Platelet-Rich Fibrin GEL As Scaffold For Pulp Regeneration In Vitro Studies

Objective:This in vitro study was designed to evaluate the capacity of platelet-rich fibrin (PRF) gel as scaffold for dental pulp regeneration by analyzing the three-dimensional structure and the releasing pattern of growth factors in PRF gel, investigating the effect of PRF gel on the proliferation of human dental pulp cells (hDPCs), and exploring the proper method to prepare the PRF-hDPCs complex.Methods:Part 1:PRF gel prepared by Choukroun’s protocols was evaluated by light microscope and SEM. Fresh prepared PRF gel was divided into 3 groups, named PRF, PRF lower and PRF upper. The 3 groups of PRF gel were soaked in sterile DMEM respectively. The extracts of PRF gel were collected at five experimental times:24 h,72 h,120 h,168 h and 216 h and stored at -80℃ before ELISA quantification. When all the samples were collected, quantifications of four growth factors were performed by using ELISA kits: PDGF-BB, TGF-β1, IGF-1 and FGF2.Part 2:Primary hDPCs were expanded and identified. The extract of PRF gel was collected at the time point of 7th day. The experiment group was divided into 2 groups according to the volume fraction of PRF gel extract in conditioned medium. The two groups were named 1PRF and 3PRF respectively. Cell proliferation was detected using cell counting kit-8 (CCK-8) at 24 h,48 h,72 h, 96 h,120 h,144 h and 168 h respectively.Part 3:The hDPCs in logarithmic growth phase were selected to perform the experiment. The cells were resuspended with Phosphate Buffered Saline (PBS) at the density of 105 cells/mL. During the preparation of PRF, at each experimental time, i.e. before blood centrifugation, right after blood centrifugation and 10 minutes after blood centrifugation,500 uL of cell suspension was seeded into a 5 mL vacuum blood collection tube (without anticoagulant) which was filled with fresh venous blood. The prepared PRF-hDPCs complex samples were cultured in DMEM for 7 days. All the samples were fixed for histologic processing and scanning electron microscopy evaluation.Results:1. The PRF gel can be described as composed of three main parts, a fbrin matrix,"buffy coat" and some red blood cell on the bottom. Leukocyte was mainly distributed at the "buffy coat", which was constructed by large and dense fibrin clusters. PRF gel slowly released certain amounts of PDGF-BB, TGF-β1, IGF-1 and FGF2. The quantity of growth factors released by PRF lower group was higher than PRF group (P<0.05) at most of the time. PRF upper group hardly released any growth factor.2. At 24,48,72 and 96 h, there was no significant difference of optical density (OD) between experiment groups in which hDPCs were cultured in conditioned medium containing extract of PRF gel and control group (P>0.05). At 120 h,144 h and 168 h, the OD of experiment groups was higher than that of control group (P<0.05);.there was no significant difference of OD between group 1PRF and 3PRF (P>0.05).3. Cells with bigger size than blood cells and irregular shape were found in the group in which hDPCs were added before centrifugation, and were positive for vimentin but negative for CD45 and CD68. The morphology and identification met with description of hDPCs. In the group in which hDPCs were added after centrifugation, no certain cells were observed. During the 7 days incubation, the morphology of hDPCs changed from irregular to spindle shape. Some hDPCs colonies were found in the slices of PRF-hDPCs complex, a few hDPCs were seen along the edge between the "buffy coat" and the normal fibrin network.Conclusion:Our observations confirm that the PRF gel is composed of three main parts, a fbrin matrix,"buffy coat" and some red blood cell on the bottom. PRF gel could be used as a slow releasing system of growth factors. The extract of PRF gel can accelerate the proliferation of hDPCs. We demonstrated that the most effective time point for inoculate hDPCs to PRF gel was before blood centrifugation and hDPCs experienced a favorable proliferation in the fibin matrix of PRF gel 7 days after inoculated. We conclude that PRF gel might be a proper scaffold for pulp regeneration.