Bone regeneration potential of human dental pulp stem cells derived from elderly patients and induced by a helioxanthin derivative

Background: Human dental pulp stem cells (DPSCs) have the ability to differentiate into multiple lineage cell types including adipogenic, neurogenic, and osteogenic cells. Dental pulp stem cells can be easily collected from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells. Previous studies have described the osteogenic ability of DPSCs that were isolated from only young patients. However, in fact, elderly patients more frequently require bone regenerative therapy—for example, in alveolar bone defects resulting from periodontal disease—than young patients. We previously reported that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of DPSCs derived from young patients. However, the effect of TH on the osteogenic differentiation potential of DPSCs derived from elderly patients remains unknown. Therefore, the present study aimed to examine and compare the osteogenic differentiation potential of TH-induced DPSCs from elderly patients and young patients to explore the potential clinical use of DPSCs for elderly patients. Methods: Dental pulp stem cells were obtained from the dental pulp of teeth of healthy young patients (18-39 years old) and healthy elderly patients (40-67 years old) and cultured in regular medium and osteogenic medium with or without TH. We assessed the morphological characteristics, proliferation, and osteogenic differentiation of DPSCs from both young and elderly patients. Moreover, DPSC sheets were transplanted into mouse calvarial defects to investigate osteogenesis of TH-induced DPSCs in vivo. Results: We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation and osteogenesis of DPSCs from elderly patients as well as young patients more than osteogenic conditions without TH.


Conclusions:
Our results suggested that the osteogenic differentiation potential of DPSCs from elderly patients is as high as that of DPSCs from young patients. Moreover, THinduced DPSCs show increased osteogenic differentiation potential, and they can be a useful cell source for bone regenerative therapy for not only young patients but also elderly patients.

Background
Bone regenerative therapy is in great demand nowadays for alveolar bone loss caused by periodontitis, tumor resection, traumatic injury, and dental implant therapy. In the clinical field, bone regenerative therapy is required especially for elderly patients over 40 years of age owing to the high morbidity rate of periodontitis. Mesenchymal stem cells (MSCs) derived from bone marrow (BMSCs) and adipose tissue are known to be a useful cell source for tissue engineering. However, collecting BMSCs is challenging owing to the necessity of using an invasive technique. To date, bone augmentation techniques such as bone harvesting from the iliac crest under general anesthesia, autogenous bone grafting from the mandible, and artificial bone grafting have been used for alveolar bone defect in periodontitis treatment and dental implant treatment. However, these present a high likelihood of infection and unexpected resorption.
Human dental pulp stem cells (DPSCs) were identified in 2000, and they have a high potential to proliferate, self-renew, and differentiate into multiple lineage cells such as adipogenic, neurogenic, and osteogenic cells [1][2][3]. The clonogenic and proliferative potential of DPSCs is higher than that of BMSCs. Moreover, DPSCs can be obtained from extracted teeth that are usually discarded, and the method of collecting DPSCs is noninvasive and uncomplicated without ethical issues. Thus, DPSCs have the potential to be a promising cell source for regenerative therapy.
Several studies report that the number of MSCs and stem cell functions such as the ability to self-renew decline with increasing age [4]. Furthermore, the proliferation and differentiation potential of MSCs also declines with age and after repeated passage [5][6][7][8], owing to telomere shortening, DNA damage, and epigenetic changes in transcriptional regulation [9]. Although many studies have investigated the differentiation potential of DPSCs isolated from young patients, little information is available about osteogenic differentiation potential of DPSCs derived from elderly patients. In fact, the volume of dental pulp tissue decreases with age owing to secondary dentin formation and root canal mineralization [10]. Thus, a simple and effective method of transplantation is required to overcome these limitations.
In our previous study, we showed that 4-(4-methoxyphenyl)pyrido [40,30:4,5]thieno [2,3b]pyridine-2-carboxamide (TH)-a small-molecule derivative of helioxanthin-induces osteogenic differentiation of DPSCs isolated from young patients in vitro and in vivo, using transplantation of DPSCs with cell-sheet technology. The cell-sheet technology facilitates stem cell harvest and transplantation without the need for digestive enzymes and scaffolds, resulting in minimal cell loss and damage and avoiding inflammatory responses caused by biodegradable scaffolds [11]. However, the effect of TH on the osteogenic differentiation potential of DPSCs derived from elderly patients remains unknown.
In the present study, we investigated and compared the osteogenic differentiation potential and osteogenesis of TH-induced DPSCs from elderly and young patients in vitro and in vivo to explore the potential clinical use of DPSCs.

Isolation of dental pulp stem cells
Human dental pulp stem cells were isolated from extracted teeth from healthy young (16-39-year-old) and elderly (40-76-year-old) patients (hereafter referred to as "young DPSCs" and "elderly DPSCs", respectively)-after obtaining informed consents-at Tokyo Medical University Hospital. This study was approved by the institutional ethics committee of the Faculty of Medicine, Tokyo Medical University, Japan (approval no. 3486). Dental pulp was extracted and minced into small pieces, followed by enzymatic digestion using 3 mg/mL collagenase type I (Sigma-Aldrich) for 45 min at 37 °C; single-cell suspensions were obtained by passing the cells through a 70 µm cell strainer. The isolated cells were plated at a density of 1 × 10 5 cells on 100 mm dishes and cultured in alpha-modified Eagle's medium (αMEM; Gibco/BRL) supplemented with 15 % fetal bovine serum (FBS; Biowest) and 1 % penicillin/streptomycin (P/S; Wako Pure Chemical Industries). Cells were passaged at 70 % confluence. Young and elderly DPSCs in passage 3 (P3) or P4 were used for experiments. Dental pulp stem cells were cultured in 12-well plates containing osteogenic differentiation media with and without TH. We used αMEM supplemented with 10 % FBS, 1 % P/S, and 10 nM dexamethasone (Wako Pure Chemical Industries) for osteogenic induction (osteogenic medium [OM]). We added TH (Takeda Chemical Industries) at the optimal concentration of 10 -6 M to the OM, as described previously [12].

Cell Proliferation
Dental pulp stem cells (1 × 10 5 cells) were cultured in 100 mm dishes at a density of 1 ×  Non-labelled cells were used as negative controls. Cells were analyzed using a flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software (FlowJo; FlowJo, LLC).

Alizarin red S staining
Young and elderly DPSCs were cultured in OM with or without TH for 14 days and stained with alizarin red S (Sigma-Aldrich), as described previously. In brief, cells were fixed with 10 % formaldehyde in PBS for 10 min at 4 ℃, followed by two washes with distilled water.
Next, the cells were stained in 1 % alizarin red S solution for 15 min. After staining, they were washed twice with distilled water.

Alkaline phosphatase staining
Young and elderly DPSCs were stained with alkaline phosphatase (ALP) as described previously. In brief, the cells were fixed in 70 % ethanol after they were rinsed with PBS, followed by staining for 10 min with 0.01 % naphthol AS-MX phosphate (Sigma-Aldrich), using 1 % N,N-dimethyl formamide (Wako Pure Chemical Industries) as the substrate and 0.06 % Fast BB salt (Sigma-Aldrich) as a coupler.

Reverse transcription-polymerase chain reaction analysis
Total RNA from young and elderly DPSCs cultured in regular medium (RM Dulbecco's Modified Eagle Medium [DMEM, GIBCO/BRL] supplemented with 10% FBS and 1% P/S), OM, and OM with TH was isolated using TRIzol (Invitrogen), and reverse transcription was performed using QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. Real-time polymerase chain reaction of Runx2, an osteoprogenitor maker, Alp and Colla1, early markers of osteoblast differentiation, and osteocalcin, a mature osteoblast marker, was performed in a Lightcycler 96 (Roche Diagnostics) using THUNDERBIRD SYBR qPCR Mix (Toyobo), as described previously.
GAPDH was used as an endogenous control. The primer sequences used in this study are presented in Table 1.

Transplantation of young and elderly dental pulp stem cell sheets into a mouse calvarial defect model
A mouse calvarial defect model was used for animal experiments, as described previously, following the guidelines of Animal Care and Use committee of the Faculty of Medicine, Tokyo Medical University. In brief, young and elderly DPSC sheets were cultured on temperature-responsive dishes in OM with TH treatment for 14 days, and each sheet was transplanted into the calvarial defects (3.5 mm in diameter) created in the right parietal bone using biopsy punches (Kai Corporation). Four mice were used in each experimental group. Eight weeks after transplantation, mice were sacrificed, and calvaria were harvested.

Radiological evaluation
The calvarial defects were examined by micro-computed tomography (micro-CT; SMX-90CT; Shimadzu) The scanning conditions were as follows: 90 kV, 110 µA, and a field of view (XY) of 10 mm; the resolution of one CT slice was 512 × 512 pixels. The data were reconstructed and analyzed using morphometric software TRI/3D-BON (Ratoc System Engineering). Bone volume (BV), bone mineral content (BMC), and bone mineral density (BMD) of a 5 × 5 × 3 mm 3 cuboid area located in the center of the initial defect area were calculated.

Statistical analysis
Each experiment was replicated at least three times. Statistical analysis was performed using SPSS 24.0 software (IBM); all data are expressed as mean ± SD. Statistical significance was evaluated using one-way ANOVA, and p values were calculated with Student t test. P < 0.05 or P < 0.01 was considered to indicate statistically significant differences between two groups.

Characterization of young and elderly dental pulp stem cells
At initial culture, young and elderly DPSCs showed morphologically similar spindle shape (Fig. 1A). The average number of DPSCs derived from young and elderly patients was 70 % and 42 % of the total number of cells collected, respectively ( Table 2). Proliferation potential of elderly DPSCs in P1 was significantly lower than that of young DPSCs (Fig. 1B).
However, in P2, the proliferation potential of elderly DPSCs increased and was not significantly different from that of young DPSCs, it remained at a high level in P3 (Fig. 1B).
Flow cytometry analysis indicated that young and elderly DPSCs showed a similar pattern of expression of MSC surface markers, being positive for CD29, CD44, CD73, CD81, CD90, and CD105 and negative for CD14 and CD34 (Fig. 2), which are the minimal criteria for DPSCs.

Effect of TH on osteogenic differentiation of young and elderly dental pulp stem cells
To determine the effect of TH on elderly DPSCs, we compared the osteogenic differentiation potential of TH-induced DPSCs from young and elderly patients. Alizarin red S staining showed that in both young and elderly DPSCs, higher calcification was observed in DPSCs cultured in OM with TH than in OM without TH (Fig. 3A). Moreover, ALP staining showed that young and elderly TH-induced DPSCs displayed stronger ALP activity than DPSCs in OM (Fig. 3B). Real-time PCR showed that TH upregulated osteogenic differentiation markers in DPSCs derived from both young and elderly patients, although the difference was not significant. In addition, in DPSCs from both young and elderly patients, Colla1, Alp, osteocalcin , and Runx2 were found to be more highly expressed in TH-induced DPSCs than in DPSCs cultured in OM, although the difference was not statistically significant (Fig. 3C).

In vivo osteogenesis of TH-induced young and elderly dental pulp stem cells
To determine the osteogenesis of TH-induced elderly DPSCs in vivo, we cultured young and elderly DPSCs on temperature-responsive dishes (12-well, 3.5 cm 2 /well) under osteogenic conditions with TH for 2 weeks, then harvested the DPSC sheets, and transplanted them into the calvarial defects of mice. Eight weeks after transplantation, micro-CT images were taken to compare the bone regeneration potential of young DPSCS with that of elderly DPSCS (Fig. 4A). The micro-CT images revealed that the newly formed bone was detected at the edge of the defect in the elderly DPSC group as well as in the young DPSC group, whereas no bone regeneration was observed in the control group (Fig.   4B). Quantitative evaluation indicated no significant differences in BV and BMC between young and elderly DPSC groups; however, BV and BMC in the elderly DPSC group were significantly higher than those in the control group (Fig. 4C).

Discussion
In the present study, we studied the osteogenic differentiation potential of TH-induced DPSCs from elderly patients and compared it with that of DPSCs from young patients. In comparison with BMSCs [2] and adipose tissue-derived stem cells (ADSCs) [13], DPSCs have a higher proliferative potential and ease of accessibility, and the method to harvest them is less invasive; therefore, DPSCs can be a promising cell source for regenerative medicine. The MSCs and ADSCs show an age-dependent decline in their proliferative ability and regenerating capacity [4,14,15]. In fact, the volume of pulp tissue decreases with age owing to secondary dentin formation and root canal mineralization [10]. The present study demonstrated that the number of collected cells from the pulp tissue of elderly patients was lower than that collected from young patients, which agrees with previously published data [16]. Both the quality and quantity of DPSCs are important in regenerative therapy; therefore, these age-related changes can make the clinical use of DPSCs from elderly patients challenging. However, the effect of age in bone regeneration has not yet been clear. Thus, age-independent culture methods that render the DPSCs to maintain a high proliferation and differentiation potential are desirable in tissue engineering therapy. Although some studies have reported an efficient system that induces osteogenic differentiation of DPSCs, such as producing the scaffold materials [17], and modifying the medium [18], its effect in aging patients has not yet been reported. We previously demonstrated that TH, a small osteogenic molecule, induced osteogenic differentiation of DPSCs from young patients in vivo and in vitro [12]. With this method, it is possible to culture DPSCs with a high proliferation and differentiation potential. In the present study, to assess whether TH can be used in DPSCs derived from elderly patients, the effect of TH on the osteogenic differentiation potential of DPSCs from elderly patients was examined in a mouse calvarial defect model and compared with that of DPSCs from young patients.
It has been shown that MSCs from elderly patients have a lower proliferative potential even after the initial passage [6,19]. In the present study, we demonstrated that, at initial culture and P1, the proliferation potential of DPSCs from elderly patients was significantly lower than that of DPSCs from young patients. In contrast, no significant differences were found between the proliferative ability of elderly DPSCs and young DPSCs after P2, and the proliferative ability of elderly DPSCs was maintained at a high level even at P3, indicating that once the pulp cells gain "stemness" after passaging, the high proliferation potential of DPSCs is independent of age even though the initial number of collected cells is low in elderly patients. Similar results were obtained in a previous study in which they observed a high proliferative ability of DPSCs derived from different age groups including elderly patients, and the ability was also maintained at P2 in all age groups [20].
We previously reported that the optimal concentration of TH that is the most effective on osteogenic differentiation of DPSCs is approximately 10 -6 M and that OM is necessary for osteogenic induction when DPSCs are cultured with TH [12,21]. In the present study, we used the same concentration of TH and the same culture method for both young and elderly DPSCs. Our data demonstrated that compared with OM alone, OM with TH increased-although not significantly-the expression levels of Colla1, Alp, osteocalcin, and Runx2 in both young and elderly DPSCs. Moreover, ALP staining and alizarin red S staining showed that TH induced higher ALP activity and more mineral deposition in both young and elderly groups, indicating that TH is effective in inducing osteogenic differentiation of not only young DPSCs but also elderly DPSCs.
As we previously reported, the cell sheet method using TH-induced DPSCs enables easier and safer transplantation of DPSCs [12]. Bone regenerative therapy using transplantation of DPSC sheets in combination with TH requires no scaffolds or growth factors, thus overcoming challenges associated with invasive autologous bone harvesting and ethical issues associated with allogenic bone transplantation and recombinant protein use.
Moreover, previous studies have shown that DPSCs have a higher immunosuppressive activity and ability to prevent T-cell alloreactivity than BMSCs [22,23], which can be advantageous for the clinical use of DPSC sheets. Furthermore, this finding was supported by the subsequent in vivo study using a mouse calvarial defect model. We evaluated the osteogenic potential of TH-induced DPSCs from elderly patients using a mouse calvarial defect model, which closely resembled clinical settings. Notably, transplants with elderly DPSCs and young DPSCs showed similar new bone formation at the edge of the calvarial defect. Moreover, BV and BMC in the transplants with elderly DPSCs were considerably higher than those in control, but no significant difference was found between transplants with elderly and young DPSCs, indicating that elderly DPSCs treated with TH have a high potential for osteogenic differentiation and osteogenesis, which is not inferior to that of young DPSCs. Previous studies have shown that using scaffolds increased the osteogenesis of DPSCs [24, 25] derived from young patients. However, TH improved the osteogenesis of DPSCs without any scaffolds and independent of the age of the source tissue. In conclusion, these radiological findings confirm the advantage of using TH for bone regenerative therapy to the patients irrespective of patient age.

Conclusion
Our study demonstrated that TH is effective in inducing osteogenic differentiation of DPSCs from elderly patients. Moreover, TH-induced DPSC from elderly patients have a high potential for osteogenesis as well as young patients in vivo, indicating that TH-induced DPSCs from elderly patients can be a useful cell source for bone regenerative medicine.