Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells
Introduction
The major aim of gene therapy is to effectively deliver a gene into cells for their genetic modification and functional repairing, resulting in cell-based disease therapy. Recently, a number of DNA delivery systems have been investigated to improve the efficacy of gene transfection [1], [2], [3]. There have been two major approaches proposed: the viral-mediated and non-viral-mediated gene transfections [4]. However, considering the immunological and safety issues of viral vectors, necessity in the development of non-viral vector systems has been increasingly magnified. There are several advantages of non-viral vectors, for example the lower toxicity and immune responses or no integration into the genome. Moreover, the gene transfer method with the non-viral vector is considered to be safer although the expression level is very low.
For successful tissue regeneration, the cells constituting tissues to be regenerated, such as matured, progenitor, and precursor cells, are necessary. Considering the proliferation and differentiation potential of cells, stem cells are practically promising. Among them, mesenchymal stem cells (MSC) have been widely investigated to use by themselves or combining with the scaffold for their applications to regenerative medicine. One of the future approaches with MSC is the combinational therapies with gene. Therefore, several researches have been investigated on the therapy of tissue regeneration by MSC genetically engineered. Over the last decade gene therapy has captured the scientific and public interest with the promise to deliver genes and proteins to specific tissues or to replace deficient host cell populations. Gene modification is preferred over addition of growth factor to the cell as, typically, (1) the half-life of the selected growth factor is short; (2) a single administration is usually not sufficient for a biological effect; (3) the quantities required are prohibitively expensive; and (4) continuous protein production increase the likelihood that a desired outcome will be achieved. In planning gene therapy strategies for tissue engineering, one must consider two major avenues: direct gene delivery in vivo using viral or non-viral vectors or in vitro cell mediated gene therapy. In both cases the aim is to deliver a therapeutic gene of a growth factor or cytokine, into the target tissue. Nolta et al. [5] and Bartholomew et al. [6] have demonstrated that human bone marrow stromal cells were genetically engineered to express interleukin-3 (IL-3), green fluorescent protein (GFP), and human erythropoietin by retroviral vectors and transplanted for the therapeutic applications to immunodeficient mice or were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Successful bone regeneration by MSC genetically engineered by retrovirus or adenovirus vector carrying human BMP-2, BMP-4 or BMP-7 gene has also been reported [7], [8], [9], [10], [11], [12]. Collagen matrices have been used for gene delivery and bone tissue formation [13], [14], [15], [16]. Fang et al. [15] and Bonadio et al. [16] have used collagen matrices for DNA delivery for the induction of bone formation in vivo.
Recently, a system using gene engineered urothelial cells was developed for in vivo gene therapy [17]. Cells seeded onto 3D polymer scaffolds were genetically modified, which then formed an organ-like structure with stable expression of the transgene in vivo. This paradigm provided a proof of principle for the use of tissue-engineering techniques as a means to improve methods of gene transduction. The 3D transgene cell construct can be potentially used as therapeutic cell-based gene delivery or as an in vitro model system for testing of genetic manipulations in order to understand the effects of gene expression on tissue development.
This study was undertaken to investigate the effect of 3D culture system on the enhancement of gene transfection of MSC. As a material of non-viral vector, spermine was chemically introduced to dextran to obtain a cationized dextran capable for polyion complexation of plasmid DNA. In recent publications, Azzam et al. [18], [19] reported on a new class of biodegradable polycations capable of complexing and transfecting various genes to different cell lines in relatively high yields. More than 300 different polycations were prepared starting from various natural polysaccharides and oligoamines having two to four amino groups. These cationic polysaccharides were prepared by reductive amination of the oligoamine and periodate-oxidized polysaccharides. Although most of these cationic conjugates formed stable complexes with plasmid DNAs as determined by ethidium-bromide quenching assay, only the dextran-spermine based polycations were found to be highly effective in transfecting cells in vitro. Collagen sponge reinforced by incorporation of PGA fibers was selected as the cell scaffold and was used to evaluate effect of the 3D culture system and cell scaffold type on the transfection efficiency of cationized dextran-plasmid DNA encoding BMP-2 complex on MSC.
Section snippets
Materials
An aqueous solution of type I collagen, prepared from porcine tendon by pepsin treatment (3 mg/ml, pH 3.0) in HCl, was kindly supplied by Nitta Gelatin Inc., Osaka, Japan. The non-woven fabric prepared from poly(glycolic acid) (PGA) fiber of 20 μm in diameter (0.5 mm thickness, 200–210 g/m2) was obtained from Gunze Ltd., Kyoto, Japan. DNA MW Standard Marker (1 kb DNA Ladder) was obtained from Takara Shuzo Co. Ltd., Shiga, Japan. Dextran with an average molecular weight of 40 kDa was obtained from
Preparation and characterization of cationized dextran and the complexation with plasmid DNA
Cationized dextran was prepared by reductive-amination between oxidized dextran and spermine (Fig. 2). Dextran was initially oxidized with potassium periodate and the obtained dialdehyde derivative was allowed to react under basic conditions with spermine. Three dextran-spermine based conjugates (G7TA103, G7TA107 and G7TA141) were prepared under similar conditions and characterized as illustrated in Table 1. The content of substituted spermine moieties was determined from nitrogen content (%N)
Discussion
The scaffolding materials used in tissue engineering should be biocompatible, biodegradable, and osteoinductive to accept the attachment and migration of osteoblasts. Among many materials currently used as cell scaffolds, collagen has been widely used because of the good cell compatibility and its processability into various shapes. The drawback of collagen sponge as a scaffold for cell proliferation and differentiation is its poor mechanical strength. To overcome the inherent nature of sponge,
Conclusion
Collagen sponges reinforced by incorporation of PGA fibers with an average pore size of 180 μm were fabricated and used as 3D tissue engineered scaffold. As a non-viral gene carrier, cationized dextran was enabling to form a stable complex with plasmid DNA through electrostatic interaction. The level of gene expression was enhanced by a combination technology of complexation with cationized dextran, impregnation of plasmid DNA-BMP2 into the collagen sponge reinforced with PGA fiber, and 3D
Acknowledgments
This study was performed through Special Coordination Funds for Promoting Science and Technology from the MEXT, Japan, and partially supported by research promotion bureau (No. 16-794), MEXT, Japan. We appreciate Tokyo Medical and Dental University, Japan for providing a pBacBH2.
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