p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Abstract Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR+ and p75NTR− hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR+, p75NTR− and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR+ and p75NTR− hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR+ and p75NTR− hPDLSCs. The results showed that p75NTR+ hPDLSCs demonstrated superior osteogenic capacity than p75NTR− and unsorted hPDLSCs. Differentially expressed genes between p75NTR+ and p75NTR− hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR+ hPDLSCs expressed higher ITGA1 levels than p75NTR− hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR+ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR− hPDLSCs. These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.


| INTRODUC TI ON
Mesenchymal stem cells (MSCs) derived from teeth represent a fascinating area of research in regenerative medicine due to the complex and unique developmental origin of teeth. 1 Human periodontal ligament stem cells (hPDLSCs) are odontogenic MSCs that were first identified in and isolated from periodontal ligament tissue by Seo et al in 2004. 2 hPDLSCs were reported to have the ability to self-renew and the potential to differentiate into various specialized cell types, such as osteoblasts, fibroblasts, cementoblasts, chondroblasts, adipoblasts and neuroblasts. [2][3][4][5][6] Further evidence showed that hPDLSCs had little immunogenicity, maintained a higher growth capacity and were easy to obtain from impacted third molars or premolars extracted for orthodontics, demonstrating a promising application in regenerative medicine. [5][6][7][8] Therefore, hPDLSCs have been widely investigated in recent studies. However, as a complex and heterogeneous population, hPDLSCs even within the same condition may reflect different biological properties, restricting their utilization as stem cells. 1,9 Further isolation of homogeneous hPDLSCs with high regenerative potential will greatly contribute to their applications in the future.
During tooth development, ectomesenchymal stem cells (EMSCs) originating from the cranial neural crest (CNC) differentiate into various mesenchymal cell lines, such as dental papilla cells, pre-odontoblasts and dental follicle cells, ultimately giving rise to pulp, dentine, cementum and periodontal ligaments. 10,11 Some scholars have suggested that a population of the CNC-derived progenitor cells remain in the periodontal ligaments and continue to maintain their differentiation potential, namely periodontal ligament stem cells. 12 More accurate isolation of these CNC-derived stem cells will promote their use in the studies of differentiation mechanisms and in tissue engineering applications. p75 neurotrophin receptor (p75NTR) is a well-conserved transmembrane neurotrophin/proneurotrophin receptor that belongs to the tumour necrosis factor receptor superfamily. 13,14 The role of p75NTR is not limited to the nervous system, as it may have multifarious biological functions in non-neuronal tissues during development and differentiation. 15,16 In addition, p75NTR is highly expressed in CNC-derived cells and proved to be a reliable cell surface marker for stem cells from the CNC. 17 In our previous study, p75NTR was successfully used as a cell surface marker to isolate p75NTR + EMSCs from rats. 18,19 Further research found that these p75NTR + EMSCs, as CNC-derived stem cells, displayed superior multi-lineage differentiation potential and that p75NTR was involved in the positive regulation of osteogenic differentiation of rat EMSCs. 20,21 However, few studies that isolate hPDLSCs based on p75NTR expression have been reported, and the effect and underlying mechanism of p75NTR on the osteogenic potential of hPDLSCs are also unclear.
The present study aimed to use p75NTR as a cell surface marker to isolate hPDLSCs and to further investigate the effect and underlying mechanism of p75NTR on the osteogenic potential of hPDLSCs.
We expected to find an optimized cell surface marker for the identification and purification of hPDLSCs, thus promoting their applications in regenerative medicine.

| Cell isolation and culture
Tissues were obtained under approved guidelines set by Zunyi Medical University with informed patient consent. Primary hPDLSCs were isolated from premolars extracted from 18-to 25-year-old patients undergoing orthodontic treatment. Briefly, the periodontal ligaments were dissected from the root surface and digested with 1% collagenase I at 37°C for 45 minutes and neutralized with minimum essential medium α (α-MEM; Gibco, Waltham, MA, USA) containing 10% foetal bovine serum (FBS; Gibco). Then, the cell suspension was centrifuged at 800 rpm for 5 minutes. Next, the cell pellet was resuspended in α-MEM supplemented with 10% FBS and antibiotics (100 μg/mL streptomycin and 100 μg/mL penicillin) and then cultured at 37°C in a 5% CO2 humidified incubator.

| Oil Red O staining
The specimens were washed twice with PBS, fixed in 4% paraformaldehyde for 30 minutes, soaked with 60% isopropanol for 5 minutes and stained with an Oil Red O Stain Kit (Solarbio) for 20 minutes.
After washing 3 times with double-distilled water, the specimens were observed by optical microscope.

| Alcian blue staining
The specimens were washed twice with PBS, fixed in 4% paraformaldehyde for 30 minutes and stained with an Alcian Blue Stain Kit (Solarbio) for 40 minutes. After washing three times with doubledistilled water, the specimens were observed by optical microscope.

| Alkaline phosphatase staining
The specimens were washed twice with PBS, fixed in 4% paraformaldehyde for 30 minutes and stained with an Alkaline Phosphatase Activity Kit (Beyotime) for 30 minutes. After washing three times with double-distilled water, the specimens were observed by optical microscope.

| Alizarin Red staining
The specimens were washed twice with PBS, fixed in 4% paraformaldehyde for 30 minutes and stained with Alizarin Red S solution (Solarbio) for 10 minutes. After washing 3 times with double-distilled water, the specimens were observed by optical microscope.

| Quantitative real-time polymerase chain reaction
Total RNA was extracted with RNAiso Plus ( Table 1.

| Statistical analysis
All data are expressed as the mean ± standard deviation. Statistical significance was assessed by using Student's t test for two groups or one-way analysis of variance for three or more groups. P < 0.05 was considered as statistically significant.
Moreover, flow cytometry analysis showed that MSC markers (CD44, CD73, CD90 and CD105) were highly expressed in p75NTR + , p75NTR − and unsorted hPDLSCs, while MSC-negative markers (CD45, CD34, CD11b, CD19 and HLA-DR) were expressed at low levels in the three kinds of cells ( Figure 2). Moreover, the expression rates of p75NTR were 60.59% in p75NTR + hPDLSCs, 0.55% in p75NTR − hPDLSCs and 1.31% in unsorted hPDLSCs ( Figure 2). This result was consistent with the results of the confocal laser scanning microscopy assay, in which p75NTR was significantly enhanced in p75NTR + hPDLSCs, while it was weakly expressed in p75NTR − and unsorted hPDLSCs (Figure 3).  Figure 4A,B). After 7 days of osteogenic induction, the ALP staining intensity was stronger in p75NTR + hPDLSCs than in p75NTR − and unsorted hPDLSCs ( Figure 4C). Moreover, the mRNA ( Figure 4E) and protein ( Figure 4F) levels of p75NTR, ALP and RUNX2 were increased in p75NTR + , p75NTR − and unsorted hPDLSCs, although the mRNA and protein levels of p75NTR, ALP and RUNX2 elevated more in p75NTR + hPDLSCs than in p75NTR − and unsorted hPDLSCs. After 21 days of osteogenic induction, Alizarin Red staining showed more mineralized nodules in p75NTR + hPDLSCs than in p75NTR − and unsorted hPDLSCs ( Figure 4D).

| ITGA1 silencing inhibited osteogenic differentiation in P75NTR + hPDLSCs
The confocal laser scanning microscopy assay showed that ITGA1 was weakly expressed in p75NTR + hPDLSCs transfected with ITGA1 siRNA, whereas it was significantly enhanced in p75NTR + hPDLSCs transfected with NC siRNA ( Figure 6A). After 3 days of osteogenic induction, the mRNA ( Figure 6C) and protein ( Figure 6D) levels of ITGA1, p75NTR, ALP and RUNX2 were found to be lower in p75NTR + hPDLSCs transfected with ITGA1 siRNA than in p75NTR + hPDLSCs transfected with NC siRNA. Moreover, the ALP staining intensity was weaker in p75NTR + hPDLSCs transfected with ITGA1 siRNA than in p75NTR + hPDLSCs transfected with NC siRNA ( Figure 6B).

| D ISCUSS I ON
The biological role of p75NTR is controversial because it may trigger multiple cellular responses, such as proliferation, migration, apoptosis and differentiation. 22 In this study, we successfully isolated p75NTR + hPDLSCs by fluorescence-activated cell sorting, indicating that p75NTR can be used as a cell surface marker to isolate CNC-derived hPDLSCs. In addition, the proportion of p75NTR + hPDLSCs was as low as only 0.92%. This low percentage is consistent with the results of bone mesenchymal stem cells, of which less than 1% are p75NTR + . [30][31][32] We subsequently induced adipogenesis, chondrogenesis and osteogenesis in P75NTR + , p75NTR − and unsorted hPDLSCs in vitro and observed differences in their differentiation capacities. The results suggested that all three kinds of cells have the potential to differentiate into adipogenic, chondrogenic and osteogenic cell lines. Furthermore, p75NTR + hPDLSCs, as CNC-derived stem cells, possessed osteogenic potential superior to that of p75NTR − and unsorted hPDLSCs given their stronger ALP staining intensity, greater mineralized node formation and higher ALP and RUNX2 expression, while there was no significant difference between p75NTR − and unsorted hPDLSCs. These findings are in agreement with those of previous studies in which p75NTR + dental mesenchymal stem cells were demonstrated to have the greatest osteogenic potential among the cells examined, with strong induction of osteogenic markers such as RUNX2, distal-less homeobox 5 and bone gamma-carboxyglutamate protein. 33,34 To explore the underlying mechanism, we further performed RNA sequencing on p75NTR + and p75NTR − hPDLSCs and analysed the differential gene expression profiles. The results revealed that the ECM-receptor interaction signalling pathway might be closely related to the difference in osteogenic differentiation between these two types of cells.
The ECM is an ordered three-dimensional network of many large molecules, mainly consisting of proteins, polysaccharides and proteoglycans, that are synthesized by cells and secreted into the F I G U R E 5 Differential expression genes between p75NTR + and p75NTR − hPDLSCs. (A) Heatmap of differential genes. (B) Pathway mapping of differential expression genes. (C) The mRNA levels of ITGA1, ITGA7 and ITGA7 were detected by qPCR, using GAPDH as a control. *P < 0.05; ns, no significant difference components. 36 Integrins, which contain non-covalently bound αand β-subunits, are a family of glycosylated heterodimeric transmembrane adhesion receptors. 37 Engagement of integrins with ECM ligands triggers integrin clustering, which activates a number of intracellular signalling pathways to regulate cytoskeletal and ECM assembly and cell biological behaviour. 38 Accumulating evidence has shown that a crossover effect between integrins and ECM proteins is important in mediating cell proliferation and differentiation. [39][40][41] On the inside of the cell membrane, integrins may regulate the os- was regulated, the expression of p75NTR was in accordance with the changes in ITGA1 expression. Taken together, our study first demonstrated that ITGA1 promotes osteogenic differentiation of hPDLSCs under osteogenic induction conditions and that p75NTR optimizes the osteogenic potential of hPDLSCs by up-regulating ITGA1 expression via the ECM-receptor interaction signalling pathway. These findings not only prove p75NTR can be used to isolate homogeneous hPDLSCs with superior osteogenic potential but also provide a preliminary molecular mechanism for the effect of p75NTR on the osteogenic differentiation of hPDLSCs. In addition, as the reactions between ECM and receptors are mutual, the expression of p75NTR will also change with the regulation of ITGA1. Further studies are needed to confirm the exact mechanism of regulation between p75NTR and ECM.
In conclusion, p75NTR can be used to isolate CNC-derived hP-DLSCs. Moreover, p75NTR optimizes the osteogenic potential of hPDLSCs by up-regulating ITGA1 expression via the ECM-receptor interaction signalling pathway. These findings suggest that p75NTR can be used as a novel cell surface marker to identify and purify hP-DLSCs, thus promoting their applications in regenerative medicine.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.