Studies On The Efficacy And Mechanism Of HPDLCSs Combined With TGF-β3 Freeze-dried Sponge To Repair Skull Defected In Rats

Objectives:Cranial defects pose a variety of complications,seriously affecting the life of patients,and even life-threatening.It still represents a common and significant clinical problem.In recent years,with the development of stem cell therapy,it has brought hope to repair large-scale cranial defects.Based on long-term research in our laboratory,we found that human periodontal ligament stem cells(h PDLSC)have stronger osteogenic differentiation properties under the action of TGF-β3.In this study,a new type of human-like collagen type I(RHC)/ chitosan(CS)freeze-dried sponge was used as a carrier for both of h PDLSCs and TGF-β3 to explore the critical size skull injury repair of rats and its molecular mechanism.It provides a new way for stem cell therapy research of bone defect repair as well as providing data reference for clinic.Methods:(1)The primary h PDLSCs were extracted by tissue block-enzyme digestion combined method,and the characteristics of h PDLSCs were identified by flow cytometry and induction of multi-directional differentiation.The migration of h PDLSCs on Transwell plates was used to study the recruitment effect of TGF-β3 on h PDLSCs.Alkaline phosphatase(ALP)activity assay and mineralization experiment were conducted to study the effect of TGF-β3 induction on osteogenic differentiation of h PDLSCs.(2)TGF-β3 / RHC/CS(TRC)freeze-dried sponges with different CS contents were prepared by vacuum freeze-drying and soaking adsorption.SEM observed the morphology of freeze-dried sponge.Ethanol replacement method,compression performance test,in vitro degradation test,water vapor transmission test,water absorption test and moisture retention test were used to test the porosity,mechanical properties,degradability,material exchange capacity,water absorption and moisture retention of the freeze-dried sponge to obtain the better CS content of the TRC freeze-dried sponge.WB detected the expression changes of COL I、COL II、ALP、TGF-βRI、TGF-βRII、Runx2、P-P38 and P38 to investigate the molecular mechanism of TGF-β3 inducing osteogenic differentiation of h PDLSCs.(3)SEM、MTT and live cell staining methods were used to detect the survival and proliferation of h PDLSCs cells on TRC freeze-dried sponge.The proliferation and osteogenic differentiation activity of h PDLSCs by TRC freeze-dried sponge were studied by CCK-8 assay and ALP assay.Tissue compatibility and degradation of TRC lyophilized sponge were studied by subcutaneous implantation in rats.CM-Dil labeling was used to observe the survival of h PDLSCs loaded by TRC sponge after transplantation in rats.We established a rat skull critical bone defect model for pharmacodynamic studies.The skull bone defect repair in rats was investegated by mirco-CT scanning,HE staining and Masson staining.Immunohistochemical staining was used to study the expression of COL I,Runx2 and BMP-2.Results:(1)The extracted h PDLSCs have a positive rate of more than 98 % for the MSC markers CD105、CD166、CD44 and CD90 the expression rate of hematopoietic stem cell markers CD34 、 CD45 and HLA-DR are less than 2 %,showing the ability of multidirectional differentiation.TGF-β3 dose not affect the proliferation of h PDLSCs,but can recruit h PDLSCs.The activity of ALP detection and alizarin red staining results show that TGF-β3(20 nmol/L)significantly increase the ALP activity of h PDLSCs(induction 14 d,P < 0.01,vs Control)and promote mineralization of h PDLSCs(induction 21,28 d,P < 0.01,vs Control).At the same time,TGF-β3 up-regulates the expression of COL I、ALP、Runx2、TGF-βRI、 TGF-βRII and Pp38 / t-p38 proteins in h PDLSCs.(2)1 % TRC freeze-dried sponges have shrunk without a three-dimensional structure,while 2 % or 3 % TRC freeze-dried sponges can form a porous three-dimensional structure.In addition,3 % TRC freeze-dried sponge has better mechanical properties,but 2 % TRC freeze-dried sponge has better degradability,water absorption,substance exchange ability,and moisture retention.(3)Cytotoxicity experiment,Calcein-AM / PI staining,and SEM observation show that TRC sponge has good cell compatibility.CCK-8 and ALP activity detection experiments reveal that TRC sponge can promote the proliferation and osteogenic differentiation of h PDLSCs(P < 0.05).TRC sponge implantation site is normal and no obvious inflammatory reaction is found in subcutaneous implantation experiments.Besides,TRC sponge can be degraded completely at 90 d in the implantation site.h PDLSCs labeled by CM-Dil fluorescence can miantian fluorescence in rats for at least 21 d.Acute toxicity experiments show that TRC sponge has no acute biological toxicity.The critical bone defect model of the SD rat skull was successfully established and the tissue specimens were harvested at 12 weeks after the operation.There is obvious tissue regeneration in the implant site of TRC sponge,while the non-implanted material defect region is only observed a transparent film.Miroc-CT results show that the TRC-h group have a significantly smaller area of bone defect than other groups(P < 0.01).HE and Masson staining results show that the TRC-h group have a large amount of tissue filling in the bone defect,and there are blood vessels growing around the material.Moreover,a large amount of thick bone tissue is generated,which are dyed blue or red.Immunohistochemical results indicate that the expression of Runx2、BMP-2 and COL I in cells surrounding bone defect in TRC-h group is higher than that in other groups.Conclusion:(1)h PDLSCs with high purity and multi-directional differentiation ability were isolated Successfully.TGF-β3 can recruit h PDLSCs without effect on their proliferation.(2)TRC freeze-dried sponge with 2% CS concentration not only has good physical-chemical characters,but also has good biocompatibility,osteoinductivity and proper degradation rate in vivo.h PDLSCs seeded on TRC freeze-dried sponges can survive in rats for at least 21 days.h PDLSCs combined with TRC freeze-dried sponges promotes bone regeneration in the critical size bone defect of the rat skull.Moreover,TGF-β3 can promote osteogenic differentiation of h PDLSCs cells,which may be related to the activation of TGF-β signaling pathway and signal transduction with P38 as the target.