Mass Transport via Naturally Branched Scaffolds Maintains Viability of a Reconstituted Model of Connective Tissue

Abstract

We developed two types of cell scaffolds with naturally branched capillary-like networks for reconstituting fibroblast-incorporated connective tissue-like structures. The fibrous root substratum was prepared by fixing pieces of roots of a rice plant with glutaraldhyde, washing them sufficiently with distilled water and 70% ethyl alcohol and drying each one of them in a culture dish. The cotton gauze-incorporated collagen substratum was prepared by immersing a cut of cotton gauze in type-I collagen solution and drying it in a culture dish. Human dermal fibroblasts adhered well to the above substrata, proliferated well, and formed multilayers of cells in the presence of L-ascorbic acid 2-phosphate. Fibroblast sheets thus formed could be physically removed from each dish together with the scaffold. When the detached fibroblast sheet was cultured on the nonadhesive substratum to which cells cannot attach, it gradually self-assembled and finally formed a three-dimensionally reconstituted multicellular mass (3-DRMM) involving the fibrous root or the cotton gauze-incorporated collagen fibrils. The roots could function as paths of culture medium. The cotton fibers also functioned as such paths and were able to circulate culture medium within the 3-DRMM by connecting the end of the gauze with a medium flow system. Pyknotic and necrotic cells were histologically observed in a multicellular spheroid with a diameter of 600 µm, cultured for 21 days, which had been prepared and cultured by a conventional method without capillary-like networks and medium circulation. In contrast, no such necrotic cells were seen in the same-day-cultured 3-DRMMs with capillary-like networks, in spite of the fact that their sizes were much larger than the spheroid. Electron microscopic survey revealed the presence of assemblies of collagen fibrils between cells in the 3-DRMM involving the cotton gauze-incorporated collagen fibrils but not in the conventional multicellular spheroids. The 3-DRMM with capillary-like networks appears to be a model of connective tissues that approximates their status in the living body very well.