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In 2004, Shen et al. developed a safe and efficient gene transfer system using a DNA-apatite composite layer. We have recently succeeded in improving further the gene transfer efficiency by immobilizing a cell adhesion molecule laminin, in a DNA-apatite composite layer. In this study, we showed that not only laminin but fibronectin immobilized in a DNA-apatite composite layer enhances cell adhesion and cell spreading on the layer, thereby markedly improving the gene transfer efficiency. Therefore, the immobilization of a cell adhesion molecule in a DNA-apatite composite layer is crucial for improving the gene transfer efficiency. By using fibronectin instead of laminin and optimizing the condition to prepare the fibronectin-DNA-apatite composite layer, the amount (weight) of cell adhesion molecule required was reduced to approximately one-fourth while retaining the relatively high gene transfer efficiency. It was also shown that the resulting fibronectin-DNA-apatite composite layer prepared under the optimized condition mediated the area-specific gene transfer on its surface, that is, DNA was preferentially transferred to the cells adhering to the surface of the fibronectin-DNA-apatite composite layer. The present gene transfer system with potential for area-specific transfection and advantages of safety and relatively high efficiency would be useful in tissue engineering applications, gene therapy, and production of transfection microarrays.

A low efficiency has long been the most critical problem of conventional gene-transferring systems using calcium phosphates, and this was successfully improved on by using a laminin-DNA-apatite composite (LD-Ap) layer. The genetransferring efficiency of the LD-Ap surface was 1-2 orders of magnitude higher than that of a DNA-calcium phosphate composite surface. This is because laminin enhances cell adhesion and spreading, and this provides regions of high DNA concentration between a cell and the LD-Ap surface. The efficiency of gene transfer of the LD-Ap surface was equivalent to, or even higher than that mediated using a commercial lipid-based transfection reagent applied using the manufacture's recommended optimum conditions. In addition, the gene-transferring efficiency of our system could be controlled by changing the laminin and DNA content in the LD-Ap layer. Moreover, our system is composed of highly safe reagents: apatite, DNA and laminin, all of which are present in the human body. Hence, the LD-Ap surface, which enhances cell attachment on its surface, and mediates a safe, highly efficient and controllable gene transfer, is highly applicable to tissue engineering and gene therapy applications. Gene Therapy (2007) An efficient and safe gene-transferring system is a key technology in tissue engineering and gene therapy applications. 1-12 A gene-transferring system mediated by a particulate DNA-calcium phosphate composite 1-3 is safer than viral 4-6 and lipid-based 7-9 systems, but its transferring efficiency is comparatively low. To enhance the gene-transferring efficiency, a surface-mediated genetransferring system derived from a DNA-apatite composite (D-Ap) layer has been developed. 12 In this system, regions of high DNA concentration are produced locally around cells, and this enhances the gene-transferring efficiency. In this work, the cell adhesion molecule, laminin, 13,14 was immobilized on a D-Ap layer to enhance further the gene-transferring efficiency by strengthening a cell's attachment and facilitating its spreading on a coating's surface. The gene-transferring efficiency was 1-2 orders of magnitude higher on the resulting laminin-DNA-apatite composite (LD-Ap) layer than the gene-transferring efficiency on a D-Ap layer. In this paper, we present an advanced gene-transferring system that has a high efficiency, is safe and has a great potential for use in tissue engineering and gene therapy applications.An LD-Ap layer was coated on a polymer surface in a metastable supersaturated calcium phosphate solution at neutral pH, and ambient pressure and temperature. [15][16][17][18] As controls, an apatite (Ap) layer, a D-Ap layer and a laminin-apatite composite (L-Ap) layer were also coated on the polymer surface. The coating layers were formed by immersing the polymer sample, that had an amorphous calcium phosphate (ACP) layer deposited on its surface, in four different coating solutions: a calcium phosphate solution (CP solution) [15][16][17][18] (for the Ap layer), a CP solution supplemented with DN...

Background We have recently developed a safe and efficient gene transfer system using a laminin-DNA-apatite composite layer. The objectives of the present study were to fully characterize and optimize the laminin-DNA-apatite composite layer in relation to the efficiency of gene transfer and to demonstrate the feasibility of the composite layer in the induction of cell differentiation.

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