cytochalasin-d and ilomastat

Although platelet-rich plasma (PRP) has been proposed as a therapeutic tool to enhance wound repair, the cellular and molecular mechanisms stimulated by this agent are still not completely understood. The present study was designed to characterize the effects of PRP and platelet-poor plasma (PPP) supernatants on cell responses involved in gingival tissue repair.. We studied the response of human gingival fibroblasts (HGF) to PRP and PPP fractions on: matrix contraction, cell migration, myofibroblastic differentiation, production of matrix components and proteolytic enzymes. PRP and PPP were obtained from donors using a commercial kit. Matrix contraction was evaluated by means of collagen lattices in the presence of matrix metalloproteinase (MMP) and actin polymerization inhibitors. The production of matrix molecules and proteinases was assessed through Western-blot. RhoA activity was evaluated through a pull-down assay. Actin distribution and focal adhesions were assessed through immunofluorescence. Transforming growth factor-beta (TGF-β) was quantified through ELISA.. Both PRP and PPP stimulated human gingival fibroblasts-populated collagen gel contraction and Ilomastat and cytochalasin D inhibited this response. PRP and PPP also stimulated MT1-MMP and TIMP-2 production, RhoA activation and actin cytoskeleton remodeling, cell migration/invasion and myofibroblastic differentiation. TGF-β1 was found at significantly higher concentrations in PRP than in PPP.. Both PRP and PPP promote wound tissue remodeling and contraction through the stimulation of actin remodeling, the activity of MMPs, promotion of cell migration, and myofibroblastic differentiation. The similar biological responses induced by PRP and PPP suggest that both platelet-derived fractions may exert a positive effect on gingival repair.

Topics: Actins; Adult; Blotting, Western; Cell Differentiation; Cell Movement; Cells, Cultured; Cytochalasin D; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Gingiva; Humans; Hydroxamic Acids; Indoles; Intercellular Signaling Peptides and Proteins; Male; Matrix Metalloproteinases; Platelet-Rich Plasma; Statistics, Nonparametric; Transforming Growth Factor beta; Wound Healing

Mechanical tension and contracture are two related facets of tissue biology. This study assessed the effect of ilomastat, a broad-spectrum matrix metalloprotease (MMP) inhibitor, on generation of tension by Dupuytren's disease fibroblasts. Nodule and cord-derived fibroblasts were isolated from five patients with Dupuytren's disease; flexor retinaculum acted as the control. A culture force monitor (CFM) provided an in vitro model of tissue organization to assess development of mechanical tension, lattice contraction and spatial remodelling by fibroblasts. Responses to ilomastat were compared to treatment with a control peptide. Nodule and cord-derived fibroblasts exhibited a two-fold increase in tension compared with flexor retinaculum. Ilomastat significantly inhibited development of tension by nodule and cord but not flexor retinaculum derived fibroblasts at 100 microM. These results imply that MMP activity mediates regulation of tensile strength by Dupuytren's disease fibroblasts and may be an important therapeutic target in patients with Dupuytren's disease.

Topics: Cells, Cultured; Cytochalasin D; Dupuytren Contracture; Enzyme Inhibitors; Fibroblasts; Humans; Hydroxamic Acids; Indoles; Matrix Metalloproteinase Inhibitors; Nucleic Acid Synthesis Inhibitors