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Silk fibroin (SF) and fiber alignment were introduced into polycaprolactone (PCL)-based electrospun nanofibers as chemical and physical cues for tendon tissue engineering applications. The physicochemical properties of random PCL (RP) nanofibers, random PCL/SF (RPSF) nanofibers and aligned PCL/SF (APSF) nanofibers were characterized for fiber orientation and SF blending effects. An in vitro cell culture with rabbit dermal fibroblasts (RDFBs) on nanofibers indicated that SF promotes cell proliferation to a higher extent than fiber alignment. Cells aligned in the direction of fiber axes could be confirmed through scanning electron microscopy (SEM) observation and cytoskeleton staining. The quantitative real-time polymerase chain reaction (qRT-PCR) experiments indicated up-regulated gene expression of tendon marker proteins (type I collagen (Col I), fibronectin and biglycan) on APSF nanofibers and tendon reconstruction was confirmed from Col III gene expression. Animal experiments with Achilles tendon defect repairs in rabbits were carried out with RPSF and APSF scaffolds. The beneficial effects of fiber alignment were verified from histological and immunohistochemical staining, where cell migration and extracellular matrix protein deposition tend to stretch in a parallel direction along the axial direction of APSF nanofibers with enhanced Col I and tenascin C production. Biomechanical testing indicated the tensile stiffness and maximum load of cell-seeded APSF scaffolds were 60.2 and 81.3% of normal tendon values, respectively, which are significantly higher than cell-seeded RPSF or acellular APSF and RPSF scaffolds. These results suggest that APSF nanofiber scaffolds combined with RDFBs have the potential to repair the gap defects of Achilles tendons in vivo and to effectively restore the function and structure of tendons.

To develop a pH-sensitive dual targeting magnetic nanocarrier for chemo-phototherapy in cancer treatment, we prepared magnetic graphene oxide (MGO) by depositing Fe3O4 magnetic nanoparticles on graphene oxide (GO) through chemical co-precipitation. MGO was modified with polyethylene glycol (PEG) and cetuximab (CET, an epidermal growth factor receptor (EGFR) monoclonal antibody) to obtain MGO-PEG-CET. Since EGFR was highly expressed on the tumor cell surface, MGO-PEG-CET was used for dual targeted delivery an anticancer drug doxorubicin (DOX). The physico-chemical properties of MGO-PEG-CET were fully characterized by dynamic light scattering, transmission electron microscopy, X-ray diffraction, Fourier transform Infrared spectroscopy, thermogravimetric analysis, and superconducting quantum interference device. Drug loading experiments revealed that DOX adsorption followed the Langmuir isotherm with a maximal drug loading capacity of 6.35 mg/mg, while DOX release was pH-dependent with more DOX released at pH 5.5 than pH 7.4. Using quantum-dots labeled nanocarriers and confocal microscopy, intracellular uptakes of MGO-PEG-CET by high EGFR-expressing CT-26 murine colorectal cells was confirmed to be more efficient than MGO. This cellular uptake could be inhibited by pre-incubation with CET, which confirmed the receptor-mediated endocytosis of MGO-PEG-CET. Magnetic targeted killing of CT-26 was demonstrated in vitro through magnetic guidance of MGO-PEG-CET/DOX, while the photothermal effect could be confirmed in vivo and in vitro after exposure of MGO-PEG-CET to near-infrared (NIR) laser light. In addition, the biocompatibility tests indicated MGO-PEG-CET showed no cytotoxicity toward fibroblasts and elicited minimum hemolysis. In vitro cytotoxicity tests showed the half maximal inhibitory concentration (IC50) value of MGO-PEG-CET/DOX toward CT-26 cells was 1.48 µg/mL, which was lower than that of MGO-PEG/DOX (2.64 µg/mL). The IC50 value could be further reduced to 1.17 µg/mL after combining with photothermal therapy by NIR laser light exposure. Using subcutaneously implanted CT-26 cells in BALB/c mice, in vivo anti-tumor studies indicated the relative tumor volumes at day 14 were 12.1 for control (normal saline), 10.1 for DOX, 9.5 for MGO-PEG-CET/DOX, 5.8 for MGO-PEG-CET/DOX + magnet, and 0.42 for MGO-PEG-CET/DOX + magnet + laser. Therefore, the dual targeting MGO-PEG-CET/DOX could be suggested as an effective drug delivery system for anticancer therapy, which showed a 29-fold increase in therapeutic efficacy compared with control by combining chemotherapy with photothermal therapy.

Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at −16 °C. The crosslinking density and the mechanical properties of the cryogel matrix could be tuned by adjusting the content of GlcN used during cryogel preparation. In general, incorporation of GlcN did not influence scaffold pore size and ultimate compressive strain but increased porosity. The GH-GlcN16 cryogel showed the highest swelling ratio and degradation rate in hyaluronidase and collagenase solutions. On the contrary, the Young’s modulus, storage modulus, ultimate compressive stress, energy dissipation level, and rate of stress relaxation decreased by increasing the GlcN content in the cryogel. The release of GlcN from the scaffolds in the culture medium of chondrocytes could be sustained for 21 days for GH-GlcN16 in contrast to only 7 days for GH-GlcN9. In vitro cell culture experiments using rabbit articular chondrocytes revealed that GlcN incorporation affected cell proliferation, morphology, and maintenance of chondrogenic phenotype. Overall, GH-GlcN16 showed the best performance in maintaining chondrogenic phenotype with reduced cell proliferation rate but enhanced glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Quantitative real-time polymerase chain reaction also showed time-dependent up-regulation of cartilage-specific marker genes (COL II, aggrecan and Sox9) for GH-GlcN16. Implantation of chondrocytes/GH-GlcN16 constructs into full-thickness articular cartilage defects of rabbits could regenerate neocartilage with positive staining for GAGs and COL II. The GH-GlcN16 cryogel will be suitable as a scaffold for the treatment of articular cartilage defects.

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To improve intraperitoneal chemotherapy and to prevent postsurgical peritoneal adhesion, we aimed to develop a drug delivery strategy for controlled release of a chemotherapeutic drug from the intraperitoneally injected thermosensitive poly(N-isopropylacrylamide)-based hydrogel (HACPN), which is also endowed with peritoneal anti-adhesion properties. Anticancer drug doxorubicin (DOX) was loaded into the hydrogel (HACPN-DOX) to investigate the chemotherapeutic and adhesion barrier effects in vivo. A burst release followed by sustained release of DOX from HACPN-DOX was found due to gradual degradation of the hydrogel. Cell culture studies demonstrated the cytotoxicity of released DOX toward CT-26 mouse colon carcinoma cells in vitro. Using peritoneal carcinomatosis animal model in BALB/c mice with intraperitoneally injected CT-26 cells, animals treated with HACPN-DOX revealed the best antitumor efficacy judging from tumor weight and volume, survival rate, and bioluminescence signal intensity when compared with treatment with free DOX at the same drug dosage. HACPN (or HACPN-DOX) also significantly reduced the risk of postoperative peritoneal adhesion, which was generated by sidewall defect-cecum abrasion in tumor-bearing BALB/c mice, from gross and histology analyses. This study could create a paradigm to combine controlled drug release with barrier function in a single drug-loaded injectable hydrogel to enhance the intraperitoneal chemotherapeutic efficacy while simultaneously preventing postsurgical adhesion.

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