Periodontal treatment is performed to achieve a biologically acceptable exposed root surface. Root planing removes the part of the root surface that contains inflammation-inducing substances. Previous studies reported that lipopolysaccharide (LPS) existed on superficial exposed root surfaces and deep root planing was not necessary for periodontal treatment. However, recently, it was reported that not only LPS but also various pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs) cause biological immune responses. Advanced therapies, such as periodontal tissue regeneration, use periodontal ligament stem cells (PDL-MSCs), but the influence of substances on the PDL-MSCs on the root surface is unclear.
In this study, we analyzed the permeability of the inflammation-inducing substance on the root surface to determine a suitable root surface for optimal periodontal regeneration.
Extracted teeth were planed, and the shaving extracts from root surfaces were used to measure the bacterial genome. The IL-1β expression in THP-1 cells stimulated by the shavings was measured and the production pathway of IL-1β was investigated using inhibitors. PDL-MSCs were used to examine cell attachment and proliferation on the planed root surface.
By laser microscope, the root planed depth of the periodontitis-affected exposed root surface was 32.2±3.86 μm after the first stroke, 51.9±9.31 μm after the second, 85.4±10.2 μm after the third, and 96.2±4.64 μm after the fourth stroke. Bacterial genome and IL-1β mRNA expression were detected from all shaving extracts up to 8 strokes. The 3-stroke planing showed a significantly high number of PDL-MSCs on the planed root surface. PAMPs and DAMPs in the shaving extracts were involved in the IL-1β production pathway.
These results indicated that bacterial penetration was observed up to at least the eighth stroke and 3-stroke root planing was necessary for the adherence of the PDL-MSCs.
Our research highlights the need for further studies by setting a standard for the ideal preparation of a root surface to receive periodontal stem cells for regenerative procedures.
Periodontitis is a disease caused by the loss of supporting tissue due to inflammation of the periodontal tissue and eventually loss of teeth 1). The treatment for periodontitis aims to control the inflammation of the gingival tissue. One of the various methods of controlling gingival inflammation is root planing. It prepares the outer layer of the exposed root surface to make it biologically acceptable and cures the periodontal tissue of inflammation. Root planing includes scaling and removing a thin layer of a root surface that contains an inflammation-inducing substance 2). In this study, we have discussed the appropriate depth of root planing treatment, i.e., how much of the exposed root must be removed to facilitate optimal periodontal regeneration.
Recent reports have suggested that the main harmful component present on the root surface is the endotoxin lipopolysaccharide (LPS) of gram-negative bacteria. The studies determined the adverse effect of LPS on the body by quantifying it on the root surface 3-10). The results of these studies showed that LPS was present only at the outer layer of the root surface and had not penetrated into the deeper layer 3,5,8-10). Moore et al. quantified LPS by performing limulus amebocyte lysate (LAL) assay using 9 periodontally compromised teeth from 2 periodontitis patients and 9 healthy teeth from 9 healthy subjects, and reported that LPS on the root surface could be removed by washing with water (39%) and by polishing with a rotary cleaning tool (60%) for 1 minute 4). Nyman et al. concluded that the healing observed after scaling root planing (SRP) and polishing the exposed root surfaces was comparable in canine and human periodontal surgery studies and that excessive SRP was not necessary 11,12). However, these studies showed no significant difference in epithelial adhesion or periodontal regeneration.
Oda et al. found that LPS penetrated about 55 μm into the root surface and reported that it was necessary to scale the cement until that depth 13). Oshima et al. examined the influence of exposed root surface debris on cell growth and reported that the LPS layer of 30 μm inhibited the cell growth 14). Hatfield et al. reported that root fragments from teeth with periodontal disease induced irreversible morphologic changes in the cells of the culture, but healthy teeth did not produce such changes 15).
In recent years, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) have been identified as causing inflammatory response in the living body 16). Innate immunity patterns recognize signs of infection and tissue damage with a limited number of receptors. The specific and common constituents of foreign microorganisms are called PAMPs, while the tissue components released from damaged cells are called DAMPs. The cellular pattern recognition receptors (PRRs) detected in PAMPs and DAMPs lead to the production of cytokines 17,18). Thus, the innate immune system monitors the presence of PAMPs/DAMPs and is protective against infectious non-self and tissue-damaging non-self agents.
Besides LPS, various substances derived from pathogenic microorganisms act as ligands with PRRs on the bacterial cell surface to cause an immunological response. However, it is unclear how deep these harmful substances penetrate the root surface, and it is not known to what extent the root surface should be planed to achieve periodontal regeneration.
Mesenchymal stromal cells are required for periodontal tissue regeneration 19). Our group previously reported that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models 20) and human clinical studies 21). Regeneration of periodontal tissue requires the suppression of inflammatory immune cells and the colonization of PDL-MSCs on the root surface.
The purpose of this study is to achieve a suitable root surface for optimal periodontal regeneration. We analyzed the permeability of the inflammation-inducing substances present on the root surface and investigated the attachment and proliferation of PDL-MSCs under various conditions on the root surface.
This study was conducted under the approval of the Tokyo Medical and Dental University (TMDU) Ethics Review Board (#D2016-091). A total of 50 teeth extracted at the Department of Periodontology and Oral Surgery in TMDU were used, of which 30 teeth were extracted due to periodontal disease without preoperative antibiotics and 20 healthy teeth were obtained from orthodontic extractions. Exposed root surfaces of periodontitis-affected teeth and unexposed root surfaces of healthy teeth were used for the following experiment. Healthy teeth have no attachment loss. Wisdom teeth and root canal-treated teeth were excluded. The teeth were extracted from patients who were in a generally healthy condition.
Measurement of root planing depth on the root surfaceTwo periodontal teeth and 2 healthy teeth were used in this experiment. After tooth extraction, the teeth were washed with sterile saline and the plaque on the root surface was wiped with sterile gauze. Using 5× magnifying glasses, the calculus was removed carefully with a sterile 1/2 Gracey curette (Hu-Friedy, Chicago, USA). The root planing site was a flat root surface 4-6 mm from the cementoenamel junction (CEJ). The target site was planed by one investigator (CK), using 5× magnifying glasses and a 1/2 Gracey curette with 2 N lateral pressure; the pressure was checked using a force gauge (A&D, Inc., Tokyo, Japan). The root planing depth was measured using a laser microscope OLS4000 LEXT (Olympus, Tokyo, Japan). The root planing depth was defined as the distance from the root surface to the deepest portion of the root as seen in the vertical cross-section. Root planing was done up to 8 strokes.
Collection of root surface substances by root planingTwenty periodontal teeth and 10 healthy teeth were used in this experiment. The teeth were prepared and planed using up to 8 strokes in the manner described above. All the shaved substances were collected in tubes containing 100 μl of phosphate-buffered saline (PBS) (Fujifilm Wako, Osaka, Japan). The tube was shaken by a vibrator for 3 minutes to grind the pieces into fine particles. After centrifugation at 10000 rpm for 5 minutes, the supernatant was collected and used for the experiment. As a control, a healthy root surface was similarly planed up to 1-3 strokes.
Cell cultureThe THP-1 (JCRB0112.1) cell line was purchased from the Culture Resource Laboratory JCRB Cell Bank. RPMI-1640 medium (containing L-glutamine and phenol red) (Fujifilm Wako) with 10% fetal bovine serum (FBS) (Gibco Carlsbad, CA, USA), and 1% Penicillin-Streptomycin Mixed Solution (NACALAI TESQUE, INC., Kyoto, Japan) was used and the cells were cultured in a 37 °C incubator with 5% CO2 and 95% air.
The method for collection of PDL-MSCs was according to the steps defined by Iwata et al. 21). The medium used was D-MEM high Glucose (containing L-glutamine and phenol red) (Fujifilm Wako), 10% FBS (Gibco Carlsbad), and 1% Penicillin-Streptomycin Mixed Solution (NACALAI TESQUE, INC.). The cells were cultured in a 37 °C incubator with 5% CO2 and 95% air.
ReagentsPorphyromonas gingivalis LPS of 1 μg/ml (standard) (InvivoGen, San Diego, CA, USA) was used for stimulation. The reagents for the inhibitor were 20 μg/ml and 0.2 μg/ml of polymyxin B sulfate (Fujifilm Wako), an LPS inhibitor, 5 μg/ml and 0.5 μg/ml of MCC 950 (Funakoshi, Tokyo, Japan), which inhibits inflammasome, and 10 μg/ml and 0.1 μg/ml of Shikonin (Funakoshi), a nuclear factor kappa B (NFκB) inhibitor.
DNA extraction and real-time PCRThe presence of the bacterial genome in each stroke layer was quantified by the real-time PCR method. The extraction of the bacterial genome from shaved substances was performed using the DNeasy Mini Kit (QIAGEN, Hilden, Germany). The amount of genome was examined by quantitative PCR using the THUNDERBIRD SYBR qPCR Mix (Toyobo) and all the bacterial primers 22) under the protocol of Thermal Cycler Dice Real Time System II (Takara-bio). Using artificial synthetic gene expression as a standard curve for quantitative PCR, the following primers were set:
Forward: 5'-GTG STG CAY GGY TGT CGT CA-3'
Reverse: 5'-ACG TCR TCC MCA CCT TCC TC-3'
RNA extraction and real-time RT-PCRThe THP-1 cells were stimulated by root-shaved stimulants. Inhibitors were added 30 minutes before stimulation. After 2, 6, 12, and 24 hours, mRNA was collected using the RNeasy Mini Kit (QIAGEN). The IL-1β and GAPDH mRNA expressions were measured by quantitative PCR. RNA-direct™ SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) was used under the protocol of Thermal Cycler Dice Real Time System II (Takara-bio, #TX711, Otsu, Japan). Using artificial synthetic gene expression as a standard curve for quantitative PCR, the following primers were set:
IL-1β Forward: 5'-ATG ATG CTT ATT ACA GTG CAA-3'
IL-1β Reverse: 5'-GTC GGA GAT TCG TAG CTG GA-3'
GAPDH Forward: 5'-GCA CCG TCA AGG CTG AGA AC-3'
GAPDH Reverse: 5'-TGG TGA AGA CGC CAG TGG A-3'
IL-1β production measurement by ELISAThe THP-1 cells were stimulated with the periodontitis-affected exposed root-shaved stimulants. Inhibitors were added 30 minutes before stimulation. After 6, 12, 24, and 48 hours, the supernatant was collected. The IL-1β Colorimetric Sandwich ELISA kit (protein tech, Illinois, USA) was used as per the recommended protocol, and IL-1β was quantified.
Cell attachment and proliferation experimentThis experiment was conducted with the approval of the TMDU Ethics Review Board (#D2016-037). PDL-MSCs were taken from root surfaces of 3 teeth obtained from orthodontic extractions. The PDL-MSCs were used to examine cell attachment and proliferation on the planed root surface. The PDL-MSCs were applied to the planed root surface, and their degree of colonization was examined. The root surfaces of 8 periodontitis-affected and 8 healthy teeth were divided into small pieces measuring about 5 mm × 5 mm × 1 mm using high-speed air turbine with a dental burr. Root surface areas of 1 mm × 2 mm were planed up to 4 strokes using a 1/2 Gracey curette. These root surfaces were sterilized at 121 °C for 20 minutes. The sterilized surface pieces were placed facing upwards on a 48-well plate (FALCON, Franklin Lakes, NJ, USA), and 5.0 × 104 cells/ml of PDL-MSCs were seeded. After 48 hours, the cells were fixed for 10 minutes with 4% paraformaldehyde PBS (Fujifilm Wako) and treated with 0.3% PBST for 10 minutes. They were then stained with Fluoromount-G™, with DAPI (Affymetrix Inc., San Diego, CA, USA). Fluorescence was observed by EVOS (Thermo Fisher Scientific Inc., California, USA). The number of cells on the root surface was counted by identifying the number of stained nuclei.
Statistical analysisFor statistical analysis, the In Stat 4.0 software (GraphPad Software, US) was used. One-way analysis of variance (ANOVA) test with Tukey-Kramer post hoc test was used to make multiple comparisons, and p=0.05 was set as the significance level.
The healthy root surface and the periodontitis-affected exposed root surface were planed with a hand scaler, and the root planing depth was measured by a laser microscope.
The healthy root surface was planed till 32.2±3.86 μm after the first stroke, 51.9±9.31 μm after the second, 85.4±10.2 μm after the third, 96.2±4.64 μm after the fourth, 105±1.70 μm after the fifth, 115±1.86 μm after the sixth, 121±7.17 μm after the seventh, and 126±10.8 μm after the eighth stroke (Fig. 1A).
The exposed root surface with periodontal disease was planed till 34.8±4.32 μm after the first stroke, 54.0±4.15 μm after the second, 83.5±7.38 μm after the third, 93.3±4.28 μm after the fourth, 100±4.83 μm after the fifth, 111±4.83 μm after the sixth, 121±10.4 μm after the seventh, and 130±7.41 μm after the eighth stroke (Fig. 1B).
There was no significant difference in the root planing depth between the healthy tooth and the periodontally compromised tooth.
Measurement of the root planing depth of the exposed root surface with a hand scaler.
(A) The healthy root surface was planed till 32.2±3.86 μm at the first stroke, 51.9±9.31 μm at the second, 85.4±10.2 μm at the third, 96.2±4.64 μm at the fourth, 105±1.70 μm at the fifth, 115±1.86 μm at the sixth, 121±7.17 μm at the seventh, and 126±10.8 μm at the eighth stroke. (B) The exposed root surface with periodontal disease was planed till 34.8±4.32 μm at the first stroke, 54.0±4.15 μm at the second, 83.5±7.38 μm at the third, 93.3±4.28 μm at the fourth, 100±4.83 μm at the fifth, 111±4.83 μm at the sixth, 121±10.4 μm at the seventh, and 130±7.41 μm at the eighth stroke.
Even in a no-template negative control, a small amount of bacterial genome contained in recombinant polymerase such as Taq polymerase was amplified by PCR. The amount of bacterial genome measured on the periodontitis-affected exposed root surface and healthy root surface is shown in Fig. 2. On the exposed root surface, the first stroke shavings showed the largest value. Although the amount of bacterial genome decreased with increasing depth, some levels were detected up to the eighth stroke. The amount of genome at strokes 1, 2, and 3 was significantly different from the genome amounts after other strokes respectively, whereas the comparison of strokes 4-8 showed no significant difference. A little bacterial genome was detected from healthy root surface shavings.
The amount of total bacterial genome in the debris of exposed root surfaces.
The first, second, and third stroke at the periodontitis-affected exposed root surface were significantly different from those at the healthy root surfaces; a denotes the significant difference with stroke 1, b denotes the significant difference with stroke 2, and c denotes the significant difference with stroke 3 by Tukey-Kramer post-hoc test (P＜0.05). The bacterial genome was detected up to the eighth stroke. Almost no bacterial genome was detected from healthy root surfaces.
After 2, 6, 12, and 24 hours, the amount of IL-1β mRNA expression in THP-1 cells, which was stimulated by debris on the periodontitis-affected exposed root surface, was examined. The expression level peaked after 2 hours, decreased at 6 hours and 12 hours, and increased again after 24 hours (Fig. 3). After 24 hours, the IL-1β mRNA expression increased again owing to the autocrine effect. The stimulation of IL-1β mRNA expressions in the THP-1 cells, which occurred due to the debris on the exposed root surfaces, is shown in Fig. 4. The IL-1β mRNA expression by stimulation after the first and second strokes showed significantly higher values than the degree of expression after other strokes. Although the expression decreased with each stroke, some level of mRNA expression was observed up to the eighth stroke. However, stimulation with healthy root surface did not show any IL-1β mRNA expression in the THP-1 cells.
The level of IL-1β mRNA expression was the highest after 2 hours.
The THP-1 cells were stimulated with the debris of the exposed root surface at 0, 2, 6, 12, and 24 hours. After 24 hours, the IL-1β mRNA expression increased again due to autocrine effect.
The IL-1β mRNA expression level in THP-1 cells by stimulation of exposed root surfaces.
LPS of 1 μg/ml was used for stimulation. The THP-1 cells were stimulated for 2 hours. IL-1β mRNA expression by stimulation with 1-2 strokes showed significantly higher values on the periodontitis-affected exposed root surfaces than on the healthy exposed root surfaces; a denotes the significant difference with stroke 1 and b denotes the significant difference with stroke 2 by Tukey-Kramer post-hoc test (P＜0.05). Although the expression decreased with increasing depth, mRNA expression was observed up to the eighth stroke. Stimulation with healthy root surface did almost not show IL-1β mRNA expression.
IL-1β in the supernatant was measured after stimulation due to root shaving. IL-1β production from THP-1 cells was 16.0 pg/ml after 6 hours of stimulation, 44.6 pg/ml after 12 hours, 83.7 pg/ml after 24 hours, and 240 pg/ml after 48 hours. IL-1β production increased in a time-dependent manner (Fig. 5).
IL-1β production increased in a time-dependent manner.
The THP-1 cells were stimulated due to the debris of the exposed root surface at 0, 6, 12, 24, and 48 hours. The control was not stimulated.
The IL-1β mRNA expression in THP-1 cells, which was stimulated by root shaving stimulation and/or various inhibitors, is shown in Fig. 6. Polymyxin B, an LPS inhibitor, significantly reduced the expression level of IL-1β mRNA (Fig. 6A). MCC950, which inhibits inflammasome, did not alter mRNA expression (Fig. 6B). Shikonin, an NFκB inhibitor, significantly reduced the IL-1β mRNA expression (Fig. 6C).
IL-1β mRNA expression level in THP-1 cells by stimulation of the exposed root surfaces with an inhibitor.
The THP-1 cells were stimulated with the debris of the exposed root surface for 2 hours. The concentration of (A) Polymyxin B was 20 μg/ml or 0.2 μg/ml, (B) MCC950 was 50 μg/ml or 0.5 μg/ml, and (C) Shikonin was 10 or 0.1 μg/ml. The level of IL-1β mRNA expression significantly reduced with the use of Polymyxin B and Shikonin (P＜0.05). Control was not stimulated after the inhibitor was added. Only the cells with no inhibitor were stimulated. The inhibitor alone did not express IL-1β mRNA. Shikonin was diluted in DMSO, which was not involved in the expression of IL-1β mRNA.
Polymyxin B, MCC950, and Shikonin significantly reduced the amount of IL-1β secretion from the THP-1 cells (Fig. 7).
IL-1β production in the THP-1 cells by stimulation of exposed root surfaces with an inhibitor.
The THP-1 cells were stimulated due to the debris of the exposed root surface for 24 hours. The concentration of (A) Polymyxin B was 20 μg/ml or 0.2 μg/ml, (B) MCC950 was 50 μg/ml or 0.5 μg/ml, and (C) Shikonin was 10 μg/ml or 0.1 μg/ml. The amount of IL-1β production significantly reduced using Polymyxin B, MCC950 and Shikonin. (P＜0.05). Shikonin was diluted in DMSO, which did not induce IL-1β expression. Control was not stimulated after the inhibitor was added. Only the cells with no inhibitor were stimulated. The inhibitor alone did not produce IL-1β. Shikonin was diluted in DMSO, which was not involved in the production of IL-1β.
On the healthy root surface, the volume of PDL-MSCs was 306±9.20 cell/mm2 at no stroke, 396±45.1 cell/mm2 after the first, 209±12.2 cell/mm2 after the second, and 421±24.4 cell/mm2 after the third stroke (Fig. 8A). As compared to the number of cells on the unplaned root surface, the number of PDL-MSCs increased significantly after the first stroke, decreased significantly after the second stroke, and increased significantly again after the third stroke. In the exposed root surface of periodontitis-affected teeth, the number of PDL-MSCs was 332±17.4 cell/mm2 at no stroke, 340±52.5 cell/mm2 after the first, 260±10.5 cell/mm2 after the second, 545±24.4 cells/mm2 after the third, and 232±18.2 cell/mm2 after the fourth stroke (Fig. 8B). The data are representative of three individual experiments. After the third stroke, the number of PDL-MSCs significantly increased in volume. A comparison of PDL-MSC numbers is shown in Fig. 9A, B.
The nucleus of PDL-MSCs on the exposed root surface was stained with DAPI. A total of 5.0^104 cells were seeded and cultured for 48 hours.
After fixation with DAPI, the cells were observed under a fluorescence microscope. (A) Cells on the healthy root surface after root planing. (B) Cells on the periodontitis-affected exposed root surface after root planing.
The number of periodontal ligament cells on the exposed root surface.
Periodontal ligament cells were cultured on healthy root surface and periodontitis-affected exposed root surface for 48h. 5.0^104 cells were seeded. The theoretical value was the quantity which all 5.0^104 cells landed on the plate. (A) The number of periodontal ligament cells on the healthy root surface and (B) the periodontitis-affected exposed root surface increased significantly in first stroke relative to on the unstroked root surface, decreased significantly in the second stroke (P＜0.05). It increased significantly again at the third stroke. There was no significant difference in the number of the cells between the healthy root surface and the periodontitis-affected exposed root surface.
There are many reports on the measurement of root planing depth in the literature. Ritz et al. reported a root planing depth of 60.2-264 μm by hand scaler in 12 strokes; they used a substance loss measuring device to measure the depth 23). Berkstein et al. reported an average root planing depth of 27.1 μm using a digital micrometer 24). In the present study, we used a laser microscope to measure the root planing depth. This device can measure a fineness of 0.2 μm and give an accurate depth value. From the results, both the healthy root surface and periodontitis-affected exposed root surface showed about 35 μm depth at the first stroke, 20 μm at the second, 30 μm at the third, and 10 μm after the fourth stroke. There was no significant difference in the root planed depth between the healthy root surface and the periodontitis-affected exposed root surface. We considered that the amount of root planing per stroke might not be dependent on the morbidity of periodontal disease.
The thickness of the cementum is reported to be 30-120 μm 25). The average cementum thicknesses are 95 μm at the age of 20 years and 215 μm at the age of 60 years 26). After 3 root planing strokes that plane the root till a depth of 80 μm, the cementum might get obliterated and the dentin might be exposed. The reason why only 10 μm was planed after 4 strokes might be because the dentin had been accidentally planed. The thickness of cementum varies depending on its position; it is thicker apically and thinner cervically. In this experiment, a site 4-6 mm from the CEJ was chosen for SRP. Further studies need to conducted using SRP sites greater than 6 mm from the CEJ.
Real-time PCR was used to measure the amount of bacterial genome on the root surfaces. Our results showed that more bacterial genome was detected in each periodontitis-affected exposed root fragment than healthy root surface. These data support previous reports that state that bacterial penetration into the cementum was detected around the cemento-dentinal junction 27) and it might penetrate the dentinal tubules 28). The reason why PCR detected the bacterial genome in healthy teeth might be that the PCR enzyme was derived from bacteria, and therefore, a small amount of bacterial genome was contained in the PCR enzyme.
The amounts of bacterial DNA in the first, second and third stroke shavings were significantly higher than the bacteria in the fourth stroke shaving. Since bacterial genomes can cause inflammation via toll-like receptor (TLR) 9 in human cells 29), a 3-stroke root planing procedure might be necessary for reducing inflammation from bacterial genomes.
IL-1β is a potent inflammatory cytokine in the immune response and is involved in the inflammation of periodontal tissues 30). Montenegro et al. reported that bacterial components in calculus induced IL-1β production in monocytes and neutrophils 31). In this study, we measured the IL-1β production in THP-1 cells, which was stimulated by bacterial components in root fragments.
Our results showed that the expression of IL-1β mRNA was detected in every stroke stimulation of the periodontitis-affected teeth, but almost not in the shavings of the healthy root surface. IL-1β mRNA expression was significantly high after the first and second strokes. To reduce the IL-1β mRNA expression, 2-stroke root planing might be necessary.
In this experiment, IL-1β was used as an indicator of cellular inflammation due to innate immunity. In tissues affected by periodontitis, acquired immunity is developed and a large number of B-cells are present32). Regarding the establishment of periodontitis, it is necessary to examine not only the formation of innate immunity but also the development of acquired immunity.
Inhibitors polymyxin B sulfate, MCC950, and Shikonin were used to examine the effect on IL-1β production. Polymyxin B sulfate binds to the lipid A portion of LPS and neutralizes it 33), MCC950 suppresses the inflammasome by inhibiting NLRP3 34), and Shikonin inhibits LPS and NFκB35).
In IL-1β production, the pathogenic-factor-mediated NFκB induces transcription from the pro-IL-1 beta gene via TLRs. DAMPs activate caspase-1 via inflammasome to cleave the IL-1β precursor to form mature IL-1β 36,37).
The results of our mRNA experiment with inhibitors showed that polymyxin B sulfate and Shikonin suppressed the expression amount of IL-1β mRNA in a concentration-dependent manner. Among the bacterial factors of the root shavings, LPS was involved in IL-1β expression. LPS activates NFκB to produce IL-1β via TLR4. Olson et al. reported that LPS-free diseased cementum shavings did not affect the development of the hepatocyte growth factor (HGF) 38). The neutralizing effect of LPS is considered to suppress IL-1β mRNA expression and reduce innate immunity. Moreover, bacterial components may induce acquired immunity as well as innate immunity. To suppress the acquired immunity, the bacterial population should be reduced as much as possible.
Similar to polymyxin B sulfate and Shikonin, MCC950 also suppressed IL-1β protein secretion. MCC950 inhibits NLRP3 and suppresses the inflammasome 34). Dead cells on the root surface and shavings of cementum can cause biological reactions such as DAMPs. Since IL-1β protein production was suppressed by MCC950, PAMPs and DAMPs may be implicated as harmful substances found on the root surface. It is important to reduce inflammation by removing PAMPs and DAMPs from the root surface. During root planing, the debris should be washed away and removed completely.
The PDL-MSCs were used to examine cell attachment and proliferation on the planed root surface. No difference was observed in the number of cells 1 day after the seeding of PDL-MSCs (data not shown); therefore, the measurements were recorded after 2 days. The reason why no difference was seen after a day is that the amount of the seeded floating stem cells may have landed on the root surface in equal proportions.
Based on our results, the difference in the number of stem cells after 2 days depends on the rate of cell proliferation. There was no significant difference in the number of PDL-MSCs on the root surface between periodontitis-affected teeth and healthy teeth. Therefore, the adhesion and proliferation of PDL-MSCs, regardless of the degree of morbidity of periodontitis, is likely to be a more important factor in the properties of the root surface.
The number of cells on the root surface decreased at the second stroke and increased after three strokes in both the healthy root surface and periodontitis-affected root surface. The number of cells decreased after the fourth stroke and was most abundant when at the third stroke. In periodontal ligament regeneration, it is necessary to ensure cell migration and fix them to the root surface. It is considered necessary to perform firm root planing up to 3 strokes. If 4 or more strokes are planed, the dentin on the inner layer of the cementum may be exposed, resulting in unfavorable root surface properties where adherence of stem cells is difficult to establish. However, since the thickness of cementum is not uniform, it is also necessary to consider clinical symptoms. Moreover, this research was conducted at a site 4-6 mm from CEJ, and it was thought that the amount of root planing might differ at a place shallower or deeper than this.
A limitation of this study was that it only evaluated inflammatory responses in a series of in vitro experiments. In actual clinical practice, healing of the organism occurs despite salivary bacteria constantly invading the root surface. It is necessary to consider the number of root planing procedures that may be acceptable to a living body and the time it may take to heal between procedures. Since this study was performed at a site 4-6 mm from the CEJ on an exposed root surface in limited terms, further studies on root planing in regenerative therapy, on a different site and on human patients in dental practice, may be necessary.
We concluded that PAMPs and DAMPs were part of the inflammation-inducing substances present on the exposed root surface. The substance causing inflammation at the root surface penetrated the root up to the eighth root planing stroke. Also, planing of up to 3 strokes was effective in ensuring proper attachment of PDL-MSCs to the root surface. Our research highlights the need for further studies by setting a standard for the ideal preparation of a root surface to receive periodontal stem cells for regenerative procedures.
We thank Dr. Shigeru Oda for his kind support and advice provided in this study.
This work was supported by grants-in-aid for Scientific Research (No. 19K10124) from the Japan Society for the Promotion of Science.
Conflict of Interest: None.
