METTL3 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells through IGF2BP1-Mediated Regulation of Runx2 Stability.

N6-Methyladenosine (m6A) has been reported to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) remains unclear. Here, we found that methyltransferase-like 3 (METTL3) was up-regulated synchronously with m6A during the osteogenic differentiation of hPDLSCs. Functionally, lentivirus-mediated knockdown of METTL3 in hPDLSCs impaired osteogenic potential. Mechanistic analysis further showed that METTL3 knockdown decreased m6A methylation and reduced IGF2BP1-mediated stability of runt-related transcription factor 2 (Runx2) mRNA, which in turn inhibited osteogenic differentiation. Therefore, METTL3-based m6A modification favored osteogenic differentiation of hPDLSCs through IGF2BP1-mediated Runx2 mRNA stability. Our study shed light on the critical roles of m6A on regulation of osteogenic differentiation in hPDLSCs and served novel therapeutic approaches in vital periodontitis therapy.

Association between periodontitis and endometriosis: a bidirectional Mendelian randomization study.

Introduction: A potential association between periodontitis and endometriosis has been indicated in previous observational studies. Nevertheless, the causal link between these two disorders has not been clarified. Methods: Based on publicly available genome-wide association study (GWAS) summary datasets, we conducted a bidirectional Mendelian randomization (MR) study to investigate the relationship between periodontitis and endometriosis and its subtypes. Single nucleotide polymorphisms (SNPs) strongly associated with candidate exposures at the genome-wide significance level (P < 5 × 10-8) were selected as instrumental variables (IVs). The inverse variance-weighted regression (IVW) was performed to estimate the causal effect of periodontitis on endometriosis. We further conducted two sensitivity analyses, MR-Egger and weighted median, to test the validity of our findings. The main results were replicated via data from the UK Biobank. Finally, a reverse MR analysis was performed to evaluate the possibility of reverse causality. Results: The IVW method suggested that periodontitis was positively associated with endometriosis of the pelvic peritoneum (OR = 1.079, 95% CI = 1.016 to 1.146, P = 0.014). No causal association was indicated between periodontitis and other subtypes of endometriosis. In reversed analyses, no causal association between endometriosis or its subtypes and periodontitis was found. Conclusions: Our study provided genetic evidence on the causal relationship between periodontitis and endometriosis of the pelvic peritoneum. More studies are necessary to explore the underlying mechanisms.

LncRNA urothelial cancer associated 1 promotes the osteogenic differentiation of human periodontal ligament stem cells by regulating the miR-96-5p/Osx axis.

OBJECTIVES: To investigate the expression of long non-coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) in human periodontal ligament stem cells (hPDLSCs), its effect on osteogenic differentiation of hPDLSCs and its mechanism. DESIGN: The expression of osteogenic genes Osx, Runx2, Ocn and Opn was explored by qPCR. Protein expression in hPDLSCs was estimated by Western blot. The osteogenic differentiation of hPDLSCs was detected by Alizarin red staining assays. The interaction between UCA1 and miR-96-5p was explored by RNA pulldown assay and dual luciferase assay. The interaction between miR-96-5p and Osx 3'-UTR was measured by dual luciferase assay. RESULTS: The expression of UCA1 and miR-96-5p was negatively correlated in hPDLSCs. During the osteogenic differentiation of hPDLSCs, the expression of UCA1 was increased, while the expression of miR-96-5p was decreased. Knockdown of UCA1 in hPDLSCs inhibited osteogenic differentiation but induced upregulation of miR-96-5p expression, and vice versa. In addition, miR-96-5p partially reversed the positive effect of UCA1 on osteogenic differentiation of hPDLSCs. Notably, UCA1 was identified as a miR-96-5p sponge, and miR-96-5p targeted Osx. CONCLUSIONS: Our results demonstrated that the novel UCA1/miR-96-5p/Osx pathway regulates osteogenic differentiation of hPDLSCs and sheds new insights and targets for periodontitis therapeutics.

The Association between Tooth Loss and Insulin Resistance Mediated by Diet Quality and Systemic Immunoinflammatory Index.

(1) Background: Both tooth loss and diabetes have high global prevalence, and both have a significant influence on patients' general health and quality of life. Previous research has indicated a possible connection between tooth loss and diabetes, but it has been unclear whether tooth loss has an effect on the development of diabetes and how it affects it. We aim to investigate the relationship between insulin resistance (IR) and tooth loss and examine how the systemic immune-inflammation index (SII) level and diet quality mediate it. (2) Methods: The cross-sectional study data were obtained from the National Health and Nutrition Examination Survey (NHANES). After describing and comparing baseline data, we used regression models to evaluate the relationship between IR and tooth loss, diet quality and tooth loss and IR, SII and tooth loss and IR. Furthermore, we applied bootstrapping to test the mediation effect of diet quality and SII between tooth loss and IR. Diet quality is reflected by the HEI (Healthy Eating Index)-2015 score. (3) Results: The total number of subjects included was 8197, with 3861 individuals belonging to the IR group (HOMA-IR ≥ 2.5) and 4336 in the non-IR group (HOMA-IR < 2.5). In the model with all covariates adjusted, tooth loss in the fourth quartile was found to be positively correlated with an increase in HOMA-IR (OR = 1.301; 95% confidence interval (CI) = [1.102, 1.537]; p < 0.001) compared to the first quartile; tooth loss in the fourth quartile correlated with the HEI-2015 score compared to the first quantile (ß = -0.121, 95% CI = [-4.839, -2.974], p < 0.001); and the highest number of tooth loss was found to have a significant effect on SII (ß = 0.032; 95%CI = [1.777, 47.448]; p < 0.05). Compared to average diet quality, best diet quality acts as a safeguard against elevated HOMA-IR (OR = 0.776; 95% CI = [0.641, 0.939]; p < 0.01); inadequate diet quality is a risk factor (OR = 1.267; 95%CI = [1.138, 1.411]; p < 0.001) conversely. Meanwhile, it can be seen that compared with the first quantile of SII, the highest score is significantly correlated with the higher incidence of IR (OR = 1.363; 95%CI = [1.179, 1.575]; p < 0.001). Diet quality and SII played a partial mediating role in the relationship between HOMA-IR and tooth loss, and the mediating effect ratio for the total effect value was 4.731% and 4.576%, respectively. The mediating effect of SII and diet quality in the association of the relationship between HOMA-IR and tooth loss both was 0.003 (95%CI = [0.001, 0.004]). (4) Conclusions: Our study revealed the relationship between IR and tooth loss, and further explored the mediating role of SII and diet quality between the number of missing teeth and IR, emphasizing that improving diet quality and reducing SII can effectively prevent and treat IR and related diseases. It provides new theoretical support for the study of IR mechanisms and new ideas and approaches to deal with related diseases.

Retinol-Binding Protein 4 and Visfatin Levels in Patients with Periodontitis and Obesity/Overweight: A Systematic Review and Meta-Analysis.

Prior studies demonstrated an equivocal conclusion about the association between the level of retinol-binding protein 4 (RBP4)/visfatin and periodontitis patients with obesity. The aim of our study (Prospero ID: CRD42023469058) was to systematically review the available articles linking the biofluid levels of RBP4/visfatin to the comorbidity of periodontitis and obesity. Clinical trials were screened in accordance with specific inclusion criteria from seven databases up to November 2023. A quality assessment was performed with the Newcastle-Ottawa Scale and ROBINS-I tools for observational and interventional trials, respectively. The standard mean difference (SMD) with a 95% confidence interval (CI) related to the RBP4 level was recorded; the other indicators related to the visfatin level were measured via the mean difference (MD) with the corresponding 95% CI, and Fisher's Z transformation was measured to reveal the association using Review Manager 5.4. The current evidence was based on five observational studies and two interventional studies. All of them were included in the systematic review, and six of them were in the meta-analysis. Statistical analysis indicated that there was no significant difference in the circulating levels of RBP4 in the periodontitis patients with obesity or without, who were labeled as OP or NP, respectively (155 OP-107 NP: SMD = 1.38; 95% CI: -0.18-2.94, p = 0.08), as well as the periodontal healthy patients with a normal weight, who were labelled as NnP (116 OP-79 NnP: SMD = 6.76; 95% CI: -5.34-18.87, p = 0.27). Meanwhile, a significant higher level of serum visfatin was found in the OP patients than that of the NP (86 OP-45 NP: MD = 4.21; 95% CI: 2.65-5.77, p < 0.00001)/NnP (164 OP-88 NnP: MD = 13.02; 95% CI: 7.34-18.70, p < 0.00001) group. In addition, a positive association was observed between the serum RBP4 and body mass index/clinical attachment loss (CAL). And, then, there was a positive association between the serum visfatin and periodontal parameters, including the probing depth, CAL, and plaque index, as well as metabolic parameters, including the total cholesterol, triglycerides, fasting blood glucose, and low-density lipoprotein cholesterol. Here, the circulating RBP4 level was not independently related to the comorbidity of periodontitis and obesity, while serum visfatin was significantly associated with periodontitis and obesity. Notably, the positive association between circulating RBP4/visfatin and the periodontal parameters/metabolic parameters firmly suggested that the higher severity of the obese or periodontal status was associated with an elevated level of serum visfatin or RBP4 in the OP group. With more rigorous longitudinal research, the exact causations between RBP4/visfatin and the patients affected by obesity and periodontitis could be disentangled. RBP4 and visfatin might be novel, enlightening prospective bio-indexes for the targeted treatment of comorbidities.

Effect of non-surgical periodontal treatment on cytokines/adipocytokines levels among periodontitis patients with or without obesity: a systematic review and meta-analysis.

BACKGROUND: The objective of this systematic review and meta-analysis was to evaluate the effects of non-surgical periodontal therapy (NSPT) on inflammatory-related cytokines/adipocytokines in periodontitis patients with or without obesity. METHODS: We followed the preferred reporting items for systematic reviews and meta-analyses statement and registered the study (CRD42022375331) in the Prospective International Register of Systematic Reviews. We screened randomized-controlled trials and controlled clinical trials from six databases up to December 2022. Quality assessment was performed with RoB-2 and ROBINS-I tools for randomized trials and non-randomized trials, respectively. Meta-analysis was carried out using a random-effect model. RESULTS: We included seventeen references in the systematic analysis, and sixteen in the meta-analysis. Baseline results of pro-inflammatory biomarkers, including serum interleukin (IL)-6, serum and gingival crevicular fluid (GCF), tumor necrosis factor (TNF)-a, serum C-reactive protein (CRP)/hs-CRP, and serum and GCF resistin, were higher in obesity subjects than in normal weight subjects. The effect of NSPT with respect to levels of cytokines/adipocytokines, including IL-6, TNF-a, CRP/hs-CRP, resistin, adiponectin, leptin and retinol binding protein 4 (RBP4), were then analyzed in the systematic and meta-analysis. After three months of NSPT, serum (MD = -0.54, CI = -0.62 - -0.46), and GCF (MD = -2.70, CI = -4.77 - -0.63) levels of IL-6, along with the serum RBP4 (MD = -0.39, CI = -0.68-0.10) decreased in periodontitis individuals with obesity. NSPT also improved GCF adiponectin levels after three months (MD = 2.37, CI = 0.29 - 4.45) in periodontitis individuals without obesity. CONCLUSIONS: Obese status altered the baseline levels of cytokines/adipocytokines (serum IL-6, serum and GCF TNF-a, serum CRP/hs-CRP, and serum and GCF resistin). Then NSPT can shift the levels of specific pro-inflammatory mediators and anti-inflammatory mediators in biological fluids, both in obesity and non-obesity individuals. NSPT can reduce serum and GCF IL-6 levels together with serum RBP4 level in individuals with obesity after 3 months, besides, there is no sufficient evidence to prove that obese patients have a statistically significant decrease in the levels of other cytokines compared to patients with normal weight. NSPT can also increase GCF adiponectin level in normal weight individuals after 3 months. Our findings imply the potential ideal follow-up intervals and sensitive biomarkers for clinical bioanalysis in personalized decision-making of effect of NSPT due to patients' BMI value.

Long non­coding RNA SNHG5 promotes osteogenic differentiation of human periodontal ligament stem cells via mediating miR­23b­3p/Runx2 axis.

The treatment of bone loss due to periodontitis has posed a great challenge for physicians for decades. Therefore, it is of extraordinary significance to identify an effective regeneration scheme for alveolar bone. This study aimed to investigate long non-coding RNA (lncRNA) small nucleolar RNA host gene 5 (SNHG5) whether sponges microRNA-23b-3p (miR-23b-3p) to achieve the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Results revealed that the expression of SNHG5 was upregulated whereas that of miR-23b-3p was downregulated in osteogenic hPDLSCs. Alizarin red staining assays and qRT-PCR demonstrated that SNHG5 silencing or miR-23b-3p overexpression inhibits hPDLSCs osteogenic differentiation and vice versa. In addition, miR-23b-3p partially abolished the promotive effect of SNHG5 on osteogenic differentiation of hPDLSCs. Dual luciferase report and RNA pulldown assay verified that miR-23b-3p is a regulatory target of SNHG5 and that Runx2 is a gene target of miR-23b-3p. In brief, the results demonstrate that SNHG5 promotes the osteogenic differentiation of hPDLSCs by regulating the miR-23b-3p/Runx2 axis. Our study provides novel mechanistic insights into the critical role of lncRNA SNHG5 as a miR-23b-3p sponge to regulate Runx2 expression in hPDLSCs and may serve as a potential therapeutics target for periodontitis.

A novel strategy combining Mini-CEX and OSCE to assess standardized training of professional postgraduates in department of prosthodontics.

OBJECTIVE: Mini clinical evaluation exercise (mini-CEX) and objective structured clinical examination (OSCE) are widely acknowledged as effective measures of resident standardization training (RST) in European and American countries. However, in China primary mini-CEX and OSCE forms are mainly limited in undergraduate clinical examination. Little knowledge is available regarding the validity and right way of mini-CEX /OSCE evaluation system in advanced dental clinical education so far. This study aimed to explore whether combination of mini-CEX and OSCE represents a global-dimension assessment for postgraduate clinical competence in RST. METHODS: Postgraduates who received RST from June 2017 to June 2019 were selected and evaluated by modified mini-CEX/OSCE scales. Each student received evaluations at least twice in the initial and final stages of training (tested every 4 months). A questionnaire was conducted to investigate the satisfaction with the arrangement of RST. RESULTS: Mini-CEX/OSCE test results indicated that postgraduates have significantly improved their comprehensive competence in RST projects in the department of prosthodontics (P < 0.05). Compared to other master of Stomatology students, postgraduates taking up prosthodontics master's degree have made more progresses through a training period of up to 1 year and four sessions of face-to-face feedback tutoring (P < 0.05). Survey results revealed high level of satisfaction on clinical practice evaluation. CONCLUSION: Modified mini-CEX/OSCE combined evaluation system is an effective and reliable assessment tool for clinical comprehensive ability in the RST of professional graduates and can fully highlight their respective advantages on the improvement of students' clinical competency, especially after several rounds of assessments.

Reactive Oxygen Species Enlightened Therapeutic Strategy for Oral and Maxillofacial Diseases-Art of Destruction and Reconstruction.

Reactive oxygen species (ROS) are byproducts of cell metabolism produced by living cells and signal mediators in biological processes. As unstable and highly reactive oxygen-derived molecules, excessive ROS production and defective oxidant clearance, or both, are associated with the pathogenesis of several conditions. Among them, ROS are widely involved in oral and maxillofacial diseases, such as periodontitis, as well as other infectious diseases or chronic inflammation, temporomandibular joint disorders, oral mucosal lesions, trigeminal neuralgia, muscle fatigue, and oral cancer. The purpose of this paper is to outline how ROS contribute to the pathophysiology of oral and maxillofacial regions, with an emphasis on oral infectious diseases represented by periodontitis and mucosal diseases represented by oral ulcers and how to effectively utilize and eliminate ROS in these pathological processes, as well as to review recent research on the potential targets and interventions of cutting-edge antioxidant materials. The PubMed, Web of Science, and Embase databases were searched using the MesH terms "oral and maxillofacial diseases", "reactive oxygen species", and "antioxidant materials". Irrelevant, obsolete, imprecise, and repetitive articles were excluded through screening of titles, abstracts, and eventually full content. The full-text data of the selected articles are, therefore, summarized using selection criteria. While there are various emerging biomaterials used as drugs themselves or delivery systems, more attention was paid to antioxidant drugs with broad application prospects and rigorous prophase animal experimental results.

Researching progress on bio-reactive electrogenic materials with electrophysiological activity for enhanced bone regeneration.

Bone tissues are dynamically reconstructed during the entire life cycle phase, which is an exquisitely regulated process controlled by intracellular and intercellular signals transmitted through physicochemical and biochemical stimulation. Recently, the role of electrical activity in promoting bone regeneration has attracted great attention, making the design, fabrication, and selection of bioelectric bio-reactive materials a focus. Under specific conditions, piezoelectric, photoelectric, magnetoelectric, acoustoelectric, and thermoelectric materials can generate bioelectric signals similar to those of natural tissues and stimulate osteogenesis-related signaling pathways to enhance the regeneration of bone defects, which can be used for designing novel smart biological materials for engineering tissue regeneration. However, literature summarizing studies relevant to bioelectric materials for bone regeneration is rare to our knowledge. Consequently, this review is mainly focused on the biological mechanism of electrical stimulation in the regeneration of bone defects, the current state and future prospects of piezoelectric materials, and other bioelectric active materials suitable for bone tissue engineering in recent studies, aiming to provide a theoretical basis for novel clinical treatment strategies for bone defects.

Light limitation inducing overcompensatory growth of cyanobacteria and function of serine/threonine kinase (STK) genes involved.

The rapid overcompensatory growth that appears when cyanobacteria are supplied with adequate resources after a period of resource deprivation might contribute to the occurrence of cyanobacterial blooms. We investigated the changing characteristics of overcompensatory growth and serine/threonine kinase (STK) genes expression of cyanobacterium Microcystis aeruginosa in response to light limitation. The results showed M. aeruginosa exhibited overcompensatory growth for 2 days after light recovery, during which the increase in growth was inversely related to light intensity. Expression of STK genes, such as spkD, was upregulated significantly at 0.5-4 h after light recovery (P < 0.05). To investigate the function of STK genes in the overcompensatory growth, M. aeruginosa spkD was heterologously expressed in Synechocystis. Transgenic Synechocystis exhibited greater and longer overcompensatory growth than wild-type Synechocystis after light recovery. Relative expression levels of STK genes in transgenic Synechocystis were significantly higher than those in wild-type Synechocystis at 24 h of light recovery (P < 0.05). Heterologous expression of Microcystis spkD might stimulate overcompensatory growth of Synechocystis by affecting its STK gene expression.

A novel multifunctional carbon aerogel-coated platform for osteosarcoma therapy and enhanced bone regeneration.

Nowadays, groundbreaking strategies are urgently needed to address drug resistance, osteolysis, bone defects and other predicaments impeding the therapeutic efficacy of osteosarcoma. Among them, photothermal therapy (PTT), using systematically administrated nanoagents, exhibits attractive therapeutic efficacy, yet is powerless in bone defect regeneration. Herein, a novel multifunctional beta-tricalcium phosphate (ß-Ca3(PO4)2, ß-TCP) bioceramic platform-coated with carbon aerogel (CA), which was initially developed for tumor therapy, was fabricated. On account of the desirable photothermal capabilities of CA, sufficient hyperthermia is generated under the irradiation of an 808 nm laser to achieve a thorough ablation of osteosarcoma tumors. Furthermore, CA-coated surfaces provide extra roughness and a higher specific surface area, which promoted the protein recruitment ability and osteogenesis via a fibronectin (FN)-mediated signaling pathway. The photothermal therapeutic efficacy and osteogenesis capability of CA-coated ß-TCP-C suggests a novel approach for the treatment of osteosarcoma and provides provoking inspiration for the prospective bio-application of CA.

Microcystins distribution, bioaccumulation, and Microcystis genotype succession in a fish culture pond.

In freshwater aquaculture ponds, cyanobacterial blooms and microcystins (MCs) pollution have attracted considerable attention due to their toxic effects. To provide an insight into cyanobacterial problems in aquaculture ponds, MCs distribution, bioaccumulation, and Microcystis genotype succession in a fishpond were investigated from May 2017 to November 2017. The distribution of MCs in filtered water, seston, and sediment varied considerably among months. MCs concentrations in filtered water, seston, and sediment ranged from 1.16 to 3.66 µg/L, 0.64 to 13.98 µg/g DW, and 1.34 to 5.90 µg/g DW, respectively. In addition, chemical oxygen demand was positively correlated with sestonic MCs concentrations. MCs concentrations accumulated in different tissues of market-size fish were in the order of liver > kidney > intestine > muscle. MCs content in muscle was 4.3 times higher than the WHO recommended tolerable daily intake level. Twenty-four ITS genotypes of Microcystis were identified from a total of 653 sequences. During the survey period, considerable genotype variation and rapid genotype succession were observed and dominant genotype was absent. A redundancy analysis revealed that Microcystis genotypes could significantly influence the variations in the proportions of the potentially toxic Microcystis, which could in turn influence the MCs concentrations in seston.

Effects of Bacillus subtilis on the growth, colony maintenance, and attached bacterial community composition of colonial cyanobacteria.

In freshwater aquaculture ponds, application of algicidal Bacillus is a promising way in the control of cyanobacterial blooms. To best understand Bacillus algicidal characters and mechanisms in the field, different-sized colonial cyanobacteria were isolated from an aquaculture pond, and the effects of B. subtilis on their growth, colony maintenance, and colony-attached bacterial community composition were investigated. The results showed that B. subtilis could inhibit the growth of colonial cyanobacteria. Bigger-sized colonies isolated from the field could spontaneously disintegrate into smaller-sized colonies in the laboratory. Algicidal B. subtilis could accelerate the disintegration of colonies and decrease colony size. B. subtilis not only decreased the colony-attached bacterial community diversity but also changed its composition. B. subtilis increased the relative abundances of some attached bacterial genera, including Pseudomonas, Shewanella, Bacillus, Shinella, Rhizobium, and Ensifer. These bacteria with algicidal, microcystin-degrading, and flocculating activities might be an important contributor to algicidal effects of B. subtilis on colonial cyanobacteria.

Effects of toxic Microcystis genotypes on natural colony formation and mechanism involved.

The cyanobacterium Microcystis occurs as colonies of different sizes with varying abundance of toxic genotypes versus non-toxic genotypes under natural conditions. To investigate the effects of toxic Microcystis genotypes on natural colony formation, samples collected from the mainstream of Haihe River from July to October 2015 were sieved into four colony classes with sizes of <8 µm, 8-20 µm, 20-90 µm and >90 µm. Each colony size class was analyzed for the proportion of toxic Microcystis genotypes, and microcystins (MCs) cellular production of toxic genotypes. The results showed the smallest size class of Microcystis colonies (<8 µm) showed the lowest proportion of toxic genotypes and the highest MC-RR and MC-YR cellular production. With the increasing colony sizes, the proportion of toxic Microcystis genotypes increased but the MC-RR and MC-YR cellular production decreased. A negative correlation between the MCs cellular production and the proportion of toxic genotypes was observed in all four colony size classes, suggesting that the less there were toxic Microcystis cells able to produce MCs, the more each toxic cell needed to produce that molecule. Toxic Microcystis played an important role in the colony formation in natural waters via producing MCs.

Heat Transfer Behavior across the Dentino-Enamel Junction in the Human Tooth.

During eating, the teeth usually endure the sharply temperature changes because of different foods. It is of importance to investigate the heat transfer and heat dissipation behavior of the dentino-enamel junction (DEJ) of human tooth since dentine and enamel have different thermophysical properties. The spatial and temporal temperature distributions on the enamel, dentine, and pulpal chamber of both the human tooth and its discontinuous boundaries, were measured using infrared thermography using a stepped temperature increase on the outer boundary of enamel crowns. The thermal diffusivities for enamel and dentine were deduced from the time dependent temperature change at the enamel and dentine layers. The thermal conductivities for enamel and dentine were calculated to be 0.81 Wm-1K-1 and 0.48 Wm-1K-1 respectively. The observed temperature discontinuities across the interfaces between enamel, dentine and pulp-chamber layers were due to the difference of thermal conductivities at interfaces rather than to the phase transformation. The temperature gradient distributes continuously across the enamel and dentine layers and their junction below a temperature of 42°C, whilst a negative thermal resistance is observed at interfaces above 42°C. These results suggest that the microstructure of the dentin-enamel junction (DEJ) junction play an important role in tooth heat transfer and protects the pulp from heat damage.