The Effect Of Concentrate Growth Factors On Biological Behavior Of Human Dental Pulp Cells In Vitro And In Vivo

Background: Dental pulp has four main functions: forming function,nutrition function,sensory function,defense function.The healthy dental pulp tissue is important for the biological function and long-term maintenance of teeth.Direct pulp capping should be a important way to preserve vital pulp.The selection of the appropriate pulp material is an important step of direct pulp capping treatment,which can affect the prognosis of the subjected teeth.Calcium hydroxide and mineral trioxide aggregate(MTA,mineral trioxide aggregate)are commonly used for direct pulp capping at present,however,they still exist their own deficiencies.Calcium hydroxide may cause inflammation of the pulp and calcification of the pulp.MTA also has many disadvantages,such as its long curing time,not easy to shape,tooth discoloration,high cost.So,finding out a new better direct pulp capping material is necessary.Concentrate growth factors(CGF,concentrated growth factors)as a new generation of platelet-derived concentrate products,have good biological compatibility and can be obtained from autologous peripheral blood.They are rich in abundant growth factors,have the functions of the anti-inflammation and tissue repair promotion.CGF may be a potential material for direct pulp capping,so we hypothesized that it can be applied in direct pulp capping treatment alone or in combination.Objective:The objectives of this study were to explore the effect of CGF on the viability,apoptosis,proliferation and mineralization of human dental pulp cells(hDPCs),as well as the reparative dentine formation and inflammatory reaction after CGF direct pulp capping in animal models,thus the feasibility of CGF applied in direct pulp capping material was evaluated.Methods:Healthy permanent teeth extracted for orthodontic treatment purpose were collected in outpatient clinic of stomatological hospital of Chongqing medical university.Human pulp tissues were obtained,and hDPCs were isolated using the modified tissue enzyme digestion method.Peripheral venous blood of healthy volunteers was collected and CGF was extracted using centrifugation technique.HDPCs were treated with CGF and MTA,respectively.The cell apoptosis,proliferation,cell cycle and ALP activity were analyzed after 1,3,and 7 days.Cell proliferations were assessed using cell counting kit-8.Osteo-/odontogenic differentiation of hDPCs was evaluated by measurement of alkaline phosphatase activity(ALP).Cell cycles of treated cells were assessed using flow cytometry.Cell apoptosis was evaluated by Annexin V assay.A total of sixteen teeth from two 10-month-old beagles were chose and buccal V class cavities were prepared until about 0.5 mm diameter pulp exposures were made,thus direct pulp capping animal research model was established.The CGF and the MTA were used for direct pulp capping,chemical curing glass ionomer and 3M composite resin were subsequently filled to restore the cavities.After 2 months,X rays were used to observe periapical lesions.Teeth are pulled out after euthanasia of animals and fixed with paraformaldehyde.Micro-CT was used to detect the formation of dentin bridge after direct pulp capping and Hematoxylin-Eosin(HE,Hematoxylin and Eosin)staining was used to detect dental pulp tissue inflammation.The experimental data was statistically analyzed using rank sum test and One-way ANOVA analysis.Results:Compared with the MTA group,CGF significantly promoted cell proliferation and increased the proportion of S-phase cells and ALP activity on day 3 and 7(P < 0.01).CGF increased the ALP activity compared with control group(P < 0.05).Besides,h DPCs apoptotic rates decreased in CGF group(P < 0.05)in comparison with MTA group.The results of X-ray and cone-beam CT showed that both CGF and MTA rarely caused periapical lesions,and Micro CT examination showed that the formation of calcified bridge in CGF group was weaker than MTA group.Histological examination showed no obvious inflammatory response after CGF and MTA direct pulp capping.Conclusions:CGF is a non-toxic autologous biological material with good biocompatibility and bioactivity.CGF was demonstrated to promote cell proliferation and have an anti-apoptosis fuction in vitro and cause no inflammatory reaction of dental pulp tissue in vivo,which has the application potential as a direct pulp capping material.In future,CGF can be used in direct pulp capping treatment alone or in combination with other agents.