Type I collagen gene suppresses tumor growth and invasion of malignant human glioma cells.

BACKGROUND: Invasion is a hallmark of a malignant tumor, such as a glioma, and the progression is followed by the interaction of tumor cells with an extracellular matrix (ECM). This study examined the role of type I collagen in the invasion of the malignant human glioma cell line T98G by the introduction of the human collagen type I alpha1 (HCOL1A1) gene. RESULTS: The cells overexpressing HCOL1A1 were in a cluster, whereas the control cells were scattered. Overexpression of HCOL1A1 significantly suppressed the motility and invasion of the tumor cells. The glioma cell growth was markedly inhibited in vitro and in vivo by the overexpression of HCOL1A1; in particular, tumorigenicity completely regressed in nude mice. Furthermore, the HCOL1A1 gene induced apoptosis in glioma cells. CONCLUSION: These results indicate that HCOL1A1 have a suppressive biological function in glioma progression and that the introduction of HCOL1A1 provides the basis of a novel therapeutic approach for the treatment of malignant human glioma.

Potential of atelocollagen-mediated systemic antisense therapeutics for inflammatory disease.

To study the possibility of using atelocollagen as an oligonucleotide (ODN) delivery carrier in vivo, the activity of formulated antisense ODN targeted against the intercellular adhesion molecule-1 (ICAM-1) mRNA was investigated in an allergic dermatitis model in mice. The allergic dermatitis was elicited in one ear of animals sensitized by treatment with 2,4-dinitrofluorobenzene. Antisense ODN was given to the animals as a single intravenous injection of formulation containing atelocollagen. Antisense activity was determined by measurement of ear thickness, histopathology, and immunohistochemistry 24 hr after the initiation of the dermatitis. Antisense activity was found to increase according to the concentration of atelocollagen in the formulation. The effect mediated by the ODN formulated with 0.05% atelocollagen was more than 50 times greater than that provided by ODN infusion, although the levels of ODN formulated with atelocollagen dropped below that of the 24-hr infusion group within 30 min. The formulated ODN could suppress inflammatory progression by treatment at 8 hr after the ear challenge when inflammation had already commenced at the challenged site. Moreover, antisense activity was noted even when the formulated ODN was injected 3 days before the initiation of inflammation. These data demonstrate that atelocollagen can enhance antisense activity remarkably and that the sustainable antisense activity mediated by the formulation of ODN with atelocollagen could completely change the strategy of antisense therapeutics.

Atelocollagen for protein and gene delivery.

Recent progress in recombinant gene technology and cell culture technology has made it possible to use protein and polynucleotides as effective drugs. However, because of their short half-lives in the body and the necessity of delivering to target site, those substances do not always exhibit good potency as expected. Therefore, delivery systems of such drugs are important research subjects in the field of pharmacology, and to prolong the effect of these drugs, many studies are being conducted to control the release of proteins and polynucleotides from various carrier materials. Collagen is one of the most useful carrier materials for this purpose. In this article, we report on the controlled release of protein drugs using collagen, focusing on a new drug delivery system (DDS), the Minipellet, as our basic technology. Then we introduce our recent work about gene therapy using collagen-based DDS. Basic formulation study showed that collagen DDS protects DNA degradation from both chemical cleavage and enzymatic digestion. A single injection of collagen DDS containing plasmid DNA produced physiologically significant levels of gene-encoding proteins in the local site and systemic circulation of animals and resulted in prolonged biological effects. These results suggest that collagen DDS containing plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Also, variety kinds of application of collagen DDS for gene therapy using adenovirus vector, antisense DNA and DNA vaccine, will be discussed.

In Vitro Orientation of Fibroblasts and Myoblasts on Aligned Collagen Film.

A collagen film in which the collagen fibers were aligned was prepared and characterized by scanning electron microscopy. Cell orientation on this film was studied in vitro using human fibroblasts and chick embryo myoblasts. Ninety-four percent of innoculated fibroblasts were aligned along the direction of the collagen fiber. The cell orientation was disturbed when cytochalasin B or colchicine was added to the culture medium. The myoblasts showed a similar alignment along the direction of collagen fiber. The scanning electron microscopic observation revealed that none of the cytoplasmic extensions had consistent relationships to the direction of collagen fiber. Myoblast fusion was accelerated on the aligned membrane as compared to a randomly oriented film, suggesting some role of contact guidance in muscle cell differentiation.

Design of long-acting formulation of protein drugs with a double-layer structure and its application to rhG-CSF.

In order to design a sustained-release formulation of protein drugs characterized by excellent long-acting properties without an initial burst, a new double-layer minipellet (DL-MP) in which the lateral side of a matrix-type sustained-release formulation 'minipellet' using collagen as a carrier was coated with collagen was designed, and its performance was evaluated. In a DL-MP using bovine serum albumin (BSA) as a model drug, the initial burst observed with a single-layer minipellet (SL-MP) was effectively inhibited in an in vitro release test, and the addition of additives such as chondroitin sulfate (CS) permitted control of release rate. This formulation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was then prepared, and its characteristics were determined in normal rats. It was found that blood rhG-CSF concentration was maintained for about 1 week after administration of a DL-MP with additional CS, with persistent increase in white cell count. The results of this study indicated that DL-MP was useful as a long-acting formulation of rhG-CSF characterized by excellent long acting properties without an initial burst.

The role of atelocollagen-based cell transfection array in high-throughput screening of gene functions and in drug discovery.

The human genome project has been completed, but the function of many genes is unknown. It is, therefore, necessary to elucidate the function of a large number of genes within a short time. To achieve this goal, materials are needed that condense or package DNA into nano-particles that can easily be taken up by cells and would allow DNA to be retained without degradation. Atelocollagen is a reliable carrier for gene delivery because it is considered safe and appropriate for practical use. We developed a basic technique for high-throughput gene transfer and expression screening by pre-coating a multi-well plate with an Atelocollagen/DNA complex in which cells are then seeded. Complexes with a nano-particle form were efficiently transduced into cells without the use of additional transfection reagents, and they allowed for long-term gene expression. The complex spotted onto the well of a plate was stable for a long period and allowed the cells to transduce and express reporter genes. We also showed that the present method with Atelocollagen-based gene transfer is applicable to gene medicines, such as antisense ODNs, siRNA, and adenovirus vectors. These results suggest that an Atelocollagen-based cell transfection array may be appropriate for general use in the high-throughput screening of large sets of gene medicines with functions in mammalian cells.

Biodegradable honeycomb collagen scaffold for dermal tissue engineering.

Tissue engineering requires a mechanically stable, biocompatible, and biodegradable scaffold that permits cell adherence and proliferation, allows preservation of cell-specific properties, and suitable for surgical implantations. In this study, honeycomb collagen sheet was used for three-dimensional (3D) cultures of human skin fibroblasts and characterized as an effective and suitable scaffold for dermal tissue engineering. About 1-mm-thick honeycomb collagen sheets, prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation, and sterilized by heat, were placed on the proliferating fibroblasts on day 3 of the culture. The cells attached quickly to the collagen scaffold, proliferated inside the honeycomb pores, and formed a structure similar to dermis within 60 days. On day 60, total cellular DNA content of the 3D cultures was 12-fold higher when compared with the 2D control cultures without the scaffold. Measurement of procollagen type I in the media demonstrated a 20-fold increase. Scanning electron microscopy of the 3D cultures showed a well-formed structure similar to dermis and biodegradation of the honeycomb collagen scaffold. Our study proved that honeycomb collagen sheet is a mechanically stable, biocompatible and biodegradable scaffold for dermal tissue engineering, and also potentially useful for other cell-based therapies and tissue engineering applications.

Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds.

Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The adult mesenchymal stem cells can be differentiated into multilineage cells, such as adipocytes, chondrocytes, or osteoblasts when cultured with specific growth factors. In the present investigation, we have studied the effect of honeycomb collagen scaffolds for the adhesion, differentiation and proliferation of bone marrow-derived mesenchymal stem cells into osteoblasts. Mesenchymal stem cells were isolated from 6-week old albino rat femur bone marrow, and cultured in alpha-MEM medium without beta-glycerophosphate and dexamethasone. Honeycomb collagen discs were prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation and sterilized by heat. The honeycomb discs were placed on the culture dishes before seeding the stem cells. The cells attached quickly to the honeycomb collagen scaffold, differentiated and proliferated into osteoblasts. The differentiated osteoblasts were characterized by morphological examination and alkaline phosphatase activity. The osteoblasts also synthesized calcium-deficient hydroxyapatite (pseudo-hydroxyapatite) crystals in the culture. The mineralization was confirmed by Von Kossa staining and the crystals were analyzed by X-ray diffraction. Light microscopy and DNA measurements showed that the differentiated osteoblasts multiplied into several layers on the honeycomb collagen scaffold. The results demonstrated that the honeycomb collagen sponge is an excellent scaffold for the differentiation and proliferation of mesenchymal stem cells into osteoblasts. The data further proved that honeycomb collagen is an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy.