Stromal cell‐derived factor‐1/Exendin‐4 cotherapy facilitates the proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo

Abstract Objectives Stromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo. Methods Cell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo. Results SDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4+ cells and CD90+/CD34‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo. Conclusions SDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.


| INTRODUC TI ON
Periodontitis is associated with many chronic diseases therefore represents a major global health problem. 1 Present therapies for periodontitis such as subgingival scaling and root planning, can only remove inflammation and halt disease progression while failing to accomplish bone tissue reconstruction. Therefore, novel approaches are in high demands to achieve periodontal bone regeneration. 2 Mesenchymal stem cells (MSCs) are regarded as the key element for tissue repair due to their potential to regenerate injured or pathologically damaged tissues and restore into a normal and healthy state. 3 With the development of stem cell therapies, endogenous MSCs recruitment which harnesses the innate regenerative potential of the body suggests new effective therapeutic approaches. 4 Chemokines are signalling molecules that can enhance cell migration, regulate immune responses and promote wound healing. 5 Stromal cell-derived factor-1 (SDF-1) belongs to the chemokine family, can promote the proliferation and migration of various MSCs in vitro by activating C-X-C chemokine receptor type 4 (CXCR4). [6][7][8] In vivo experiments evidenced that topical application of SDF-1 recruited MSCs to the wound area and promoted local vascular regeneration. 9,10 The defect area of periodontitis with insufficient cell number and poor cell activity is extremely unfavourable for subsequent periodontal bone regeneration. SDF-1 could mobilize bone marrow mesenchymal stem cells (BMSCs) to periodontal defects by activating SDF-1/CXCR4 signal axis. 7 Mounting evidences demonstrated that the introduce of SDF-1 enhanced the recruitment of endogenous cells to defects for regeneration. [10][11][12] It is obvious that SDF-1 is closely associated with the migration and growth of MSCs. However, SDF-1 does not always enhance proper osteogenic differentiation of these cells. 13 BMSCs transfected with SDF-1 could only promote osteogenic differentiation of the cells in early-stage, but not middle/late stage in vitro. 14 The application of SDF-1 alone is insufficient for favourable bone regeneration. 15 The optimal method to potentiate periodontal bone regeneration is to recruit a sufficient number of endogenous stem cells by SDF-1 and to promote committed differentiation by other growth factors or drugs.
Exendin-4 (EX-4), a full agonist of glucagon-like peptide-1 receptor (GLP-1R), has the ability to promote insulin secretion and release, inhibit glucagon secretion, and delay gastric emptying. 16 EX-4 is widely used in clinical treatment of type 2 diabetes (T2DM) for its half-life is longer than natural glucagon-like peptide-1 (GLP-1). 17 EX-4 could control blood sugar, exert anti-inflammatory, anti-oxidant and protective effects on cardiomyocyte metabolism, and promote the proliferation and migration of MSCs. [18][19][20][21] Recently, studies have confirmed that EX-4 has the capability to promote osteogenic differentiation, inhibit adipogenic differentiation of a variety of stem/precursor cells and promote bone formation to repair bone defects. [22][23][24] More importantly, the number of CXCR4 + MSCs increased by EX-4 and the PI3K/AKT-SDF-1/CXCR4 signalling pathway has been reported to play an important role in EX-4-mediated MSCs mobilization. 21 Accordingly, both the recruitment effect of SDF-1 and the osteogenic differentiation capability of MSCs could effectively be enhanced by EX-4. 21 Therefore, the application of SDF-1 together with EX-4 might be an effective strategy to augment periodontal bone regeneration.
The purpose of this study was to evaluate the effects of SDF-1/ EX-4 cotherapy on the proliferation, migration and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro.

Afterwards, local application of SDF-1 and systemic injection of EX-4
were applied to a rat periodontal bone defect model. Endogenous cells recruitment, early osteolclasteogenesis and bone regeneration were evaluated to verify whether the cotherapy will provide a new strategic option for in situ periodontal bone regeneration.

| Cell culture
The study protocol was approved by the School and Hospital of Stomatology, Shandong University (Protocol Number: GR201603), and written informed consent was obtained. Human PDLSCs were isolated according to our previous study. 6 Culture, osteogenic and adipogenic differentiation capabilities of human PDLSCs were performed as described in supplemental materials. Experiments were performed in sextuplicate (N = 6).

| Cell migration assay
The migratory effect of SDF-1 and EX-4 on PDLSCs was evaluated by an 8 µm transwell chamber (Corning). PDLSCs were seeded onto the upper chamber at a density of 5 × 10 4 cells/well and cultured in maintenance media. The lower plates were supplemented in 500 µL maintenance media with SDF-1, EX-4, or SDF-1+EX-4. 500 µL maintenance media served as a negative control (NC) and 500 µL basic media served as a positive control (PC). After 20 hours, cells that had migrated through the membrane were fixed with 4% paraformaldehyde (Sigma Aldrich) and stained with 0.1% crystal violet (Solarbio). Then cells were observed under a microscope and six randomly selected high-power microscopic fields (×200) per filter were counted. Experiments were performed in triplicate (N = 3).

| Cell osteogenic differentiation assay
PDLSCs were seeded in 6-well plates at a density of 2 ×

| Alizarin Red S staining
After 21-day induction, PDLSCs were fixed with 4% paraformaldehyde and extracellular matrix calcification was estimated by 2% Alizarin Red S (pH 4.3, Sigma Aldrich). 10% (w/v) cetylpyridinium chloride (CPC, Solarbio) and 10 mmol/L sodium phosphate solution were used to quantify the relative amount of calcium. The absorbance was measured at 562 nm wavelength.

| Alkaline phosphatase activity assay
After 7, 14 days induction, PDLSCs were lysed with 1% Triton-X (Solarbio) for 30 minutes. Protein concentration of collected cell lysates was measured by BCA assay (Solarbio). Alkaline phosphatase (ALP) activity in these lysates was detected according to assay kit (Nanjing Jiancheng Bioengineering Institute). The absorbance was measured at 520 nm wavelength.  Table 1.

| Western blotting assay
Cells were seeded in 6-well plates at a density of 3 ×

| Effect of ERK inhibitor U0126 on SDF-1 and EX-4 mediated PDLSC proliferation
A specific ERK inhibitor U0126 was used to clarify the role of ERK signalling pathway in SDF-1+EX-4 mediated PDLSC proliferation.
Briefly, PDLSCs were seeded in 96-well plates and cultured in basic media. After 24 hours, cells were cultured in maintenance media or maintenance media with SDF-1+EX-4, SDF-1+EX-4+U0126 or U0126 for 1, 3 or 5 days. Cell proliferation was quantified using a CCK8 kit as described above. Experiments were performed in sextuplicate (N = 6).

| Preparation of collagen membranes
Medical collagen membranes (Zhenghai Biotechnology) were cut into small pieces, placed in 96-well plates and cultured with 100 μL SDF-1 (50 μg/mL) or phosphate-buffered saline (PBS, Hyclone) according to our previous study. 11 The soaked collagen scaffolds were incubated at 4°C overnight before grafting.

| Preparation of periodontal defect model
The were performed as previously described. 11 Briefly, after general anaesthesia, we exposed the mandible surface of the rat and removed the buccal periodontal support tissues of the mandibular first molar roots. The periodontal fenestration defects (length × width × depth: 5 mm × 4 mm × 1 mm) located 1 mm below the crest of the alveolar bone and 1 mm behind the front of the mandible were prepared.
Collagen membranes loaded with SDF-1 or PBS were implanted into the defect. The masseter muscle, skin was reset and sutured.
Penicillin sodium (160 000 IU/mL) was administered daily for 5 days after surgery. The rats were sacrificed under general anaesthesia at 3 days, 1, 2, 4 and 8 weeks after surgery, and fixed with 4% paraformaldehyde solution by cardiac perfusion. The mandibles were harvested for the following experiments.

| Micro-CT analysis
The samples of mandibles were scanned in three dimensions (3D) by a micro-CT scanner (PerkinElmer). The effective voxel size was 50 μm in high-resolution mode. The results were performed 3D reconstruction using Quantum GX micro-CT software (PerkinElmer).

| Histological analysis
The bilateral mandibles of each rat were decalcified with 10% ethylenediaminetetraacetate (EDTA, Solarbio), dehydrated with a series of graded ethanol solutions and embedded in paraffin wax.
The embedded mandibles were cut in a buccal-lingual direction to acquire transverse sections (5 μm thick). The prepared sections were stained with haematoxylin and eosin (H&E, Solarbio).
The samples were observed under a BX53 microscope (Olympus Corporation) and measured with Image pro-plus 6.0 Software (Media Cybernetics).

| Immunofluorescence staining
The mandible sections were incubated with antigen retrieval solu-

| TRAP staining
Sections were stained with a leucocyte acid phosphatase kit (Solarbio) according to the manufacturer's instructions, nuclei were visualized with methyl green. The number of TRAP + cells was counted and measured by Image pro-plus 6.0 software.

| Immunohistochemical staining
The blocked sections were incubated with rabbit anti-ALP an- photographed under a microscope. The mean optical density (OD) of ALP and Col I staining was measured by Image pro-plus 6.0 software.

| Statistical analysis
All data were expressed as mean ± standard deviation (SD) and performed at least triplicates. Tests were analysed using graphpad prIsm software (version 6, MacKiev Software) and differences among more than two groups were analysed by one-way or two-way analysis of variance followed by Tukey's honestly significant difference comparison test. Value of P < .05 was defined as significant.
Meanwhile, EX-4 presented no effect on PDLSC proliferation compared with control group (P > .05). The results showed that SDF-1/ EX-4 cotherapy dramatically enhanced the proliferation of PDLSCs. SDF-1 or EX-4 also promoted PDLSC migration compared with NC (P < .001), and the promotion effect of SDF-1 on cell migration was stronger than that in EX-4 group (P < .001). These results indicated that SDF-1/EX-4 cotherapy enhanced PDLSC migration.

| SDF-1/EX-4 cotherapy promoted osteogenesis
ALP expression was significantly increased in SDF-1, EX-4 or SDF-1+EX-4 groups than that in control group at week 1 (P < .01), while there was no significant difference between SDF-1 and EX-4 groups (P > .05; Figure 9A,B). ALP expression was reduced obviously at week Several pathways have been reported to be involved in cell proliferation and migration, including ERK and PI3K/AKT pathways. 28,29 ERK signalling pathway plays an important role in regulating SDF-1 induced cellular functions. 29,30 Since ERK signalling pathway also plays a pivotal role in EX-4 mediated adipose-derived mesenchymal stem cells (ADSCs) and preosteoblast proliferation, here we investi- Bone remodelling depends on the coordination of bone resorption by osteoclasts and bone matrix synthesis by osteoblasts, thus osteoclasts play a vital role in the process of bone repair. 43,44 At the early stage of healing, osteoclasts create resorption pits, F I G U R E 6 SDF-1/EX-4 cotherapy significantly promoted the migration of CXCR4 + cells in defect areas. A, Immunofluorescence staining of CXCR4 (green) in the mandible sections at day 3, week 1, 2 and 4 post-surgery (×400). Scale bar: 50 μm. B, The number of CXCR4 + cells in SDF-1+EX-4 group was significantly larger than that in control, SDF-1 or EX-4 groups. The number of CXCR4 + cells peaked at week 1, reduced at week 2 and hardly detected at week 4. * P < .05, ** P < .01 and *** P < .001 growth factors such as TGFβ are released from the bone matrix.
The growth factors may recruit MSCs and osteoblast progenitors, promote these cells proliferation, differentiation with matrix formation and mineralization to fill resorbed bone area. 44,45 We investigated the effects of SDF-1 or/and EX-4 on osteoclastogenesis in vivo and our TRAP staining results showed that both However, which mechanism plays the key role in the periodontal bone regeneration remains to be elucidated.