The Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Dental Pulp in Modular Alginate-Gelatin/Nano-Hydroxyapatite Microcapsules

Background Microcapsule is considered as a promising 3D microenvironment for Bone Tissue Engineering (BTE) applications. Microencapsulation of cells in an appropriate scaffold not only protects cells against excess stress but also promotes cell proliferation and differentiation. Methods In the current study, human Dental Pulp Stem Cells (hDPSCs) were cultivated in Alginate/Gelatin (Alg/Gel) and Alginate/Gelatin/nano-Hydroxyapatite (Alg/Gel/nHA) microcapsules. The proliferation and osteogenic differentiation of these cells were evaluated by MTT assay, qRT-PCR, Alkaline phosphatase, and Alizarine Red S. Results The results revealed that microencapsulation by Alg/Gel/nHA could improve cell proliferation and induce osteogenic differentiation. The cells cultured in the Alg/Gel and Alg/Gel/nHA microcapsules after 28 days showed 2.5-fold and 4-fold more activity of BMP-2 gene expression in comparison with the cells cultured on the polystyrene surface as a control group. The nHA addition to hDPSCs-laden Alg/Gel microcapsule could also up-regulate the bone-related gene expressions of osteocalcin, osteonectin, and RUNX-2 during a 28-day culture period. Calcium deposition and ALP activities of the cells were obsereved in accordance with the proliferation results as well as the gene expression analysis. Conclusion The study demonstrated that microencapsulation of hDPSCs inside Alg/Gel/nHA hydrogel can be a potential approach for regenerative dentistry in the near future.


Introduction
Congenital and acquired bone defects and diseases result in signi cant morbidity and reduction of quality of life for wide range of patients. Bone defects in critical sizes remain challenging for dentists and orthopedic surgeons (1). Allogenic and xenogenic bone grafts are available but infection transmission and limited e cacy are the main disadvantages (2). The autologous bone grafts are commonly used for these defects and considered as a gold standard method (3). However, the limitations of these grafts such as donor site morbidity as well as increased operative time have restricted the method application (4). Designing natural and synthetic scaffolds with the ability of delivering growth factors for stem cell growth and differentiation is a novel strategy desires to overcome these drawbacks in the Bone Tissue Engineering (BTE) (5,6). Although cell-based therapies play an important role in regenerative medicine, there is a major concern for e cient delivery and resistance of transferred cells after injection or implantation. One of the novel advanced methods as a solution can be tissue modi cation with the fabrication of modular block (7).
The appropriate scaffold as a key part of tissue engineering should provide cell survival, induce cell bioactivity, and enhance cell retention in implanted sites. Encapsulation of cells with various polymers protects them from injection forces and immune system reactions while allowing the bidirectional diffusion of nutrients and wastes (8)(9)(10). Microcapsules as spherical microcarriers have been recently used for providing a three-dimensional hydrophilic microenvironment for supporting and transferring of In our previous studies, osteogenic potential of Alginate/Gelatin/nano-hydroxyapatite (Alg/Gel/nHA) microcapsule for modular bone formation was evaluated by using the osteoblastic cell line (6,44). In the current study, Alginate/Gelatin (Alg/Gel) and Alginate/Gelatin/nano-hydroxyapatite (Alg/Gel/nHA) microcapsules containing hDPSCs were used to investigate the in uence of 3D spherical scaffold and nano-hydroxyapatite (nHA) on cell proliferation and osteogenic differentiation of the stem cells for bone regeneration. This study would illuminate the hDPSCs phenotype in 3D microbeads and its potential toward the formation of modular bone tissues for future in vivo studies.

Isolation and adhesion of human dental pulp stem cells (hDPSCs)
This study was approved by the stem cell research center of Tabriz University of Medical Sciences under the code of TBZMED.REC.1396.654. All experimental protocols were in compliance with the Helsinki declaration.
Human dental pulp stem cells were isolated from extracted permanent teeth according to orthodontic treatment in the Oral and Maxillofacial Surgery Department of Dental Faculty of Tabriz University of Medical Sciences. All participants signed the written consent after being informed about the objective of the study.
Isolation and characterization of the hDPSCs were performed as our previous study (25). Brie y, after teeth extraction under local anesthesia, teeth were split with a chisel and the extracted pulp tissue was divided into small pieces. Pulp pieces were then digested in 3 mg/mL type I collagenase and 4 mg/ml dispase for 40 min at 37°C. After centrifuging, the harvested cells were cultured to reach 80% con uence.
Dental pulp stem cells were transferred to 75 cc asks containing DMEM high glucose ampli ed with 10% fetal bovine serum (FBS), 100 U/mL penicillin/ streptomycin, and 1% amphotericin B for proliferation.

Microcapsule fabrication and analysis
Powder of gelatin and nano-hydroxyapatite were sterilized by autoclaving and alginic acid sodium salt was suspended in ethanol 70% and kept under the laminar hood for 24h.
Cell microcapsulation was carried out according to our previous study (7). Concisely, 2% w/v sodium alginate with and without 2% w/v nHA and sterilized gelatin were dissolved in calcium-free Krebs Ringer HEPES-buffered saline (CF-KRH, pH = 7.4). Microcapsules preparation was carried out by the voltage power supply and syringe pump (Vita Teb, Iran). 2×10 6 cells/ml were mixed with alginate (1%) solutions containing gelatin (1.25%) with or without nHA (1%) and loaded into syringes equipped to 30-gage needles. Extruded microcapsules were dropped in CF-KRH containing 100 mM BaCl 2 as a crosslinking solution. The voltage and extrusion ow rate were considered 8kV and 0.08 ml/min, respectively. Continuously, the formed microcapsules were washed by CF-KRH buffer twice to remove unbounded barium ions. Then, both prepared Alginate-Gelatin (Alg/Gel) and Alginate-Gelatin/Nano-Hydroxyapatite (Alg/Gel/nHA) microcapsules were transferred to distinct asks containing the culture medium. The asks were then incubated under the atmosphere of 5% CO 2 at 37 °C and monitored for 28 days for more cell experiments and morphology evaluation by microscope (Olympus IX71). The culture medium was refreshed every 3 days. The surface morphology of the microcapsule samples was also observed by scanning electron microscopy (SEM, Tescan MW2300).

Proliferation and differentiation of hDPSCs
2.4.1. Metabolic activity and live/dead assays Human dental pulp stem microbeads were transferred to 96-well plates to determine metabolic activity in 7, 14, and 21 days. 5 mg/ml MTT solution was added to each well. The plates were incubated under the atmosphere of 5% CO 2 at 37 °C for 4 h. Then, DMSO was added to wells and the absorbance was measured by the UV-160 spectrophotometer (BioQuest) at 570 nm. Dental pulp stem cells cultured on polystyrene surface (PS) were considered as control. The absorbance values were normalized with considering the number of microcapsules per sample and the experiments were carried out in triplicates and the data was reported as the mean ± SD.
To live/dead assay, after rinsing microcapsulated cells by PBS, these microcapsules were incubated in saline containing calcein-AM and propidium iodide (PI) at 37°C, as described elsewhere (45). The microcapsules were then washed with PBS for three times again after incubating in 5% carbon dioxide for 45 minutes. Finally, the microcapsules containing hDPSCs were observed under the uorescence microscope (Olympus, IX71) at wavelengths of 488 nm (green, living cells) and 543 nm (red, dead cells).

qRT-PCR
The osteogenic gene expression was evaluated after 21 and 28 days. After washing microcapsules by PBS, samples were fractured gently. Then, Trizol reagent was used for the extraction of total RNA according to the manufacture instruction. The gel electrophoresis and Nanodrop (Thermo Scienti c, Waltham, MA, USA) were applied for the determination of the yield and value of extracted RNA. 1μg of total RNA was used for cDNA synthesis by CDNA synthesis kit (YektaTajhizAzma, Iran). Synthesized cDNA, syber green master mix, and designed primers were mixed based on the manufacture instruction. Primer sequences, melting temperature, and amplicon size of designed primers were offered in Table 1. Dental pulp stem cells without microcapsules were used as the control group and all the experiments were repeated three times.  (46) tests were carried out for hDPScs microcapsulation after 21 days. For alkaline phosphatase staining, BCIP/NBT solution was prepared and added over non-medium microcapsules before incubating in a dark situation. Then microcapsules were kept in formalin solution (10%) for one min. Finally, the microcapsules were washed by PBS buffer and photographed. To quantify ALP activity, according to the method described elsewhere (44), 21 days after microcapsulation of hDPSCs the microcapsules were rinsed with PBS and centrifuged at 1200 rpm for 20 min. Then, the supernatant was collected and the ALP activity was detected using ALP assay Kit (DIALAB, Austria) according to the manufacturer protocol. ALP activity was measured by evaluating the absorbance at 405 nm by the spectrophotometer and normalized with total protein content. The experiments were carried out in triplicate and hDPSCs cultured on the conventional polystyrene culture surface was used as control.

Alizarin Red S Staining
Calcium deposition of hDPSCsin the modular structure was assessed by alizarin red S staining after 21 days. Shortly, microcapsules were removed from the ask and washed three times by HEPES buffer and xed by 3.6% (v/v) formaldehyde solution for 15 min at 25°C. 2% alizarin red liquid (pH: 4.1-4.3) was then poured over the samples in a dark place and maintained for 15 min. Finally, the stained microcapsules were washed by deionized water several times and examined under the microscope.

Statistical Analysis
The data were determined as the mean ± standard deviation and comprised by using One-way ANOVA and Tukey Test analysis with Prism software (version 8.0, GraphPad, San Diego, CA, USA). P-value<0.05 was considered statistically signi cant.

Results
3.1. In uence of hydrogel composition on microcapsule stability and microcapsulated hDPSCs proliferation Figure 1 reveals the surface morphology of both hydrogel microcapsules of Alg/Gel (Fig. 1A) and Alg/Gel/nHA (Fig. 1B) as well as the appearance of the proliferated hDPSCs in the microcapsules (Fig.   1C). Interestingly, the Alg/Gel microcapsules containing nHA showed a rough and more compact surface morphology in comparison with the Alg/Gel microcapsules ( Fig. 1 A-B) revealing microcapsule stability can be improved when nHA is added to the alginate-based hydrogels.
Live and dead assay (Fig. 1C) cell proliferation in majority of the fabricated microcapsules. However, some of the cells died inside the bigger aggregates of the microcapsules. The cell viability in the microcarrier structures can be affected by the sizes of more than 400 μm (47). The highly hydrated 3-D alginate-based microbeads, as shown in Fig. 1C, could provide an immobilized matrix for the cells with a permeable membrane for waste, nutrients, and oxygen transmissions. Therefore, the average diameter of microcapsules was examined by BEL view software (ver.6.2) (data are not shown). More than 65% of the microcapsules at both cases had 300±40µm diameter leading to su cient nutrient and waste transmissions to the inner core cells. The microscopy images of microcapsules were shown in Figure 2.
Mitochondrial activity as a vital cue demonstrates the viability of cells. Human dental pulp stem cells were cultured in both nHA modi ed and unmodi ed Alg/Gel microcapsules ( g3). Unmicrocapsuled stem cells cultured on the conventional PS were considered as a control group. Figure 3 shows the proliferation of hDPSCs in the microcapsules and without microcapsules for a 3-week culture period. The proliferation of stem cells increased in the microcapsules and on the PS during this period. Dental pulp stem cells in the microcapsules revealed statistically more mitochondrial activities as compared to the control group during the culture period (P < 0.05). Interestingly, the microcapsules containing nano-hydroxyapatite showed signi cant increases in all experiment times. Using nano-hydroxyapatite in microbeads increased the mitochondrial activity of the stem cells 1.26 times (P < 0.05) per microcapsules after 3 weeks. These outcomes can be accomplished that nano-hydroxyapatite plays an important role in the proliferation of dental pulp stem cells.

In uence of microcapsule composition on bone-related gene expressions of microencapsulated hDPSCs
The qRT-PCR analysis was performed to evaluate the effect of Alg/Gel and Alg/Gel/nHA microbeads on the expression levels of the key genes in bone and dentin formation including osteocalcin, BMP2, RunX2, osteonectin, and DSPP after 21 and 28 days.
According to the results shown in Figures 4 and 5, the expression levels of all the genes increased in the hDPSCs, which were microcapsulated in the Alg/Gel and Alg/Gel/nHA as compared with the control.
BMP-2 levels on day 21 signi cantly increased only in the Alg/Gel/nHA group. However, on day 28, the expression levels were signi cantly higher in both groups comparing the control group. By comparing BMP-2 expression in two distinct days, it could gure out that the expressions of this osteogenic gene in microbeads, especially in the presence of nano-hydroxyapatite were up-regulated. 21 days after microcapsulation, osteocalcin expression in both microcapsule groups was signi cantly higher than the control group. Although the expression levels in the Alg/Gel/nHA hydrogel microcapsule were higher than the Alg/Gel groups after 21 and 28 days, this difference was statistically signi cant only on the 28 th day (P < 0.05).
After 21 days, osteonectin expression in the Alg/Gel microcapsules was in the range of the control sample, while a signi cant increase of the gene expression was observed in the Alg/Gel/nHA group. However, after 28 days, both microcapsule groups showed signi cant increases in the osteonectin expression levels in comparison with the stem cells without microcapsules.
In a like manner, it can be imagined that the concentrations of both osteocalcin and osteonectin can be intensi ed by the passing of time. These results provided great con dence in Alg/Gel/nHA microcapsule su ciency to create bone volume in in vivo experiments.
RUNX-2 expression of the hDPSCs in the Alg/Gel/nHA microcapsules showed a 3.5-fold increase as compared to the control sample on the 21stday, the value of which was about 2.6-fold for the Alg-Gel microcapsule. However, after 28 days, the signi cant RUNX-2 up-regulation was observed in the Alg/Gel/nHA group which was 5.1-fold more than the control group (P < 0.01).
The results revealed that a three-dimensional microenvironment that was created by alginate-based microcapsules containing nHA could up-regulate RUNX-2 expression considerably in comparison with the conventional two-dimensional control group.
As shown in Figure 4, DSPP expression in both the Alg/Gel/nHA and Alg/Gel microcapsule groups showed no signi cant differences between the groups after 21 days. The expression of the DSPP gene on the 28th day for both the microcapsule groups, however, was signi cantly increased, the value of which was 2.5-fold and 3-fold higher than the control group,. The low up-regulation of this gene as a classic marker of odontogenic differentiation compared to other osteogenic genes demonstrated the useful application of microcapsulation of hDPSCs with Alg/Gel/nHA in bone tissue engineering.

In uence of microcapsule composition on hDPSCs osteogenic marker production and mineralization
To validate the presence of osteocalcin and bone sialoprotein, two distinct stainings were carried out. The enzymatic activity can be asserted directly for the dyed cells. As shown in Figure 6.A, blue-color spots have been shown in Figure 6.A indicated the positive ALP activity in the xed days. Intensi ed hued microbeads with nano-hydroxyapatite presence aggravate signi cantly hydrolase enzyme expression, motivating ALP expression at the surface of the hDPSCs, as compared to the Alg/Gel microbeads.
Quanti cation of alkaline phosphatase measured in the microcapsules revealed further corroborating evidence for the hDPSCs differentiation. According to the ALP activity shown in Figure 6.B, differentiation of the hDPSCs to osteoblast-like cells increased signi cantly 2.7-fold and 3-fold in the Alg/Gel and Alg/Gel/nHA microcapsules, respectively. The increases in the ALP levels as early bone formation indicator in the Alg/Gel/nHA were statistically higher as compared to the Alg/Gel microbeads (P < 0.05). In fact, microspheres create three-dimensional space for cells to be in the exposure of factors that accelerate differentiation as well as retain cells phenotype. The quantitative analysis con rmed that ALP enzyme activity increased remarkably in the alginate-based microcapsules in comparison to non-3D scaffolds.
As also illustrated in Figure 6.A for calcium deposition, arrows show red-colored surroundings nodules, which demonstrate mineralized cells in the microcapsules. Hydroxyapatite particles in the presence of Alg/Gel composition increased calcium deposition on days 21.
The results also con rmed the formation of mineralized matrix and osteoblastic differentiation in both the microcapsules.
Consequently, it can be deduced that the 3-D microsphere network provided a network for cells to precipitate calcium and relevant enzymes content overtime to speed up modular formation.

Discussion
Microcapsules are widely used in tissue engineering including regeneration of different tissues and organs such as liver, cartilage, skin, neural tissue, and bone (9,(48)(49)(50)(51)(52). These microstructures with different characteristicsincluding immunoisolation, micrometer size, and providing 3D microenvironment considered a promising approach in regenerative medicine (9,53). These modular microcarriers could directly be injected and transplanted to the defect side (54). This property is important for the reconstruction of hard tissues. The injectable microcarriers could adjust to bone defects with irregular shape and geometry, occupying the available spaces, precluding the invagination of the adjacent tissues and promoting tissue repairing (55,56). As shown in graphical abstract, the current study aimed to use the available source of stem cells in the oral cavity and microcapsulating of theses cells for modular tissue engineering approach of bone defects in the oral and maxillofacial region.
The main ndings of this study is the high osteogenic differentiation capacities of hDPSCs in Alg/Gel/nHA microcapsules. Moreover, both ALP and Alizarin red staining of micro carriers showed a greater extent of mineral deposition in nano-hydroxyl apatite modi ed microcapsules.
In the current study we investigated the osteogenic potential of hDPSCs as an available source of mesenchymal stem cells in Alg/Gel/nHA microcapsules for the rst time.
Alginate is a natural biopolymer that has been widely used for drug delivery, dental impression materials, and tissue engineering (4,57).
Gelatin is derived from collagen, the major organic component of the extracellular matrix of bone tissue, with adequate properties for bone tissue regeneration and revascularization (58,59).
In the structure of Alg/Gel microcapsules, a rapid reaction between carboxyl groups of alginate and barium cations makes it possible to form an ion-crosslinking hydrogel. Microcapsules were cross-linked by the ionotropic gelation in the presence of BaCl 2 that has shown high stability and low degradability in comparison to CaCl 2 (60). Indeed, adding nHA to the system increases the crosslinking of the polymeric network, leading to an enhanced homogeneity and strength of the microcapsules (55).
Nano-hydroxyapatite (nHA) as the major inorganic mineral part of bone could increase the homogeneity and strength of microcapsules. Moreover, nHA increased the crosslinking of the polymeric network (55).
These features besides, the facilitation of bone formation in the presence of nHA turn it to the important component of BTE (27).
The interaction between calcium ions of nHA and G-blocks of alginate makes a strong matrix with the rougher surface which improves cell adhesion, proliferation, and differentiation compared to smooth alginate surface. In the current study, the proliferation of hDPSCs in the Alg/Gel/nHA microcapsules was higher than Alg/Gel and control groups. Moreover, the cell viability in the micro-carrier structures was in uenced by the size and mass too, which can diffuse inner layers (47). The viability and proliferation of hDPSCs in Alg/Gel and Alg/Gel/nHA microcapsules increased during the study. This viability is a result of an appropriate bead size, which could provide an immobilized matrix with a permeable membrane that facilitates waste, nutrients, and oxygen transmission for cells. This could avoid the crowd of toxic wast and lead to appropriate activity and proliferation of cells. These results were in accordance with other previous studies that used microcapsulation methods for cell transferring (7,27).
The aim of BTE is not only to provide 3D structutres and cell proliferation without cell cytotoxicity or foreign body reaction but also the regeneration of new bone by provoking osteogenic differentiation of stem cells (4,61).
In the oral and maxillofacial regions for the regeneration of bone defects, the non-invasive injectable methods for stem cell delivery are more useful (62). The osteo/odontogenic differentiation of hDPSCs on various scaffolds was evaluated previously (41,43,63,64). However, none of these scaffolds evaluated non-invasive injectable carriers for these cells. In the current study, we transferred these accessible stem cells in the oral region to spherical injectable microcapsules, which could be useful for bone regeneration of this area.
The osteogenic differentiation of hDPSCs in Alg/Gel and Alg/Gel/nHA group was assessed by the relative expression of BMP-2, Osteocalcin, Osteonectin, and RUNX-2. Various extracellular ligands such as BMPs, WNTs, and FGFs controlled osteogenic differentiation of different multipotent mesenchymal stem cells (65). These ligands direct the three main stages of osteogenic differentiation, which associate with the expression of some genes like BMP-2, RunX-2, Osteocalcin, and Osteonectin (46).
The expression of BMP-2 as the most important growth factor in bone formation is known as an early indicator of calci ed tissue generation and osteoblastic differentiation (61,66). The expression of this factor was signi cantly higher in Alg/Gel/nHA group after 21 and 28 days.
Osteocalcin and osteonectin are noncollagenous proteins in the extracellular matrix of bone. Osteocalcin has an important role in the maturation of mineralized tissues and the regulation of osteogenic differentiation of mesenchymal stem cells (67). The microcapsulation of hDPSCs has positive effects on the expression levels of these genes. However, these effects were greater in Alg/Gel/nHA group.
RUNX-2 is known as a critical transcription factor associated with bone formation and plays an important role in the differentiation of pre-osteoblastic cells to mature osteoblasts. Also, this factor upregulates the VEGF factor which is important in the angiogenesis of bone tissue (29,68). The upregulation of this gene as a late indicator of osteogenesis was higher than other genes after 21 and 28 days.
Moreover, DSPP, a classic odontogenic differentiation marker, gene expression was assessed in order to investigate any odontogenic differentiation of hDPSCs in microcapsules. As demonstrated in results, the up-regulation of this gene was not increased as well as osteogenic genes in microcapsules, which indicates the osteogenic potential of Alg/Gel/nHA microcapsules rather than odontogenic potential.
In general, hDPSCs in Alg/Gel/nHA microcapsules showed higher expression of these genes after 21 and 28 days. These results were in compliant with other previous studies, which evaluated the different alginate-nano hydroxyapatite-based scaffolds on osteogenic differentiation of stem cells (55,61,69).
To evaluate the mineralized deposition of hDPSCs in Alg/Gel/nHA microcapsules Alkaline phosphatase and Alizarin red staining were performed in the current study. The formation of mineralized nodules as shown in our results indicate the late stage of osteogenic differentiation. In summary, our results indicated the notable enhancement in mineralization of hDPSCs in Alg/Gel/nHA microcapsules after 21 days.
The combination of calcium with alizarin red makes orange nodules, which showed mineralization sites (41). These nodules were clearly observed in Alg/Gel/nHA group.
ALP enzyme activity as a major factor in the beginning phase of bone matrix mineralization was quanti ed by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol (70).This enzyme is a universal marker for the indication of mineralization. ALP deposition occurs during osteoblast maturation and bone matrix mineralization (71). Therefore, the ALP activity could demonstrate the biological activity of osteoblasts (41). Moreover, The formation of the bone-like mineralized tissue due to calcium deposition is essential to bonding of newly regenerated bone with the former bone tissue (55,72).
The results observed for physical and osteoconductive properties of nHA particles in the hDPSCs-laden alginate hydrogels were in agreement with the reported results of other previous studies (6,26,41,44), indicating the importance of nHA bioactivity in the composite hydrogel.
In general, our results showed that the proliferation of hDPSCs increased and the expression of all osteogenesis genes upregulated in nano-hydroxyapatite containing microcapsules. Moreover the calcium deposition was increased in the presence of nHA. These results are in compliance with other studies, which used these microcapsules on different stem cells (45,48,73). However, this study evaluated these modular microcapsules in osteogenic differentiation of hDPSCs for the rst time, based on authors' information.

Conclusion
The cell viability and proliferation of hDPSCS in both Alg/Gel and Alg/Gel/nHA microcapsules increased. Beside, differentiation of hDPSCs was immoderately intensi ed in the presence of nano-hydroxyapatite.
In the engineering of hard mineralized tissues, the structure of the carriers must be hardened in order to simulate the real extracellular matrix. The presence of nano-hydroxyapatite particles in microcapsules simulated the real architecture of bone tissue. In addition, these engineered scaffolds allowed the cells to maintain their original phenotype in the absence of inducers, which is important for future in vivo application.