original article recombinant human amelogenin reverses … · recombinant human amelogenin reverses...

Int J Clin Exp Med 2016;9(12):23276-23284www.ijcem.com /ISSN:1940-5901/IJCEM0033362

Original Article Recombinant human amelogenin reverses osteogenesis suppression induced by an inflammatory microenvironment in human periodontal ligament cells

Jiachen Dong*, Zhongchen Song*, Rong Shu, Song Li, Mengjun Sun, Zhikai Lin

Department of Periodontology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. *Equal contributors.

Received June 6, 2016; Accepted August 22, 2016; Epub December 15, 2016; Published December 30, 2016

Abstract: Objective: The purpose of our study was to investigate the effects of recombinant human amelogenin (rHAm) on the osteogenic differentiation of PDLCs and to examine the role of Wnt/β-catenin signaling in PDLCs in the inflammatory microenvironment in vitro. Study Design: The proliferation rate of PDLCs was tested by MTT assay. The osteogenic differentiation of PDLCs under treatment with P. gingivalis LPS or rHAm was examined by real-time PCR and Western blotting. The gene and protein expression of Wnt signaling molecules was detected by real-time PCR and Western blotting, respectively. Data were analyzed statistically using the SPSS13.0 software package. Results: Our results show that 20 µg/mL rHAm can promote the proliferation of PDLCs, and 10 µg/mL P. gingivalis LPS has an inhibitory effect on these cells. The expression of osteogenic factors is significantly decreased in the 10 µg/mL P. gingivalis LPS group but is increased in the 20 µg/mL rHAm group. ALP activity also can be improved by rHAm under an inflammatory microenvironment. After treatment with P. gingivalis LPS, the mRNA expression of Wnt1, LRP5/6 and β-catenin in PDLCs decreases. Conclusion: Our data indicate that rHAm could improve the activ-ity of osteogenic differentiation under an inflammatory microenvironment.

Keywords: Human periodontal ligament cells, inflammatory microenvironment, lipopolysaccharide, recombinant human amelogenin, Wnt signaling

Introduction

Periodontitis is a prevalent disease worldwide and is mostly caused by oral microbial biofilms, which lead to progressive destruction of the periodontal tissues, including the gingiva, peri-odontal ligament, and alveolar bone. Treatment of periodontal tissues defects is an important therapeutic goal of periodontist. An approach that promotes periodontal regeneration despite periodontium defects is the key to the treat-ment of periodontitis. Periodontal ligament cells (PDLCs), the dominant cell type in the peri-odontal ligament, are widely used in periodon-tal tissue regeneration. PDLCs are capable of osteogenic differentiation, which can elevate ALP activity and form osteoid [1].

Enamel matrix proteins (EMPs), which are pro-duced by the ameloblast cells, can induce peri-odontal regeneration of new bone, cementum, and dentin [2]. Amelogenin comprises 90% of

EMPs. Veis et al showed that a specific low-molecular-mass amelogenin splice product, leucine-rich amelogenin peptide (LRAP), is ass- ociated with osteoblast differentiation. Other investigators also showed that LRAP could enhance osteogenic induction of mouse embry-onic stem (ES) cells and induce cementogene-sis and osteogenesis [3, 4]. Enamel matrix derivative (EMD) significantly enhances osteo-blast differentiation, mineralization [5], prolif-eration and migration of PDLCs and human gin-gival fibroblasts (GF) [6].

There are various factors that affect osteoblast differentiation of PDLCs including inflamma-tion, hypoxia and compressive stress. The inflammatory microenvironment is an impor-tant factor that we will explore further in this study.

Lipopolysaccharide (LPS) is derived from the cell wall of Gram-negative bacterial, including

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rHAm reverses osteogenesis suppression induces by LPS in hPDLCs

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Porphyromonas gingivalis, Prevotella interme-dia, and Fusobacterium nucleatum. LPS may stimulate host cells or cause direct destruction to periodontal tissues [7], which is known to induce not only an inflammatory response but bone resorption as well. LPS plays an important role in the progression of periodontitis. When osteoblasts are stimulated with LPS, the expression of receptor activator of NF-κB ligand (RANKL) is increased, and the expression of osteoprotegerin (OPG) is down-regulated [8].

Various signaling pathways regulate the pro-gression of tissue regeneration. Wnt/β-catenin signaling regulates the differentiation of osteo-blast progenitor cells [9]. The Wnt/β-catenin signaling includes the canonical Wnt pathway, the noncanonical Wnt-planar cell polarity path-way and the Wnt-calcium pathway [10]. Several Wnt proteins, such as Wnt1, 3a, 4, 5, 10b, and 13, have been shown to play a crucial role in osteoblast formation [11]. Wnt proteins are secreted glycoproteins that signal through bind-ing to a co-receptor complex formed by proteins of the frizzed (FZD) family and the lipoprotein receptor-related 5/6 proteins (LRP5/6). Canon- ical Wnt signaling stimulates cytoplasmic β-catenin to enter the nucleus and regulate tar-get gene expression [12].

Several previous studies have shown that enamel matrix proteins influence the differenti-ation of several types of cells. However, the role of rHAm in osteogenic differentiation of PDLCs under an inflammatory microenvironment re- mains unclear.

The purpose of this study was to explore the effect of rHAm and P. gingivalis LPS on the pro-liferation of human PDLCs. We also examined the influence of rHAm on osteogenic differenti-ation of human PDLCs under an inflammatory microenvironment in vitro and investigated the role of Wnt/β-catenin signaling in this process.

Materials and methods

Human PDLC isolation and culture

Human PDLCs were obtained from healthy human premolar, extracted for orthodontic treatment. Written consent was obtained from patients prior to conducting the study. Ethical approval was obtained from the Ethics commit-tee of the Ninth People’s Hospital, Shanghai

Jiao Tong University. PDLCs were isolated and cultured according to the method by Song et al [13]. Periodontal ligament tissues were scraped off the middle third of the root surface and cul-tured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum in humidified air containing 5% CO2 at 37°C. DMEM was changed every 5 days until conflu-ent cells were formed. After four to five subcul-tures, homogeneous, slim, spindle-shaped cells were obtained.

MTT assay

The proliferation of PDLCs under 0, 10, and 20 µg/mL rHAm and 0, 1, and 10 µg/mL P. gingiva-lis LPS was tested using an MTT assay. Samples were cultured for 8 days, and at every time point, MTT solution was added to each well and the samples were incubated for 4 hours to form formazan. The formazan was dissolved by dimethyl sulfoxide and density detected at a wavelength of 490 nm.

Quantitative real-time polymerase chain reac-tion (PCR)

The mRNA was extracted from all the groups and analyzed in order to detect changes in the osteogenic and Wnt signaling related genes under different PDLCs environments. We examined the following osteogenic and Wnt sig-naling factors: ALP, Collagen-I, BMP2, RUNX2, Wnt1, LRP5, LRP6 and β-catenin. The expres-sion levels of the target genes were normalized to that of the housekeeping gene glyceralde-hyde-3-phosphate dehydrogenase (GAPDH). At 1, 3, 6, and 12 hours and 1, 3, 5, and 7 days after treatment, the total mRNA was extracted by Trizol. cDNA was synthesized using a reverse transcription Kit (TaKaRa, Bio Inc, Otsu, Japan). GAPDH mRNA levels served as an internal control. Real-time PCR analysis was performed in a 20 µl reaction system. Changes in the expression of target genes were analyzed by a 2-ΔΔCt method: ΔΔCt=(Cttarget-CtGAPDH)sample-(Cttarget- CtGAPDH)control. We used an untreated sample as the contrast group.

The primers used in this study are as follows: LP: forward: 5’-TCAAACCGAGATACAAGCAC-3, and reverse: 5’-CCACCAGCAAGAAGAAGC-3’; RUNX2: forward: 5’-GCCTTCAAGGTGGTAGC- CC-3’, and reverse: 5’-CGTTACCCGCCATGACA- GTA-3’; Collagen-I: forward: 5’-TCCAACGAGATC-


23278 Int J Clin Exp Med 2016;9(12):23276-23284

GAGATCC-3’, and reverse: 5’-AAGCCGAATTCC- TGGTCT-3’; BMP2: forward: 5’-CCTCAGCAGA- GCTTCAGGTT-3’, and reverse: 5’-CCAACCTGGT- GTCCAAAAGT-3’; GAPDH: forward: 5’-TTCGAC- AGTCAGCCGCATCTT-3’, and reverse: 5’-ATCCG- TTGACTCCGACCTTCA-3’; Wnt1: forward: 5’-ATC- CTGCACAGCGTGAGTG-3’, and reverse: 5’-GATA- AACGCCGTTTCTCGAC-3’; LRP5: 5’-GACTCCTTC- CCCGACTGTATC-3, and reverse: 5’-AGAGAGAG- AGGATGATGCCAAT-3’; LRP6: 5’-AGCCTGTGGG- ACTTACTGTGTT-3, and reverse: 5’-CACAAGG- GTGCTGTCTGTATTC-3’; β-catenin: 5’-GCCAAGT- GGGTGGTATAGAGG-3, and reverse: 5’-GTGGGA- TGGTGGGTGTAAGAG-3’.

Western blot analysis

Total protein lysate was extracted using a cell protein extraction reagent containing a prote-ase and phosphatase inhibitor cocktails (Kang- Chen Bio-tech Inc, Shanghai, People’s Republic of China). Protein concentration was deter-mined using a BCA assay kit (Pierce, Rockford, IL, USA). An equal concentration of protein was separated on a 15% SDS-PAGE gel and then transferred to a PVDF membrane that was then blocked with 5% skim milk and incubated over-night at 4°C with the following primary antibod-ies: alkaline phosphatase (Abcam, Cambridge,

Figure 1. PDLCs were stimulated by different concentrations of rHAm (A) and P. gingivalis LPS (B) for 8 days, and the proliferation was measured by MTT assay. There is a significant increase in the 20 µg/mL rHAm group compared to the control group from day 2 to day 8 (A) and a decrease in the 10 µg/m P. gingivalis LPS group compared with control group from day 1 to day 8 (B). After 14 days of culture, human PDLCs were fixed and stained with an alkaline phosphatase activity kit. The expression of ALP activity in descending order is as follows: 20 µg/mL rHAm group, the control group, the 20 µg/mL rHAm+10 µg/mL P. gingivalis LPS group, and the 10 µg/mL P. gingivalis LPS group (C). (*P<0.05, **P<0.01 with respect to the control group; #P<0.05, ##P<0.01 with respect to the 10 µg/mL P. gingivalis LPS group).


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MA, USA), Collagen-I (Abcam), BMP2 (Abcam), RUNX2 (Abcam), Wnt1 (Abcam), LRP5/6 (Abcam), β-catenin (Abcam), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then incubated with appropri-ate secondary antibodies, and protein bands were detected using a chemiluminescence detection system.

Alkaline phosphatase activity

PDLCs were cultured in 12-well plates at a den-sity of 2.5×104 cells per mL. After 14 days of culture, the PDLCs were fixed and stained using an alkaline phosphatase kit (Shanghai Hongqiao Medical Reagent Company, Shanghai, People’s Republic of China).

Statistical analysis

The Student’s t-test was used to evaluate if the differences between two groups of data are significant. A P value <0.05 is considered significant.

Results

MTT assay for cell proliferation

The activity of the mitochondria respiratory chain was examined by MTT assay, which is used as a measure for proliferation in viable cells. The results of the MTT assay show that 10 µg/mL P. gingivalis LPS inhibits the prolifer-ation of human PDLCs and that 20 µg/mL rHAm promotes the proliferation of human PDLCs. There are significant differences between the 10 µg/mL P. gingivalis LPS group, the 20 µg/mL rHAm group and the control group from day 2 to day 8 (Figure 1A, 1B).

The expression of osteogenesis-related genes

In our experimental set up, 20 µg/mL rHAm up-regulated the expression of osteogenesis-relat-ed genes, but the opposite result emerged in the 10 µg/mL P. gingivalis LPS group (Figure 2). Treatment with 20 µg/mL rHAm significantly

Figure 2. The osteogenesis-related genes ALP, Collagen-I, BMP2, RUNX2 were measured by real-time PCR. ALP, Collagen-I, RUNX2, BMP2 gene expression levels in the 20 µg/mL rHAm group was higher than in the control group. The 10 µg/mL P. gingivalis LPS has a lower gene expression than the control group. The protective effects of rHAm rescued the lowered gene expression caused by inflammation. (*P<0.05, **P<0.01 with respect to the control group; #P<0.05, ##P<0.01 with respect to the 10 µg/mL P. gingivalis LPS group).


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up-regulated ALP mRNA levels at day 5 and day 7 compared with the control group (P<0.01). The gene expression level of Collagen-I, RUNX2 and BMP2 in the 20 µg/mL rHAm treated group was higher than that of the control group at 1, 3, 5, and 7 days. In general, for the osteogene-sis-related genes (ALP, Collagen-I, BMP2, and RUNX2), the 10 µg/mL P. gingivalis LPS group had a lower gene expression than the control group. After treatment with 10 µg/mL P. gingi-valis LPS and 20 µg/mL rHAm together, the mRNA expression of ALP, Collagen-I, BMP2 and RUNX2 was up-regulated compared to the 10 µg/mL P. gingivalis LPS-only group.

The expression of osteogenesis-related pro-teins

To determine the involvement of rHAm and P. gingivalis LPS in the osteogenic potential of PDLCs, antibodies against ALP, Collagen-I, BMP2, RUNX2 and β-actin were prepared. The results of the western blot analysis show that 20 µg/mL rHAm effectively increased the expression of ALP, Collagen-I, BMP2 and RUNX2 at 1, 3, 5, 7 days. In contrast, 10 µg/mL P. gingivalis LPS decreased the expression of these factors. As shown in Figure 3, ALP, Collagen-I, BMP2 and RUNX2 protein levels were increased in the presence of rHAm plus P. gingivalis LPS compared to the 10 µg/mL P. gingivalis LPS-only group.

Alkaline phosphatase activity

PDLCs were plated in six-well plates and cul-tured in osteogenic media. On day 14, ALP expression was enhanced in the 20 µg/mL rHAm group and decreased in the 10 µg/mL P. gingivalis LPS group. A marked increase in ALP activity in the 20 µg/mL rHAm and 10 µg/mL P. gingivalis LPS group compared with the 10 µg/mL P. gingivalis LPS group was also observed (Figure 1C).

The canonical Wnt/β-catenin signaling path-way

To determine whether rHAm and P. gingivalis LPS influence osteogenic progress through Wnt/β-catenin signaling, we examined the mRNA expression of several related genes. Wnt1, LRP5, LRP6 and β-catenin expression was significantly increased in the 20 µg/mL rHAm group compared to the control group at

1, 3, 6, 12 hours. PDLCs treated with P. gingivalis LPS at a concentration of 10 µg/mL reduced the gene expression of Wnt1 at 1 and 3 hours, β-catenin at 12 hours and LRP5/6 at 6 and 12 hours. Conversely, the inhibitory effect of 10 µg/mL P. gingivalis LPS was abol-ished by cotreatment with 20 µg/mL rHAm at the same time points (Figure 4A). Protein expression of LRP5/6 was up-regulated in the 20 µg/mL rHAm group compared to the control group at 6 hours and down-regulated in the 10 µg/mL P. gingivalis LPS group. At other time points, the expression levels of these proteins were not obvious (Figure 4B).

Discussion

It is well known that the periodontal ligament is a dense soft connective tissue that contains progenitor cells that can differentiate into fibro-blast, osteoblast and cementoblast cells [14]. In 2007, Lallier concluded that periodontal liga-ment cells grown in culture represent an imma-ture form of periodontal ligament fibroblasts [15]. In vitro, periodontal ligament cells have been shown to have the ability to undergo osteogenesis, as well as form mineralized nod-ules. Our study reports the effects of rHAm on human PDLCs osteogenesis in an inflammatory microenvironment. The results of our study demonstrate that PDLCs have a great osteo-genic capacity and that rHAm can enhance the expression of bone markers in PDLCs under an inflammatory microenvironment.

Enamel proteins are traditionally separated into amelogenins and non-amelogenins. Ame- logenin comprises almost 90% of the total enamel protein and has an important role in enamel crystal formation [16]. A previous study demonstrated that enhancement of the ALP activity in PDLCs occurs by treatment with EMD [17]. Li [18] found that amelogenin significantly promotes the proliferation of PDLCs. In our study, the proliferation of PDLCs is significantly enhanced when the cells are treated with 20 µg/mL rHAm compared to the control group. The gene expression and protein levels of ALP, BMP2, Collagen-I and RUNX2 are significantly up-regulated in PDLCs treated with 20 µg/mL rHAm. These results demonstrate that rHAm can effectively promote proliferation and bone formation via PDLCs.

Inflammation in periodontal ligaments may enhance the formation of deep periodontal


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Figure 3. Protein expression is significantly enhanced in the 20 µg/mL rHAm group. Osteogenic differentiation of PDLCs under an inflammatory environment is strongly reduced at 1, 3, 5, and 7 days. Protein levels are increased in the presence of rHAm plus P. gingivalis LPS, compared to the 10 µg/mL P. gingivalis LPS group.


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Figure 4. A: The expression of Wnt/β-catenin signaling related genes is significantly increased in the 20 µg/mL rHAm group compared to the control group at 1, 3, 5, and 12 hours. 10 µg/mL P. gingivalis LPS reduces the gene expression levels of Wnt1 at 1 and 3 hours, β-catenin at 1 and 2 hours and LRP5/6 at 6 and 12 hours; at these same time points, the reducing effect could be countered by the addition of 20 µg/mL rHAm. B: Protein expression of LRP5/6 is strongly activated at 6 hours by 20 µg/mL rHAm, but is reduced under an inflammatory microenvironment.


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pockets, and the absorption of alveolar bone may even facilitate the progress of periodonti-tis. P. gingivalis LPS has been implicated as a major pathogen in the development and pro-gression of periodontal disease. We stimulated an inflammatory microenvironment by treating PDLCs with P. gingivalis LPS. An MTT assay was used to detect proliferation of PDLCs under dif-ferent concentrations of P. gingivalis LPS. Our data suggests that 10 µg/mL P. gingivalis LPS significantly decreases the proliferation rate of PDLCs. In recent years, researchers have focused more attention on the production of pro-inflammatory cytokines stimulated by P. gingivalis LPS, rather than on the relationship between LPS and osteoblast differentiation. Li found that LPS decreases the osteogenic dif-ferentiation of periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs) [19]. The results of this study showed that 10 µg/mL P. gingivalis LPS is capable of down-reg-ulating osteogenesis-related genes and pro-teins expressed in PDLCs, and the same effects were seen in ALP activity. Few studies have focused on the relationship between inflammatory circumstance and the regeneration of periodontal tissue. Our results show that rHAm can compensate for the reduction of bone for-mation caused by P. gingivalis LPS at the gene expression and protein level. This result indi-cates that rHAm has the ability to induce bone regeneration under an inflammatory microenvironment.

It is well known that Wnt/β-catenin signaling is dynamically active in tooth-forming regions at various stages of tooth development and plays a complex role in this progress [12]. Wnt/β-catenin signaling has been shown to control stem cells proliferation and differentiation [20]. Canonical Wnt/β-catenin signaling is required for bone formation [21]. The β-catenin mediat-ed transcription is necessary for the differenti-ation of osteoblast progenitors. Jung’s study mimicked the effect of Wnt by adding LiCl to PDLCs and found that canonical Wnt signaling positively affects the differentiation of PDLCs into osteoblasts [22]. Similar results appeared in a study by Rungnapa [23]. In our study, we found rHAm up-regulates Wnt pathway factors but that P. gingivalis LPS down-regulates them. After treating PDLCs with rHAm and 10 µg/mL P. gingivalis LPS, there is an increase in Wnt pathway factors compared to the 10 µg/mL P.

gingivalis LPS-only treatment. The Wnt pathway is complex. Our findings imply that P. gingivalis LPS stimulated Wnt signaling at the protein level, but still inhibited osteogenic differentia-tion. Other pathways must be influencing this inhibition. Chen et al [24] Speculated that GSK-3β regulates osteogenesis of PDLSCs, but β-catenin signaling is not the most important regulatory network response to inflammation and leads to defective osteogenic differentia-tion. A better understanding of this unknown mechanism will help our understanding of the periodontal regeneration of PDLCs and other kinds of stem cells.

In conclusion, our study has provided a model for the application of rHAm and shown that PDLCs have the capability of osteogenic differ-entiation in an inflammatory microenvironment via rHAm. Inflammation, hypoxia and pressure microenvironments often occur simultaneously in periodontitis. Our future studies will be focused on understanding other signaling path-ways that may have a vital impact on enhancing bone regeneration and repair in inflammatory and other microenvironments.

Acknowledgements

This work was supported by the National Na- ture Science Foundation of China (Project No. 81271156, 81570977) and Biomedical Engi- neering Cross Research Foundation of Shang- hai Jiao Tong University (Project No. YG20- 15MS04).

Disclosure of conflict of interest

None.

Address correspondence to: Rong Shu, Depart- ment of Periodontology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, No. 500 Qu Xi Road, Huangpu District, Shanghai, China. Tel: +86-021-23271699-5510; E-mail: [email protected]

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original article recombinant human amelogenin reverses … · recombinant human amelogenin reverses...

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