Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets
© The Author(s). 2017
Received: 22 October 2016
Accepted: 4 March 2017
Published: 17 April 2017
Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets.
PDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration.
The PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix.
Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets and could enhance their clinical utility in tissue regeneration.
KeywordsCryopreservation Periodontal ligament stem cell Cell sheet
Tissue engineering based on biocompatible and biodegradable scaffolds has emerged as an attractive field in regenerative medicine over the past several decades . Despite recent advances, clinical application of implantable scaffolds remains limited due to several major drawbacks, including insufficient cell proliferation and adhesion, undesirable stimulation of the local inflammatory response, difficulty in balancing cell proliferation with scaffold degradation, and the inability to generate functionally competent tissues . To address these limitations, cell sheet engineering, in which one or multiple layers of intact cell sheets are grown on and subsequently detached from a thermosensitive surface without the use of a scaffold, has been developed as an alternative approach for tissue regeneration. Compared to the scaffold-based approach, cell sheet engineering offers the obvious advantage of well-preserved endogenous extracellular matrix (ECM) and cellular junctions which greatly increases the chances of success in transplantation . Cell sheet engineering has been applied to the construction of soft and hard tissues alike, including myocardial tissues [4, 5], liver tissues , bone , and blood vessels , etc. Previously, we reported the development of an effective and reliable method based on vitamin C (Vc) treatment that facilitated the construction of highly viable and functional periodontal ligament stem cell (PDLSC) sheets and the subsequent regeneration of periodontal tissues . Vc has been shown to promote the generation of collagen and other ECM constituents [10–12], as well as to mimic the in vivo physiological environment. Besides, when supplied to the culture medium, it can act as a growth promoter to stimulate cell proliferation . We then successfully employed PDLSC sheets to regenerate a functional bio-root structure for artificial crown restoration . However, clinical application of PDLSC sheets was limited by the fact that their laboratory preparation was very time consuming. In fact, using conventional methods, it would require at least 10 days to grow the cell sheets in our laboratory which precluded any therapeutic usage in the event of medical emergency.
Cryopreservation has been extensively studied as a viable solution to the long-term storage of various biomaterials, such as oocytes , stem cells [16, 17], vascular tissues , and embryos . Recently, several groups demonstrated the feasibility of obtaining viable PDLSCs from frozen periodontal ligament tissues or intact whole teeth [20, 21]. However, to the best of our knowledge there has been no comprehensive study on the impact of cryopreservation on PDLSC sheets. To this end, our current study aims to investigate whether cryopreservation could affect the structural integrity and physiological function of PDLSC sheets. We also compared the proliferative capacities and differentiation potentials of PDLSCs derived from cryopreserved or freshly prepared cell sheets. Finally, we examined whether the cryopreserved PDLSC sheets could be used as implants for tissue regeneration in a mouse model.
All protocols for the handling of human tissues were approved by the Research Ethics Committee of Shandong University (No. MECSDUMS2012087). Informed consent was obtained from the donors and their parents. The animal study was reviewed and approved by the Committee on the Ethics of Animal Experiments of Shandong University (No. ECAESDUSM2012075).
Extracted human impacted third molars were collected from 16 subjects being treated at the Department of Oral and Maxillofacial Surgery, Stomatological Hospital of Shandong University. Human PDLSCs were isolated from the root surface and digested in a solution of 3 mg/mL collagenase type I (Sigma-Aldrich, USA) and 4 mg/mL dispase (Sigma-Aldrich) for 1 h at 37 °C as previously reported . PDLSCs were cultured at 37 °C under 5% carbon dioxide in 25 cm2 flasks (Corning, USA) using alpha-modified Eagle’s medium (α-MEM; Invitrogen, USA) supplemented with 15% fetal bovine serum (FBS; Invitrogen), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were then passaged two to three times in the same medium before being used for the growth of cell sheets.
Preparation of PDLSC sheets
To induce the growth of cell sheets, PDLSCs were cultured in 60-mm culture dishes (Corning, USA) with Vc (Sigma-Aldrich) added to the PDLSC culture to a final concentration of 20 μg/mL . After reaching confluency in 2–3 days, the PDLSCs were cultivated for an additional 10–14 days until the cells at the edge of the dishes started to wrap up, indicating the formation of cell sheets. The intact sheets were isolated with a blunt blade under humidified conditions when they reached an average thickness of around two layers of cells. The cell sheet will then shrink to about 15 mm due to the elasticity. The isolated cell sheets were assigned to the cryopreservation group (cPDLSC) or the control group (freshly prepared PDLSC; fPDLSC) for the following experiments. Paired cell sheets (cPDLSC sheet and fPDLSC sheet) were prepared from molars of the same subject. The cPDLSC sheets were submitted to the cryopreservation and thawing procedures described below, whereas the ones in the control group were freshly prepared and directly used for subsequent studies.
Cryopresevation and thawing of PDLSC sheets
All experiment procedures pertaining to the cryopreservation and recovery of cell sheets were conducted under a sterile environment, either on a clean bench or with the containers wrapped in parafilm (Bemis® Flexible Packaging) to prevent contamination.
For cryopreservation, the cell sheets were directly equilibrated in a pre-chilled solution mix of 90% FBS and 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 4 °C. The subsequent chilling and freezing was performed in a controlled manner using a programmable freezer (Taiyo-Toyo Sanso Co., Japan). The temperature drop rate was –0.5 °C /min from 4 °C to –20 °C, and –1 °C /min from –20 °C to –80 °C. After 24 h incubation at –80 °C, the cryovials were submerged in liquid nitrogen. Woods et al. reported that differences in viability were not statistically significant comparing 1 week to 1 month to 6 months ; in the present study, we stored the sheets in liquid nitrogen for 3 months.
For thawing, the cryovials were retrieved from the liquid nitrogen, rapidly immersed in a 37 °C water bath and gently agitated until the cryopreservation medium was completely melted. The cell sheet was then transferred to tubes containing 5 mL α-MEM supplemented with 15% FBS. The tubes were placed into the shaker, gently agitated at 1000 rpm for 5 min at 37 °C, and then centrifuged at 1000 rpm for 5 min. After the supernatant was discarded, the pelleted cell sheets were submitted to the same procedures mentioned above. Finally, the cell sheets were carefully transferred to culture plates each containing 5 mL of the same culture medium as described above and cultivated at 37 °C under 5% CO2.
Immunohistochemistry of cryopreserved PDLSC sheets
A series of 5-μm thick cryosections of cPDLSC sheets were prepared and incubated at room temperature for 60 min in 50 mM Tris-buffered saline with 0.4% Triton X-100 (TBS-T; pH 7.2) containing 5% bovine serum albumin (BSA). For the detection of fibronectin, the cells were stained overnight with 1:500 anti-fibronectin (Sigma-Aldrich) diluted in TBS-T containing 1% BSA. After washing to remove the unbound primary antibodies, the cells were then incubated at room temperature for 1 h with 1:200 fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin M (Chemicon, USA) diluted in TBS-T containing 1% BSA. For the detection of integrin, 1:500 anti-integrin (Sigma-Aldrich) and 1:200 FITC-labeled goat anti-mouse immunoglobulin M (Chemicon) were used as the primary and secondary antibodies, respectively. For the detection of collagen type I, 1:1000 anti-collagen type I (Sigma-Aldrich) and 1:200 phycoerythrin-labeled goat anti-mouse immunoglobulin G (Chemicon) were used as the primary and secondary antibodies, respectively. The fluorescently labeled cell sheets were viewed under a confocal laser scanning microscope (Carl Zeiss LSM700, Germany).
Cell viability assay
After thawing, the viability of the PDLSC sheets was assessed by the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen). Briefly, the cell sheets were washed with phosphate-buffered saline (PBS) and then the cell sheets were mechanically disrupted, followed by incubation for 45 min at room temperature in 150 μL combined Live/Dead solution provided in the kit. Imaging was performed using a microscope (Olympus IX71, Japan) at 10× magnification. Ten fields of microscope were used for statistics of the cell viability rate. Live cells emitted green fluorescence due to the cleavage of membrane-permeated calcein AM by intracellular esterases, whereas dead cells were characterized by their emission of red fluorescence generated from the labeling of nucleic acids by membrane-impermeable ethidium homodimer-1.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
PDLSC sheets were mechanically disrupted. The mechanically disrupted cells and the fresh PDLSCs (fPDLSCs) were seeded into 96-well plates at a cell density of 1 × 104 cells/mL, followed by cultivation in α-MEM with 15% FBS at 37 °C under 5% carbon dioxide for 24 h, 48 h, and 72 h, respectively. Subsequently, the culture medium was replaced with 5 mg/mL of MTT solution (Sigma-Aldrich) diluted in PBS. The plates were incubated again for 4 h at 37 °C and a volume of 150 μL DMSO was added to each well. The plates were then agitated for 10 min to ensure the dissolution of any remaining crystals. Optical density (OD) was measured at 490 nm (A 490).
Multilineage differentiation potential
List of primers used in real time polymerase chain reaction
G-banded karyotype analysis was employed to examine the chromosomal stability of cPDLSC and fPDLSC sheets. Preparation of chromosomes was performed using a previously described protocol with minor modifications . In brief, the cell sheets were disrupted and the resultant cells were detached by being treated with a final concentration of 0.2 μL/mL colcemid for 2.5 h. Then, 8 mL hypotonic solution containing 0.075 M KCl was added and the resulting cell suspension was incubated for 30 min at 37 °C. The cells were then fixed using methanol-acetic acid (3:1) fixative and centrifuged at 1500 rpm for 10 min. The fixation experiment and the subsequent centrifugation was performed a total of three times. The pellet was re-suspended in fresh fixative (as above) and spread on slides. A drop of the suspension was carefully placed onto a wet microscope slide and allowed to dry under moderate humidity (around 50%). Giemsa banding (GTG-banding) was also performed using a previously described protocol with minor modifications . In brief, slides were incubated in trypsin solution for 8–10 s, rinsed in normal saline (sodium chloride 0.9%) three times, then stained in 10% Giemsa stain (Sigma-Aldrich) in phosphate buffer (pH 6.8) for 1.5 min. Slides were then rinsed in phosphate buffer (pH 6.8) three times, dried, and mounted in Entellan mountant (Sigma-Aldrich). The chromosome number, chromosome length, kinetochore position, and G-band position were observed according to the ISCN 2005 standard .
Animal model and transplantation of cell sheets
A total of 24 female nude mice were raised under specific pathogen-free conditions up to 5 weeks. The mice were then anesthetized via an intraperitoneal injection of 10% chloral hydrate at a dose of 0.003 mL/g body weight. Nude mice were randomly assigned to two experiment groups that were transplanted with cPDLSC and fPDLSC sheets, respectively. The prepared cell sheets were transplanted subcutaneously into the dorsal site of nude mice as previously described . All animals were sacrificed 4 weeks after transplantation. The regenerated tissue samples were isolated, fixed with 4% paraformaldehyde, and subjected to histological examinations.
Sirius Red staining
Sirius Red staining was conducted as previously described . Briefly, the tissue sections were dewaxed, dipped into water, stained with 1 g/L Picric Acid-Sirius Red at 37 °C for 1 h, and then washed with water. The sections were mounted and viewed under a polarized light microscope (Leica DM5000 B, Germany) and darkfield images were obtained.
Student’s t test was used to analyze the differences between experimental groups. P < 0.05 was considered statistically significant. All experiments were performed in triplicate.
The ECM in cPDLSC sheets was not disrupted by freezing or thawing
Cryopreservation and thawing had no detectable impact on the viability of PDLSCs
Cryopreservation and thawing did not negatively affect the proliferative capacities of PDLSCs
MTT value (mean ± standard deviation)
0.46 ± 0.04
0.49 ± 0.04
0.56 ± 0.03
0.45 ± 0.03
0.50 ± 0.03
0.53 ± 0.03
0.40 ± 0.05
0.48 ± 0.04
0.53 ± 0.04
cPDLSC sheets maintained their multilineage differentiation potential
Cryopreserved PDLSCs show no alterations of karyotype
cPDLSC sheets regenerated tissue in nude mice
Our investigation found that cPDLSC sheets can preserve PDLSC architecture similar to fPDLSC sheets in terms of cell proliferation rate, cell viability, karyotype analysis, and multilineage differentiation potential, which is set to herald the coming of cell-sheet products entering the clinical arena.
In order to obtain many cell-sheet products, cryopreservation takes on an important part of the process. DMSO  is one of the most commonly used cryoprotectants to aid in the long-term storage of viable biomaterials due to its ability to penetrate the cell membrane and reduce the formation of ice crystals during the freezing process . There is also evidence that the electrostatic interaction between DMSO and the phospholipid components of the cell membrane is an important contributor to the cryoprotective effects of the former . However, similar to many other cryoprotective agents, DMSO can exhibit cytotoxicity, particularly at a high concentration over 40% (5.1 M) [29–31]. It was shown that the use of 1–1.5 M DMSO provided the optimal protection in the cryopreservation of both dental pulp stem cells and tissues . This finding was consistent with the choice of Kaku et al. of 10% (1.3 M) DMSO as the cryoprotectant for the long-term storage of whole teeth at –150 °C . Based on these results, we prepared a cryopreservation solution consisting of 90% FBS and 10% DMSO to minimize cell damage and loss of viability during freezing. To our gratification, the cryopreserved cell sheets did not show any noticeable disruption of the ECM structure or loss of viability and proliferative potential. Consistent with our findings, Kaku et al.  and Vasconcelos et al.  showed that long-term storage of periodontal ligament cells and cryopreserved periodontal ligament-derived undifferentiated mesenchymal cells in 10% (1.3 M) DMSO did not negatively affect their in vitro proliferative capacities or cell viability. These results demonstrated the feasibility of cryopreservation as a potential solution to the long-term storage of PDLSC sheets.
The proliferative capacity of pre-differentiated stem cells is widely employed to predict the clinical outcome in patients receiving tissue and/or cell sheet transplant. In the current study, MTT assay was used to compare the proliferation rates of PDLSCs derived from cryopreserved cell sheets, freshly prepared sheets, and fPDLSCs. Interestingly, although no statistically significant difference was observed among the three groups of stem cells at 48 h and 72 h following their initial inoculation in the growth medium, PDLSCs that had undergone cryopreservation were found to proliferate at a slightly slower rate than the freshly prepared PDLSCs during the first 24 h. It is noteworthy that a similar delay was detected in the periodontal regeneration of cryopreserved molar transplants during a 4-week period after the operation . Taken together, we demonstrated that, although PDLSCs freshly exiting the freezing stage require a period of adaptation to become sufficiently metabolically competent for growth, they retained their proliferative potentials once fully recovered.
Multilineage differentiation potential is one of the distinguishing features of mesenchymal stem cells. Although cryopreservation of adipose-derived stem cells [34, 35] and bone marrow-derived mesenchymal stem cells  has been achieved without the loss or alteration of their multipotent properties, no investigation has been conducted on PDLSCs. Based on our preliminary findings  and current study, the ability of cPDLSC sheets and fPDLSC sheets to differentiate into osteoblasts was demonstrated by both von Kossa staining and the detection of several osteogenesis-related regulator and effector genes, including RUNX2, OSX, and OCN. On the other hand, when induced in an adipogenic induction medium, both types of cell sheet were also shown to readily undergo adipogenesis. Oil red O staining and the detection of several adipogenesis-related regulator and effector genes, such as PPARγ2 and LPL, confirmed similar levels of intracellular triglyceride accumulation in the differentiated adipocytes derived from cPDLSC sheets and fPDLSC sheets. Furthermore, no significant difference in the expression of key lipogenic genes was observed by RT-PCR. Therefore, the experimental findings suggested that cryopreserved PDLSC sheets were able to maintain their ability to differentiate into multiple lineages.
Karyotype analysis is frequently performed on cryopreserved tissues to assess the impact of freezing on chromosomal stability and integrity, features that are often associated with oncogenesis . Our current study found no obvious karyotypic re-arrangement for the cPDLSC sheets, which was consistent with the report of Ding et al. that cryopreserved stem cells from apical papilla retained the normal karyotype . Similarly, Imaizumi et al.  and López et al.  both observed no chromosomal structural abnormalities in cryopreserved human induced pluripotent stem cells and adipose-derived stem cells, respectively. Recently, it was shown that the differentiation potency of induced pluripotent stem cells was correlated with their telomere length . These findings, coupled with our experiment data, offer convincing evidence that our cPDLSC sheets could maintain similar differentiation capacity as fPDLSC sheets.
Cell-ECM interactions play a critical role in tissue regeneration by producing extracellular signals that can stimulate cell proliferation and matrix remodeling . ECMs have also been established to be able to directly interact with a wide range of cell surface receptors and soluble factors through which various aspects of cellular activities are tightly modulated . In our current study, the cPDLSC sheets were shown to be able to preserve ECM and its components, including fibronectin, type I collagen, and integrin, etc., in the absence of additional matrix or support. After 4 weeks being transplanted subcutaneously into the dorsal site of nude mice, the transplanted cPDLSC sheets formed bone-like matrix which exhibited a dense and interconnected network of collagen I and III, similar to that observed in tissues generated from freshly prepared cell sheets (Fig. 6). Taken together, cPDLSC sheets can be a feasible alternative for fPDLSC sheets, with an opportunity to use them in tissue regeneration.
Our combined experimental data indicated that cryopreservation maintained the structural integrity and functional viability of PDLSC sheets. Therefore, the cryopreservation method, coupled with the Vc-based induction protocol that we have previously developed for cell sheet generation, could potentially serve as a solution for tissue regeneration. Further research is needed to better understand the long-term clinical effect of our cell-sheet engineering method.
Bovine serum albumin
Cryopreservation periodontal ligament stem cell
Fetal bovine serum
Freshly prepared periodontal ligament stem cell
Hematoxylin and eosin
Periodontal ligament stem cell
Peroxisome proliferating activated receptor γ
Reverse-transcription polymerase chain reaction
Runt-related transcription factor 2
Tris-buffered saline with 0.4% Triton X-100
Alpha-modified Eagle’s medium
We gratefully acknowledge the technical support from the Director of Shandong Provincial Key Laboratory of Oral Tissue Regeneration. We acknowledge all the laboratory members for their contributions.
This study was supported by grants from National Natural Science Foundation of China (81470709, 11202118); Construction Engineering Special Fund of “Taishan Scholars” (tsqn20161068, ts201511106); Jinan Scientific Research Program (201302030); and Key research and development project of Shandong Province (2015GGH318018).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
FW conceived and designed the experiments. ML performed the experiments. CF and XG assisted the experiments. ML, CF, XG, and QH analyzed the data. ML wrote the paper. FW revised the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethical approval and consent to participate
PDLSCs were obtained from the extracted teeth of donors from which informed consent had been obtained. All protocols for handling dental tissues were performed in accordance with relevant guidelines and regulations. All protocols for the handling of human tissues were approved by the Research Ethics Committee of Shandong University (No. MECSDUMS2012087). The animal study was reviewed and approved by the Committee on the Ethics of Animal Experiments of Shandong University (No. ECAESDUSM2012075).
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