Accepted Manuscript Title: Effects of Cryopreservation on the Characteristics of Dental Pulp Stem Cells of Intact Deciduous Teeth Author: Daniele Lindemann Sefanie B. Werle Daniela Steffens Franklin Garcia-Godoy Patricia Pranke Luciano Casagrande PII: DOI: Reference:

S0003-9969(14)00088-0 http://dx.doi.org/doi:10.1016/j.archoralbio.2014.04.008 AOB 3167

To appear in:

Archives of Oral Biology

Received date: Revised date: Accepted date:

20-12-2013 8-4-2014 13-4-2014

Please cite this article as: Lindemann D, Werle SB, Steffens D, Garcia-Godoy F, Pranke P, Casagrande L, Effects of Cryopreservation on the Characteristics of Dental Pulp Stem Cells of Intact Deciduous Teeth, Archives of Oral Biology (2014), http://dx.doi.org/10.1016/j.archoralbio.2014.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 Title: Effects of Cryopreservation on the Characteristics of Dental Pulp Stem Cells of Intact Deciduous Teeth

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Daniele Lindemann Corresponding author: Prof. Luciano Casagrande Federal University of Rio Grande do Sul Pediatric Dentistry Ramiro Barcelos 2492 90035003 Porto Alegre, RS Brazil Phone: +555133085493 Fax: +555133085010 E-mail: [email protected]

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Order of Authors: Daniele Lindemann, DDS, MS; Sefanie B Werle, DDS, MS; Daniela Steffens, MS; Franklin Garcia-Godoy, DDS, MS, PhD; Patricia Pranke, MS, PhD; Luciano Casagrande, DDS, MS, PhD

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2 Abstract

Objectives: The aim of this study was to isolate and cultivate cells from the pulp of 7-

cryopreservation on Dental Pulp Stem Cell (DPSC) characteristics.

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days cryopreserved intact deciduous human teeth and evaluate the effect of

Design: Twenty-six deciduous teeth were collected and allocated in two groups:

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immediate cell isolation (Non-cryopreserved group) and intact cryopreserved

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(Cryopreserved group). The teeth were cryopreserved in dimethyl-sulfoxide solution and recovered after seven days. Success rate of isolation, proliferation, surface

capacity, and morphology were evaluated.

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markers (CD14, CD29, CD34, CD45, CD73, CD90, HLA-DR), differentiation

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Results: Isolation success rate was 61% and 30% for non-cryopreserved and cryopreserved groups respectively. There were no statistical differences between the groups for the tested surface markers. The cells in both groups were capable of

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differentiating into three mesenchymal lineages. No statistical differences between

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the groups were observed through the time course proliferation assay (0, 1, 3, 5 and

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7 days), however the mean time between isolation and the fifth passage was shorter for the non-cryopreserved group (p=0.035). The morphology of the cells was considered altered in the cryopreserved group. Conclusion: DPSCs were obtained from cryopreserved intact deciduous teeth without changes in the immunophenotypical characteristics and differentiation ability; however, lower culture rates, proliferation potential, and morphological alterations were observed in relation to the control group.

Keywords: Stem cells; Dental pulp; Deciduous teeth; Cryopreservation.

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3 Introduction Mesenchymal stem cells (MSCs) have been identified and isolated from almost every postnatal tissue in the human organism1, even in Dental Pulp of Permanent2 and

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Deciduous Teeth3. Dental Pulp Stem Cells (DPSCs), especially from deciduous teeth, have become an attractive postnatal source for research in regenerative

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medicine, because they exhibit proliferation and differentiation abilities4-8 and their

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obtainment causes minimal discomfort to the donor 9.

Cultivating cell lineages indefinitely is expensive and time consuming and equally

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risky for contamination and spontaneous differentiation10. However, most of the knowledge generated so far concerns the use of cells which are isolated and

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cultivated immediately after tooth extraction. Considering that the procedures for processing dental pulp cells is unlikely to happen immediately after tooth extraction in a clinical environment, research in dental tissue storage should be addressed11. It

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has been shown that stem cells normally survive in low temperatures as long as they

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are dispersed in cryoprotectants12-14. Some researchers have studied cells that have

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been isolated and cultivated from dental pulp of permanent teeth after pulp tissue cryopreservation12,15-17. Nevertheless, little is known about methods and alternatives for the stabilization of dental pulp cells during cryopreservation of intact deciduous teeth.

The aim of this study was to cryopreserve for 7 days intact human deciduous teeth,

isolate and cultivate cells from the pulp after thawing and evaluate them for mesenchymal stem cell characteristics, comparing them to pulp cells from noncryopreserved deciduous teeth. The null hypothesis was that cryopreservation of intact deciduous teeth had no effect on the characteristics of dental pulp stem cells. Materials and Methods

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4 Human subjects

Thirteen patients, aged 9 to 11 years-old, with two sound deciduous teeth with at least 1/3 of physiologic root resorption7 were selected. Each patient contributed with

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two teeth, one to each group. Procedures with patients were conducted in

accordance with the Helsinki Declaration and were approved by the Federal

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University of Rio Grande do Sul (UFRGS) Ethics Committee (n. 20865).

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Cryopreservation of Intact Deciduous Human Teeth and Cell Culture

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After the extraction, the teeth were placed into a falcon tube containing 2 mL of Dulbecco’s modified Eagle medium (DMEM) supplemented by 10% fetal bovine

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serum (FBS) (Sigma- Aldrich, St. Louis, MO), 100U/mL penicillin, 100mg/mL streptomycin and 0.45mg/mL gentamicin (Gibco, Grand Island, NY). The teeth were

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allocated into two groups: control (non-cryopreserved) and test (cryopreserved).

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Teeth allocated for cryopreservation were dropped into 2mL vials containing 1.5 mL dimethylsulfoxide (DMSO, Sigma- Aldrich, St. Louis, MO) and FBS (1:9) at 4ºC. The

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vials were cooled (4°C/1 hour) for the cryoprotectant to penetrate into the soft tissue through the resorbed area. The vials were then subjected to a control-freeze method (Mr. Frosty – Nalgene) with a -1°C /min cooling rate in an -80°C freezer (VIP Series

Ultra-Low Temperature Freezer, Sanyo Scientific, Bensenville, IL) for 24 hours. The

following day, the samples were transferred to a -196°C liquid nitrogen tank, where they remained for seven days.

After the freezing period, the vials were thawed in a 37°C water bath for 1 minute. The pulp tissue was separated from the dentin and the cells were retrieved by enzymatic digestion (0.2% type I collagenase solution Gibco, Grand Island, NY) for 60 minutes. During the first two weeks, the culture medium was supplemented by 20% FBS 18. The passages were performed when the cultures reached 90%

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5 confluence using a 0.5% trypsin-EDTA solution (Sigma –Aldrich, St Louis, MO) for 5 minutes. The cell suspensions were plated (5.000 cells/cm2) and cultivated at 37°C and in a 5% CO2 environment. The cells in the fifth passage were used for the experiments. Non-cryopreserved teeth went through the same isolation and

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cultivation protocols.

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Flow cytometry analysis

A total of 1x105 cells of each sample were incubated with the following types of

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conjugated antibodies: CD14/FITC, CD29/PE, CD34/PE, CD45/FITC, CD73/PE, CD90/FITC and HLA-DR/FITC, according to the International Society of Cellular

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Therapy 19. Unstained cells and mice IgG isotype controls conjugated with PE and FITC were used as controls. Only living cells were analyzed by excluding

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dead/apoptotic cells that were positive for 7AAD (7-Amino Actinomycin D Invitrogen). Two-cell surface markers were evaluated simultaneously using the

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monoclonal antibodies against antigens. The data was acquired using the FACS –

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Aria flow cytometer (BD Bioscience) and 10.000 events were analyzed (FACSDiva 6.1.3 - BD Bioscience).

Proliferation assay

A water-soluble tetrazolium monosodium salt cell counting (WST-8, Sigma–Aldrich,

St. Louis, MO) was used for colorimetric cell viability and proliferation assays. A total of 2.5x103 cells/well of each sample were seeded onto 3 wells of a 24 well plate and

incubated with 1mL culture medium. The analysis was performed on day 1, 3, 5 and 7 after seeding, according to the manufacturer’s instructions. Proliferation was also evaluated by using the mean time between the isolation and the fifth passage.

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6 Multipotent assay

Each culture from DPSCs (1x104) were seeded in triplicate onto 12-well plates, and

replaced by one of three differentiation-inducing medium as follows: -

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cultivated in regular medium until confluence reached 70%. The medium was then

osteogenic differentiation: DMEM/HEPES supplemented with 10% FBS; 0.1%

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dexamethasone, 10-5 mol/L; 10% b- glycerophosphate, 10nmol/L; and

-

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ascorbic acid 2-phosphate, 5mg/mL.

chondrogenic differentiation: DMEM/HEPES supplemented with 6.25mg/mL

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bovine insulin, 50nmol/L ascorbic acid 2-phosphate and 10ng/mL transforming growth factor – beta 1 (Millipore, Tokyo, Japan). adipogenic differentiation: Iscove’s Modified Dulbecco’s Medium (Sigma-

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-

Aldrich, St. Louis, MO) supplemented with 10% FBS; 1μL/mL bovine insulin;

1mL/10mL.

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5μM/L rosiglitazone; 10-7 M/L dexamethasone, 5μM/L and indomethacin

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DPSCs were maintained in these conditions for up to 28 days. During this period, the

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cultures were evaluated every three days and when alterations on morphology were observed, the culture was washed with deionized water and fixed in 4% paraformaldehyde for 20 minutes (osteogenic and chondrogenic) or 1 hour (adipogenic). Alizarin red was used to stain calcium depositions in osteogenic differentiation. Alcian blue was used to stain extracellular matrix in chondrogenic differentiation and Oil-Red-O to stain lipid vacuoles in adipogenic differentiation. The cells cultured in a regular culture medium were used as control.

Morphology

The morphology of the cells was observed under confocal microscopy. DPSCs (1x104) were cultivated and, after 14 days of incubation, for both groups (n=2),

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7 washed with PBS 1x, fixed with 4% paraformaldehyde (30 minutes) and permeabilized with Triton X-100 0.1% (10 minutes). The DPSCs were stained with 50µg/mL rhodamine conjugated phalloidin (40 minutes), washed with PBS 1x and stained with 0.5 µg/ml of DAPI (1 minute). Photographs representing the different

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samples were obtained using a FV1000 Olympus confocal microscope.

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Statistical Analysis

The means for each surface marker were submitted to one-way ANOVA.

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Time-course proliferation assay was analyzed using Poisson regression for the comparison within the groups and Mann-Whitney’s test and Wilcoxon’s test, for the

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comparison between the groups. Student’s t test was used to compare the meantime between isolation and the fifth passage.

The statistical analysis was performed using the statistical software STATA 9.0

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(Stata Corp, College Station, USA) and Statistical Package for Social Sciences

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version 20.0 (SPSS Inc., Chicago, IL, USA, 2011) at 5% significance level.

Results

Culture Establishment

Cell culture was considered a successful outcome when the grouped cells were seen at the base of the well within 30 days after isolation and reached confluence for the passage. Four of the 13 cryopreserved teeth (30%) established culture. One culture was contaminated after the first passage; the other 3 were used in the experiments. The average time from day one to the first passage was 33.5±7.9 days. For the noncryopreserved group, 8 out of 13 teeth (61%) established culture, 5 were used in the experiments, and the average time for the first passage was 27.1±15.9 days.

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8 Flow cytometry

More than 94% of the cells in each culture were positive for CD29, CD 73 and CD90; and 99 % were negative for hematopoietic and endothelial markers (CD14, CD34,

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CD45, HLA-DR). No statistical difference (p >0.05) was observed between the groups. When the 7AAD was analyzed (Figure 1), it was observed that the

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cryopreserved sample showed a higher number of 7AAD positive cells (26.5 %)

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compared with the non-cryopreserved cells (6.6 %).

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Proliferation assay

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No statistical differences between the groups were observed through the time course proliferation assay (Table 1). When the analysis was made within the groups, there was no differences for cryopreserved group (p=0.762), while for non-cryopreserved

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group there was a tendency to increase the absorbance (proliferation) with time

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(p=0.028). There was statistical difference (p=0.035) in the mean time between

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isolation and the fifth passage for the non-cryopreserved (49.3 ± 16.5 days) and cryopreserved (87.6 ± 21.2 days) groups. The average number of cells per flask (175 cm2) of culture (P5) was 7335.910 (±3273.835) for the non-cryopreserved group and

1506.717 (±166.586) for the cryopreserved group.

Multipotent assay

Cultures from the cryopreserved teeth grown in the presence of an osteo-inductive medium for 28 days showed the ability to form Alizarin red positive condensed nodules of calcium. These deposits were sparsely scattered over the cells. Cultures from the non-cryopreserved teeth, also positive for Alizarin red, produced wide sheets of calcified deposits over the adherent layer in 21 days of induction.

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9 Twenty-eight days were necessary for the cells of both groups to show the formation of an Alcian blue stained extracellular matrix (chondrogenic differentiation). The adipogenic potential was also seen in the cultures, although the oil red positive lipid clusters were very tiny and sparsely grouped in 28 days. Negative control

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consisting of the cells of both groups cultured in a regular culture medium showed no

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differentiation (Figure 2).

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Morphology

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DPSCs with a round shaped cytoplasm were seen in the cultures of the cryopreserved teeth. The non-cryopreserved group presented cells with spindle

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shaped cytoplasm (Figure 3).

Discussion

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The successful isolation of DPSCs for up to 120 hours after extraction was already

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reported15, however, the isolation success decreases with time. The long term culture

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of DPSCs could cause some complications, such as the decrease in the ability of differentiation, senescence and apoptosis induced by consecutive passages 20,21, the possibility of genetic alterations and the costs involved 22. Since the exfoliation process of deciduous teeth occurs usually between 5 and 13 years of age, the cryopreservation is presented as an alternative to maintain the viability of stem cells until their use is required, allowing long-term storage 17.

The results showed that DPSCs could be obtained from cryopreserved deciduous teeth with 30% culture rate, whereas from non-cryopreserved teeth, this rate increases to 61%. A previous study observed 20% isolation rate from cryopreserved permanent teeth16. The lower isolation success rate in the cryopreserved group may be associated to the freezing process, by the ice crystals forming. Although, in order to prevent the

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10 damage by ice crystals presents in low temperatures, cryoprotectant (DMSO) was added to the freezing solution 12. When the intact tooth is cryopreserved, the crown makes insulation 22 and the cryoprotectant penetrates through the area of resorption. In this study, only teeth with, at least, 1/3 of physiologic root resorption were used.

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First, because have been reported that is not possible to isolate stem cells from pulp of deciduous teeth with no visible resorption 7; second, because the resorption area

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allow a greater diffusion of cryoprotectant solution. A previous study showed no

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differences in the isolation rate between resorption stages 18. However, futures

studies are needed to evaluate the freezing solution diffusion through the pulp.

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The cells in both groups met all the criteria established for MSCs regarding adherence to plastic, presence of surface markers for mesenchymal stem cells, and

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differentiate into osteoblasts, chondroblasts and adipocyte 19. The results from flow cytometry assay showed that cryopreservation had no effect on the

with a previous study 23.

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immunophenotypical expression of the cell surface antigen markers, corroborating

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No statistical difference was found between the groups in the time course

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proliferation assay. A previous study 7 showed that cells needed one day to accommodate in the wells and more three days to duplicate or triplicate in number. However, the lower isolation rates reduced the sample size (cryopreserved group) and this might explain the absence of statistical differences in the proliferation assay, moreover, 7 days may not be enough time to show proliferation differences for this sample size.

When the average time between isolation and the fifth passage was calculated, the mean for the cryopreserved group (87 days) was approximately 44% higher than noncryopreserved (49 days), suggesting a lower proliferation rate for cryopreserved group, which is in accordance with a previous study 22. Moreover, when considered the average number of cells per flask (175cm2) of culture at fifth passage, after

seeded 875.000 cells for both groups (5x103 cells/cm2), the lower proliferation for

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11 cryopreserved group was evident, since only a mean of 1.506.717 cells were obtained for this group, while the non-cryopreserved group yielded 7.335.910 cells. The proliferation capacity is a determining factor to considered SHED as a potential source for tissue engineering 24 since the number of stem cells obtained after

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isolation is very low 3. Thus, alterations in the proliferation capacity, as shown in the cryopreserved group, may limit its applications, since millions of stem cells are

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typically required for therapeutic purpose 25. The great amount of cells needed for

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clinical trials may constitute one of the reasons to explain the slow research progress with patients4. Maybe, the supplementation of culture medium with 20% FBS would

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increase this growth rate, however, there is a currently tendency to decrease the animal supplementation in stem cells culture to enable the clinic use 8,26.

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The cells from both groups possessed the ability to differentiate into the tested phenotypes. For both groups, the most evident differentiation was to the osteogenic lineage. This result might be due to the influence of the local environment, from

28,29

. In this way, the DPSCs are the focus of research for

, moreover, a relative small amount of cells (approximately

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pulp regeneration

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phenotype of the stem cells

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where the cells originate, reflecting the importance of the niche in establishing the

1x106 cells) are required for this strategy of tissue engineering 28,29,30. During the induction period, stem cells from both groups showed visible less differentiation products for adipogenic differentiation. Miura et al. also reported that after 5 weeks of induction only 5% of the DPSC cultures showed the potential to develop into fat cells 3.

The cell morphology was analyzed at 14th day after seeding, for both groups. In this period, the confluences of the groups were different, since the groups presented different growth potential, as mention before. Under confocal microscopy, the cells from the cryopreserved group showed different morphology patterns, presenting a round shaped cytoplasm. The altered morphology was also found in a previous

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12 study22. This morphology suggests cellular senescence or apoptosis, corroborating the highest 7AAD staining (26%), a nuclear stain for apoptosis 31, in cryopreserved group. A recent study reported 5.4% 7AAD positive cells (third passage) for bone marrow stem cells 32, result similar to the noncryopreserved group in the present

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study, which showed 6.6% 7AAD positive cell.  

These culture characteristics from cells of cryopreserved teeth, like as altered

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morphology and low proliferation rate, limit their application in tissue engineering at

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this moment, especially for therapeutic purposes 25. On the other hand, some modifications in the protocols, such as the use of a magnetic field freezer to

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cryopreserve intact teeth 23 or a laser piercing technique 22 to produce microchannels in enamel and dentin are found in the literature and show better isolation rates.

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However, if any additional procedure is adopted before freezing, the storage technique becomes more difficult, if not impossible, for most of the clinicians at the time of the tooth extraction.

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This study shows that it is possible to retrieve DPSCs from intact cryopreserved

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human deciduous teeth. Further studies should be conducted to improve this

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method, since using this protocol the cells obtained presenting a low proliferation rate compared to cells from the non-cryopreserved teeth.

Acknowledgments

The authors thank Pedro Chagastelles and Andrea Galuppo, for their assistance in the flow citometry procedures and are grateful to National Council for Scientific and Technological Development - CNPq (process n. 478778/2011-2) for the financial support.

Conflict of interest statement

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13 There are no conflicts of interest.

References

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4. D’Aquino R, De Rosa A, Laino G, Caruso F, Guida L, Rullo R, et al. Human dental

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15 their tissues of origin for banking and clinical use. Cryobiology2009;59(2):150-7. 17. Ma L, Makino Y, Yamaza H, Akiyama K, Hoshino Y, Song G, et al. Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem

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23. Lee SY, Chiang PC, Tsai YH, Tsai SY, Jeng JH, Kawata T, et al. Effects of cryopreservation of intact teeth on the isolated dental pulp stem cells. J Endod 2010;36(8):1336-40. 24. Wang X, Sha XJ, Li GH, Yang FS, Ji K, Wen LY, et al. Comparative

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16 characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol2012;57(9):1231-40. 25. Haque N, Rahman MT, Abu Kasim NH and Alabsi AM. Hypoxic culture conditions a

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31. Philpott NJ, Turner AJ, Scopes J, Westby M, Marsh JC, Gordon-Smith EC, et al. Use of 7-Amino Actinomycin D in Identifying Apoptosis: Simplicity of Use and Broad Spectrum of Application Compared With Other Techniques. Blood1996;87(6):224451. 32. Saini U1, Gumina RJ, Wolfe B, Kuppusamy ML, Kuppusamy P, Boudoulas KD. Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation. J Cell Biochem2013;114(11):2612-

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18 Figure captions:

Fig 1 Cytometry analysis from the cryopreserved sample showing 26.5% of

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the cells positive for 7AAD, suggesting that they are dead or apoptotic cells. (B) Cytometry analysis from the non cryopreserved sample showing 6.6% of

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the cells positive for 7AAD, suggesting that they are dead or apoptotic cells.

(C) Cytometric evaluation. Representation of the percentage values by means

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and standard deviation obtained by analyzing the percentage expression of

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the markers of all samples

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Fig 2 Multipotent assay. (A-D) Non-Cryopreserved group. (A) Dental Pulp Stem Cell culture without induction medium for negative control. (B)

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Osteogenic differentiation showing wide calcium sheets stained with Alizarin

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red. (C) Chondrogenic differentiation visualized by Alcian blue staining of the glycosaminoglicans deposits. (D) Adipogenic differentiation shown by Oil red

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staining of lipid vacuoles. (E-H) Cryopreserved group. (E) DPSC culture with regular culture medium. (F) Osteogenic differentiation showing sparsely scattered calcium deposits stained with Alizarin red. (G) Chondrogenic differentiation visualized by Alcian blue staining of the small glycosaminoglicans deposits. (H) Adipogenic differentiation shown by Oil red staining of rare lipid vacuoles (yellow arrows)

Fig 3 Morphology assay. (A-C) Non-Cryopreserved group. (A and B) Confocal microscopy. (C) Inverted microscopy image. (D-F) Cryopreserved group. (D and E) Confocal microscopy. (F) Inverted microscopy image

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19 Table 1. A) Means (SD) of proliferation assay by WST-8 on day 1, 3, 5 and 7 after seeding. Day 1

Day 3

Day 5

Day 7

Cryo

0.31 (0.0)

0.33 (0.0)

0.35 (0.0)

0.4 (0.0)

0.44 (0.11)

Non-Cryo

0.33 (0.0)

0.39 (0.0)

0.50 (0.2)

1.15 (1.0)

1.29 (0.96)

p

0.45

0.29

0.29

0.18

0.10

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Day 0

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Figure(s)

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Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth.

The aim of this study was to isolate and cultivate cells from the pulp of 7-day-cryopreserved intact deciduous human teeth and evaluate the effect of ...
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