Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector.

Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.

ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure.

The aim of this study was to identify the signal transduction pathways and mechano-transducers that play critical roles in the processes induced by changes in cyclic hydrostatic pressure and fluid shear in 3-dimensional (3D) culture systems. Mesenchymal stem cells were loaded into a polymeric scaffold and divided into three groups according to the stress treatment: static, fluid shear, and hydrostatic pressure with fluid shear. Cells were exposed daily to a hydrostatic pressure of 0.2 MPa for 1 min followed by 14 min rest with fluid flow at 30 rpm. Protein extracts were analyzed by Western blot for extracellular signal-regulated kinase 1/2 (ERK1/2). The complexes were cultured under the mechanical stimuli for 21 days with or without phospho-ERK1/2 inhibitor (U0126) and evaluated by RT-PCR, calcium contents, and immunohistochemistry. Under conditions of mechanical stimulation, the activation of ERK1/2 was sustained or increased with time. U0126 suppressed mechanical stimuli-induced expression of osteocalcin. In addition, calcium contents and the degrees of osteocalcin and osteopontin staining were decreased by this inhibitor. These results demonstrate that mechanical stimuli, particularly hydrostatic pressure with fluid shear, enhance osteogenesis in 3D culture systems via ERK1/2 activation.

A proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell.

Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.

Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts.

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.

Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation.

Gain-of-function mutations of KIT are common genetic events in gastrointestinal stromal tumors (GISTs). To investigate the molecular characteristics of KIT mutations in GISTs, 20 GISTs (14 GISTs with KIT mutation and 6 GISTs without KIT mutation) were analyzed by two-dimensional electrophoresis and matrix-associated laser desorption ionization mass spectrophotometry-time of flight. Comparative analysis of the respective spot patterns on two-dimensional electrophoresis showed that HMGB1, an intranuclear protein that interacts with several transcription factors and plays a role in tumor metastasis after its secretion, was overexpressed in GISTs with KIT mutation. All of the 14 GISTs with KIT mutation, and only 2 of 6 GISTs without KIT mutation, revealed HMGB1 expression. Of the GISTs with KIT mutation, 12 (86%) showed strong expression of HMGB1, more than three times higher in intensity than the maximum observed in the 6 GISTs without KIT mutation by two-dimensional electrophoresis analysis. The overexpression of HMGB1 was further supported by Western blot analysis, and directly related to matrix metalloproteinase 2 overexpression. Our results indicate that the overexpression of HMGB1 is common in GISTs and is related to the KIT mutation, and that this may play a role in the tumorigenesis of GISTs because overexpressed HMGB1 could accelerate genes related to tumor growth and invasion.