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Effects of connective tissue growth factor on human periodontal ligament fibroblasts
Archives of Oral Biology 84 (2017) 37–44
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Research Paper
Effects of connective tissue growth factor on human periodontal ligament fibroblasts Xuejing Duana,1, Mei Jia,1, Fengying Dengb, Zhe Sunb, Zhiyong Lina, a b
MARK
⁎
School of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China School of Stomatology, Shandong University, Jinan, Shandong Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Connective tissue growth factor Human periodontal ligament fibroblasts Periodontal regeneration Concentrations
Objective: The aim of this study was to evaluate the effects of different concentrations of connective tissue growth factor (CTGF) on human periodontal ligament fibroblasts(HPLFs). Design: HPLFs were cultured and identified. Then, different concentrations of CTGF (1, 5, 10, 50, 100 ng/ml) were added to the HPLF culture. Next, CCK-8 assays, alkaline phosphatase (ALP) assays, hydroxyproline determination, alizarin red staining methods, Transwell chambers and real-time PCR methods were applied to observe the effects of CTGF on the proliferation, ALP activity, synthesis of collagen, formation of mineralized nodules and migration. We also studied expression of ALP, fiber link protein (FN), integrin-binding sialoprotein (IBSP), osteocalcin (OC), and integrin beta 1 (ITGB1) mRNA by HPLFs. Statistical significance was assumed if P < 0.05 or P < 0.01. Results: The addition of CTGF (1, 5, 10 ng/ml) remarkably promoted the proliferation and collagen synthesis of HPLFs compared with controls. CTGF (1, 5, 10, 50 ng/ml) improved ALP activity of HPLFs, and at all concentrations, CTGF (1, 5, 10, 50, 100 ng/ml) improved the expression of ALP, FN, IBSP and ITGB1 mRNA. In addition, CTGF (1, 5, 10, 50, 100 ng/ml) promoted the migration of HPLFs, which was dose-dependent, with maximal promotion in the 10 ng/ml group (P < 0.05 or P < 0.01). Conclusions: Thus, in a certain range of concentrations, CTGF can promote the biological effects, including proliferation, migration and collagen synthesis of HPLFs, to promote the differentiation of HPLFs in the process of osteogenesis.
1. Introduction As one of the most common infectious diseases, periodontal disease (PD) causes substantial destruction of alveolar bone, periodontal ligament (PDL), and gingiva, leaving the oral environment exposed and allowing initiation of root contamination and tooth loss. PDL, a type of tooth-supporting structure and non-mineralized connective tissue between alveolar bone and cementum, is composed of heterogeneous cell populations, including osteoclasts, cementoblasts, fibroblasts, osteoblasts, mast cells, mesenchymal cells, and phagocytes (Beertsen, McCulloch, & Sodek, 1997). PDL appears to be actively involved in remodeling of alveolar bone, has shock-absorbing properties against mechanical stress, and prevents the tooth and alveolar bone from being damaged during mastication. Thus, PDL has an important functional role in the maintenance and renewal of periodontal tissues (Wu, Zhang,
Wang, Zhang, & Tan, 2015) and enables teeth to move via periodontal regeneration during orthodontic treatment (Yashiro et al., 2014). Among the cells present in the periodontium, periodontal ligament fibroblasts (PLFs) are the most abundant and play the most important role on the development, repair and regeneration of periodontal tissues (Choe et al., 2012). It has been proven that PLFs display biological characteristics similar to those of bone marrow-derived undifferentiated mesenchymal cells (Ren et al., 2015). In addition to constantly producing new dental cement and principal fibers and reconstructing alveolar bone, PLFs also synthesize the extracellular matrix, which holds the cells and plays special biological roles in cell conglutination, transportation and mineralization (Choe et al., 2012; Zhang et al., 2013). Being naturally osteogenic, PLFs are capable of differentiating into osteoblasts (Heo, Lee, & Lee, 2010). Furthermore, it has been found that PLFs are able to perceive the mechanical signals
Abbreviations: CTGF, connective tissue growth factor; HPLFs, human periodontal ligament fibroblasts; ALP, alkaline phosphatase; FN, fiber link protein; IBSP, integrin binding sialoprotein; OC, osteocalcin; ITGB1, integrin beta 1; PDL, periodontal ligament; PLFs, periodontal ligament fibroblasts; ECM, extracellular matrix; TGF-β, transforming growth factor-β ⁎ Corresponding author at: School of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, No 51, Jing Seven and Wei Six Road, Jinan, Shandong, China. E-mail address: [email protected] (Z. Lin). 1 These authors contributed to the work equally and should be regarded as co-first authors. http://dx.doi.org/10.1016/j.archoralbio.2017.09.010 Received 9 January 2017; Received in revised form 28 August 2017; Accepted 16 September 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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Fig. 1. The morphology, immunohistochemical staining and proliferation of HPLFs. A: HPLFs were either fusiform or dendroid in shape, with full cell bodies, clear nuclei, and two or three fine cytoplasmic processes. (40×). B: The proliferation curve of HPLFs. It was similar to an upside down ‘S’ with the arrest, lgarithmic growth, and plateau phases. C: Vimentin was positive in the cytoplasm of HPLFs (20×). D: Keratin was not expressed in HPLFs (20×).
regulation of various biological processes associated with fibrogenesis, including proliferation, differentiation, chemotaxis, cellular adhesion, migration, production of extracellular matrix (ECM) and angiogenesis, especially in promotion of deposition of several ECM proteins, such as fibronectin, collagen, and tenascin C (Yang et al., 2015; Zhang, Meng, Zhu, Liu, & Deng, 2014). Furthermore, CTGF also contributes to the proliferation and differentiation of mouse periodontal ligament-derived cells and a human periodontal ligament stem/progenitor cell line (Asano et al., 2005; Yuda et al., 2015). However, to date, there has been no effort to determine the effects of different concentrations of CTGF, if any, on the biological activities of human PDL fibroblastic cells (HPLFs). This study was performed to investigate the biological effects of HPLFs, aiming to provide a new research direction for dental treatment of periodontal disease and to offer a new growth factor for periodontal disease clinical drug research and development.
and exhibit a number of the phenotypic characteristics of osteoblasts under stress stimulation, including osteocalcin (OCN), osteopontin (OPN), Osteriox (also known as SP7), sialoprotein (BSP), collagen type I (Col I), and activating transcription factor 4 (ATF4). The quality of PLFs is related to the differentiation of osteoblasts and remodeling of periodontal tissue (Ren et al., 2015). Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN family and is a secreted matricellular and multifunctional protein. The CCN family was named after the first three members, Cyr61 (CCN1), CTGF (CCN2) and Nov (CCN3), were identified. It also includes CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP3) (Klaassen, van Geest, Kuiper, van Noorden, & Schlingemann, 2015). It has been shown that CCN family members are involved in regulation of cell proliferation, differentiation, migration, extracellular matrix remodeling and apoptosis (Chen & Lau, 2009). CTGF was originally identified as a growth factor-inducible immediate early gene in mouse fibroblasts and in human vascular endothelial cells and is the most studied member of this family (Cheng, Chang, Fang, Sun, & Leung, 2015), which was found to promote the chemotaxis and mitosis of fibroblasts. While its expression level in various tissues under the normal physiological state is lower, CTGF expression can be up-regulated greatly in many diseases, especially in fibrotic and cancerous tissues or under high mechanical stress load stimulation (Chaqour & GoppeltStruebe, 2006; Lomas et al., 2011; Reich, Maziel, Ashkenazi, & Ornan, 2010; Wang et al., 2011). Moreover, it was recently reported that CTGF/CCN2 gene expression in human PDL cells is up-regulated by stretch loading (Yuda et al., 2015). CTGF has been implicated in a number of more complex biological processes, including angiogenesis, osteogenesis, chondrogenesis, fibrosis, wound healing, and tumorigenesis (Arnott et al., 2011). It has been shown that CTGF is involved in
2. Materials and methods 2.1. HPLFs culture and identification Dental premolars that were free of dental caries or periodontal disease but extracted due to orthodontic treatment were collected. Immediately after extraction, the teeth were immersed in DMEM (Gibco, USA) containing double antibiotics (100 μg/ml penicillin G and 100 μg/ml streptomycin; Sijiqing Company, Hangzhou, China) and washed 10 times with PBS (pH 7.2) under sterile conditions. Then, the periodontal ligament tissues attached to the middle third of the roots were curetted gently by a surgical scalpel, minced and placed in 24-well plates. The plates were maintained at 37 °C in an atmosphere of 95% air 38
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and 5% CO2 in DMEM containing 10% FBS (Gibco BRL, USA). After reaching 80% confluence, HPLFs were digested with a mixture of 0.25% (w/v) trypsin and 0.02% (w/v) EDTA and subcultured at a 1:3 ratio. Cellular morphologies of HPLFs were observed by inverted phase contrast microscopy and then HPLFs were identified by vimentin and keratin immunostaining.
M2 multifunctional ELISA plate reader (Molecular Devices, USA) at 450 nm. 2.4. ALP activity test HPLFs were plated at a density of 2 × 104/ml in a 96-well culture plate. Different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml) were added to each well, and four duplicate wells were set up for each sample. All groups were incubated for 7, 14 and 21 days and then were washed three times with PBS. Then, 1% TritonX-100 (Sigma, USA) was used to dissolve the cells, and the lysates with the ALP assay working solution were incubated for 4 min. After incubation, the absorbance of p-nitrophenol was read at 560 nm in an ELISA plate reader. Double distilled water was used as the blank group, and phenol standard solution was used as the standard group.
2.2. Transwell chamber migration assay DMEM containing different concentrations of CTGF (0, 1,5, 10, 50, and 100 ng/ml)was added to the lower well of the Transwell chambers (Corning Company, America), and 200 μl of resuspended cells was added to the upper surface. Transwell chambers containing cells were then incubated at 37 °C with 5% CO2 for 24 h. The upper layer of the Transwell chamber was then removed, and the lower surface was washed 3 times with PBS, fixed with 5% paraformaldehyde for 5–10 min and then stained with 0.1% crystal violet for 1–2 min. Nuclei of migrated cells on the lower surfaces were stained and were observed by fluorescence microscopy. The number of migrating cells in each microscope field (6100) was also counted.
2.5. Total RNA isolation and real-time PCR (RT-PCR) Total RNA was extracted from cells using Trizol reagent(Invitrogen, USA)for conventional reverse transcription. RT-PCR was used to detect the expression of alkaline phosphatase (ALP), fibronectin (FN), bone sialoprotein (IBSP), osteocalcin (OC) and integrin beta 1 (ITGB1) by HPLFs. 1 μg of RNA was reverse transcribed with a SuperScript firststrand synthesis system (Invitrogen, Carlsbad, CA) following the manufacturer’s recommendations. RT-PCR reactions were prepared utilizing iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The sequences of the primers included the following: β-actin (Forward: 5′CAAGTACCAGCGAGCAGCTT-3′, Reverse: 5′-AAAGCCGAGCTGCCA GAGTT-3′); ALP (Forward: 5′-AACGGTCCAGGCTATGTGCT-3′, Reverse: 5′- CTGCTGACTGCTGCCGATAC-3′); FN (Forward: 5′- GCCAGATGA TGAGCTGCAC-3′, Reverse: 5′-GAGCAAATGGCACCGAGATA-3′); IBSP (Forward: 5′-CTGGCACAGGGTATACAGGGTTAG-3′, Reverse: 5′GCCTCTGTGCTGTTGGTACTGGT-3′); ITGB1 (Forward: 5′-GGTTTCACT TTGCTGGAGATGG-3′, Reverse: 5′-CAGTTTCTGGACAAGGTGAG
2.3. CCK-8 assay HPLFs (1 × 104 cells/ml, fifth passage) were plated in a 96-well culture plate at 100 μl/well in DMEM containing 10% FBS. Three duplicate wells were set up for each sample. After 24 h, the culture medium was replaced by serum-free DMEM for another 12 h. Subsequently, HPLFs were cultured in DMEM containing 10% FBS with different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml). Cells cultured on basic media (DMEM with 10% FBS) were the control group, and DMEM with 10 ng & z.urule;ml CTGF was the blank group. After 24, 48 and 72 h incubation, 10 ui CCK-8 (Nanjing Jiancheng Bioengineering Institute, China) was added to each well and incubated for 4 h. The growth state of the HPLFs was measured with a SpectraMax
Fig. 2. Effects of different concentrations of CTGF on the migration ability of HPLFs(40×). A: HPLFs migration of control group; B: HPLFs migration of 1 ng/ml CTGF group; C: HPLFs migration of 5 ng/ml CTGF group; D: HPLFs migration of 10 ng/ml CTGF group; E: HPLFs migration of 50 ng/ml CTGF group; F: HPLFs migration of 100 ng/ml CTGF group; G: Effects of different concentrations of CTGF on the migration ability of HPLFs. And when the mass concentration was 10 ng/ml, CTGF could promote the mineralization greatly (P < 0.01). *P < 0.05, **P < 0.01.
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Fig. 3. Effects of different concentrations of CTGF on the proliferation of HPLFs. The proliferation curves of HPLFs in different groups. Compared with the control group, CTGF (1, 5, and 10 ng/ml) significantly promoted the proliferation of HPLFs after 24, 48 and 72 h (P < 0.05 or P < 0.01). However, CTGF at a concentration of 100 ng/ml had an inhibitory effect.The effects of CTGF were all dose-dependent, with maximal proliferation in the 10 ng/ml group. *P < 0.05, **P < 0.01.
package (SAS, Cary, NC). All the data were expressed as the mean ± standard deviation.
CAATA-3′); and OC (Forward: 5′-GGCAGCGAGGTAGTGAAGAGA-3′, Reverse: 5′-CTCCTGAAGCCGATGTGG-3′). β-actin gene expression was detected for normalization purposes.
3. Results 2.6. Hydroxyproline determination 3.1. HPLFs culture and proliferation assay HPLFs were plated at a density of 5 × 104/ml in a 96-well culture plate and incubated for 24 h at 37 °C with 5% CO2. Then, the previous culture solution was discarded, serum free DMEM was added, and HPLFs were incubated for an additional 12 h. Subsequently, 1 ml substrate containing different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml) was added to each well. Each group contained four duplicate wells. After 24 h of incubation, we collected 0.5 ml of supernatant from each well and determined hydroxyproline levels following kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The OD of each tube was measured using an ELISA plate reader at 550 nm.
HPLFs began to migrate from the edge of the tissues after 8–10 days of culture. HPLFs were fusiform in shape and in good condition after being passaged. The cells were arranged in a sarciniform or swirl pattern (Fig. 1A). The proliferation curve of HPLFs was an S-shaped curve, with arrest, logarithmic growth and plateau phases (Fig. 1B). IHC testing of the HPLFs (third passage) revealed that the cytoplasm was positive for vimentin (yellow-brown color) (Fig. 1C); however, keratin was not expressed in the cytoplasm (Fig. 1D). 3.2. Effects of different concentrations of CTGF on the migration of HPLFs Cell migration ability increased with higher CTGF concentrations and exhibited a marked concentration-dependence (Fig. 2A–F). Further studies (Fig. 2G) showed that there were statistically significant differences in the number of migrating cells among all CTGF groups (1,5, 10, 50, and 100 ng/ml) compared to the control group after 24 h incubation (P < 0.05 or P < 0.01).
2.7. Alizarin red S staining HPLFs were plated at a density of 2 × 104/ml in six 24-well culture plates at 1 ml/well. The culture solution was discarded, and HPLFs were washed 3 times with PBS after 24 h incubation. A 1-ml aliquot of mineralized induced solution containing 10% FBS, 1 × 10−8mol/l dexamethasone (Sigma, USA), 10 mmol/l β-sodium glycerophosphate and 50 g/ml DMEM was added to the blank group, while mineralized induced solution containing different concentrations of CTGF(1, 5, 10, 50, 100 ng/ml)was added to the other five 24-well culture plates. All were incubated 21 days and then washed 3 times with PBS. An aliquot of 500 μl 4% paraformaldehyde was added to each well, and HPLFs were fixed for 30 min. After that, the 4% paraformaldehyde was removed and replaced with sterile 2% alizarin red (Sigma, USA) solution and incubated for 30 min at room temperature. Finally, the 2% alizarin red solution was discarded, and HPLFs were washed 3 times with double distilled water. The numbers of newly formed calcium nodules in each well were then counted under an inverted phase contrast microscope (Leica MPS30, Germany).
3.3. Effects of different concentrations of CTGF on the proliferation of HPLFs The effects of different concentrations of CTGF on the proliferation of HPLFs are illustrated in Fig. 3. Compared with the control group, Table 1 Effects of different concentrations of CTGF on the ALP activity of HPLFs ( x ± s). Concentration (ng/ ml)
Time (d) 7
0 (control) 1 5 10 50 100
2.8. Statistical analysis Statistically significant differences (P < 0.05 or P < 0.01) between the various groups were measured using ANOVA. All statistical analyses were carried out using an SPSS 22.0 statistical software
0.222 0.252 0.298 0.507 0.286 0.168
14 ± ± ± ± ± ±
0.003 0.006* 0.005* 0.013* 0.012* 0.009*
0.254 0.276 0.354 0.520 0.364 0.174
21 ± ± ± ± ± ±
0.009 0.003 0.012* 0.016** 0.019* 0.015*
Compared with the control group, *P < 0.05, **P < 0.01.
40
0.262 0.294 0.341 0.506 0.357 0.163
± ± ± ± ± ±
0.007 0.012 0.016* 0.018** 0.015* 0.011*
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Fig. 4. Effects of different concentrations of CTGF on the expressions of ALP, FN, IBSP, OC, ITGB1 mRNA of HPLFs(*P < 0.05)A: the expressions of ALP mRNA in different groups; B: the expressions of FN mRNA in different groups; C: the expressions of IBSP mRNA in different groups; D: the expressions of OC mRNA in different groups; E: the expressions of ITGB mRNA in different groups. In addition, the results showed that the expression of ALP, FN, IBSP, OC and ITGB1 mRNA followed normal distribution and reached a peak when the concentration of CTGF was 10 ng/ ml (ALP, IBSP, OC and ITGB1) or 5 ng/ml (FN).
followed normal distribution and reached a peak when the concentration of CTGF was 10 ng/ml (ALP, IBSP, OC and ITGB1) or 5 ng/ml (FN).
CTGF (1, 5, and 10 ng/ml) significantly promoted the proliferation of HPLFs after 24, 48 and 72 h (P < 0.05 or P < 0.01). The effects of CTGF were all dose-dependent, with maximal proliferation in the 10 ng/ml group. There was no statistically significant difference between the 50 ng/ml CTGF group and the control group (P > 0.05). However, CTGF at a concentration of 100 ng/ml had an inhibitory effect. Thus, the lower concentrations of CTGF stimulated proliferation of HPLFs, while higher concentrations inhibited proliferation (P < 0.05). The 10 ng/ml concentration of CTGF promoted the highest proliferation rate for the periodontal ligament fibroblasts (Fig. 3).
3.6. Effects of different concentrations of CTGF on the secretion of hydroxyproline by HPLFs Hydroxyproline is a specific amino acid component of collagen, which can be used to estimate collagen content and act as an important indicator of collagen quality (Kuttan & Radhakrishnan, 1973). The effects of different concentrations of CTGF on the synthesis of hydroxyproline by HPLFs are illustrated in Table 2. Compared with the control group, CTGF (1, 5, 10 ng/ml) significantly enhanced the secretion of hydroxyproline by HPLFs after 24 h (P < 0.05 or P < 0.01). The effects of CTGF were dose-dependent at all concentrations, with maximal promotion in the 10 ng/ml group. There was no statistically significant difference between the 50 and 100 ng/ml CTGF groups and the control group (P > 0.05).
3.4. Effects of different concentrations of CTGF on the ALP activity of HPLFs The effects of different concentrations of CTGF on the ALP activity of HPLFs are illustrated in Table 1. Compared with the control, CTGF (5, 10, 50 ng/ml) significantly increased ALP activity of HPLFs after 7, 14 and 21 days (P < 0.05 or P < 0.01). The effects of CTGF were all dose-dependent with maximal promotion of ALP activity in the 10 ng/ ml group. However, CTGF at a concentration of 100 ng/ml had a significant inhibitory effect on the ALP activity of HPLFs.
Table 2 Effects of different concentrations of CTGF on the synthesis of collagen of HPLFs ( x ± s).
3.5. Effects of different concentrations of CTGF on the expression of ALP, FN, IBSP, OC, and ITGB1 mRNA by HPLFs RT-PCR was used to measure the mRNA expression levels of ALP, FN, IBSP, OC and ITGB1 by HPLFs cultured in different media after 14 days. The results showed that CTGF, in a range of concentrations, increased the mRNA expressions levels. In addition, the results (Fig. 4) showed that the expression of ALP, FN, IBSP, OC and ITGB1 mRNA
Concentration(ng/ml)
Hydroxyproline(ug/ml)
0 (control) 1 5 10 50 100
1.655 2.221 2.967 3.762 1.524 1.376
± ± ± ± ± ±
0.345 0.356* 0.415* 0.313** 0.189# 0.217#
Compared with the control group, *P < 0.05, **P < 0.01, #P > 0.05.
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Fig. 5. Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs (20×). A:The formation of mineralized nodules in control group; B: The formation of mineralized nodules in 1 ng/mg CTGF group; C:The formation of mineralized nodules in 5 ng/mg CTGF group; D: The formation of mineralized nodules in 10 ng/mg CTGF group; E: The formation of mineralized nodules in 50 ng/mg CTGF group; F: The formation of mineralized nodules in 1000 ng/mg CTGF group;G: Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs. And when the mass concentration was 10 ng/ml, CTGF could promote the mineralization greatly (P < 0.01). *P < 0.05, **P < 0.01.
(Nowak, Popow-Wozniak, Raznikiewicz, & Malicka-Blaszkiewicz, 2009), which is important for periodontium regeneration. CTGF can also induce chemotactic migration in many kinds of cells in vitro, such as certain epithelial cells, vascular endothelial cells and vascular smooth muscle cells (Cicha and Goppelt-Struebe, 2009; Jun and Lau, 2011). Our study showed that cell migration ability increases when the CTGF concentration is higher, which supports a role for CTGF in periodontium regeneration. HPLFs have the potential to differentiate into cementoblasts and osteoblasts which are needed for cementum and alveolar bone formation, respectively (Basdra & Komposch, 1997; Seo et al., 2004; Tomokiyo et al., 2008). Studies have reported higher gene expression of OC, IBSP and TGFβ receptor in alkaline phosphatase-positive subpopulations of PDLFs (Murakami, Kojima, Nagasawa, Kobayashi, & Ishikawa, 2003). These molecules all play important roles in osseous development, induction, maintenance and repair. In addition, ALP is also important for differentiating osteoblast-like cells, and its degree of activity reflects the mineralization ability of tissues and cells and underlies the formation of osteogenic properties (Pae, Kim, Kim, & Woo, 2011). Our study revealed that CTGF could promote ALP activity, the formation of mineralized nodules and the expression of IBSP, OC and ALP mRNA. However, the promotion of CTGF decreased when the concentration was high. These studies suggest that CTGF may play a role in the commitment of mesenchymal progenitors to the osteogenic lineage, which is important for periodontal regeneration. That effect was dose-dependent. As we know, the most important component of periodontium is collagen fibers, which are mainly composed of Col-I. Col-I of periodontium does not only maintain the stability of organizational structure but is also necessary for periodontium to perform its physiological function and carry out complete physiological and structural regeneration (Ivanovski, Li, Daley, & Bartold, 2000). The level of Col-I expression may directly reflect the degree of periodontium regeneration. In addition, it is also an important component of bone matrix and
3.7. Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs After culture for 21 days, Alizarin red S staining of HPLFs showed different calcium nodules in all groups (Fig. 5A–F). Six mineralization rate comparisons between experimental groups showed statistically significant differences (P < 0.05). CTGF (10 ng/ml) promoted mineralization greatly compared to controls (P < 0.01) (Fig. 5G).
4. Discussion CTGF has been shown to be an autocrine mediator of transforming growth factor-β (TGF-β) in fibroblasts. Previously, studies reported that TGF-β stimulation of fibroblast proliferation, collagen synthesis, and most recently, myofibroblast differentiation is mediated via CTGF-dependent pathways (Grotendorst & Duncan, 2005). Cell proliferation is one of the most important factors in maintaining the balance of cell numbers and maintaining normal organ structure (Zhang et al., 2013). In this study, we showed that CTGF, within a certain concentration range, can promote the proliferation of HPLFs after 7, 14 and 21 days, while high concentration inhibits proliferation. We also showed that the effect is dose-dependent. These findings are in accordance with other studies. A great number of experimental studies have previously demonstrated that CTGF can stimulate growth of multiple cell types (Bai et al., 2011; Chu, Chang, Prakash, & Kuo, 2008; Wu, Wu, Lu, Dong, & Chen, 2006); however, the overall number of cells decreases when the concentration is too high. Those studies suggested that the effects of CTGF on HPLFs are directly related to the concentration of CTGF. Migration of fibroblasts is a key step in the process of periodontium regeneration. Phanish, Winn, and Dockrell (2010) found that CTGF may promote the migration of mesangial cells after trauma. In addition, in the wound healing process, fibroblasts can chemotactically migrate to specific locations, secrete cytokines and synthesize extracellular matrix 42
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K., & Dockrell, M. E. (2010). Connective tissue growth factor(CTGF, CCN2)–A marker, mediator and therapeutic target for renal fibrosis. Nephron Experimental Nephrology, 114(3), e83–e92. Reich, A., Maziel, S. S., Ashkenazi, Z., & Ornan, E. M. (2010). Involvement of matrix metalloproteinases in the growth plate response to physiological mechanical load. Journal of Applied Physiology (1985), 108(1), 172–180. Ren, D., Wei, F., Hu, L., Yang, S., Wang, C., & Yuan, X. (2015). Phosphorylation of Runx2, induced by cyclic mechanical tension via ERK1/2 pathway, contributes to osteodifferentiation of human periodontal ligament fibroblasts. Journal of Cellular Physiology, 230(10), 2426–2436. Seo, B. M., Miura, M., Gronthos, S., Bartold, P. M., Batouli, S., Brahim, J., et al. (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 364(9429), 149–155. Shi-Wen, X., Leask, A., & Abraham, D. (2008). Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine and Growth Factor Reviews, 19(2), 133–144. Tomokiyo, A., Maeda, H., Fujii, S., Wada, N., Shima, K., & Akamine, A. (2008). Development of a multipotent clonal human periodontal ligament cell line. Differentiation, 76(4), 337–347. Verrecchia, F., & Mauviel, A. (2007). Transforming growth factor-beta and fibrosis. World Journal of Gastroenterology, 13(22), 3056–3062. Wang, H. Q., Bai, L., Shen, B. R., Yan, Z. Q., & Jiang, Z. L. (2007). Coculture with endothelial cells enhances vascular smooth muscle cell adhesion and spreading via activation of beta1-integrin and phosphatidylinositol 3-kinase/Akt. European Journal of Cell Biology, 86(1), 51–62. Wang, Q., Usinger, W., Nichols, B., Gray, J., Xu, L., Seeley, T. W., et al. (2011). Cooperative interaction of CTGF and TGF-beta in animal models of fibrotic disease. Fibrogenesis Tissue Repair, 4(1), 4. Wu, S. H., Wu, X. H., Lu, C., Dong, L., & Chen, Z. Q. (2006). Lipoxin A4 inhibits proliferation of human lung fibroblasts induced by connective tissue growth factor. American Journal of Respiratory Cell and Molecular Biology, 34(1), 65–72. Wu, X., Zhang, G., Wang, X., Zhang, P., & Tan, Y. (2015). Endotoxin tolerance induction in human periodontal ligament fibroblasts stimulated with different bacterial
is one of the significant indications of collagen formation. FN is also a necessary component of periodontium extracellular matrix. ITGB1 is a widely expressed integrin with critical roles in mediating cell-ECM interactions. It forms a receptor for many extracellular-matrix proteins including fibronectin, laminin, collagen, epiligrin and thrombospondin. Previous research found that ITGB1 mediates adherence of PLFs to the extracellular matrix (Kasaj et al., 2008), and the activation of ITGB1 subunit contributes to the activation of other signaling pathways (Wang, Bai, Shen, Yan, & Jiang, 2007). Consequently, we selected Col-I, FN and ITGB1 as markers of the extracellular matrix in this study. Verrecchia and Mauviel (2007) discovered that CTGF stimulates the synthesis of collagen and promotes the production and accumulation of the extracellular matrix by TGF-β-mediated or autocrine regulation. Additionally, Shi found that the interaction between CTGF and the integrin β3 subunit could promote the expression of fibronectin (Shi-Wen, Leask, & Abraham, 2008). Our study demonstrates that CTGF could significantly promote the synthesis of collagen by HPLFs, while higher concentrations of CTGF would inhibit it. Furthermore, RT-PCR analysis showed that the concentrations of CTGF in a certain range could promote the expression of FN and ITGB1 mRNA, and the difference was statistically significant (P < 0.05). Those studies indicate that CTGF could increase the expression of Col-I and FN; that is, CTGF could increase the expression of extracellular matrix by HPLFs. The effects were dose-independent. Those studies further suggest the optimal application of CTGF in periodontal regeneration. CTGF could not only improve the migration, proliferation and differntiation of HPLFs, but also enhance the collagen formation and cellular matrix mineraliztion. Compared with other factors which were applied in periodontal regeneration such as Emdogain, PDGF, and PRF, CTGF, CTGF can significantly enhance the expression of collagen I and III, and regeneration of polysaccharides and periostin, especially high-density of periostin gathering in collagen fiber (Asano et al., 2005). These indicated CTGF may promote the mature pariodontium-like tissue regeneration. In conclusion, various concentrations of CTGF could promote the biological activity of HPLFs, including the proliferation, migration, collagen synthesis and differentiation in the process of osteogenesis. All effects are dose-dependent and exhibit a regular pattern of effects on a number of possible mechanisms. Our findings thus provide an important guide to the application of CTGF in periodontal therapy. However, further research on the mechanisms of the effects of CTGF on HPLFs is required. Conflicts of interest There are no conflicts of interest. Fund This research is supported by Shandong province science and technology development plans (Grant No:2012G0021849) and the National Natural Science Foundation of China (Grant No:81200789). Acknowlegement We would like to acknowledge Xiaoyang Sun for his assistance in article preparation. References Arnott, J. A., Lambi, A. G., Mundy, C., Hendesi, H., Pixley, R. A., Owen, T. A., et al. (2011). The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Critical Reviews in Eukaryotic Gene Expression, 21(1), 43–69. Asano, M., Kubota, S., Nakanishi, T., Nishida, T., Yamaai, T., Yosimichi, G., et al. (2005). Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells. Cell Communication and Signaling, 3, 11. Bai, Y. C., Kang, Q., Luo, Q., Wu, D. Q., Ye, W. X., Lin, X. M., et al. (2011). Role of
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Effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of a human periodontal ligament stem/progenitor cell line. Journal of Cellular Physiology, 230(1), 150–159. Zhang, C., Meng, X., Zhu, Z., Liu, J., & Deng, A. (2004). Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biology International, 28(12), 863–873. Zhang, H. Y., Liu, R., Xing, Y. J., Xu, P., Li, Y., & Li, C. J. (2013). Effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts in vitro. Experimental and Therapeutic Medicine, 6(6), 1553–1559.
lipopolysaccharides. Archives of Oral Biology, 60(3), 463–470. Yang, Z., Sun, Z., Liu, H., Ren, Y., Shao, D., Zhang, W., et al. (2015). Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis. Molecular Medicine Reports, 12(1), 1091–1097. Yashiro, Y., Nomura, Y., Kanazashi, M., Noda, K., Hanada, N., & Nakamura, Y. (2014). Function of chemokine (CXC motif) ligand 12 in periodontal ligament fibroblasts. Public Library Of Science, 9(5), e95676. Yuda, A., Maeda, H., Fujii, S., Monnouchi, S., Yamamoto, N., Wada, N., et al. (2015).
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Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
Research Paper
Effects of connective tissue growth factor on human periodontal ligament fibroblasts Xuejing Duana,1, Mei Jia,1, Fengying Dengb, Zhe Sunb, Zhiyong Lina, a b
MARK
⁎
School of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China School of Stomatology, Shandong University, Jinan, Shandong Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Connective tissue growth factor Human periodontal ligament fibroblasts Periodontal regeneration Concentrations
Objective: The aim of this study was to evaluate the effects of different concentrations of connective tissue growth factor (CTGF) on human periodontal ligament fibroblasts(HPLFs). Design: HPLFs were cultured and identified. Then, different concentrations of CTGF (1, 5, 10, 50, 100 ng/ml) were added to the HPLF culture. Next, CCK-8 assays, alkaline phosphatase (ALP) assays, hydroxyproline determination, alizarin red staining methods, Transwell chambers and real-time PCR methods were applied to observe the effects of CTGF on the proliferation, ALP activity, synthesis of collagen, formation of mineralized nodules and migration. We also studied expression of ALP, fiber link protein (FN), integrin-binding sialoprotein (IBSP), osteocalcin (OC), and integrin beta 1 (ITGB1) mRNA by HPLFs. Statistical significance was assumed if P < 0.05 or P < 0.01. Results: The addition of CTGF (1, 5, 10 ng/ml) remarkably promoted the proliferation and collagen synthesis of HPLFs compared with controls. CTGF (1, 5, 10, 50 ng/ml) improved ALP activity of HPLFs, and at all concentrations, CTGF (1, 5, 10, 50, 100 ng/ml) improved the expression of ALP, FN, IBSP and ITGB1 mRNA. In addition, CTGF (1, 5, 10, 50, 100 ng/ml) promoted the migration of HPLFs, which was dose-dependent, with maximal promotion in the 10 ng/ml group (P < 0.05 or P < 0.01). Conclusions: Thus, in a certain range of concentrations, CTGF can promote the biological effects, including proliferation, migration and collagen synthesis of HPLFs, to promote the differentiation of HPLFs in the process of osteogenesis.
1. Introduction As one of the most common infectious diseases, periodontal disease (PD) causes substantial destruction of alveolar bone, periodontal ligament (PDL), and gingiva, leaving the oral environment exposed and allowing initiation of root contamination and tooth loss. PDL, a type of tooth-supporting structure and non-mineralized connective tissue between alveolar bone and cementum, is composed of heterogeneous cell populations, including osteoclasts, cementoblasts, fibroblasts, osteoblasts, mast cells, mesenchymal cells, and phagocytes (Beertsen, McCulloch, & Sodek, 1997). PDL appears to be actively involved in remodeling of alveolar bone, has shock-absorbing properties against mechanical stress, and prevents the tooth and alveolar bone from being damaged during mastication. Thus, PDL has an important functional role in the maintenance and renewal of periodontal tissues (Wu, Zhang,
Wang, Zhang, & Tan, 2015) and enables teeth to move via periodontal regeneration during orthodontic treatment (Yashiro et al., 2014). Among the cells present in the periodontium, periodontal ligament fibroblasts (PLFs) are the most abundant and play the most important role on the development, repair and regeneration of periodontal tissues (Choe et al., 2012). It has been proven that PLFs display biological characteristics similar to those of bone marrow-derived undifferentiated mesenchymal cells (Ren et al., 2015). In addition to constantly producing new dental cement and principal fibers and reconstructing alveolar bone, PLFs also synthesize the extracellular matrix, which holds the cells and plays special biological roles in cell conglutination, transportation and mineralization (Choe et al., 2012; Zhang et al., 2013). Being naturally osteogenic, PLFs are capable of differentiating into osteoblasts (Heo, Lee, & Lee, 2010). Furthermore, it has been found that PLFs are able to perceive the mechanical signals
Abbreviations: CTGF, connective tissue growth factor; HPLFs, human periodontal ligament fibroblasts; ALP, alkaline phosphatase; FN, fiber link protein; IBSP, integrin binding sialoprotein; OC, osteocalcin; ITGB1, integrin beta 1; PDL, periodontal ligament; PLFs, periodontal ligament fibroblasts; ECM, extracellular matrix; TGF-β, transforming growth factor-β ⁎ Corresponding author at: School of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, No 51, Jing Seven and Wei Six Road, Jinan, Shandong, China. E-mail address: [email protected] (Z. Lin). 1 These authors contributed to the work equally and should be regarded as co-first authors. http://dx.doi.org/10.1016/j.archoralbio.2017.09.010 Received 9 January 2017; Received in revised form 28 August 2017; Accepted 16 September 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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Fig. 1. The morphology, immunohistochemical staining and proliferation of HPLFs. A: HPLFs were either fusiform or dendroid in shape, with full cell bodies, clear nuclei, and two or three fine cytoplasmic processes. (40×). B: The proliferation curve of HPLFs. It was similar to an upside down ‘S’ with the arrest, lgarithmic growth, and plateau phases. C: Vimentin was positive in the cytoplasm of HPLFs (20×). D: Keratin was not expressed in HPLFs (20×).
regulation of various biological processes associated with fibrogenesis, including proliferation, differentiation, chemotaxis, cellular adhesion, migration, production of extracellular matrix (ECM) and angiogenesis, especially in promotion of deposition of several ECM proteins, such as fibronectin, collagen, and tenascin C (Yang et al., 2015; Zhang, Meng, Zhu, Liu, & Deng, 2014). Furthermore, CTGF also contributes to the proliferation and differentiation of mouse periodontal ligament-derived cells and a human periodontal ligament stem/progenitor cell line (Asano et al., 2005; Yuda et al., 2015). However, to date, there has been no effort to determine the effects of different concentrations of CTGF, if any, on the biological activities of human PDL fibroblastic cells (HPLFs). This study was performed to investigate the biological effects of HPLFs, aiming to provide a new research direction for dental treatment of periodontal disease and to offer a new growth factor for periodontal disease clinical drug research and development.
and exhibit a number of the phenotypic characteristics of osteoblasts under stress stimulation, including osteocalcin (OCN), osteopontin (OPN), Osteriox (also known as SP7), sialoprotein (BSP), collagen type I (Col I), and activating transcription factor 4 (ATF4). The quality of PLFs is related to the differentiation of osteoblasts and remodeling of periodontal tissue (Ren et al., 2015). Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN family and is a secreted matricellular and multifunctional protein. The CCN family was named after the first three members, Cyr61 (CCN1), CTGF (CCN2) and Nov (CCN3), were identified. It also includes CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP3) (Klaassen, van Geest, Kuiper, van Noorden, & Schlingemann, 2015). It has been shown that CCN family members are involved in regulation of cell proliferation, differentiation, migration, extracellular matrix remodeling and apoptosis (Chen & Lau, 2009). CTGF was originally identified as a growth factor-inducible immediate early gene in mouse fibroblasts and in human vascular endothelial cells and is the most studied member of this family (Cheng, Chang, Fang, Sun, & Leung, 2015), which was found to promote the chemotaxis and mitosis of fibroblasts. While its expression level in various tissues under the normal physiological state is lower, CTGF expression can be up-regulated greatly in many diseases, especially in fibrotic and cancerous tissues or under high mechanical stress load stimulation (Chaqour & GoppeltStruebe, 2006; Lomas et al., 2011; Reich, Maziel, Ashkenazi, & Ornan, 2010; Wang et al., 2011). Moreover, it was recently reported that CTGF/CCN2 gene expression in human PDL cells is up-regulated by stretch loading (Yuda et al., 2015). CTGF has been implicated in a number of more complex biological processes, including angiogenesis, osteogenesis, chondrogenesis, fibrosis, wound healing, and tumorigenesis (Arnott et al., 2011). It has been shown that CTGF is involved in
2. Materials and methods 2.1. HPLFs culture and identification Dental premolars that were free of dental caries or periodontal disease but extracted due to orthodontic treatment were collected. Immediately after extraction, the teeth were immersed in DMEM (Gibco, USA) containing double antibiotics (100 μg/ml penicillin G and 100 μg/ml streptomycin; Sijiqing Company, Hangzhou, China) and washed 10 times with PBS (pH 7.2) under sterile conditions. Then, the periodontal ligament tissues attached to the middle third of the roots were curetted gently by a surgical scalpel, minced and placed in 24-well plates. The plates were maintained at 37 °C in an atmosphere of 95% air 38
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and 5% CO2 in DMEM containing 10% FBS (Gibco BRL, USA). After reaching 80% confluence, HPLFs were digested with a mixture of 0.25% (w/v) trypsin and 0.02% (w/v) EDTA and subcultured at a 1:3 ratio. Cellular morphologies of HPLFs were observed by inverted phase contrast microscopy and then HPLFs were identified by vimentin and keratin immunostaining.
M2 multifunctional ELISA plate reader (Molecular Devices, USA) at 450 nm. 2.4. ALP activity test HPLFs were plated at a density of 2 × 104/ml in a 96-well culture plate. Different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml) were added to each well, and four duplicate wells were set up for each sample. All groups were incubated for 7, 14 and 21 days and then were washed three times with PBS. Then, 1% TritonX-100 (Sigma, USA) was used to dissolve the cells, and the lysates with the ALP assay working solution were incubated for 4 min. After incubation, the absorbance of p-nitrophenol was read at 560 nm in an ELISA plate reader. Double distilled water was used as the blank group, and phenol standard solution was used as the standard group.
2.2. Transwell chamber migration assay DMEM containing different concentrations of CTGF (0, 1,5, 10, 50, and 100 ng/ml)was added to the lower well of the Transwell chambers (Corning Company, America), and 200 μl of resuspended cells was added to the upper surface. Transwell chambers containing cells were then incubated at 37 °C with 5% CO2 for 24 h. The upper layer of the Transwell chamber was then removed, and the lower surface was washed 3 times with PBS, fixed with 5% paraformaldehyde for 5–10 min and then stained with 0.1% crystal violet for 1–2 min. Nuclei of migrated cells on the lower surfaces were stained and were observed by fluorescence microscopy. The number of migrating cells in each microscope field (6100) was also counted.
2.5. Total RNA isolation and real-time PCR (RT-PCR) Total RNA was extracted from cells using Trizol reagent(Invitrogen, USA)for conventional reverse transcription. RT-PCR was used to detect the expression of alkaline phosphatase (ALP), fibronectin (FN), bone sialoprotein (IBSP), osteocalcin (OC) and integrin beta 1 (ITGB1) by HPLFs. 1 μg of RNA was reverse transcribed with a SuperScript firststrand synthesis system (Invitrogen, Carlsbad, CA) following the manufacturer’s recommendations. RT-PCR reactions were prepared utilizing iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The sequences of the primers included the following: β-actin (Forward: 5′CAAGTACCAGCGAGCAGCTT-3′, Reverse: 5′-AAAGCCGAGCTGCCA GAGTT-3′); ALP (Forward: 5′-AACGGTCCAGGCTATGTGCT-3′, Reverse: 5′- CTGCTGACTGCTGCCGATAC-3′); FN (Forward: 5′- GCCAGATGA TGAGCTGCAC-3′, Reverse: 5′-GAGCAAATGGCACCGAGATA-3′); IBSP (Forward: 5′-CTGGCACAGGGTATACAGGGTTAG-3′, Reverse: 5′GCCTCTGTGCTGTTGGTACTGGT-3′); ITGB1 (Forward: 5′-GGTTTCACT TTGCTGGAGATGG-3′, Reverse: 5′-CAGTTTCTGGACAAGGTGAG
2.3. CCK-8 assay HPLFs (1 × 104 cells/ml, fifth passage) were plated in a 96-well culture plate at 100 μl/well in DMEM containing 10% FBS. Three duplicate wells were set up for each sample. After 24 h, the culture medium was replaced by serum-free DMEM for another 12 h. Subsequently, HPLFs were cultured in DMEM containing 10% FBS with different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml). Cells cultured on basic media (DMEM with 10% FBS) were the control group, and DMEM with 10 ng & z.urule;ml CTGF was the blank group. After 24, 48 and 72 h incubation, 10 ui CCK-8 (Nanjing Jiancheng Bioengineering Institute, China) was added to each well and incubated for 4 h. The growth state of the HPLFs was measured with a SpectraMax
Fig. 2. Effects of different concentrations of CTGF on the migration ability of HPLFs(40×). A: HPLFs migration of control group; B: HPLFs migration of 1 ng/ml CTGF group; C: HPLFs migration of 5 ng/ml CTGF group; D: HPLFs migration of 10 ng/ml CTGF group; E: HPLFs migration of 50 ng/ml CTGF group; F: HPLFs migration of 100 ng/ml CTGF group; G: Effects of different concentrations of CTGF on the migration ability of HPLFs. And when the mass concentration was 10 ng/ml, CTGF could promote the mineralization greatly (P < 0.01). *P < 0.05, **P < 0.01.
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Fig. 3. Effects of different concentrations of CTGF on the proliferation of HPLFs. The proliferation curves of HPLFs in different groups. Compared with the control group, CTGF (1, 5, and 10 ng/ml) significantly promoted the proliferation of HPLFs after 24, 48 and 72 h (P < 0.05 or P < 0.01). However, CTGF at a concentration of 100 ng/ml had an inhibitory effect.The effects of CTGF were all dose-dependent, with maximal proliferation in the 10 ng/ml group. *P < 0.05, **P < 0.01.
package (SAS, Cary, NC). All the data were expressed as the mean ± standard deviation.
CAATA-3′); and OC (Forward: 5′-GGCAGCGAGGTAGTGAAGAGA-3′, Reverse: 5′-CTCCTGAAGCCGATGTGG-3′). β-actin gene expression was detected for normalization purposes.
3. Results 2.6. Hydroxyproline determination 3.1. HPLFs culture and proliferation assay HPLFs were plated at a density of 5 × 104/ml in a 96-well culture plate and incubated for 24 h at 37 °C with 5% CO2. Then, the previous culture solution was discarded, serum free DMEM was added, and HPLFs were incubated for an additional 12 h. Subsequently, 1 ml substrate containing different concentrations of CTGF(1, 5, 10, 50, and 100 ng/ml) was added to each well. Each group contained four duplicate wells. After 24 h of incubation, we collected 0.5 ml of supernatant from each well and determined hydroxyproline levels following kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The OD of each tube was measured using an ELISA plate reader at 550 nm.
HPLFs began to migrate from the edge of the tissues after 8–10 days of culture. HPLFs were fusiform in shape and in good condition after being passaged. The cells were arranged in a sarciniform or swirl pattern (Fig. 1A). The proliferation curve of HPLFs was an S-shaped curve, with arrest, logarithmic growth and plateau phases (Fig. 1B). IHC testing of the HPLFs (third passage) revealed that the cytoplasm was positive for vimentin (yellow-brown color) (Fig. 1C); however, keratin was not expressed in the cytoplasm (Fig. 1D). 3.2. Effects of different concentrations of CTGF on the migration of HPLFs Cell migration ability increased with higher CTGF concentrations and exhibited a marked concentration-dependence (Fig. 2A–F). Further studies (Fig. 2G) showed that there were statistically significant differences in the number of migrating cells among all CTGF groups (1,5, 10, 50, and 100 ng/ml) compared to the control group after 24 h incubation (P < 0.05 or P < 0.01).
2.7. Alizarin red S staining HPLFs were plated at a density of 2 × 104/ml in six 24-well culture plates at 1 ml/well. The culture solution was discarded, and HPLFs were washed 3 times with PBS after 24 h incubation. A 1-ml aliquot of mineralized induced solution containing 10% FBS, 1 × 10−8mol/l dexamethasone (Sigma, USA), 10 mmol/l β-sodium glycerophosphate and 50 g/ml DMEM was added to the blank group, while mineralized induced solution containing different concentrations of CTGF(1, 5, 10, 50, 100 ng/ml)was added to the other five 24-well culture plates. All were incubated 21 days and then washed 3 times with PBS. An aliquot of 500 μl 4% paraformaldehyde was added to each well, and HPLFs were fixed for 30 min. After that, the 4% paraformaldehyde was removed and replaced with sterile 2% alizarin red (Sigma, USA) solution and incubated for 30 min at room temperature. Finally, the 2% alizarin red solution was discarded, and HPLFs were washed 3 times with double distilled water. The numbers of newly formed calcium nodules in each well were then counted under an inverted phase contrast microscope (Leica MPS30, Germany).
3.3. Effects of different concentrations of CTGF on the proliferation of HPLFs The effects of different concentrations of CTGF on the proliferation of HPLFs are illustrated in Fig. 3. Compared with the control group, Table 1 Effects of different concentrations of CTGF on the ALP activity of HPLFs ( x ± s). Concentration (ng/ ml)
Time (d) 7
0 (control) 1 5 10 50 100
2.8. Statistical analysis Statistically significant differences (P < 0.05 or P < 0.01) between the various groups were measured using ANOVA. All statistical analyses were carried out using an SPSS 22.0 statistical software
0.222 0.252 0.298 0.507 0.286 0.168
14 ± ± ± ± ± ±
0.003 0.006* 0.005* 0.013* 0.012* 0.009*
0.254 0.276 0.354 0.520 0.364 0.174
21 ± ± ± ± ± ±
0.009 0.003 0.012* 0.016** 0.019* 0.015*
Compared with the control group, *P < 0.05, **P < 0.01.
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0.262 0.294 0.341 0.506 0.357 0.163
± ± ± ± ± ±
0.007 0.012 0.016* 0.018** 0.015* 0.011*
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Fig. 4. Effects of different concentrations of CTGF on the expressions of ALP, FN, IBSP, OC, ITGB1 mRNA of HPLFs(*P < 0.05)A: the expressions of ALP mRNA in different groups; B: the expressions of FN mRNA in different groups; C: the expressions of IBSP mRNA in different groups; D: the expressions of OC mRNA in different groups; E: the expressions of ITGB mRNA in different groups. In addition, the results showed that the expression of ALP, FN, IBSP, OC and ITGB1 mRNA followed normal distribution and reached a peak when the concentration of CTGF was 10 ng/ ml (ALP, IBSP, OC and ITGB1) or 5 ng/ml (FN).
followed normal distribution and reached a peak when the concentration of CTGF was 10 ng/ml (ALP, IBSP, OC and ITGB1) or 5 ng/ml (FN).
CTGF (1, 5, and 10 ng/ml) significantly promoted the proliferation of HPLFs after 24, 48 and 72 h (P < 0.05 or P < 0.01). The effects of CTGF were all dose-dependent, with maximal proliferation in the 10 ng/ml group. There was no statistically significant difference between the 50 ng/ml CTGF group and the control group (P > 0.05). However, CTGF at a concentration of 100 ng/ml had an inhibitory effect. Thus, the lower concentrations of CTGF stimulated proliferation of HPLFs, while higher concentrations inhibited proliferation (P < 0.05). The 10 ng/ml concentration of CTGF promoted the highest proliferation rate for the periodontal ligament fibroblasts (Fig. 3).
3.6. Effects of different concentrations of CTGF on the secretion of hydroxyproline by HPLFs Hydroxyproline is a specific amino acid component of collagen, which can be used to estimate collagen content and act as an important indicator of collagen quality (Kuttan & Radhakrishnan, 1973). The effects of different concentrations of CTGF on the synthesis of hydroxyproline by HPLFs are illustrated in Table 2. Compared with the control group, CTGF (1, 5, 10 ng/ml) significantly enhanced the secretion of hydroxyproline by HPLFs after 24 h (P < 0.05 or P < 0.01). The effects of CTGF were dose-dependent at all concentrations, with maximal promotion in the 10 ng/ml group. There was no statistically significant difference between the 50 and 100 ng/ml CTGF groups and the control group (P > 0.05).
3.4. Effects of different concentrations of CTGF on the ALP activity of HPLFs The effects of different concentrations of CTGF on the ALP activity of HPLFs are illustrated in Table 1. Compared with the control, CTGF (5, 10, 50 ng/ml) significantly increased ALP activity of HPLFs after 7, 14 and 21 days (P < 0.05 or P < 0.01). The effects of CTGF were all dose-dependent with maximal promotion of ALP activity in the 10 ng/ ml group. However, CTGF at a concentration of 100 ng/ml had a significant inhibitory effect on the ALP activity of HPLFs.
Table 2 Effects of different concentrations of CTGF on the synthesis of collagen of HPLFs ( x ± s).
3.5. Effects of different concentrations of CTGF on the expression of ALP, FN, IBSP, OC, and ITGB1 mRNA by HPLFs RT-PCR was used to measure the mRNA expression levels of ALP, FN, IBSP, OC and ITGB1 by HPLFs cultured in different media after 14 days. The results showed that CTGF, in a range of concentrations, increased the mRNA expressions levels. In addition, the results (Fig. 4) showed that the expression of ALP, FN, IBSP, OC and ITGB1 mRNA
Concentration(ng/ml)
Hydroxyproline(ug/ml)
0 (control) 1 5 10 50 100
1.655 2.221 2.967 3.762 1.524 1.376
± ± ± ± ± ±
0.345 0.356* 0.415* 0.313** 0.189# 0.217#
Compared with the control group, *P < 0.05, **P < 0.01, #P > 0.05.
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Fig. 5. Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs (20×). A:The formation of mineralized nodules in control group; B: The formation of mineralized nodules in 1 ng/mg CTGF group; C:The formation of mineralized nodules in 5 ng/mg CTGF group; D: The formation of mineralized nodules in 10 ng/mg CTGF group; E: The formation of mineralized nodules in 50 ng/mg CTGF group; F: The formation of mineralized nodules in 1000 ng/mg CTGF group;G: Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs. And when the mass concentration was 10 ng/ml, CTGF could promote the mineralization greatly (P < 0.01). *P < 0.05, **P < 0.01.
(Nowak, Popow-Wozniak, Raznikiewicz, & Malicka-Blaszkiewicz, 2009), which is important for periodontium regeneration. CTGF can also induce chemotactic migration in many kinds of cells in vitro, such as certain epithelial cells, vascular endothelial cells and vascular smooth muscle cells (Cicha and Goppelt-Struebe, 2009; Jun and Lau, 2011). Our study showed that cell migration ability increases when the CTGF concentration is higher, which supports a role for CTGF in periodontium regeneration. HPLFs have the potential to differentiate into cementoblasts and osteoblasts which are needed for cementum and alveolar bone formation, respectively (Basdra & Komposch, 1997; Seo et al., 2004; Tomokiyo et al., 2008). Studies have reported higher gene expression of OC, IBSP and TGFβ receptor in alkaline phosphatase-positive subpopulations of PDLFs (Murakami, Kojima, Nagasawa, Kobayashi, & Ishikawa, 2003). These molecules all play important roles in osseous development, induction, maintenance and repair. In addition, ALP is also important for differentiating osteoblast-like cells, and its degree of activity reflects the mineralization ability of tissues and cells and underlies the formation of osteogenic properties (Pae, Kim, Kim, & Woo, 2011). Our study revealed that CTGF could promote ALP activity, the formation of mineralized nodules and the expression of IBSP, OC and ALP mRNA. However, the promotion of CTGF decreased when the concentration was high. These studies suggest that CTGF may play a role in the commitment of mesenchymal progenitors to the osteogenic lineage, which is important for periodontal regeneration. That effect was dose-dependent. As we know, the most important component of periodontium is collagen fibers, which are mainly composed of Col-I. Col-I of periodontium does not only maintain the stability of organizational structure but is also necessary for periodontium to perform its physiological function and carry out complete physiological and structural regeneration (Ivanovski, Li, Daley, & Bartold, 2000). The level of Col-I expression may directly reflect the degree of periodontium regeneration. In addition, it is also an important component of bone matrix and
3.7. Effects of different concentrations of CTGF on the formation of mineralized nodules of HPLFs After culture for 21 days, Alizarin red S staining of HPLFs showed different calcium nodules in all groups (Fig. 5A–F). Six mineralization rate comparisons between experimental groups showed statistically significant differences (P < 0.05). CTGF (10 ng/ml) promoted mineralization greatly compared to controls (P < 0.01) (Fig. 5G).
4. Discussion CTGF has been shown to be an autocrine mediator of transforming growth factor-β (TGF-β) in fibroblasts. Previously, studies reported that TGF-β stimulation of fibroblast proliferation, collagen synthesis, and most recently, myofibroblast differentiation is mediated via CTGF-dependent pathways (Grotendorst & Duncan, 2005). Cell proliferation is one of the most important factors in maintaining the balance of cell numbers and maintaining normal organ structure (Zhang et al., 2013). In this study, we showed that CTGF, within a certain concentration range, can promote the proliferation of HPLFs after 7, 14 and 21 days, while high concentration inhibits proliferation. We also showed that the effect is dose-dependent. These findings are in accordance with other studies. A great number of experimental studies have previously demonstrated that CTGF can stimulate growth of multiple cell types (Bai et al., 2011; Chu, Chang, Prakash, & Kuo, 2008; Wu, Wu, Lu, Dong, & Chen, 2006); however, the overall number of cells decreases when the concentration is too high. Those studies suggested that the effects of CTGF on HPLFs are directly related to the concentration of CTGF. Migration of fibroblasts is a key step in the process of periodontium regeneration. Phanish, Winn, and Dockrell (2010) found that CTGF may promote the migration of mesangial cells after trauma. In addition, in the wound healing process, fibroblasts can chemotactically migrate to specific locations, secrete cytokines and synthesize extracellular matrix 42
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is one of the significant indications of collagen formation. FN is also a necessary component of periodontium extracellular matrix. ITGB1 is a widely expressed integrin with critical roles in mediating cell-ECM interactions. It forms a receptor for many extracellular-matrix proteins including fibronectin, laminin, collagen, epiligrin and thrombospondin. Previous research found that ITGB1 mediates adherence of PLFs to the extracellular matrix (Kasaj et al., 2008), and the activation of ITGB1 subunit contributes to the activation of other signaling pathways (Wang, Bai, Shen, Yan, & Jiang, 2007). Consequently, we selected Col-I, FN and ITGB1 as markers of the extracellular matrix in this study. Verrecchia and Mauviel (2007) discovered that CTGF stimulates the synthesis of collagen and promotes the production and accumulation of the extracellular matrix by TGF-β-mediated or autocrine regulation. Additionally, Shi found that the interaction between CTGF and the integrin β3 subunit could promote the expression of fibronectin (Shi-Wen, Leask, & Abraham, 2008). Our study demonstrates that CTGF could significantly promote the synthesis of collagen by HPLFs, while higher concentrations of CTGF would inhibit it. Furthermore, RT-PCR analysis showed that the concentrations of CTGF in a certain range could promote the expression of FN and ITGB1 mRNA, and the difference was statistically significant (P < 0.05). Those studies indicate that CTGF could increase the expression of Col-I and FN; that is, CTGF could increase the expression of extracellular matrix by HPLFs. The effects were dose-independent. Those studies further suggest the optimal application of CTGF in periodontal regeneration. CTGF could not only improve the migration, proliferation and differntiation of HPLFs, but also enhance the collagen formation and cellular matrix mineraliztion. Compared with other factors which were applied in periodontal regeneration such as Emdogain, PDGF, and PRF, CTGF, CTGF can significantly enhance the expression of collagen I and III, and regeneration of polysaccharides and periostin, especially high-density of periostin gathering in collagen fiber (Asano et al., 2005). These indicated CTGF may promote the mature pariodontium-like tissue regeneration. In conclusion, various concentrations of CTGF could promote the biological activity of HPLFs, including the proliferation, migration, collagen synthesis and differentiation in the process of osteogenesis. All effects are dose-dependent and exhibit a regular pattern of effects on a number of possible mechanisms. Our findings thus provide an important guide to the application of CTGF in periodontal therapy. However, further research on the mechanisms of the effects of CTGF on HPLFs is required. Conflicts of interest There are no conflicts of interest. Fund This research is supported by Shandong province science and technology development plans (Grant No:2012G0021849) and the National Natural Science Foundation of China (Grant No:81200789). Acknowlegement We would like to acknowledge Xiaoyang Sun for his assistance in article preparation. References Arnott, J. A., Lambi, A. G., Mundy, C., Hendesi, H., Pixley, R. A., Owen, T. A., et al. (2011). The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Critical Reviews in Eukaryotic Gene Expression, 21(1), 43–69. Asano, M., Kubota, S., Nakanishi, T., Nishida, T., Yamaai, T., Yosimichi, G., et al. (2005). Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells. Cell Communication and Signaling, 3, 11. Bai, Y. C., Kang, Q., Luo, Q., Wu, D. Q., Ye, W. X., Lin, X. M., et al. (2011). Role of
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Effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of a human periodontal ligament stem/progenitor cell line. Journal of Cellular Physiology, 230(1), 150–159. Zhang, C., Meng, X., Zhu, Z., Liu, J., & Deng, A. (2004). Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biology International, 28(12), 863–873. Zhang, H. Y., Liu, R., Xing, Y. J., Xu, P., Li, Y., & Li, C. J. (2013). Effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts in vitro. Experimental and Therapeutic Medicine, 6(6), 1553–1559.
lipopolysaccharides. Archives of Oral Biology, 60(3), 463–470. Yang, Z., Sun, Z., Liu, H., Ren, Y., Shao, D., Zhang, W., et al. (2015). Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis. Molecular Medicine Reports, 12(1), 1091–1097. Yashiro, Y., Nomura, Y., Kanazashi, M., Noda, K., Hanada, N., & Nakamura, Y. (2014). Function of chemokine (CXC motif) ligand 12 in periodontal ligament fibroblasts. Public Library Of Science, 9(5), e95676. Yuda, A., Maeda, H., Fujii, S., Monnouchi, S., Yamamoto, N., Wada, N., et al. (2015).
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