Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues ? A Systematic Review

biological properties (Cell survival rate (CSR), proliferation, differentiation, maintenance of stem cell markers) of stem cells obtained from dental tissues (DSC) post-thaw. An electronic search was carried out within PubMed and ISI Web Science by using specific keyword. Two independent reviewers read the titles and abstracts of all reports respecting predetermined inclusion/exclusion criteria. Data were extracted considering the biological properties of previously cryopreserved DSCs and previously cryopreserved dental tissues. DSCs cryopreserved as soon as possible after their isolation presents a CSR quite similar to the non-cryopreserved DSC. Dimethyl sulfoxide (DMSO) [10%] showed good results related to cell recovery post-thaw to cryopreserve cells and tissues for periods of up to 2 years. The cryopreservation of DSC in a mechanical freezer (-80°C) allows the recovery of stem cells post-thaw. The facilities producing magnetic field (MF), demand a lower concentration of cryoprotectant, but their use is not dispensable. It is possible to isolate and cryopreserve dental pulp stem cell (DPSC) from healthy and diseased vital teeth. Cryopreservation of dental tissues for late DSC isolation, combined with MF dispensability, could be valuable to reduce costs and improve the logistics to develop teeth banks. Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues? A Systematic Review


Introduction
The discovery and isolation of responsive mesenchymal stem cells (MSC) from dental tissues (1-4) raised the possibility for developing new regenerative therapies.Such MSC populations possess high proliferative, self-renewal, and multipotency, expressing the ability to differentiate in tissues, such as fat, bone, cartilage, and neural cells.MSCs could proliferate in vitro, maintaining their differentiation capacity, for a limited period, ranging for 15 to 50 population doublings (5).Currently it is clear that the number of passaging reduces the differentiation potential and the capacity of proliferation (6).Cryopreservation comprises a key method to keep MSC at early passages maintaining their biological properties.
Cryopreservation relies on a complex balance established by a well-controlled cooling rate and the cryoprotectant agent's (CA) concentration (7,8).Thus, a proper CA should allow water to leave the cell slowly enough to avoid impairment on cells' membranes, but sufficiently fast to avoid ice crystals formation inside the cell.Dimethyl sulfoxide (DMSO) -(CH 3 ) 2 SO -a polar aprotic solvent, is widely applied as CA.Due to its hydrophilic nature, DMSO induces the water output in an ideal speed reducing the heat stress during transition from liquid to solid state (8)(9)(10)(11).
However, DMSO has been shown to be cytotoxic, since DMSO can decrease the MSC's ability to proliferate and differentiate post-thaw (11).Thus, researchers have been testing alternative substances, such as glycerol and ethyleneglycol and sugars such as sucrose and trehalose, due to their reduced cytotoxicity as CA (12).Such CAs are widely used in vitrification techniques, which are based on eliminating the phase-glazing glass transition through a fast-freezing process.After that, the solution is converted into an amorphous solid, which should be free from ice crystal.In addition, new specialized facilities, such as magnetic field freezers (13)(14)(15)(16) and programed freezers (12), have been applied to improve cell biological properties and minimize the toxic effects of CA.
There is a lack of information regarding the behavior of well-characterized cryopreserved MSC from dental tissues (dental stem cells -DSC).MSCs come from tissues with specific-related characteristics containing a heterogeneous cells population (5), thus making the definition of the best CA or the best facilities to cryopreserve cells and tissues a hard task.Davies et al. (17) have shown that the effects of in vitro expansion, after 10% DMSO cryopreservation, on the viability and differentiation capacity is cell-dependent.The objective of this study has been to systematically review the literature in order to identify the influence of different cryopreservation protocols over the biological properties of dental stem cells post-thaw.

Material and Methods
This systematic review was carried out according to PRISMA statement (18).To structure the research question As for exclusion criteria, the presented systematic review did not include studies evaluating the viability of cryopreserved MSC from tissues other than dental pulp, periodontal ligaments, apical papilla, and tooth germ.
Regarding the information sources, studies were identified by searching electronic databases, including PubMed (PM) and the ISI Web of Science® (IWS), up to April 2015.The keywords selected included relevant entry terms associated with each MeSH Term database and the search was developed considering the relevant entry terms associated with each MeSH Term (Table 1).
With respect to selection of studies and data collection, pre-selected keywords were combined to retrieve the studies (Table 2).The records retrieved have been uploaded into the ENDNOTE® basic (www.myendnoteweb.com) to delete the duplicated ones.Two independent reviewers read the titles and abstracts of all found reports considering the inclusion criteria to perform later the full report evaluation.For data collection, the full version of all included studies were obtained and the data were extracted (Table 3).If any disagreement was found in relation to the inclusion of some study, the reviewers discussed the matter to obtain consensus.

Results
The initial search (Fig. 1) resulted in 1773 initial records corresponding to 482 individual, from which 32 were selected by evaluating their title and abstract for complete reading.After that, 21 papers were selected for the data extraction (Table 3).Dental pulp stem cells (DPSC) were the most cryopreserved DSC (52%).DMSO has been applied as CA in 100% of the selected studies, at concentrations  between 3% and 20% (16,19,20).Only 19% of the included studies performed the cryopreservation for periods higher than one year (20)(21)(22)(23).Specialized facilities (magnetic or programed freezers) were applied in 19% of the studies (12)(13)(14)16).
Biological Properties of Previously Cryopreserved Dental Stem Cells: Selected studies (Table 3) showed that the applied cryopreservation protocols, in periods ranging from 24h up to 2 years, have been allowed to recover DSC presenting suitable biological properties (5).DMSO in a  (14,(20)(21)(22)24).Besides, it was possible to cryopreserve DPSC by the fast-freezing technique (-80 °C) for up to 1 year, thus eliminating the pre-freezing steps (20).Lower DMSO concentration 3% (DPSC) and 5% (Human Tooth Germ Stem Cells -HTGSC) were effective to maintain cell biological properties when cryopreserved into a magnetic field freezer (14) or by adding NaB (20 µg) to CA (19).DSC cryopreserved with 10% glycerol and 10% ethylene glycol (12,25) presented similar cell biological properties when compared to those cryopreserved with 10% DMSO.When Pluronic 188 (F68) was incorporated into 10% DMSO, a slight improvement in HTGSC viability was reported (26).Biological Properties of Stem Cells Isolated from Previously Cryopreserved Dental Tissues: Selected studies depicted to be possible isolate viable DSC from previously cryopreserved dental tissues.DPSC isolated from cryopreserved dental pulps , for up to 2 years in N 2 (11,13,23,27,28), has been able to retain their ability to differentiate and express mesenchymal stem cell surface markers.In addition, pulp tissues' cryopreservation, for up to 6 months at -85°C mechanical freezer, has been shown to be a viable option to store DPSC (27).10% or 15% DMSO has not been deleterious for DSC biological properties, being better than vitrification agents such as glycerol and ethylene glycol (27).However, a modified cryoprotectant (0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol) allowed > 70% of the survival rate of human follicle stem cells after 3 months of tissue storage (12).The cells from cryopreserved human dental follicles (HDFSC) expressed mesenchymal stem cell markers in a manner similar to fresh cells (12).Moreover, it has been possible to isolate viable DPSCs from diseased 6 month-cryopreserved pulp tissue (8); DPSCs has been able to maintain their biological properties and differentiate toward hepatic cells (28).DPSC obtained from whole-tooth cryopreserved dental pulps has been shown a small recuperation rate (20%) (8,27).However, DPSC has been successfully recovered when the whole tooth has been submitted to a previous treatment with a Nd:YAG laser beam to generate microchannels across the enamel and dentin layers, improving the cryoprotectant diffusion until the pulp tissue (29).

Discussion
The presented systematic review has shown If DSC are cryopreserved after their isolation, then the biological properties such as CSR, adhesion ability, high proliferation rate, and differentiation ability (multipotency), remains quite similar to those observed in non-cryopreserved DSC.All of included studies applied DMSO in different concentration and 10% DMSO was the most-applied CA, providing post-thaw CSR above 90%.Interestingly, we could observe that DMSO concentrations under 10% were suitable for DPSC cryopreservation, thus allowing DPSC survival (-85°C or N 2 -196°C) (27) and normal karyotype maintenance (30).
The drawback related to DMSO consists on its inherent cytotoxicity, which would be detrimental for cell viability (31); thus it has been emphasized the need to reduce DMSO concentrations to ensure maximum cell yield post-thaw.However, Kumar et al. (20) after evaluating eight different DMSO-based cryopreservation for DPSC (Table 3), could recover, in every tested protocols, DPSC able to differentiate in adipose-, osteoblast-and neural-like cells.They could conclude that uncontrolled freezing (fast freezing) at -80°C has been as effective as controlled freezing (slow freezing), independently of DMSO concentration (20).Furthermore, 15% DMSO allowed the survival of DPSC from previously cryopreserved dental pulp tissues [-80°C mechanical freezer or N 2 -196°C for 6 months] (27).In both studies (20,27), every evaluated cell population has been retained their stemness surface markers (CD-90, CD-105, CD-73, CD-34, CD-45, CD-11b, CD-19, and HLA-DR) post-thaw depicting multipotency ability.DMSO concentrations up to 5% were effective (CSR ≥ 90%) when associated to chemical reagents or specific facilities producing magnetic fields (14,19).Demirci et al. (19) showed that 5% DMSO supplemented with NaB provided a CSR similar to 10% DMSO, when HTGSC has been cryopreserved.
Ice crystal formation starts in temperatures between 0 and 4°C (31) generating a weak electric current which is able to disrupt cell membranes affecting CSR post-thaw.Magnetic fields could exert a positive influence during MSC cryopreservation since prevent ice crystal formation by allowing the water molecules to be instantly frozen.DSC's CSR in a DMSO-free environment has been increased 2 or 2.5 fold, depending on the configuration of the magnetic field (16).The addition of trehalose as CA's additive has not been improved DMSO's performance during cryopreservation of dental pulp or intact teeth under magnetic field (15).Thus, considering the limitations related to the selected studies, is possible to hypothesize that lower DMSO concentration and shorter preequilibration time are needed when cryopreservation is performed in magnetic-emitting field freezer.In fact, Lee Huang et al. (14) has been shown that DPSC's biological properties post-thaw, such as viability, adhesion, proliferation, MSC surface markers expression and multipotency, were quite similar after cryopreservation with 10% DMSO in a regular freezer or 3% DMSO, FBS-free medium, in a magnetic field; both DSC population presented lower rates of apoptotic cells, suggesting a freezing process with less cell damage under magnetic fields.
Another important factor to be considered during cryopreservation is the fetal bovine serum (FBS) concentration applied.FBS possess a central role in maintaining biological properties reducing the risk of cell damage during freezing and thawing cycles (32).FBS comprises a complex mixture of growth factors, proteins, carbohydrates, cytokines, and indispensable nutrients for cell development in vitro (33).In fact, high FBS concentration (90%) has induced less cell damage when compared to concentration under 90% concentration in animal cells (32).However, there is a concern regarding FBS due to risk related to pathogens transmissions and internalization of animal's proteins, which can unleash antigenic responses in the patient post-DSC implantation (34).FBS content protein remain present in human cells after consecutive cell washings (34) and may change the cell surface markers even as induce unforeseen modification of cell biology (35).Due to such characteristics, FBS is not recommended in studies aiming to perform Stem Cell-Based Therapy (SC-BT) for clinical transition.A free-FBS cell-freezing (DMEM, 10% hetastarch, human albumin, DMSO) allowed for cryopreservation of swine DSCs for one year.Cryopreserved swine DSCs were positive for CD90, CD105, and CD146 (22).Besides, a commercially available FBS-medium (Cryostor-CS-10), worked as well as DMEM containing 10% FBS (27).
The cryopreservation of previous isolated DSCs seems to be a process that maintains the proliferation and differentiation ability of these cells.It was interesting to note that is possible to store the teeth, after extraction, for up to 120 h in PBS (-4°C) to obtain high proliferative DPSCs (36).It was also possible to recover 70% of DPSCs from a cryopreserved whole tooth after one month in liquid nitrogen (36).Conversely, other studies showed that only small rates (around 20%) of viable DPSCs could be retrieved from cryopreserved healthy (27) or diseased teeth (8).To be effective, the CA must penetrate by passive diffusion into the whole tissue (27,36), which is affected by the surface area available for CA contact.A tooth is constituted by highly mineralized tissues surrounding the dental pulp, which presents just the apical foramen for CA access the pulp.To obtain viable cells from the pulp tissue, the apical foramen from human teeth should have a minimum dimension of 9.42 mm2 (37) to allow preservation agents to be effective.Cells isolated from the apical (root) portion of rat teeth showed a high CSR when compared with cells from the other two sections-the middle and the coronal (15).Another alternative to improve the CA diffusion is to build up artificial openings to improve the CA diffusion through dental tissues.Gioventu et al. (29) showed that cells from cryopreserved teeth, submitted to previous laser-piercing (the coronal portion), showed mesenchymal stem cells' morphology, immunophenotype, viability, and a proliferation rate similar to those cells from non-cryopreserved teeth.
This systematic review depicted that DSC could be cryopreserved, mainly with DMSO [10% -20%], for periods up to 2 years maintaining their high proliferation rate, multipotency, karyotype and stem cells surface markers.The whole tooth cryopreservation (to isolate DSC) seemed to be a not reliable method and future investigation are needed in this filed.On the other hand, the cryopreservation of the intact pulp tissue seemed to constitute an attractive and reliable source to isolate DPSC.It could be valuable since avoids the immediate stem isolation before cryopreservation.However, evaluated cryopreservation times evaluated in selected studies has been too short; just 19% of the included studies have been evaluated the cryopreservation for periods longer than one year.Thus, the behavior of DSC in long times storage cannot be securely predicted.The conclusions presented in this systematic review should be interpreted with caution.

Figure 1 .
Figure 1.Flowchart from selected studies

Table 1 .
Database syntax applied to investigate the selected MeSH terms and its respective entry terms.TS= (At the ISI Web of Knowledge it field searches into abstracts, keywords, and article titles)

Table 2 .
Keywords combination and records recovered in each database.