| Background and objection:Stem cells are unique cell populations with self-renewal and multi-directional differentiation potential. In 2000, Gronthos and his colleagues for the first time found some precursor cells with self-renewal and multi-directional differentiation potential existed in dental pulp for the first time and named them human dental pulp stem cells (hDPSCs).The find of hDPSCs provides a new idea for pulp injury and repair mechanism research, and also brings a big breakthrough for tissue regeneration engineering. Since the hDPSCs can obtained form the third molars from patients who have the need to extract them or the need of routine orthodontic treatments.Becauses the cells are came from the patients themselves,there are no immune rejection, and because of hDPSCs has multi-directional differentiation potential, which means hDPSCs can be induced to differentiate into odontoblast-like cells, makes it become the first choice of tissues regeneration research.Hypoxia is life’s basic phenomenon, the oxygen volume fraction of dental pulp tissue in normal physiological conditions significantly lower than the air oxygen volume fraction of 21%.And since the dental pulp tissue are surrounded with the dentin,which is hard tissue, when stimulated by inflammatory factors, the pressure of root canal rose and then the vascular permeability increased, leading to more lower oxygen volume fraction. hDPSCs plays an important role in the repair of dental pulp tissue.Currently most studies of hDPSCs are performed under the normoxia of 21% oxygen volume fraction. However, the oxygen volume fraction of dental pulp tissue was significantly lower than oxygen volume fraction of air, in either physiological or pathological status.And a large number of researches showed that different oxygen volume fraction can significantly affect the biology characteristics and functions of stem cells.So study the biological characteristics of hDPSCs under hypoxia environment can further clarify the role of hDPSCs in the dental pulp repair and regeneration medicine.To date,there is only a few researches about the effect of the hypoxia on human dental pulp stem cells biological characteristics, and there are much differences among the existing research results.This study is aimed to study the effect of hypxoia on the proliferation, migration, osteo-/odontogenic differentiation and angiogenesis characteristic of hDPSCs, in order to further clarify the biological characteristics of dental pulp stem cells.This paper is composed of the following four chaptersChapter 1 The isolation and identification of hDPSCsWe used tissue explant collagenase method to isolate human dental pulp stem cells from young permanent teeth. Flow cytometry analysis was used to measure cell surface molecule expression pattern.The colony forming capacity and multilineage differentiation capability of hDPSCs were also examined.Chapter 2 The effect of hypoxia on hDPSCs proliferation and migrationhDPSCs were cultured under hypoxia (3% O2) and normoxia (21% O2), the cell surface molecule expression pattern and cell cycle distribution and apoptisis of both groups was revealed by flow cytometry analysis.The growth rate of hypoxia group hDPSCs and normoxia group hDPSCs was examined by MTT assay at 4 different time points(day 1, day 2, day 3, day 4 after culture). The migration capacity of hypoxia group hDPSCs and normoxia group hDPSCs was evaluated by Transwell assay.Chapter 3 The effect of hypoxia on odontogenic differentiation of hDPSCsWe used qRT-PCR to reveal the expression level of osteo-/odontogenic differentiation related genes in both groups including ALP, DMP-1,DSPP mRNA following 7days,14days and 21 days of mineralization induction.Chapter 4 The effect of hypoxia on angiogenic factors expression of hDPSCsqRT-PCR was used to detected expression level of angiogenic factors VEGF,SDF-1 mRNA of hDPSCs in both groups.We collected culture supernatants of hDPSCs in hypoxia group and normoxia group at 1 day,2day,3day, and ELISA was used to compare the two group protein secretion of VEGF.Materials and methodshDPSCs isolation and cultureHuman dental pulp was obtained from complete third molar extracted due to impaction or orthodontic of healthy subjects (age range from 18 to 25) at oral and maxillofacial surgery department of Nanfang Hospital. Informed consent was obtained from each patient. After extraction, the teeth were put into DMEM medium containing Penicillin-Streptomycin and sent to laboratory immediately(within 4h). Tissue explant collagenase method was uesd to isolate human dental pulp stem cells. The teeth were splited along the groove around the cemento-enamel junction. The pulp tissue was taken out in super clean bench (2 mm pulp close to the apical were removed) and placed in sterile PBS buffer containing Penicillin-Streptomycin,washed with PBS at least 3 times.The pulp tissue was cut into pieces about 1 mm×1 mm×1 mm size,then digested in 3 mg/mL collagenase type I at 37℃ for 10-15 min,followed by centrifugation at 800 rpm for 3 min.Abandon the supernatant,and transfer the tissues to a 25mm2 culture flask and evenly spread.Turn the culture flask and add 3mL cell culture medium with 10%FBS,100 units/mL penicillin and 100 mg/mL streptomycin into it. Turn the culture flask again after 24 h to let the cell culture medium cover the explants,and incubated at 37℃ with 5% CO2 in humidified incubator. Cell culture medium was changed every 3 days.Colony-forming assayThe cells were seeded in 6 wells-plate with 100 cells per well. After 14 days culture, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cell colonies were observed by the inverted microscope (50 or more cells were defiend as a clone).Cell phenotype analysis by flow cytometry analysisAt least 1×106 cells were prepared in PBS for the test. The cell phenotype analysis was performed by flow cytometric detection of CD29/PE, CD34/PE CD90/PE,CD45/PE and CD44/FITC according to the manufacturers’ instructions.The analysis of hDPSCs multilineage differentiation potential in vitroFor osteogenic differentiation, hDPSCs were seeded into 6 wells-plate with 2×104 cells per well and cultured to 80% confluence. Osteogenic differentiation was induced by culturing hDPSCs in mineralized solution for 21 days. Then the induced cells were fixed with 4% paraformaldehyde for 10-15 min at room temperature and then stained with 2% Alizarin Red for 10 min. After that,the cell was washed by PBS for 3 times and observed by inverted microscope.For adipogenic differentiation, hDPSCs were seeded into 6 well cell cultrue cluster with 2×104 per well and cultured to 80% confluence. Adipogenic differentiation was induced by culturing hDPSCs in complete medium supplemented with 0.5 mM methylisobutylxanthine,0.5 mM hydrocortisone, and 60 mM indomethacin for 21 days.The hDPSCs were fixed in 4% paraformaldehyde for 10-15 min at room temperature and then stained with Oil Red O for 10 min. After that,the cell was washed by PBS for 3 times and observed by inverted microscope.For chondrogenic differentiation, hDPSCs were seeded into 6 well cell cultrue cluster with 2×104 per well and cultured to 80% confluence. Chondrogenic differentiation was induced by culturing hDPSCs with chondrogenic differentiation medium supplemented with 10 ng/mL transforming growth factor-β3(TGF-P3) for 21 days. The hDPSCs cells were fixed in 4% paraformaldehyde for 10-15 min at room temperature and then stained with Alcian Blue for 2 h. After that,the cell was washed by PBS for 3 times and observed by inverted microscope.Cell proliferation assay by MTTFor MTT assays, the hDPSCs at logarithmic phase were seeded in 96-well plates at 2×103 in 100μL medium per well. Cell proliferation was detected at the indicated time-points.20μL3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)at a concentration of 5 mg/mLwas then added into each culture well. Then the cells were incubated for 4 h at 37℃, the supernatant was discarded, and 200 μL dimethyl sulfoxide (DMSO)was added into each well. The absorbance of each well was measured by using microculture plate reader with a test wavelength of 490 nm.Cell activity by LIVE/DEAD assayshDPSCs were cultured under hypoxia supplied with 3% O2 or cultured under normoxia with 21% O2 for 24 h,48 h,72h,respectively. Then each cell sample were stained with PI/Calcein-AM fluorescent dye(PI with a final concentration of 4 μmol/L, Calcein-AM with a final concentration of 2 μmol/L) for 15-30 min at room temperature, then abandoned the dyeing liquid, washed by PBS twice, observed under the inverted microscope (dead cells can be stained by PI,observed as red, living cells can be stained by Calcein-AM, observed as green and yellow).Cell cycle analysis by flow cytometry analysishDPSCs were cultured under hypoxia supplied with 3% O2 or cultured under normoxia with 21% O2 for 24 hours were collected and mixed with 1 mL cold 70% alcohol at-20℃ for 48 h to fix cells. The cells were then washed with PBS for two times, then stained with 1 mg/mL propidium iodide(PI) at 4℃ for 30 minutes, and analyzed on fluorescence activated cell sorter with excitation wavelength of 488 nm and emission wavelength of 610 nm. The fraction of hDPSCs in Gi, S, and G2/M phases of cell cycle was determined.Cell apoptisis analysis by flow cytometry analysishDPSCs were cultured under hypoxia supplied with 3% O2 or cultured under normoxia with 21% O2 for 24 hours were collected by pancreatic enzyme digestion and centrifugation. Washed cells with Precooling PBS twice, and took 500 uL cells to mix wih 5 μL Annexin V-EGFP, and blended them, then add 5μL Propidium Iodide into the mixture,also blendeed,incubated away from light for 5-15 min at room temperature, and analyzed on fluorescence activated cell sorter with excitation wavelength of 488 nm and emission wavelength of 530 nm.Transwell migration assayThe migration activities of hypoxia group hDPSCs and normoxia group hDPSCs were measured by using transwell cell culture chambers. Following 24 hours serum starvation, cells were collected and resuspended in DMEM without containing FBS. hDPSCs were added into the upper transwell chamber.The migration of hDPSCs were stimulated by the addition of DMEM+10% FBS to the lower chamber. Then the transwell cell culture chambers were incubated in hypoxia with 3% O2 or normoxia with 21% O2 respectively.After 24 hours the cells that migrated through the holes were fixed with methanol and stained with 0.1% crystal violet. Cells in upper transwell chamber were moved out by cotton swabs. Cells migrated through the holes were counted in six random fields under a inverted microscope and calculated the average number of cells per field.qRT-PCRTotal RNA extraction of hDPSCs was using TRIzol according to the manufacturer’s instructions. First-strand cDNA was generated using the PrimeScript(?) RT reagent Kit. The cDNA was amplified with SYBR(?) Premix DimerEraserTM using an LightCycler 480 realtime PCR system, and glyceraldehydes-3-phosphatedehydrogenase (GAPDH) was used as the internal control to normalize for the difference in amount of total RNA in each sample.ELISAhDPSCs were cultured under hypoxia with 3% O2 or cultured under normoxia with 21% O2 for 24 hours,48 hours,72 hours respectively. Then supernatant of each cell sample were detected by ELISA kit for VEGF concentration (each sample were detected for 3 times).The ELISA detection process was in strict accordance with the ELISA kit manufacturers’ instructions.Statistical analysisThe data were analyzed and expressed as the mean±tandard deviation. Statistical significance was evaluated by independent samples t-test using SPSS 13.0 software. Statistical significance was set at P< 0.05.Results1. hDPSCs were successfully separated and culturedWe successfully expanded cells from human dental pulp by tissue explant collagenase method. To detect the phenotype and biological characteristics of the cells,we performed Colony-forming assay and verified that the obtained cells can form cell colony in culture.The positive rate of CD34,CD45 were 0.01%,0.49%, which were hematopoietic surface markers.However, the positive rate of CD29,CD44,CD90 were 99.97%,100%,99.92%, which were MSC cell surface markers.To evaluate the multipotent differentiation capacity of hDPSCs,we treated hDPSCs with different differentiation inducing medium.For osteogenic differentiation, mineralization nodules could be detected in the presence of mineralization medium after 21 days of culture.For adipogenic differentiation, the accumulation of lipid-rich vacuoles was visualized within cells by Oil Red O staining. For Chondrogenic differentiation, the accretion of sulfated matrix stained with Alcian Blue was apparent.The results revealed that the obtained cells could be considered as hDPSCs.2. The effect of hypoxia on hDPSCs proliferation and migration.This experiment used flow cytometry to detect hDPSCs surface markers of hypoxia and normoxia group, the results show that the cells in both group cells have high expression of CD29 and CD44, CD90, which are mesenchymal stem cell surface molecular markers,and low expression of CD34, CD45,which are hematopoietic stem cell surface molecular markers.This confirmed that hDPSCs cultured under hypoxia can maintain the cell phenotype of mesenchymal stem cells.Flow cytometry was also performed to detect the cell cycle distribution and apoptosis of hDPSCs in hypoxia group and normoxia group, and the results showed that there is no significant difference between two groups. MTT assays was uesd to compared cell proliferation of the two group, the statistical results showed that the hypoxia group hDPSCs has significantly higher cell proliferation ablility than normoxia group. Transwell experimental results showed that the hypoxia group had a significantly higher migration ability than normoxia group.3. The effect of hypoxia on odontogenic differentiation of hDPSCsBoth hypoxia group hDPSCs and normoxia group hDPSCs were cultured in mineralized medium for 7days,14days,21 days. The expression level of ALP, DSPP, DMP-1 mRNA was detected by quantitive realtime PCR analysis. The data of 7 days showed that the odontogenic differentiation gene ALP, DSPP, DMP-1 mRNA expressed significantly lower in hypoxia group than normoxia group. The data of 14 days showed that the ALP, DSPP, DMP-1 mRNA expressed higher in hypoxia group than normoxia group. And the data of 21 days showed that the ALP, DSPP, DMP-1 mRNA expressed higher in hypoxia group than normoxia group.4. The effect of hypoxia on the angiogenic factors expression of hDPSCsWe used qRT-PCR and ELISA to detect angiogenic factors expression of hypoxia group and normoxia group at different time-points (24 hours,48 hours,72 hours). The results showed that a significant expression rise of VEGF mRNA in hypoxia group, and the expression increased most significantly at 48 hours.At the same time, the ELISA results also showed that hypoxia group had a significantly higher secretion of VEGF protein than normoxia group.But there was no significant difference between hypoxia group and normoxia group of SDF-1 mRNA expression.Conclusions1. hDPSCs were successfully isolated and cultured by tissue explant collagenase method. The obtained cells with clone formation ability had the mesenchymal stem cells phenotype and biological properties,and also multilineage differentiation potential.2. After cultured under hypoxia,the hDPSCs maintained the mesenchymal stem cells phenotype, and still had a good cell activity, meanwhile the proliferation and migration ability were enhanced.3. Hypoxia significantly inhibited the odontogenic differentiation related genes expressions of hDPSCs in the early, but with the mineralized induced time prolonged, the inhibition was abated, and turned to promote the expression of those genes, which prompted that the effect of hypoxia on hDPSCs odontogenic differentiation is more complex.4. Hypoxia increased the VEGF expression of hDPSCs, so we speculated that hypoxia may have a role in promoting the angiogenic characteristics of hDPSCs. |
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