**2. Results**

#### *2.1. Human Dental Pulp Harbours a Population of Adherent Cells with MSC Characteristics*

Accumulating evidence indicates that the dental pulp contains a unique population of adherent cells with mesenchymal characteristics, including adhesion to plastic surfaces, fibroblast-like morphology, lack of expression of CD14, CD34 or CD45 and potential to di fferentiate into osteoblasts in vitro [18,43]. Thus, in the current study, we developed and optimized the protocol for the isolation of such cells with the corresponding characteristics of MSCs (Figure 1a). The dental pulp extracted from the permanent teeth of healthy human donors was subjected to enzymatic digestion to isolate a mixture of di fferent populations of cells, which were further seeded on cell culture plates. After 10 days, we obtained a population of adherent cells that were proliferated further. Moreover, we also isolated

UC-MSCs from human UC Wharton's Jelly tissue (Figure 1b) by employing a similar approach, which were used as a "classic" control MSCs for comparison with the dental pulp- derived cells in vitro.

**Figure 1.** Isolation procedure and morphology of dental pulp stem cells (DPSCs) and umbilical cord Wharton's jelly-derived mesenchymal stem/stromal cells (UC-MSCs). (**a**) Isolation of DPSCs from pulp tissue. The upper part of the tooth was drilled and the dental pulp was extracted. The dental pulp was enzymatically digested by a mixture of collagenase I and dispase. The isolated cells and tissue sections were seeded onto cell culture plates in a complete cell culture medium. On day 10 post-seeding, non-adherent cells and tissue pieces were removed. (**b**) Isolation of UC-MSCs from Wharton's jelly. The umbilical cord was washed with PBS to remove residual cord blood, and arteries and vein were further dissected. Wharton's Jelly tissue was cut into 12 mm pieces and placed on the tissue culture dishes in a complete cell culture medium. On day five post-seeding, non-adherent cells and tissue pieces were removed. (**c**) Representative images of the morphology of DPSCs (left) and UC-MSCs (right). Scale bars: 50 μm.

We observed that both DPSCs, as well as UC-MSCs, demonstrated adhesion to plastic surfaces when maintained under standard culture conditions in vitro. Dental pulp-derived cells exhibit a morphology of spindled-shaped, fibroblast-like cells, similar to UC-MSCs (Figure 1c). By using a flow cytometry platform, we analysed the antigenic profile of isolated cells following the minimal criteria for defining multipotent mesenchymal stem/stromal cells published by the ISCT [3].

We demonstrated that the population of dental pulp-derived cells isolated in this study exhibited a high expression of MSC-specific markers, such as CD29, CD44, CD73, CD90, CD105 and Stro-1, and does not express markers specific to hematopoietic cells, such as CD45, CD14, CD34 or HLA-DR antigen (Figure 2a). UC-MSCs also express antigens typical for MSCs, such as CD29, CD44, CD73, CD90 and Stro-1, and, in parallel, do not possess CD45, CD14, CD34 and HLA-DR antigens on their surface that are considered markers of hematopoietic cells (Figure 2b). Thus, the multiantigenic phenotype of the dental pulp-derived cells is similar to the antigenic profile of UC-MSCs as shown in Figure 2c. Thus, based on their ability to adhere to plastic surfaces and antigenic profiles, we confirmed the identity of the isolated dental pulp-derived cells as previously described, representing the subpopulation of MSCs.

**Figure 2.** Antigenic profile of DPSCs and UC-MSCs with flow cytometry. Expression of MSC-negative markers (D45, CD14, CD34), MSC- positive markers (CD29, CD44, CD73, CD90, CD105, Stro-1), and HLA-DR antigen on DPSCs and UC-MSCs. (**a**) Representative histograms of the expression of analysed

antigens on DPSCs. (**b**) Representative histograms of the expression of analysed antigens on UC-MSCs. The peaks of unstained cells (grey) were overlaid with the peak for analysed antigen (violet). (**c**) Quantitative data representing the percentage content of DPSCs or UC-MSCs positive for analysed antigens. Results are presented as mean ± SD, *n* = 3.

#### *2.2. DPSCs Exhibit Wide Di*ff*erentiation Potential In Vitro*

In the next step, to answer the question about the biological potential of DPSCs with respect to their pro-regenerative ability in injured tissues, we first analysed the tri-lineage di fferentiation potential of such cells compared to UC-MSCs in vitro. For that purpose, the DPSCs and UC-MSCs were di fferentiated into osteoblasts, chondroblasts, and adipocytes after 7, 14 and 21 days in tissue-specific di fferentiation media. We observed that both DPSCs and UC-MSCs exhibit tri-lineage di fferentiation potential (as shown in Figures 3 and 4, respectively), which also confirmed their MSC phenotype as defined by minimal criteria recommended by ISCT [3].

In the case of osteogenic di fferentiation, we analysed the expression of osteogenesis-related genes during the di fferentiation process of both MSC populations, such as Runx2, osteocalcin and osteopontin, in comparison with the control (undi fferentiated) cells, which were cultured under standard culture conditions. We observed that the expression levels of transcription factor Runx2 and osteocalcin (a marker of bone formation) were comparable between DPSCs and UC-MSCs, whereas the fold change in expression of osteopontin (a protein expressed in maturated bone tissue) was elevated in UC-MSCs, notably on the 14th-day post-stimulation (Figure 3a, Table S1). Real-time RT-PCR results obtained for both MSC populations were compared with those of the control (undi fferentiated) cells cultured in a standard cell culture medium (mRNA levels in such cells were calculated as 1.0).

The histochemical staining of cells di fferentiated into osteoblasts demonstrated larger deposits of calcium phosphate (indicated by red-coloured deposits of calcium phosphate) that were observed following DPSC di fferentiation when compared to the di fferentiation of UC-MSCs. Moreover, the deposits were observed earlier (at 14 days) in the case of DPSC osteogenic di fferentiation compared to those with di fferentiation of UC-MSCs (Figure 4). The comparable expression of the genes between DPSCs and UC-MSCs along with the higher formation of calcium phosphate deposits following DPSC di fferentiation may demonstrate a higher osteogenic di fferentiation potential of the DPSCs compared to that of the UC-MSCs.

The DPSCs, as well as UC-MSCs, were successfully di fferentiated into chondroblasts in vitro (Figures 3b and 4, respectively). In the case of DPSCs, we observed increased expression of *Sox9* transcription factor mRNA on days 7 and 14 of di fferentiation, compared to that in the undi fferentiated cells, which confirmed their chondrogenic di fferentiation potential. However, the expression of *Sox9* gene was higher in UC-MSCs in comparison with DPSCs. We did not observe any significant change in the expression of *Col2A1* between both types of cells, while the fold change in the expression of *Col10A1* was higher in the UC-MSCs compared to that in the DPSCs (Figure 3b, Table S2). Recent evidence indicates that *Col10A1* is a marker of hypertrophic chondrocytes, which may be implicated as the principal factor driving bone growth. It has also been observed in skeletal dysplasia and osteoarthritis disorders [44]. The histochemical staining of DPSCs and UC-MSCs that were di fferentiated into chondroblasts indicated extracellular secretion of sulphated proteoglycans (indicated by blue coloured staining) by both types of MSCs, and the kinetics of di fferentiation seemed to be similar between both populations of SCs (Figure 4).

**Figure 3.** Comparison of tri-lineage differentiation potential of DPSCs and UC-MSCs by real-time RT-PCR. (**a**) Quantitative analysis of mRNA expression for osteogenesis related genes (osteocalcin, osteopontin, *Runx2*) in DPSCs (left) and UC-MSCs (right). (**b**) Quantitative analysis of mRNA expression for chondrogenesis related genes (*Acan, Col10A1, Col2A1, Sox9*) in DPSCs (left) and UC-MSCs (right). (**c**) Quantitative analysis of mRNA expression for adipogenesis related genes (*CEBP*<sup>α</sup>*, PPAR*γ) in DPSCs (left) and UC-MSCs (right). Cells were cultured in a StemPro osteogenesis differentiation kit, StemPro chondrogenesis differentiation kit, and StemPro adipogenesis differentiation kit for 7, 14, and 21 days, respectively. Fold differences in expression (2−ddCt) of analysed genes in control cells cultured in standard cell culture medium (undifferentiated) were calculated as 1.0 and marked by a solid line. Graphs present different scales. Results are presented as mean ± standard error of the mean (SEM), *n* = 3 (every sample prepared for each DPSCs line derived from each donor were run in duplicates); *t*-test, (\*) *p* < 0.05 vs. undifferentiated cells.

**Figure 4.** Tri-lineage differentiation potential of DPSCs and UC-MSCs in an in vitro culture demonstrated by histochemical staining. (**a**) Representative images of DPSCs differentiated into osteoblasts, chondroblasts and adipocytes. (**b**) Representative images of UC-MSCs differentiated into osteoblasts, chondroblasts, and adipocytes. DPSCs and UC-MSCs were cultured in a StemPro osteogenesis differentiation kit, StemPro chondrogenesis differentiation kit, or StemPro adipogenesis differentiation kit. On days 7, 14, and 21 of differentiation, DPSCs and UC-MSCs were fixed with paraformaldehyde and stained with Alizarin Red S (red staining of calcium phosphate deposits that are a characteristic of osteogenic differentiation), Alcian Blue (blue staining of sulphated proteoglycans that are a characteristic of chondrogenic differentiation) or Oil Red O (brownish red oil droplets that are a characteristic of adipogenic differentiation). Scale bars: 50 μm.

When focusing on adipogenic di fferentiation, the mRNA expression corresponding to *PPAR*γ adipogenesis-related transcription factor on day 21 as well as *CEBP*α protein expression on day 14 was significantly higher in the UC-MSCs in comparison with that in the DPSCs (Figure 3c, Table S3). Moreover, the presence of oil droplets indicating an ongoing process of adipogenesis was typically observed in both cell fractions on the 14th day of di fferentiation. The results considering the level of demonstrate higher adipogenic di fferentiation of UC-MSCs compared to DPSCs.

Taken together, our first analyses confirmed that DPSCs may be successfully di fferentiated into osteoblasts, chondroblasts, and adipocytes similar to other "classic" MSC populations such as UC-MSCs. The histochemical analyses of the final cell phenotypes confirmed a higher ability of DPSCs to di fferentiate into osteoblasts compared to UC-MSCs, which are primarily restricted to chondrogenic and adipogenic di fferentiation.

To establish whether DPSCs exhibit any cardiomyogenic potential in vitro, they were cultured in cardiomyogenesis stimulating medium as previously described [6]. We analysed mRNA expression of cardiac markers such as *Gata-4, Nkx2.5* and *Myl2c* after 7, 14 and 21 days following the induction of di fferentiation. We observed that the expression of these genes was markedly elevated after 7 and 21 days of cardiomyogenic di fferentiation induction in UC-MSCs than that in DPSCs (Figure 5a and Table S4). Moreover, both types of MSCs express intranuclear cardiac transcription factor *Gata-4* as well as cytoplasmic structural protein troponin T-C after seven days of di fferentiation (Figure 6). This may sugges<sup>t</sup> that DPSCs possess the lower capacity for cardiac cell phenotypes, when culture under lineage-specific conditions, in comparison with other MSC fractions, such as UC-MSCs.

**Figure 5.** Comparison of cardiomyogenic and endothelial differentiation potential of DPSCs and UC-MSCs by Real-Time PCR. (**a**) Quantitative analysis of mRNA expression of cardiomyogenesis

related genes (*Gata-4, Nkx2.5, Myl2c*) in DPSCs (left) and UC-MSCs (right). Cells were cultured in DMEM/F12 supplemented with 2% FBS and 10 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL vascular endothelial growth factor (VEGF) and 10 ng/mL transforming growth factor β1 (TGF-β1) for 7, 14 and 21 days. (**b**) Quantitative analysis of mRNA expression for endothelial related genes (*Gata-2, Tie-2*, VE-cadherin) in DPSCs (left) and UC-MSCs (right). Cells were cultured in EGM-2MV endothelial cell growth medium for 7, 14 and 21 days. Fold differences in the expression (2−ddCt) of analysed genes in control cells cultured in standard cell culture medium (undifferentiated) were calculated as 1.0 and marked by a solid line. Graphs present different scales. Results are presented as mean ± SEM, *n* = 3 (every sample prepared for each DPSCs line from each donor was run in duplicate); *t*-test, *p* < 0.05 vs. undifferentiated cells.

**Figure 6.** Cardiomyogenic and endothelial differentiation of DPSCs and UC-MSCs in vitro on day 7. (**a**) Representative images of cardiomyogenic and endothelial marker expression in DPSCs. (**b**) Representative images of cardiomyogenic and endothelial marker expression in UC-MSCs. In the case of cardiomyogenic differentiation, cells were cultured in DMEM/F12 supplemented with 2% FBS and 10 ng/mL bFGF, 10 ng/mL VEGF and 10 ng/mL TGF-β1. On day 7, cells were fixed, permeabilized, and stained against intranuclear transcription factor *Gata-4* (Alexa Fluor 488, green) and troponin T-C (Alexa Fluor 546, red), whereas nuclei were co-stained with DAPI (blue). In the case of endothelial differentiation, cells were cultured in EGM-2MV. On day 7, cells were fixed, permeabilized, and stained against intranuclear transcription factor *Gata-2* (Alexa Fluor 488, green) and VE-cadherin (Alexa Fluor 546, red), whereas nuclei were co-stained with DAPI (blue). Cells were analysed with Leica DMI6000B ver. AF7000 fluorescent microscope. Scale bars: 100 μm.

To assess the potential angiogenic differentiation capacity of DPSCs, in comparison with UC-MSCs, we launched a long-term culture of these cells in proangiogenic medium containing VEGF (EGM-2MV). The angiogenic potential was examined both at the mRNA and protein levels. The highest expression of angiogenesis-related genes (*Gata-2, Tie2*, VE-cadherin) was observed in UC-MSCs on day 7 of differentiation, whereas the DPSCs were unresponsive to proangiogenic stimulation (expression of the proangiogenic genes was at the same level as in unstimulated DPSCs; Figure 5b, Table S5). Interestingly, the enhanced expression of these genes was also observed in UC-MSCs on the 14th and 21st days of proangiogenic stimulation. However, in the immunocytochemical staining, we did not observe any prominent expression of proangiogenic transcription factor Gata-2 or cell membrane protein VE-cadherin supporting angiogenesis (Figure 6).

Collectively, we observed the following features of DPSCs as cells that (i) exhibit higher osteogenic differentiation capacity, (ii) demonstrate comparable chondrogenic and adipogenic differentiation potential, and (iii) possess limited ability for cardiac or endothelial phenotype, in comparison with other "classic" MSCs (UC-MSCs).

#### *2.3. 3D Encapsulated DPSCs Exhibit Higher Di*ff*erentiation Capacity into Osteoblasts in Vitro*

As shown previously, the DPSCs exhibit a higher osteogenic potential compared to the UC-MSCs. Based on the fact that osteogenic differentiation leading to bone formation is a process that takes place in the regular in vivo 3D niche of developing organism [45], we encapsulated the DPSCs in a hydrogel matrix to mimic such 3D niche in vitro and further analysed their morphology, proliferation, metabolic activity, and osteogenic potential in both normoxic or hypoxic culture environment.

Young's moduli of hydrogel matrix measured using AFM were normally distributed (assessed by the Shapiro–Wilk test) with a mean value of E = 3.69 ± 1.49 kPa (Figure 7a). Elasticity maps demonstrate a heterogeneous distribution of elastic modulus (Figure 7b,c), thus providing more realistic conditions for cell growth. The resulting Young's modulus was in the range of physiological tissue elasticity (~1–100 kPa, [34]) demonstrating rather highly deformable substrate properties. For proliferating cells encapsulated inside hydrogels, such gel does not constitute a barrier. Cells may be able to generate protrusive forces during cellular divisions and can be released into the surrounding environment [46].

We further observed that the DPSCs encapsulated in the 3D hydrogel exhibited a round shape 24 h post mixing, whereas certain flattened cells exhibiting a spindle-shaped morphology were observed after 48 h, indicating a phenotypical change. In contrast, DPSCs seeded on cell culture plates (2D) exclusively exhibited spindle-shaped morphology at both time points as expected in standard 2D conditions (Figure 7d,e). On the first day of 2D and 3D cultures, the relative proliferation of DPSCs in an environment containing 2% or approximately 18% of O2 was the same (Figure 7f). Between days 2 and 7 after culture initiation, we observed a greater relative proliferation of DPSCs in 2D culture compared to that in the 3D culture. However, we did not observe any influence of O2 concentration on the proliferation ratio of DPSCs in both 2D and 3D culture conditions at fixed-time intervals (Figure 7f). The increased proliferation of DPSCs in 2D culture was correlated with the higher metabolic activity of these cells (Figure 7g). DPSCs in 3D culture exhibited lower metabolic activity along with their lower proliferation. We also did not observe any significant impact of O2 concentration on the levels of ATP production in DPSCs in 2D and 3D cell culture (Figure 7g).

**Figure 7.** Mechanical properties of the hydrogel matrix and their impact on morphology, proliferation, and metabolic activity of DPSCs. (**a**) Young's modulus distributions of hydrogel matrix by AFM. (**b**) Exemplary force curve recorded on the hydrogel. (**c**) Exemplary elasticity map of peptide hydrogel. (**d**) Morphology of DPSCs encapsulated in hydrogel (3D) or seeded on the surfaces coated with gelatin (2D) at 24 and 48 h post-seeding. DPSCs were cultured in DMEM/F12 supplemented with 10% FBS and cultured under standard culture conditions (5% CO2, normoxia). (**e**) Morphology of DPSCs encapsulated in hydrogel (3D culture) on day 4 post-seeding. Cells were stained with fluorescein diacetate and analysed with Leica DMI6000B ver. AF7000 fluorescent microscope to visualize their morphology. (**f**) The proliferation of DPSCs in 2D and 3D cultures in the environment containing about 18% or 2% O2 analysed every 24 h until day 7. The analyses were conducted using the Cell Counting Kit-8. (**g**) Metabolic activity of DPSCs in 3D and 2D culture in the environment containing about 18% or 2% O2 measured every 24 h until day 7. The analyses were conducted using the ATP Lite Luminescence assay kit. The results from proliferation and metabolic activity are presented as mean ± SEM, *n* = 3 (every sample prepared for each DPSCs line from each donor was analysed in triplicate).

In the next step, we conducted in vitro osteogenic differentiation of DPSCs in 2D or 3D culture in the presence of 2% or approximately 18% of O2. After seven days of stimulation, the concentration of mRNA for Runx2 was elevated by three times in DPSCs cultured in both 2D and 3D conditions in the presence of hypoxia (2% O2) in comparison with undifferentiated cells. The elevated expression of Runx2 in 3D culture in an environment containing 2% O2 was sustained up to 14 days after stimulation. Importantly, under such culture conditions, we observed the highest fold change in the expression of mRNA corresponding to Runx2 as well as Col1A, at every analysed experimental time point (Figure 8a). Hypoxic microenvironment (2% O2) also stimulated the expression of a gene encoding osteopontin on day 7 in DPSCs in 2D culture (Figure S2). Recent evidence indicates that osteopontin regulates matrix remodelling and tissue calcification and may also be implicated in the pathophysiological process such as osteoporosis [47]. After seven days of differentiation, despite the increased expression of Runx2, we did not observe prominent calcium phosphate deposits in cells cultured in an environment containing 2% O2. The most explicit deposits of calcium phosphate were observed after 21 days of differentiation: in case of 3D culture, we observed large rounded deposits of calcium phosphate especially in the presence of 2% O2, whereas in case of 2D culture, these deposits were more prominent in the presence of approximately 18% O2 (Figure 8b).

**Figure 8.** Osteogenic differentiation of DPSCs encapsulated in hydrogel (3D) or seeded on the surface coated with gelatin (2D) cultured in hypoxic (2% O2) or normoxic (about 18% O2) environment. (**a**) Quantitative analysis of mRNA expression for osteogenesis associated genes (*Col1A, Runx2*) in DPSCs on days 7, 14, and 21 of differentiation. Fold change in the expression of analysed genes in control cells

before differentiation was calculated as 1.0 and marked by a solid line. Results are presented as mean ± SEM, *n* = 3 (every sample was analysed in duplicate). *p* < 0.05 (*t*-test). (**b**) Representative images of DPSCs differentiated into osteoblasts on days 7, 14, and 21 days of differentiation. Panel "Background" contains the representative images of 2D and 3D surfaces (standards plastic dish and hydrogel without cells, respectively) stained with Alizarin Red S solution to visualize background staining (on day 7 post surface preparation). Panels "Day 7", "Day 14", "Day 21" demonstrate representative images of DPSCs cultured in StemPro osteogenesis differentiation kit. On days 7, 14 and 21, DPSCs were fixed with paraformaldehyde and stained with Alizarin Red S (red staining of deposits of calcium phosphate is a characteristic for osteogenic differentiation).

Taken together, the results indicate that 3D cell encapsulation as well as the low concentration of O2 resembling conditions in the stem cell niches may favour osteogenic differentiation of DPSCs in an in vitro environment.
