Effects Of Modified Platelet-rich Plasma On The Proliferation And Osteogenic Differentiation Of Stem Cells From Human Exfoliated Deciduous Teeth

Many common diseases in oral medicine fields such as periodontal disease, tumor and trauma often result in bone defects. There are two main methods for the treatment of bone defects presently:autogenous bone graft and artifactitious allogeneic or xenogeneic bone transplantation. The former osteogenic effect is good, but the quantity of transplanted bone is limited. In addition, the increased operative sites bring in additional trauma, which is generally more difficult to be accepted by patients. The latter osteogenic effect is poor, and the time of bone remodeling is long. With the establishment of the concept of regenerative medicine and development of tissue engineering technology in recent years, stem cells (seed cells) from patient bodies are used to construct defect tissues in vitro, and then they were transplanted to the defect sites of the patient body tissues to reconstruct by this model, which is a development direction to solve the problem of bone tissue defect treatment. At present, it has been reported that in vitro constructed tissue engineering bone complex can be implanted into animal bodies and form new bone with normal bone tissue morphology by using bone marrow mesenchymal stem cells. Construction of tissue engineering bone mainly includes three factors:stem cells (seed cells), scaffold materials and growth factors promoting osteogenesis.Mesenchymal stem cells (MSCs) are generally considered to be the ideal seed cells for tissue engineering bone. So far, BMMSCs have been used in most of the research and clinical application. BMMSCs are a kind of stem cells located in bone marrow. Research has demonstrated that BMMSCs can differentiate into skeletogenous cells, chondrocytes, fibroblasts, adipocytes, mesodermal tissue cells and many mesodermal tissue cells originated from mesenchymal tissues under in vitro appropriate culture conditions. However, extraction of bone marrow is an invasive operation and it brings in some pain to donors. In addition, common diseases causing Jaw bone defects in clinical application include periodontal disease and tumors occurred mainly in adult and elderly people, but the proliferation and differentiation abilities of BMMSCs of the elderly are significantly reduced, which are not suitable to be used as seed cells for bone tissue engineering. Therefore, it is particularly important to select other sources of MSCs.In2000, Gronthos prepared the third human healthy molar pulp into single cell suspension and cultured the cells by using enzymatic digestion method to obtain the cells with cell clone formation ability and high proliferation ability, which were named human dental pulp stem cells (DPSCs). In2003, Miura isolated and obtained stem cells with highly proliferative and multilineage differentiation abilities from exfoliated deciduous teeth dental pulp, which were named stem cells from human exfoliated deciduous teeth (SHED). SHED, DPSCs and BMMSCs were compared, and it was revealed that these three kinds of cells all had characteristics of mesenchymal stem cells including fibroblast like morphology and expression of stem cell surface markers. Gronthos has used the research methods of BMMSCs and conducted contrastive study on DPSCs and BMMSCs. Under the same inoculation density, clone formation rate of DPSCs was22～70clones per10000cells, which was significantly higher than that of the clone formation rate of BMMSCs,2.4-3.1clones per10000cells. The data indicate that adult dental pulp stem cells have high colony forming ability, and it can be deduced that content of stem cells in dental pulp tissues may be higher than that of bone marrow. Miura found in his research that12-20cells of SHED obtained from exfoliated deciduous teeth could form adhesion colony. Compared with that of BMMSCs and DPSCs, SHED had a higher proliferative capacity. Gene expression profile revealed that there were4386genes with more than two times differences in SHED and DPSCs. In respect to cell proliferation and extracellular matrix secretion pathways, the expression of SHED was significantly higher than that of DPSCs. SHED can differentiate into osteoblasts by induction culture in vitro and form three-dimensional woven bone like structures. Miura also found that SHED had osteoinductive properties. Although SHED inoculated in nude mice cannot be directly differentiated into osteoblasts, they can induce host cells to differentiate into osteoblasts, which is different from the stem cells from DPSCs and other dental tissues in some degree. In view of the fact that SHED may have a strong proliferation and multilineage differentiation potential and they may have properties of inducing bone regeneration, human exfoliated deciduous dental pulp may serve as potential mesenchymal stem cell niches, and they probably are ideal osteoblast source library. Mineralized tissues formed by SHED may be used in bone regeneration, bone transplantation and other clinical treatments.MSCs need to be expanded in vitro during preparatory phase of clinical application. Fetal bovine serum (FBS) is the most commonly used substance in cell culture, and it provides nutrients, growth factors and hormones and other biologically active substances for cell proliferation and other biological behaviors. However, application of FBS may be related to ethics, science and security issues. FBS is obtained from fetal bovine body, and pregnant cows are needed to be killed. The current preparation method of FBS will lead to deaths of a lot of fetal calves. Constituents of FBS are uncertainty, and there are tremendous differences of various components in each batch, so it is easy to cause errors in scientific research, affecting the repeatability of tests. As a kind of exogenous serum, FBS used in clinic may cause immunological rejection. FBS may even spreads bovine viruses, bacteria and other pathogens to people. American FDA has strictly limited the stem cell in vitro culture system applied in clinical treatments. Therefore, fetal bovine serum is not suitable for in vitro culture of mesenchymal stem cells used for clinical application.Platelet-rich plasma (PRP) is platelet concentrate obtained by centrifugation from autologous whole blood, and PRP usually contains a lot of platelets and small amounts of other blood cells. Platelets contain abundant growth factors, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), insulin-like growth factor, transforming growth factor β (TGF-β) and vascular endothelial growth factor, which play an important role in the proliferation process of cells.In the field of oral medicine, PRP has been applied to the treatment of maxillofacial bone defect and guiding periodontal tissue regeneration. PRP was respectively mixed with BMMSCs, DPSCs or SHED and implanted into mandible defect areas. Bone quantity in bone defect area increased dramatically after8weeks. Cancer patients with jaw bone necrosis can be conducted combined treatment of operation, chemotherapy, laser and PRP, which could effectively promote the healing of surgical wound. For the young permanent teeth with pulp necrosis, platelet rich fibrin membrane could be used as apical barrier combined with the use of MTA. On one hand, it can prevent MTA super filling. On the other hand, growth factor secreted by platelets can promote proliferation of osteoblast, gingival fibroblast and periodontal ligament cells and induce the formation of apical foramen. PRP was mixed with BMMSCs, DPSC or SHED respectively and implanted into extraction sockets, which were implanted into bodies after8weeks. It was observed that implants could form better osseointegration and vascularization after16weeks.A series of basic research confirmed that PRP can promote proliferation, migration and differentiation of mesenchymal stem cells, osteoblasts and fibroblasts, which have concentration specificity and cell specificity.Compared with FBS, PRP can promote the proliferation of human MSCs, reduce the time of cell fusion, increase sizes of cell colony forming unit (CFU), and can maintain their osteogenic, chondrogenic and adipogenic differentiation ability, and maintain immunosuppressive activity. Flow cytometry examination showed that MSCs expressed high levels of PDGF-A, PDGF-B, bFGF, TGF-p and IGF-1receptor, suggesting that PDGF, bFGF, TGF-β and1GF-1in platelets can play roles in MSCs. Further experiments confirmed that PDGF, FGF and TGF-P signaling pathways played an important role in the proliferation and differentiation process of MSCs, which selectively inhibited PDGF receptor kinase. Bone cell formation was significantly reduced, and there was no mineralized nodule formation. At the same time, the cell proliferation rate was significantly reduced. When TGF-β, PDGF and bFGF were added to the serum free cell culture medium, MSCs could maintain long fusiform and survived5generations, in which the proliferation of MSC showed no obvious difference with that of DMEM medium containing10%FBS, and the cells kept ability to differentiate into various cells. PDGF in PRP may be the key factor to promote the proliferation and migration of MSCs possibly through affecting cell mitosis by the ERK signaling pathway.PRP can promote the proliferation of DPSCs. Compared with that of FBS, the proliferation enhancing ability of PRP is stronger, and it does not change the immunophenotype of DPSCs, CFU and directional differentiation ability. PRP promotes the proliferation of human dental pulp cells and protein synthesis through PI3K/AKT and MAPK signaling pathways. DPSCs treated with PRP express high levels of odontoblast gene and osteogenic gene by up-regulating the expression of osteopontin (OPG) and alkaline phosphatase (ALP) and promoting mineralization. In a certain range of concentration, proliferation ability of DPSCs is dependent on PRP in a concentration dependent manner, but high concentration of PRP inhibits the proliferation of DPSCs. Due to the different sources and preparation methods of PRP, it is unable to determine the optimum concentration. Our preliminary study showed that10～30%PRP could significantly improve the activity of ALP in dental pulp cells, of which20%concentration was especially obvious.10～30%PRP significantly promoted the formation of mineralized nodules from dental pulp cells induced and mineralized after10days, in which the formed mineralized nodules were maximum from dental pulp cells induced and mineralized after20days with10%concentration PRP.The various components in PRP were analyzed. PDGF and IGF-1could promote the proliferation of dental pulp cells. aFGF, IGF-1and IGF-2promote the synthesis of extracellular matrix. TGF-β,PDGF, aFGF and bFGF may be involved in the regulation of transformation of dental pulp cells into odontoblasts. Three kinds of PDGF subtypes are further subdivided, in which PDGF-AA has inhibitory effect on DSP synthesis of dental pulp cells, but PDGF-AB and PDGF-BB shows an accelerating effect.Based on the above research status at home and abroad and the prophase research results in the present study, we put forward the following working proposals:(1) The modified PRP in a certain concentration can promote better proliferation of SHED in vitro;(2) The modified PRP can replace fetal bovine serum and become culture medium for stem cell rapid amplification in vitro needed in tissue engineering bone;(3) Under osteogenic induction conditions, platelet-derived growth factors could promote differentiation of SHED into osteoblast in vitro. To verify the above assumptions, the modified PRP with different concentrations were used in the in vitro amplification and culture of SHED and research on osteogenic induction and differentiation in the present study.The present paper mainly includes the following four chapters:The first chapter:isolation, culture and identification of deciduous dental pulp stem cellsAfter informed consents from the parents, retained deciduous teeth with non dental pulp diseases of Children of6to8years old were removed. Deciduous dental pulp stem cells were isolated and cultured by enzyme digestion methods. After HE staining, cells mostly demonstrated long fusiform and fibroblast like morphology. A few cells were polygon or oval, and nucleus had large volume and deep dyeing, and staining of cytoplasm was shallow. Cell proliferation was detected by CCK-8method. Cell growth curve was drawn. The results showed that cell number increased with prolonged cell culture time. At the third to fourth day, cells entered the logarithmic growth phase. Expressions of cell surface keratin and vimentin were examined by immunocytochemical technique, and the results demonstrated that they were keratin-negative and vimentin-positive, indicating that the cells derived from mesenchymal tissues. Cell surface markers of stem cells were detected by flow cytometry, and the results showed that99.69%cells were CD44positive, and90.13%of the cells were CD105positive, but the cells were CD34negative by antibody detection, indicating that the cells conformed to the characteristics of mesenchymal stem cells; The cells were cultured in the mineralization and induction medium for30days. Alizarin red staining results illustrated that there were mass formation of mineralized nodules, indicating that SHED have ability to differentiate into osteoblasts. The cells were cultured in adipogenic induction medium for continuous21days, and oil red-O staining results demonstrated that there were transparent high brightness points, showing that SHED have the ability of differentiation into adipocytes direction.The proliferation ability of deciduous dental pulp stem cells obtained in this experiment was strong. The deciduous dental pulp stem cells expressed stem cell specific markers and could differentiate into osteogenic and adipogenic directions under different induced conditions, which are good in vitro cell models for further experiments.The second chapter:preparation and activation of modified platelet-rich plasma0.1unit platelets with good testing results harvested by human AB type machine were collected. Heparin was added into platelets. The platelet suspensions containing heparin were conducted centrifugation with a speed of3×103r/min for20min, some supernatant was discarded. The platelet concentration was adjusted at1×1012platelets/L. Platelets were pipetted repeatedly with tubes and resuspended and prepared into platelet-rich plasma. PRP was packed into freezing tubes and immersed in liquid nitrogen tank for5min. The PRP were quickly taken out and immersed in37℃water bath box for5min, which was repeated three times to make sure that platelets were fully frozen and thawed and lysed.3×103r/min centrifugation was conducted for20min to remove platelet sediments, and then platelets were filtered with0.2μm filtering membranes. ELISA method was used for quantitative detection of PDGF and TGF-β concentrations in platelet-rich plasma.Compared with that of the PRP preparation and activation methods in previous studies, the present investigation improved two aspects. One is that most experiments used twice centrifugal method to prepare platelet concentrates, and the present study employed automatic blood cell analyzer to select and harvest platelets, which effectively avoided pollution of the other blood cells. The second point is that bovine thrombin or calcium chloride was used to activate PRP in all the previous studies. Taking into account that bovine thrombin was exogenous protein, it may cause dysfunction of blood coagulation and immune rejection. Calcium ion in the calcium chloride may cause potential interference to the subsequent experiments, especially the induced mineralization experiments. Therefore, the experiment used the physical method of liquid nitrogen freeze-thaw to lyse platelets so that a variety of growth factors in platelets could be released. The concentration of PDGF-AA was19.159μg/L, and the concentration of TGF-β was57.163μg/L measured by ELISA in the preparation and activation of PRP by the modified method, which were consistent with the concentrations of main growth factors in PRP prepared by other researchers.The third chapter:research on the effects of modified platelet-rich plasma on cell proliferation of deciduous dental pulp stem cellsThe fourth generations of SHED were divided into4experimental groups and one control group according to the different culture conditions. In the experimental group,2%,5%,10%and20%PRP were added into a-MEM cell culture medium, respectively. In the control group,10%PRP was added into a-MEM cell culture medium. Cells were cultured in96well plates with a concentration of1×104/mL and200μL/well. There were four replicates in each group. At the first day to seventh day of cell culture, CCK-8method was used to measure OD value of450nm wave length after incubation for2h. Cell growth curve was drawn. SPSS18.0software was used in statistical analysis. This study showed that PRP could promote proliferation of SHED, which is related to a large number of growth factors in PRP. These growth factors alone or in combination with others can promote the proliferation of SHED. The roles of different concentrations of PRP in promoting proliferation of SHED are not the same, in which the proliferation ability of2%PRP was the strongest. During the whole cell culture period, there was no statistical difference in proliferation enhancing ability for2%PRP and10%FBS. In the middle and late stages of cell culture in5%PRP, there was no statistical difference in proliferation enhancing ability on SHED between PRP group and the control group, while high concentrations of PRP inhibited growth of SHED, which is similar to our previous findings that the modified PRP showed proliferation enhancing effects on human dental pulp cells. Preliminary experimental study found that the effects of modified PRP on proliferation promoting effect of human dental pulp cells were concentration dependent.1%-10%modified PRP significantly increased the proliferation activity of human dental pulp cells, especially10%concentration of PRP. In a certain range of concentration, proliferation ability of DPSCs positively correlated with PRP in a concentration dependent manner; while high concentrations of PRP inhibited the proliferation of DPSCs. Jianmin Duan and other researchers believed that this concentration specificity may be related to the produced prostaglandin E2(PGE2) by promotion of dental pulp cells. In other words, low concentration of PRP promotes production of proper amount of PGE2by pulp cells and thus promotes cell proliferation, while high concentration of PRP promotes production of excess amount of PGE2by pulp cells and thus inhibits cell proliferation. In addition, inhibition of high concentration of PRP on the proliferation of dental pulp cells also may be associated with plasma components. There may be substances in plasma showed anti platelet growth factor effects, and the washed platelets after removal of plasma promote stronger cell proliferation.The results of this study showed that PRP may replace FBS for amplification of SHED in vitro and thus increase clinical safety of tissue engineering products used in clinical applications. The fourth chapter:research on effects of modified platelet-rich plasma on osteoblasts and differentiation of deciduous dental pulp stem cells.The fourth generations of SHED with good growth conditions were divided into4experimental groups and one control group according to the different culture conditions. In the experimental group,1%,2%,5%and10%modified PRP were added in the a-MEM culture medium, respectively. In the control group,10%FBS was added into the a-MEM medium. Mineralization induction fluid was added into every group to induce and mineralize. Cells were cultured in96well plates with a density of5×103/well. After culturing for24h when cells adhered on cell culture flask walls, different cell culture medium were added to the cells. After culturing for2d,4d, and6d, OD values of various cells in wells were detected by enzyme reader instrument at a wavelength of520nm according to the ALP kit instructions to detect the activity of ALP in cells. Alkaline phosphatase is an important substance involved in metabolism and regeneration of bone and other mineralized tissues. The role of alkaline phosphatase in hard tissue formation and enhancing calcification has been confirmed. The activity of ALP is an important index of differentiation and maturation of osteoblast like cells, the activity level of which can reflect mineralization ability of different tissues and cells as well as the trend to differentiate into osteogenic direction. The present experimental results showed that different concentrations of modified PRP all could enhance the activity of alkaline phosphatase of SHED, especially on the sixth day, on which there were significant differences between all the concentration of PRP groups compared with that of the control group (P<0.05). Specifically, the effects of the2%concentration of modified PRP on promoting ALP activity of SHED are the maximum(P<0.01).Five different cell culture media were used to culture SHED. After minera lization and induction of SHED for7d, Q-PCR was employed to examine mRNA contents of RUNX2and osteocalcin. Differences of mRNA contents of RUNX2and osteocalcin of SHED in various groups were compared and analyzed. RUNX2and osteocalcin are important factors related to mineralization, and they are also important indice for detecting cell mineralization. In the present investigation, qRT-PCR method was used to detect effects of different concentrations of modified PRP on the expression changes of RUNX2and osteocalcin mRNA in SHED, which directly reflects the osteogenic promoting ability of the modified PRP on SHED. Results illustrated that the enhancing effects of the modified PRP on expression of RUNX2and osteocalcin mRNA in SHED were concentration specific. At the seventh day of mineralization and induction, the expression contents of RUNX2and osteocalcin mRNA in SHED cultured in2%modified PRP were dramatically higher than that of10%FBS, and the expression contents of RUNX2and osteocalcin mRNA in SHED cultured in10%modified PRP were dramatically lower than that of10%FBS.The results of this study showed that a certain concentration of the modified PRP displayed enhancing effects on osteogenic differentiation of SHED, and the strong proliferation and osteogenic differentiation capabilities were just the characteristics of SHED as seed cells required in tissue engineering. The present results provide some experimental basis for better application of SHED in bone/tooth tissue engineering.To sum up, selecting SHED with strong in vitro amplification ability and osteogenic capability and the modified platelet-rich plasma for in vitro cell culture may solve the existed problem presently in the restriction of construction of in vitro tissue engineering bone so as to improve the efficiency of establishment of in vitro tissue engineering bone, reduce cost, and increase the safety of clinical treatment.