Influence of mesenchymal stem cells niche in their ostegenic fate on different surfaces.

: Bone innate ability to repair without scaring is surpassed by major bone damage. Current gold-standard strategies do not achieve a full recovery of 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, are influenced by the tissue source. The aim of this study is to define the MSC source and construct (MSC and scaffold combination) most interesting for its clinical application in bone regeneration. iTRAQ generated the hypothesis that anatomical proximity to bone has a direct effect on MSC phenotype. MSCs 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. 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. We propose DPSCs as a suitable MSCs for bone regeneration cell-based strategies.


Introduction
Bone regeneration and bone remodelling can be considered as two sides of the same coin. While bone remodelling is a life-long process, bone regeneration occurs mainly during bone healing. [1] It is known that for small fractures or injuries, the bone has an innate ability to repair without scaring. [2] These regenerative processes are largely surpassed by major bone damage such as skeletal reconstruction surgeries, bone defects of varied origins (traumatic, infectious and tumoral) or congenital skeletal dysplasias. Current orthopaedic surgery strategies are mostly bone grafting (both autologous and allogenic) and osteodistraction. [3] They can be combined with the use of growth factors or osteoinductive scaffolds. However, the successful recovery of the bone biomechanical properties is limited. To bypass these limitations, novel strategies have been developed. Among those, hybrid materials consisting of osteoprogenitor cells, like mesenchymal stem cells (MSCs), and bioactive ceramic scaffolds have been proposed as promising tools. [4] 3 of 21 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, project title "Estudio del comportamiento funcional en scaffolds biocerámicos de células madres mesenquimales obtenidas de distintos orígenes" code nº 13/247-E, PI Dr. Benjamín Fernández-Gutiérrez Servicio de Reumatología Hospital Clínico San Carlos, el Comité informa favorablemente Madrid 08 Julio 2013) and the principles expressed in the Declaration of Helsinki. Discs of CaPs bioceramics used in this study were synthesised, sintered, and polished if needed. They were kindly supplied by Instituto de Cerámica y Vidrio (ICV-CSIC). Discs had a diameter of 18mm and 4mm thickness. Sintering temperature employed for β-TCP was 1130 ºC and for HA was 1250 ºC.

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% CO2 atmosphere. The medium was changed every 3 days until cell confluence at passage 3.

Cell characterization
In order to confirm that cells satisfied the minimal criteria for the definition of MSC proposed by the International Society for Cellular Therapy, [26] flow cytometry and histochemistry assays were carried out as we previously described. [27] 2.5. Biological features on plastic surface 2.5.1. MSC proliferation rate During cell expansion, cell proliferation was evaluated by calculating the population doubling time (Dt), which is defined by: Dt= T ln2/ln(Nf/Ni) Where "T" represents the time elapsed between determinations of the final number of cells (Nf) obtained from an initial cell number (Ni).

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 quantified, in terms of percentage, with ImageJ 1.43v software (National Institutes of Health, free available: https://imagej.nih.gov/ij/index.html).
2.6. Cell behaviour on CaPs 2.6.1. 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 Scientific, 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 fixation with a phosphate buffer solution containing 4% paraformaldehyde and 2.5% glutaraldehyde for 30 minutes. After fixation, 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, finally, 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 final 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 Scientific, Cat#37629), following the manufacturer instructions, and measuring the absorbance at 405nm.

Cell behaviour on CaPs
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 significance p<0.05 was considered statistically significant. 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. Results obtained revealed the identification of 1012 unique proteins in samples. Fifty of those proteins display statistically significant differences ( Table 2). Among those, 5 proteins have been previously associated with the osteoblast differentiation process: PALLD, HSPA5/GRP78, FLNA, IGFBP3 and DSTN.

Biological features on plastic surface
The proliferation rate and osteogenic potential of MSCs when cultured on plastic surfaces were measured. Figure 1 shows the proliferation rate results. BM-MSCs and ASCs showed identical doubling time while DPSCs proliferated more rapidly (ASCs=10 days, BM-MSCs=10 days, DPSCs=1.76 days, p=0.0002). Furthermore, Alizarin Red staining showed that DPSCs presented a much more extended stained area than the other sources (Figure 2), and its quantification ratified the significant differences between MSCs (ASCs=9.4210%, BM-MSCs=39.7150%, DPSCs=72.5965%, p=0.0038).

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.

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 influence the osteogenic potential is required. Anatomical localization of the cells used is among those characteristics. To our knowledge, this is the first 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. Specifically, results obtained after comparing these locations in healthy individuals were analysed in a free-hypothesis context. Fifty proteins were identified ( 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 flux. [29][30][31][32] In contrast, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 November 2020 doi:10.20944/preprints202011.0349.v1 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 cartilage. This supports the hypothesis that an anatomical proximity to bone has a direct effect on MSC phenotype in terms of increased osteogenic commitment.
To confirm this hypothesis, we analysed the biological behaviour 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 defined as MSCs ( Figure S1). [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 cellbased 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 (Figure 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 red-coloured. Microscopy images show that DPSCs exhibit the most intense staining, and its quantification evidence significant differences ( Figure 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 confirms 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 cellfriendly than HA (Figure 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 fluids, an initial interaction occurs between the living bone and tissue and the implant surface. In this sense, the use of materials filled 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 significant at 7 days ( Figure  4). SEM images (Figures 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. Specific images of the pores existing in both CaPs proof a pore successfully colonization by all MSCs after 24 hours (Figure 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 (Figure 8) mismatch viability results ( Figure 3): while β-TCP appears to be the best in terms of viability, HA obtained the highest values in ALP activity. These 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 significant. Interestingly, this pattern in ALP activity also appears in recent studies that compare multi-sources derived MSCs seeded on plastic surfaces, although these studies obtained significant differences. [20,21] The absence of significance in our results could be due to the osteoinductive properties that both CaPs present, which the plastic surface lacks. [44,45] Since DPSCs present the highest metabolic activity during osteogenesis on both CaPs ( Figure S2), we consider conceivable that CaPs enhance the osteogenesis preferentially on DPSCs over other MSCs. In relation to biomaterial-cells combinations, the highest increase in ALP activity, although not significant, was obtained by DPSCs+HA combination.

Conclusions
In summary, our results point to DPSCs as ideal cell in bone regeneration scenario. Within bone regeneration, DPSCs might be especially beneficial 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 Identifier: 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 field of dentistry and bone regeneration. [47,48] Supplementary Materials: Figure S1: Characterization of MSCs, Figure S2: Cell activity during induced osteogenesis.