With the increase in interest for using electrospun nanofibers for skin tissue formation, it has become essential to establish the correlation between nanofiber configurations and skin cell responses. In this regard, the present study was aimed at understanding how nanofiber matrices, especially their chemical composition, regulate the phenotype of dermal fibroblasts in a transforming growth factor (TGF)-β1 rich milieu close to the native wound-healing microenvironment. Cultures of human dermal fibroblasts on fibrinogen- and collagen-containing electrospun nanofiber matrices revealed that with the presence of exogenous TGF-β1 the fibroblasts on fibrinogen matrices exhibited a differentiation phenotype, characterized by lower proliferation, faster migration and higher expression of α-smooth muscle actin (α-SMA), in contrast to the proliferation phenotype on collagen matrices. Such distinct cellular responses are a result of the differential activation of TGF-β1/Smad signaling in fibroblasts on different nanofiber matrices, with marked elevation of TGF-β1 receptor I and Smad2/3 phosphorylation on the fibrinogen fibers. Blockade of integrin αVβ3 with an inhibitor (Cilengitide) showed a decreased migration and expression of α-SMA in fibroblasts along with a reduced Smad3 phosphorylation, confirming the involvement of integrin αVβ3 in TGF-β1-induced fibroblast differentiation on the fibrinogen-containing nanofibers. These findings demonstrate the regulatory effects of nanofiber composition on the TGF-β1-dependent wound-healing activities of skin fibroblasts through the integrin-mediated signaling pathway. Meanwhile, these results also point out the importance of recreating the wound microenvironment for in vitro studies on materials–cell interactions.

There are many natural examples of smart structures that are able to change conformations and functionalities responding to the external stimuli. The responsiveness is directly related to their unique structures. In the design of new materials, it is crucial to endow these materials with the capabilities to change structures and functionalities under the external stimuli. In this research, virus-mimicking protein nanogels with temperature-induced reversible structures and redox responsiveness are synthesized by cross-linking a thermally responsive polymer poly(di(ethylene glycol) methyl ether methacrylate-co-2-(2-pyridyldisulfide) ethyl methacrylate) with reduced bovine serum albumin (BSA) molecules through thiol–disulfide exchange reaction. The lower critical solution temperature (LCST) and sizes of the nanogels can be controlled by controlling the reaction conditions. The nanogels are able to change their structures responding to the temperature change. Below the LCST, BSA molecules are embedded inside the nanogels and protected by the polymer chains. Above the LCST, polymer chains collapse forming the cores, and BSA moves to the shells to stabilize the nanogels. The disulfide-cross-linked nanogels are dissociated in the presence of glutathione. In vitro cytotoxicity assays and cell uptake assays demonstrate that the nanogels show low toxicity toward 3T3, 293T, and MCF-7 cells and can be internalized into the MCF-7 cells. The nanogels will find applications in protein delivery.Keywords: bovine serum albumin; core−shell structure; nanogels; thiol−disulfide exchange reaction;

Repairing damaged annulus fibrosus (AF) is one of the most challenging topics for treating intervertebral disc (IVD) disease. Tissue engineering combining scaffolds with cells provides a promising solution. However, fabricating scaffolds with a circumferentially oriented fibrous structure similar to native AF remains a big challenge. In this study, we present an effective and convenient wet spinning strategy for fabricating an AF scaffold composed of circumferentially oriented poly(ε-caprolactone) microfibers. Cell culture experiments demonstrated that this scaffold could support AF cell attachment, proliferation and infiltration as confirmed by scanning electron microscopy (SEM), confocal microscopy, live/dead staining and a MTT assay, respectively. Histological, immunohistochemical staining, biochemical quantitative analysis and RT-PCR showed that the AF cells (AFCs) inside scaffolds could spread along the microfiber direction and secrete an AF-related extracellular matrix (e.g., glycosaminoglycans, collagen type I and II) which also oriented along the microfiber direction. As a result, the compressive and tensile properties were enhanced with increasing culture time. These results demonstrate the feasibility of using this new wet-spun microfibrous oriented scaffold for AFCs culture, and the potential application for regeneration of AF.

Repairing osteochondral defects (OCD) remains a formidable challenge due to the high complexity of native osteochondral tissue and the limited self-repair capability of cartilage. Osteochondral tissue engineering is a promising strategy for the treatment of OCD. In this study, we fabricated a novel integrated trilayered scaffold using silk fibroin and hydroxyapatite by combining paraffin-sphere leaching with a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique. This biomimetic scaffold is characterized by three layers: a chondral layer with a longitudinally oriented microtubular structure, a bony layer with a 3D porous structure and an intermediate layer with a dense structure. Live/dead and CCK-8 tests indicated that this scaffold possesses good biocompatibility for supporting the growth, proliferation, and infiltration of adipose-derived stem cells (ADSCs). Histological and immunohistochemical stainings and real-time polymerase chain reaction (RT-PCR) confirmed that the ADSCs could be induced to differentiate toward chondrocytes or osteoblasts in vitro at chondral and bony layers in the presence of chondrogenic- or osteogenic-induced culture medium, respectively. Moreover, the intermediate layer could play an isolating role for preventing the cells within the chondral and bony layers from mixing with each other. In conclusion, the trilayered and integrated osteochondral scaffolds can effectively support cartilage and bone tissue generation in vitro and are potentially applicable for OC tissue engineering in vivo.Keywords: adipose-derived stem cells; calcified cartilage layer; integrated scaffold; osteochondral tissue engineering; silk fibroin

•A novel 3D silk fibroin biphasic scaffold for integrated IVD tissue engineering was fabricated by sequentially using the paraffin sphere-leaching method and the phase separation method.•With this technique, the pore size, porosity and mechanical property of the scaffold were controllable, moreover, the high interconnected AF and NP phases integrated tightly.•This tightly integrated silk biphasic scaffold with good biocompatibility provides a suitable 3-D environment for IVD cells to adhere, migrate and proliferate.The intervertebral disc (IVD) tissue engineering construct provides a promising approach for the treatment of symptomatic degenerative disc diseases. In this study, a novel 3D silk fibroin biphasic scaffold for integrated IVD tissue engineering was fabricated by sequentially using the paraffin sphere-leaching method for annulus fibrosus (AF) phase and the phase separation method for nucleus pulposus (NP) phase. Both phases were perfectly integrated and possessed a highly interconnected porous structure with pore size of 220±23.1 μm for AF and 90±17.8 μm for NP phase. Furthermore, the scaffolds were found to have a high porosity of 91% and 93% for AF and NP phases, respectively. In addition, this silk biphasic scaffold had a relative high compressive modulus under wet conditions (150.7±6.8 kPa). Rabbit AF and NP cells could attach, grow and further penetrate into the scaffold and distribute uniformly. This silk fibroin biphasic IVD scaffold emerges as a potential candidate for IVD tissue engineering.

•The oriented PCL microfiber scaffolds were fabricated by a new wet-spinning system using blended solution of edible oil and hexane as the non-solvent coagulation bath.•The fiber diameter and porosity of these microfiber scaffolds are controllable by tune the spun condition.•Smooth muscle cells could grow in an oriented fashion along the fibers and infiltrate the scaffold as well as maintain SMCs phenotype.The tissue engineered blood vessel requires oriented fibrous scaffold as template to guide smooth muscle cells (SMCs) oriented growth and infiltration. It is a considerable challenge to fabricate this kind of fibrous scaffold. In this letter, we developed a new wet-spinning system that involves a blended solution of edible oil and hexane as a non-solvent coagulation bath to fabricate oriented poly(ε-caprolactone) (PCL) microfiber scaffold. The fiber diameter and porosity could be controlled in the range of 7 to 27 μm and 68 to 82%, respectively. SMCs could grow in an oriented fashion along the fibers and infiltrate inside the scaffold as well as maintain SMCs phenotype. This oriented microfiber scaffold has a promising potential for regenerating blood vessels and other fibrous tissues such as tendon, ligament and intervertebral discs.

Poly(ε-caprolactone) (PCL) is widely adopted as an ingredient for tissue engineering scaffolds. To improve its cell affinity, in this study, we developed a new method to introduce bioactive RGD peptides onto the surface of PCL via condensation reaction between 2-cyanobenzothiazole (CBT) and D-cysteine. The PCL fibrous membranes were prepared by electrospinning, and RGD functionalization was characterized by fluorescence microscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA). As expected, our results demonstrated the successful RGD immobilization on the surface of PCL. RGD modification improved the hydrophilicity of PCL, changing their WCA from 112.20° to 38.35°. Cell adhesion, spreading and proliferation of 3T3 fibroblasts were also enhanced. We therefore believe that the methods reported in this study was facile and effective for functional modification of the hydrophobic PCL scaffolds. The moderate reaction conditions are also suitable for covalent immobilization of bioactive molecules onto PCL.

•Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold.•The scaffold has appropriate mechanical property, porosity and pore size•The scaffold supports growth and infiltration of nucleus pulposus cells.•Nucleus pulposus cells can secrete extracellular matrix in the scaffolds.•The scaffold is a potential candidate for tissue engineered nucleus pulposus.Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue.

Nanogels are promising carriers for the delivery of anti-cancer drugs for cancer therapy. We report in this study on a Janus nanogel system formed by mixing a prodrug of Taxol (PEGylated Taxol) and a copolymer of PLGA–PEG–PLGA. The Janus nanogels have good stability over months in aqueous solutions and the freeze-dried powder of nanogels can be re-dispersed instantly in aqueous solutions. The Janus nanogels show an enhanced inhibition effect on tumor growth in a mice breast cancer model probably due to the enhanced uptake of the nano-sized materials by the EPR effect. What is more, the nanogels can also serve as physical carriers to co-deliver other anti-cancer drugs such as doxorubicin to further improve the anti-cancer efficacy. The results obtained from H&E staining and TUNEL assay also support the observation of tumor growth inhibition. These results suggest the potential of this novel delivery system for cancer therapy.

Molecular hydrogels of therapeutic agents are a novel kind of self-delivery system that can sustain release of drugs or pro-drugs. We have previously developed a molecular hydrogelator of folic acid (FA)–Taxol conjugate triggered by phosphatase. In this paper, we report a novel molecular hydrogelator system of FA–Taxol conjugates with improved synthetic strategy. The hydrogels are formed by the reduction of disulfide bond by glutathione (GSH). These hydrogels could sustain release of Taxol through ester bond hydrolysis. Compared with intravenous (i.v.) injection of clinically used Taxol® with four times the dosage, our hydrogel could inhibit tumor growth more efficiently by a single dose of intra-tumor (i.t.) administration. These observations suggested the big potential of this novel gelation system of Taxol for cancer therapy.

A novel approach for vascular grafts to achieve rapid endothelialization is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via EPC-specific antibodies, aptamer, or peptides that specifically bind to EPCs. Inspired by this idea, the electrospun poly(epsilon-caprolactone) (PCL) mats were modified with zwitterionic poly(carboxybetaine methacrylate) (PCBMA) and phage display-selected-EPC-specific peptide, TPSLEQRTVYAK (TPS). We tested the physical and chemical properties, cyto-compatibility, and platelet adhesion of the modified material, and investigated the specificity of the functionalized surface for capturing EPCs. The results indicated that PCL modified with zwitterionic PCBMA and TPS peptide showed improved hydrophilicity without morphology change and damage of the mats. Furthermore, the modified material supported adherence and growth of vascular cells and resisted platelets adhesion. The surfaces also specifically captured EPCs rather than bone marrow mesenchymal stem cells and human umbilical vein endothelial cells. This surface-functionalized PCL graft may offer new opportunities for designing new vascular grafts.Highlights► Surface of electrospun PCL fibers was dually functionalized with PCBMA and TPS peptide. ► Modified surface of electrospun PCL fibers showed effective inhibition of platelet adhesion. ► The conjugation of TPS provided specific cell binding toward EPC.

With the increase in interest for using electrospun nanofibers for skin tissue formation, it has become essential to establish the correlation between nanofiber configurations and skin cell responses. In this regard, the present study was aimed at understanding how nanofiber matrices, especially their chemical composition, regulate the phenotype of dermal fibroblasts in a transforming growth factor (TGF)-β1 rich milieu close to the native wound-healing microenvironment. Cultures of human dermal fibroblasts on fibrinogen- and collagen-containing electrospun nanofiber matrices revealed that with the presence of exogenous TGF-β1 the fibroblasts on fibrinogen matrices exhibited a differentiation phenotype, characterized by lower proliferation, faster migration and higher expression of α-smooth muscle actin (α-SMA), in contrast to the proliferation phenotype on collagen matrices. Such distinct cellular responses are a result of the differential activation of TGF-β1/Smad signaling in fibroblasts on different nanofiber matrices, with marked elevation of TGF-β1 receptor I and Smad2/3 phosphorylation on the fibrinogen fibers. Blockade of integrin αVβ3 with an inhibitor (Cilengitide) showed a decreased migration and expression of α-SMA in fibroblasts along with a reduced Smad3 phosphorylation, confirming the involvement of integrin αVβ3 in TGF-β1-induced fibroblast differentiation on the fibrinogen-containing nanofibers. These findings demonstrate the regulatory effects of nanofiber composition on the TGF-β1-dependent wound-healing activities of skin fibroblasts through the integrin-mediated signaling pathway. Meanwhile, these results also point out the importance of recreating the wound microenvironment for in vitro studies on materials–cell interactions.

Molecular hydrogels of therapeutic agents are a novel kind of self-delivery system that can sustain release of drugs or pro-drugs. We have previously developed a molecular hydrogelator of folic acid (FA)–Taxol conjugate triggered by phosphatase. In this paper, we report a novel molecular hydrogelator system of FA–Taxol conjugates with improved synthetic strategy. The hydrogels are formed by the reduction of disulfide bond by glutathione (GSH). These hydrogels could sustain release of Taxol through ester bond hydrolysis. Compared with intravenous (i.v.) injection of clinically used Taxol® with four times the dosage, our hydrogel could inhibit tumor growth more efficiently by a single dose of intra-tumor (i.t.) administration. These observations suggested the big potential of this novel gelation system of Taxol for cancer therapy.