Effect of substrate on the response of 3-dimensional periodontal ligament tissue to compressive stimulation

Abstract

Purpose: The purpose of this study was to investigate the influence of substrates under the three-dimensional periodontal ligament (3D PDL) tissue on its biological functions after compressive stimulation.

Methods: A 3D PDL tissue was created using a poly(L-lactic acid) (PLLA) porous scaffold impregnated with human periodontal ligament fibroblasts (hPDLFs). It was then placed on a polyetheretherketone (PEEK) substrate, which has a comparable elastic modulus to bone and was compressed (25 g/cm²) for 1, 3, and 7 days. The morphology and biological functions of the hPDLFs in the 3D PDL tissue on the PEEK substrate were evaluated and compared with those on the polystyrene (PS) substrate.

Results: Compressive forces for the PLLA porous scaffold on the PEEK substrate were higher than those on the PS substrate. hPDLFs were present throughout the PLLA porous scaffold and there was no morphological change upon compressive stimulation. Increased expression of osteoclastogenic genes was observed after 3 days of compressive stimulation, while the level of these genes was increased by changing the substrate under the PDL tissue from PS to PEEK.

Conclusion: The substrate placed under the PDL tissue during compressive stimulation affects the biological functions of hPDLFs.

Introduction

Orthodontic tooth movement is a central mechanism in orthodontic treatment that appropriately aligns the tooth by applying a continuous force onto a tooth surface. Its mechanism has been actively explored from the cellular to physical levels and has been shown to cause bone remodeling around the tooth as a result of mechanical stimulation: this includes bone formation on the tension side and bone resorption on the compression side [1]. Periodontal ligament (PDL) tissue is the connective tissue between the tooth and alveolar bone (Fig. 1A) that acts as a cushion to dissipate and transform the mechanical stimulation from the tooth during orthodontic treatment [2].

Based on these findings, in vitro two-dimensional (2D) models with cells from PDL tissue have been developed to simulate orthodontic tooth movement and easily identify the cellular biological event [3]. Moreover, three-dimensional (3D) culture systems have recently been introduced to in vitro models of orthodontic tooth movement to bridge the gap between conventional 2D cell culture systems and in vivo conditions. These 3D culture systems are expected to more precisely reflect the in vivo environment and more closely reflect in vivo conditions [4]. Several approaches to 3D cell culture systems for orthodontic tooth movement have been developed. These include spheroid [5]; collagen hydrogel [6]; and porous synthetic polymer scaffolds, poly(lactic-co-glycolic acid) (PLGA) [7] or poly(L-lactic acid) (PLLA) [8], used to culture human periodontal ligament fibroblasts (hPDLFs).

When compressive stimulation is applied on a tooth’s side, the force applied to the PDL tissue affects the neighboring alveolar bone. However, in previous studies, compressive stimulation was applied to 3D PDL tissue on a polystyrene tissue culture plate whose stiffness varies from that of bone tissue. Moreover, no studies have been conducted on the effect of the substrate on 3D PDL tissue upon compressive stimulation.

Polyetheretherketone (PEEK) is a super engineering plastic widely used as implants for bone and joint replacements, dental restorations, and spinal cages [9,10,11]. Moreover, the elastic modulus of PEEK is similar to that of bone tissue [12]. Using PEEK sheet as a substrate for 3D PDL tissue to mimic the mechanical environment might lead to a precise evaluation of orthodontic tooth movement. Bioinert properties of PEEK are one of its advantages over hydroxyapatite, a bioactive bone-like substrate, in simple exploration of its influence on mechanical properties [9].

In this study, 3D PDL tissue was created by impregnating hPDLFs into a PLLA porous scaffold, as previously prepared [8,13]. Furthermore, the effect of the substrate under the 3D tissue on the biological functions of hPDLFs upon compressive stimulation was investigated.

Fig. 1 (A) Schematic illustration of orthodontic tooth movement. (B) In vitro orthodontic tooth movement model prepared in the present study with focus on anatomical feature of periodontal ligament-bone tissue on the compression side

Materials and Methods

Fabrication of PLLA porous scaffold

A porous scaffold of PLLA was prepared using conventional solvent casting and particulate leaching methods [8]. Pellets of PLLA (MW 240,000, BMG Inc., Kyoto, Japan) were dissolved in dichloromethane (DCM; 5% wt/v; Nacalai Tesque Inc., Kyoto, Japan) for 2 h. Sodium chloride (NaCl) particles (75-150 µm) obtained by separating NaCl granules with sieves were then mixed with the DCM solution containing PLLA (volume ratio; NaCl:PLLA = 1:2). The mixture (0.2 mL) was cast into a snap tube (15 mmφ) and allowed to stand at room temperature for 24 h. The resulting PLLA matrix was washed three times with 30% ethanol and distilled water for 10 min each to remove the residual DCM and NaCl to obtain the PLLA porous scaffold with a diameter and thickness of 15 mm and 400 µm, respectively. The obtained scaffolds were sterilized with ethylene gas (EOG) and stored at room temperature. The morphology of PLLA porous scaffold was observed using a scanning electron microscope (SEM) (S-4800; Hitachi High-Tech Corp., Tokyo, Japan) under the accelerating voltage of 5 kV after osmium coating (HPC-20, Vacuum Device Co., Ltd., Mito, Japan).

Surface treatment of PLLA porous scaffold

The surface of PLLA porous scaffold is too hydrophobic for culture medium containing cells to penetrate, thus preventing homogenous distribution of cells within the scaffold. Therefore, the surface treatment of PLLA porous scaffold to improve water-wettability is necessary for the homogenous cellular distribution. The contact angle measurement is an indicator of surface treatment. The PLLA porous scaffold was soaked in 0.05 vol% ammonia solution (Nacalai Tesque Inc.) at room temperature for 16 h. Then the scaffold was washed thrice for 10 min each with Dulbecco’s phosphate-buffered saline (PBS; Nacalai Tesque Inc.). It was then washed twice with culture medium Dulbecco’s modified Eagle’s medium (Fujifilm Co., Ltd., Tokyo, Japan) supplemented with 10 vol% fetal bovine serum (Nichirei Inc., Tokyo, Japan) and 1 vol% penicillin/streptomycin (Nacalai Tesque Inc.) for 10 min each. The scaffold was stored in the culture medium. The wettability of the PLLA porous scaffold before and after surface treatment with ammonia was evaluated by capturing water droplets and measuring the contact angles using an apparatus (LSE-ME2, Nick Corp., Saitama, Japan). The experiment was carried out three times independently.

Compression test of the PLLA porous scaffold on the PEEK substrate

The PLLA porous scaffold sandwiched with an upper cover glass (15 mmφ, Matsunami Glass Ind., Ltd., Kishiwada, Japan) and a lower PEEK or polystyrene (PS) substrate was pressed using an autograph (AGS-X, Shimadzu Co., Kyoto, Japan) with a compressive probe moving at a speed of 0.05 mm/min. The forces at compressive distances were recorded with a software (Trapezium Lite X, Shimadzu Co.). The experiment was carried out three times independently.

Creation of the 3D periodontal ligament tissue

hPDLFs (CLCC-7049; Lonza Co., Basel, Swiss) with passage numbers from 4 to 6 were used in this study. The PLLA scaffold after surface treatment was placed on a 24-well plate and the hPDLFs (1 × 10⁵ cells) were seeded into the center of the scaffold and incubated for 48 h to obtain a 3D PDL tissue. After washing with PBS and fixing with 2% glutaraldehyde (Nacalai Tesque Inc.) for 10 min, the sample was freeze-dried with tert-butyl alcohol (VFD-21S; Vacuum Device Co., Ltd.) and observed by SEM (S-4800) under the accelerating voltage of 5 kV as described above. The experiment was carried out three times independently.

Application of compressive stimulation on PDL tissue on a PEEK substrate

The PDL tissue was transferred to a fresh 12-well plate and placed on a PEEK substrate (15 mmφ; 1 mm height, As One Corp., Osaka, Japan) sterilized with EOG. Compressive stimulation (25 g/cm²) was applied to the PDL tissue on the PEEK substrate by placing a lead-filled snap tube (15 mmφ) through a cover glass for 1, 3, and 7 days (Fig. 1B). PDL tissue compressed on a different substrate (PS substrate) and that not compressed were used as controls. After compressive stimulation, the morphology of PDL tissue on each substrate was observed by SEM (S-4800) under the accelerating voltage of 5 kV as described above. The experiment was carried out three times independently.

The viability of hPDLFs on the PDL tissue after compressive stimulation was evaluated using the Cellstain double staining kit (Dojindo Corp., Kamimashiki, Japan) [14]. After washing with PBS for 10 min, the PDL tissue was cultured with Calcein-AM/Propidium iodide solution at 37°C for 30 min. The stained tissue was washed once with PBS and observed via a fluorescence microscope (BZ-X800; Keyence Corp., Tokyo, Japan).

Total RNA was isolated from hPDLFs in PDL tissue after compressive stimulation using a RNeasy mini kit (Qiagen Co., Hilden, Germany), and reverse transcription was carried out using a SuperScript IV VILO Master Mix kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) to obtain cDNA. Real-time PCR was performed on a Step One Plus real-time PCR System (Thermo Fisher Scientific Inc.) by mixing solutions containing the obtained cDNA, TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific Inc.), TaqMan Gene Expression Assay for receptor activator of nuclear factor kappa-Β ligand (Rankl, Hs00243522_m1), Osteoprotegerin (OPG, Hs00900358_m1), Cyclooxygenase-2 (COX-2, Hs00153133_m1), and interleukin-6 (IL-6, Hs00174131_m1) (Thermo Fisher Scientific Inc.), and TaqMan GAPDH (glyceraldehyde-3-phosphate dehydrogenase) Control Reagents (Thermo Fisher Scientific Inc.). The relative gene expression of the GAPDH gene was calculated using the ∆∆Ct method. The expression level of each gene in the Control group at day 1 was expressed as 1. The experiment was carried out three times independently.

Statistical analysis

All results were presented as mean ± standard deviation. Statistical analyses were all performed on GraphPad Prism version 8 (GraphPad Software Inc., San Diego, CA. USA). After confirming the normality of distribution by the Shapiro-Wilk test, one-way analysis of variance (ANOVA) with Tukey-Kramer test was applied to assess the statistical significance.

Results

Characterization of the PLLA porous scaffold

When observed on SEM, the PLLA porous scaffold had a thickness of 400 µm and pore size of 75-150 µm (Fig. 2). The pore size corresponded to the size of the NaCl particles used as a porogen, suggesting that the PLLA porous scaffold was successfully prepared by the conventional salt leaching method. Figure 3 shows the water wettability of PLLA porous scaffold before and after treatment with ammonia. The ammonia treatment improved the wettability of the PLLA porous scaffold. Figure 4 shows the compressive strain curve of the PLLA porous scaffold placed on the PS or PEEK substrate. The force at the compressive distance for the PLLA porous scaffold on the PEEK substrate was higher than that when placed on the PS substrate.

Fig. 2 Representative SEM images of PLLA porous scaffold on top (A) and side views (B)

Fig. 3 Representative images (A) and contact angles of a water droplet on PLLA porous scaffold before and after treatment with ammonia (B)

Fig. 4 Force-displacement curve of PLLA porous scaffold placed on PS and PEEK substrates

Morphological and biological evaluations of PDL tissue cultured under compressive stimulation

The morphology of the PDL tissue cultured under compressive stimulation was observed on SEM (Fig. 5). hPDLFs were observed to proliferate in the PLLA porous scaffold and secreted extracellular matrix with culture time. The morphological change of PDL tissue was independent of compressive stimulation and the difference in substrate placed under the tissue.

The viability of hPDLFs in PDL tissue cultured under compressive stimulation was evaluated by the Live/Dead assay (Fig. 6). Irrespective of the culture duration and compression, most hPDLFs in the PLLA porous scaffold were alive. No difference was observed between the PS and PEEK groups.

Profiles of expression levels of several genes (RANKL, OPG, COX-2, and IL-6) responsive to mechanical stimulation were evaluated for hPDLFs in PDL tissue cultured under compressive stimulation (Fig. 7). A slight change in gene expression level was observed in the control group during culture. In contrast, the gene expression levels were increased under compressive stimulation, especially after 3 days of compression. The extent of increment of gene expression level for the PEEK group was higher than that for the PS group.

Fig. 5 Representative SEM images of PDL tissue with or without compressive stimulation for 1, 3, and 7 days. Control group: images of PDL tissue without compressive stimulation. PS or PEEK groups: images of PDL tissue placed on PS or PEEK substrates with compressive stimulation. Arrows A and B indicate the extracellular matrix and attached hPDLFs, respectively.

Fig. 6 Representative images of Live/Dead assay for hPDLFs in PDL tissue with and without compressive stimulation for 1, 3, and 7 days. Control group: images of PDL tissue without compressive stimulation. PS or PEEK groups: images of PDL tissue placed on PS or PEEK substrates with compressive stimulation

Fig. 7 Relative expression of genes (A) RANKL, (B) OPG, (C) RANKL/OPG, (D) COX-2, and (E) IL-6 for hPDLFs in PDL tissue with and without compressive stimulation for 1, 3, and 7 days. Control group: relative gene expressions of PDL tissue without compressive stimulation. PS or PEEK groups: relative gene expressions of PDL tissue placed on PS or PEEK substrates with compressive stimulation. The expression level of each gene in the Control group at day 1 was expressed as 1. The P-values of Shapiro-Wilk test to certify the normality of distribution before one-way ANOVA for the Control, PS, and PEEK groups were (A) 0.2129, 0.1096, 0.9380, (B) 0.5744, 0.8764, 0.7302, (C) 0.5611, 0.9883, 0.9966, (D) 0.3345, 0.4999, 0.9033, and (E) 0.7587, 0.0220, 0.7770. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001: ANOVA with Tukey-Kramer test, vs groups indicated at the corresponding time

Discussion

The present study prepared a 3D PDL tissue consisting of PLLA porous scaffold with hPDLFs and showed that the substrate placed under the PDL tissue during compressive stimulation affected the biological functions of the hPDLFs. Numerous trials have been performed aiming at understanding orthodontic tooth movement and investigating their biological functions under mechanical stimulation using 3D PDL tissues [15,16]. However, their results are still controversial and no perfect PDL tissue has been developed so far. This could be due to a lack of consideration of the mechanical environment surrounding the PDL tissue. When applied to PDL tissue in vivo, the compressive power propagates to the surrounding alveolar bone. This is the first report investigating the influence of the substrate surrounding the PDL tissue on the biological functions of hPDLFs after compressive stimulation using a PEEK substrate having a comparable elastic modulus to bone.

In the present study, a PLLA porous scaffold was prepared for the PDL tissue. Its thickness was adjusted based on the anatomical thickness of the PDL [17]. It was reported that 75-150 µm pore sizes were favorable for culturing well-shaped spindle-shaped PDL cells in a 3D model [13]. Therefore, in the present study, a PLLA porous scaffold with a pore size of 75-150 µm was prepared by the conventional salt leaching method. The volume ratio of NaCl and PLLA to prepare the porous scaffold was decided using a preliminary experiment for structure integrity. SEM observation revealed that hPDLFs were present throughout the scaffold (Fig. 5) secondary to the improvement of water wettability by ammonia treatment (Fig. 3).

Based on a previous report on the 3D PDL tissue model, the applied force for compressive stimulation was set at 25 g/cm² [18]. It has been shown that this force is nearly equal to the blood pressure in terminal capillaries of the PDL and is suitable for orthodontic tooth movement [19]. The Live/Dead assay (Fig. 6) illustrated that most hPDLFs were alive in PLLA porous scaffolds under compressive stimulation of 25 g/cm², suggesting that this condition is useful in subsequent gene expression studies.

Cells in the PDL tissue secrete osteoclastogenic cytokines in response to mechanical stress to regulate alveolar bone remodeling around the teeth, which induces orthodontic tooth movement. RANKL, a member of the tumor necrosis factor ligand family, is an important regulatory molecule for osteoclast genesis [20]. OPG is a soluble decoy receptor for RANKL that acts as a negative regulator of RANKL-induced osteoclast differentiation and function [21]. RANKL/OPG is a contributing factor to bone resorption [22]. COX-2 is an enzyme responsible for prostaglandin formation, which is primarily involved in inflammatory responses, including periodontal inflammatory responses. IL-6 is a multifunctional cytokine that plays an important role in bone resorption during orthodontic tooth movement via the activation of osteoclasts [23]. In the present study, these factors were selected to investigate the biological functions of hPDLFs in PDL tissue.

As previously reported, an increased level of genes (RANKL, OPG, COX-2, and IL-6) at 3 days was observed under the culture of compressive stimulation (Fig. 7) [24,25,26,27]. Additionally, the level of these genes was increased when changing the substrate from PS to PEEK. This could be due to better propagation of compressive force to hPDLFs in PDL tissue using the PEEK substrate on compression test (Fig. 4). Therefore, the PEEK substrate gathers the compressive force in PLLA porous scaffold, leading to increased gene expression.

The present study demonstrates the importance of the type of substrate under the PDL tissue in in vitro models for orthodontic tooth movement. However, further investigations are needed to develop the best in vitro model for orthodontic tooth movement. First, the orthodontic tooth movement was induced by interaction with various cells in the PDL tissue [16]. Co-culture of hPDLFs with osteoblasts, osteoclasts, macrophages and more in the scaffold could help create the best in vitro model. Second, the bare PEEK substrate only provides stiffness comparable to bone. PEEK with bioactive properties, like hydroxyapatite coating, can facilitate the interaction with cells in the PDL tissue to provide a site for bone remodeling. Finally, novel imaging systems are required to evaluate these suggestive yet complicated in vitro models for orthodontic tooth movement.

Abbreviations

2D: two-dimensional; 3D: three-dimensional; COX-2: Cyclooxygenase-2; DCM: dichloromethane; EOG: ethylene gas; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hPDLFs: human periodontal ligament fibroblasts; IL-6: interleukin-6; NaCl: Sodium chloride; OPG: Osteoprotegerin; PBS: phosphate-buffered saline; PDL: periodontal ligament; PEEK: polyetheretherketone; PLLA: poly(L-lactic acid); PS: polystyrene; RANKL: receptor activator of nuclear factor kappa-Β ligand; SEM: scanning electron microscope

Ethical Statements

Not applicable

Conflicts of Interest

The authors have no conflicts of interest relevant to this article.

Funding

Not applicable

Author Contributions

CN: data curation, formal analysis, investigation, methodology, validation, visualization, writing – original draft preparation, writing – review and editing.; JIJ: conceptualization, methodology, project administration, resources, supervision, validation, visualization, writing – original draft preparation, writing – review and editing.; RZ: data curation, formal analysis, investigation, methodology, writing – review and editing.; YH: conceptualization, methodology, resources, writing – review and editing.; AN: conceptualization, resources, supervision, writing – review and editing. All authors read and approved the final version of the manuscript.

ORCID iD

¹⁾CN: [e-mail], https://orcid.org/0009-0008-4343-0031

²⁾JIJ*: [e-mail], https://orcid.org/0009-0008-4518-1577

¹⁾RZ: [e-mail], https://orcid.org/0009-0007-2107-9351

²⁾YH: [e-mail], https://orcid.org/0000-0002-7082-5804

¹⁾AN: [e-mail], https://orcid.org/0000-0001-9295-2532

Data Availability Statements

The data that support the findings of this study are available from the corresponding author, JIJ, upon reasonable request.

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