Orientation of Gingival Fibroblasts in Simulated Periodontal Spaces in Vitro Containing Collagen Gels

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Abstract
The present study examined the orientation of cultured human gingival fibroblasts in simulated periodontal spaces in vitro containing three dimensional hydrated collagen gels. Extracted human teeth were root planed followed by root resection and root canal instrumentation. The middle and cervical thirds of each root were cut transversely to create 600μm thick sections. Cortical bovine bone was cut, sectioned, and contoured to create bone rings 600 μm thick with an internal diameter large enough to accommodate a root slice leaving a circumferential space varying from approximately 0.1 to 1.0 mm. Root slices and bone rings were incubated in an enzyme solution to remove all remaining soft tissues and then completely demineralized in EDTA (18%) for 72 hours. Human gingival fibroblasts (HGF) were plated to confluency in tissue culture dishes. The dentin slices were then gently placed over the HGF monolayer along with bone rings around them to create simulated periodontal spaces. Five days later, when initial cell attachment to the dentin and root slices had occurred, a collagen gel was poured in the space. The cultures were maintained for six weeks and were then processed for transmission electron microscopy. The HGF appeared to have formed multilayered cell sheets extending from the periphery of the root slices to the inner surface of bone rings. The HGF had apparently attached to both the bone and root surfaces. There was a close interaction of cells with the matrix fibrils of the gel. The cells and matrix fibrils were oriented parallel to each other. The present findings suggest that HGF grown in simulated periodontal spaces containing a collagen gel in vitro can form oriented, multilayered cell sheets and some degree of physical rearrangement of matrix fibrils occurs during this process.