Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model.

Dental caries is caused by acids released from bacterial biofilms. However, the in vivo formation of initial biofilms in relation to caries remains largely unexplored. The aim of this study was to compare the oral microbiome during the initial phase of bacterial colonization for individuals with (CC) and without (NC) cavitated dentin caries lesions. Bovine enamel slabs on acrylic splints were worn by the volunteers (CC: 14, NC: 13) for in situ biofilm formation (2 h, 4 h, 8 h, 1 ml saliva as reference). Sequencing of the V1/V2 regions of the 16S rRNA gene was performed (MiSeq). The relative abundances of individual operational taxonomic units (OTUs) were compared between samples from the CC group and the NC group. Random forests models were furthermore trained to separate the groups. While the overall heterogeneity did not differ substantially between CC and NC individuals, several individual OTUs were found to have significantly different relative abundances. For the 8 h samples, most of the significant OTUs showed higher relative abundances in the CC group, while the majority of significant OTUs in the saliva samples were more abundant in the NC group. Furthermore, using OTU signatures enabled a separation between both groups, with area-under-the-curve (AUC) values of ~0.8. In summary, the results suggest that initial oral biofilms provide the potential to differentiate between CC and NC individuals.

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva.

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S. mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy-based single-cell force spectroscopy measurements of S. mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S. mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S. mutans cells compared to S. carnosus cells. Our results demonstrate that S. mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm-forming capability of that species and hence the production of caries-provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries-reducing techniques.

Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients.

AIM: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. METHODS: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4',6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. RESULTS: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. CONCLUSION: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Albumin-lysozyme interactions: Cooperative adsorption on titanium and enzymatic activity.

The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase.

Enzymes in the in-situ pellicle of children with different caries activity.

The present study investigated, for the first time, enzymes in the in-situ pellicle of children. Furthermore, it was purposed to detect glucosyltransferase (GTF) isoforms in each child's pellicle. Twenty-four children (5-9 yr of age) participated in the study. Twelve were caries free with no decayed, missing, and filled teeth (dmft), whereas 12 had active caries (dmft ≥ 2, indicating at least two untreated carious lesions). Bovine enamel slabs, fixed on individual upper jaw splints, were utilized for pellicle formation in situ. After 3 and 30 min, samples were tested for amylase, lysozyme, and peroxidase activities; total GTF activity was examined only in the 30-min pellicle. Gold-immunolabelling was used to quantify the GTF B, C, and D isoforms in the pellicle by transmission electron microscopy (TEM). All enzymes tested were detected in the children's in-situ pellicle in an active conformation, and there were no significant differences in their levels of activity between caries-free and caries-active children. All GTF isoforms were found to be randomly distributed within all pellicle layers, althoug GTF B was only detected very sporadically. A significantly higher amount of GTF D was detected in the pellicle of caries-active children. Pellicle formation in children is characterized by uniformity and selectivity. Glucosyltransferase D might represent a possible biomarker for high caries risk in children.

Cleaning of biomaterial surfaces: protein removal by different solvents.

The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM.

Folate is related to phosphorylated neurofilament-H and P-tau (Ser396) in rat brain.

Protein phosphatase PP2A dephosphorylates phosphorylated tau (P-tau) and neurofilaments (pNFs). PP2A is S-adenosylmethionine (SAM)-dependent and might thus link methylation with neurodegeneration. Low SAM and increased S-adenosylhomocysteine (SAH) can enhance the risk of dementia. We studied the effect of hyperhomocysteinemia on P-tau (Ser396), pNF-H (heavy chain), and PP2A-activity and level (the C subunit) in rat brain. Wistar rats (total n=55) were fed either on a standard, a homocystine 1.7% or a methionine 2.4%-rich diet for 5 months. P-tau was tested in 21 frontal cortex tissue slices using immuno-fluorescence. Concentrations of pNF-H and the activity and level of PP2A were measured in brain extracts. Concentrations of homocysteine, SAM and SAH strongly increased in plasma of rats on the modified diets. The diets caused lowering of plasma folate and vitamin B12 and a significant increase in P-tau (Ser396) in brain tissues but PP2A activity and level were unchanged. Plasma folate correlated to brain tissue PP2A activity (r=0.28), pNF-H (r=-0.30), and P-tau (Ser396) staining (r=-0.57) all p<0.05. Phosphorylation of brain functional proteins was related to folate. The effect of the diet on P-tau and pNF-H seemed not to be explained by a lower activity or protein level of PP2A. Folate might prove protective against multiple steps in the process of neurodegeneration.

Protein adhesion on dental surfaces-a combined surface analytical approach.

Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.

The cardiac effects of prolonged vitamin B12 and folate deficiency in rats.

In the recent past, hyperhomocysteinemia (HHCY) has been linked to chronic heart failure. Folate and vitamin B12 deficiencies are the common causes of HHCY. The impact of these vitamins on cardiac function and morphology has scarcely been investigated. The aim of this study was to conduct an analysis of the cardiac effect of folate and vitamin B12 deficiency in vivo. Two groups of rats, a control (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10), were fed for 12 weeks with a folate and vitamin B12-free diet or an equicaloric control diet. Plasma and tissue concentrations of HCY, S-adenosyl-homocysteine (SAH), S-adenosyl-methionine (SAM), and brain natriuretic peptide (BNP) were measured. Moreover, echocardiographic and histomorphometric analyses were performed. VitDef animals developed a significant HHCY (Co vs VitDef: 6.8 +/- 2.7 vs 61.1 +/- 12.8 micromol/l, P < 0.001). Fractional shortening, left ventricular dimension at end-diastole and end-systole, posterior wall thickness, perivascular collagen, mast cell number, and BNP tissue levels were comparable in VitDef and Co animals. Interstitial collagen (Co vs VitDef: 6.8 +/- 3.0 vs 4.5 +/- 2.1%, P < 0.05), plasma BNP (Co vs VitDef: 180 +/- 80 vs 70 +/- 60 ng/l, P < 0.05), and tissue HCY (Co vs VitDef: 0.13 +/- 0.07 vs 0.07 +/- 0.04 micromol/g protein, P < 0.05) were lower in VitDef animals. Folate and vitamin B12 deficiency do not affect cardiac function and morphology.

Experimental folate and vitamin B12 deficiency does not alter bone quality in rats.

Hyperhomocysteinemia (HHCY) has been linked to fragility fractures and osteoporosis. Folate and vitamin B(12) deficiencies are among the main causes of HHCY. However, the impact of these vitamins on bone health has been poorly studied. This study analyzed the effect of folate and vitamin B(12) deficiency on bone in rats. We used two groups of rats: a control group (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10). VitDef animals were fed for 12 wk with a folate- and vitamin B(12)-free diet. Co animals received an equicaloric control diet. Tissue and plasma concentrations of homocysteine (HCY), S-adenosyl-homocysteine (SAH), and S-adenosyl-methionine (SAM) were measured. Bone quality was assessed by biomechanical testing (maximum force of an axial compression test; F(max)), histomorphometry (bone area/total area; B.Ar./T.Ar.], and the measurement of biochemical bone turnover markers (osteocalcin, collagen I C-terminal cross-laps [CTX]). VitDef animals developed significant HHCY (Co versus VitDef: 6.8 +/- 2.7 versus 61.1 +/- 12.8 microM, p < 0.001) that was accompanied by a high plasma concentration of SAH (Co versus VitDef: 24.1 +/- 5.9 versus 86.4 +/- 44.3 nM, p < 0.001). However, bone tissue concentrations of HCY, SAH, and SAM were similar in the two groups. Fmax, B.Ar./T.Ar., OC, and CTX did not differ between VitDef and Co animals, indicating that bone quality was not affected. Folate and vitamin B(12) deficiency induces distinct HHCY but has no effect on bone health in otherwise healthy adult rats. The unchanged HCY metabolism in bone is the most probable explanation for the missing effect of the vitamin-free diet on bone.

Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone.

BACKGROUND: Recently, hyperhomocysteinemia (HHCY) has been suggested to have adverse effects on bone. This study investigated if an experimental HHCY in rats induces an accumulation of homocysteine (HCY) in bone tissue that is accompanied by bone loss and reduced bone strength. MATERIAL AND METHODS: HHCY was induced in healthy rats by either a methionine (Meth)- or a homocystine (Homo)-enriched diet and compared with controls. Homocystine is the product of two disulfide linked HCY molecules. Tissue and plasma concentrations of HCY, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were measured. Bones were assessed by biomechanical testing, histomorphometry, microCT and the measurement of biochemical bone turnover markers in plasma. RESULTS: Meth and Homo animals developed a significant HHCY that was accompanied by a tissue specific accumulation of HCY (1300 to 2000% vs. controls). 65% of HCY in bone was bound to collagen of the extracellular matrix. The SAH / SAM-ratio in bone and plasma of Meth and Homo animals exhibited a tissue specific increase indicating a reduced methylation capacity. Accumulation of HCY in bone was characterized by a distinct reduction of cancellous bone (proximal femur: -25 to -35%; distal femur -56 to -58%, proximal tibia: -28 to -43%). Accordingly, bone strength was significantly reduced (-9 to -12%). CONCLUSION: A tissue specific accumulation of HCY in bone may be a promising mechanism explaining adverse effects of HHCY on bone. A reduced methylation capacity of bone cells might be another relevant pathomechanism.

Stimulation of osteoblast activity by homocysteine.

Homocysteine (HCY) has recently been linked to fragility fractures. Moreover, HCY activates osteoclasts. Little is known about the effect of HCY on activity of human osteoblasts (OBs). We hypothesized that HCY decreases the activity of OBs. Osteoblasts obtained from trabecular human bone specimens of eight donors were cultured with conditioned medium. Culture medium was adjusted to 0, 100, 500, 1000 and 2000 microM HCY. After 14 days alkaline phosphatase (AP) activity, pro-collagen type I N-terminal peptide (PINP) and osteocalcin (OC) secretion in the supernatant were measured. After 20 days the formation of mineralized matrix was analyzed. HCY-stimulated AP activity gradually (100 microM HCY: 118%, P=0.006; 500 microM HCY: 125%, P<0.001). At 1000 and 2000 microM HCY the increase of AP activity was reversible (1000 microM HCY: 106%, P=0.317; 2000 microM HCY: 102%, P<0.737). The PINP secretion was also stimulated by HCY reaching a maximum of 260+/-154 microg/l at 500 micromol/l versus 205+/-94 microg/l in controls. After 20 days of culture the formation of bone matrix was increased at 100 and 500 microM HCY. OC secretion was not significantly changed. The results of the present study consistently demonstrate a moderate stimulation of primary human OB activity by increasing concentrations of HCY. However, the magnitude of this effect seems to be less pronounced than recent observations on primary human osteoclasts, suggesting a dysbalance between OBs and osteoclasts in favour of osteoclasts.

The role of hyperhomocysteinemia as well as folate, vitamin B(6) and B(12) deficiencies in osteoporosis: a systematic review.

Hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. Recent epidemiological, clinical and experimental studies provide a growing body of data, which is reviewed in this article. Epidemiological and (randomized) clinical trials suggest that HHCY increases fracture risk, but has minor effects on bone mineral density. Measurement of biochemical bone turnover markers indicates a shift of bone metabolism towards bone resorption. Animal studies confirm these observations showing a reduced bone quality and stimulation of bone resorption in hyperhomocysteinemic animals. Homocysteine (HCY) has been found to accumulate in bone by collagen binding. Cell culture studies demonstrate that high HCY levels stimulate osteoclasts but not osteoblasts, indicating again a shift of bone metabolism towards bone resorption. Regarding B-vitamins, only a few in vivo studies with equivocal results have been published. However, two large cell culture studies confirm the results obtained with exogenous HCY administration. In addition, HHCY seems to have adverse affects on extracellular bone matrix by disturbing collagen crosslinking. In conclusion, existing data suggest that HHCY (and possibly B-vitamin deficiencies) adversely affects bone quality by a stimulation of bone resorption and disturbance of collagen crosslinking.

The effect of B-vitamins on biochemical bone turnover markers and bone mineral density in osteoporotic patients: a 1-year double blind placebo controlled trial.

BACKGROUND: Hyperhomocysteinemia is a new risk factor for osteoporosis. This study analyzed the effect of a homocysteine (HCY)-lowering treatment in osteoporotic individuals. METHODS: Osteoporotic subjects (n=47, 55-82 years) were treated with either a combination of 2.5 mg folate, 0.5 mg vitamin B(12) and 25 mg vitamin B(6) or placebo. Bone mineral density (BMD) at lumbar spine and hip was measured at baseline and after 1 year. Urinary desoxypyridinoline cross-links (DPD) and plasma levels of tartrate resistant acid phosphatase (TRAP), C-terminal cross-links of collagen I (CTx), pro-collagen type I N-terminal peptide (PINP) and osteocalcin (OC) were measured after 0, 4, 8 and 12 months. RESULTS: B-vitamin supplementation significantly reduced HCY (0 vs. 12 months: 13.6+/-4.8 vs. 8.9+/-2.4 micromol/L). Placebo treatment had no effect on HCY (0 vs. 12 months: 12.0+/-3.4 vs. 12.7+/-3.9 micromol/L). BMD, TRAP, CTx, OC and PINP did not change throughout the study in both groups. Vitamin treatment decreased urinary DPD by -13% (p<0.01) after 8 and 12 months. In a sub-group analysis of hyperhomocysteinemic subjects (HCY>15 mumol/L, n=8), B-vitamin treatment tended to increase BMD at the lumbar spine, with a t-score from -2.7 to -1.7, and to decrease OC and PINP by approximately 50%. CONCLUSIONS: B-vitamin supplementation had no consistent effects on bone turnover or BMD. However, the situation may be different in patients with hyperhomocysteinemia.

Accumulation of homocysteine by decreasing concentrations of folate, vitamin B12 and B6 does not influence the activity of human osteoblasts in vitro.

BACKGROUND: Homocysteine (HCY) has recently been linked to fragility fractures. Elevated circulating HCY is mainly caused by folate, vitamin B12 and B6 deficiencies. However, little is known about the effect of these vitamins on the activity of osteoblasts. We hypothesized that decreasing concentrations of folate, vitamin B12 and B6 decrease osteoblasts activity by accumulation of HCY. METHODS: Osteoblasts obtained from trabecular human bone specimens of 8 donors were cultured with decreasing concentrations of folate, vitamin B12 and B6. Vitamin concentrations were modified in combination or one vitamin only (8 repetitions x 8 donors, n=64). After 14 days alkaline phosphatase (AP) activity, pro-collagen type I N-terminal peptide (PINP) and osteocalcin secretion in the supernatant was measured. After 20 days, the formation of mineralized matrix was analyzed. RESULTS: Decreasing B-vitamin concentrations induced a significant accumulation of HCY in the supernatant reaching up to 160%. The increase in HCY was not accompanied by changes of AP, osteocalcin and PINP. Moreover, mineralized matrix formation was not affected. CONCLUSION: Accumulation of HCY by decreasing concentrations of folate, vitamin B12 and B6 does not affect the activity of human osteoblasts. Consequently, other mechanisms have to be responsible for the reduced bone quality in hyperhomocysteinemic subjects.

Experimental hyperhomocysteinemia reduces bone quality in rats.

BACKGROUND: Recently, hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. This study investigated if HHCY is a causal osteoporotic factor in vivo. METHODS: We used 3 groups of rats: a control group (n = 20), a moderate HHCY group (induced by a 2.4% methionine-enriched diet, n = 10), and an intermediate HHCY group (induced by a 2% homocystine-enriched diet, n = 10). We measured bone fragility [maximum force of an axial compression test (F(max))], bone area as percentage of total area (BAr/TAr, histomorphometry), and biochemical bone turnover markers [osteocalcin (OC) and collagen I C-terminal crosslaps (CTx)]. RESULTS: Compared with controls, 3 months of moderate or intermediate HHCY increased mean (SD) bone fragility at the femoral neck by 18% (6%) in methionine-fed (P = 0.001) and 36% (13%) in homocystine-fed rats (P <0.001). Mean (SD) BAr/TAr at the distal femur in methionine and homocystine groups was decreased by 45% (21%; P = 0.001) and 93% (9%; P = 0.001), respectively. At the femoral neck, BAr/TAr was decreased by 19% (11%; P <0.001) and 55% (19%; P <0.001). At the lumbar spine, the reduction of BAr/TAr was 17% (23%; P = 0.099) and 44% (19%; P <0.001). Plasma OC (bone formation marker) was decreased by 23% (20%; P = 0.006) and 34% (21%; P <0.001). Plasma CTx (bone resorption marker) did not differ between groups. CONCLUSION: Bone quality is consistently decreased in the presence of increased circulating homocysteine. The results provide evidence that HHCY is a causal osteoporotic factor.

Hyperhomocysteinemia and myocardial expression of brain natriuretic peptide in rats.

BACKGROUND: Hyperhomocysteinemia (HHcy) has been linked to impaired left ventricular function and clinical class in patients with chronic heart failure. We hypothesized that HHcy stimulates myocardial brain natriuretic peptide (BNP) expression and induces adverse left ventricular remodeling. METHODS: We randomized 50 rats into 5 groups. Groups Co1 and Co2 (controls) received a typical diet. Groups Meth, Hcy1, and Hcy2 were fed the same diet supplemented with 2.4% methionine, 1% homocystine, and 2% homocystine, respectively. After 12 weeks, we measured total plasma homocysteine (tHcy) and BNP in plasma and tissue, and we performed histomorphometric analyses. RESULTS: All animals had comparable baseline body weight [mean (SD) 234 (26) g] and total circulating Hcy [4.7 (1.7) micromol/L]. After 12 weeks of treatment, total circulating Hcy increased in Meth, Hcy1, and Hcy2 [27.3 (8.8), 40.6 (7.0), and 54.0 (46.0) micromol/L, respectively] and remained unchanged in Co1 and Co2. Serum BNP significantly increased in 1 of 10 animals in Meth, 3 of 10 animals in Hcy1, and 3 of 10 animals in Hcy2. Median (25th-75th percentile) BNP tissue concentrations in Hcy1 and Hcy2 were 55% higher than in the corresponding controls [Co1 vs Hcy1, 225 (186-263) vs 338 (262-410) pg/mg protein, P = 0.05; Co2 vs Hcy2, 179 (107-261) vs 308 (192-429) pg/mg protein, P = 0.12]. In the Meth group, BNP expression was comparable to that of controls [200 (159-235) vs 225 (186-263) pg/mg protein, P = 0.32]. The percentage of perivascular and interstitial collagen and mast cell infiltration were comparable in all groups, indicating no adverse cardiac remodeling. CONCLUSION: Three months of intermediate HHcy stimulated increased cardiac BNP expression that was not accompanied by adverse cardiac remodeling.