Alveolar blood clots and platelet-rich fibrin induce in vitro fibroblast proliferation and migration

Abstract

Wound healing process comprises a complex network of cells and molecules that are regulated in order to pursue tissue regeneration. Our study focused on the capacity of alveolar blood clots (ABCs), platelet-rich fibrin (PRF) and plasma rich in growth factors (PRGF) to induce in vitro fibroblasts proliferation and migration as a measure of alveolar regeneration. Using cellular impedance with xCELLigence technology we quantified the proliferation and the migration capacity of L929 fibroblast standard cell line in the presence of 4 different ABCs and 3 different PRFs harvested from healthy individuals during standard tooth extraction. We obtained a clear cellular proliferation induced by the compounds mainly after 24 h of cultivation, in a dose-dependent manner. After 48 h of cultivation we registered activated proliferation, but slightly decreased compared to the 24 h profile. Our data confirm that the presence of the blood clot is involved in the regenerative processes. The migratory capacity of fibroblasts was statistically activated by the PL compounds while not affected by the tested PRFs. The chemical mediators present within the blood clot, either produced by inflammatory cells captive within, or by endothelial or mesenchymal cells induced fibroblastic proliferation and subsequent collagen deposition.

Keywords: alveolar blood clot; cellular impedance; fibroblasts; plasma rich in growth factors; platelet-rich fibrin; tissue regeneration; wound healing.

Figure 1.

L929 cell line maintained in culture; active proliferation after 24 h post-seeding; ×20 magnification in inverse microscopy.

Figure 2.

Cell migration takes place from the upper chamber (chamber A) through a membrane that allows only migratory phenotype cells to transverse the membrane and adhere to the impedance electrodes on the lower part of the membrane. Cells migrate towards the lower chamber (chamber B) where the chemoattractants reside. The adherence capacity is quantified as cellular index registered by the microelectrodes similar to the proliferation assay.

Figure 3.

(A) Fibroblasts were seeded at a density of 0.33×10⁶ cells/IBIDI plate in a final volume of 1.7 ml. (B) After 24 h of cultivation the silicon frame is removed and (C) in the plate the remaining gap measures 500 µm.

Figure 4.

L929 fibroblast proliferation registered for 47 h of cultivation in ABC in various serial dilution compared to controls cultivated in plain culture medium (control 1) and culture medium with serum supplements (control 2). Black arrow depicts the moment ABCs in the presented dilutions are introduced in the experimental system (example of an individual experiment CI - mean of triplicates). ABC, alveolar blood clot.

Figure 5.

Cellular index (CI) calculated for cultivated fibroblasts in individual ABC (nos. 2, 3, 5, 6) compared to plain cell culture medium (control 1), cell culture medium supplemented with 10% horse serum (control 2). A, 2 h; B, 24 h; C, 48 h of cultivation (CI of mean ± SD for 3 different experiments). p<0.001 when comparing controls with all the corresponding compounds. ABC, alveolar blood clot.

Figure 6.

Fibroblast migration registered for 24 h in the presence of PRGF1 and 2, PRF2-4 compared to control 1 (fibroblasts migrating toward complete culture medium) and control 2 (fibroblasts migrating toward plain medium). Representation of CI mean for triplicates.

Figure 7.

Fibroblast migration registered for 44 h in the presence of PRGF1 and 2, PRF2-4 compared to control 1 (fibroblasts migrating toward complete culture medium) and control 2 (fibroblasts migrating toward plain medium). Representation of CI mean for triplicates.

Figure 8.

Cellular index (CI) calculated for cultivated fibroblasts in compounds for 24 h (A) and 48 h (B) compared to control 1 (fibroblasts migrating toward plain medium) and control 2 (fibroblasts migrating toward complete medium). CI of mean ± SD for 3 different experiments.