Protease profiles of cells isolated from regenerative membranes are associated with clinical outcomes.

Guided tissue regeneration (GTR) is a clinical procedure used to restore the attachment apparatus of periodontally diseased teeth. Guided bone regeneration (GBR) is a similar procedure used to augment bone of edentulous ridges. Both therapies enhance the ability of regenerative cells to repopulate wounds by using expanded polytetrafluoroethylene (ePTFE) membranes to exclude gingival fibroblasts and keratinocytes from the healing site. Cells were isolated from 12 membranes used in each procedure and screened for the ability to form mineralized nodules in vitro, a property of cells with osteogenic potential. Using zymography and reverse zymography, low-passage isolates of cells which formed nodules were examined for the expression of gelatinolytic and caseinolytic proteases as well as for proteinase inhibitors. These molecular data were then compared with clinical outcomes for each procedure. Cells isolated from regenerative membranes exhibited variable expression of 72 kDa gelatinase, fibroblast collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP-1), and other unidentified proteases. The greatest proportion of clinical failures was associated with GTR therapy. Cells from GTR membranes which did not exhibit gains in clinical attachment often exhibited aberrant proteinase profiles. When compared with cells from GBR procedures, most cells from GTR procedures also secreted lower amounts of TIMP-1. The study shows that cells isolated from regenerative procedures produce degradative enzymes in vitro which may be related to the success or failure of the regenerative process in vivo. Generally, cells from unsuccessful GTR procedures produced low molecular weight gelatinases not associated with cells from successful cases.

Cytokine production by cells adherent to regenerative membranes.

Since cytokines play a critical role in tissue regeneration, we have assayed cytokine production by cells from tissue adherent to regenerative membranes. Cells were recovered from Gore-tex membranes in guided tissue regeneration (GTR) procedures to regenerate that attachment apparatus around teeth and from Gore-tex augmentation membranes (GTAM) used for guided gone regeneration (GBR) procedures in edentulous ridge augmentation with or without implant placement. Cells were screened for mineralized nodule formation in vitro to mRNA analysis to demonstrate that they could form mineralized tissue. Production in interleukin-1 alpha (IL-1 alpha) interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) was evaluated by reverse transcribed-polymerase chain reaction (RT-PCR) of mRNA from rescued regenerative cells, human gingival fibroblasts and periodontal ligament (PDL) cells. Both the gingival fibroblast and PDL cells isolates produced all 4 cytokines. However, the cell isolates from the regenerative membranes had various profiles of cytokine expression. Most GTR cell isolates were positive for all 4 cytokines. IL-1 beta was produced by all 6 GTR cell isolates but was not detected at the same number of cycles of RT-PCR amplification in any of the 6 GBR cell isolates. IL-1 beta transcripts were also not observed in cells derived from a direct biopsy of GBR tissue. Cells were recovered from unexposed GBR membranes did not produce detectable amounts of IFN-gamma, whereas cells recovered from exposed GBR and all GTR membranes produced IFN-gamma. These findings indicate that cells from regenerative tissues express different cytokines and that exposure to the tissue to the oral cavity during healing may modulate this expression.