Hydrogels have been widely used as cell-laden vehicles for therapeutic transplantation in regenerative medicine. Although the advantages of biocompatibility and injectability for in situ grafting have made hydrogel a superior candidate in tissue engineering, there remain challenges in long-term efficacy of tissue development using hydrogel, especially when more sophisticated applications are demanded. The major bottleneck lies in environmental constraints for neo-tissue generation in the gel bulk such as proliferation of encapsulated cells (colonies) per se and also accommodation of their endogenously produced extracellular matrices. In this study, we endeavor to develop an innovative tissue engineering system to overcome these drawbacks through a novel microcavitary hydrogel (MCG)-based scaffolding technology and a novel phase transfer cell culture (PTCC) strategy to enable phenotypically bona fide neo-tissue formation in an injectable artificial graft. For this purpose, microspherical cavities are created in cell-encapsulating hydrogel bulk via a retarded dissolution of coencapsulated gelatin microspheres. Based on proliferation and affinity selection, the encapsulated cell colonies adjacent to the gel-cavity interface will spontaneously outgrow the hydrogel phase and sprout into cavities, enabling neo-tissue islets to fill up the voids and further expand throughout the whole system for full tissue regeneration. The design of MCG-PTCC strategy was elicited from an observation of a spontaneous dynamic outgrowth of chondrocytes from the edge of a cell-laden hydrogel construct over prolonged cultivation--a phenomenon named edge flourish. This MCG-PTCC strategy potentially introduce a new application to hydrogels in the field of regenerative medicine through elevation of its role as a cell vehicle to a three-dimensional transplantable growth-guiding platform for further development of newly generated tissues that better fulfill the demanding criteria of scaffolds in therapeutic tissue regeneration.

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.

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In this study, transgenic effects of combination of transforming growth factor (TGF) beta3 and shRNA silencing type I collagen (Col I) on chondrogenesis of synovium-derived mesenchymal stem cells (SMSCs) were evaluated. SMSCs were infected with recombinant adenoviruses encoding TGF beta 3 (Ad-TGF beta 3) and/or anti-Col I shRNA (Ad-shRNA) separately, simultaneously (Ad-combination), or conjugately (Ad-double, mediated by one vector encoding both). The transduced SMSCs were encapsulated in alginate hydrogel and cultured for 30 days in chondrogenic medium. The expression of cartilaginous extracellular matrix components was investigated by quantitative real-time RT-PCR (qRT-PCR) and histological staining. qRT-PCR showed an up-regulation in chondrocytes marker genes such as type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) in Ad-TGF beta 3, Ad-double, and Ad-combination groups on day 30. Whereas, Ad-TGF beta 3 treatment induced significant elevation in Col I, which could be largely resisted by anti-Col I shRNA functionality. Histological and immunohistochemical staining results were consistent with our qRT-PCR data. These results demonstrate that the application of combinational adenoviral vector-mediated transgenic TGF beta 3 and shRNA targeting Col I possesses the potential in promoting the chondrogenic differentiation of SMSCs as well as inhibiting the formation of fibrocartilage.