mimic the hardness of tumor tissues without reducing the porosity. In this study, a new 3D loofah-inspired scaffold was developed for prostate cancer cell culture. Since the loofah sponge structure of the spacer fabric, the composite scaffolds had a compression modulus similar to that of natural prostate tumor tissue at a lower hydrogel concentration (0.25% W/V), and also, endowed the scaffold with high porosity (85 ± 2.52%) for mass transfer. The results of in vitro cell experiments showed that the composite scaffold can support tumor cells to form clusters in a short time (3 days). Preliminary chemosensitivity analysis showed that the drug resistance of the composite scaffold was significantly higher than that of two-dimensional (2D) culture and COL scaffold. Therefore, the 3D tumor cell culture scaffold with bionic structures has the potential to be used as a tumor drug screening model.

Hierarchical Nanostructured Electrospun Membrane with Periosteum-Mimic Microenvironment for Enhanced Bone Regeneration.

An ideal periosteum substitute should be able to mimic the periosteum microenvironment that continuously provides growth factors, recruits osteoblasts, and subsequent extracellular matrix (ECM) mineralization to accelerate bone regeneration. Here, a calcium-binding peptide-loaded poly(ε-caprolactone) (PCL) electrospun membrane modified by the shish-kebab structure that can mimic the periosteum microenvironment was developed as a bionic periosteum. The calcium-binding peptide formed by the negatively charged heptaglutamate domain (E7) in the E7-BMP-2 with calcium ion in the tricalcium phosphate sol (TCP sol) through electrostatic chelation not only extended the release cycle of E7-BMP-2 but also promoted the biomineralization of the bionic periosteum. Cell experiments showed that the bionic periosteum could significantly improve the osteogenic differentiation of the rat-bone marrow-derived mesenchymal stem cells (rBMSCs) through both chemical composition and physical structure. The in vivo evaluation of the bionic periosteum confirmed the inherent osteogenesis of this periosteum microenvironment, which could promote the regeneration of vascularized bone tissue. Therefore, the hierarchical nanostructured electrospun membrane with periosteum-mimic microenvironment is a promising periosteum substitute for the treatment of bone defects.

Chitosan/gelatin-tannic acid decorated porous tape suture with multifunctionality for tendon healing.

The inferior tendon healing after surgery is inextricably linked to the surgical suture. Poor load transfer along the suture often results in a high tendon re-tear rate. Besides, the severe inflammation and infection induced by sutures even cause a second surgery. Herein, to alleviate the above-mentioned issues, a multifunctional suture was fabricated by decorating chitosan/gelatin-tannic acid (CS/GE-TA) on the porous tape suture. The porous tape suture ensured the required mechanical properties and sufficient space for tissue integration. Compared to the pristine suture, the CS/GE-TA decorated suture (TA100) presented a 332% increase in pull-out force from the tendon, indicating potentially decreased re-tear rates. Meanwhile, TA100 showed superior anti-inflammatory and antibacterial performances. In vivo experiments further proved that TA100 could not only reduce inflammatory action but also facilitate collagen deposition and blood vessel formation. These results indicate that the multifunctional sutures are promising candidates for accelerating tendon healing.

[Research Progress of Antibacterial Biliary Stent].

Biliary stent has been widely used in the treatment of biliary stricture and obstruction, it can relieve the pain of patients effectively, but bacterial infection and stent obstruction are still troublesome after surgery. We introduce the mechanism of infection and stent blockage caused by bacterial invasion after biliary stent implantation, and expound the formation mechanism of bacterial biofilm and bile sludge in this review. Antibacterial biliary stent is an effective way to inhibit biliary tract infection, the literatures on antibacterial modification of biliary stent with different antibacterial methods in domestic and abroad are reviewed, and the research prospect of antibacterial biliary stent is summarized and prospected.

ß-Tricalcium phosphate contained beaded-fiber scaffolds characterized by high early osteoinductive activity for vascularized bone regeneration.

The calcium phosphate component and surface topology of a scaffold are considered the two main factors that influence osteogenic differentiation. This research reports a one-step but effective scaffold preparation method that can regulate the morphology of nanofibers and control the distribution and release behavior of calcium phosphate nanoparticles (CaPs). Two beaded-on-string CaPs-loaded electrospun scaffolds (PT7.5 and PT4.5) with composite microstructures of microbeads and nanofibers were fabricated by adjusting the concentration of the electrospinning solution. The presence of the composite microstructure was conducive to the surface exposure and sustained release of bioactive components, which in turn could significantly promote the biomineralization and protein adsorption of the scaffold. A study of the human umbilical vein endothelial cells (HUVECs) and rat-bone marrow-derived mesenchymal stem cells (rBMSCs) revealed that cells cultured on scaffolds with composite microstructures (especially PT4.5) could enhance tube formation of the HUVECs and osteogenic differentiation of rBMSCs. The PT4.5 with significantly different microbead and nanofiber sizes presented the high potential to improve the early osteoinductive activity and angiogenesis of the CaPs-loaded electrospun scaffold and expand its advantage in bone regeneration.

Complete biosynthesis of the potential medicine icaritin by engineered Saccharomyces cerevisiae and Escherichia coli.

Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase III clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum (EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4'-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria (higher pH than cytoplasm) of the 8-prenylkaempferol-producing yeast strain or co-culturing the 8-prenylkaempferol-producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.

Innovative textile structures designed to prevent type III endoleaks in endovascular stent-grafts.

The damage caused to the fabric of endovascular stent-grafts most often occurs at the contact zones between the fabrics where they are attached to the apices of Z-shaped stents as a result of normal physiologic pulsatile movement within angulated vessels in vivo. Although design improvements were made over the years, the risks were not fully eliminated even with the newer M-shaped stent reconfiguration. In this study, we proposed to create and manufacture a novel fabric for stent-grafts with specifically designed reinforced zones to enhance resistance to fabric abrasion. These reinforced zones are set at the vicinity of the apices of the Z-shaped stents and between two adjacent Z-shaped stents where folding and pleating are commonly observed to occur in angulated vessels. Three innovative weaving structures with two different types of yarns and two controls were designed and prepared. Two commonly used commercial devices supported, respectively, with ringed stents, and Z-shaped stents were selected as the references for comparison. Textile structures including thickness, water permeability, mechanical properties, (more specifically tensile and bursting stress, as well as fatigue simulation) were tested on all fabrics. Compared to commercially available plain weaves, the fabrics with locally reinforced zones showed improved mechanical characteristics and fatigue resistant properties. A fabric designed with specifically reinforced zones has now clearly been shown to effectively reduce the abrasion caused by the apices of Z-shaped stents. However, further optimization may still be possible.

The unprecedented diversity of UGT94-family UDP-glycosyltransferases in Panax plants and their contribution to ginsenoside biosynthesis.

More than 150 ginsenosides have been isolated and identified from Panax plants. Ginsenosides with different glycosylation degrees have demonstrated different chemical properties and bioactivity. In this study, we systematically cloned and characterized 46 UGT94 family UDP-glycosyltransferases (UGT94s) from a mixed Panax ginseng/callus cDNA sample with high amino acid identity. These UGT94s were found to catalyze sugar chain elongation at C3-O-Glc and/or C20-O-Glc of protopanaxadiol (PPD)-type, C20-O-Glc or C6-O-Glc of protopanaxatriol (PPT)-type or both C3-O-Glc of PPD-type and C6-O-Glc of PPT-type or C20-O-Glc of PPD-type and PPT-type ginsenosides with different efficiencies. We also cloned 26 and 51 UGT94s from individual P. ginseng and P. notoginseng plants, respectively; our characterization results suggest that there is a group of UGT94s with high amino acid identity but diverse functions or catalyzing activities even within individual plants. These UGT94s were classified into three clades of the phylogenetic tree and consistent with their catalytic function. Based on these UGT94s, we elucidated the biosynthetic pathway of a group of ginsenosides. Our present results reveal a series of UGTs involved in second sugar chain elongation of saponins in Panax plants, and provide a scientific basis for understanding the diverse evolution mechanisms of UGT94s among plants.

Construction and application of textile-based tissue engineering scaffolds: a review.

Tissue engineering (TE) provides a practicable method for tissue and organ repair or substitution. As the most important component of TE, a scaffold plays a critical role in providing a growing environment for cell proliferation and functional differentiation as well as good mechanical support. And the restorative effects are greatly dependent upon the nature of the scaffold including the composition, morphology, structure, and mechanical performance. Medical textiles have been widely employed in the clinic for a long time and are being extensively investigated as TE scaffolds. However, unfortunately, the advantages of textile technology cannot be fully exploited in tissue regeneration due to the ignoring of the diversity of fabric structures. Therefore, this review focuses on textile-based scaffolds, emphasizing the significance of the fabric design and the resultant characteristics of cell behavior and extracellular matrix reconstruction. The structure and mechanical behavior of the fabrics constructed by various textile techniques for different tissue repairs are summarized. Furthermore, the prospect of structural design in the TE scaffold preparation was anticipated, including profiled fibers and some unique and complex textile structures. Hopefully, the readers of this review would appreciate the importance of structural design of the scaffold and the usefulness of textile-based TE scaffolds in tissue regeneration.

Tricalcium Phosphate Sol-Incorporated Poly(ε-caprolactone) Membrane with Improved Mechanical and Osteoinductive Activity as an Artificial Periosteum.

The periosteum plays a very important role in bone remodeling and regeneration due to its excellent osteogenic ability. However, in bone defects, the periosteum is inevitably damaged, has poor self-repair ability, and requires artificial materials as a substitute. This study is aimed to fabricate a highly bioactive poly(ε-caprolactone)/tricalcium phosphate sol (PCL/TCP sol) hybrid membrane as an artificial periosteum covering the surface of the bone defect to enhance bone regeneration. Three kinds of PCL membranes with different TCP contents were prepared and marked as P20T1 (4.8 wt %), P10T1 (9.1 wt %), and P5T1 (16.7 wt %). The physicochemical properties' evaluation confirmed that TCP sol was homogeneously dispersed in the PCL nanofibers. Compared with P5T1, samples P10T1 and P20T1 had enhanced the mechanical properties and a moderately hydrophilic surface (67.3 ± 2.4° for P20T1 and 48.9 ± 4.1° for P10T1). The biomineralization of hybrid membranes was significantly improved compared to the PCL membrane. Moreover, hybrid membranes significantly upregulated the rat bone marrow mesenchymal stem cells' (rBMSCs) response (proliferation and osteogenic differentiation) to them, and P10T1 showed better surface properties (hydrophilicity, bioactivity, and biomineralization) than P20T1. Thus, sample P10T1 with the best properties in this study has great potential as an artificial periosteum to accelerate bone regeneration.

Long-term anticoagulation and selective cells adhesion surface via combination of covalent grafting and layer by layer assembly.

Surface modification by long-term active component is essential for biocompatible polymers-based vascular grafts to prevent thrombus formation and reduce intimal hyperplasia. In this study, a simple approach was developed to immobilize bioactive heparin to the surface of ε-polycaprolactone (PCL) grafts through a two-step strategy combining covalent grafting and layer by layer assembly of polyelectrolytes. The performance of heparinized PCL was evaluated in vitro, including the release behavior of heparin, anticoagulation and different types of cells adhesion characteristic. A sustained-release of heparin was achieved by this immobilization strategy. Surface remaining heparin was up to 1.10 µg cm-2 on the modified PCL after release in vitro for 30 d. Specifically, the heparinized PCL has the long-term ability to prevent adhesion of blood cells and thrombus formation, and significantly inhibit the adhesion of smooth muscle cells. The two-step strategy provides a simple and general route to incorporate heparin on PCL graft surface. The surface heparinized PCL demonstrated in this work can be a useful material platform for biodegradable vascular stent graft.

[Structural control and characterization of hierarchically structured fibrous scaffolds].

OBJECTIVE: To prepare hierarchically structured fibrous scaffolds with different morphologies, and to explore the additional dimensionality for tuning the physicochemical properties of the scaffolds and the effect of their hemocompatibility and cytocompatibility. METHODS: Electrospinning poly (e-caprolactone) (PCL)/polyvinylpyrrolidone (PVP) bicomponent fibers (PCL∶PVP mass ratios were 8∶2 and 5∶5 respectively), and the surface porous fibrous scaffolds were prepared by extracting PVP components. The scaffolds were labeled PCL-P8 and PCL-P5 respectively according to the mass ratio of polymer. In addition, shish-kebab (SK) structured scaffolds with different kebab sizes were created by solution incubation method, which use electrospun PCL fibers as shish while PCL chains in solution crystallizes on the fiber surface. The PCL fibrous scaffolds with smooth surface was established as control group. The hierarchically structured fibrous scaffolds were characterized by field emission scanning electron microspore, water contact angle tests, and differential scanning calorimeter (DSC) experiments. The venous blood of New Zealand white rabbits was taken and hemolysis and coagulation tests were used to characterize the blood compatibility of the scaffolds. The proliferation of the pig iliac artery endothelial cell (PIEC) on the scaffolds was detected by cell counting kit 8 (CCK-8) method, and the biocompatibility of the scaffolds was evaluated. RESULTS: Field emission scanning electron microscopy showed that porous morphology appeared on the surface of PCL/PVP bicomponent fibers after extracting PVP. In addition, SK structure with periodic arrangement was successfully prepared by solution induction, and the longer the crystallization time, the larger the lamellar size and periodic distance. The contact angle and DSC measurements showed that when compared with smooth PCL fiber scaffolds, the crystallinity of PCL surface porous fibrous scaffolds and PCL-SK fibrous scaffolds increased, while the hydrophobicity of PCL-SK fibrous scaffolds increased, but the hydrophobicity of PCL porous scaffolds did not change significantly. The hemolysis test showed that the hemolysis rate of PCL surface porous fibrous scaffolds and PCL-SK fibrous scaffolds was higher than that of PCL fibrous scaffolds. According to American Society of Materials and Tests (ASTM) F756-08 standard, all scaffolds were non-hemolytic materials and were suitable for blood contact materials. Coagulation test showed that the coagulation index of PCL surface porous fibrous scaffolds and PCL-SK fibrous scaffolds was higher than that of PCL fibrous scaffolds at 5 and 10 minutes of culture. CCK-8 assay showed that both hierarchically structured fibrous scaffolds were more conducive to PIEC proliferation than PCL fibrous scaffold. CONCLUSION: Based on electrospinning technology, solution-induced and blend phase separation methods can be used to construct multi-scale fiber scaffolds with different morphologies, which can not only regulate the surface physicochemical properties of the scaffolds, but also have good blood compatibility and biocompatibility. The hierarchically structured fibrous scaffolds have high application potential in the field of tissue engineering.

Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency.

Synthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3-OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.