Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

The Human Mesenchymal Stromal Cell-Derived Osteocyte Capacity to Modulate Dendritic Cell Functions Is Strictly Dependent on the Culture System.

In vitro differentiation of mesenchymal stromal cells (MSC) into osteocytes (human differentiated osteogenic cells, hDOC) before implantation has been proposed to optimize bone regeneration. However, a deep characterization of the immunological properties of DOC, including their effect on dendritic cell (DC) function, is not available. DOC can be used either as cellular suspension (detached, Det-DOC) or as adherent cells implanted on scaffolds (adherent, Adh-DOC). By mimicking in vitro these two different routes of administration, we show that both Det-DOC and Adh-DOC can modulate DC functions. Specifically, the weak downregulation of CD80 and CD86 caused by Det-DOC on DC surface results in a weak modulation of DC functions, which indeed retain a high capacity to induce T-cell proliferation and to generate CD4(+)CD25(+)Foxp3(+) T cells. Moreover, Det-DOC enhance the DC capacity to differentiate CD4(+)CD161(+)CD196(+) Th17-cells by upregulating IL-6 secretion. Conversely, Adh-DOC strongly suppress DC functions by a profound downregulation of CD80 and CD86 on DC as well as by the inhibition of TGF-β production. In conclusion, we demonstrate that different types of DOC cell preparation may have a different impact on the modulation of the host immune system. This finding may have relevant implications for the design of cell-based tissue-engineering strategies.

Osteocyte Density in the Peri-Implant Bone of Immediately Loaded and Submerged Dental Implants

Background: The role of osteocytes in bone structure and function remains partially unresolved. Their participation in mechanotransduction, i.e., the conversion of a physical stimulus into a cellular response, has been hypothesized. The present study was an evaluation of the osteocyte density in the peri-implant bone of immediately loaded and submerged dental implants. Methods: Fourteen male patients were included in the study; all of them were partially edentulous and needed a posterior mandibular restoration. Implants were inserted in these areas; half of the sample was loaded immediately (included in a fixed provisional prosthesis on the same day as implant surgery), whereas the other half was left to heal submerged. Fourteen implants (seven immediately loaded and seven unloaded) were retrieved with a trephine after a healing period of 8 weeks. The specimens were treated to obtain thin ground sections, and histomorphometry was used to evaluate the osteocyte index in the peri-implant bone. Results: A higher and statistically significant number of osteocytes was found in the peri-implant bone around immediately loaded implants (P=0.0081). A correlation between the percentage of bone-implant contact and osteocyte density was found for immediately loaded implants (P=0.0480) but not for submerged implants (P=0.2667). Conclusion: The higher number of osteocytes in the peri-implant bone around immediately loaded implants could be related to the functional adaptation required by the loading stimulus, which also explains the hypothesized involvement of the osteocytes in the maintenance of the bone matrix. J Periodontol 2009;80:499-504.

Repression of osteocyte Wnt/beta-catenin signaling is an early event in the progression of renal osteodystrophy

Chronic kidney diseasemineral bone disorder (CKD-MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft-tissue calcification. Emerging evidence suggests that features of CKD-MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild-type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild-type mice. To capture the early molecular and cellular events in the progression of CKD-MBD we examined cell-specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor-Ser33/37-beta-catenin was observed both in mouse and human bones. Overall repression of Wnt/beta-catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF-?B ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late-stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD-MBD and suggest that repression of the Wnt/beta-catenin pathway is involved in the pathogenesis of renal osteodystrophy. (C) 2012 American Society for Bone and Mineral Research.

The behaviour of osteocyte dendritic processes in bone under cyclic load (il comportamento dei processi dendritici degli osteociti nel tessuto osseo sottoposti a carico ciclico)

Bone is continually being removed and replaced through the actions of basic multicellular units (BMU). This constant upkeep is necessary to remove microdamage formed naturally due to fatigue and thus maintain the integrity of the bone. The repair process in bone is targeted, meaning that a BMU travels directly to the site of damage and repairs it. It is still unclear how targeted remodelling is stimulated and directed but it is highly likely that osteocytes play a role. A number of theories have been advanced to explain the microcrack osteocyte interaction but no complete mechanism has been demonstrated. Osteocytes are connected to each other by dendritic processes. The “scissors model" proposed that the rupture of these processes where they cross microcracks signals the degree of damage and the urgency of the necessary repair. In its original form it was proposed that under applied compressive loading, microcrack faces will be pressed together and undergo relative shear movement. If this movement is greater than the width of an osteocyte process, then the process will be cut in a “scissors like" motion, releasing RANKL, a cytokine known to be essential in the formation of osteoclasts from pre-osteoclasts. The main aim of this thesis was to investigate this theoretical model with a specific focus on microscopy and finite element modelling. Previous studies had proved that cyclic stress was necessary for osteocyte process rupture to occur. This was a divergence from the original “scissors model" which had proposed that the cutting of cell material occurred in one single action. The present thesis is the first study to show fatigue failure in cellular processes spanning naturally occurring cracks and it's the first study to estimate the cyclic strain range and relate it to the number of cycles to failure, for any type of cell. Rupture due to shear movement was ruled out as microcrack closing never occurred, as a result of plastic deformation of the bone. Fatigue failure was found to occur due to cyclic tensile stress in the locality of the damage. The strain range necessary for osteocyte process rupture was quantified. It was found that the lower the process strain range the greater the number of cycles to cell process failure. FEM modelling allowed to predict stress in the vicinity of an osteocyte process and to analyse its interaction with the bone surrounding it: simulations revealed evident creep effects in bone during cyclic loading. This thesis confirms and dismisses aspects of the “scissors model". The observations support the model as a viable mechanism of microcrack detection by the osteocyte network, albeit in a slightly modified form where cyclic loading is necessary and the method of rupture is fatigue failure due to cyclic tensile motion. An in depth study was performed focusing on microscopy analysis of naturally occurring cracks in bone and FEM simulation analysis of an osteocyte process spanning a microcrack in bone under cyclic load.

Osteocyte lacunar density and area in newly formed bone of the augmented sinus

OBJECTIVES Osteocytes, the most common cells of the bone, are buried in lacunae. Density and area of the osteocyte lacunae change with increasing maturation of the newly formed bone. Evaluation of osteocyte lacunae can therefore provide insights into the process of graft consolidation. MATERIALS AND METHODS Here, we determined the osteocyte lacunar density (number of osteocyte lacunae per bone area; N.Ot/BAr) and the osteocyte lacunar area in μm(2) (Lac.Ar) in histological specimens 6 and 12 weeks after the sinuses of 10 minipigs were augmented with Bio-Oss(®) , a deproteinized bovine bone mineral, and Ostim(®) , an aqueous paste of synthetic nanoparticular hydroxyapatite. The region of interest was defined by the following criteria: (i) >1 mm from the host bone, (ii) >0.5 mm from the sinus mucosa, (iii) minimum area of 0.2 mm(2) , and (iv) bone tissue spanning at least two bone substitute particles. RESULTS The overall osteocyte lacunar density was significantly higher in the Bio-Oss(®) group than in the Ostim(®) group and decreased during the observation period at a similar range in both groups. The osteocyte lacunar area was smaller in the Bio-Oss(®) group than the Ostim(®) group but there was no significant change within the groups over time. CONCLUSIONS These results suggest that bone substitutes affect the osteocyte lacunar density and the osteocyte lacunar area in the newly formed bone within the augmented sinus in this particular model situation. These measures can provide insights into the maturation of newly formed bone in the augmented sinus.

High fat-fed diabetic mice present with profound alterations of the osteocyte network

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Is Nitric Oxide a Mediator of the Effects of Low-Intensity Electrical Stimulation on Bone in Ovariectomized Rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvo et al., Calcif Tissue Int 84:502-509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by l-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200-220 g) were divided into six groups: sham, sham-l-NAME (6 mg/kg/day), OVX, OVX-l-NAME, OVX-LIES, and OVX-LIES-l-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of l-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). l-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both l-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats l-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.

Low-Intensity Electrical Stimulation Counteracts the Effects of Ovariectomy on Bone Tissue of Rats: Effects on Bone Microarchitecture, Viability of Osteocytes, and Nitric Oxide Expression

Low Intensity Electrical Stimulation (LIES) has been used for bone repair, but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone structure and remodeling, NOS expression and osteocyte viability in ovariectomized (OVX) rats. Thirty rats (200-220 g) were divided into 3 groups: SHAM, OVX, and OVX subjected to LIES (OVX + LIES) for 12 weeks. Following the protocol, rats were sacrificed and tibias were collected for histomorphometric analysis and immunohistochemical detection of endothelial NO synthase (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). OVX rats showed significant (p < 0.05 vs. SHAM) decreased bone volume (10% vs. 25%) and trabecular number (1.7 vs. 3.9), and increased eroded surfaces (4.7% vs. 3.2%) and mineralization surfaces (15.9% vs. 7.7%). In contrast, after LIES, all these parameters were significantly different from OVX but not different from SHAM. eNOS and iNOS were similarly expressed in subperiosteal regions of tibiae cortices of SHAM, not expressed in OVX, and similarly expressed in OVX + LIES when compared to SHAM. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX + LIES (8%). Our results suggest that LIES counteracts some effects of OVX on bone tissue preserving bone structure and microarchitecture, iNOS and eNOS expression, and osteocyte viability.

Tetraploidía en Ceratophrys (Anura, Leptodactylidae), análisis del registro fósil

It has been confirmed the existence of the tetraploid counterpart of the genus Ceratophrys Wied, 1824 (extint at the present time) in Argentina and Brazil since the Pliocene, and the apparition of the octaploidy in the Upper Pleistocene - Holocene. Fossil material of the diploid form, distinctive of the Chacoan District, has been reported from the horizons of the Montehermosense Formation in south-east of the province of Buenos Aires, Argentina. The coexistence of diploid-tetraploid and tetraploid-octaploid forms in the same province was documented.

Histomorphometric Evaluation of Bioceramic Molecular Impregnated and Dual Acid-Etched Implant Surfaces in the Human Posterior Maxilla

Background: Physical and bioceramic incorporation surface treatments at the nanometer scale showed higher means of bone-to-implant contact (BIC) and torque values compared with surface topography at the micrometer scale; however, the literature concerning the effect of nanometer scale parameters is sparse. Purpose: The aim of this study was to evaluate the influence of two different implant surfaces on the percentage bone-to-implant contact (BIC%) and bone osteocyte density in the human posterior maxilla after 2 months of unloaded healing. Materials and Methods: The implants utilized presented dual acid-etched (DAE) surface and a bioceramic molecular impregnated treatment (Ossean(R), Intra-Lock International, Boca Raton, FL, USA) serving as control and test, respectively. Ten subjects (59 1 9 years of age) received two implants (one of each surface) during conventional implant surgery in the posterior maxilla. After the non-loaded period of 2 months, the implants and the surrounding tissue were removed by means of a trephine and were non-decalcified processed for ground sectioning and analysis of BIC%, bone density in threaded area (BA%), and osteocyte index (Oi). Results: Two DAE implants were found to be clinically unstable at time of retrieval. Histometric evaluation showed significantly higher BIC% and Oi for the test compared to the control surface (p < .05), and that BA% was not significantly different between groups. Wilcoxon matched pairs test was used to compare the differences of histomorphometric variables between implant surfaces. The significance test was conducted at a 5% level of significance. Conclusion: The histological data suggest that the bioceramic molecular impregnated surface-treated implants positively modulated bone healing at early implantation times compared to the DAE surface.

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

Tumor Necrosis Factor alpha and Interleukin-1 beta Modulate Calcium and Nitric Oxide Signaling in Mechanically Stimulated Osteocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of simvastatin on bone regeneration of tibial defects and blood cholesterol level in rats

The purpose of this study was to evaluate the effects of simvastatin, by oral or subcutaneous administration, on tibial defects regeneration and blood cholesterol level in rats. A surgical defect was made on the right tibia of 40 male animals assigned to 4 groups (n=10), based on two routes of administration and on the use or not of simvastatin: subcutaneous injection of simvastatin (7 mg/kg) (group AT) or only the vehicle of drug suspension (group AC), above the defect area, for 5 days; and 20 mg/kg of simvastatin macerated on water (group BT) or only water (group BC), orally, daily, during the whole observation period. The animals were sacrificed after 15 or 30 days, when blood samples were analyzed to check plasma cholesterol levels. Tibiae were removed and, after decalcification and routine laboratorial processing, histological and histomorphometrical analyses were carried out. ANOVA was used for statistical analysis at 5% signficance level. The histological and histomorphometrical analyses showed significant differences only between the experimental periods (p<0.05). Animals sacrificed after 30 days showed better bone repair (p<0.05). There was no statistically significant difference (p>0.05) for blood cholesterol levels between the groups. In conclusion, simvastatin administration either orally or subcutaneously did not improve bone repair of experimental tibial defects and did not alter blood cholesterol levels in rats.

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.