Influence of Mesenchymal Stem Cell Sources on Their Regenerative Capacities on Different Surfaces

Current gold-standard strategies for bone regeneration do not achieve the optimal recovery of bone biomechanical properties. To bypass these limitations, tissue engineering techniques based on hybrid materials made up of osteoprogenitor cells—such as mesenchymal stem cells (MSCs)—and bioactive ceramic scaffolds—such as calcium phosphate-based (CaPs) bioceramics—seem promising. The biological properties of MSCs are influenced by the tissue source. This study aims to define the optimal MSC source and construct (i.e., the MSC–CaP combination) for clinical application in bone regeneration. A previous iTRAQ analysis 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, then cultured both on a plastic surface and on CaPs (hydroxyapatite and β-tricalcium phosphate), to compare their biological features. On plastic, MSCs isolated from dental pulp (DPSCs) presented the highest proliferation capacity and the greatest osteogenic potential. On both CaPs, DPSCs demonstrated the greatest capacity to colonise the bioceramics. Furthermore, the results demonstrated a trend that DPSCs had the most robust increase in ALP activity. Regarding CaPs, β-tricalcium phosphate obtained the best viability results, while hydroxyapatite had the highest ALP activity values. Therefore, we propose DPSCs as suitable MSCs for cell-based bone regeneration strategies.


Introduction
Bone regeneration and bone remodelling can be considered two sides of the same coin. While bone remodelling is a life-long process, bone regeneration occurs mainly during bone healing [1]. It is described that, for small fractures or injuries, the bone has an innate ability to repair without scarring [2]. These regenerative processes are largely surpassed by major bone damage, such as skeletal reconstruction surgeries, bone defects of various origins (e.g., traumatic, infectious, or tumoral), or congenital skeletal dysplasias. Current orthopaedic surgery strategies are mostly bone grafting (both autologous and allogeneic) and osteodistraction [3]. They can be combined with the use of growth factors or osteoinductive scaffolds. However, the successful recovery of the biomechanical properties of bone is limited. To bypass these limitations, novel strategies have been developed. Among these, hybrid materials consisting of osteoprogenitor cells, such mesenchymal stem cells (MSCs) and bioactive ceramic scaffolds, have been proposed as promising tools [4].

Samples
MSCs from three different localisations (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. Discs of CaPs bioceramics used in this study were synthesised, sintered, and polished (if needed). They were kindly supplied by the Instituto de Cerámica y Vidrio (ICV-CSIC). The discs had a diameter of 18 mm and 4 mm thickness. The sintering temperature employed for β-TCP was 1130 • C, and that for HA was 1250 • C [25,26].

Cell Isolation and Culture
ASCs were obtained from adipose tissue after surgical biopsies, according to Yang et al. [27]. DPSCs were isolated after dental pulp mechanical extraction from wisdom exodontias, as described by Huang et al. [28]. 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. [29].
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. [30]. Cell cultures were expanded at 37 • C in a 5% CO 2 atmosphere. The medium was changed every 3 days until cell confluence at passage 3.

Cell Characterisation
In order to confirm that cells satisfied the minimal criteria for the definition of MSC proposed by the International Society for Cellular Therapy [31], flow cytometry and histochemistry assays were carried out, as we have previously described [32].  Prior to cell seeding, the scaffolds were previously submerged in growth medium for 24 h, then seeded with 1 mL of a cellular suspension containing 50,000 cells.
Monitoring of cell viability was conducted using the colourimetric indicator Ala-marBlue™ (Cat#Y00-100. Thermo Fisher Scientific, Waltham, MA USA). The variation of absorbance at 570 nm was measured at 24 h, 4 days, and 7 days using a Heales MB-580 microplate reader (Shenzhen Heales Technology Development Co. Ltd., Shenzhen, China). The amount of absorbance corresponded to cell metabolic activity.

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

ALP Activity
Early osteoblast differentiation was evaluated by measuring the alkaline phosphatase (ALP) activity, which is expressed just before the matrix mineralisation occurs, and its role as an osteogenic activity marker is established [33]. The evaluation was made using 24-well plates, and three conditions for each sample were evaluated: cell in plastic with growth medium as an internal control, and cells seeded on HA with osteogenic medium or β-TCP with osteogenic medium. Duplicates for each experimental condition were made.
Osteogenesis progression was measured between 24 h and 7 days. At these time points, media were discarded, and ceramic discs recovered, washed with PBS, stored at −20 • C, and soaked in lysis buffer (0.1 wt%, Triton-X 100, 1 mM MgCl 2 , 0.1 mM ZnCl 2 ). 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 405 nm.

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism version 7.00 software for Windows (GraphPad Software, La Jolla, CA, USA; www.graphpad.com, accessed on 5 November 2020). We used one-way ANOVA followed by Bonferroni post-hoc tests and two-tailed paired/unpaired Student t-tests for comparison of normal variables. The level of significance p < 0.05 was considered statistically significant. Each experiment was performed with replicates.

iTRAQ Results Analysis
The difference in protein components between MSCs from subchondral bone and cartilage were analysed using an iTRAQ-based comparative analysis. The results obtained revealed the identification of 1012 unique proteins in the samples. Fifty of these proteins displayed statistically significant differences (Table 2). Among those, five 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 grown on β-TCP and HA scaffolds, were studied by SEM at both 24 h and 7 days after culture. In all cases, cells did not show visual signs of cytotoxicity. Cells were visualised in their normal shape and size.

Discussion
In the bone regeneration context, therapies based on the use of osteoprogenitor cells have shown promise. However, the full therapeutic potential of these techniques has not yet been achieved. A full understanding of how different biological aspects influence the osteogenic potential is required. The anatomical localisation of the cells used is among those characteristics. To our knowledge, this is the first comparative work that has analysed a possible osteogenic commitment depending on the anatomic localisation of non-commercial human MSCs from bone marrow, adipose tissue, and dental pulp seeded both on a 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 analysis, where MSCs from subchondral bone and cartilage were compared in an osteoarthritis (OA) study. OA is a condition characterised by excessive bone growth, with the main affected tissues being subchondral bone and cartilage. Specifically, the results obtained after comparing these locations in healthy individuals were analysed in a freehypothesis context. Fifty proteins were identified ( Table 2). Five of those 50 proteins have been communicated as being related to the osteogenic process. FLNA, HSPA5/GRP78, and PALLD were up-regulated in subchondral bone, and their expression has been correlated with osteoblast differentiation, as they contribute to the stabilisation of cytoskeleton, which is necessary for the osteogenesis and regulation of protein folding and calcium flux [34][35][36][37]. In contrast, DSTN and IBP3-inhibitors of osteoblast differentiation [38,39]-were downregulated in MSCs isolated from the 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 anatomical proximity to bone has a direct effect on MSC phenotype, in terms of increased osteogenic commitment. Further studies are required to clarify whether this commitment is due to cell location or cell inherent properties.
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 A1) [31].
On plastic surfaces, proliferative ability and osteogenic potential were studied. Evaluating cell proliferation is important for cell-based therapies, as it has been communicated that stem cell therapy failures are likely to be due to a massive cell death occurring after cell transplantation [40]. Our results established that DPSCs have the highest proliferation rate ( Figure 1A), in line with previous studies [19,24]. Furthermore, it was observed that DPSCs presented a smaller size. As DPSCs have a higher proliferation rate, this smaller size is likely a consequence, due to the indirect relationship between proliferation and cell size [41]. Alizarin Red staining is commonly used as an osteogenic differentiation indicator, as mineralized nodules are red-coloured. Microscopy images show that DPSCs exhibited the most intense staining, with its quantification evidencing significant differences ( Figure 1B). These results indicate that DPSCs presented the most osteogenic capacity in vitro, followed by BM-MSCs and ASCs as the least osteogenic. This staining pattern confirms and extends the results obtained by Tamaki et al. [19]. Furthermore, it has been described that DPSCs likely have an advantage for osteogenic differentiation over other MSCs [22], as DPSCs only differentiate into osteoblasts at high passages [42].
Once the constructs were generated, we studied their cell viability, colonisation ability, and osteogenic capacity. The viability test showed that β-TCP was more cell-friendly than HA (Figure 2A,C). 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 [43]. Regarding MSCs, the viability test was favourable for DPSCs at 24 h and 4 days, compared to ASCs or BM-MSCs; however, these differences were not significant at 7 days ( Figure 2B). SEM images (Figures 3 and 4) were coherent, as only DPSCs developed a cell layer on HA; whereas, on β-TCP, all three source-derived MSCs achieved this. CaPs are porous, so pore colonisation by the cells is required for the optimal colonisation of the CaP. Specific images of the pores existing in both CaPs prove the pores were successfully colonised by all MSCs after 24 h ( Figure 5). Furthermore, cytotoxicity was not observed in the studied samples. Remarkably, DPSCs showed the greatest proliferation ability on both plastic and bioceramic surfaces. This feature provides an advantage for bone regeneration, as high MSCs density enhances osteogenic differentiation [44]. These results, together with those on plastic surfaces and previous studies, highlight the features of DPSCs for dental and bone regeneration applications [21,45].
The interplay of the tissue engineering triad (cells, signalling 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 pulpdentin complex and bone defects. In the literature, it is established that the mechanical properties of the scaffold influence cell processes such as proliferation and differentiation [46]. Recent data have implied that β-TCP is a bioactive and biocompatible material, capable of enhancing the proliferation, migration, and adhesion of DPSCs. Moreover, recent data have been conclusive about the higher levels of osteogenic and odontogenic differentiation markers related to DPSCs, 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 [47].
Regarding osteogenic potential, the ALP activity test results ( Figure 6A) mismatched with the viability results ( Figure 2A): while β-TCP appeared to be the best in terms of viability, HA obtained the highest values in ALP activity. These results indicate that a combination of materials may be more effective. Referring to cell sources, ASCs and BM-MSCs presented similar absolute values, which were slightly superior to that of DPSCs, but not significant ( Figure 6B). Interestingly, this pattern in ALP activity was obtained in recent studies comparing multisource-derived MSCs seeded on plastic surfaces, although authors communicated significant differences [23,24]. The absence of significance in our results could be due to the osteoinductive properties that both CaPs present, which the plastic surface lacked [48,49]. As DPSCs presented the highest metabolic activity during osteogenesis on both CaPs ( Figure A2), we consider it conceivable that CaPs can enhance osteogenesis preferentially in DPSCs over other MSCs.

Conclusions
Our results indicate DPSCs as the ideal cell for bone regeneration scenarios. Within bone regeneration, DPSCs are potentially 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 regarding the use of DPSCs for periodontal regeneration have been published [50]. Moreover, a combination of the best viability obtained with β-TCP and the enhanced osteogenic capacity of HA may be appropriate. Future studies are necessary to obtain the best combination of cells and biomaterials, together with other signalling enhancers or inhibitors and different proteins that have been demonstrated in the field of dentistry and bone regeneration [51].