jan. 2007, volume 4, no.1 (serial no.26)

8/14/2019 Jan. 2007, Volume 4, No.1 (Serial No.26)

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Jan. 2007, Volume 4, No.1 (Serial No.26) Journal of US-China Medical Science , ISSN1548-6648, US A

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The Effects of Human Peripheral Blood Mononuclear Cells

on the Proliferation and Differentiation of Human

Periodontal Ligament Fibroblast Cells

WANG Yan1,2

, DONG Rui3, LIANG Li

1, LI Yong-ming

1, HUANG Jian

4, WANG Wen-qing

5, DING Yin

1

(1. Department of Orthodontics, College of Stomatology, the Fourth Military Medical University, Xian 710032;

2. Department of Stomatology of Tangdu Hospital, the Fourth Military Medical University, Xian 710038;

3. Department of Pathology, College of Stomatology, the Fourth Military Medical University,Xian 710032;

4. Department of Social Science, the Fourth Military Medical University,Xian 710032;

5. Department of Hematology of Xijing Hospital, the Fourth Military Medical University,

Xian 710032)

Abstract: Objective In order to illuminate the potential effect of periodontal vascular system on the

remodeling of the periodontal tissues, the author investigated the effect of human peripheral blood mononuclear

cells (PBMCs) on human periodontal ligament cells (PDL cells). Methods The co-culture system of PDL cells

and human PBMCs separated by transwell was established. PDL cells proliferation was determined by direct cell

counting and Proliferating cell nuclear antigen (PCNA) labeling index (LI). The alkaline phosphatase (ALPase)

activity of PDL cells was evaluated by enzyme kinetics methods after 1, 3, 5, 7 days. Results After co-culture, the

number of PDL cells was 4.5 104

after 3 days and 8.5 104

after 5 days. Compared with the control group, the

difference between the two groups and control group was significant (p


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The Effects of Human Peripheral Blood Mononuclear Cells on the Proliferation and Differentiation of Human PeriodontalLigament Fibroblast Cells

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only for the formation and maintenance of the PDL but also for the repair, remodelling and regeneration of the

adjacent alveolar bone and cementum[3]

PDL fibroblasts also exhibit characteristics of osteoblasts. These

fibroblasts show high basal alkaline phosphatase (ALPase) activity[4]

, which is important during hard tissue

mineralization and might be related to the mechanisms of alveolar bone or cementum mineralization. Moreover,

ALPase positive cells in the bone marrow have been reported to be associated with osteoclastogenesis[5]

.

The periodontal vascular system changes when orthodontic therapeutic mechanical stress is applied to the

teeth[6-9]

. The changes in the microvasculature related to the reorganization of periodontal tissue during tooth

movement were observed to evaluate the role of periodontal vasculature. Two main experimental methods have

been applied to test the response of the periodontal blood flow to strain/stress changes in the periodontal tissue:

perfusion with fluorescent microspheres[10, 11]

and laser-Doppler flowmetry[12-14]

. They considered that blood

vessels responded rapidly to the environmental changes in the periodontal membrane, adjusting their morphology

to functional changes. These vascular changes preceded the deposition or resorption of bone. On the tension side,

vascularization occurred at the sites of bone formation and fibroblast proliferation. While on the pressure side,

vascularization caused proliferation of osteoblasts and fibroblasts, resulting in regeneration of the PDL. Thesevascular changes seemed to contribute to the regeneration of the periodontal membrane

[11]. The results showed

that periodontal vascular system plays an important role in the reconstruction of periodontal tissues during

orthodontic tooth movement. But, the mechanism how the vascular system participates in the orthodontic tooth

movement is not yet clear.

Histologically, the periodontal vascular system changes with the application of orthodontic therapeutic

mechanical stress to the tooth. Some researches showed that vascular permeability increases in both the pressure

and tension sides following the application of orthodontic force. Infiltrating blood cells, lymphocytes and

monocytes, may affect the cell function in the oral microenvironment in vivo. Therefore, it is necessary to

examine the interaction between PDL cells and PBMCs.

In order to illuminate the potential effect of periodontal vascular system on the remodeling of the periodontal

tissues, we established a co-culture system of PDL cells and human PBMCs separated by transwell and then

analyzed the effect of human peripheral blood mononuclear cells on the proliferation and differentiation of human

periodontal ligament fibroblast cells. ALPase activity was used as a marker of PDL phenotypic differentiation.

MATERIALS AND METHODS

1. Samples

Periodontal ligament samples and peripheral blood samples were obtained with informed consent.

Experiments were performed using PDL cells and PBMCs from three and two different donors, respectively.

2. Primary Culture of Human Periodontal Ligament Fibroblast-like Cells (PDL Cells)

Pieces of periodontal ligament were obtained only from the middle of tooth roots extracted for orthodontic

reasons to exclude the intermixture of gingivae and dental pulp. They were then cultured in alpha-modified

minimal essential medium (a-MEM) (GibcoBRL, USA)containing 20%(v/v) fetal bovine serum (FBS) (Hyclone,

USA) and five-fold-reinforced antibiotics (500U/ml penicillin, 500 g/mlstreptomycin) in 35-mm primary culture

dishes. Cells that grew out from the extracts were passaged in 10% FBS a-MEM supplemented with antibiotics

(100U/ml penicillin, 100 g/ml streptomycin). PDL cells were used in these experiments at passage four to six.

3. Preparation of Peripheral Blood Mononuclear Cells (PBMCs)


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Heparin-treated human peripheral blood was obtained from healthy adult donors. The cells were collected by

gradient centrifugation (1.077g/ml, 3000rpm, 4, 30min) and then resuspended in 10% FBS a-MEM

supplemented with antibiotics (100U/ml penicillin, 100 g/mlstreptomycin).

4. Co-culture of PBMCs and PDL Cells Separated by a Membrane Filter[15]

PDL cells were pre-cultured in 24-well plates at a density of 1104cells/well for 24h until confluent, then

Millicell culture plate well inserts(Millipore Products Division, USA) seeded with PBMCs (2 105cells/well) were

inserted. This method allowed co-culture of PBMCs and PDL cells without the two cell types coming into contact,

but with interaction of soluble factors produced by the cells.

5. PDL Cells Proliferation Assay

PDL cells proliferation was determined by direct cell counting and proliferating cell nuclear antigen (PCNA)

labeling index (LI). PDL cells were cultured in 24-well plates at a density of 1 104cells/well for 24h until

confluent, then co-culture of PBMCs and PDL cells separated by a membrane filter for 1, 3, 5, 7 days.PDL cells

were trypsinized and cell numbers were determined by trypan blue dye exclusion method using hemocytometer.

Then the growth cure was done.PCNA labeling index (LI) was detected by immunostaining using mouse anti-PCNA monoclonal antibody

(Antibody Diagnostica Inc., USA). The cultured PDL cells which co-culture with PBMCs for 1,3,5,7 days were

fixed with methanol and acetone (3:1) and treated with 3% H2O2 to quench endogenous peroxidase. Cells were

incubated with blocking buffer (PBS containing 10% neonate bovine serum) and then incubated overnight at 4 C

with a 1:100 dilution ofmouse anti-PCNA monoclonal antibody. The negative control was prepared in an identical

manner except that the primary antibody was replaced with normal serum. Biotin-goat anti-mouse IgG (Sigma,

USA) was used as secondary antibody. Nuclei that were brown to black were counted as positive cells. The

number of PCNA positive cells was counted in 30 random high-power fields. Data were expressed as the

percentage of PCNA positive cells per total number of cells.

6. Measurement of Alkaline Phosphatase (ALPase) Activity

PDL cells were rinsed twice with PBS after removal of the culture media, and then 1ml of 10mM Tris-HCl

buffer, pH 8.5, was added. The cell layer was scraped off the plates and homogenized by sonication three times on

ice (10s each time). ALP activity was quantified by colorimetric assay using ALP kit (Sigma). Briefly, 150 l of

substrate mixture consisting of 0.2% (w/v) or 5mM para-nitrophenyl phosphate disodium in 1M diethanolamine

HCl at pH 9.83 was added to 50 l of each thawed lysate in 96-well plates. The plates were incubated at 37C for

15min and the reaction terminated by adding 200 l of 2M NaOH, 0.2mM EDTA. ALP activity was measured by

the absorbance at 490nm (A490) using lysate buffer as blank. A standard curve was prepared using known

concentrations of para-nitrophenol and NaOH/EDTA as blank. The International Units (U/l) of enzyme activity in

experimental samples was calculated from standard curve of Sigma units.7. Statistical Analysis

All values are expressed as meanSD. Statistical analysis of the results was performed using the Student's

paired ttest. A p value of less than 0.05 was considered statistically significant.

RESULTS

1. Human PBMCs Induced the Proliferation of PDL Cells

After co-culture, the number of PDL cells were 4.5 104

after 3 days and 8.5104

after 5 days .Compared


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with the control group, the difference between the two groups and control group was significant (p


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PBMCs on ALP activity in PDL cells, PBMCs were prepared using the Ficoll-Isopaque centrifugation method and

then cultured indirectly with PDL cells. A Millipore membrane with a pore size of 0.45mm was inserted between

the PDL cells and PBMCs to prevent direct contact between the two cell types, while enabling the free diffusion

of soluble factors produced by the cells. This experiment was performed using PDL cells and PBMCs derived

from three and two different donors, respectively. Representative results are shown. ALPase activity in PDL cells

was reduced by co-culture with PBMCs. The suppression of ALPase activity was detected after 3, 5, 7 days in

culture (Fig. 3).

DISCUSSION

The periodontal vascular system changes when orthodontic therapeutic mechanical stress is applied to the

teeth. The changes in the microvasculature related to the reorganization of periodontal tissue during tooth

movement were observed to evaluate the role of periodontal vasculature. Two main experimental methods have

been applied to test the response of the periodontal blood flow to strain/stress changes in the periodontal tissue:

perfusion with fluorescent microspheres [10, 11] and laser-Doppler flowmetry[12-14]. They considered that blood

vessels responded rapidly to the environmental changes in the periodontal membrane, adjusting their morphology

to functional changes. These vascular changes preceded the deposition or resorption of bone. On the tension side,

vascularization occurred at the sites of bone formation and fibroblast proliferation. While on the pressure side,

vascularization caused proliferation of osteoblasts and fibroblasts, resulting in regeneration of the PDL. These

vascular changes seemed to contribute to the regeneration of the periodontal membrane[16]

. The results showed

that periodontal vascular system plays an important role in the reconstruction of periodontal tissues during

orthodontic tooth movement. But, the mechanism how the vascular system participates in the orthodontic tooth

movement is not yet clear.

Periodontal tissue is composed of various cell types. PDL cells consist of fibroblasts, endothelial cells,

cementoblasts, osteoblasts, osteoclasts, tissue macrophages, Malassezs epithelial rests, and their precursors. In the

present study, we focused on the PDL fibroblasts, because fibroblasts are the predominant cell type in the PDL.

Some researches showed that vascular permeability increases in both the pressure and tension sides following the

application of orthodontic force. Macrophages occurred consistently near blood vessels both in areas of tension

and in areas of resorption. These are multipotent cells that obviously influence the remodeling process. At the

same time, the increase in blood flow following orthodontic force application was found to continue for a week,

after which it subsided to control values[17]

. But infiltrating blood cells, such as lymphocytes and monocytes, and

systemic hormones derived from the circulation might affect the cell function in the oral microenvironment in

vivo. Therefore, it is necessary to examine the interaction between PDL cells and PBMCs.

First, we established a co-culture system. This method allowed co-culture of PBMCs and PDL cells without

the two cell types coming into contact, but with interaction of soluble factors produced by the cells. We examined

the effect of human PBMCs on the proliferation of PDL cells by direct cell counting and PCNA labeling index

(LI). This is the first report to demonstrate that Human PBMCs induced the proliferation of PDL cells. Next, we

also examined the functional differentiation of PDL cells is affected by PBMCs, ALPase activity which was

closely associated with bone cell function, was analyzed. The result showed that the ALPase activity of PDL cells

was inhibited by co-culture with PBMCs. We concluded that human PBMCs could induce the proliferation of

PDL cells, while restrain the differentiation of PDL cells.


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The results showed that some soluble factor(s) released by PBMCs and/or PDL cells resulted in proliferation

and differentiation of PDL cells in this culture system. Cytokines and growth factors are important locally-acting

regulators of cell function[9, 18-20]

. PBMCs could secrets many cytokines which act on the adjacent tissues or cells

by autocrine or paracrine manner. At the same time, PDL cells are capable of synthesizing and secreting some of

these factors. Finally, autocrine and paracrine factors were mixed, as in the local microenvironment in vivo.

Therefore, we can conclude that periodontal vascular system take part in the remodeling of periodontal tissues by

inducing proliferation but restraining differentiation of PDL cells. So, vascularization should be the early reaction

during orthodontic tooth movement. Vascular changes were consistent with the regeneration of periodontium. But

further studies are required to determine the molecular mechanism responsible for this effect.

REFERENCES

[1] Lekic, P. & McCulloch, C.A. Periodontal Ligament Cell Population: The Central Role of Fibroblasts in Creating a UniqueTissue.Anat Rec, 1996, 245(2): 327-341.

[2] Pitaru, S., McCulloch, C.A., Narayanan, S.A. Cellular Origins and Differentiation Control Mechanisms during PeriodontalDevelopment and Wound Healing.J Periodontal Res , 1994, 29(2): 81-94.[3] Lackler, K.P., Cochran, D.L., Hoang, A.M ., et al. Development of an in vitro Wound Healing Model for Periodontal Cells.JPeriodontol, 2000, 71(2): 226-237.

[4] Yamashita, Y., Sato, M., Noguchi, T. Alkaline Phosphatase in the Periodontal Ligament of the Rabbit and Macaque Monkey .Arch Oral Biol, 1987, 32(9): 677-678.

[5] Kondo, Y., Irie, K., Ikegame, M., et al. Role of Stromal Cells in Osteoclast Differentiation in Bone Marrow. J Bone MinerMetab, 2001, 19(6): 352-358.

[6] Rygh, P., Bowling, K., Hovlandsdal, L., et al. Activation of the Vascular System: A Main Mediator of Periodontal FiberRemodeling in Orthodontic Tooth Movement.Am J Orthod, 1986, 89(6): 453-468.

[7] Cooper, S.M. & Sims, M.R. Evidence of Acute Inflammation in the Periodontal Ligament Subsequent to Orthodontic ToothMovement in Rats.Aust Orthod J, 1989, 11(2): 107-109.

[8] Vandevska-radunovic, V. Neural Modulation of Inflammatory Reactions in Dental Tissues Incident to Orthodontic ToothMovement. A Review of the Literature.Eur J Orthod, 1999, 21(3): 231-247.

[9] Davidovitch, Z., Nicolay, O.F., Ngan, P.W., et al. Neurotransmitters, Cytokines, and the Control of Alveolar Bone Remodelingin Orthodontics .Dent Clin North Am, 1988, 32(3): 411-435.

[10] Kvinnsland, S., Heyeraas, K., Ofjord, E.S. Effect of Experimental Tooth Movement on Periodontal and Pulpal Blood Flow.EurJ Orthod, 1989, 11(3): 200-205.

[11] Attal, U., Blaushild, N., Brin, I., et al. Histomorphometric Study of the Periodontal Vasculature during and after ExperimentalTipping of the Rat Incisor.Arch Oral Biol, 2001, 46(10): 891-900.

[12] Yamaguchi, K., Nanda, R.S., Kawata, T. Effect of Orthodontic Forces on Blood Flow in Human Gingiva .Angle Orthod, 1991,61(3): 193-204.

[13] McDonald, F. & Pitt Ford, T.R. Blood Flow Changes in Permanent Maxillary Canines during Retraction .Eur J Orthod, 1994,16(1): 1-9.

[14] Barwick, P.J. & Ramsay, D.S. Effect of Brief Intrusive Force on Human Pulpal Blood Flow .Am J Orthod Dentofacial Orthop,1996, 110(3): 273-279.

[15] Kanzaki, H., Chiba, M., Shimizu, Y., et al. Periodontal Ligament Cells under Mechanical Stress Induce Osteoclastogenesis byReceptor Activator of Nuclear Factor KappaB Ligand Up-regulation via Prostaglandin E2 Synthesis .J Bone Miner Res, 2002,

17(2): 210-220.

[16] Hosoyama, M. Changes in the Microvascular Pattern of the Periodontium in an Experimental Tooth Movement.Nippon KyoseiShika Gakkai Zasshi, 1989, 48(4): 425-442.

[17] Vandevska-Radunovic, V., Kristiansen, A.B., Heyeraas, K.J., et al. Changes in Blood Circulation in Teeth and SupportingTissues Incident to Experimental Tooth Movement.Eur J Orthod, 1994, 16(5): 361-369.

[18] Carter, D.H. & Sloan, P. The Fibrous Architecture of the Rat Periodontal Ligament in Cryosections Examined by ScanningElectron Microscopy.Arch Oral Biol, 1994, 39(11): 949-953.

[19] Blom, S., Holmstrup, P., Dabelsteen, E. A Comparison of the Effect of Epidermal Growth Factor, Platelet-derived GrowthFactor, and Fibroblast Growth Factor on Rat Periodontal Ligament Fibroblast-like Cells' DNA Synthesis and Morphology .J

Periodontol, 1994, 65(5): 373-378.

[20] Brady, T.A., Piesco, N.P., Buckley, M.J., et al. Autoregulation of Periodontal Ligament Cell Phenotype and Functions byTransforming Growth Factor-beta1.J Dent Res , 1998, 77(10): 1779-1790.

(Edited by Jane Chen, Bonny)

jan. 2007, volume 4, no.1 (serial no.26)

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