Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/
Title
Combined effects of systemic parathyroid hormone (1‑34) and locally delivered neutral self‑
assembling peptide hydrogel in the treatment of periodontal defects: An experimental in vivo investigation
Author(s) Alternative
Yoshida, W; Matsugami, D; Murakami, T; Bizenjima, T; Imamura, K; Seshima, F; Saito, A
Journal Journal of clinical periodontology, 46(10): 1030‑
1040
URL http://hdl.handle.net/10130/5214
Right
This is the peer reviewed version of the following article: J Clin Periodontol. 2019 Oct;46(10):1030‑
1040., which has been published in final form at https://doi.org/10.1111/jcpe.13170. This article may be used for non‑commercial purposes in
accordance with Wiley Terms and Conditions for Use of Self‑Archived Versions.
Description
1 Original Article
Combined effects of systemic parathyroid hormone (1-34) and locally delivered neutral self-assembling peptide hydrogel in the treatment of periodontal defects: An experimental in vivo investigation
Wataru Yoshida1, Daisuke Matsugami1, Tasuku Murakami1, Takahiro Bizenjima1, Kentaro Imamura1,2 Fumi Seshima1, Atsushi Saito1,2
1Department of Periodontology, Tokyo Dental College, Tokyo Japan
2Oral Health Science Center, Tokyo Dental College, Tokyo Japan
Running Title: PTH and SPG-178 in periodontal healing
Correspondence address: Atsushi Saito, DDS, PhD Department of Periodontology, Tokyo Dental College
2-9-18 Kanda-Misakicho, Chiyoda-ku, Tokyo 101-0061, Japan Phone; +81-3-6380-9171 Fax: +81-3-6380-9172
E-mail: [email protected]
Acknowledgements
The authors thank Takahiro Takeuchi, DDS, PhD and Akira Yamaguchi, DDS, PhD for technical guidance and helpful discussions, and Mr. Katsumi Tadokoro for technical assistance. This study was supported by KAKENHI Grant numbers 16K20678 from the
2
Japan Society for Promotion of Science and a grant from Multidisciplinary Research Center for Jaw Disease (MRCJD), Tokyo Dental College, Tokyo, Japan (a MEXT Private University Research Branding Project).
3 Abstract
Aim: To evaluate in vivo combination therapy of systemic parathyroid hormone (PTH) and locally delivered neutral self-assembling peptide (SAP) hydrogel for treatment of periodontal defects.
Materials and Methods: Viability/proliferation of rat periodontal ligament cells in a neutral SAP nanofiber hydrogel (SPG-178) was evaluated using WST-1 assay.
Periodontal defects were created mesially to the maxillary first molars in 40 Wistar rats.
Defects were filled with 1.5% SPG-178 or left unfilled. Animals received PTH (1-34) or saline injections every 2days. Microcomputed tomography, histological, and
immunohistochemical examinations were used to evaluate healing.
Results: At 72 hr, cells in 1.5% SPG-178 showed increased viability/proliferation compared to cells in 0.8% SPG-178 or untreated controls. In vivo, systemic PTH resulted in significantly greater bone volume in the Unfilled group at 4 wk than in the saline control (p < 0.0001). A significantly greater bone volume was observed in the PTH/SPG-178 and PTH/Unfilled groups compared to Saline/SPG-178 group (p = 0.004-0.0003). Histologically, greater bone formation was observed in the PTH/SPG- 178 at 4 wk than in other groups. In the PTH/SPG-178 group, increased proportions of PCNA-, VEGF-, and Osterix-positive cells were observed in the treated sites.
Conclusions: These findings suggest that intermittent systemic PTH and local delivery of neutral SAP hydrogel enhanced periodontal healing.
Keywords: periodontitis, parathyroid hormone, regeneration, self-assembling peptide
4 Clinical Relevance
Scientific rationale for the study: Self-assembling peptide (SAP) hydrogels have been tested for the use in tissue regeneration. Currently, information is limited on the
effectiveness of the novel SAP hydrogel, SPG-178, in tissue regeneration. We
investigated in vivo effects of systemic parathyroid hormone (PTH) in combination with locally delivered SPG-178 on the healing of periodontal defects.
Principal findings: PTH/SPG-178 combination therapy promoted healing of surgical periodontal defects in rats, possibly by increasing cell proliferation, angiogenesis and osteoblastic differentiation.
Practical implications: SPG-178 is promising as a scaffold for periodontal
regeneration, and its use in combination with an anabolic agent may further enhance regeneration.
5 Introduction
Regeneration of periodontal tissue damaged by periodontitis is the ultimate goal of periodontal treatment, and various regeneration methods have been studied and implemented clinically (Kao, Nares, & Reynolds, 2015; Susin & Wikesjö, 2013).
However, currently available regenerative therapies have limitations towards achieving complete and functional periodontal tissue regeneration, especially that of new bone formation for severe periodontal defects.
Parathyroid hormone (PTH) plays an essential role in regulating phosphorous (P) and calcium (Ca) levels during bone remodeling (Alkhiary et al., 2005). PTH has been used in the treatment of osteoporosis to increase bone density and prevent fractures (Metcalf, Aspray, & McCloskey, 2017). It has been reported for rat femurs that intermittent PTH therapy is better at increasing bone mass than that of continuous PTH therapy (Hock &
Gera, 1992). Furthermore, data from animal studies (Barros, Silva, Somerman, &
Nociti, 2003; Tokunaga et al., 2011; Vasconcelos et al., 2014) and human studies in patients with periodontitis (Bashutski, Eber, et al., 2010) showed that intermittent PTH treatment results in improved bone healing.
Recently, new artificial biomimetic matrices have been used to regenerate tissues (Rubert Perez et al., 2015). Among the materials used are self-assembling peptides (SAPs). The complete sequence of a self-assembling peptide was originally found in alternating hydrophobic and hydrophilic residues in zuotin (Zhang, Holmes, Lockshin,
& Rich, 1993), which exhibits a stable β-sheet structure that undergoes self-assembly into nanofibers (Ando et al., 2018).
SAP possess similar biological features to extracellular matrix (Kumada & Zhang, 2010; Zhang et al., 1995). SAP hydrogels provide a favorable microenvironment for the
6
migration and proliferation of various cells (Bradshaw et al., 2014). Attempts have been made to use SAPs for the treatment of various bone defects (Misawa et al., 2006;
Nakahara et al., 2010). Previously, using a pre-clinical model, our research group reported that locally delivered SAP hydrogel (RADA16) enhances healing of
periodontal defects (Takeuchi et al., 2016). RADA16 has a low pH of approximately 3–
4. Although a pre-neutralization procedure was used and no obvious detrimental effects on the cells or tissues were observed, it may be more preferable in clinical situations to use a hydrogel with a neutral pH (Komatsu, Nagai, Naruse, & Kimata, 2014). Ando et al. (2016) developed a novel SAP hydrogel, SPG-178 (Self-assembling Peptide Gel, amino acid sequence #178; [CH3CONH]-RLDLRLALRLDLR-[CONH2]; R = arginine, L = leucine, D = aspartic acid, A = alanine), as a scaffold material. The stability of SPG-178 at neutral pH (the isoelectric point, at which a protein has a zero net charge and reaches minimum solubility) contributes to the biocompatibility of the SAP hydrogel (Ando et al., 2016).
Given this background information, we hypothesized that the combined use of systemic PTH and locally delivered neutral SAP hydrogel (SPG-178) would enhance periodontal healing, especially that of new bone formation. This study aimed to assess in vivo the effects of intermittent systemic PTH administration and a novel neutral SAP
hydrogel on the healing of surgically-created periodontal defects.
7 Materials and Methods
SAP hydrogel
SPG-178, an SAP hydrogel with neutral pH (6.5–7.5), was obtained from Menicon (PanaceaGel®; Nagoya, Japan). Scanning electron microscopy (SEM) was used to assess the morphological features of 0.8% and 1.5% SPG-178, according to the method of Takeuchi et al. (2016).
Animals
Male Wistar rats (n = 40; 10 weeks old, 250 –300 g) were purchased from Sankyo Labo Service (Tokyo, Japan). The animals were maintained under standard laboratory
conditions. The study adhered to the ARRIVE guidelines and the experimental protocol conformed to the Treatment of Experimental Animals at Tokyo Dental College (no.
302202).
Cell viability/proliferation assay
Periodontal ligament (PDL) cells were isolated from rat incisors as previously described (Inoue et al., 1986). Viability/proliferation of rat PDL (rPDL) cells in 0.8% and 1.5%
SPG-178 was assessed using a WST-1 assay (Takara Bio, Otsu, Japan), according to the method of Takeuchi et al. (2016).
Surgical procedures and PTH administration
The animals were assigned to one of the following four subgroups: PTH/SPG-178 (n = 10), PTH/Unfilled (n = 10), Saline/SPG-178 (n = 10), and Saline/Unfilled (n = 10) (Fig.
8
S1a). Standardized periodontal defects (Oortgiesen et al., 2012) (2.0 × 2.0 × 1.7 mm) were surgically created bilaterally under general and local anesthesia according to the method by Bizenjima et al. (2015). The roots of first molars (M1) were carefully denuded of their PDL and cementum. The defects in the SPG-178-treated groups received 50 µl of 1.5% SPG-178 while the defects in Unfilled groups were left unfilled.
The flaps were closed and sutured. Acetaminophen was used for pain control.
The administration of PTH was performed essentially as described by Vasconcelos et al. (2014). Animals in the PTH group received intermittent subcutaneous injections of recombinant human PTH (1-34) (Sigma-Aldrich, St. Louis, MO) at 40µg/kg every 2 days for 2 weeks or 4 weeks. Animals in the Saline group were given only vehicle (sterile saline) each time. The day of surgery and first PTH administration was designated as day 0.
Microcomputed tomography analysis
For evaluation, the animals were anaesthetized and 4% paraformaldehyde was used for cardiovascular perfusion. Systemic anabolic effects of PTH were assessed by subjecting femurs (n = 3/group) to microcomputed tomography (micro-CT) using the method described by Gohin et al. (2016). Bone healing in the periodontal defect was analyzed using the micro-CT system as described previously (Bizenjima et al., 2015; Takeuchi et al., 2016). Image data were analyzed using structural analysis software (Spplemental file; Appendix S1).
Histological analysis
9
Histological analysis was performed as described previously (Takeuchi et al., 2016).
Briefly, the maxillae were bisected at the palatal median line. The specimens were fixed in buffered 4% paraformaldehyde for 24 hr, decalcified in 10% EDTA at 4°C for 3 wk, and then embedded in paraffin. Each specimen was cut (thickness 5 µm) and several sections from the central part of the root in the defect were analyzed using
haematoxylin-eosin or Azan–Mallory staining.
Histomorphometric analysis
Histomorphometric assessment of epithelial down-growth was performed using a light microscope and histomorphometric software by the method of Bizenjima et al. (2015) (Supplemental file; Appendix S2)
The angulation of PDL-like fibers on the root planed surface of M1 was analyzed by the method of Park et al. (2012).
Immunohistochemistry
After deparaffinization, sections were incubated in 3% hydrogen peroxide with
methanol for 30 min at room temperature. Analysis of proliferating cell nuclear antigen (PCNA) was performed as described previously (Bizenjima et al., 2015). Detection of vascular endothelial growth factor (VEGF) and Osterix (Osx) was carried out using anti-VEGF monoclonal antibodies and anti-Osx polyclonal antibodies, respectively.
For quantitative analysis, each periodontal defect was compartmentalized into three regions (Root side, Bone side, and Middle area) and the number of PCNA-, VEGF-, and Osx-positive cells was counted in each region. The Root side was defined as the area mesial to the M1 root above the newly formed bone (Takeuchi et al., 2016). The Bone
10
side was defined as the area of the superior border of the existing bone (Nagayasu et al., 2015). The Middle area was the region between the Bone side and Root side on the imaginary line of the bottom of the defect. Detailed methods for staining and quantitative analysis are presented in the Supplemental file (Supplemental file;
Appendix S3).
Statistical analysis
Inter-group comparisons for the WST-1 assay were made by one-way analysis of variance (ANOVA) with Tukey post hoc test. Inter-group comparisons for the micro- CT, histomorphometric and immunohistochemical analyses were made by two-way analysis of variance (ANOVA) with Tukey post hoc test or Kruskal-Wallis test Dunn’s post-hoc test. The analysis was performed using Prism software (ver 7.05, GraphPad, La Jolla, CA, USA). Statistical significance was set at p < 0.05.
11 Results
SPG-178 Structure
SPG-178 exhibited nanofiber structure with nanopores (mean 5–200 nm) (Supplemental file; Fig. S2). The nanofibers formed a partial mesh structure with a variable mesh size reaching up to 500 nm. A finer network structure was observed in the 1.5% SPG-178 compared to that of the 0.8% SPG-178.
Effect of PTH and SPG-178 on cell viability/proliferation
rPDL cells in the 1.5% SPG-178 group demonstrated a rapid increase in
viability/proliferation at 72 hr (Supplemental file; Fig. S3). In contrast, cells in the 0.8%
SPG-178 or the control showed only a moderate increase. At 72 h, the cell
viability/proliferation of the 1.5% SPG-178 group was significantly greater than that of the control group (p < 0.001) or 0.8% SPG-178 group (p < 0.001).
Micro-CT analysis of defect healing
Except for two rats that died during the creation of defects and two that died from postoperative bleeding, post-surgical wound healing was generally uneventful for all groups.
In structural analysis of the femoral metaphysis, in the PTH administration groups, there was a trend for increase in the bone volume fraction (BV/TV) and decrease in trabecular separation over time. PTH administration yielded approximately 30%
increase in BV/TV and 60% decrease in trabecular separation compared to the saline control at 4 wk (Supplemental file; Fig. S4). These findings confirmed that systemic intermittent PTH administration exerted an effect on the structure of trabecular bone.
12
Sagittal images from micro-CT are shown in Fig. 1a and Fig. 1f. At 4 wk
postoperative, new bone formation was evident in the PTH/SPG-178 group (Fig. 1f).
Quantitative analysis at 2 wk (Fig. 1b–1e) and 4 wk postoperative (Fig. 1g–1j) indicated a trend for increases in BV/TV and trabecular thickness and decreases in trabecular separation for all groups.
At 2 wk postoperative, BV/TVs were significantly greater in the PTH/SPG-178 group (p < 0.0001), PTH/Unfilled group (p = 0.001), and Saline/SPG-178 group (p = 0.004) than in the Saline/Unfilled group (Fig. 1b). At 4wk postoperative, BV/TVs were significantly greater in the PTH/SPG-178 group and PTH/Unfilled group (p < 0.0001) than in the Saline/Unfilled group (Fig. 1g). BV/TVs were significantly greater in the PTH/SPG-178 group (p = 0.0003) and PTH/Unfilled group (p = 0.004) than in the Saline/SPG-178 group. The trabecular thickness in the PTH/SPG-178 group at 4 wk postoperative was significantly greater than that in the Saline/Unfilled group (p = 0.0013) and Saline/SPG-178 group (p = 0.0179) (Fig. 1h). A significantly greater trabecular number was observed in the PTH/SPG-178 group at 2 wk postoperative than in the Saline/Unfilled group (p = 0.0003) and Saline/SPG-178 group (p = 0.0491) (Fig.
1d). The trabecular separation was significantly smaller at 2 wk postoperative in the PTH/SPG-178 group (p < 0.0001), PTH/Unfilled group (p = 0.0022), and Saline/SPG- 178 group (p = 0.0095) than in the Saline/Unfilled group (Fig. 1e). At 4 wk
postoperative, the trabecular separation was significantly smaller in the PTH/SPG-178 group and PTH/Unfilled group (p < 0.0001) than in the Saline/Unfilled group (Fig. 1j).
The value was significantly smaller in PTH/SPG-178 group (p = 0.027) and PTH/Unfilled group (p = 0.0282) than in the Saline/SPG-178 group.
13 Histological observations
Histological overviews are shown in Fig. 2. At 2 wk postoperative, the previous defect spaces were filled with connective tissue in all groups (Fig. 2a–2d). No obvious signs of inflammation were noted. New bone formation had initiated from the root side of the defect in the PTH/SPG-178, PTH/Unfilled, and Saline/SPG-178 groups at 2 wk postoperative. Bone formation appeared to be greater in the PTH/SPG-178 group than in the other groups (Fig. 2d). Little bone formation was observed in the Saline/Unfilled group (Fig. 2a). At 4 wk postoperative, greater bone formation was observed in the PTH/SPG-178 group than in the other groups (Fig. 2h). New bone formation in the Saline/Unfilled group was limited (Fig. 2e).
Histomorphometric analysis
At 2 wk postoperative, epithelial down-growth in the PTH/SPG-178 group was limited compared to that in the Saline/Unfilled group (Supplemental file; Fig. S5a–S5d). At 4 wk postoperative, progression of down-growth was observed (Fig. S5e–S5h). The junctional epithelium measurements were consistent with this observation, showing significant differences between the groups at 2 wk and 4 wk postoperative (Fig. S5i and S5j).
Representative micrographs of Azan-Mallory staining of the areas near the defect bottom are shown in Fig. 3a–3h. PDL-like bundles in all groups were almost parallel to the root surfaces at 2 wk postoperative. No cementum formation was observed. At 4 wk postoperative, the PDL-like bundles ran obliquely in the PTH/SPG-178 group and Saline/SPG-178 group, whereas the ligaments ran near parallel to the root in the other groups (Fig. 3f and 3h). A thin layer of newly formed cementum was observed only in
14
the PTH/SPG-178 group and Saline/SPG-178 at 4 wk postoperative.
Next, angulation of the fiber bundles was measured (Fig. 3i and 3j). At 2 wk postoperative, the fiber angulation in the PTH/SPG-178 group was significantly greater than that in other groups (p < 0.0001). At 4 wk postoperative, the mean angulation in the PTH/SPG-178 group was significantly greater than that in other groups. Angulation in the Saline/SPG-178 group was significantly greater than that in the PTH/Unfilled group (p = 0.0465) and Saline/Unfilled group (p < 0.0001).
Immunohistochemical analysis
PCNA-positive cells were observed in the area in the Root side, Bone side, and Middle area (Fig. 4). At 2 wk postoperative, SPG-178 and/or PTH treatment yielded a greater proportion of PCNA-positive cells in almost all areas (Table 1). At 4 wk postoperative, in the root side, the proportion of PCNA-positive cells in the PTH/SPG-178 group was significantly greater than that in the other groups.
In the PTH and/or SPG-178 groups, VEGF-positive cells were mostly observed near blood vessels in the connective tissues (Fig. 4) and capillary ingrowth was evident. At 2 wk postoperative, in the Root side, SPG-178 and/or PTH treatment yielded a greater proportion of VEGF-positive cells (Table 2). At 4 wk postoperative, in the Root side and Bone side, the proportion of VEGF-positive cells in the PTH/SPG-178 group was significantly greater than that in the Saline/Unfilled group.
In the PTH and/or SPG-178 groups, Osx-positive cells appeared to be increased for both the Root side and Bone side at 2 wk postoperative (Supplemental file; Fig. S6). At 2 wk and 4 wk postoperative, the proportion of Osx-positive cells in the PTH/SPG-178
15
group was significantly greater than that in the Saline/Unfilled group in the Root side and Bone side (Table 3).
16 Discussion
In the current study, we demonstrated, for the first time, that the systemic intermittent administration of PTH in combination with the local application of the SAP nanofiber hydrogel SPG-178 promoted periodontal healing in vivo. Moreover, the combination therapy reduced epithelial migration and promoted new bone formation. These effects can potentially be attributed to increased cell proliferation, angiogenesis and
differentiation.
Micro-CT and histological observations demonstrated that the systemic intermittent PTH improved bone formation in periodontal defects at 2 wk and 4 wk postoperative.
Previously, it was shown that intermittent PTH treatment improves alveolar bone levels affected by periodontitis in rats and humans (Barros et al., 2003; Bashutski et al., 2010;
Marques et al., 2005; Vasconcelos et al., 2014). The cyclic adenosine monophosphate (cAMP)–protein kinase signaling pathway response to PTH 1 receptor (PTH1R) activation is considered to be the main mechanism that mediates the anabolic action of PTH in bone (Swarthout, D’Alonzo, Selvamurugan, & Partridge, 2002; Yang, Guo, Divieti, & Bringhurst, 2006). PTH stimulates endothelial VEGF expression through PKA and PKC pathways (Rashid, Bernheim, Green, & Benchetrit, 2008). In the present study, the administration of PTH resulted in a significantly greater numbers of PCNA- and VEGF-positive cells than did Saline administration. Similarly, some studies have shown that intermittent PTH treatment induces differentiation of fibroblasts and osteoblasts and increases expression of VEGF-positive cells (Nagayatsu-Tanaka et al., 2015; Wang et al., 2014). In addition, VEGF facilitates the proliferation of endothelial cells and promotes angiogenesis (Unlü, Güneri, Hekimgil, Yeşilbek, & Boyacioğlu, 2003). In bone metabolism, VEGF facilitates osteoblastic differentiation (Liu et al.,
17
2012). Osx is one of the master transcriptional regulators of osteogenesis (Lian et al.,2006). It has been reported that PTH accelerates bone formation by enhancing the differentiation of progenitor cells to an osteoblastic phenotype, possibly by increasing Osx expression (Kaback et al., 2008). In the present study, an enhanced expression of Osx-positive cells was observed by PTH administration. These results collectively suggest that PTH enhances bone healing in periodontal defects by promoting PDL cell proliferation, angiogenesis and osteoblast differentiation via certain signaling pathways.
It has been shown that endothelial and mesenchymal progenitor cells form a vascular network in the microenvironment provided by SAP hydrogel (Allen, Melero- Martin, & Bischoff, 2011). In the present study, the expression of VEGF-positive cells was more pronounced in the SPG-178 group than in the Unfilled group at both 2 wk and 4 wk postoperative. SPG-178 forms an antiparallel β-sheet structure as its secondary structure in aqueous solution (Komatsu, Nagai, Naruse, & Kimata, 2014). The peptide self-assembles to form nanofibers and further forms a stable net structure. The in vivo efficacy of SPG-178 as a local hemostatic material has also been reported (Komatsu, Nagai, Naruse, & Kimata, 2014). The clot filling the remainder of the wound space is eventually replaced by the newly forming bone. Thus, the promotion of periodontal healing in the SPG-178 group at 2 wk postoperative is considered to be due to the SPG- 178 providing an environment for stable clots and neovascularization.
At 2 wk postoperative, a significantly greater mean BV/TV value was observed in the Saline/SPG-178 group than in the Saline/Unfilled group, although no significant difference was observed at 4 wk postoperative. It has been reported that SPG-178 hydrogel facilitates the proliferation of neurons and myoblasts in vitro (Ando et al., 2016; Nagai, Yokoi, Kaihara, & Naruse, 2012). It has also been shown to be suitable as
18
a scaffold to support dental pulp stem cell proliferation and osteogenic differentiation at the start of the culture (Tsukamoto et al., 2017). Ando et al. (2018) showed that the level of Osx mRNA in MC3T3-E1 cells in SPG-178 was significantly increased
compared to that in control at 14 days. Therefore, it is considered that SPG-178 function as an appropriate scaffold for the growth of various cells in periodontal tissue during the early stage of healing.
Interestingly, in the PTH/SPG-178 group, elevated cellular activity was confirmed on the root side by the immunohistochemical analysis at 4 wk postoperative. In addition, the newly formed bone adjacent to the root was more pronounced in the combination treatment group, than in the other groups. It is speculated that the majority of cells on the apical side consisted of PDL-derived cells while those on the bone side were osteoblastic cells. During periodontal regeneration, the proliferation and migration of PDL fibroblasts on the root surface prevent migration of cells from the gingiva or alveolar bone (Herr, Matsuura, Lin, Genco, & Cho, 1995). In our study, rPDL cells incubated with 1.5% SPG-178 showed elevated viability/proliferation compared to that of the control group at 72 hr. In a previous study, the SAP hydrogel RADA16 promoted human PDL fibroblasts to proliferate and migrate into the hydrogel (Kumada & Zhang, 2010). It is possible that SPG-178 and PTH enhanced the proliferative activity of PDL cells during the early stage of healing.
In an in vitro experiment, PTH was shown to increase the production of cAMP in both freshly isolated PDL tissues and cultured PDL cells (Nojima et al., 1990).
However, neither ALP activity nor PTH‐dependent cAMP production was noted in gingival tissues or cultured gingival fibroblasts. From these results, it is suggested that the combination treatment of PTH and SPG-178 promoted connective tissue attachment
19
and decreased epithelial down-growth by increasing the activity of PDL cells.
Newly formed cementum-like structures were observed in Azan–Mallory stained samples from the SPG-178 group and PTH/SPG-178 combination group at 4 wk postoperative. It has been reported that cementoblasts express PTH1R (Berry et al., 2006). It is possible that the PTH induced the differentiation of cementoblasts with activation of PTH1R in cells in the defect sites. Mature cementum is related to the maintenance or replacement of periodontal fibrils (Stern, 1964). During the
development of PDL, principal fibers align coronally from cementum to bone to form the oblique bundles (Cho & Garant, 2000). In the present study, at 4 wk postoperative, the PDL-like bundles in the PTH/SPG-178 group ran obliquely to the root and the mean angulation of the PDL-like collagen bundles (51.5°) was similar to that of the native PDL-fibers (59.1°) and in a previous study (54.9°) (Park et al., 2012). These findings suggest that functional periodontal ligament was regenerated by PTH/SPG-178 therapy.
It is also important to note that the use of SPG-178 alone yielded a significantly greater angulation value compared to the PTH administration alone. This may support the contribution of SPG-178 in the reformation of PDL.
There are relevant limitations to the present study. Because the results were from early stages, further studies with longer time courses are needed to determine whether SPG-178/PTH therapy accelerates the healing process and truly induces complete regeneration. A more detailed mechanism by which SPG-178 or the combination therapy enhances periodontal healing remains to be clarified. Additionally, how the results in this pre-clinical model translate to periodontal healing in humans needs to be clarified. In clinical situations, systemic administration of PTH for the sole purpose of periodontal treatment may not be justified. Local administration may be an alternative
20
method, but it is challenging to develop a system able to deliver PTH at appropriate doses to achieve anabolic effects (Bashutski et al., 2012).
Despite these limitations, this study provides important information on the effects of a combination therapy consisting of an anabolic agent and a biocompatible scaffold for the healing of periodontal tissue. The SAP nanofiber hydrogel, SPG-178, may prove to be an effective scaffold for use in periodontal regeneration and has a potential to yield even greater effects when combined with an appropriate biological agent.
Conflict of Interest
The SPG-178 was supplied by Menicon Co, Ltd, Nagoya, Japan, under a material transfer agreement. The authors declare that there are no conflicts of interest in this study. Only authors are responsible for the content and writing of this paper.
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27 Figure legends
Figure 1. Two-dimensional images in micro-CT and quantitative analysis by 3-D structural analysis software.
(a, f) Sagittal images from micro-CT. Enhanced new bone formation can be observed in the PTH/SPG-178 group at 2 wk and 4 wk postoperative. (b-e, g-j) Quantitative analysis of micro-CT images by a 3-D structural analysis software (TRI/3D-BON). Box in black, saline group; Box in blue, PTH group. Bone volume fraction (b, g), trabecular thickness (c, h), trabecular number (d, i) and separation (e, j) of ROI were compared between groups. Data shown as box-and-whiskers plot with minimum, maximum, median and 25th and 75th percentiles (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey post hoc test.
Figure 2. Histopathological overview (H&E staining).
At 2 wk postoperative, spaces in the defect are filled with newly formed connective tissue. (a) New bone formation is very limited in the Saline/Unfilled group. (b-d) In the PTH/SPG-178, PTH/Unfilled and Saline/SPG-178 groups, newly formed bone can be observed from the tooth side of the intrabony defect.
(e) At 4 wk postoperative, new bone formation was minimal in the Saline/Unfilled group. (h) The extent of newly formed bone in the defect area in the PTH/SPG-178 group appeared to be greater than other groups. Arrows indicate newly formed bone.
(original magnification ×25, bar = 500 µm).
Figure 3. Healing of PDL.
28
(a-h) Representative photomicrographs of the root area near the bottom of defect. (a-d) At 2 wk postoperative (upper panel), collagen bundles run near parallel to the surfaces in all groups. (f, h) At 4 wk postoperative, in Saline/SPG-178 and PTH/SPG-178 groups, PDL-like collagen bundles are well-aligned and obliquely inserted onto the root surface similar to native PDL with signs of thin-layer of cementogenesis (indicated by an arrow). (e, g) In other groups, collagen bundles ran near parallel to the root surfaces.
(Azan–Mallory’s stain, original magnification original magnification ×400; bar = 100 μm).
(i, j) The angulation of the fibers at the apical extent of instrumentation on M1 root was observed under ×200 and analyzed by Image J software at 2 wk and 4 wk postoperative.
Box in black, saline group; Box in blue, PTH group. Data shown as box-and-whiskers plot with minimum, maximum, median and 25th and 75th percentiles (n = 10). *p <
0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey post hoc test.
Figure 4. Representative photomicrographs of immunohistochemical staining for PCNA and VEGF at 2 wk postoperative.
PCNA-positive cells and VEGF-positive cells are observed in Root side (a), Bone side (b), and middle area (c) (see the schematic in the upper right). A brown coloration indicates a PCNA-positive or VEGF-positive reaction. In all area, the proportion of PCNA- or VEGF-positive cells in the PTH/SPG-178 group appear to be greater than that in the Saline/Unfilled group. (PCNA, VEGF and counterstaining with Mayer’s haematoxylin stain, original magnification ×200; bar = 50 m).
29 Supplemental files
Figure S1. In vivo experimental protocol and surgical creation of periodontal defects.
(a) Experimental protocol and groups. (b) Incision design (dotted line) (c) After raising a full-thickness flaps, standardized periodontal defects were created mesially of the maxillary first molars (M1). (d) A surgical template was used to create each defect (2.0×2.0×1.7 mm). (e) Defects received 1.5 % SPG-178. (f) Flaps were closed using resorbable sutures. (g) Micro-CT image of the created periodontal defect.
Figure S2. Characteristics and SEM analysis of the neutral self-assembling peptide hydrogel, SPG-178.
(a) 1.5% and (b) 0.8%.
Figure S3. Viability of rat periodontal ligament (rPDL) cells over time.
rPDL cells were seeded onto the SPG-178 (0.8%), SPG-178 (1.5%) with MEM or medium alone (control) and allow to grow for up to 72 h. WST-1 assay was used to determine cell viability (metabolism) at indicated time points. In each group, a reference absorbance at 450 nm was subtracted from the absorbance for each sample, and the values relative to those at 0 h were shown. Data shown as mean ± SD (n = 6),
Significantly greater than the control group (†††p < 0.001) or SPG-178 (0.8%) (‡‡p <
0.001), by one-way ANOVA with Tukey post hoc test.
Figure S4. Quantitative analysis of femurs formation by micro-CT by 3-D structural analysis software (TRI/3D-BON). The volume of new bone (a), trabecular thickness (b),
30
trabecular number (c) and trabecular separation (d) were compared between groups. 2 weeks (blank bar), 4 weeks (filled bar). Data shown as mean ± SD (n = 3).
Figure S5. Histological assessment of epithelial down-growth.
(a-d and e-h) The apical extent of epithelial down-growth (indicated by arrows) was compared at 2 wk and 4 wk postoperative. Blue arrowheads indicate CEJ. (a, d) At 2 wk postoperative, epithelial down-growth along the root surface in the PTH/SPG-178 group appears to be less than that in the Saline/Unfilled group. (e, f, g) At 4 wk, a general trend for progression of the down-growth can be observed (original magnification ×50;
bar = 200 μm). (i, j) Epithelial down-growth (%) was calculated as (the length between the most coronal and most apical aspects of the junctional epithelium on the root surface) / (the distance between CEJ and the bottom of the defect). Box in black, saline group; Box in blue, PTH group. Data shown as box-and-whiskers plot with minimum, maximum, median and 25th and 75th percentiles (n = 10). *p < 0.05, **p < 0.01, ***p
< 0.001, ****p < 0.0001 by two-way ANOVA with Tukey post hoc test.
Figure S6. Representative photomicrographs of immunohistochemical staining for Osterix at 2 wk postoperative.
Osterix-positive cells are observed in Root side (a) and Bone side (b). A brown coloration indicates an Osterix-positive reaction. In both areas, the proportion of Osterix-positive cells in the PTH/SPG-178 group was significantly greater than that in the Saline/Unfilled group. (Osterix and counterstaining with Mayer’s haematoxylin stain, original magnification ×200; bar = 50 m).
Appendix S1.
31 Detailed methods for Micro-CT analysis.
Appendix S2.
Detailed methods for histomorphometric analysis.
Appendix S3.
Detailed methods for Immunohistochemistry.
32 Figure 1.
33 Figure 2.
34 Figure 3.
35 Figure 4.
36 Supplemental files
Figure S1.
37 Figure S2.
38 Figure S3.
39 Figure S4.
40 Figure S5.
41 Figure S6.
42 Table 1. Proportion of PCNA-positive cells
Data shown as mean ± SD (n=10) of PCNA-positive cells / total cells (%) in the count area (Root side, Bone side, Middle area). PCNA; Proliferating cell nuclear antigen a; significantly different from the Saline/Unfilled group, b; significantly different from the Saline/SPG-178 group, c; significantly different from the PTH/Unfilled group, by two-way ANOVA with Tukey post hoc test (p < 0.05 – 0.0001).
Group Saline/Unfilled Saline/SPG-178 PTH/Unfilled PTH/SPG-178
2 wk
Root side 18.1 ± 5.3 26.8 ± 7.1 a 29.3 ± 3.8 a 32.8 ± 5.6 a Bone side 18.7 ± 5.9 25.1 ± 6.2 26.1 ± 6.1 a 30.7 ± 6.0 a Middle area 14.1 ± 5.7 23.8 ± 4.0 a 26.6 ± 4.2 a 27.7 ± 8.1 a
4 wk
Root side 17.9 ± 4.5 21.8 ± 5.8 24.0 ± 5.8 31.3 ± 6.4 a,b,c Bone side 22.9 ± 4.3 25.1 ± 5.8 25.2 ± 6.0 26.9 ± 4.0 Middle area 19.6 ± 4.3 21.4 ± 4.5 21.4 ± 5.9 26.9 ± 10.7 a
43 Table 2. Proportion of VEGF-positive cells
Group Saline/Unfilled Saline/SPG-178 PTH/Unfilled PTH/SPG-178
2 wk
Root side 7.7 ± 1.6 13.7 ± 1.5 a 12.4 ± 3.7 a 16.4 ± 3.1 a Bone side 7.2 ± 2.6 12.0 ± 3.2 10.8 ± 4.0 17.1 ± 6.9 a Middle area 7.7 ± 2.4 10.6 ± 2.4 10.3 ± 4.9 10.9 ± 2.6
4 wk
Root side 10.3 ± 1.0 17.4 ± 4.5 20.1 ± 6.7 22.7 ± 4.8 a Bone side 11.2 ± 3.8 15.6 ± 7.6 18.0 ± 8.1 23.1 ± 7.3 Middle area 11.9 ± 5.5 13.7 ± 4.0 16.5 ± 7.4 17.3 ± 8.1 Data shown as mean ± SD (n=6) of VEGF-positive cells / total cells (%) in the count area (Root side, Bone side, Middle area). VEGF; vascular endothelial growth factor
a; significantly different from the Saline/Unfilled group by two-way ANOVA with Tukey post hoc test (p < 0.05 – 0.0001).
44 Table 3. Proportion of Osx-positive cells
Group Saline/Unfilled Saline/SPG-178 PTH/Unfilled PTH/SPG-178 2 wk Root side 4.1 ± 0.6 8.3 ± 3.7 9.2 ± 1.9 17.9 ± 5.0a
Bone side 7.8 ± 2.3 8.9 ± 2.3 13.1 ± 5.3 18.0 ± 3.5a 4 wk Root side 13.8 ± 7.0 13.9 ± 2.5 21.6 ± 5.1 25.0 ± 5.4a Bone side 7.9 ± 4.7 14.4 ± 2.7 16.1 ± 2.4 18.5 ± 3.9a Data shown as mean ± SD (n=6) of Osx-positive cells / total cells (%) in the count area (Root side and Bone side). Osx; Osterix
a; significantly different from the Saline/Unfilled group by Kruskal-Wallis test with Dunn’s post-hoc test (p < 0.05 – 0.001).
