Salivary Biomarkers and Their Application in the Diagnosis and Monitoring of the Most Common Oral Pathologies.

Saliva is a highly versatile biological fluid that is easy to gather in a non-invasive manner-and the results of its analysis complement clinical and histopathological findings in the diagnosis of multiple diseases. The objective of this review was to offer an update on the contribution of salivary biomarkers to the diagnosis and prognosis of diseases of the oral cavity, including oral lichen planus, periodontitis, Sjögren's syndrome, oral leukoplakia, peri-implantitis, and medication-related osteonecrosis of the jaw. Salivary biomarkers such as interleukins, growth factors, enzymes, and other biomolecules have proven useful in the diagnosis and follow-up of these diseases, facilitating the early evaluation of malignization risk and the monitoring of disease progression and response to treatment. However, further studies are required to identify new biomarkers and verify their reported role in the diagnosis and/or prognosis of oral diseases.

Mitochondrial stress and redox failure in steroid-associated osteonecrosis.

The purpose of the role of antioxidant enzymes and mitochondria in the developmental mechanism of steroid-associated osteonecrosis in the femur. In the present study Japanese white rabbits (mean weight 3.5kg) were injected into the gluteus with methylprednisolone (MP) 20mg/kg, and killed after 3 days (MP3 group), 5 days (MP5 group), and 14 days (MP14 group) (n=3 each). As a Control group (C group) Japanese white rabbits not administered MP were used. In experiment 1, the expression of the antioxidant enzymes Superoxide dismutade (SOD) and catalase was compared in liver, kidney, heart, humerus, and femur in C group, and the presence/absence of mitochondria transcription factor A (TFAM) expression was determined by Western blotting (WB) and used to evaluate the number of mitochondria and their function. In experiment 2, the presence/absence of necrosis was determined by immunohistochemistry, while changes in the expression of SOD, catalase, and TFAM in the femur after steroid administration were determined by Western blotting (WB). In experiment 1, intense expression of all of SOD, catalase, and TFAM was found in the liver, kidney, and heart as compared to the humerus and femur. In experiment 2, the expression of all of SOD, catalase, and TFAM in MP3 and MP5 groups was decreased on WB as compared with C group, while in MP14 group a tendency to improvement was seen. Accordingly, steroid-associated mitochondrial injury and redox failure are concluded to be important elements implicated in the pathogenesis of osteonecrosis.

Asparaginase Potentiates Glucocorticoid-Induced Osteonecrosis in a Mouse Model.

Osteonecrosis is a common dose-limiting toxicity of glucocorticoids. Data from clinical trials suggest that other medications can increase the risk of glucocorticoid-induced osteonecrosis. Here we utilized a mouse model to study the effect of asparaginase treatment on dexamethasone-induced osteonecrosis. Mice receiving asparaginase along with dexamethasone had a higher rate of osteonecrosis than those receiving only dexamethasone after 6 weeks of treatment (44% vs. 10%, P = 0.006). Similarly, epiphyseal arteriopathy, which we have shown to be an initiating event for osteonecrosis, was observed in 58% of mice receiving asparaginase and dexamethasone compared to 17% of mice receiving dexamethasone only (P = 0.007). As in the clinic, greater exposure to asparaginase was associated with greater plasma exposure to dexamethasone (P = 0.0001). This model also recapitulated other clinical risk factors for osteonecrosis, including age at start of treatment, and association with the systemic exposure to dexamethasone (P = 0.027) and asparaginase (P = 0.036). We conclude that asparaginase can potentiate the osteonecrotic effect of glucocorticoids.

Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits.

Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid-associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c-src] gene). This study was designed to prove our hypothesis that blocking the VEGF-Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti-VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast-enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro-computed tomography (µCT)-based trabecular architecture with finite element analysis (FEA), µCT-based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti-VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF-mediated vascular hyperpermeability but preserved VEGF-induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti-VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D µCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF-Src signaling pathway by specific Src siRNA was able to prevent steroid-associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy.

iNOS expression and osteocyte apoptosis in idiopathic, non-traumatic osteonecrosis.

BACKGROUND AND PURPOSE: Non-traumatic osteonecrosis is a progressive disease with multiple etiologies. It affects younger individuals more and more, often leading to total hip arthroplasty. We investigated whether there is a correlation between inducible nitric oxide synthase (iNOS) expression and osteocyte apoptosis in non-traumatic osteonecrosis. PATIENTS AND METHODS: We collected and studied 20 human idiopathic, non-traumatic osteonecrosis femoral heads. Subchondral bone samples in the non-sclerotic region (n = 30), collected from osteoarthritis patients, were used as controls. Spontaneously hypertensive rats were used as a model for osteonecrosis in the study. We used scanning electron microscopy, TUNEL assay, and immunohistochemical staining to study osteocyte changes and apoptosis. RESULTS: The morphology of osteocytes in the areas close to the necrotic region changed and the number of apoptotic osteocytes increased in comparison with the same region in control groups. The expression of iNOS and cytochrome C in osteocytes increased while Bax expression was not detectable in osteonecrosis samples. Using spontaneously hypertensive rats, we found a positive correlation between iNOS expression and osteocyte apoptosis in the osteonecrotic region. iNOS inhibitor (aminoguanidine) added to the drinking water for 5 weeks reduced the production of iNOS and osteonecrosis compared to a control group without aminoguanidine. INTERPRETATION: Our findings show that increased iNOS expression can lead to osteocyte apopotosis in idiopathic, non-traumatic osteonecrosis and that an iNOS inhibitor may prevent the progression of the disease.

Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: new implications for osteonecrosis treatment?

Elevated hydrogen peroxide (H2O2) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H2O2 stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H2O2-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H2O2-induced cell damage. These results confirmed that H2O2-activated AMPK is pro-cell survival. We observed that H2O2 induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H2O2-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H2O2 in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H2O2-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H2O2 in these cells. Based on these results, we concluded that H2O2-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.

Histologic findings of femoral heads from patients with Gaucher disease treated with enzyme replacement.

OBJECTIVES: To assess correlations of patient demographics, including enzyme replacement therapy (ERT) with bone histology, to facilitate decisions of whether and when to perform hip replacement surgery in patients with Gaucher disease. METHODS: We examined the histology of surgically removed femoral heads and categorized findings by the presence or extent of osteonecrosis, Gaucher cell (GC) infiltration, and bone regeneration qualifiers using a tripartite histology-based scoring system. RESULTS: Twenty-two patients with 26 bone specimens were evaluated. Seventeen patients (77%) were splenectomized, 16 (73%) received ERT, and 12 (55%) had the putatively milder genotype (N370S/N370S), with the rest putatively at increased risk for skeletal disease (N370S/other). The 3 histology subscores were applicable to all specimens. Osteonecrotic bone was seen in 19 of 26 (73%); osteoarthritis was seen in all cartilage specimens. Gaucher cell infiltration was not correlated with demographics or disease severity. A trend was noted between reduced GC infiltration and ERT (ρ = 0.407), but regeneration qualifiers were not correlated with ERT or other features. CONCLUSIONS: Histologic findings of GC infiltration and bone regeneration qualifiers did not correlate with demographics or with exposure to ERT. Most specimens unexpectedly showed good regenerative responses to osteonecrosis despite heavy GC infiltration.

An aromatase polymorphism (g.132810C>T) predicts risk of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: Bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) is an unpredictable, debilitating adverse effect. Recently, genetic polymorphisms have arisen as promising tools to identify patients with a higher risk of drug-related adverse events. AIM: We aimed to examine the association between the aromatase polymorphism g.132810C>T, and the estrogen receptor polymorphisms g.156705T>C and g.156751A>G, and the risk of BP-related ONJ. METHODS: Eighty-three subjects were included in the study. A clinical and radiological examination was conducted on oncologic subjects treated with zoledronic acid. Subjects with histologically confirmed ONJ were included in the test group (n = 30) whereas subjects with good oral health were included in control group (n = 53). Aromatase and estrogen receptor polymorphisms from blood samples were analyzed. RESULTS: The aromatase g.132810C>T polymorphism displayed an over-representation of the TT genotype in the test group (36.67 vs 16.98%; p < 0.05). There was no significant difference in either estrogen receptor polymorphism genotype frequency between the test and control groups. CONCLUSION: Our data suggest a role for the g.132810C>T polymorphism in predicting ONJ risk. These results can pave the way to the personalization of BP therapy, based on individual genotype.

Osseous manifestations of adult Gaucher disease in the era of enzyme replacement therapy.

Enzyme replacement therapy (ERT) for Gaucher disease with mannose-terminated glucocerebrosidase has proved its therapeutic position with salutary effects on hematologic abnormalities, visceral infiltration, and quality of life. The frequency of new bone complications is reduced but not eliminated. Established osteonecrosis is beyond salvage. A systematic description of the burden of bone manifestations, persisting despite ERT, should inform future remedial strategies. Thus, we conducted this study to quantify the burden of residual skeletal disease and to explore putative relationships between clinical, radiologic, and biochemical factors and bone sequelae associated with disability.Consecutive adult patients attending 3 referral centers in the United Kingdom were invited to participate. A representative group of 100 patients agreed to a structured interview, clinical examination, radiologic review, and completion of questionnaires. Osteonecrosis was evident in 43%, Erlenmeyer flask deformity in 59%, fragility fracture in 28%, osteomyelitis in 6%, and lytic lesions in 4%. Mobility was impaired in 32% of patients, while 15% experienced significant pain. The EuroQol 5D (EQ5D) quality of life summary measure was reduced and was associated with osteonecrosis and fragility fracture. Eight patients experienced new osteonecrosis after the start of ERT, though the presentation and evolution were often atypical. Nine patients had been treated from childhood and had an excellent outcome. Osteonecrosis was associated with age of presentation and with splenectomy-indeed, we observed a strong temporal association between splenectomy and incidence of osteonecrosis.The biomarkers PARC/CCL18 and chitotriosidase were associated with prevalent osteonecrosis, and, in particular, with osteonecrosis occurring despite treatment. This study documents significant residual skeletal pathology and disability in patients in the mature phase of their treatment in a developed region. The temporal association between splenectomy and osteonecrosis implies causation. The relationship between clinical and biochemical markers and existing bone complications sets the scene for future prospective studies that will focus on management strategies informed by credible assessment of risk.

Effect of simvastatin on steroid-induced osteonecrosis evidenced by the serum lipid level and hepatic cytochrome P4503A in a rabbit model.

OBJECTIVE: This study was designed to investigate the efficacy of lipid-lowering agents in preventing steroid-induced osteonecrosis and the mechanism by which they do so in a rabbit model. METHODS: Female Japanese white rabbits were randomly allocated to receive probucol (group P), pravastatin (group PS), simvastatin (group SS), or saline (group C) for 6 weeks (n = 15 in groups P, PS, and SS; n = 30 in group C). Methylprednisolone (20 mg/kg) was injected at 3 weeks after starting treatment, and the femurs were histologically examined bilaterally 3 weeks after methylprednisolone injection. Midazolam clearance was measured before treatment and before methylprednisolone injection to determine hepatic cytochrome P4503A (CYP3A) levels. RESULTS: The incidence of osteonecrosis in the proximal metaphysis of the femurs in groups PS and SS was significantly lower than in group C (P < 0.05 and P < 0.0001, respectively), whereas it did not differ between groups P and C. It was significantly lower in group SS than in group PS (P < 0.05). Plasma concentrations of lipids (low-density lipoprotein, triglyceride, free fatty acid, and total cholesterol) in groups P, PS, and SS were significantly lower than in group C; and hepatic CYP3A levels were significantly higher in group SS than in groups P or PS after treatment (P < 0.005 for both). CONCLUSIONS: Simvastatin and pravastatin significantly reduced the incidence of steroid-induced osteonecrosis in rabbits. Simvastatin was more effective in reducing the incidence of the disease, and increased CYP3A activity is a possible mechanism for this effect.

Bisphosphonate-related osteonecrosis of the jaw is associated with polymorphisms of the cytochrome P450 CYP2C8 in multiple myeloma: a genome-wide single nucleotide polymorphism analysis.

We have explored the potential role of genetics in the development of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients under bisphosphonate therapy. A genome-wide association study was performed using 500 568 single nucleotide polymorphisms (SNPs) in 2 series of homogeneously treated MM patients, one with ONJ (22 MM cases) and another without ONJ (65 matched MM controls). Four SNPs (rs1934951, rs1934980, rs1341162, and rs17110453) mapped within the cytochrome P450-2C gene (CYP2C8) showed a different distribution between cases and controls with statistically significant differences (P = 1.07 x 10(-6), P = 4.231 x 10(-6), P = 6.22 x 10(-6), and P = 2.15 x 10(-6), respectively). SNP rs1934951 was significantly associated with a higher risk of ONJ development even after Bonferroni correction (P corrected value = .02). Genotyping results displayed an overrepresentation of the T allele in cases compared with controls (48% vs 12%). Thus, individuals homozygous for the T allele had an increased likelihood of developing ONJ (odds ratio 12.75, 95% confidence interval 3.7-43.5).

Low hepatic cytochrome P450 3A activity is a risk for corticosteroid-induced osteonecrosis.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is one of the major side effects of corticosteroid therapy. Because corticosteroids are metabolized by hepatic cytochrome P450 (CYP) 3A, a low endogenous activity of this enzyme may contribute to the pathogenesis of ONFH. The purpose of this study was to examine the possible association of hepatic CYP3A activity and the susceptibility to ONFH in patients treated with corticosteroids. METHODS: In this prospective controlled study we measured the clearance of intravenous midazolam (0.25 mg/kg) to estimate hepatic CYP3A activity in patients with steroid-induced ONFH (n = 26), patients with alcohol-related ONFH (n = 29), and non-ONFH control patients (n = 75) undergoing orthopedic surgery. Midazolam clearance was compared between the groups, and the relationship between the level of hepatic CYP3A activity and the prevalence of ONFH was evaluated by multivariate analysis. RESULTS: Midazolam clearance in patients with steroid-induced ONFH was significantly lower than that in control patients and patients with alcohol-related ONFH (7.7 +/- 1.8 mL x kg(-1) x min(-1) versus 11.4 +/- 3.5 mL x kg(-1) x min(-1) and 10.5 +/- 2.8 mL x kg(-1) x min(-1), respectively; P < .001). Patients with low midazolam clearance (<9.5 mL x kg(-1) x min(-1)) had a 9-fold greater risk for steroid-induced ONFH (adjusted odds ratio, 9.08 [95% confidence interval, 2.79-29.6]; P < .001). Midazolam clearance did not show a significant correlation with the prevalence of alcohol-related ONFH. CONCLUSIONS: Low hepatic CYP3A activity may significantly contribute to the risk for steroid-induced ONFH.

Increased numbers of osteoclasts expressing cysteine proteinase cathepsin K in patients with infected osteoradionecrosis and bisphosphonate-associated osteonecrosis--a paradoxical observation?

Erosion of the bone is a common problem in patients suffering from infected osteoradionecrosis (IORN) or bisphosphonate-associated osteonecrosis (BON). Besides inflammatory mechanisms and infectious agents, osteoclasts were recently suggested to be specifically involved in the osteolytic mechanisms. We therefore investigated the immunohistochemical labeling of the cysteine proteinase cathepsin K, which plays a central role in osteoclast-mediated bone resorption, in tissue specimens of patients with BON (n=9; four male, five female; mean age=69.2 years) and IORN (n=10; nine male, one female; mean age=60.5 years), and compared the results with control specimens (n=8; six male, two female; mean age=60 years). In all cases, osteoclasts were the predominant cell type expressing cathepsin K. For semiquantitative analysis, we therefore defined osteoclasts as multinuclear giant cells attached to the bone and expressing cathepsin K. Significantly higher numbers of osteoclasts were found for both types of osteonecrosis when compared with the control group (BON+IORN vs controls: P=0.0000036). Within the pathological lesions, IORN cases exhibited significantly less osteoclasts than BON (P=0.00097). Our study verified increased numbers of osteoclasts in patients suffering from BON and IORN. Although it is known that bisphosphonates (and to a lesser extent, irradiation, too) decrease osteoclast function, these findings suggest a critical involvement of osteoclasts in the mechanisms of bone destruction in the respective lesions.

Investigation of alcohol metabolizing enzyme genes in Chinese alcoholics with avascular necrosis of hip joint, pancreatitis and cirrhosis of the liver.

AIMS AND METHODS: Alcoholism may cause a range of diseases including avascular necrosis of the hip joint (AVN), cirrhosis of the liver, pancreatitis and oesophageal carcinoma. Chinese alcoholic patients diagnosed with AVN have a higher incidence of cirrhosis than of acute pancreatitis or oesophageal cancer. Thus, the aim of this study was to investigate genetic differences in polymorphisms of the alcohol-metabolizing enzymes ADH2, ADH3, ALDH2 and P4502E1 for subgroups of Chinese alcoholic patients, defined by diagnoses of AVN (n = 51), acute pancreatitis (n = 92) and liver cirrhosis (n = 159), and for 280 non-alcoholic patients. RESULTS: Analysis revealed that ADH2*1 allele frequency was significantly lower for the alcoholic AVN than for the cirrhosis subgroup. However, no significant difference was found between the alcoholic AVN and pancreatitis subgroups. Furthermore, ALDH2*2 prevalence was not found to differ significantly between the alcoholic subgroups. When compared with our previously published data for alcoholic patients with oesophageal carcinoma, ADH2*1 carriage was significantly less frequent for the alcoholic AVN patients in the current study. Further, ALDH2*2 carriage was significantly less frequent for the alcoholic AVN subgroup than for the oesophageal carcinoma patients. CONCLUSIONS: The allele frequencies for ADH2*1 and ALDH2*2 are different when comparing subpopulations of alcoholics defined by presence of specific alcohol-induced diseases, suggesting that genetic variation in alcohol-metabolizing enzyme genes accounts for, at least in part, the specific types of organ damage observed. We also found the combination of AVN and cirrhosis to be more prevalent than that of AVN and acute pancreatitis. In contrast, the ADH2 and ALDH2 allele frequencies for the AVN subgroup were more similar to those of the acute-pancreatitis than to the cirrhosis subgroup. These data indicate the possibility that other genetic variations may also influence the type of organ-specific complications in Chinese alcoholics.

The methylene tetrahydrofolate reductase (C677T) mutation as a potential risk factor for avascular necrosis in sickle cell disease.

Avascular necrosis (AVN) of the humeral and femoral heads is a frequent and debilitating complication of sickle cell disease. Some of the risk factors for AVN are alpha-thalassemia and age. Recently, newly discovered thrombophilia mutations have been associated with AVN in patients without sickle cell disease. We studied the frequency of the thermolabile methylene tetrahydrofolate reductase (MTHFR) variant (C677T) in adult sickle cell patients with and without AVN. The frequency of the MTHFR mutation was 35.6% in patients with AVN and 12.9% in those without AVN (p = 0.006). These data suggest that the thermolabile MTHFR variant may be a contributing risk factor for AVN in some populations with sickle cell disease.