Effect of osteogenic growth peptide on proliferation and mineralization of human dental pulp cells in vitro

doi:10.3877/cma.j.issn.1674-1366.2011.03.004

Abstract

Abstract:

Objective

To investigate the effect of osteogenic growth peptide on proliferation and mineralization of human dental pulp cells （hDPCs） in vitro.

Methods

The hDPCs were cultured in vitro with tissue-explants enzymatic digestion method.The morphological characters of the cells were observed by inverted microscope.We selected the best concentration of OGP beneficial to the proliferation and ALP activity of cultured hDPCs by using CCK-8 method and alkaline phosphatase activity test； Alizarin bordeaux and Von Kossa staining methods were used to observe the minera-lization nodules.RT-PCR test was used to detect relative RNA level of CollagenⅠ， DSPP and OCN.

Results

Tissue-explants enzymatic digestion method could cultivate hDPCs well in vitro； the maximum response was obtained with 10 ^-10 mol/L and 10^-9 mol/L by using CCK-8 method.However， the optimal concentration was 10 ^-9 mol/L by using alkaline phosphatase activity test.Alizarin bordeaux and Von Kossa staining cells cultured for 14 and 21 d，we discovered mineralization nodules in each group， but in the join group the nodules were more numerouse and larger.The results of RT-PCR test showed that mRNA level of CollagenⅠ， DSPP and OCN was higher in the join group.

Conclusions

OGP could promote the proliferation and mineralization of hDPCs in vitro， and the optimal concentration was 10^-9 mol/L when considering the two aspects.Alizarin bordeaux and Von Kossa staining methods could observe the mineralization nodules dramaticlly.Mineralization induced liquid containing OGP group could markedly improve the mineralization of hDPCs in vitro.

Key words: Osteogenic growth peptide, Human dental pulp cells, Proliferation, Mineralization

Tao-hong LV, Yan GAO, Shao-feng AN, Pei-lin ZHUANG, Guo-rong YE. Effect of osteogenic growth peptide on proliferation and mineralization of human dental pulp cells in vitro[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2011, 5(03): 252-262.

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Figures/Tables 14

References 20

1	Bab I， Gazit D， Chorev M， et al.Histone H4-related osteogenic growth peptide （OGP）： a novel circulatingstimulator of osteoblastic activity.EMBO J， 1992，11（5）：1867-1873.
2	Fazzi R， Galimberti S， Testi R， et al.Bone and bone marrow interactions： hematological activity of osteoblastic growth peptide （OGP）-derived carboxy-terminal pentapeptide.Ⅱ.Action on human hematopoietic stem cells.Leuk Res， 2002，26（9）：839-848.
3	Fazzi R， Testi R， Trasciatti S， et al.Bone and bone-marrow interactions： haematological activity of osteoblastic growth peptide（OGP）-derived carboxy-terminal pentapeptide.Mobilizing properties on whi-te blood cells and peripheral blood stem cells in mice.Leuk Res，2002，26（1）：19-27.
4	Nakao K， Itoh M， Tomita Y， et al.FGF-2 potently induces both proliferation and DSP expression in collagen type Ⅰgel cultures of adult incisor immature pulp cells.Biochem Biophys Res Commun， 2004，325（3）：1052-1059.
5	Huojia M， Muraoka N， Yoshizaki K， et al.TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development.Dev Growth Differ， 2005，47（3）：141-152.
6	Reichenmiller KM， Mattern C， Ranke MB， et al.IGFs， IGFBPs， IGF-binding sites and biochemical markers of bone metabolism during differentiation in human pulp fibroblasts.Horm Res， 2004，62（1）：33-39.
7	Greenberg Z， Gavish H， Muhlrad A， et al.Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins.J Cell Biochem， 1997，65（3）：359-367.
8	Gabarin N， Gavish H， Muhlrad A， et al.Mitogenic G （i） protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells： activation by C-terminal pentapeptide of osteogenic growth peptide ［OGP （10-14）］ and attenuation of activation by cAMP.J Cell Biochem， 2001，81（4）：594-603.
9	Robinson D， Bab I， Nevo Z.Osteogenic growth peptide regulates proliferation and osteogenic maturation of human and rabbit bone marrow st romal cells.J Bone Miner Res， 1995，10（5）：690-696.
10	徐凌，黄姣，向学熔.成骨生长肽对人牙周膜细胞增殖和碱性磷酸酶活性的影响.中国组织工程研究与临床康复， 2009，13（14）：4627-4631.
11	Fei Q， Guo C， Xu X， et al.Osteogenic growth peptide enhances the proliferation of bone marrow mesenchymal stem cells from osteoprotegerin-deficient mice by CDK2/cyclin A.Acta Biochim Biophys Sin （Shanghai）， 2010，42（11）：801-806.
12	肖毅，王建国，白增亮.成骨生长肽对大鼠骨髓基质细胞增殖和成骨分化的作用.中国骨伤， 2008，21（11）：843-845.
13	徐杨，陈统一，林飞跃，等.合成成骨生长肽对人骨髓间充质干细胞成骨转化的促进.中国组织工程研究与临床康复， 2009，13（6）：1053-1058.
14	周雯雯，王璐，徐凌.成骨生长肽对钛表面人牙周膜成纤维细胞增殖分化影响的研究.重庆医科大学学报， 2010，35（6）：873-876.
15	Brager MA， Patterson MJ， Connolly JF， et al.Osteogenic growth peptide normally stimulated by blood loss and marrow ablation has local and systemic effects on fracture healing in rats.J Orthop Res， 2000，18（1）：133-139.
16	Vanella L， Kim DH， Asprinio D， et al.HO-1 expression increases mesenchymal stem cell-derived osteoblasts but decreases adipocyte lineage.Bone， 2010，46（1）：236-243.
17	Shuqiang M， Kunzheng W， Xiaoqiang D， et al.Osteogenic growth peptide incorporated into PLGA scaffolds accelerates healing of segmental long bone defects in rabbits.J Plast Reconstr Aesthet Surg， 2008，61（12）：1558-1560.
18	Moore NM， Lin NJ， Gallant ND， et al.The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation.Biomaterials， 2010，31（7）：1604-1611.
19	Fazzi R， Galimberti S， Pacini S， et al.Bone and bone marrow interactions：hematological activity of osteo blastic growth peptide （OGP）-derived carboxy-terminal pentapeptide Ⅲ.Action on human megakaryocytopoiesis：focus on essential thrombocythemia.Leuk Res， 2004，28（10）：1097-1105.
20	Liao H， Andersson AS， Sutherland D， et al.Response of rat osteoblast-like cells to microstructured model surfaces in vitro.Biomaterials，2003，24（4）：649-654.

基因	引物序列
CollagenⅠ	Left Primer：5'-AAGAGGAAGGCCAAGTCGAG-3'
	Right Primer：5'-CACACGTCTCGGTCATGGTA-3'
DSPP	Left Primer：5'-GCCACTTTCAGTCTTCAAAGAGA-3'
	Right Primer：5'-GCCCAAATGCAAAAATATGTAA-3'
OCN	Left Primer：5'-CTCACACTCCTCGCCCTATT-3'
	Right Primer：5'-TTGGACACAAAGGCTGCAC-3'
GAPDH	Left Primer：5'-AAGGTGAAGGTCGGAGTCAA-3'
	Right Primer：5'-AAGGTGAAGGTCGGAGTCAA-3'

基因	引物序列
CollagenⅠ	Left Primer：5'-AAGAGGAAGGCCAAGTCGAG-3'
	Right Primer：5'-CACACGTCTCGGTCATGGTA-3'
DSPP	Left Primer：5'-GCCACTTTCAGTCTTCAAAGAGA-3'
	Right Primer：5'-GCCCAAATGCAAAAATATGTAA-3'
OCN	Left Primer：5'-CTCACACTCCTCGCCCTATT-3'
	Right Primer：5'-TTGGACACAAAGGCTGCAC-3'
GAPDH	Left Primer：5'-AAGGTGAAGGTCGGAGTCAA-3'
	Right Primer：5'-AAGGTGAAGGTCGGAGTCAA-3'

OGP 浓度（mol/L）	A 值
OGP 浓度（mol/L）	1 d	2 d	3 d	5 d
0	0.2485 ± 0.0518	0.3992 ± 0.0054	0.4606 ± 0.0067	0.5822 ± 0.0485
10^-7	0.2575 ± 0.0517	0.4177 ± 0.0056^a	0.4751 ± 0.0118^a	0.6178 ± 0.0197^a
10^-8	0.3241 ± 0.0542	0.4229 ± 0.0084^b	0.4804 ± 0.0105^b	0.6297 ± 0.0200^b
10^-9	0.4031 ± 0.0365^a	0.4412 ± 0.0036^b	0.5091 ± 0.0167^b	0.6465 ± 0.0126^b
10^-10	0.4057 ± 0.0530^b	0.4950 ± 0.0090^b	0.5187 ± 0.0114^b	0.6756 ± 0.0324^b
10^-11	0.3888 ± 0.0321^a	0.4318 ± 0.0060^b	0.4824 ± 0.0142^b	0.6401 ± 0.0153^b

OGP 浓度（mol/L）	A 值
OGP 浓度（mol/L）	1 d	2 d	3 d	5 d
0	0.2485 ± 0.0518	0.3992 ± 0.0054	0.4606 ± 0.0067	0.5822 ± 0.0485
10^-7	0.2575 ± 0.0517	0.4177 ± 0.0056^a	0.4751 ± 0.0118^a	0.6178 ± 0.0197^a
10^-8	0.3241 ± 0.0542	0.4229 ± 0.0084^b	0.4804 ± 0.0105^b	0.6297 ± 0.0200^b
10^-9	0.4031 ± 0.0365^a	0.4412 ± 0.0036^b	0.5091 ± 0.0167^b	0.6465 ± 0.0126^b
10^-10	0.4057 ± 0.0530^b	0.4950 ± 0.0090^b	0.5187 ± 0.0114^b	0.6756 ± 0.0324^b
10^-11	0.3888 ± 0.0321^a	0.4318 ± 0.0060^b	0.4824 ± 0.0142^b	0.6401 ± 0.0153^b

OGP 浓度（mol/L）	A 值
OGP 浓度（mol/L）	1 d	3 d	5 d	7 d	10 d
0	0.1500 ± 0.0041	0.1599 ± 0.0045	0.1612 ± 0.0047	0.2046 ± 0.0026	0.6285 ± 0.0666
10^-7	0.1577 ± 0.0061^a	0.1603 ± 0.0075	0.1672 ± 0.0027	0.2291 ± 0.0173^a	0.8494 ± 0.1465^a
10^-8	0.1582 ± 0.0038^a	0.1677 ± 0.0055^a	0.1701 ± 0.0060^a	0.2476 ± 0.0143^b	0.8767 ± 0.1748^b
10^-9	0.1589 ± 0.0054^b	0.1694 ± 0.0055^b	0.1773 ± 0.0054^b	0.2661 ± 0.0112^b	0.9318 ± 0.1554^b
10^-10	0.1550 ± 0.0034	0.1634 ± 0.0030	0.1684 ± 0.0079^a	0.2382 ± 0.0135^b	0.8653 ± 0.1566^b
10^-11	0.1523 ± 0.0049	0.1629 ± 0.0029	0.1669 ± 0.0034	0.2361 ± 0.0208^b	0.8468 ± 0.1179^a

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