Effects Of Puerarin On Differentiation Of Human Periodontal Ligament Stem Cells Into Osteoblasts In Vitro

Periodontal diseases that lead to the destruction of periodontal tissues including periodontal ligament, cementum and alveolar bone are a major cause of tooth loss in adults and are a substantial public health burden worldwide. To regenerate periodontal tissues and reserve tooth is always a highlight for many researchers, many new approaches have been developed for treating periodontal defects, including guided tissue regeneration, growth factors and enamel matrix proteins, however, tissue engineering offers an ideal option to supplement existing treatment regimen for periodontal tissue regeneration.In recent years, many adult stem cells have been identified and successfully isolated from various tissues including bone marrow, muscle, neurtal tissue, skin, dental pulp and so on.In2004, Seo discovered the stem cells in periodontal ligament tissue for the first time, providing the direct evidence of the existence of hPDLSCs. hPDLSCs possess unique properties, they were found expressing STRO-1and CD146as potent surface markers and also highly expess the specific transcription factor of tendon cellsScleraxis. As a kind of ASC in periodontal ligament tissue, hPDLSCs have the plasticity to differentiate into osteoblastic, cementoblast-like, adipocytic cells and so on under appropriate conditions, representing a promising cell-based therapy in reconstructive dentistry for the treatment of damaged periodontium.Another important element for periodontal tissue regeneration is the effective and inductive morphogenic signals. Up to now, BMP, EMP, TGF, PDGF and estrogen have been proved to be valid osteoinductive growth factors, they have the capability to modulate proliferation and differentiation of implanted osteogenic cells. Although hormone replacement therapy is one of the treatments for postmenopausal osteoporosis, the reported side effects, such as development of hormone dependent breast and uterine cancers, have prompted the use of alternative therapies.Phytoestrogens are plant-derived non-steroidal compounds with estrogen-like activity that bind to estrogen receptors. Epidemiological studies suggested that phytoestrogens have preventive effects for breast cancer and menopausal symptoms. Compared with hormone replacement therapy, the risk for side effects seems to be low. Therefore, they have been focused as alternative treatment for prevention of postmenopausal-related diseases. Puerarin, an isoflavones extracted from the root of Pueraria Labata(Willd) Ohwi, a wild creeper leguminous plant, is one of the earliest and most important crude herbs used in Chinese medicine for various medicinal purposes. For the past several decades, puerarin has been used for the treatment of hypertension and angina pectoris in China. Recently, researchers have paid more attention to puerarin because of its possible roles in the prevention of osteoporosis. Studies showed that isoflavones such as daidzein and puerarin possess a structural similarity to estrogen and bind to the ER in several tissues such as uterus and skeleton, and are effective in preventing bone loss in ovariectomized mice and postmenopausal women. What’s more, they stimulated osteogenesis at low concentrations and inhibited osteogenesis at high concentrations in osteoblasts or osteoprogenitor cells, they are also effective in increasing cell growth, ALP activities and BMP in osteoblasts. Further more, Cai found that puerarin can promote the umbilical cord MSCs to differentiate into osteoblasts and has an effect on the proliferation of umbilical cord MSCs. Li studied the effect of puerarin on the bone formation and the bone resorption in vitro, and discovered that puerarin can suppress the bone resorption and promote the bone formation, and the former effect is stronger than the latter. Jin injected puerarin into the submucosa of the residual ridge after tooth extraction, and proved that residual ridge resorption can be inhibited and the height of the alveolar bone conserved by submucoal injection of puerarin.Although numerous studies in vivo and in vitro have shown that puerarin can promote osteoprogenitor cells differentiate into osteoblasts, it has been obscure whether it aslo affect the differentiation of hPDLSCs into osteoblasts.In this experiment, we want to investigate if puerarin can promote hPDLSCs differentiate into osteoblasts, to learn the possible mechanism of induction with puerarin, and to provide a basis for periodontal tissue regeneration.Chapter One:Isolation and Identification of HPDLSCsObjectives:to isolate and identify the hPDLSCs in vitro. Methods:1. Human premolars were obtained from healthy patients for orthodontic reasons after obtaining the patients’approval and informed consent(donor age:12-16years). Periodontal ligament tissues were gently scraped from the middle portion of the root surface, minced into1mm×1mm×1mm cubes, and then digested in a solution of3mg/mL collagenase type I and4mg/mL dispase for1h at37℃, then centrifuge at1000rpm for5min, the sedimentation were grown in a-minimum essential medium(a-MEM) supplemented with10%(v/v) fetal bovine serum (FBS),0.292mg/mL glutamine,100U/mL penicillin and100ug/mL streptomycin. The cultures were incubated at37℃in a humidified atmosphere with5%CO2. STRO-1+stem cells were prepared using immunomagnetic beads according to the manufacturer’s instructions. After washing, bead-positive cells were subsequently seeded into12-well culture dishes at37℃in5%CO2. The hPDLSCs from this passage were used for further study.2. Identification of hPDLSCs:The expression of stem cell surface markers STRO-land CD146, vimentin and keratin were detected in the hPDLSCs by immunofluorescence assay.Results:1. Morphological characteristics of hPDLSCs:STRO-1+hPDLCs isolated from periodontal ligament tissue by immunomagnetic beads swim out after cultured for3-6days, and the cells aren’t significantly different in cell morphology, the majority are fibrous long shuttle, and a small number of the cells presented as polygonal, spindle-shaped or oval-shaped. The cells grow fast and begin to converge after7-14days.2. Results of immunofluorescence assay:Immunofluorescence assay showed that cells positively express stem cell surface markers_STRO-1and CD146, and positive for vimentin staining, negative for keratin staining. It is confirmed that the cultured cells derived from the mesoderm.Chapter Two:The Influences of Puerarin on the Proliferation and Viability of HPDLSCsObjectives;To observe whether the concentrations that test groups choose have toxicity on hPDLSCs.Methods:1. Puerarin medium for test groups: According to the previous investments, we choose these three concentrations:0.01mmol/L,0.1mmol/L and1mmol/L. Dissolve the puerarin powder into DMSO completely, and then add to a-MEM medium to form relevant concentrations. Non-puerarin was set as blank group.2. The influences on the porliefration of hPDLSCs by puerarin were evaluated by MTT colorimetric assay.Results:To determine the effects of puerarin on hPDLSCs growth, MTT assays were used. Puerarin treatment differentially affected cell proliferation in a dose-dependent manner. In particular, at a concentration of0.01mmol/L, puerarin incrementally promoted proliferation. Puerarin at0.1mmol/L and1mmol/L did not significantly affect cell proliferation, but all of the three concentrations did no harm to hPDLSCs’ viability.Chapter Three:The Effects of Puerarin on the Differentiation of HPDLSCs into Osteoblasts in vitroObjectives:To study the influence of puerarin on the osteogenic differentiation of hPDLSCs.Methods:Expression of osteogenic mineralized-related proteins and calcified nodules were analyzed between puerarin-treated groups and blank group and the positive control group by irnmunofluorescence assay, ALP kit, real-time polymerase chain reaction and alizarin red staining, including COL-I, ALP, OPN and OCN.Results:1. Immunofluorescence assay showed that hPDLSCs positively express osteo-relative markers_COL-I and OPN after exposing to puerarin for7and14days, respectively. 2. The results of ALP kit showed that after7days’culture with puerarin, the ALP levels were significantly increased compared with the blank group.3. The results of RT-PCR showed that the relative expression levels of ALP and OCN in the puerarin-treated group were much higher than the blank group on7days and14days, separately.4. Alizarin red staining showed that lots of calcified nodules were formed in the puerarin-treated group and positive group after14days.Conclusions:1. Puerarin can promote hPDLSCs proliferation at an appropriate concentration.2. Expression of mineral-associated proteins were significantly increased in the hPDLSCs induced by puerarin. This maybe one of the mechanisms of the hPDLSCs differentiation into osteoblasts.3. Differentiation potential of the hPDLSCs was increased obviously after induced by puerarin.