Dental Pulp Stem Cells: an Alternative Source of Mesenchymal Stem Cells with Potential Osteogenic Regenerative Capacities

Background: Bone innate ability to repair without scaring is surpassed by major bone damage. Current gold-standard strategies do not achieve a full recovery of the bone biomechanical properties. To bypass these limitations, tissue engineering techniques based on hybrid materials made up of osteoprogenitor cells, like mesenchymal stem cells (MSCs), and bioactive ceramic scaffolds, like calcium phosphate-based (CaPs), are promising. Biological properties of the MSCs, including osteogenic potential, are inuenced by the tissue source. The aim of this study is to dene the MSC source and construct (MSC and scaffold combination) most interesting for its clinical application in the context of bone regeneration. Methods: MSCs of 9 healthy donors were isolated from adipose tissue, bone marrow and dental pulp. MSCs were cultured both on plastic surface and on CaPs (hydroxyapatite and β-tricalcium phosphate) to compare their biological features: proliferation rate, osteogenic potential, cell viability and activity, ability to colonize the CaPs and ALP activity. Results: iTRAQ results generated the hypothesis that anatomical proximity to bone has a direct effect on MSC phenotype. On plastic, MSCs isolated from dental pulp (DPSCs) were the MSCs with the highest proliferation capacity and the greatest osteogenic potential. On both CaPs, DPSCs are the MSCs with the greatest capacity to colonize bioceramics. Furthermore, results show a trend for DPSCs are the MSCs with the most robust increase in the ALP activity. Conclusion: Based in our results, we propose DPSCs as a suitable MSCs for bone/dental regeneration cell-based strategies.

osteogenesis in animal models has been described. [13] MSCs isolated from dental pulp (DPSCs) are usually isolated after surgical removal of wisdom teeth, so they are considered as a non-invasive source of MSCs. DPSCs present a high proliferative capacity and easily differentiate into odontoblasts, osteoblasts and chondrocytes. [14] DPSCs have been recently proposed to be used in regenerative therapies for bone diseases, among other condition. [15] Considering the osteogenic potential as a property in uenced by the tissue, several comparative studies of MSCs derived from various sources on different surfaces have been published. [16][17][18][19][20][21] Despite these, a clari cation is still needed regarding the in uence of the cell source and surface in the osteogenic potential. Osteogenic potential maximization in a context of the bone-regeneration process would be an important step. The aim of this study is to de ne the MSC source and construct (MSC and scaffold combination) with the highest osteogenic potential.

Methods
Proteomic analysis by iTRAQ labelling Data obtained in a previous proteomic analysis where MSCs from subchondral bone and cartilage were compared (not published). Speci cally, results obtained after comparing these locations in healthy individuals were analysed.
Brie y, MSCs were obtained surgically from subchondral bone and cartilage of a donor without osteoporotic or osteoarthritis signs. Total protein content was isolated, dried in air and then re-suspended in 25μl of iTRAQ dissolution buffer (ABSciex, Foster City, CA, USA). Protein concentrations were determined by Bradford assay (Sigma-Aldrich, St. Louis, MO, USA). TRAQ labelling (ABSciex, Foster City, CA, USA) was performed with SB(116) and C(117) mass tags and desalted with home-made C-18 Stage-tips. Fractions separated in a nanoLC system (Tempo, Eksigent) were automatically deposited on a MALDI plate and analysed by MSMS (4800 MALDI-TOF/TOF system. ABSciex). Relative quantitative analysis was done using ProteinPilot software (ABSciex) using the Paragon™ Algorithm for protein identi cation and quanti cation. Only proteins identi ed with at least 95% con dence, a Prot Score of at least 1.3, a p-value ≤ 0.05 and ratio (≠1) were considered as modulated.

Samples
MSCs from 3 different localizations (bone marrow, dental pulp, and adipose tissue), obtained from healthy donors (Table 1), were used for in vitro biological studies. Written informed consent was obtained from all donors before sample collection. The study was approved following the guidelines of the institutional ethics committee (Comité Ético de Investigación Clínica Hospital Clínico San Carlos) and the principles expressed in the Declaration of Helsinki. Cell isolation and culture ASCs were obtained from adipose tissue after surgical biopsies, according to Yang et al. [22] DPSCs were isolated after dental pulp mechanical extraction from wisdom exodontias, as described by Huang et al. [23] Finally, BM-MSCs were obtained from femoral channel aspirates of bone marrow, taken during joint replacement surgery, in a Ficoll density gradient and cultured directly as described by Gudlevicine et al. [24] Once isolated, cells were expanded in growth medium: DMEM supplemented with 10% FBS and antibiotics; DPSCs required 20% FBS instead of the 10% usually established as described by Alkhalil et al. [25] Cell cultures were expanded at 37ºC in a 5% CO 2 atmosphere. The medium was changed every 3 days until cell con uence at passage 3.

Cell characterization
In order to con rm that cells satis ed the minimal criteria for the de nition of MSC proposed by the International Society for Cellular Therapy, [26] ow cytometry and histochemistry assays were carried out as we previously described. [27] Biological features on plastic surface Osteogenic commitment of each MSC Alizarin Red staining was carried out along culture on plastic. The coloured area of cell cultures after alizarin red staining was quanti ed, in terms of percentage, with ImageJ 1.43v software (National Institutes of Health, free available: https://imagej.nih.gov/ij/index.html).

Cell behaviour on CaPs
Cell activity/viability Prior to cell seeding, the scaffolds were previously submerged in growth medium for 24 hours and then seeded with 1ml of a cellular suspension containing 50 000 cells.
Monitorization of cell viability was performed using the colourimetric indicator AlamarBlue™ (Cat#Y00-100. Thermo Fisher Scienti c, Waltham, MA USA). The variation of absorbance at 570nm was measured at 24 hours, 4 and 7 days using Heales MB-580 microplate reader (Shenzhen Heales Technology Development Co. Ltd. Guangdong. China). The amount of absorbance corresponds to cell metabolic activity.

Scanning Electronic Microscopy (SEM)
The cellular organization, adhesion and colonization of the scaffolds were assessed at 24 hours and 7 days. Each sample was subjected to xation with a phosphate buffer solution containing 4% paraformaldehyde and 2.5% glutaraldehyde for 30 minutes. After xation, samples were washed 3 times with phosphate-buffered saline (PBS) for 20 minutes followed by incubation for 45 minutes with a solution of 1% osmium tetraoxide and, nally, washed again with PBS 3 times for 10 minutes. The next step was the dehydration of the samples by immersing them in increasing ethanol concentrations: 30%, 50%, 70%, 96% and 100%. The nal step was to introduce the samples into the critical-point device and cover them with vaporized gold. Samples were observed and analysed by scanning electron microscopy (SEM JEM 6400, JEOL, Japan).

ALP activity
Early osteoblast differentiation was evaluated by measuring the alkaline phosphatase (ALP) activity, which is expressed just before the matrix mineralization occurs and its role as osteogenic activity marker is established [28]. The evaluation was made using 24-well plates and 3 conditions for each sample were evaluated: cell in plastic with growth medium as an internal control; cells seeded on HA with osteogenic medium and β-TCP with osteogenic medium. Duplicates for each experimental condition were made.
Osteogenesis progression was measured between 24h and 7 days, at this time points media was discarded and ceramic discs recovered, washed with PBS and stored at -20ºC soaked in lysis buffer (0.1 wt%, Triton-X 100, 1 mM MgCl2, 0.1 mM ZnCl2). The ALP activity was determined by a colourimetric method using a commercial kit (Thermo Fisher Scienti c, Cat#37629), following the manufacturer instructions, and measuring the absorbance at 405nm.

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 7.00 for Windows, (GraphPad Software, La Jolla California USA, www.graphpad.com). We have used two-tailed paired/unpaired Student's t-test for comparison of normal variables. Normal variables were presented as mean±SD (standard deviation). The level of signi cance p<0.05 was considered statistically signi cant. Each experiment was done with replicates.

iTRAQ results analysis
The different protein components between MSCs from subchondral bone and cartilage were analysed using an iTRAQ-based comparative analysis. We have previously performed this iTRAQ in the context of osteoarthritis vs control patients and in this case we have analysed quantitative differences between subchondral bone and cartilage only from healthy control individuals.
Results obtained revealed the identi cation of 1012 unique proteins in samples. Fifty of those proteins display statistically signi cant differences (Table 2). Among those, 5 proteins have been previously associated with the osteoblast differentiation process: PALLD, HSPA5/GRP78, FLNA, IGFBP3 and DSTN.

Scanning Electronic Microscopy
Cell morphology and behaviour when growing on β-TCP and HA scaffolds were studied by SEM at both 24 hours and 7 days after culture. In all cases, cells do not show visual signs of cytotoxicity. Cells can be visualized in its normal shape and size.
According to cell localization (Fig. 9), each localization shows a signi cant increase in ALP activity (ASCs, t 1

Discussion
In bone regeneration context, therapies based on using osteoprogenitor cells are promising. However, the full therapeutic potential of these techniques has not been achieved. A full understanding of how different biological aspects in uence the osteogenic potential is required. Anatomical localization of the cells used is among those characteristics. To our knowledge, this is the rst comparative work that analyses a possible osteogenic commitment depending on the anatomic localization of non-commercial human MSCs from bone marrow, adipose tissue and dental pulp, seeded both on plastic surface and CaPs (β-TCP and HA).
The starting point of this work was the analysis of the results obtained in a previous iTRAQ where MSCs from subchondral bone and cartilage were compared in an osteoarthritis (OA) study. OA is a condition characterized by excessive bone growth, and the main affected tissues are subchondral bone and cartilage. Speci cally, results obtained after comparing these locations in healthy individuals were analysed in a free-hypothesis context. Fifty proteins were identi ed ( Table 2). Five out of those 50 proteins have been communicated as related to osteogenic process. FLNA, HSPA5/GRP78 and PALLD were up-regulated in subchondral bone and its expression correlated with osteoblast differentiation as they contribute to the stabilization of cytoskeleton, which is necessary for the osteogenesis, and regulate protein folding and calcium ux. [29][30][31][32] In contrast, DSTN and IBP3, inhibitors of the osteoblast differentiation, [33,34] were down-regulated in MSCs isolated from subchondral bone. Considering this background, MScs from subchondral bone display a more established commitment to osteogenesis compared to MSCs from cartigale. This supports the hyptothesis that an anatomical proximity to bone has a direct effect on MSC phenotype in terms of increased osteogenic commitment.
To con rm this hypothesis, we analysed the biological behiviour of MSCs isolated from locations with different proximity to bone (adipose tissue, bone marrow and dental pulp) on different surfaces. All cells used in this work met the minimal criteria to be de ned as MSCs (see Additional le 1). [26] When cultured on plastic surfaces, two biological characteristics of MSCs were evaluated: proliferative ability and osteogenic potential. Evaluating the cell proliferation is important for cell-based therapies since it has been communicated that failures on stem cell therapies is likely to be due to a massive cell death occurring after cell transplantation. [35] Our in vitro results established that DPSCs have the highest proliferation rate (Fig. 1), in line with previous studies. [36] Besides, it was observed that DPSCs present a smaller size. Since DPSCs have a higher proliferation rate, it is likely this smaller size is a consequence of this due to the indirect relationship between proliferation and cell size. [37,38] Alizarin Red staining is commonly used as an osteogenic differentiation indicator as mineralized nodules are redcoloured. Microscopy images show that DPSCs exhibit the most intense staining, and its quanti cation evidence signi cant differences (Fig. 2). These results indicate that DPSCs present the most in vitro osteogenic capacity, followed by BM-MSCs and ASCs as the least osteogenic. This staining pattern con rms and extends results obtained by Tamaki et al. [16] Furthermore, it has been described that DPSCs likely have an advantage for osteogenic differentiation over other MSCs, [19] and DPSCs only differentiate into osteoblasts at high passages. [39] Once MSCs were seeded on the scaffolds and constructs generated, it was studied their cell viability, colonization ability, and osteogenic capacity. Viability test showed that β-TCP is more cell-friendly than HA (Fig. 3). Both β-TCP and HA are biomaterials commonly used in bone tissue engineering and dentistry to treat bone defects. Implant surface quality is a major factor in biocompatibility. When the surface of the implanted biomaterial is exposed to tissue uids, an initial interaction occurs between the living bone and tissue and the implant surface. In this sense, the use of materials lled with tricalcium phosphate appears promising following the observation that more living cells are in this material. [40] Regarding MSCs, viability test is favourable for DPSCs at 24hs and 4 days comparing to ASCs or BM-MSCs. These differences are not signi cant at 7 days (Fig. 4).
SEM images (Figs. 5 and 6) are coherent since only DPSCs developed cell layer on HA whereas on β-TCP the three source-derived MSCs did it. CaPs are porous, so it is required pore colonization by the cells for an optimal colonization of the CaP. Speci c images of the pores existing in both CaPs proof a pore successfully colonization by all MSCs after 24 hours (Fig. 7). Besides, cytotoxicity was not appreciated in the studied samples. It is remarkable that DPSCs have shown to be the MSCs with the greatest proliferation ability on both plastic surface and bioceramics. This feature is an advantage for bone regeneration as a high MSCs density enhances the osteogenic differentiation. [41] These results indicate, together with other studies, that DPSCs could be the optimal stem cells for dental and bone regeneration. [18,42] The interplay of the tissue engineering triad (cells, signaling molecules, and scaffolds) is essential for recapitulation of the biological events of tissue regeneration. These elements have been used either separately or in combination for the reconstitution of the pulp-dentin complex and bone defects. Recent data imply that β-TCP is a bioactive and biocompatible material capable of enhancing DPSCs proliferation, migration, and adhesion. Moreover, recent data are conclusive about DPSCs higher levels of osteogenic and odontogenic differentiation markers such as COLI, DSPP, OC, RUNX2, and DMP-1. Our results suggest, in accordance with the literature, that DPSCs may be a valuable tool in the context of dental and bone regeneration. [43] Regarding osteogenic potential, ALP activity test results (Fig. 8) mismatch viability results (Fig. 3): while β-TCP appears to be the best in terms of viability, HA obtained the highest values in ALP activity. Theses results indicate that a combination of materials would be more effective. Referring to cell sources, ASCs and BM-MSCs present similar absolute values, that are slightly superior to DPSCs values but no signi cant. Interestingly, this pattern in ALP activity also appears in recent studies that compare multisources derived MSCs seeded on plastic surfaces, [20,21] although these studies obtained signi cant differences. The absence of signi cance in our results could be due to the osteoinductive properties that both CaPs present, [44,45]

Conclusions
In summary, our results point to DPSCs as ideal cell in bone regeneration scenario. Within bone regeneration, DPSCs might be especially bene cial in periodontal regeneration. Supporting this, a Phase 3 clinical study using DPSCs for alveolar cleft lip and palate repair has recently been initiated (ClinicalTrials.gov Identi er: NCT03766217), and promising results of the using DPSCs for periodontal regeneration has been published. [46] Moreover, a combination of the best viability of β-TCP and the enhanced osteogenic capacity of HA would be appropriate. Other studies will be necessaries to obtain the best combination of cells and biomaterials together with other signaling enhancers or inhibitors as different proteins have really demonstrated in the eld of dentistry and bone regeneration. [47,48]  Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests Funding This work was supported by grants RIER (Instituto de Salud Carlos III) and FIS PI13/01863 (Instituto de Salud Carlos III).
Authors' contributions AM and EV performed the experiments and collected and assembled data. EM, AG-C, MJP-S, PP and CB provied study materials or patients. AM, LA and LR-R performed the data analysis and interpretated the results. AM writed the manuscript. JRL and BF-G planned the study and reviewed and edited the paper. All authors read and approved the nal manuscript.