Increased Osteoblast Activity Research Articles

The Effect of Pulsed Electromagnetic Field Exposure on Fracture Healing Through the Wnt Signal Pathway

Pulsed Electromagnetic Fields (PEMF) is reported to encourage the healing of nonunion fractures. However, the mechanism by which this occurs is still not known. Wnt signaling pathways are believed to be important signaling pathways in bone formation. This study will evaluate the healing of delayed union femur fracture, given PEMF exposure. 48 Spraque Dawley rats were fracturized at the left femoral shaft. These rats were randomized into two groups: A control group (24 rats) and the PEMF group (24 rats). The PEMF group was given PEMF exposure of 1.6 mT, with a frequency of 50 Hz for 4 h every day for 5, 10, 18 and 28 days, while the control group was not given PEMF exposure. Consequently, on days 5, 10, 18 and 28 days after fracture, 6 rats from each group were sacrificed. Callus bone was used for histological and RT-PCR examination on the expression of Wnt10b, Wnt5a and β-catenin. Blood samples were taken to examine Alkaline Phosphatase (ALP) activity using the ELISA method. Hematoxylin Eosin (HE) staining results showed that in the initial phase of healing, fibrous tissue in the fracture gap of the PEMF group was less compared to the control group. In the PEMF group, ALP activity increased significantly on day 10. This is thought to be related to an increase in osteoblast activity in a bone matrix formation. Furthermore, RT-PCR examination results showed that Wnt10b, Wnt5a and β-catenin gene expression was higher in the PEMF group compared to the control group. It can be concluded that PEMF exposure is thought to accelerate delayed union fracture healing through the Wnt signal pathway.

Deletion of PPARγ in Mesenchymal Lineage Cells Protects Against Aging-Induced Cortical Bone Loss in Mice.

Bone loss in aging is linked with chronic low-grade inflammation and the accumulation of marrowfat in animals and humans. Peroxisome proliferator-activated receptor gamma (PPARγ), an adipogenic regulator, plays key roles in these biological processes. However, studies of the roles of PPARγ in age-related bone loss and inflammation are lacking. We hypothesized that deletion of PPARγ in bone marrow mesenchymal lineage cells would reduce bone loss with aging, potentially through a reduction in fat-generated inflammatory responses and an increase in osteoblastic activity. In the present study, we show that mice deficient of PPARγ in Dermo1-expressing mesenchymal lineage cells (Dermo1-Cre:PPARγ fl/fl) have reduced fat mass and increased cortical bone thickness but that deficiency of PPARγ had limited effect on protection of trabecular bone with aging as demonstrated by dual-energy X-ray absorptiometry, µCT, and histomorphometric analyses. Conditional knockout of PPARγ reduced serum concentrations of adipokines, including adiponectin, resistin, and leptin, and reduced marrow stromal cell expression levels of inflammation-related genes. Inflammation genes involved in the interferon signaling pathway were reduced the most. These results demonstrate that disruption of the master adipogenic regulator, PPARγ, has a certain protective effect on aging-induced bone loss, suggesting that regulation of adipose function and modulation of interferon signaling are among the key mechanisms by which PPARγ regulates bone homeostasis during aging process.

Targeted overexpression of the long noncoding RNA ODSM can regulate osteoblast function in vitro and in vivo

Ameliorating bone loss caused by mechanical unloading is a substantial clinical challenge, and the role of noncoding RNAs in this process has attracted increasing attention. In this study, we found that the long noncoding RNA osteoblast differentiation-related lncRNA under simulated microgravity (lncRNA ODSM) could inhibit osteoblast apoptosis and promote osteoblast mineralization in vitro. The increased expression level of the lncRNA ODSM partially reduced apoptosis and promoted differentiation in MC3T3-E1 cells under microgravity unloading conditions, and the effect was partially dependent on miR-139-3p. LncRNA ODSM supplementation in hindlimb-unloaded mice caused a decrease in the number of apoptotic cells in bone tissue and an increase in osteoblast activity. Furthermore, targeted overexpression of the lncRNA ODSM in osteoblasts partially reversed bone loss induced by mechanical unloading at the microstructural and biomechanical levels. These findings are the first to suggest the potential value of the lncRNA ODSM in osteoporosis therapy and the treatment of pathological osteopenia.

Aspalathin from Aspalathus linearis (rooibos) reduces osteoclast activity and increases osteoblast activity in vitro

Abstract Bone remodelling in a healthy body is in constant balance, maintaining an adaptive and robust skeletal system. In osteoporosis this balance is disrupted with the rates of osteoclastic bone resorption exceeding osteoblastic bone formation, resulting in lower bone mineral density. Rooibos tea, is a popular South African drink made from Aspalathus linearis leaves grown in the Western Cape. This tea is rich in phenolic compounds which have been widely investigated in recent years as a potential treatment for many ailments. In this study, aspalathin, a phenolic compound found exclusively in rooibos, increases osteoblast formation and function including increased osteoblast marker expression and mineralisation. In addition, aspalathin decreased differentiation and function of osteoclasts as well as reducing osteoclast formation in an osteoclast/osteoblast co-culture model. These results illustrate bone-protective effects of aspalathin in vitro through the reduction of osteoclast activity and promotion of osteoblast activity, with potential applications in the maintenance of bone density.

Monosodium Glutamate Supplementation Improves Bone Status in Mice Under Moderate Protein Restriction.

ABSTRACTAdequate protein intake during development is critical to ensure optimal bone gain and to attain a higher peak bone mass later. Using a mild protein restriction model in Balb/C mice consuming 6% of their total energy intake as soy protein (LP‐SOY)—for which we observed a significantly lower femoral cortical thickness, bone volume, trabecular number, and thickness reduction—we evaluated the effects of monosodium glutamate (MSG) supplementation at different concentrations (0.5, 1, 5, 10, and 20 g/kg of diet) on bone characteristics in LP‐SOY‐fed mice. After 6 and 12 weeks, LP‐SOY‐fed mice had lower BMD and reduced body weight related to lower lean mass, which was associated with a reduced IGF‐1 level. The negative effect of the LP‐SOY diet on BMD correlated with impaired bone formation. MSG supplementation, at 5, 10, and 20 g/kg of diet, and PTH injection, used as a positive control, were able to improve BMD and to increase osteoblast activity markers (P1NP and osteocalcin), as well as glutamine plasma concentration. An analysis of bone microarchitecture found that cortical bone was less sensitive to protein restriction than trabecular bone, and that MSG ingestion was able to preserve bone quality through an increase of collagen synthesis, although it did not allow normal bone growth. Our study reinforces the view that glutamate can act as a functional amino acid for bone physiology and support clinical investigation of glutamate supplementation in adults characterized by poor bone status, notably as a result of insufficient protein intake. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The Effect of Sulforaphane on the Activity and Mineralization of Osteoblasts under Oxidative Stress

Sulforaphane (SFN) is considered an antioxidant agent, but the biological effects on hypoxia-treated osteoblasts remain unclear. Therefore, the aims of this study were to investigate the effects of SFN on the activity and mineralization of osteoblasts in hypoxia. Osteoblasts were treated with hypoxia with or without SFN, and apoptosis was assayed with caspase 3 Activity Assay Kit and flow cytometer. The levels of reactive oxygen species (ROS) were measured with DCFH-DA. The levels of glutathione (GSH) and glutathione disulphide were determined by the o-phthalaldehyde fluorimetric assay. Mineralization of Osteoblasts was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the relative proteins levels were examined by Western blotting. Our results showed that SFN reduced the hypoxia-mediated apoptosis and ROS levels in osteoblasts. The utilization of SFN improved the inhibitory effect of osteoblast mineralization by hypoxia. Additionally, the effect of alleviating hypoxia by SFN will be an increase in osteoblast activity. These findings clarify the effects of SFN on hypoxia-treated osteogenesis and will help identify novel therapeutic strategies for the protection of skeletal health.

The effect of luteolin in prevention of periodontal disease in Wistar rats.

Periodontal disease is the chronic infectious disease of the periodontium. Because of irreversibility, prevention of disease is one of the most important goals of periodontal treatment. The aim of this study was to evaluate the effect of luteolin, a powerful anti-inflammatory agent, on the prevention of experimental periodontitis by determining morphological and histological tissue alterations. This study consisted of 28 rats and four experimental groups: healthy control group (C, n = 6); periodontitis group (P, n = 6); periodontitis and 50 mg/kg luteolin administered group (L-50, n = 8); and periodontitis and 100 mg/kg luteolin administered group (L-100, n = 8). Experimental periodontitis was induced via ligature method around lower right first molar teeth. All rats were euthanized 11 days after. The severity of periodontal destruction was determined by measuring alveolar bone loss under a stereomicroscope. Osteoblast and inflammatory cell counts were counted on hematoxylin-eosin-stained slides and osteoclasts were counted on tartrate-resistant acid phosphatase-stained slides. The levels of inducible nitric oxide synthase (iNOS), bone morphogenetic protein (BMP)-2, matrix metalloproteinase (MMP)-8, tissue inhibitor of MMP (TIMP)-1, receptor activator of nuclear factor κB ligand (RANKL), and osteoprotegerin (OPG) were determined by immunohistochemistry. The highest alveolar bone loss was observed in the periodontitis group and the luteolin administration decreased bone loss in both groups. Osteoblast cell number was higher and osteoclast and inflammatory cell numbers were lower in the P group compared to C, L-50, and L-100 groups. Luteolin, dose-dependently increased osteoblast cell counts. Luteolin attenuated periodontal inflammation in both L-50 and L-100 groups. Like osteoblast cell numbers, BMP-2 expressions were also elevated in luteolin groups. Both doses of luteolin significantly increased TIMP-1 and BMP-2 expressions and decreased MMP-8 levels. iNOS expressions increased in P group and L-100 significantly decreased iNOS levels. RANKL increased and OPG decreased in P group and 100 mg/kg luteolin increased OPG and decreased RANKL levels significantly. Within the limits of present experimental study, luteolin successfully improved periodontal health in a ligature-induced experimental periodontitis model in Wistar rats. The decrease in inflammation, osteoclastic and collagenase activity and increase in osteoblastic activity are possibly involved in this process.

Increase in Osteocalcin Following Testosterone Therapy in Men With Type 2 Diabetes and Subnormal Free Testosterone.

ContextOne-third of men with type 2 diabetes have subnormal free testosterone concentrations. We evaluated the following: (i) whether bone mineral density (BMD) and bone strength are affected by gonadal status in type 2 diabetes and (ii) the effect of testosterone replacement on markers of osteoblast and osteoclast activity.DesignThis is a secondary analysis of a previously completed, randomized, placebo-controlled trial. Ninety-four men with type 2 diabetes were recruited; 44 had subnormal free testosterone concentrations. Men with subnormal free testosterone concentrations were randomized to receive intramuscular injections of testosterone or placebo every 2 weeks for 22 weeks. Dual energy X-ray absorptiometry scans were performed at baseline and at 23 weeks.ResultsMen with subnormal free testosterone had similar BMD compared with men with normal free testosterone. However, bone strength indices were lower in men with subnormal free testosterone. BMD was related to free estradiol concentrations (r = 0.37, P = 0.004 at hip), whereas bone strength was related to free testosterone concentrations (r = 0.41, P < 0.001). Testosterone replacement increased osteocalcin concentrations [mean change (95% CI), 3.52 (0.45, 6.59), P = 0.008]. C-Terminal telopeptide (CTx) concentrations also increased at 15 weeks but reverted to baseline following that. There were no changes in other bone turnover markers or BMD.ConclusionWe conclude that testosterone replacement resulted in an increase in osteocalcin and a transient increase in CTx, indicating an increase in osteoblastic activity and transient increase in bone breakdown. Therefore, a major action of testosterone is to increase bone turnover in men with type 2 diabetes.

Combined low-dose LiCl and LY294002 for the treatment of osteoporosis in ovariectomized rats

BackgroundTo provide a low-toxicity and high-efficacy clinical treatment for osteoporosis via a novel combination of LiCl and LY294002.MethodsThe protein levels of p-AKT, AKT, p-GSK3β, GSK3β, β-catenin, p-β-catenin, and NFATC1 were measured in osteoblasts and osteoclasts by Western blot. ALP activity and TRACP activity were measured using the corresponding kit. The levels of BALP, PINP, CTX, and TRACP-5b were determined in accordance with the requirements of the ELISA kits. Microstructural analysis was performed on the left distal femur using microcomputed tomography.ResultsTreatment with the combination of LiCl and LY294002 led to a markedly increased osteoblast activity but significantly decreased osteoclast differentiation and bone absorption capacity compared with the treatment with LiCl or LY294002 alone (P < 0.01). In serum, the low-dose combination of LiCl and LY294002 significantly enhanced BALP levels (P < 0.01) and significantly decreased PINP, TRACP-5b, and CTX levels (P < 0.01) compared with the application of either drug alone.ConclusionsThis study indicates that drug combinations directed at multiple targets could be used for osteoporosis treatment by promoting osteoblast proliferation and inhibiting differentiation with high efficiency.

A Pyk2 inhibitor incorporated into a PEGDA-gelatin hydrogel promotes osteoblast activity and mineral deposition

Pyk2 is a non-receptor tyrosine kinase that belongs to the family of focal adhesion kinases. Studies from our laboratory and others demonstrated that mice lacking the Pyk2 gene (Ptk2B) have high bone mass, which was due to increased osteoblast activity, as well as decreased osteoclast activity. It was previously reported that a chemical inhibitor that targets both Pyk2 and its homolog FAK, led to increased bone formation in ovariectomized rats. In the current study, we developed a hydrogel containing poly(ethylene glycol) diacrylate (PEGDA) and gelatin which was curable by visible-light and was suitable for the delivery of small molecules, including a Pyk2-targeted chemical inhibitor. We characterized several critical properties of the hydrogel, including viscosity, gelation time, swelling, degradation, and drug release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin (P1000:G10) exhibited Bingham fluid behavior that can resist free flowing before in situ polymerization, making it suitable for use as an injectable carrier in open wound applications. The P1000:G10 hydrogel was cytocompatible and displayed a more delayed drug release behavior than other hydrogels we tested. Importantly, the Pyk2-inhibitor-hydrogel retained its inhibitory activity against the Pyk2 tyrosine kinase, and promoted osteoblast activity and mineral deposition in vitro. Overall, our findings suggest that a Pyk2-inhibitor based hydrogel may be suitable for the treatment of craniofacial and appendicular skeletal defects and targeted bone regeneration.

Effect of strontium substitution on the material properties and osteogenic potential of 3D powder printed magnesium phosphate scaffolds

3D powder printing is a versatile method for the fabrication of individual bone implants and was used for the processing of in vivo degradable ceramic scaffolds based on ammonium magnesium phosphate hexahydrate (struvite). In this study, synergetic effects could be achieved by the substitution of magnesium phosphate cements with strontium carbonate. This substitution resulted in 8.2 wt%, 16.4 wt%, and 24.6 wt% Sr2+ doped scaffolds, with a 1.9–3.1 times increased radiopacity compared to pure struvite. The maximal compressive strength of (16.1 ± 1.1) MPa found for strontium substituted magnesium phosphate was in the range of cancelleous bone, which makes these 3D printed structures suitable for medical application in low-load-bearing bone areas. In an ion release study over a course of 18 days, the release of strontium, magnesium, calcium, and phosphate ions from scaffolds was analyzed by means of inductively coupled plasma mass spectrometry. Independent of the scaffold composition the Mg2+ concentrations (83–499 mg/l) continuously increased in the cell media. The Sr2+ release varied between 4.3 μg/day and 15.1 μg/day per g scaffold, corresponding to a Sr2+ concentration in media between 1.14 mg/l and 7.24 mg/l. Moreover, decreasing calcium and phosphate concentrations indicated the precipitation of an amorphous calcium phosphate phase. The superior osteogenic properties of strontium substituted magnesium phosphate, e.g. the increase of osteoblast activity and cell number and the simultaneous suppression of osteoclast differentiation could be verified in vitro by means of WST-assay, TRAP-staining, and SEM imaging.

Dynamics of bone metabolism markers in oral fluid after surgical treatment of chronic periodontal disease with various osteoplastic materials

The aim of the work was to assess the balance between osteosynthetic and osteodestructive processes in the oral cavity in patients with chronic generalized periodontitis (CGP) of moderate and severe severity in the dynamics of a remote period of patchwork using various bone-plastic materials (BPM) based on xenogeneic hydroxyapatite (HA), β-tricalcium phosphate (TCP). 123 patients with CGP were examined. Depending on the type of BPM, two groups were distinguished by composition in the course of patchwork operations. Patients of group 1 (n=61) received BPM on the basis of xenogeneic HA, 2 groups (n=62) on the basis of β-TCP. Initially and after surgery at 8 and 12 months in the oral fluid, the concentration of osteoprotegerin (OPG), the ligand of the soluble activator of the kappa B nucleation factor (sRANKL), the bone isoenzyme of alkaline phosphatase (BAP), was determined by an immunoenzyme method. As a result of the study, it was found that in patients with moderate-bodied CGP after surgical treatment, restriction of osteoclast activity with a decrease in sRANKL due to an increase in OPG in the oral fluid, an increase in osteoblast activity with BAP secretion is most pronounced when using β-TCP-based BPM at 8 months. After surgery with an average severity of the disease. Less pronounced changes in bone metabolism in the oral cavity are detected with the use of BPM on the basis of HA. In severe CGP, changes in bone homeostasis in the oral cavity after scrappy operations with BPM insertion based on TCP or HA are not expressed and are observed only after 12 months. Thus, the most effective changes in bone metabolism develop with the use of TCP as BPM.

Effects of nicotine on markers of bone turnover in ovariectomized rats.

IntroductionOsteoporosis is characterized by low bone mass and density, as well as change in microarchitecture of bone tissue leading to decreased bone strength. In vitro research shows nicotine can increase osteoblast activity and proliferation, also suppress osteoclast activity. Therefore we explore nicotine anti-resorptive property by in vivo true experimental and randomized posttest only controlled group research that was conducted in 18-20 weeks old Rattus norvegicus.MethodsTwenty-five female rats were divided into five groups, with 5 rats per group. The first group represented normal rats (Sham), while the second to fifth group underwent bilateral ovariectomy. The second group serves as positive control group (ovariectomy-only/OVX). The third to fifth group serve as dose 1 (P1-0.25mg/kg), dose 2 (P2-0.5 mg/kg), and Dose 3 (P3-0.75 mg/kg) treatment group receiving daily per-oral nicotine for 28 days, started 3 weeks post- ovariectomy. After 28 days treatment, the serum was checked.ResultsNicotine has dose-dependent manner on serum osteocalcin and serum DPD level. Level of osteocalcin in P2 group was significantly lower (Mann-Whitney, p = 0.008) compared to OVX group (59.4% lower). Level of DPD in all group was not significantly different (ANOVA, p < 0.05) but shows lowest level in P2 group. For serum calcitonin level, there's no significant different between groups.ConclusionNicotine at right low-dose might be able to inhibit osteoclast activity, thus open a possibility of anti-resorptive property of nicotine.

Alveolar Ridge Preservation: A Histomorphometric Analysis

Objective: This study presents the histomorphometric findings after tooth extraction with and without Alveolar Ridge Preservation (ARP) with a collagen cone filling the socket in combination with a collagen membrane covering the socket. Materials and Methods: In a controlled randomized clinical study, 10 patients were treated with the combination material after tooth extraction. In 10 patients, the extraction sockets were left to heal without further intervention. Soft tissue, new bone formation, bone quality and bone remodeling, blood flow vascularization, and inflammation were evaluated histomorphometrically. This was performed (semi-) quantitatively using a blinded protocol. Results: The statistical evaluation showed no significant difference for any parameter. When the combination material was used, more pronounced remodeling, increased osteoblast activity, and increased vascularization were demonstrated based on the histomorphometric findings. In contrast, there were reduced levels of osteogenesis and less mineralization. There was slightly more bundle bone in patients with ARP. Conclusion: The histomorphometric analysis of ARP with a combination material consisting of a collagen cone and a collagen membrane showed no significant differences in terms of new bone formation and bone quality. Descriptively, however, different manifestations were seen that might benefit from being documented using larger samples and being tested for clinical relevance.

SMAD1/5 signaling in osteoclasts regulates bone formation via coupling factors.

Bone remodeling occurs via coupling between bone resorption by osteoclasts and bone formation by osteoblasts. The mechanisms that regulate osteoclast signals to osteoblasts are not well understood. Published studies have reported that BMP signaling in osteoclasts regulate osteoclast coupling targets. To investigate the necessity of canonical BMP signaling on osteoclast differentiation and coupling, we mated Smad1fl/fl; Smad5fl/fl mice to c-Fms-Cre mice. We analyzed male mice at 3 months of age to determine the skeletal phenotype of the Smad1fl/fl; Smad5fl/fl;c-Fms-Cre (SMAD1/5 cKO) mice. There was a 1.2-fold decrease in trabecular BV/TV in SMAD1/5 cKO. Analyses of osteoclast serum markers in SMAD1/5 cKO mice, showed a significant increase in CTX-1 (1.5 fold) and TRAP ELISA (3 fold) compared to control mice. In these same mice, there was a 1.3-fold increase in cortical thickness. Consistent with the increase in cortical thickness, we found a 3-fold increase in osteoblast activity as measured by P1NIP ELISA assay from SMAD1/5 cKO mice. To explain the changes in cortical thickness and P1NP activity, we determined conditioned media from SMAD1/5 cKO osteoclast cultures enhanced mineralization of an osteoblast cell line and coupling factors expressed by osteoclasts that regulate osteoblast activity Wnt1 (4.5-fold increase), Gja1 (3-fold increase) and Sphk1 (1.5-fold increase) were all upregulated in osteoclasts from SMAD1/5 cKO compared to control osteoclasts. Lastly osteoclasts treated with dorsomorphin, a chemical inhibitor of SMAD1/5 signaling, demonstrates an increase in Wnt1 and Gja1 expression similar to the SMAD1/5 cKO mice. Previous studies demonstrated that TGF-β signaling in osteoclasts leads to increases in WNT1 expression by osteoclasts. Therefore, our data suggest that TGF-β and BMP signaling pathways in osteoclasts could act in an antagonistic fashion to regulate osteoblast activity through WNT1 and other coupling factors.

Ursolic Acid Prevents Retinoic Acid-Induced Bone Loss in Rats.

To examine the effects of ursolic acid (UA) on mitigating retinoic acid (RA)-induced osteoporosis in rats. Fifty female Sprague-Dawley rats were randomly divided into the control group (n=10) and the osteoporosis group (n=40). The 40 osteoporosis rats were induced by 75 mg/(kg•d) RA once daily for 2 weeks, and then were randomly assigned to vehicle control (model), low-, middle-, and high-dose UA [(UA-L, UA-M, UA-H; 30, 60, 120 mg/(kg•d), respectively] groups (10 rats each). UA were administered once daily to the rats from the 3rd weeks for up to 4 weeks by gavage. Bone turnover markers [serum alkaline phosphatase (ALP), osteocalcin (OCN), urine deoxypyridinoline (DPD)] and other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), urine phosphorus (U-P), and bone mineral density (BMD) of the femur, 4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture, were measured. The osteoporosis in rats was successfully induced by RA. Compared with the model group, UA-M and UA-H significantly reversed the RA-induced changes in S-P, U-Ca, U-P, ALP, OCN and urine DPD ratio and markedly enhanced the BMD of right femur, 4th lumbar vertebra and tibia (Plt;0.05 or Plt;0.01). Further, biomechanical test and microcomputed tomography evaluation also showed that UA-H drastically improved biomechanical properties and trabecular microarchitecture (Plt;0.05 or Plt;0.01). UA could promote bone formation, increase osteoblastic activity and reduce osteoclastic activity in rats, indicating that UA might be a potential therapeutic of RA-induced acute osteoporosis.

Hydroxyapatite calvaria graft repair in experimental diabetes mellitus in rats

Among the systemic conditions that impact negatively on the planning and execution of surgical procedures, diabetes mellitus (DM) is the primary clinical condition responsible for complications. This study investigated bone formation in critical defects surgically filled with hydroxyapatite (HA) in diabetic rats. A descriptive, randomized sample and blinded analysis were conducted to test bone regeneration in critical bone defects surgically performed in rat calvaria. Twenty adult male Wistar rats were randomly divided into two groups: control, normoglycemic animals (CG); and test, streptozotocin-induced hyperglycemic animals (TG). A circular bone defect was filled with HA and maintained subperiosteally. The clinical parameters evaluated were body weight, water and food intake, fasting blood glucose, and bone alkaline phosphatase. Bone-grafted area samples were submitted for histomorphometric and stereological analysis. The TG showed a significantly higher rate of new bone formation compared with the CG, sacrificed 15 days after surgery (p < 0.0001). However, at the end of the study, there was no significant difference in the amount of bone formed between groups (p = 0.077). In parallel, with the increase in osteoblastic activity observed in the TG by the measurement of systemic bone alkaline phosphatase (p = 0.016), the analysis of polarized microscopy and stereology demonstrated a lower level collagen maturation and mineralization in the TG. Quantitatively, the TG showed significantly better results for bone gain in the first 15 days. Qualitative assessments, however, showed fewer collagen fibers and bone maturation in the TG compared with the CG both at 15 and 45 days. Therefore, the postoperative evaluation of bone grafts with HA in hyperglycemic situations should consider the systemic and local effects of this condition on the quality of bone repair, rather than identifying the filling or stability of the grafted area after the process. We conclude that clinically detectable bone repair in diabetic animal models submitted to hydroxyapatite grafts may be satisfactory in the early stages. However, hyperglycemia compromises the quality of the newly formed bone and the collagen cross-linking involved in this process.

Morphometric and histopathological evaluation of the effect of grape seed proanthocyanidin on alveolar bone loss in experimental diabetes and periodontitis.

Grape seed proanthocyanidine extract (GSPE) is a strong antioxidant derived from the grape seeds (Vitis vinifera, Terral J.F.) and has a polyphenolic structure with a wide range of biological activity. The aim of the present study was to evaluate the effects of GSPE on alveolar bone loss and histopathological changes in rats with diabetes mellitus and ligature-induced periodontitis. Forty rats were divided into 6 study groups. Control (C, 6 rats) group, periodontitis (P, 6 rats) group, diabetes (D, 6 rats) group, diabetes and periodontitis (D+P, 6 rats) group, diabetes, periodontitis and 100mg/kg/day GSPE (GSPE-100, 8 rats), and diabetes, periodontitis and 200mg/kg/day GSPE (GSPE-200, 8 rats) group. Diabetes mellitus was induced by intraperitoneal injection of a single dose of streptozotocin (60mg/kg). Periodontitis was induced via ligation method. Silk ligatures were placed at the mandibular right first molars. GSPE was administered by oral gavage. After 30days, all rats were killed. Alveolar bone loss was measured morphometrically via a stereomicroscope. For histopathological analyses, Alizarin red staining, and matrix metalloproteinase (MMP)-8, vascular endothelial growth factor and hypoxia inducible factor (HIF)-1α immunohistochemistry were performed. Tartrate-resistant acid phosphatase-positive osteoclast cells and relative total inflammatory cells were also determined. The highest alveolar bone loss was observed in the D+P group (P<.05). GSP-200 group decreased alveolar bone loss (P<.05). The D+P group had the highest osteoclast counts, but the difference was not significant compared to the P, GSPE-100 and GSPE-200 groups (P>.05). The inflammation in the D+P group was also higher than the other groups (P<.05). The osteoblast numbers increased in the GSPE-100 and GSPE-200 groups compared to the P and D+P groups (P<.05). MMP-8 and HIF-1α levels were highest in the D+P group and GSPE significantly decreased these levels (P<.05). Within the limits of this animal study, it can be suggested that GSPE administration may decrease periodontal inflammation and alveolar bone loss via decreasing MMP-8 and HIF-1α levels and increase osteoblastic activity in diabetic rats with experimental periodontitis.

Functions of vitamin D in bone.

Vitamin D, synthesized in the skin or absorbed from the diet, undergoes multi-step enzymatic conversion to its active form, 1,25-dihydroxy vitamin D [1,25(OH)2D], followed by interaction with the vitamin D receptor (VDR), to modulate target gene expression. Loss-of function mutations in the genes encoding the enzymes regulating these processes, or in the VDR, result in human diseases, which have demonstrated the paramount role of 1,25(OH)2D in mineral and skeletal homeostasis. Mouse genetics has been used to create disease phenocopies which have produced considerable insight into the mechanisms of 1,25(OH)2D regulation of mineral and skeletal metabolism. Hypophosphatemia resulting from 1,25(OH)2D deficiency or resistance can inhibit apoptosis in hypertrophic chondrocytes leading to abnormal development of the cartilaginous growth plate in rickets. Decreased 1,25(OH)2D may also cause decreased vascular invasion associated with reduced chondroclast and osteoclast activity and thereby contribute to growth plate abnormalities. Reduced 1,25(OH)2D-mediated intestinal and renal calcium transport can reduce calcium availability, increase parathyroid hormone secretion and phosphaturia, and impair mineral availability for normal matrix mineralization, resulting in reduced growth plate mineralization and osteomalacia. 1,25(OH)2D may exert an anabolic effect in bone, apparently via the VDR in mature osteoblasts, by increasing osteoblast activity and reducing osteoclast activity. High ambient levels of exogenous 1,25(OH)2D, or of elevated endogenous 1,25(OH)2D in the presence of reduced calcium balance, can enhance bone resorption, and apparently prevent mineral deposition in bone. These actions demonstrate the critical role of vitamin D in regulating skeletal homeostasis both indirectly and directly via the 1,25(OH)2D/VDR system.

Spontaneous reossification of the sella in transsphenoidal reoperation associated with strontium ranelate.

Spontaneous reossification of the sellar floor after transsphenoidal surgery has been rarely reported. Strontium ranelate, a divalent strontium salt, has been shown to increase bone formation, increasing osteoblast activity. We describe an unusual case of a young patient with Cushing’s disease who was treated with strontium ranelate for low bone mass who experienced spontaneous sellar reossification after transsphenoidal surgery. A 21-year-old male presented with Cushing’s features. His past medical history included delayed puberty diagnosed at 16 years, treated with testosterone for 3 years without further work-up. He was diagnosed with Cushing’s disease initially treated with transsphenoidal surgery, which was not curative. The patient did not come to follow-up visits for more than 1 year. He was prescribed strontium ranelate 2 g orally once daily for low bone mass by an outside endocrinologist, which he received for more than 1 year. Two years after first surgery he was reevaluated and persisted with active Cushing’s disease. Magnetic resonance image revealed a left 4 mm hypointense mass, with sphenoid sinus occupation by a hyperintense material. At repeated transsphenoidal surgery, sellar bone had a very hard consistency; surgery was complicated and the patient died. Sellar reossification negatively impacted surgery outcomes in this patient. While this entity is possible after transsphenoidal surgery, it remains unclear whether strontium ranelate could have affected sellar ossification.Learning points:Delayed puberty can be a manifestation of Cushing’s syndrome. A complete history, physical examination and appropriate work-up should be performed before initiating any treatment.Sellar reossification should always be taken into account when considering repeated transsphenoidal surgery. Detailed preoperative evaluation of bony structures by computed tomography ought to be performed in all cases of reoperation.We speculate if strontium ranelate may have affected bone mineralization at the sellar floor. We strongly recommend that indications for prescribing this drug should be carefully followed.