Three-dimensional collagen gel is commonly used as a differentation substrate for various cell lines because it supports the formation of differentiated structures like the glands in colon carcinoma cells48 or duct like structures in mammary tumor cells.49 Culturing skin fibroblasts inside a collagenous environment has also been recognized as a valuable tool for studying collagen fiber reorganization during wound healing.50
Collagen gels are usually prepared from acidic solutions rich in type I collagen. When neutralized, the solution gelates during incubation at 37°C. Prior to gelation, the cells are mixed within the solution and trapped into this three-dimensional collagen environment. The two types of collagen gels typically used in in vitro studies are floating, where the gel is detached from sides of the cell culture wells, and anchored, where the gel adheres to the sides (Fig. 8.2).
Skin fibroblasts inside a floating collagen gel can be used to mimic the formation of dermis.50 When embedded inside the three-dimensional collagenous matrix, originally loose network of collagen fibers becomes a dense tissue like structure.51 This ability to reorganize collagen fibers can be measured by assessing gel reductions in the area. The force needed to retract the gel is derived from the spreading and elongation of cells inside the gel.52,53 Adherence of cells attempting to migrate on the proximal collagen fibers on them leads to rearrangement of the collagen network, producing a tightly packaged collagen gel. Called collagen gel contraction this process requires an intact cytoskeleton and the presence of serum. This model has been used to mimic wound healing and has many quantifiable effects on cell behavior.50 Actin stress fibers are needed for a proper wound contraction, and in this model fibroblasts also start to resemble myofibroblasts with their large bundles of actin stress fibers.54 There is a decline in cellular DNA synthesis, indicating that the cells do not proliferate under these conditions.55 The simultaneous decrease in collagen mRNA is accompanied by the increased expression of MMP-1.56 Growth factors responsible for stimulating the wound healing process, of which TGF-p and PDGF are examples, have been used to enhance the ability of fibroblasts to contract collagen gels. 57, 58
Another model for cell collagen interaction is to embed the cells in an anchored collagen gel,50 which morphologically resembles granulation tissue rather than dermis. Inside these gels fibroblasts continue to synthesize DNA and increase in cell number.55
In our own studies we have shown that osteogenic cell lines in general behave like fibroblasts when inside this collagen lattice. With some exceptions, they promote the contraction of gels, a process which TGF-p enhances.46 Osteogenic cell number, do not increase inside floating gels,59 and thus these phenomena are not restricted to fibroblasts.
Type I collagen is the most abundant matrix molecule in bone and therefore, culturing osteogenic cells inside three-dimensional collagenous environment mimics their normal growth conditions. Reorganization of the collagenous matrix around cells may also use similar mechanisms to bone fracture healing. This model enables researchers to test the effects of growth factors such as TGF-p in the modulation of osteoblast metabolism and behavior. Experimental approaches like this facilitates the study of individual collagen receptors that is not possible with fibroblasts. Given the fundamentally similar behavior of fibroblasts and osteogenic cells inside collagen gels, we suggest that observations conducted with our research model may be relevant with other mesenchymal cells as well.
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