Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs) may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1) has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH) has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+) and STRO-1(−) hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+) hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+) hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R) than STRO-1(−) hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+) hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+) hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+) hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.


Introduction
Periodontitis is the most common cause of irreversible destruction of periodontal tissue and tooth loss in adults worldwide. However, conventional periodontal therapy for the treatment of periodontal tissue damage does not induce the regeneration of periodontal supporting tissue [1,2]. Therefore, the development of regenerative therapies for the treatment of periodontal disease has become a major challenge [3].
Several studies have suggested that mesenchymal stem cells (MSCs) may represent promising therapies for the functional repair of defect and injury caused by periodontitis [4,5]. Recently, dental tissue-derived stem cell therapy has been shown to be a promising new method for the regeneration of periodontal tissue [3]. Periodontal ligament stem cells (PDLSCs), a unique population of MSCs found in periodontal ligaments, are easy to obtain and can be induced into osteoblast-like cells and adipocytes in vitro [6]. In addition, PDLSCs are currently the most favorable candidates for the treatment of advanced periodontitis, showing superiority when compared with dental pulp stem cells (DPSCs) and periapical follicular stem cells (PAFSCs) [7]. Moreover, in a swine model, researchers transplanted human PDLSCs (hPDLSCs) into alveolar bone defect areas and found that periodontal tissue was eventually repaired and regenerated [4], supporting the potential of these cells in tissue regeneration.
Parathyroid hormone (PTH), a peptide hormone produced at the endoplasmic reticulum, regulates calcium and phosphorus metabolism and mineral homeostasis [12]. A recent study showed that PTH binds to the PTH receptor on MSCs, committing MSCs to the osteoblast lineage and promoting bone formation [13]. Intermittent administration of PTH  has been shown to increase bone-to-implant contact, induce new bone formation around the implants, and increase bone mineral density [14]. Furthermore, PTH (1-34) stimulates new bone formation, leading to enhanced periodontal healing [15].
In this study, we hypothesized that PTH would affect the osteogenic capacity of PDLSCs and show the potential for application as a cell source in periodontitis treatment. Therefore, we isolated and characterized STRO-1(+) hPDLSCs, compared the levels of PTH receptor in STRO-1(+) and STRO-1(−) PDLSCs, and evaluated the effects of PTH treatment on the differentiation capacity of STRO-1(+) PDLSCs.

Cell Culture and PTH Treatment.
Healthy premolars were collected from 10 adults (15-20 years of age; five men and five women) for orthodontic purposes at the Department of Oral and Maxillofacial Surgery, the Affiliated Stomatological Hospital of Sun Yat-sen University. All participants provided informed consent for the collection and use of their tissues. The protocols were approved by the University Ethics Committee.
PDLSCs were isolated and cultured as previously reported [6]. Briefly, periodontal tissue was gently separated from the surface of the middle third of the root and then digested with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco-BRL, Gaithersburg, MD, USA) at 37 ∘ C for 1 h. Colonyforming cells were collected and then cultured in alpha modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 g/mL streptomycin, 100 U/mL penicillin (Hyclone, Logan, UT, USA), 200 M l-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 5 mM l-glutamine (Gibco-BRL) at 37 ∘ C in an atmosphere containing 5% CO 2 . The medium was changed every 3 days. Cells were used at passages 3-5. For intermittent PTH treatment, cells were treated with 10 −12 M PTH (1-34) (Sigma-Aldrich) for 6 h, followed by another 6 h treatment after 32 h. Cells were harvested at various times after treatment with 10 −12 M PTH (1-34) for RNA and protein isolation. Carlsbad, CA, USA) at room temperature for 1 h in the dark. Cells were incubated with STRO-1 IgM (1 : 10; Life Technologies Corp.) at room temperature for 1 h and then incubated with PE-conjugated anti-IgM (1 : 500; Life Technologies Corp.) for 30 min in the dark. After incubation, cells were washed three times with PBS and resuspended in 300 L PBS. Flow cytometry was carried out using a BD Accuri C6 (Becton Dickinson Biosciences). Data were analyzed using CF Low Plus Software (Becton Dickinson Biosciences).
2.3. Pluripotency of hPDLSCs. PDLSCs (passage 3) were seeded at a density of 1 × 10 5 cells/well in 12-well plates. For osteogenic differentiation, after reaching 70-80% confluence, cells were cultured in osteogenic-induction medium (Cyagen Biosciences Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Three weeks later, cultures were fixed with 4% paraformaldehyde for 15 min and stained with Alizarin red; quantification of mineralization was performed as previously described [16]. For adipogenic differentiation, cells were cultured in adipoinductive medium (Cyagen Biosciences Inc.) according to the manufacturer's protocol. Two weeks later, cells were fixed with 4% paraformaldehyde for 30 min and stained with Oil Red O.

Immunomagnetic Cell
Sorting. hPDLSCs were collected at passage 3, and STRO-1(+) cells were isolated using Dynabeads rat anti-mouse IgM (Life Technologies) according to the manufacturer's instructions. Briefly, 1 × 10 7 cells were resuspended in 1 mL PBS with 0.1% bovine serum albumin, 2 mM EDTA, and 25 L washed rat anti-mouse magnetic Dynabeads. Next, 10 L of anti-human STRO-1 IgM antibodies (Life Technologies) was added, and cells were incubated at 4 ∘ C for 1 h. The tube was then placed in a magnet for 2 min, and the bead-bound STRO-1(+) cells and bead-free STRO-1(−) cells were isolated.

Alkaline
Phosphatase (ALP) Activity. ALP activity was measured after 8 days of osteogenic differentiation. Cells were harvested with lysis buffer (20 mM Tris-HCl, 150 mM MgCl 2 , 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride). Total protein concentrations were determined using a Pierce BCA Protein Assay Kit (Life Technologies). Fifty microliters of supernatant was added to 50 L p-nitrophenyl phosphate hexahydrate (1 g/L; Sigma-Aldrich) containing 1 mM MgCl 2 , and the mixture was incubated at 37 ∘ C for 30 min. Absorption at 405 nm was measured in duplicate well with a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). ALP activity per total protein ( g) represented the millimoles of p-nitrophenol released after a 30 min incubation at 37 ∘ C.

Characterization of hPDLSCs.
hPDLSCs were isolated from the root surface of premolars and cultured. The colonyforming hPDLSCs displayed a uniform fibroblast-like morphology (Figure 1(a)). Culture-expanded hPDLSCs exhibited osteogenic and adipogenic differentiation potential, as shown by the formation of mineralized nodules and lipid-containing adipocytes (Figure 1(b)).
To test this hypothesis, we treated the cells with PTH intermittently and detected changes in the expression of osteogenesis-related genes. After 8 days of PTH treatment and osteogenic induction, the expression levels of osteogenesis-related genes were quantified by RT-qPCR and western blotting. After induction, the expressions of PTH1R, RUNX2, and SP7 were upregulated by PTH treatment (Figure 4(a)). Moreover, intermittent PTH treatment elevated SP7 expression in both induced and uninduced cells, and PTH increased the expression of PTH1R, creating a positive feedback loop (Figure 4(a)).
We then used ALP activity assays to determine whether PTH affected osteogenic differentiation. PTH-treated cells exhibited stronger osteogenic differentiation potential ( < 0.01; Figure 4(b)). After 22 days of induction, the formation of mineralized nodules was observed and measured. Control cells formed small mineralized nodules in terms of both area and density. In contrast, in cells treated with osteogenic medium and PTH, the formation of mineralized nodules was significantly increased in terms of both area and intensity   ( Figure 4(c)). Taken together, our data showed that intermittent treatment with PTH significantly enhanced osteogenesis in STRO-1(+) hPDLSCs.

Discussion
In this study, we aimed to determine whether PTH affected the osteogenic capacity of STRO-1(+) PDLSCs in order to analyze the potential applications of these cells in the treatment of periodontitis. Our results showed that PTH could stimulate osteogenesis in STRO-1(+) hPDLSCs, suggesting that this population of cells could be used in tissue regeneration to repair periodontitis-dependent bone damage.
We isolated PDLSCs from periodontal ligaments and evaluated their biological and immunological properties. Colony-forming cells expressed the surface markers CD90, CD105, and CD166 at high frequencies (95%) but rarely expressed CD34 (less than 2%). Moreover, the cells exhibited osteogenic and adipogenic differentiation potential. These results indicated that we had successfully isolated PDLSCs, according to the criteria presented in the International Society for Cellular Therapy position statement [6,20]. With the capacity for self-renewal and differentiation into osteoblastlike cells, PDLSCs have become a promising source for regeneration of tissue in the treatment of bone defects [4,21].
Previous studies have found that intermittent or lowdose PTH (1-34) enhances bone repair and increases bone turnover [31,32]. In our study, we used human STRO-1(+) PDLSCs as a model to explore the roles of PTH in osteogenesis. We found that PTH (1-34) increased the expression of osteoblast-related genes and the mineralization capacity of the cells, supporting the future applications of PTH in periodontitis treatment. Further studies are needed to examine the effects of PTH on periodontal tissue regeneration in vivo by transplantation of PTH-treated STRO-1(+) PDLSCs into alveolar bone defect areas in an appropriate animal model. The functions of PTH are mediated by a G protein coupled receptor, referred to as PTH1R, the PTH-1 receptor, which regulates skeletal development, bone turnover, and mineral ion homeostasis [33,34]. In continuous treatment, PTH caused severe bone loss and inhibits bone related protein expression [35,36], while intermittent administration of PTH enhanced bone formation [37]. These studies have shown that PTH may be a double-edged sword, suggesting that the molecular mechanisms of PTH may be highly complicated [38]. The time-dependent action of PTH might be due to differences in the signal transduction systems, indicating a synchronized regulation of cAMP/PKA and PKC signaling after PTH stimulation [39]. While the PKA pathway acts in response to short exposure to PTH, the PKC pathway is mainly involved at longer times of exposure in an antagonist mode [40,41]. In our study, STRO-1(+) PDLSCs were treated under the intermittent administration of PTH, which might be mediated predominantly by cAMP/PKA pathway. Interestingly, our data showed that PTH upregulated its receptor PTH1R in STRO-1(+) PDLSCs, which may result in the establishment of a positive feedback loop. The observed effects could be explained by the feedback of PKA signals on PTH1R expression levels, ligand sensitivity, and crosstalk with other downstream signaling pathways [42]. However, the nature of interactions between PTH responsive signaling systems in STRO-1(+) PDLSCs remains to be further elucidated.
Runx2 is a key transcription factor involved in the process of osteogenesis and directs multipotent mesenchymal cells to the osteoblast lineage [43]. During the process of osteogenesis, Sp7 has proved to be a direct downstream target of RUNX2, and these two proteins physically interacted and synergistically activated osteogenic genes [44,45]. In our current study, PTH (1-34) treatment leads to a significant increase of RUNX2 and Sp7 after osteogenic induction. The anabolic effects of PTH on osteoblast-specific transcription factors (Runx2 and Sp7) could be attributed to the predominant activation of cAMP/PKA signal transduction [46][47][48]. However, our data showed that the enhancement of Sp7 expression by PTH was even more robust in both uninduced and induced STRO-1(+) cells than that of Runx2, indicating a Runx2 independent activation of Sp7 gene expression [49]. It is possible that other genes upstream of Sp7 may also be affected by PTH, or effects of PTH may be directly mediated through Osx and independently of Runx2 [50,51]. Further studies are needed to fully elucidate the specific mechanisms involved in these processes.
In summary, we achieved successful culture of hPDLSCs and separation of STRO-1(+) and STRO-1(−) cells. Our additional experiments suggested that STRO-1(+) hPDLSCs had higher sensitivity to PTH and stronger osteogenic ability, which may play a major role in bone regulation in the presence of PTH. These data provide a deeper understanding of the effects of PTH on the regeneration of periodontal support tissues and may facilitate the development of improved biomaterials for periodontal stem cell treatment.

Conclusion
STRO-1(+) hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity of STRO-1(+) hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis. Our results provided evidences for applications of PTH in stem cell therapy for periodontitis diseases.

Competing Interests
The authors have no competing interests to declare.

Authors' Contributions
Xiaoxiao Wang and Yanlan Wang contributed equally to this work.