Odontogenesis and neuronal differentiation characteristics of periodontal ligament stem cells from beagle dog

Abstract Periodontal ligament stem cells (PDLSCs) from beagle dogs had the characteristics of multi‐directional differentiation and had great application potential in tissue engineering and cell regenerative medicine. In this study, we analysed the odontogenesis and neuronal differentiation characteristics of PDLSCs in vitro. Results showed that the calcined tooth powder (CTP) and silver nanoparticles (AgNPs) additives could induce the PDLSCs into odontogenesis differentiation; besides, the immunofluorescence staining identified that the high dosage calcined tooth powder (400 μg/mL) significantly facilitated the odontogenesis associated with BMP4 expression. While the nutritional factor (L‐glutamine, NGF (nerve growth factor), bFGF (basic fibroblast growth factor), IGF‐1 (insulin‐like growth factor‐1) and EGF (epidermal growth factor)) additives were prior to induce the PDLSCs into neuronal differentiation. Simultaneously, PDLSCs had high proliferation ability with the different supplemented additives. Importantly, the Western blot results also proved the BMP4 and SMAD1 proteins were highly expressed in the induced odontoblast, while the SOX1, NCAM1, GFAP and VEGFA proteins were all obviously expressed in the induced neurons. Hence, PDLSCs had characteristics of both odontogenesis and neuronal differentiation.


| INTRODUC TI ON
With the rise of tissue engineering, the construction of biological substitutes such as biological tissues, organs and materials, which were used to rescue or/restore various forms of damaged or defective human tissues during recent years, has become a hotspot in the research of tissues engineering. 1,2 In endodontic treatment, there were shortcomings of the conventional pulp capping material, calcium hydroxide 3 ; hence, the dental stem cells isolated from teeth and nearby tissues had been attracted more and more attention in investigating their utilities in dental prosthesis by researchers due to their self-renewal and multilineage differentiation potential. 4 Previous studies had showed that periodontal ligament fibroblasts (PDLFs) had great potential in maintaining and repairing the periodontal tissues. 5,6 While the PDLSCs were obtained from the beagle dog periodontal ligament (PDL) of teeth freshly extracted, which had been recognized as a useful cell source for periodontal tissue regeneration. 7 They had the characteristics of easy to access and cryopreserved conveniently; besides, studies identified that they could induce the formation of new blood vessels and improve osteogenic. 8,9 In vitro culture studies, it had been reported that they could differentiate into odontoblasts, osteoblasts, chondrocytes and neurons [10][11][12] ; hence, the PDLSCs had great utility in regenerative medicine.
The calcined tooth powder (CTP) was obtained from beagle exfoliated teeth with 300°C calcine temperature, which completely removed the organic components and wiped out the antigenicity; the study of Jintao Wu, etc, 13 had found that CTP promoted hDPSCs into osteogenic and odontogenic differentiation. While the growth factors, NGF, 14 bFGF, EGF 15 and IGF-1, 16 had been identified playing crucial roles in the differentiation of MSCs into neuronal lineage and enhance cell viability, hence, to our knowledge, in this study, we attempted to study the characteristics of PDLSCs deriving from beagle dog differentiate into odontoblasts with CTP supplementary additives; besides, the nutritional and growth factors (L-glutamine, NGF, bFGF, IGF-1 and EGF) were also added as supplementary additives to induce PDLSCs into neural differentiation, both of which were in the purpose of providing important insights of PDLSCs in the application of dentin/pulp tissue regeneration for future endodontic treatment.

| PDLSCs isolation and culture
The molars were collected from the beagle dog of 12 months old.
The collected teeth were washed with PBS and obtained the periodontal membrane, then digested them in a solution of type I collagenase (3 mg/mL) (catlog: C0130, sigma) for 30-60 minutes at 37°C; afterwards, the digestion process was inactivated with 20% foetal bovine serum (FBS). Then, the digested cell suspensions were centrifuged at 300 g for 10 minutes, twice to remove the tissues debris; afterwards, the PDLSCs at a density of 3.0 × 10 5 cells were seeded in each 25 cm 2 plate with cell culture DMEM medium supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin; besides, the calcined tooth powder (CTP) at a concentration of 100 and 400 μg/mL was dividedly added into the cell culture medium, and the silver nanoparticles (AgNps) (catlog: JL-8001) 100 μmol/L concentration were also administrated into each cell culture medium.

| Cell morphology
The PDLSCs were cultured for 7 days in 6-well plates, and one group cells were treated with CTP (100 μg/mL) and AgNps (100 μmol/L), and the other one group cells were treated with CTP (400 μg/mL) and AgNps (100 μmol/L); the induced cell morphology was visualized at the seventh days.

| Immunofluorescence
The PDLSCs were cultivated like above, and one group cells in each well were treated with CTP (100 μg/mL) and AgNps (100 μmol/L), and the other one group cells in each well were treated with CTP (400 μg/mL) and AgNps (100 μmol/L); after 7 days cultivation, the cells in each well were washed with PBS; then, they were fixed with 4% paraformaldehyde for 30 minutes; and afterwards, they were permeabilized with 0.25% Triton-100 for 10 minutes at room temperature, then washed them with PBS for three times and, later, blocked them with 10% BSA for 60 minutes at 37°C.

| Cell colony formation assay
The PDLSCs were cultured for 7 days in 6-well plates, and one group cells were treated with CTP (400 μg/mL) and AgNps (100 μmol/L), and the other one group cells were treated with CTP (400 μg/mL), AgNps (100 μmol/L) and L-glutamine, NGF, bFGF, IGF-1, EGF growth factors. After 7 days cultivation, the cells were washed with PBS and fixed in 4% paraformaldehyde for 30 mins; then, they were stained with crystal violet for 30 minutes at room temperature; afterwards, the number of proliferated cells was counted with Image J software; besides, all assays were performed in triplicate.

| Western blot
The PDLSCs at a density of 3.0 × 10 5 cells were seeded into each 25 cm 2 plate with cell culture DMEM medium supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin; besides, the CTP, 400 μg/mL, and the AgNps, 100 μmol/L, concentrations were all administrated into each cell culture medium. Additionally, the nutritional growth factors, 200 μg/mL L-glutamine, 10 μg/mL beta-NGF, 10 μg/mL EGF, 10 μg/mL FGF and 10 μg/mL IGF-1, were all placed into one group cell culture medium; then, all the cultured cells were incubated at 37°C in 5% CO 2 incubator, when the cells grew well during the fifth to seventh day, washed them with PBS for three times and lysed them in RIPA buffer (Beyotime) containing 1 mmol/L protease inhibitor phenylmethanesulfonyl (PMSF). Subsequently, the proteins were denatured with hot water and measured the proteins concentration with BCA method; then, the proteins were isolated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); afterwards, the protein gel was transferred into polyvinylidene fluoride (PVDF; Millipore) membranes with a wet transfer apparatus (Bio-Rad).

| Statistical analysis
The differences of the data were analysed with one-way ANOVA method in the SPSS 19.0 statistical software (IBM, Corp.). The statistically significance was P < .05. And, the differences displayed in graphs were analysed with Prism 6.0 software (GraphPad Software), and data were presented in mean ± standard deviation.

| PDLSCs with high proliferation ability into odontogenesis differentiation and neuronal differentiation
To explore the induced cell proliferation ability, the crystal violet assay and cell colony formation assay were all conducted, and results showed that both the odontogenesis-like differentiated cells and neuronal differentiated cells were all with high proliferation ability from the third day to seventh day ( Figure 3A); the cell viability curve of the seven days cultivated period was logarithmic growth trend ( Figure 3B); moreover, the cell colony assay also identified the cells with high proliferation ability ( Figure 4A), and the difference was significant in the seventh day compared with that in the third day ( Figure 4B). Furthermore, the Western blot results revealed the CTP and AgNPs significantly induced the PDLSCs into odontogenesis-like cell differentiation, for the proteins BMP4 and SMAD1, and were highly expressed. However, the L-glutamine, NGF, bFGF, IGF-1 and EGF growth factors administration promoted the proteins expression of neural cell-associated markers, SOX1, NCAM1, SMAD1, GFAP and VEGFA; therefore, PDLSCs had the ability of neuronal cell differentiation ( Figure 5).

| D ISCUSS I ON
From the study, we had provided the evidence that the calcined tooth powder (CTP) and silver nanoparticles (AgNPs) additives had the ability to induce the PDLSCs differentiate into odontogenesis, while the nutritional factor (L-glutamine, NGF, bFGF, IGF-1 and EGF) additives were prior to induce the PDLSCs into neuronal differentiation. The CTP ingredients were major tooth calcium powder and hyaluronic acid, which were suitable for the migration and osteogenic differentiation of dental stem cells 17 ; besides, its' good biological compatibility made it to be a useful therapy for dental regeneration. 18 While the AgNps is effective for antibiotic-resistant bacterial strains, which was able to accelerate the osteogenesis of urine-derived stem cells through activating the RhoA signalling pathway. 19 Additionally, the growth factor mixtures (NGF, bFGF, IGF-1 and EGF) were benefit for the cell proliferation and differentiation [14][15][16] ; hence, there is a possibility that the utility of PDLSCs for dental restoration could enhance the teeth sensitivity and perceptivity. In our study, with the cell crystal violet assay and clonogenic assay in vitro, results showed that the induced odontoblast and neural cells had high cell viability and proliferation ability, which further confirmed PDLSCs to be ideal candidates for

E TH I C A L A PPROVA L
The study was followed the Ethical legals of animals.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available if necessary.