Enhanced Antimicrobial Properties of Polymeric Denture Materials Modified with Zein-Coated Inorganic Nanoparticles.

Background: Polymeric denture materials can be susceptible to colonization by oral microorganisms. Zein-coated magnesium oxide nanoparticles (zMgO NPs) demonstrate antimicrobial activity. The aim of this study was to investigate the antimicrobial effect and adherence of different oral microorganisms on hybrid polymeric denture materials incorporated with zMgO NPs. Methods: Five types of polymeric denture materials were used. A total of 480 disc-shaped specimens were divided by material type (n=96/grp), then subdivided by zMgO NPs concentration: control with no nanoparticles and other groups with zMgO NPs concentrations of 0.3%, 0.5% and 1% by weight. Characterization of the polymeric denture materials incorporating zMgO NPs was done, and the antimicrobial activity of all groups was tested against four types of microorganisms: 1) Streptococcus mutans, 2) Staphylococcus aureus, 3) Enterococcus faecalis and 4) Candida albicans. The samples underwent an adherence test and an agar diffusion test. Experiments were done in triplicates. Results: The characterization of the hybrid samples revealed variation in the molecular composition, as well as a uniform distribution of the zMgO NPs in the polymeric denture materials. All hybrid polymeric denture materials groups induced a statistically significant antimicrobial activity, while the control groups showed the least antimicrobial activity. The agar diffusion test revealed no release of the zMgO NPs from the hybrid samples, indicating the NPs did not seep out of the matrix. Conclusion: The zMgO NPs were effective in reducing the adherence of the tested microorganisms and enhancing the antimicrobial activity of the polymeric denture materials. This antimicrobial effect with the polymeric dentures could aid in resisting microbial issues such as denture stomatitis.

Face-to-face and e-learning during the COVID-19 pandemic: A Pakistani dental undergraduates perspective.

BACKGROUND: Globally, educational institutes have been obliged to make a quick transition from traditional face-to-face teaching to e-learning during the coronavirus disease 2019 (COVID-19) pandemic, which has purportedly created various barriers to achieving desired learning outcomes. OBJECTIVE: The aim of our study was to assess the perceptions of undergraduate dental students regarding online and physical learning in Pakistan. METHODS: This cross-sectional study was conducted from January to March 2022. The study comprised dental students from 1st to 4th years who were above the age of 18 and of either gender. The Google Forms questionnaire was created to evaluate the quality and efficiency of online and physical learning among dentistry undergraduates. RESULTS: In a survey of 246 students, 70.3% strongly agreed that they would prefer face-to-face instruction over e-learning. There was a statistically significant difference in the improvement of students' technical skills (p = 0.01), accessibility to instructors (p = 0.01) and classmates (p = 0.02), value of technology (p = 0.03), improvement of oral communication skills (p = 0.01), promotion of hybrid model learning after the pandemic (p = 0.01), and utilization of tutorial services (p = 0.04) between different year of study. CONCLUSION: Pakistani dental students indicated satisfaction with numerous aspects of e-learning, such as technical skills, accessibility to instructors and classmates, the value of technology, improvement of oral communication skills, promotion of hybrid model learning, and utilization of tutorial services compared to face-to-face education concerning the year of study.

Management of Concomitant Intrusion and Complicated Crown-Root Fracture Injury of Maxillary Central Incisors in a Child.

Dental intrusions are a severe type of injury because they impact the neurovascular supply of the tooth as well as the supporting tissues which predispose the tooth to necrosis and root resorption. Management of these injuries requires repositioning of the teeth under close monitoring to avoid complications. The management becomes more comprehensive when an intrusion is combined with other injuries, such as a crown-root fracture. This case report represents a 4-year follow-up of a child who suffered from a concomitant injury of intrusion and complicated crown-root fracture to the maxillary immature permanent central incisors. The management involved a multidisciplinary approach including endodontics, pedodontics, orthodontics, periodontics, and prosthodontics. Given the guidelines of dental trauma and the circumstances of the case, the fractured teeth were root canal treated, filled with a bioceramic plug and gutta-percha, and then restored with posts/cores and temporary crowns. The intrusion was managed initially by passive eruption followed by an active orthodontic eruption, after which the teeth were restored with permanent ceramic crowns. Throughout the course of treatment, the teeth showed no complications of root resorption or ankylosis, although one tooth developed a periapical infection which was managed by apical surgery. At the 4-year follow-up, the teeth revealed healthy periodontium and good esthetics.

Cytotoxic effects of dose dependent inorganic magnesium oxide nanoparticles on the reproductive organs of rats.

INTRODUCTION: Magnesium oxide nanoparticles (MgO NPs) have a variety of applications that have contributed to their elevated popularity, however, the safety and toxic effects on humans are also of concern with these increased applications. There is insufficient data regarding the effect of MgO NPs on reproductive organs, which are crucial aspects to the body's vital physiological functions. The present study was undertaken in male and female rats to assess the reproductive toxicological potential of two doses (low versus high) of MgO NPs on testicular and ovarian tissues. The toxicity was evaluated using histological, hormonal, and oxidative parameters. MATERIAL AND METHODS: In this work, magnesium oxide nanoparticles (MgO NPs) were synthesized by the sol-gel route and were characterized by X ray diffraction analysis (XRD) and Fourier transform infra-red spectroscopy (FTIR). Forty-eight adult Wister albino rats were used in this experiment which were divided into groups of male and female, and then further into control, low dose MgO NPs, and high dose MgO NPs. The low dose used was 131.5 mg/kg b.w. (1/10 LD50) while the high dose used was 263 mg/kg b.w. (1/5 LD50). All doses were given orally by gastric tube. After 4 weeks, blood samples were collected to investigate the level of sex hormones and both ovarian and testicular tissues were examined for variable oxidative parameters and histopathological changes by light microscopy. RESULTS: The obtained findings showed that high dose of MgO NPs produced considerable changes in sex hormones and stress parameters in both male and female rats in comparison to the low dose and control groups. Histomorphometric analysis demonstrated the presence of histopathological alterations in the testicular and ovarian tissues. CONCLUSION: The results of this study showed dose-dependent adverse effects of MgO NPs on the testis and ovary both functionally and histopathologically as compared to the control rats.

Assessment of Oral Hygiene, Gingival, and Periodontal Health, and Teeth Eruption among Type 1 Diabetic Saudi Children.

Aims and objectives: To determine the differences between type 1 diabetic children and healthy children regarding oral hygiene, gingival and periodontal health, and permanent teeth eruption.Materials and methods: A case-control study was conducted on 80 children (40 type 1 diabetic children and 40 healthy children) aged 6-12-year-old. The groups were further divided into subgroups (early and late mixed dentition). All study aspects were examined clinically using the simplified oral hygiene index, Löe and Silness gingival index, clinical attachment loss (CAL), and Logan and Kronfeld stages for tooth eruption. The data were analyzed using Fisher's exact test, chi-squared test, and logistic regression models. A p-value of ≤0.05 was the threshold for statistical significance. Results: No significant difference was found between diabetic and healthy children regarding oral hygiene and gingival health. Most children had poor oral hygiene (52.5% in the case group and 60% in the control group), with fair gingival health (70% in the case group and 55% in the control group). Diabetic children had significantly (p = 0.05) more periodontitis than healthy children. Teeth in the advanced stage of the eruption were significantly higher in diabetic than control subjects (p = 0.048 in stage V and p = 0.003 in stage VI). Older diabetic children in late mixed dentition exhibited accelerated eruption. Conclusion: Periodontitis was significantly more common in diabetic than in healthy children. The advanced stage of the eruption was significantly higher in diabetic than in control subjects. Clinical significance: Type 1 diabetic children had more periodontal disease and advanced stage of permanent teeth eruption compared to healthy children. Therefore, periodic dental evaluation and a strong preventive plan for diabetic children is crucial. How to cite this article: Mandura RA, El Meligy OA, Attar MH, et al. Assessment of Oral Hygiene, Gingival, and Periodontal Health, and Teeth Eruption among Type 1 Diabetic Saudi Children. Int J Clin Pediatr Dent 2022;15(6):711-716.

Gingival health and oral hygiene practices among high school children in Saudi Arabia.

BACKGROUND: Gingivitis is a site-specific inflammatory condition initiated by dental biofilm accumulation. The accumulation of dental plaque on the gingival margin triggers inflammatory effects that can become chronic. In addition to its local effect, gingival inflammation has recently been suggested to have an impact on general health. OBJECTIVE: Determine the prevalence of gingivitis and its relationship to oral hygiene practices in high school children in Saudi Arabia. DESIGN: Cross-sectional. SETTING: High schools from different regions in Saudi Arabia. PATIENTS AND METHODS: Periodontal examinations were conducted on a randomly selected sample of high school children between the ages of 15 and 19 years. Gingival and plaque indices, probing depth, clinical attachment level, oral hygiene practices and sociodemographic characteristics were recorded. Data were analyzed using descriptive statistics, chi-square and the independent t test. MAIN OUTCOME MEASURE: Prevalence of gingivitis as defined by mean gingival index. SAMPLE SIZE: 2435 high school students. RESULTS: Twenty-one percent of the sample had slight gingivitis, 42.3% had moderate, and 1.8% had severe. Gender, toothbrushing, tongue brushing, plaque index, and the percentage of pocket depth (PD) ≥4 mm showed a significant relationship with the severity of gingivitis. Almost 39.3% of females had a healthy periodontal status when compared to males (30.7%). Thirty-five percent (35.5%) of students who brushed their teeth had a healthy periodontium compared to 26.9% who did not brush. The mean plaque index was significantly higher in students with severe gingivitis when compared to students with healthy periodontium (2.4 vs. 0.79, respectively). CONCLUSION: Gingivitis prevalence was high compared with Western countries in a nationally representative sample of high school students in Saudi Arabia and was influenced by oral hygiene practices. LIMITATIONS: The half-mouth study design may underestimate disease prevalence. Data on oral hygiene practices was self-reported and may thus have been affected by social desirability bias. CONFLICT OF INTEREST: None.

Oral and Dental Health Status among Adolescents with Limited Access to Dental Care Services in Jeddah.

The purpose of this study was to assess the prevalence and associated factors of dental caries and periodontal diseases among 14⁻19-year-old schoolchildren with limited access to dental care services. A cross sectional study design was conducted during field visits to seven governmental schools in Al-Khomrah district, South Jeddah, over the period from September 2015 to May 2016. Clinical examinations and administered questionnaires were carried out in mobile dental clinics. The dentists carried out oral examinations using the dental caries index (DMFT), the simplified oral hygiene index (OHI-S), and the community periodontal index for treatment needs (CPITN). Statistical analyses were performed using SPSS 20. A total of 734 schoolchildren were examined. The prevalence of decayed teeth was 79.7% and was significantly higher among boys (88.9%) than girls (69.0%). About 11% of students had missing teeth, with a significantly higher figure among females than males (15.9% versus 7.3%); 19.8% of students had filled teeth. Moreover, a DMFT of seven or more was significantly more prevalent among males (43.3%) than females (26.8%), while the percentage of females with sound teeth was significantly higher than for males (20.4% and 9.6% respectively). The CPITN revealed 0, 1 and 2 scores among 14.6%, 78.2%, and 41.6% respectively. Males had a significantly higher percentage of healthy periodontal condition (23.8%) than females (3.8%). Dental caries prevalence was moderate to high, calculus and gingival bleeding were widespread among schoolchildren, and were more prevalent among students with low socioeconomic status.

OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

TNFα contributes to diabetes impaired angiogenesis in fracture healing.

Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.

Comparison Between Dexamethasone and Ibuprofen for Postoperative Pain Prevention and Control After Surgical Implant Placement: A Double-Masked, Parallel-Group, Placebo-Controlled Randomized Clinical Trial.

BACKGROUND: Postoperative pain is a potential adverse side effect of oral surgeries, and attempts should be made to prevent or minimize it. This study compares efficacy of preemptive ibuprofen and dexamethasone protocols for pain prevention or control after surgical implant placement. METHODS: This prospective, double-masked, parallel-group, placebo-controlled, randomized clinical trial included 117 patients with planned dental implant placement. Patients were assigned to receive one of three different protocols: 1) 600 mg ibuprofen 1 hour before surgery and another 600 mg 6 hours after the first dose; 2) 4 mg dexamethasone 1 hour before surgery and another 4 mg 6 hours after the first dose; or 3) placebo. Rescue medication (1,000 mg acetaminophen) was made available to each patient, and they were instructed to take it as necessary. Pain intensity was evaluated via a 101-point numeric rating scale and a visual analog scale, and discomfort was evaluated using a four-point verbal rating scale hourly for the first 8 hours after surgery and three times daily for the following 3 days. RESULTS: Ibuprofen and dexamethasone significantly reduced pain (Kruskal-Wallis; P <0.05) up to 3 days after surgery and discomfort (P <0.05) up to 2 days after surgery compared with placebo treatment. Both treatments reduced the number of painkillers taken and increased time before the first painkiller was taken (P <0.01). CONCLUSION: Steroidal dexamethasone is as effective as non-steroidal ibuprofen for preventing or controlling postoperative pain and discomfort after surgical implant placement.

Periodontal findings in systemic lupus erythematosus patients and healthy controls.

OBJECTIVES: To compare periodontal findings in systemic lupus erythematosus (SLE) patients and healthy controls, and to determine, whether there is a correlation between periodontal parameters and SLE biomarkers. METHODS: This cross-sectional study was conducted in the Faculty of Dentistry, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia between November 2012 and February 2014. Twenty-five participants diagnosed with SLE and 50 healthy controls were selected. Periodontal assessment consisted of clinical attachment level (CAL), probing depth (PD), bleeding on probing, and plaque scores. For the SLE group, several laboratory tests were obtained, such as, white blood cell count, hemoglobin level, platelet count, anti-nuclear antibody, anti-double-stranded DNA antibody, calcium level, and vitamin D. RESULTS: Periodontal findings in SLE patients and controls were not significantly different. The SLE patients who had no flare-ups for more than a year showed significant bleeding on probing and deeper PD compared with those who had flare-ups less than a year before starting the study. The SLE patients with arthritis symptoms showed more CAL than those without arthritis. In the SLE patients, no significant correlation was found between their periodontal findings and SLE biomarkers. CONCLUSION: Periodontal health was not different between SLE patients and healthy controls. In SLE patients however, flare-ups and presence of arthritis had a significant relation with periodontal health.

Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism.

AIMS/HYPOTHESIS: Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair. METHODS: Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA. RESULTS: Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes. CONCLUSIONS/INTERPRETATION: Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients.

Illicit drug abuse affects periodontal health status.

OBJECTIVE: To determine periodontal health status among drug addicts in Jeddah, Kingdom of Saudi Arabia. METHODS: Drug addiction recovery patients were recruited from Al-Amal Rehabilitation Hospital, Jeddah, Kingdom of Saudi Arabia between October and December 2012. A questionnaire was used to determine socio-demographic data, oral hygiene measures, and previous drug abuse. Full periodontal charting was carried out including probing depth, recession, attachment loss, bleeding on probing, and plaque index. RESULTS: A total of 57 male patients participated in the study. Cannabis was the drug of choice of most (66.7%) of the subjects, followed by amphetamines (52.6%), alcohol (43.9%), heroin (35.1%), and 8.8% reported using cocaine. All participants had some form of periodontitis with moderate chronic periodontitis affecting 60% of the sample, while mild periodontitis affected 29.1%, and severe periodontitis affected 10.9% of the sample. Cocaine and heroin users showed higher mean clinical attachment loss compared with non-users (p<0.05). Pocket depths of 5-6 mm were found in more than half of the sample. Cocaine users had the highest percentage (80%) of pocket depths that ranged from 5-6 mm. CONCLUSION: Illicit drug use, especially heroin and cocaine, is associated with more severe forms of periodontitis.

The role of osteoimmunology in periodontal disease.

Periodontal disease is a pathological condition that involves inflammation of the tooth supporting structures. It occurs in response to the presence of bacterial plaque on the tooth structure. The host defense system, including innate and adaptive immunity, is responsible for combating the pathologic bacteria invading the periodontal tissue. Failure to eradicate the invading pathogens will result in a continuous state of inflammation where inflammatory cells such as lymphocytes, PMNs, and macrophages will continue to produce inflammatory mediators in an effort to destroy the invaders. Unfortunately, these inflammatory mediators have a deleterious effect on the host tissue as well as foreign microbes. One of the effects of these mediators on the host is the induction of matrix degradation and bone resorption through activation of proteases and other inflammatory mediators that activate osteoclasts.

Chemokine expression is upregulated in chondrocytes in diabetic fracture healing.

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P<0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P<0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect.

Analysis of bone formation after sinus augmentation using ß-tricalcium phosphate.

Implant placement in the edentulous maxilla often represents a clinical challenge due to insufficient bone height after crestal bone resorption and maxillary sinus pneumatization. Several graft materials have been evaluated for augmenting the maxillary sinus to compensate for the lost vertical dimension. Allografts are readily available without the risk of disease transmission and the need for a second site surgery. The aim of this case series was to systematically evaluate the development and maturation of augmented bone in the maxillary sinus using beta-tricalcium phosphate. In 21 to 40 weeks post-sinus elevation, bone biopsies were taken and implants placed simultaneously. All specimens were demineralized and subjected to staining procedures (ie, Hematoxylin and Eosin [H&E], Goldner's staining, and tartrate-resistant acid phosphatase [TRAP]). Total bone increased over time, whereas the amount of graft material diminished. A lack of inflammatory reaction was noticed with the use of this graft material. In addition, TRAP staining revealed the presence of osteoclasts surrounding the remaining particles. During a 12-month follow-up, no implant failure or complications were observed.

TNF-alpha mediates diabetes-enhanced chondrocyte apoptosis during fracture healing and stimulates chondrocyte apoptosis through FOXO1.

To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor alpha (TNF-alpha) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-alpha protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-alpha, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation.

High levels of tumor necrosis factor-alpha contribute to accelerated loss of cartilage in diabetic fracture healing.

Diabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. Microarray and gene set enrichment analysis revealed an up-regulation of gene sets related to inflammation, including tumor necrosis factor (TNF) signaling in the diabetic group, when cartilage is being replaced by bone on day 16, but not on days 12 or 22. This change coincided with elevated osteoclast numbers and accelerated removal of cartilage in the diabetic group (P < 0.05), which was reflected by smaller callus size. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss, and number of TNF-alpha and receptor activator for nuclear factor kB ligand positive chondrocytes were significantly reduced (P < 0.05). The transcription factor forkhead box 01 (FOXO1) was tested for mediating TNF stimulation of osteoclastogenic and inflammatory factors in bone morphogenetic protein 2 pretreated ATDC5 and C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-alpha, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1alpha, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF-alpha promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes in vivo and increased FOXO1 DNA binding activity in diabetic fracture calluses (P < 0.05). These results suggest that diabetes-enhanced TNF-alpha increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF-alpha dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage.

Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment.

Fracture healing in diabetic individuals and in animal models of diabetes is impaired. To investigate mechanisms by which diabetes may affect fracture healing we focused on the transition from cartilage to bone, a midpoint in the fracture healing process. Femoral fractures were induced in mice rendered diabetic by multiple low dose streptozotocin treatment and compared to matching normoglycemic mice. One group of diabetic animals was treated with slow release insulin to maintain normal serum glucose levels. The results indicate that there was relatively little difference in the initial formation of the fracture callus on day 10. However, on day 16 the diabetic group had significantly smaller callus, greater loss of cartilage and enhanced osteoclastogenesis that was normalized by treatment with insulin when assessed by histomorphometric analysis. Chondrocyte apoptosis was significantly higher in diabetic mice and this increase was blocked by insulin. These changes were accompanied by diabetes-increased mRNA levels of RANKL, TNF-alpha, and ADAMTS-4 and -5 measured by real-time PCR, which was reversed by insulin treatment. On days 16 and 22 bone formation within the callus of diabetic mice was significantly less than the normoglycemic and brought to normal levels by insulin treatment. These results suggest that a significant effect of diabetes on fracture healing is increased chondrocyte apoptosis and osteoclastogenesis that accelerates the loss of cartilage and reduces the anlage for endochondral bone formation during fracture repair. That insulin reverses these effects demonstrates that they are directly related to the diabetic condition.

Diabetic complications and dysregulated innate immunity.

Diabetes mellitus is a metabolic disorder that leads to the development of a number of complications. The etiology of each diabetic complication is undoubtedly multifactorial. We will focus on one potential component that may be common in many diabetic complications, dysregulation of innate immunity associated with an increased inflammatory response. High glucose levels lead to shunting through the polyol pathway, an increase in diacylglycerol which activates protein kinase C, an increase in the release of electrons that react with oxygen molecules to form superoxides, and the non-enzymatic glycosylation of proteins that result in greater formation of advanced glycation end products. Each of these can lead to aberrant cell signalling that affects innate immunity for example, by activating the MAP kinase pathway or inducing activation of transcription factors such as NF-kappaB. This may be a common feature of several complications including periodontal disease, atherosclerosis, nephropathy, impaired healing and retinopathy. These complications are frequently associated with increased expression of inflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 and enhanced generation of reactive oxygen species. Cause and effect relationship between dysregulation of key components of innate immunity and diabetic complications in many instances have been demonstrated with the use of cytokine blockers and antioxidants.