转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响

doi:10.3969/j.issn.2095-4344.2014.28.019

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (28): 4542-4548.doi: 10.3969/j.issn.2095-4344.2014.28.019

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇下一篇

转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响

任飞¹，刘建平²，林锦梅³，周慧⁴，陈晓春¹，徐平平¹，杨勤¹

¹广东省口腔医院，广东省广州市 510260；²中山大学孙逸仙纪念医院，广东省广州市 510120；³广州医科大学医学检验中心，广东省广州市 510015；⁴珠海市口腔医院，广东省珠海市 519000

出版日期:2014-07-02 发布日期:2014-07-02
通讯作者: 刘建平，博士，教授，硕士生导师，中山大学孙逸仙纪念医院，广东省广州市 510120
作者简介:任飞，女，1974年生，贵州省贵阳市人，土家族，2007年南方医科大学毕业，硕士，主任医师，主要从事儿童口腔疾病的临床，科研及教学工作。
基金资助:
广东省科技计划项目(2009B030801253)

Effects of transforming growth factor beta 3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth

Ren Fei¹, Liu Jian-ping², Lin Jin-mei³, Zhou Hui⁴, Chen Xiao-chun¹, Xu Ping-ping¹, Yang Qin¹

¹Guangdong Provincial Stomatological Hospital, Guangzhou 510260, Guangdong Province, China;² Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China; ³Medical Testing Center, Guangzhou Medical University, Guangzhou 510015, Guangdong Province, China; ⁴Zhuhai Municipal Stomatological Hospital, Zhuhai 519000, Guangdong Province, China

Online:2014-07-02 Published:2014-07-02
Contact: Liu Jian-ping, M.D., Professor, Master’s supervisor, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China
About author:Ren Fei, Master, Chief physician, Guangdong Provincial Stomatological Hospital, Guangzhou 510260, Guangdong Province, China
Supported by:
the Guangdong Provincial Science and Technology Project, No. 2009B030801253

摘要/Abstract

摘要：

背景：目前已有研究报道转化生长因子β超家族对各类干细胞成骨矿化的作用，但转化生长因子β3与肝素联合作用后对人乳牙牙髓干细胞的增殖和矿化能力的作用仍有待研究。
目的：观察转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响。
方法：采用酶消化法将人乳牙牙髓分离培养，测定细胞集落克隆形成率，流式细胞术鉴定细胞表面标记物CD146，细胞爬片行Vimentin和STRO1免疫化学染色进行人乳牙牙髓干细胞鉴定；对体外培养的第3代人乳牙牙髓干细胞给予肝素和质量浓度1，5，25 μg/L的转化生长因子β3进行干预。MTS法测细胞生长曲线；茜素红染色；碱性磷酸酶试剂盒检测碱性磷酸酶活性的改变。
结果与结论：实验所得细胞集落克隆形成率高，细胞表面标记物CD146呈阳性，Vimentin和STRO1免疫化学呈强阳性，鉴定获得人乳牙牙髓干细胞。MTS结果显示，给予转化生长因子β3刺激人乳牙牙髓干细胞后并无明显的促增殖的作用。碱性磷酸酶活性检测结果显示，转化生长因子β3-肝素联合作用可随着浓度的增加而增强人乳牙牙髓干细胞的碱性磷酸酶活性，其中质量浓度分别为5，25 μg/L转化生长因子β3和肝素联合作用组与转化生长因子β3单独作用组、肝素单独作用组以及对照组相比显著性升高(P < 0.01)。转化生长因子β3-肝素联合作用组的茜素红染色均呈阳性，其中以5 μg/L转化生长因子β3-肝素联合作用组染色最强。结果证实，转化生长因子β3与肝素联合作用可促进人乳牙牙髓干细胞矿化能力。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

关键词: 干细胞, 培养, 转化生长因子β3, 干细胞因子及调控因子, 人乳牙牙髓干细胞, 肝素, CD146, 克隆形成率, 矿化, 茜素红染色, 碱性磷酸酶, Vimentin蛋白, STRO1蛋白

Abstract:

BACKGROUND: The role of transforming growth factor β superfamily has been reported in bone mineralization of various types of stem cells, but the effects of transforming growth factor β3 (TGF-β3) combined with heparin on proliferation and mineralization of dental pulp stem cells from human deciduous teeth remains to be studied.
OBJECTIVE: To evaluate the effects of TGF-β3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth.
METHODS: The enzyme digestion method was utilized to separately culture dental pulp stem cells from human deciduous teeth. The cell colony forming efficiency was determined. Flow cytometry was utilized to identify cell surface marker CD146. Immunochemistry for Vimentin and STRO1 was performed to measure dental pulp stem cells from human deciduous teeth. The third passage dental pulp stem cells from human deciduous teeth cultured in vitro were intervened with heparin and TGF-β3 of 1, 5, 25 μg/L mass concentration. The MTS method was applied to measure cell growth curves. Alizarin red staining was carried out. The changes in alkaline phosphatase activity were determined with alkaline phosphatase kit.
RESULTS AND CONCLUSION: The cell colony forming efficiency was high. Cells were positive for CD146, and strongly positive for Vimentin and STRO1. Dental pulp stem cells from human deciduous teeth were identified. MTS assay indicated that there was no obvious effect on promoting proliferation of dental pulp stem cells from human deciduous teeth after stimulation of TGF-β3. Detection results of alkaline phosphatase activity demonstrated that the combination of TGF-β3 and heparin could strengthen the alkaline phosphatase activity of dental pulp stem cells from human deciduous teeth with increased concentration. Alkaline phosphatase activities were significantly higher in the TGF-β3 + heparin group, TGF-β3 group and heparin group than in the control group (P < 0.01). Alizarin red staining was positive in the TGF-β3 + heparin group, and the staining was strongest in the 5 μg/L TGF-β3 + heparin group. Results indicated that TGF-β3 combined with heparin promoted mineralization of dental pulp stem cells from human deciduous teeth.

全文链接：

Key words: stem cells, dental pulp, transforming growth factor beta 3, heparin, alkaline phosphatase

中图分类号:

R394.2

任飞，刘建平，林锦梅，周慧，陈晓春，徐平平，杨勤. 转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响[J]. 中国组织工程研究, 2014, 18(28): 4542-4548.

Ren Fei, Liu Jian-ping, Lin Jin-mei, Zhou Hui, Chen Xiao-chun, Xu Ping-ping, Yang Qin. Effects of transforming growth factor beta 3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4542-4548.

图/表（结果） 3

Growth of dental pulp stem cells from human deciduous teeth

Dental pulp stem cells from human deciduous teeth slowly adhered to the wall. Four hours after culture, about 70% cells adhered. Forty-eight hours later, cells were basically confluent. Cells were small in size, exhibited a spindle shape, and had one or two processes. A few of them were irregular. Cells tightly arranged. Some cells with rapid proliferation showed colony-like growth (Figure 1A). Two weeks later, cells reached 90% confluence.

Clone formation of dental pulp stem cells from human deciduous teeth

After 14 days of incubation, the culture was terminated when cell colonies appeared under a microscope. Following crystal violet staining, ELISPOT instrument was employed for detection. The polymer with > 50 cells was considered a cell colony formation. Cells were quantified. Colony forming efficiency was calculated by the number of cell colonies/the number of incubated cells, which concluded that colony forming efficiency was averagely 12 colonies/10³ cells (Figure 1B).

Results of immunocytochemical staining of dental pulp stem cells from human deciduous teeth

Immunocytochemical staining revealed that Vimentin and STRO1 protein was strongly detected in cytoplasm (Figures 1C, D).

Results of phenotype identification of dental pulp stem cells from human deciduous teeth

Flow cytometry revealed that positive rate of CD146 was 91.06% in P2 cells (Figure 2).

Changes in growth curve of dental pulp stem cells from human deciduous teeth

As displayed in Figure 3A, with prolonged time of culture, cell number increased in each group. At 21 days of culture, no significant difference in cell proliferation was detected among control group, transforming growth factor β3 group, heparin group and transforming growth factor β3 + heparin group (P > 0.05).

Results of alizarin red staining in dental pulp stem cells from human deciduous teeth

The third passage dental pulp stem cells from human deciduous teeth cultured in vitro were separately treated with 0, 1, 5, 25 μg/L recombinant transforming growth factor β3, or separately cultured with heparin. At 21 days after culture, alizarin red staining was positive in the transforming growth factor β3 + heparin group, especially in the 5 μg/L transforming growth factor β3 + heparin group. Alizarin red staining was negative in the control, transforming growth factor β3 and heparin groups (Figure 3).

参考文献

[1]Miura M, Gronthos S, zhao M, et al. SHED: stem cells from human exfoliated deci- duous teeth. Cell Biol. 2003;100(10): 5807-5812.
[2]Hansen G, McIntire JJ, Yeung VP, et al. CD4(+)T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and inflammation. J Clin Invest. 2000;105(1):61-70.
[3]Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2 polarization in mucosal immunity. Nat Med. 2002;8(6): 567-573.
[4]Sakai VT, Zhang Z, Dong Z, et al. SHED differentiate into functional odontoblasts and endothelium. J Dent Res. 2010; 89(8):791-796.
[5]Casagrande L, Demarco FF, Zhang Z, et al. Dentin -derived BMP-2 and odontoblast differentiation. J Dent Res. 2010; 89(6):603-608.
[6]Paula-Silva FW, Ghosh A, Silva LA, et al. TNF -alpha promotes an odontoblastic phenotype in dental pulp cells. J Dent Res. 2009;88(4):339-444.
[7]Bluteau G, Luder HU, De Bari C, et al. Stem cells for tooth engineering. Eur Cell Mater. 2008;31(16):1-9.
[8]Liu J, Jin TC, Chang S, et al. Matrix and TGF-beta-related gene expression during human dental pulp stem cell (DPSC) mineralization. In Vitro Cell Dev Biol Anim. 2007;43(3-4):120-128.
[9]Wang SN, Mu JQ, Fan ZP, et al. Insulin-like growth factor 1 can promote the osteogenic differentiation and osteogenesis of stem cells from apical papilla. Stem Cell Res. 2012;8(3):346-356.
[10]Yu Y, Mu JQ, Fan ZP, et al. Insulin-like growth factor 1 enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells via ERK and JNK MAPK pathways. Histochem Cell Biol. 2012;137(4): 513-525.
[11]He HX, Jin Y, Shi JN, et al. Effects of bFGF, TGF- β on proliferation and differentiation of human dental pulp cells the ability. 2004;14(7):359-363.
[12]Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature. 2001;414:98-104.
[13]Karaoz E, Dogan BN, Aksov A, et al. Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol. 2010;133(1):95-112.
[14]Sakai VT, Zhang Z, Dong Z, et al. SHED differentiate into functional odontoblasts and endothelium. J Dent Res. 2010; 89(8):791-796.
[15]Casagrande L, Demarco FF, Zhang Z, et al. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res. 2010; 89(6):603-608.
[16]Chadioiralla K, Yochim JM, Bahuleyan B, et al. Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth. Cell Tissue Res. 2012;340(2):323-333.
[17]Wang J, Wang X, Sun Z, et al. Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells. Stem Cells Dev. 2010; 19(9): 1375-1383.
[18]Morsczeck C, Vollner F, Saugspier M, et al. Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro. Clin Oral Investig. 2010;14(4): 433-440.
[19]Gage FH. Mammalian neural stem cells. Science. 2000;287: 1433-1438.
[20]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71-74.
[21]Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science. 2000;287: 1442-1446.
[22]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-147.
[23]Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol. 2001;3:778-784.
[24]Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA. 2000;97:13625-13630.
[25]Feng MW. Kouqiang Shengwuxue. Beijing: People's Medical Publishing House. 2004
[26]Iohara K, Nakashima M, Ito M, et al. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. Dent Res. 2004;83:590-595.
[27]Bonewald LF, Mundy GR. Role of transforming growth factor beta in bone remodeling. Clin Orthop. 1990;250: 261-276.
[28]Feng YH, Diao ZH, Gao Y, et al. Ultrastructure changes of dog dental pulp cells transfected with bone morphogenetic proteins 2 in vitro. Zhongguo Zuzhi Gongcheng Yanjiu. 2012; 12(6):206-210.
[29]Yand X, Han G, Pang X, et al. Chitosan/collagen scaffold containing bone morphogenetic protein-7 Dna supports dental pulp stem cell differentiation in vitro and in vivo. J Biomed Mater Res A. 2012.
[30]Ding SS, Cui YL. heparin in the application of growth factors in the controlled release. Huaxue Jinzhan. 2008; 12(20):1998-2011.
[31]Rider CC. In Heparin/heparan sulphate binding in the TGF-beta cytokine superfamily. Biochem Soc Trans. 2006; 34(3):458-460.
[32]Lyon M, Rushton G, Gallagher JT. The Interaction of the Transforming Growth Factor- s with Heparin/Heparan Sulfate Is Isoform-specific. J Biol Chem. 1997;272(29): 18000-18006.

引言

Dental pulp stem cells from human deciduous teeth have been major seed cells for tissue engineering, have been extensively utilized in tissue engineering and treatment of oral disease. Miura et al ^[1]firstly cultured multipotential stem cells in dental pulp of caduceus human deciduous teeth in 2003, which was named as dental pulp stem cells from human deciduous teeth. They reported that dental pulp stem cells from human deciduous teeth have the potential of differentiating into osteoblasts, lipoblasts and nerve cells. Compared with dental pulp stem cells from permanent teeth, dental pulp stem cells from human deciduous teeth have higher proliferation rate. Dental pulp stem cells from human deciduous teeth implanted in nude mouse could induce the differentiation of host mouse cells into osteoblasts, forming new bones. Another advantage of this kind of stem cells is that it can be obtained from human discarded tissue, which is convenient and does not injure the body. Therefore, due to its differentiation potential and high proliferation ability, dental pulp stem cells from human deciduous teeth have been a hot focus in the field of regenerative medicine. Some scholars have verified that dental pulp stem cells from human deciduous teeth could differentiate into odontoblasts under multiple conditions^[2-6].

A previous study demonstrated that dental pulp stem cells from human deciduous teeth could apparently promote reparative dentin formation in vivo ^[7]. Numerous studies indicated that transforming growth factor β1 is a cytokine playing a key role in dental development^[8-10]. He et al^[11]confirmed that the effects of transforming growth factor β alone did not obviously positively associate with proliferative effect and concentration of dental pulp stem cells from permanent teeth, but contributed to the differentiation of dental pulp stem cells of permanent teeth.

This study explored the effects of transforming growth factor β3 on mineralization of dental pulp stem cells from human deciduous teeth, and provided experimental evidence for exploring the effects of dental pulp stem cells from human deciduous teeth on dental regeneration.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
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材料方法

Design

Cytological in vitro experiment.

Time and setting

Experiments were performed in the laboratory of Guangdong Provincial Stomatological Hospital in China from January 2011 to February 2014.

Materials

Deciduous teeth were obtained from coronal pulp of deciduous central incisor of children aged 7 or 8 years in the out-patient clinic of Guangdong Provincial Stomatological Hospital in China.

Experimental methods

Isolation and culture of dental pulp stem cells from human deciduous teeth

Healthy deciduous central incisor was collected in out-patient clinic and stored in α-MEM medium containing double antibodies. Primary cells were collected within 4 hours. Method of collecting primary cells: mass concentration 4 g/L protease was mixed with 3 g/L type I collagenase at 1:1, and the mixture was incubated in a water bath at 37 ℃ for 1 hour. 50 μL 10 g/L DNAse digestive juice was added for 30 seconds^[1]. In a 15 mL tube, tissues were washed with 10 mL Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Cell suspension was filtered with 200-mesh screen. Blocks were discarded. Cell suspension was seeded in gelatin-coated petri dish and incubated in a 5% CO₂ incubator at 37 ℃. After adherence, PBS was replaced by α-MEM + 10% fetal bovine serum for further culture. Cell growth was observed and photographed under a common microscope day by day.

Colony formation test

Cells were diluted into 100/well, and incubated for 14 days. The medium was removed from each well. Cells were washed twice with PBS or physiological saline. 200 μL of crystal violet was added in each well. The staining solution should fully cover the bottom of the well. Twenty minutes later, the 96-well plate was slowly washed with running water. After open-air drying, the number of colony was calculated.

Immunocytochemical staining

The third passage dental pulp stem cells from human deciduous teeth were seeded in 0.5% gelatin-coated slide at a concentration of 1×10⁷/L. Under 60%-80% cell concentration, the samples were fixed with 40 g/L paraformaldehyde for 15 minutes. In accordance with instructions, immunochemical staining was performed using STRO-1 antibody (1:300) and Vimentin antibody (1:200). After incubation with primary antibody at 4 ℃overnight, the samples were washed with PBS

5 minutes×3. The samples were then incubated with secondary antibody (1:40) for 30 minutes, developed with 3,3'-diaminobenzidine, counterstained with hematoxylin, and mounted with neutral resin.

Identification with flow cytometry

The third passage dental pulp stem cells from human deciduous teeth were incubated in a 6-well plate. When cells reached 80% confluence, cell suspension was obtained by digesting with trypsin. Cells were quantified. Cell concentration was adjusted to 1×10⁴/L. Experimental group was treated with 1 μL CD146-FITC. Negative control group was treated with 0.5 μL IgG1K-FITC. Blank control group was not treated with any antibody. All samples were incubated in the dark at room temperature for 20 minutes. 500 μL PBS was added in each tube for once wash, followed by centrifugation at 1 200 r/min for

5 minutes. 100 μL IC Fixation buffer was added, stirring and mixing, in the dark at room temperature for 20 minutes. Cells were resuspended with 0.2 mL PBS. Flow cytometry was used to detect positive rate of CD146.

Detection of cell growth curve

The third passage dental pulp stem cells from human deciduous teeth in the logarithmic phase were seeded in a 96-well plate at the concentration of 1×10⁷/L. Twenty-four 4 hours later, control group was normally cultured. Transforming growth factor β3 group was treated with 1, 5, 25 μg/L transforming growth factor β3. Heparin group was treated with 10 U/mL heparin. Transforming growth factor β3 + heparin groups were separately treated with 10 U/mL heparin and 1, 5, 25 μg/L transforming growth factor β3. Cells after cultured for 0, 7, 14 and 21 days were collected for detection. cellTiter96AQ single-cell proliferation assay solution was added at a proportion of 1/10. After 4 hours of incubation, absorbance values were measured at 490 nm with a microplate reader. Growth curves were drawn.

Alizarin red staining

Cells were treated 21 days. The petri dish was washed twice with PBS. The samples were fixed with 40 g/L formalin for 10 minutes, washed with distilled water three times. Cells were covered with 0.1% alizarin red-Tris-HCL (pH 8.3) solution for 30 minutes at 37 ℃ Cells were then washed with distilled water, dried, and photographed.

Quantitative detection of dental pulp stem cells from human deciduous teeth with alkaline phosphatase

The third passage dental pulp stem cells from human deciduous teeth were incubated in a 6-well plate. After adherence, cells were treated according to different groups. Nine days later, cells were washed twice with PBS, lysed with an equal volume of cell lysate, agitated with a pipette until clarification. In accordance with the instruction of alkaline phosphatase kit, alkaline phosphatase levels in cell lysate were analyzed.

Main outcome measures

After cells were processed in accordance with experimental groups, we measured (1) cell growth curve; (2) cell colony forming efficiency; (3) cell surface marker CD146 expression; (4) Vimentin protein and STRO1 protein expression; (5) cell mineralization.

Statistical analysis

Measurement data were expressed as mean ± SD, and analyzed with SPSS 19.0 software. Data of different groups were compared with one-way analysis of variance and Student-Newman-Keuls’ test. A value of P < 0.01 was considered statistically significant.

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讨论

Numerous studies confirmed that dental pulp stem cells from human deciduous teeth, similar to human dental pulp stem cells, could be induced to differentiate into nerve cells, adipocytes and odontoblasts^[12-24]. Finally, new bones and dentin were produced. Compared with human dental pulp stem cells and bone marrow stromal cells, dental pulp stem cells from human deciduous teeth have high proliferation rate and multiple differentiation ability. More importantly, deciduous teeth are derived from autologous tissue, no immune rejection, have high safety. Moreover, source is extensive. Study process did not involve in ethical constraints. Therefore, dental pulp stem cells from human deciduous teeth will be ideal source for repair of odontal tissue defects.

Stem cell differentiation can be regulated by a series of signaling molecules. Of them, transforming growth factor β superfamily plays an important role. Transforming growth factor β includes transforming growth factor β family (transforming growth factor βs) and can regulate cell growth and differentiation, and determines the formation of odontoblasts and osteoblasts of the body in embryonic stage^[25]. Transforming growth factor βs has three isomers. Of them, transforming growth factor β3 is mainly expressed in cells derived from mesenchyme. In situ hybridization suggested that transforming growth factor β1, 3 expression could be detected in mesenchyme during the bud and cap stages and in dental papillar cells. Transforming growth factor β3 specifically binds to receptors on the surface of cell membrane transforming growth factor βR III (CD105), and produces major biological function. Previous studies confirmed that transforming growth factor β and bone morphogenetic protein 2 stimulated the proliferation of human dental pulp stem cells cultured in vitro^[26-27]. In addition, transforming growth factor β1 and bone morphogenetic protein 2 and 7 could induce the differentiation of dental papilla odontoblasts^[28-29]. These data indicated that other members of transforming growth factor β superfamily not only has induced effects on the differentiation of human dental pulp stem cells, but also on dental pulp stem cells from human deciduous teeth with more strong differentiation ability and more immaturity.

In accordance with a previous method of Gronthos and clone colony forming ability^[1], dental pulp stem cells from human deciduous teeth derived from monoclonal colony were isolated and purified by limiting dilution assay. Dental pulp stem cells from human deciduous teeth were identified with specific antigen STRO-1 and CD146, vimentin, as well as colony forming efficiency.

MTS test results displayed that during 21 days of culture, the number of cells in each treatment group increased to different degrees. Interestingly, growth curves in the transforming growth factor β3 group and control group experience a logarithmic growth stage from 7 to 14 days, and entered the platform stage at 21 days. However, cells in the heparin group and transforming growth factor β3 + heparin group did not exhibit a rapid growth stage from

7 days, but grew at a growth speed below non-heparin group. Nevertheless, at 21 days, cell number in the heparin-containing groups was at a level of non-heparin group, and the growth speed did not reach a platform stage. Therefore, this study confirmed that transforming growth factor β3 of different concentrations did not obviously affect the proliferation of dental pulp stem cells from human deciduous teeth. Transforming growth factor β3 combined with heparin promoted the proliferation of dental pulp stem cells from human deciduous teeth to a certain extent.

Detection results of alkaline phosphatase activities suggested that compared with other groups, transforming growth factor β3 combined with heparin could contribute to alkaline phosphatase activities in dental pulp stem cells from human deciduous teeth. Moreover, with increased concentration of transforming growth factor β3, the promoting effects on alkaline phosphatase activities also enhanced. Alkaline phosphatase involves in the formation of sclerous tissues, promotes calcification, and plays a key effect on the formation, metabolism and regeneration of tooth and bones. Alizarin red staining results demonstrated that transforming growth factor β3 combined with heparin formed many big calcified nodules, which suggested that transforming growth factor β3 combined with heparin could contribute to the differentiation of dental pulp stem cells from human deciduous teeth, and exerted the ability of inducing cell mineralization.

Experiments found that transforming growth factor β3 alone could not affect the proliferation and differentiation of dental pulp stem cells from human deciduous teeth. However, when heparin was added, positive results were found. Studies concluded that heparin exerted a crucial effect on regulation of various biological signals^[30]. A previous study verified that heparin in intracellular matrix could provide many binding sites for transforming growth factor β superfamily^[31]. Heparin could prevent transforming growth factor βs hydrolysis by protease or chemical inactivation, effectively protected its activities, and stabilized molecular conformation^[32]. Thus, in this study, the promoting effects of transforming growth factor β3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth should be attributed to heparin and its compound, which can maintain its activities, lessen the release of growth factors, prevent overdose-induced negative effect.

In conclusion, in vitro cultured dental pulp stem cells from human deciduous teeth induced using transforming growth factor β3 and heparin in this study is a very simple model, but a complicated mechanism among growth factors may exist in vivo. Experimental results indicated that growth factors could be used to supplement the adverse reactions induced by scaffold materials in the future study of tissue engineering, which provides a laboratory evidence for the directed differentiation of dental pulp stem cells from human deciduous teeth into odontoblasts and pulpodentinal complex.

转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响

Effects of transforming growth factor beta 3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth

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