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Objective@#To establish the experimental model of rabbit mandibular anterior implant repair and evaluate the effects of transforming growth factor (TGF)-β3 and dental pulp stem cells (DPSC) in promoting the bone integration of implant.@*Methods@#The New Zealand rabbits were randomly divided into experimental group, control group and blank group (6 rabbits for each group) . In the experimental group, the implant area was filled with the mixture of TGF-β3, DPSC and Bio-oss powder. In the control group, the implant area was filled with the mixture of DPSC and Bio-oss powder. In the blank group, the implant area was filled with the mixture of phosphate buffer solution and Bio-oss powder. Eighteen New Zealand rabbits were sacrificed in 2 weeks after procedure. The treated alveolar bone tissue was observed. The bone tissue around the implant were estimated by HE staining, immunocytochemical staining and real-time quantitative PCR.@*Results@#The implants were no shedding nor loose. HE staining shows the blank group had a sparse trabecular bone and a small amount of blood vessel around the implant and no obvious new bone formation. The control group showed that the bone trabecula around the implant was sparse and slender, the osteoblasts were arranged linearly around the trabecular bone, a small amount of new bone formation was found around the implant. In the experimental group, there were more thick and dense trabecular bone around the implant, the surrounding osteoblasts were arranged in clusters. The osteoblasts were active and many new bone formed. Typical bone lacunae, bone cells and a large number of new blood vessels can be observed. Immunohistochemistry showed that the proportion of average positive area in the experimental group, control group, blank group were (24.6±5.3) %, (11.3±2.8) % and (7.6±3.8) % respectively. The expression of bone sialoprotein in experimental group were significantly higher than the other 2 groups(P=0.000). Real-time quantitative PCR results showed that the expression level of Runt-related transcription factor 2 (RUNX2), type Ⅰcollagen (COL-Ⅰ), alkaline phosphatase in the experimental group was higher than in the blank group. The expression level of RUNX2 and COL-Ⅰ in the experimental group was higher than that of the control group (P=0.023).@*Conclusions@#TGF-β3 has potential to promote the transformation of DPSC into osteoblasts, which can promote the integration of bone around the implant.
ABSTRACT
To establish the experimental model of rabbit mandibular anterior implant repair and evaluate the effects of transforming growth factor (TGF)-β3 and dental pulp stem cells (DPSC) in promoting the bone integration of implant. The New Zealand rabbits were randomly divided into experimental group, control group and blank group (6 rabbits for each group) . In the experimental group, the implant area was filled with the mixture of TGF-β3, DPSC and Bio-oss powder. In the control group, the implant area was filled with the mixture of DPSC and Bio-oss powder. In the blank group, the implant area was filled with the mixture of phosphate buffer solution and Bio-oss powder. Eighteen New Zealand rabbits were sacrificed in 2 weeks after procedure. The treated alveolar bone tissue was observed. The bone tissue around the implant were estimated by HE staining, immunocytochemical staining and real-time quantitative PCR. The implants were no shedding nor loose. HE staining shows the blank group had a sparse trabecular bone and a small amount of blood vessel around the implant and no obvious new bone formation. The control group showed that the bone trabecula around the implant was sparse and slender, the osteoblasts were arranged linearly around the trabecular bone, a small amount of new bone formation was found around the implant. In the experimental group, there were more thick and dense trabecular bone around the implant, the surrounding osteoblasts were arranged in clusters. The osteoblasts were active and many new bone formed. Typical bone lacunae, bone cells and a large number of new blood vessels can be observed. Immunohistochemistry showed that the proportion of average positive area in the experimental group, control group, blank group were (24.6±5.3) %, (11.3±2.8) % and (7.6±3.8) % respectively. The expression of bone sialoprotein in experimental group were significantly higher than the other 2 groups(0.000). Real-time quantitative PCR results showed that the expression level of Runt-related transcription factor 2 (RUNX2), type Ⅰcollagen (COL-Ⅰ), alkaline phosphatase in the experimental group was higher than in the blank group. The expression level of RUNX2 and COL-Ⅰ in the experimental group was higher than that of the control group (0.023). TGF-β3 has potential to promote the transformation of DPSC into osteoblasts, which can promote the integration of bone around the implant.
Subject(s)
Animals , Rabbits , Bone Substitutes , Therapeutic Uses , Core Binding Factor Alpha 1 Subunit , Dental Implantation, Endosseous , Dental Pulp , Cell Biology , Integrin-Binding Sialoprotein , Metabolism , Mandible , Minerals , Therapeutic Uses , Osseointegration , Osteoblasts , Cell Biology , Random Allocation , Stem Cell Transplantation , Transforming Growth Factor beta , Transforming Growth Factor beta3 , Therapeutic UsesABSTRACT
Objective To investigate the osteogenic differentiation of rabbit dental pulp stem cells (DPSCs) induced by transforming growth factor-β3 (TGF-β3) in vitro. Methods DPSCs and osteoblasts (OBs) were respectively obtained from rabbit dental pulp and skull by enzymetic digestion method. The morphology of the cells was observed by a light microscopy. Immunohistochemical staining and alizarin red staining were carried out to identify OBs. The third generation of DPSCs and OBs were divided into three groups, including DPSCs group (blank control), OBs group (positive control) and DPSCs+TGF-β3 group (experimental group). The expression of Runt-related transcription factor 2 (Runx-2) in each group was detected by immunohistochemical staining on the 5th day of culture. The activity of alkaline phosphatase (ALP) in each group was detected by assay kit on the 7th day of culture. Western Blot was used to detect the expression of the bone-specific markers Runx-2 and TGF-β3 proteins on the 1st, 3rd, 5th and 7th days of culture. Results The rabbit DPSCs were mostly long spindle-shaped with many synapses. The OBs were mostly short spindle-shaped or fibroblast-like, and plump with few synapses. The identification result showed that the DPSCs and OBs were positive. On the 5th day of culture, the expression of Runx-2 protein in the OBs group and DPSCs+TGF-β3 group showed strong positive. On the 7th day of culture, there was no significant difference in ALP activity between the above two groups (P>0.05). The results of Western Blot showed that the relative expressions of Runx-2 and TGF-β3 protein in the DPSCs group were significantly different from those in the other two groups, and the differences were statistically significant (all P<0.05). On the 7th day of culture, the relative expression of Runx-2 and TGF-β3 protein in the DPSCs+TGF-β3 group was higher than that in the other two groups, and the differences were statistically significant (all P<0.01). Conclusions TGF-β3 can promote the expression of early osteogenic specific proteins in DPSCs.
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Objective To establish an animal experimental model of immediately peri-implant bone defects in rabbit mandibular anterior teeth,and to investigate the osteogenetic capability of dental pulp stem cells (DPSCs) on bone defects around implant.Methods Eight rabbits were involved,whose bilateral mandibular anterior teeth were removed to form the bone defects with 2 mm × 3 mm area on the buccal side of tooth extraction sockets,and then the implants were implanted immediately.The rabbits were divided into two groups.The implants were Bio-oss with DPSCs and Bio-oss with phosphate buffer saline respectively for the experimental group and the control group.The bone regeneration in the defect region was evaluated by general observation,hematoxylin-eosin (HE) staining and Goldner trichrome staining and scanning electron microscope (SEM) at 4 weeks after implantation.Results The implantations were successfully conducted,and the implants were stable.There was no significant difference between the implants.The results of the HE staining and the trichrome staining showed that in the experimental group,new bone tissue and partial woven bone formed,and the properties of the newly formed bone cells were large,abundant,well differentiation,and arranging in woven pattern.While in the control group,the new bone-like tissue formation was less,and the mature cell differentiation was ordinary.The SEM results showed that there was a higher osteointegration between the new bone and the implant in the experimental group comparing with the control group.Conclusions The capability of Bio-oss with DPSCs is relatively superior to single Bio-oss in osteogenesis and osseointegration.
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Objective To analyze the acoustic features of plosive consonants in Uyghur as spoken by persons with post-palatoplasty velopharyngeal insufficiency.Methods Thirty-one Uyghur-speaking children without a speech disorder were selected as a control group,while 28 others with velopharyngeal insufficiency (VPI) formed the VPI group.All were recorded speaking 9 Uyghur words which contain the plosive consonants/b/,/d/and/g/.The consonants' duration (CD),amplitude (CA),their first formants (VF1),second formants (VF2),third formants (VF3) and fourth formants (VF4) as voiced as well as the voice onset time (VOT) were measured and analyzed using the PRAAT software for the two groups.Results The VF1,VF3 and VF4 of the plosive consonant /b/ were significantly lower for those in the VPI group than among the controls,and the CD of/b/was significantlylonger.There were also significant differences between the two groups in terms of the VF1s,VF3s and CDs of the other plosive consonants.The VOTs of the consonant/b/,/d/ and/g/ were (0.12±0.05)s,(0.10±0.03)s and (0.09±0.03) s,respectively,with an occurrence rate of up to 100％ in the control group,significantly different from those in the VPI group (P＜0.05).Conclusion The acoustic features of the three plosive consonants/b/,/d/ and /g/ can distinguish Uyghur-speaking children with velopharyngeal insufficiency from those with normal speech.
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Objective@#To investigate the effect of transforming growth factor-β3 (TGF-β3) and dental pulp stem cells (DPSC) in promoting the implant's osteointegration.@*Methods@#Thirty-three New Zealand white rabbits were randomly divided into phosphate buffer saline (PBS) group, DPSC group and TGF-β3 + DPSC group (12 rabbits/group). Two teeth from the rabbits's mandibular incisors or molars were pulled out randomly, then implant were placed in the tooth extraction site immediately. In PBS group, the implant area was filled with Bio-Oss powder 0.30 g mixed by PBS 20 μl only; while the implant area was filled with Bio-oss powder 0.30 g and 1×108/L DPSC 20 μl in DPSC group; in the the TGF-β3+DPSC group the implant area was filled with Bio-Oss powder 0.30 g mixed with 1×108/L DPSC 20 μl and 80 μg/L TGF-β3 20 μl. Eighteen New Zealand rabbits were executed in the 4 weeks and 8 weeks respectively. The treated alveolar bone tissue and implant were collected for plastic section. Alizarin red staining (ARS), immunohistochemical detection (IHC) of bone sialoprotein (BSP), osteocalcin (OC) and type Ⅰ collagen (COL-Ⅰ) were performed after 4 weeks and 8 weeks. Combined bone lamelta width (CBLW) and implant bone contact rate (IBCR), trabecular width (TW) and trabecular area percentage (TA) were observed by histomorphometric measurement.@*Results@#ARS staining: 4 weeks after the operation, the TGF-β3+ DPSC group showed more red calcified nodules than the other two groups; 8 weeks after operation, the red calcified nodule was further increased. 4 weeks after the operation, the expression of BSP, OC and COL-Ⅰ was (0.35± 0.04), (0.36 ± 0.03) and (0.39 ± 0.01) respectively in TGF-β3+ DPSC group, (0.27 ± 0.02), (0.24 ± 0.01) and (0.28±0.03) respectively in DPSC group, and (0.13±0.03), (0.15±0.02) and (0.16±0.02) respectively in PBS group. Eight weeks after operation, the expression of BSP, OC and COL-Ⅰ was (0.51±0.02), (0.49±0.03) and (0.53±0.02) respectively in TGF-β3+DPSC group, (0.35±0.02), (0.37±0.01) and (0.38±0.01) respectively in DPSC group, and (0.21±0.03), (0.19±0.01) and (0.22±0.02) respectively in PBS group. After 4 weeks and 8 weeks, the expression of BSP, OC and COL-Ⅰ in TGF-β3+DPSC group were significantly higher than the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05). Eight weeks after operation, the CBLW, IBCR, TW and TA around implant in TGF-β3+ DPSC group were significantly higher than that in the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05).@*Conclusions@#The DPSC has the potential osteogenic differentiation ability; TGF-β3 can accelerate the osteogenic differentiation of DPSC to some extent; TGF-β3 combined with DPSC can effectively promote the implant's osseointegration.
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<p><b>OBJECTIVE</b>To evaluate the biocompatibility and viability of nonionic triblock copolymer Pluronic F-127 as a cell scaffold for osteogenic differentiation of dental pulp stem cells(DPSC).</p><p><b>METHODS</b>DPSC were obtained via enzymatic digestion method and purified bylimited dilution method. The freeze dried hydrogel of 20% Pluronic F-127 was prepared and itsstructurewas observed usingscanning electron microscopy(SEM). After the encapsulation of cells of passage 3 in Pluronic F-127, the effects of hydrogel on the proliferations of DPSC were assessed with methyl thiazolyl terazolium(MTT) after one day and 3, 5, 7 days of incubations, respectively. On day 14, osteogenic abilities of DPSC encapsulated in the hydrogel were estimated by means of alizarin red S, immunocytochemical staining and real-time quantitative PCR(RT-qPCR).</p><p><b>RESULTS</b>DPSC were isolated and cultured successfully in the present study. SEM observations showed that porous structures which might be suitable for cell culture. A570 values of MTT were then normalized. A570 values of the cells in 2D cultures were 0.30±0.06, 0.30±0.17, 0.35±0.04 and 0.25±0.06 and A570 values of DPSC in 3D cultures were 0.36±0.06, 0.54±0.18, 0.70±0.10 and 0.32±0.10 on day 1, 3, 5 and 7, respectively. A570 value peaks were found on day 5 in both groups. The proliferation of 3D cultured DPSC was higher than that of 2D cultured cells(P<0.05). After 14 days of osteogenic induction, there were no calcium nodules observed in the control group and the numbers of calcium nodulesin the 2D and 3D groups had no significant difference(P>0.05). Inmmunocytochemical staining demonstrated strong expression of osteoblast marker Runt-related transcription factor 2(RUNX2), type Ⅰ collagen(Col-Ⅰ) and relatively low expression of osteocalcin(OCN). Moreover, RT-qPCR showed no differences between the relative expression of ALP, RUNX-2, OCN in the 2D and 3D groups (P>0.05), but a higher relative expression of Col-Ⅰ was observed in the 3D group(P=0.023).</p><p><b>CONCLUSIONS</b>Pluronic F-127 is a promising cell scaffold or cell carrier for the osteobalst differentiation of dental pulp stem cells.</p>