| Objective One of the main manifestations of periodontitis patients is the defect of periodontal support tissue,and the restoration of periodontal support tissue defect is an urgent clinical problem.With the creation of the concept of regenerative medicine and the development of tissue regeneration technology in recent years,it is a new development direction to solve the problem of periodontal loss by obtaining seed cells and expanding the site of tissue defect in vitro.However,the serum components used in vitro culture of stem cells are complex,which may lead to the risk of allogeneic immune response and potential zoonosis in future clinical application.In this study,human platelet lysate(HPL)was used to replace fetal bovine serum(FBS)for in vitro amplification of mesenchymal cells.Human dental pulp mesenchymal cells(human Dental Pulp Mesenchymal Cells,h DPSCs)were cultured in vitro with 5% human platelet lysate as experimental group;and 10% fetal bovine serum culture as control group.The ability of proliferation,osteogenic differentiation and bone defect in vivo was compared between the two groups.HPL is used to replace the FBS as a culture liquid,and the h DPSC is used as a theoretical basis for clinical application of the periodontal tissue engineering.Methods The pulp tissues were cultured with 5% HPL and 10% fetal bovine serum(FBS).Proliferation and toxicity detection kit(cell proliferation and toxicity test kit,cck-8 were used to detect the proliferation of cells on the 1st day,2nd day,3rd day,4th day,5th day,6th day,7th day respectively.After 14 days of osteogenic induction,alizarin red and ALP staining were used to detect osteogenic ability.The expression of alkaline phosphatase activity was quantitatively detected by ALP after osteogenic induction in vitro for 4,7 and 14 days.Four 8 mm-diameter round bone defects were made on the skull of 10 male New Zealand white rabbits,divided into four groups:Group A of 10% FBS was inoculated in the medical gelatin sponge support group;Group B of 5% HPL was inoculated in the medical gelatin sponge support group;Group C was medical gelatin sponge group;Group D is a blank group.Three-dimensional reconstruction of Micro-CT at 4 and 8 weeks after operation,analysis of the ratio of BV/TV in defect area;histological hematoxylin-eosin stain to observe that bone formation in the skull defect.All the obtained data were statistically analyzed using SPSS 22.0 software.Results The cck-8 results show that the 5% HPL group has a proliferation rate faster than the 10% FBS group on day 3 and 4.Statistical significance(P < 0.001);The results of alizalin red staining showed that the culture medium containing 5% HPL significantly promoted the formation of calcium nodules in osteogenic differentiation of h PDSCs.The results of ALP staining and quantitative analysis showed that the activity of ALP in the 5% HPL group was higher and the difference was significant(P <0.0001).Micro-CT showed that the formation of new bone in group B was more than that in group A at 4 and 8 weeks,but the osteogenic effect of C and D was not obvious.The ratio of BV/TV in bone defect area was analyzed by software.It was found that the area of new bone formation in group B was larger than that in group A,and the difference was statistically significant(P < 0.0001).The histological observation showed that there were new bone formation in group A and group B at 4 and 8 weeks,and the new bone formed by group B was well-formed,and the new bone area of group C and D was not obvious.Conclusion 5% HPL compared with 10% FBS,5% HPL can promote the in vitro proliferation of h DPSCs and the in vivo bone-forming ability.HPL can be used for the in vitro amplification of h DPSCs in place of FBS,it provides a theoretical basis for the safe application of dental pulp mesenchymal cells in clinical practice. |
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