Effects Of Liquid Phase Concentrated Growth Factors On Osteogenesis Of Human Periodontal Stem Cells

Background:Oral diseases like maxillofacial tumors,cleft palate,trauma,periodontal disease and periapical disease will lead to bone tissue defects in oral cavity,which will have a negative impact on the prognosis and the later repair treatment.Under these circumstances,how to strive for the regeneration of bone tissue and restore the function and aesthetics as much as possible,is an important task facing the stomatologists.As a technique to form new tissue and reconstruct missing tissue,tissue engineering is an ideal option to regenerate bone tissue in oral cavity.Periodontal ligament stem cells(PDLSCs)not only have good proliferation and multi-directional differentiation ability,but also have excellent immune regulation ability.They are one of the ideal seed cells for dental bone tissue engineering.How to improve the osteogenesis of PDLSCs is also a hotspot of current research.Liquid phase concentrated growth factors(LPCGF),as a new generation of platelet concentrates,contains rich blood-borne growth factor,platelets,leukocytes and CD34 positive cells.LPCGF reflects good potential of osteogenesis.But the current in vitro studies on LPCGF are rare.Also the effect of LPCGF on the osteogenesis of PDLSCs has not been elucidated.Objective:This subject intends to study the effects of LPCGF on proliferation,migration and osteogenesis of human periodontal ligament stem cells(hPDLSCs)in vitro.Then construct tissue engineering complexes using collagen sponge as scaffolds to explore the effect of LPCGF on the proliferation and osteogenesis ability of hPDLSCs on this scaffold.Hope to provide a certain experimental basis for the application of LPCGF,hPDLSCs and collagen sponge in dental bone tissue engineering.Method:(1)Enzyme digestion tissue block method combined with limited dilution method was used to isolate and culture hPDLSCs in vitro.Cell source was determined by immunohistochemical staining;CCK-8 assay and plate clones assay were performed to detect the proliferation ability;The ability of multi-directional differentiation was detected by staining.(2)The blood of healthy volunteers was drawn to prepare LPCGF.CCK-8 assay was used to choose the optimal concentration of LPCGF for proliferation.The scratch assay and transwell assay were performed to detect the effect of the LPCGF on migration of hPDLSCs.(3)After osteogenesis induction,alkaline phosphatase(ALP)staining,alizarin red staining and ALP activity assay are performed to detect the effect of LPCGF on osteogenesis of hPDLSCs.And quantitative real-time polymerase chain reaction(qRTPCR)was used to detect the expression of osteogenic genes.(4)The collagen sponge(CS)was used as the scaffold to culture cells,and the effects of LPCGF on the proliferation and osteogenic capacity of hPDLSCs cultured on the CS scaffold were explored using CCK-8 and qRT-PCR.Result:(1)The cells isolated and cultured in vitro were determined by immunohistochemical staining.The result shows that they are derived from mesenchymal,not epithelial.Also,they show good cloning ability and ability of osteogenic,adipogenic and chondrogenic differentiation.Combined with the morphology and proliferation ability,the cells we obtain are hPDLSCs.(2)Collect the venous blood into a white-capped blood collection tube without any additives,and centrifuge according to the CGF program preset by the Medifuge centrifuge.The obtained LPCGF is initially a light yellow transparent liquid.And it can be converted to a solid state at room temperature or higher.Lower concentration of LPCGF like 5%,10%,15%,20%,can promote the proliferation of hPDLSCs,and 5% is the optimal concentration for proliferation,Also,it can promote cell migration vertically and horizontally.(3)5%LPCCF promoted the osteogenic ability of hPDLSCs and significantly increased the expression of osteogenic genes like ALP,OCN,COL-1,BMP-2 and RUNX-2.(4)5%LPCGF can promote the proliferation of three-dimensional cultured hPDLSCs in CS scaffolds,and significantly improve the expression of osteogenic genes like ALP,COL-1,BMP-2 and RUNX-2.Conclusion:This research shows that 5% LPCGF can promote the proliferation,migration and osteogenesis of hPDLSCs in two-dimensional culture.The tissue engineering complexes constructed using collagen sponges,LPCGF and hPDLSCs in vitro also have good proliferation and osteogenesis potential.It is expected to be used in dental bone tissue engineering in the future.However,there is a more complex internal environment in vivo.Cells,growth factors and scaffolds are affected by many factors.Whether the complex has a consistent effect in vivo;and whether 5% is the best concentration in vivo still need more comprehensive research to verity.