Basic Research Of Leukocyte-Platelet-Rich Fibrin On Soft Tissue Healing And Inflammatory Affect Regulation

Due to trauma,inflammation,long-term missing teeth,the implant area often encounters problems of bone tissue or soft tissue defects,and various implant surgery operations,such as guided bone regeneration,maxillary sinus floor elevation,etc.,are required.Good healing of soft tissue is a prerequisite for successful implant surgery.Good soft tissue healing can block bacterial invasion and provide a good microenvironment for bone regeneration and implant osseointegration;healthy gingival cuffs help to achieve good and stable repair results,so soft tissue healing after implant surgery Whether the quality is good or bad,whether it can form a good biological closure,directly affects the success of implant surgery.In recent years,many scholars have done a lot of researches in order to speed up the healing of soft tissue and improve the quality of soft tissue healing.Clinically,local soft tissue flap treatment,autologous connective tissue free flap transplantation,autologous mucosal free flap transplantation,are used to close the wound,but the difficulty of surgery is increased,and the “second operation area” is re-opened which can increase the patient’s pain.In recent years,acellular dermal matrix has been applied to the repair of oral mucosa,but it is expensive,and it has problems such as disease transmission,and local immune response to wounds.Therefore,it is of clinical value to find a biological material that has the ability to promote the regeneration of gingival soft tissue,safe and reliable,inexpensive,avoids biosafety risks,and has certain anti-infective ability.Choukroun et al first extracted the platelet-rich fibrin(PRF).As an autologous platelet concentrates(PCs)rich in platelets and various growth factors,PRF is simple to prepare,does not require any anticoagulant and exogenous thrombin and gelling agent,and has a highly efficient tissue promotion.Regeneration ability,with good clinical application potential.Therefore,this study is to investigate the effect of PRF on soft tissue healing and inflammatory regulation and provide a theoretical basis for promoting soft tissue healing around oral implants.The specific research content includes the following aspects:1.Culture of human gingival fibroblasts and establishment of an in vitro inflammatory model.In this study,human gingival fibroblasts were cultured by tissue block enzymatic digestion,which has rapid proliferation,good cell purification effect,high success rate,and rapid and large-scale acquisition of primary cells.The establishment of an in vitro inflammatory model is the basis for subsequent research.In this study,Porphyromonas gingivalis-lipopolysaccharide was detected as a safe concentration by CCK-8 method,and Pg-LPS at 1 μg/ml was determined to be a safe concentration for our research,which did not affect the normal growth of cells.The optimal stimulation time of Pg-LPS was determined by Real-time qPCR.The results showed that 1μg/ml of Pg-LPS could stimulate the release of pro-inflammatory factors from fibroblasts and reached the peak after 3h and 6h.This time point of detection was used for the subsequent experiments.2.L-PRF structure and growth factors-release.In this experiment,the ultrastructure of L-PRF was observed by scanning electron microscopy.The concentrations of TGFβ-1,PDGF-AA and IGF-1 in L-PRF were detected by enzyme-linked immune-sorbent assay(ELISA).Scanning electron microscopy showed that L-PRF has a three-dimensional network structure rich in fibrin,which arrests a large number of platelets and leukocytes at the yellow-red junction,and continuously releases tissue healing-related growth factors(TGFβ-1,PDGF-AA)for at least 10 days.And IGF-1 has a certain long-term effect,therefore,we believe that L-PRF has great potential in regulating tissue healing.3.Effects of L-PRF on proliferation of human fibroblasts and regulation of inflammation.In this experiment,the effect of L-PRF on the proliferation of human gingival fibroblasts was examined by CCK-8 assay.The effect of L-PRF on the proliferation of senescent cells was observed by inverted-microscope.The effect of L-PRF on the release of Pg-LPS-stimulated fibroblast inflammatory factors was examined by enzyme-linked immunosorbent assay(ELISA).The results of CCK-8 showed that 100%,50% and 25% of L-PRF conditioned medium promoted the proliferation of human gingival fibroblasts in a concentration-dependent manner.Among them,100% of L-PRF conditioned medium promoted the most proliferative effect.Therefore,100% of the L-PRF conditioned medium was selected as the optimal concentration for subsequent experiments.Microscopic observation of L-PRF conditioned medium cultured aged cells showed that L-PRF can restore the biological characteristics of aged HGFCs cells and promote the proliferation of it.The ELISA results showed that IL-1β,TNF-α,and IL-6 secreted by human gingival fibroblasts were significantly increased after stimulation of human gingival fibroblasts with 1 μg/ml of Pg-LPS for 6 hours,and surprisingly After treatment with L-PRF,Pg-LPS-induced secretion of IL-1β,TNF-α,and IL-6 by human gingival fibroblasts was significantly inhibited.4.In vivo study of L-PRF involved in inflammation regulation.In this experiment,a tissue inflammation model was established on rabbits.The changes in body weight,wound healing area and tissue hematoxylin and eosin(H&E)staining were used to evaluate the regulation of L-PRF on oral tissue inflammation.Direct stimulation of oral gingiva with 50% acetic acid in this study caused oral inflammation on day 4.The area of oral mucositis reached the maximum on the fourth day after challenged with acetic acid.After the formation of oral ulcers,the experimental animals in the control group lost weight on the 7th day but recovered again on the 14 th day.In contrast,the body weight of the L-PRF treatment group increased with time,and the body weight of the experimental group and the control group was significantly different on the 7th day and the 14 th day.The ulcer area of the L-PRF treatment group was significantly smaller than that of the control group on day 7,which means that the healing process of this group was faster.On day 14,the mucosal ulcer of the L-PRF treatment group was significantly reduced and became the smallest group.Histopathological examination under HE staining showed that the specimens of the normal control before stimulation were intact and no inflammatory cell infiltration.After the stimulation,on the 7th day,the epithelial damage was obvious,and then the thickness of the epithelial layer changed,and the inflammatory cell infiltration in the control group was more serious.On day 7,the L-PRF treatment group initiated incomplete epithelial regeneration and the healing process was faster than the control group,with smaller ulcers and less inflammatory cell infiltration.On the 14 th day,the treatment group recovered substantially,and the epithelialization was nearly completed.The wound healing rate was significantly faster than that of the control group.On the 21 st day,both groups completed re-epithelialization,and the healing of the L-PRF test group was complete.5.Effects of L-PRF on tissue healing and postoperative inflammation control on implants: a randomized controlled clinical study.This study established a prospective and randomized controlled clinical study to assess the effects of leukocyte-plateletrich fibrin(L-PRF)on tissue response around implants.This clinical study was conducted at the Affiliated Stomatological Hospital of Jilin University from February 2016 to November 2017.A randomized selection of 20 patients(15 males and 5 females),with an average age of 38 years(30-44 years),planted 20 Implant.There were 10 planting sites in each group,of which group I was L-PRF group(test group)and group II was no L-PFR group(control group).Postoperative pain assessment,assessment of mucosal swelling,assessment of wound healing area,determination of bone height and periodontal pocket depth in postoperative implants,and postoperative patient satisfaction survey evaluated L-PRF for tissue healing around implants and the effects of postoperative inflammation control.The results of the study showed that L-PRF can significantly reduce postoperative pain and mucosal swelling,accelerate the wound healing rate,increase bone level and periodontal health,and patients are satisfied with the whole planting and repairing process.Therefore,these cases show the successful application of L-PRF to promote tissue healing around the implant,providing new ideas for further clinical application.In summary,L-PRF has a positive regulatory effect on tissue healing and post-operative inflammation regulation around the implant.