Eur J Omt Sii 1997: 105: 609-613Primed in UK. .41! rit^li!.^ reserved

Capyvighl O Eur J Oral Sci 1997

EUROPEAN JOURN=AL OFORAL SCIENCES

Cell proliferation in developinghuman dental pulpA combined flow cytometric andimmunohistochemical study

Casasco A. Casasco M, Calligaro A, Ferrieri G, Brctmhilla E. Strohmenger L.Alberici R, Mazzini G: Cell proliferation in developing human dental pulp.'Acombined flow cytometric and imtnunobistochemiccil study. Eur J Oral Sci 1997:105: 609-613. & Eur J Oral Sci, 1997

Infonnaiion concerning cell proiireration and differentiation in dental pulp maybe important to understand tooth response to exogenous stimuli. Since fewdata concerning human dental pulp are available, we have investigated thegrowth fraction and the localization of proliferating cells in pulp tissue of thirdtnolars of young adult human males and females, using flow cytometry andimmtinohistochemistry. Flow cytometric analysis demonstrates a lowproliferative activity of pulp tissue that appears to be confined to radicularpulp, as revealed by immtinohistochemical detection of proliferatmg cells. Nopolyploid or aneuploid cell populations could be identified, and G2-blockedcells., if any, represented a negligible cell population, Odontoblasts, cells of thesttb-odontoblastic layer, and cells of coronal pulp were found to be notproliferating under normal conditions. These data provide the basis for futureinvestigations on proliferative activity and regenerative potentiality of hurnanpulp cells in e,xpentnentai and clinical situations.

Andrea Casasco\Marco Casasco\Alberto Calligaro\Giovanni Ferrieri%Eugenio Brambilla^,Laura Strohmenger^,Roberta Alberici^ andGiuliano Mazzini^'Institute of Histology and GeneralEmbryology, University of Pavia, Pavia,^Dental Clinic, "San Paoilo" Hospitai,University of Milan, Milan, and ^Center forHistochemical Studies, National Council forResearch, Pavia, Italy

Prof, Andrea Casasco, Institute of Histology& General Embryology, University of Pavia,via Forlanini 10, i,-27100 Pavia, Italy

Telefax; + 30-382528330

Key words: cell proliferation; dental pulp;fiow cytometry; human;immunohrstochemistry

Accepted for publication July 1997

Dental pulp is a utiique lissue of ectomesenchymalorigin that is enclosed in rigid walls of deotitie.Dental pulp, which is richly provided by bloodvessels and nerves, is responsible for \'itaiity ofdentine. Moreover, odontoblasts, which are dedic-ated to dentine synthesis, differentiate from pulpcells during odontogenesis. Accordingly, pulp anddentine may be considered a single tooth cotnpon-ent, the so-called pulp-dentine organ.

It is well known that dental pulp reacts toexogenous stimuli, and the differentiation and pro-liferation of pulp cells is considered to be importantin dentine formation after bacterial, chemical andmechanical stimulation. Previous studies, mostlyemploying autoradiographic techniques, haveinvestigated cell proliferation and differentiation indeveloping as well as stimulated animal dental pulp(1-11), Nevertheless, little information is availableconcerning the proliferation of pulp cells in thehuman.

The aim of our study was to investigate prolifer-ative activity of human pulp cells using flow cyto-metry and immunohistochemistry.

Material and methodsCollection of samples of human dental pulp

Samples of dental pulp (n = 20) were obtained fromhealthy patients of both sexes. The age of thepatients ranged between 15 and 25 yr. Details con-cerning the subjects are shown in Table 1, Informedconsent was obtained from each patient.

Healthy third molars were extracted ueder localanaesthesia for orthodontic purposes. Immediatelyafter extraction, a longitudinal groove, 1 mm deep,,was made in the enatnel with a water-cooled high-speed drill. The teeth were then fractured with achisel and hammer so that the entire ptilp tissuecould be carefully removed from the pulp cavityunder a dissecting microscope. The samples forflow cytometry analysis (n= 15) were rapidly frozenin isopentane cooled by liquid nitrogen and storedat — 80"C until staining and measurement. Thesamples for immunohistochemical analysis (« = 5)were flxed in 4% paraformaldehyde in 0,1 M phos-phate buffer, pH 7,4, for 6-8 h, and routinely pro-cessed for paraffin embedding.

610 Casasco et al.

Table i

Details concerning the subjects, extracted teeth and per cent values for eaeh phase of the celt cycle in each sample. Values are expressedas per cent values I cell fractiotis j in total eetl population per each sample. The last line indicates mean ± standard error for each pha.se

of the cell cycle

Case tio.

1%345'

719

101113131415Mean ± standard

Age (yr)

m301817WIS16W17192017182222

error

Sex

M — tnaieF = female

iFFFMFFM

FFMFFF

Tooth

481838381838184828282838382838

GO-G! (%)

91,395,687,796,590,490,488,691,587,891,084,392,189,092,093,490,77 + 0,80

Cell fractions

S ('Ml)

5.32,19,61,05,35.36,84,05,54,99,74,56,05,05,35,35 + 0,59

G 2 - M (%)

3,-42,32,72,54.34,34,64.5•5.74,16,03,45,03,93,34,01 ±0,32

Flow cytometry

Samples were minced with a scalpel into smallfragrnents which were incubated in 10 ml porcinepepsin, 0,4 mg/ml in 0,1 M HCl, for 15 min at 37°Cwhile undergoing agitation. Residual clumps andfragments were nnechanically disaggregated by syr-inging. The resulting suspensions were centrifugedat 300 g for 5 min to sediment the nuclei, whichwere then washed in phosphate-buffered saline,pH 7,4 (PBS), Suspensions were centrifuged, andthe pellet stained with a solution of propidiumiodide (507ig/ml), 0,1% Nonidet P40 and type IARNAse (100 Kunitz units/ml) in PBS for 30 minat room temperature. Finally, cells were filtered toremove aggregates prior to flow analysis, DNAanalysis was performed with a Partec PAS II (Basel,Switzerland) arc lamp flow cytometer, with datadisplaj'ed as frequency histograms. At least20 000 cells were analysed for each sample. As acontrol for instrumental performance and align-ment as well as for staining protocol, we haveperformed the analysis on samples of normal peri-pheral blood in which 98-99% of cells are locatedin the GO-Gl phase of the cell cycle,

Immunohistochemistry

Samples of dental pulp were cut at 7-10 ,um, andsections were rehydrated and processed for theimmunohistochemical detection of proliferatingcells according to the indirect biotin-strcptavidinimmunoperoxidase technique, Pretreatment of sec-tions was performed as follows: (a) incubation with

0,3% hydrogen peroxide for 30 min to rernoveendogenous peroxidase activity; (b) incubationwith 0,05% trypsin for 10 mm at 37=C; (c) incuba-tion in citrate buffer, pH 6, in microwave oven for10 min to retrieve the antigen; and (d) incubationwith normal goat serum, diluted 1:20, for 30 minto reduce non-specific background staining.Sections were then incubated serially with the fol-lowing solutions for the immunostaining: (a) MIBlmonoclonal antibodies, (BioGenex, San Ramon,CA, USA) diluted 1:50, overnight at 4'C; (b) pre-diluted biotinylated goat anti-mouse IgG(Biogenex, Super Sensitive kit), for 1 h at roomtemperature; (c) pre-diluted streptavidin-biotin-yiated peroxidase complexes (BioGenex, SuperSensitive kit) for 1 h at room temperature; (d)0,03%), 3,3' diaminobetizidine tetrahydrochloride,to which hydrogen peroxide (0,02yii) was addedjust before use, for 5 min at room temperature.Each solution was prepared in 0,05 M Tris buffer,pH7,4, containing 0,1 M/1 NaCl (0,15 M Tris-buffered saline), and between each step of theimmunostaining procedure the sections werewashed in the same buffer. Some sections werelightly counterstained with haematoxyiin. The sec-tions were finally dehydrated, mounted andobserved in a Zeiss microscope equipped withNomarski differential interferetice contrast device.

Primary MIBl monoclonal antibody, a mouseIgGl, has been previously characterized (12), MIBlantibody recognizes a wax embedding resistantepitope of the Ki67 nuclear antigen (345 and395 kDa double band in Western blot analysis ofproliferating cells) that is expressed in Gl, S and

Cell proliferation in human dental pulp 611

Eig. 1. Distribution of M1B1/Ki67 antibody immunoreactivityin the ceil cycle,

G2-M phase-traversing cells, i,e, proliferating cells(12, 13; Fig, 1), Specificity controls of the immuno-histochemical reaction included the omission ofprimary antibody and the substitution of primaryantibody with non-immune sera or monoclonalantibodies from the same inimunoglobulin subclass,

Non-imtnunological reagents were purchasedfrom Sigma Chemical Co, (St, Louis, MO, USA),

ResultsFlow cytometry

Details concerning the subjects, extracted teeth andper cent values for each phase of the cell cycle ineach sample are shown in Table 1, Mean per centvalues standard error were GO-Gl =90,77 + 0,80,

S = 5,35 ±0,59 and G2-M = 4,01 ±0,32, No poiy-ploid or aneuploid cell populations were observedin any sample. As an example, a typical DNAhistogram from sample 5 is shown in Fig, 2,

Immunohistochemistry

Cells displaying immunoreactivity with MIBl anti-body were observed in all specimens studied,Immunostaining was detectable in the nucleus ofpositive cells. Cells of the odontoblast and subodon-toblastic layers as well as cells of the coronal partof pulp were consistently negative (Fig, 3A), Manyreactive cells were detectable in the periphery ofthe pulp corresponding to the growing roots(Fig, 3B), In control sections, no immunostainingcould be observed.

Discussion

In this study, we have investigated for the Orst timethe growth fraction and the localization of prolifer-ating cells in human dental pulp using flow cytome-try and imnumohistocheinistry. In our experiments,we have used third molar teeth that had beenextracted for orthodontic reasons, because they arerich in healthy pulp tissue. Previous studies haveinvestigated cell proliferation and differentiation indeveloping and mature dental pulp in the animal

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Eig. 2. Flow cytometric DNA histogram from sample 5,

612 Casasco et at

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Fig. 3. toninnohistochemical locdtzj .lot. of proliferating cells it. ,:eni I ,-'i'p I IM • i i .11 J ^ ̂ p 1;. r oiar. Coronal pulp,odontoblasts and celK of sub-odontoblastic layer display no ittimunoreaction with ttionoclonal MIBl antibody {.A.), whereas severalimtnunoreactive cells are Msibie tn the radicular part of pulp tissue (B). Indirect immunoperoxidase method. OL. odontoblastlaver. x 390.

(1-4, 7, 8). The reaction of pulp cells to exogenousstimuli has also been investigated in animal models,providing evidence that pulp response plays animportant role in tooth defence mechanisms thatoccur in clinical situations (5, 6, 9-11, 14, 15).

Ahhough autoradiographic techniques cannot beapplied to human tissue in vivo, the combined flowcytometric and immunohistochemical approachused in our study seems to provide sufficientinformation concerning the growth fraction andthe localization of proliferating cells in humandental pulp. Indeed, flow cytometry provides quant-itative data regarding the cell cycle of pulp cells,whereas immunohistochemistry demonstrateswhere and which cells are proliferating in pulptissue. The same technical approach has been previ-ously used in other studies on dental tissues in vivoand in vitro (16, 17).

The possible occurrence of G2-blocked cells inanimal dental pulp has been hypothesized (18, 19).The per cent values of G2-M phase cells comparedto S phase cells, as determined by flow cytometry,suggest that G2 cells represent a negligible, if at allpresent, cell population in human pulp. Moreover,no polyploid or aneuploid cell populations couldbe identified in our samples, thus excluding theoccurrence of endomitosis or unequal DNA distri-bution in human pulp cells.

Flow cytometric data suggest a rather low, butconsistent, proliferative activity in human pulp ofthird molars extracted from young patients.

Immunohistochemical localization of proliferatingcelis demonstrates that most of cycling cells arelocated in the region of the pulp corresponding togrowing roots. This is in agreement with animalmodels (3, 4) and corresponds with the notioti thatroots of third molar teeth are still developing inyoung subjects.

According to animal models, odontoblasts andcells of the sub-odontoblastic layers display noimmunoreaction to proliferation marker, sug-gesting that these cells have withdrawn from thecell cycle (7, 18, 20, 21). It will be interesting toclarify in human pulp which cells may enter thecycle again upon stimulation.

Acknowledgetnents - We thank Drs A. Icaro Cornaglia, M.Reguzzoni, A. Farina (University of Pavia). and Dr G. Marrotie(University of Milan) for their valuable technical help andCentro Diagnostico Italiano (CDl, Milan) for assistance insample collection. This research was supported by funds (40%)from the Italiatt Ministry of University and Scientific attdTechnological Research and by F.A.R. funds from theUniversity of Pavia to A.C. and A.C.

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