Wanqian Liu

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (ɛ-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.

The use of hyaluronan to regulate protein adsorption and cell infiltration in nanofibrous scaffolds

Electrospun nanofibers are prepared with mixtures of natural and synthetic polymers that can behave cooperatively to demonstrate combinations of mechanical, structural and biochemical properties for tissue engineering applications. However, the large surface area and inherent small pores of these structures give nanofibrous scaffolds high non-specific protein adsorption and poor cell infiltration. In this study, we developed a protein resistant and porous nanofibrous scaffold composed of hyaluronan (HA), silk fibroin (SF), and polycaprolactone (PCL) blends via one-step emulsion electrospinning. The scaffolds were characterized and evaluated for nanostructures, chemical composition, mechanical properties, hydrophilicity, and protein adsorption. Swelling and degradation studies revealed the formation of oriented pore structures within the body of the scaffolds and increasing the pore size between fibers. Addition of HA component transformed current PCL/SF components into hydrophilic fibers, which caused the suppression of non-specific protein adsorption, resulting in the reduction of fibrosis tissue thickness and macrophages adhesion in vivo. Importantly, HA-based scaffolds significantly enhanced cell infiltration in vitro and tissue ingrowth in vivo. In vitro cultivation of human primary skin fibroblasts on the HA-based scaffolds showed a significant increase in cell proliferation and filopodia protrusions, but decreased in collagen I production. Furthermore, HA and HA-based scaffolds interacted with cell surface receptor CD44 to activate TGF-β1/MMPs signaling pathways that conducive to cell migration. These findings suggest that such an HA-based nanofibrous scaffold resists protein adsorption and enhances cell infiltration, may offer possibilities to overcome the limitations of electrospinning technology.

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.

A review of gradient stiffness hydrogels used in tissue engineering and regenerative medicine

Substrate stiffness is known to impact characteristics including cell differentiation, proliferation, migration and apoptosis. Hydrogels are polymeric materials distinguished by high water content and diverse physical properties. Gradient stiffness hydrogels are designed by the need to develop biologically friendly materials as extracellular matrix (ECM) alternatives to replace the separated and narrow-ranged hydrogel substrates. Important new discoveries in cell behaviors have been realized with model gradient stiffness hydrogel systems from the two-dimensional (2D) to three-dimensional (3D) scale. Basic and clinical applications for gradient stiffness hydrogels in tissue engineering and regenerative medicine continue to drive the development of stiffness and structure varied hydrogels. Given the importance of gradient stiffness hydrogels in basic research and biomedical applications, there is a clear need for systems for gradient stiffness hydrogel design strategies and their applications. This review will highlight past work in the field of gradient stiffness hydrogels fabrication methods, mechanical property test, applications as well as areas for future study.

Comparative study of normal and rheumatoid arthritis fibroblast-like synoviocytes proliferation under cyclic mechanical stretch: role of prostaglandin E2.

Fibroblast-like synoviocytes (FLSs) are one of the main contributors of prostaglandin E2 (PGE2) in the hyperplastic synovium of rheumatoid arthritis (RA) patients. cyclooxygenase-2 (COX-2)/PGE2 pathway is involved in the proliferation of several cell types. We have previously shown that mechanical stretch affects COX-2 and PGE2 production in human RA FLSs; however, its role in cell proliferation remains to be elucidated. In this study, a comparison is drawn between human RA and normal FLSs to understand the role of mechanical stretch and PGE2 on the proliferation of FLSs. The results showed that physiological level (6%, 1 Hz) of cyclic mechanical stretch significantly decreased the proliferation of RA FLSs but not normal FLSs, while the induction of apoptosis was not observed by stretch in either RA or normal FLSs. IL-1β (5 ng/ml)-induced COX-2/PGE2 levels are downregulated by stretch in RA FLSs only. Further investigation showed that high concentration (100 and 500 ng/ml) of PGE2 significantly induced cell proliferation only in RA FLSs, and this induction failed to be suppressed by stretch. In conclusion, this study demonstrated that elevated levels of PGE2 in the synovial cavity are involved in the proliferation of RA FLSs, and cyclic mechanical stretch regulates the RA synovial hyperplasia.

The effect of silk gland sericin protein incorporation into electrospun polycaprolactone nanofibers on in vitro and in vivo characteristics

The application of silk fibroin is a promising approach for designing biomaterials. However, silk sericin (SS) protein has not attracted much attention in the field of biomaterials as a natural biopolymer due to its immune responses, weak structural properties and high solubility. In this study, fifth instar silkworm (B. mori) middle gland extracted sericin protein and polycaprolactone (PCL) blends nanofibrous scaffolds were successfully fabricated via an emulsion electrospinning technique. PCL/SS nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Water contact angle and tensile measurements indicated that the PCL/SS scaffolds exhibited improved mechanical properties, as well as more favorable wettability, than that obtained from PCL alone. We also analyzed the effect of SS content in blends on cell morphology and proliferation of human primary skin fibroblasts (FEK4 cells) within 1-5 days. The results showed that cell proliferation significantly increased in the appropriate ratio of PCL/SS blends while showing more elongated cellular morphology. The mRNA gene expression of transforming growth factor β1 (TGF-β1) and collagen I were up-regulated in PCL/SS scaffolds. Furthermore, in vivo experiments suggested that low fibrosis tissue formation and macrophages adhesion of the PCL/SS nanofibrous scaffolds reveal its potential as future biocompatible scaffolds for tissue engineering.

Integration of QSAR modelling and QM/MM analysis to investigate functional food peptides with antihypertensive activity

Antihypertensive peptides derived from dietary proteins have long been recognised as an important source of developing functional foods with blood pressure-lowering effect. However, most of such peptides exhibit diverse tastes, such as sweet, bitter, sour and salty, which is a non-negligible aspect considered in the food development process. In the present study, several predictive quantitative structure–activity relationship (QSAR) models that correlate peptides structural features with their multi-bioactivities and bitter taste are established at both sequence and structure levels, and the models are then used to conduct extrapolation on thousands of randomly generated, structurally diverse peptides with chain lengths ranging from two to six amino acid residues. Based on the statistical results gained from QSAR modelling, the relationship between the antihypertensive activity and bitter taste of peptides at different sequence lengths is investigated in detail. Moreover, the structural basis, energetic property and biological implication underlying peptide interactions with angiotensin-converting enzyme (ACE), a key target of antihypertensive therapy, are analysed at a complex three-dimensional structure level by using a high-level hybrid quantum mechanics/molecular mechanics scheme. It is found that (a) bitter taste is highly dependent on peptide length, whereas ACE inhibitory potency has only a modest correlation with the length, (b) dipeptides and tripeptides perform a moderate relationship between their ACE inhibition and bitterness, but the relationship could not be observed for those peptides of more than three amino acid residues and (c) the increase in sequence length does not cause peptides to exhibit substantial enhancement of antihypertensive activity; this is particularly significant for longer peptides such as pentapeptides and hexapeptides.

The use of mechano growth factor to prevent cartilage degeneration in knee osteoarthritis

Previous studies have attached more importance to growth factors in treating cartilage degeneration and osteoarthritis (OA). Here, the capability of mechano growth factor‐C24E (MGF) to prevent osteoarthritic cartilage degeneration was evaluated in vitro and in vivo. Using in vitro cultured human OA chondrocytes treated with 10–60‐ng/ml MGF for 12 hr, we detected the cell proliferation, migration, and anabolism of OA chondrocytes. The unfolded protein response and the protein characteristic of OA pathology, such as transforming growth factor β, SMAD family member 3, and hypoxia‐inducible factor 2α of OA chondrocytes, were also detected by western blotting. Furthermore, protein kinase RNA‐like endoplasmic reticulum kinase was knocked down via small interfering RNA to illuminate the potential mechanism of MGFs treatment of OA. In a rabbit knee joint OA model, cartilage degeneration was inhibited after 2 weeks of treatment with 0.1–10‐μg/ml MGF. This study demonstrated that MGF treatment can inhibit the pathological apoptosis of OA chondrocytes and promote the proliferation, migration, and matrix synthesis of the chondrocytes. The results also demonstrate that the degeneration of OA cartilage can be delayed by MGF treatment partially via unfolded protein response regulated by protein kinase RNA‐like endoplasmic reticulum kinase and suggest a potential therapeutic application of MGF for OA treatment.

Effect of substrate stiffness on hepatocyte migration and cellular Young's modulus

Hepatic fibrosis progress accompanied by an unbalanced extracellular matrix (ECM) degradation and deposition leads to an increased tissue stiffness. Hepatocytes interplay with all intrahepatic cell populations inside the liver. However, how hepatocytes migration and cellular Youngs modulus influenced by the substrate stiffness are not well understood. Here, we established a stiffness‐controllable in vitro cell culture model by using a polyvinyl alcohol (PVA) hydrogel that mimicked the same physical stiffness as a fibrotic liver. Three levels of stiffness were used in our experiment that corresponded to the stiffness levels found in normal liver tissue (4.5 kPa), the early (19 kPa) and late stages (37 kPa) of fibrotic liver tissues. Cytoskeleton of hepatocyte was influenced by substrate stiffness. Soft substrate promoted the cellular migration and directionality. The cellular Youngs modulus firstly increased and then decreased with increasing substrate stiffness. Integrin‐β1 and β‐catenin expression on cytomembrane were up‐regulated and down‐regulated with the increase of substrate stiffness, respectively. Our data not only suggested that hepatocytes were sensitive to substrate stiffness, but also suggested that there may be a potential relationship among substrate stiffness, cellular Youngs modulus and the dynamic balance of integrin‐β1 and β‐catenin pathways. These results may provide us a new insight in mechanism investigation of mechano‐dependent diseases, especially like fibrosis related diseases.

Gsdma3 regulates hair follicle differentiation via Wnt5a-mediated non-canonical Wnt signaling pathway

Hair follicle is a mini-organ that consists of complex but well-organized structures, which are differentiated from hair follicle progenitor or stem cells. How non-canonical Wnt signaling pathway is involved in regulating hair follicle differentiation remains elusive. Here we showed that Wnt5a regulates hair follicle differentiation through an epithelial-mesenchymal interaction mechanism in mice. We first observed that Wnt5a is expressed in the epithelial and dermal papilla cells during hair follicle development and growth. For the upstream of Wnt5a, RT-PCR and immunohistochemistry staining showed that Wnt5a expression is significantly decreased in the Gsdma3-mutant mice in vivo. Overexpression of Gsdma3 results in a significantly increased expression of Wnt5a in the cultured epidermal cells in vitro. We also checked the downstream factors of Wnt5a by adenovirus-mediated overexpression of Wnt5a to the dermal papilla cells isolated from the mouse whisker. We found that overexpression of Wnt5a suppresses canonical Wnt signaling pathway effectors such as β-catenin and Lef1. In addition, genes involved in maintaining cell quiescent state are also significantly decreased in their expression to the DP cells which were treated by Wnt5a. Our study indicates that Wnt5a mediates epithelia-expressed Gsdma3 to influence DP cell behaviors, which in turn regulate hair follicle epithelia differentiation in mice.