Tartrate-resistant Acid Phosphate Research Articles

Hippo signaling protein MST1 regulates osteoclast differentiation by interacting with integrin linked kinase (ILK) and modulating actin structures

Hippo signaling is implicated in balancing cell proliferation, differentiation and death in multiple organs. However, its role in specific bone cell types such as osteoclasts, and its significance in maintaining overall bone tissue homeostasis remain largely unknown. In this study, we investigated the role of the Hippo pathway in osteoclast differentiation. Human primary monocyte cells were treated with receptor activator nuclear factor kappaB ligand (RANKL) and evaluated for osteoclast differentiation by marker protein analysis, tartrate-resistant acid phosphate (TRAP) and resorption assays. Our results showed that Ste20-like kinase 1 (MST1) underwent the maximum change after RANKL treatments and is negatively associated with osteoclast differentiation. Furthermore, proteomic approaches involving coimmunoprecipitation and mass spectrometry identified MST1 interaction with integrin-linked kinase (ILK) which is lost during RANKL induced differentiation. Finally, using RNAi and ectopic expression experiments we observed that MST1-ILK interaction negatively inhibits osteoclast differentiation at the level of actin ring structure formation, which is facilitated by ILK. Together, our data highlight a role for the Hippo pathway protein, MST1, in negatively regulating osteoclast differentiation through its interaction with integrin signaling. Given that integrin signaling is progressively implicated in pathological osteolysis, augmenting this pathway could have therapeutic implications.

Development of animal model for Bisphosphonates-related osteonecrosis of the jaw (BRONJ).

BackgroundThe aim of this study is to develop a rat model of bisphosphonates-related osteonecrosis of the jaw (BRONJ) that would be verified with clinical, radiological and histological examination, and to confirm the influence of concurrent bisphosphonates and steroids use upon the occurrence and aggravation of BRONJ.MethodsTwenty seven rats were divided into 3 groups; Saline group (I), Zoledronate group (II), Zoledronate and Dexamethasone group (III). Rats got weekly intraperitoneal injection for 4 times and extraction of left maxillary and mandibular 1st, 2nd molars were followed. Consecutive injections were performed, and blood sampling for measurements of C-terminal crosslinked telopeptide of type I collagen and tartrate-resistant acid phosphate 5b rats were performed at the time of 2, 4 and 8 weeks. And then, rats were sacrificed and evaluated clinically, radiologically and histologically.Results12/18 (66.6 %) of experimental group were diagnosed as BRONJ. There was no significant difference in incidence between zoledronate alone group (ll) and concurrent use of zoledronate and dexamethasone group (lll).ConclusionsConcurrent use of bisphosphonates and steroids increase incidence of BRONJ compared to saline group (l). Zoledronate alone group (ll) and concurrent use of zoledronate and dexamethasone group (lll) shows same incidence of BRONJ. Based on this study, the rat treated with bisphosphonates and steroids can be considered a novel, reliable and reproducible model to understand pathology of BRONJ.

Effect of Control-released Basic Fibroblast Growth Factor Incorporated in β-Tricalcium Phosphate for Murine Cranial Model.

Background:β-Tricalcium phosphate (β-TCP) is used clinically as a bone substitute, but complete osteoinduction is slow. Basic fibroblast growth factor (bFGF) is important in bone regeneration, but the biological effects are very limited because of the short half-life of the free form. Incorporation in gelatin allows slow release of growth factors during degradation. The present study evaluated whether control-released bFGF incorporated in β-TCP can promote bone regeneration in a murine cranial defect model.Methods:Bilateral cranial defects of 4 mm in diameter were made in 10-week-old male Sprague-Dawley rats treated as follows: group 1, 20 μl saline as control; group 2, β-TCP disk in 20 μl saline; group 3, β-TCP disk in 50 μg bFGF solution; and group 4, β-TCP disk in 50 μg bFGF-containing gelatin hydrogel (n = 6 each). Histological and imaging analyses were performed at 1, 2, and 4 weeks after surgery.Results:The computed tomography value was lower in groups 3 and 4, whereas the rate of osteogenesis was higher histologically in group 4 than in the other groups. The appearance of tartrate-resistant acid phosphate–positive cells and osteocalcin-positive cells and disappearance of osteopontin-positive cells occurred earlier in group 4 than in the other groups.Conclusions:These findings suggest that control-released bFGF incorporated in β-TCP can accelerate bone regeneration in the murine cranial defect model and may be promising for the clinical treatment of cranial defects.

Abstract B77: Curcumin deteriorates trabecular and cortical bone in mice bearing metastatic Lewis lung carcinoma

Abstract Bone is a major target of metastasis for many malignancies; curcumin has been studied for its role in cancer prevention including early phase clinical trials for its efficacy and safe use with cancer patients. The present study investigated the effects of dietary supplementation with curcumin (2% and 4%) on (1) pulmonary metastasis of Lewis lung carcinoma and (2) bone microstructural changes in female C57BL/6 mice. Curcumin significantly increased the size of lung metastases, which was accompanied with significant increases in plasma concentrations of angiogenic factors (e.g. angiogenin, VEGF) and inflammatory cytokines (e.g. IL-1β, MCP-1). Morphometric analysis showed that curcumin at 2% and 4% dietary levels significantly reduced bone volume to total volume ratio (BV/TV), connective density (Conn.D.) and trabecular number (Tb.N.) and significantly increased structure model index (SMI, an indicator of the plate- and rod-like geometry of trabecular structure) and trabecular separation (Tb.Sp.) in vertebral bodies, and at the 4% level curcumin significantly reduced BV/TV and Tb.N. and at either level it significantly reduced trabecular thickness (Tb.Th.) in distal femurs compared with the controls. In the mid-shaft of femur, curcumin at the 4% level significantly reduced cortical bone area to total area ratio (Ct.Ar/Tt.Ar.) and cortical thickness (Ct.Th.) compared with the controls. Furthermore, curcumin feeding resulted in dose-dependent decreases in plasma concentrations of osteocalcin (a marker of bone formation) and increases plasma tartrate-resistant acid phosphate 5b (a marker of bone resorption) compared with the controls, indicating that curcumin may deteriorate trabecular and cortical bone by disrupting the balance of bone remodeling. As skeleton is a favored site of metastasis for many cancers, our results that curcumin (1) enhances metastatic growth and (2) results in osteolytic deterioration of bone suggest the need of assessment of bone structural changes for curcumin cancer prevention studies, particularly for human trials with cancer patients who are the risk of malignant spread, to determine the curcumin effects and its interaction with malignancy on skeleton health. Citation Format: Lin Yan. Curcumin deteriorates trabecular and cortical bone in mice bearing metastatic Lewis lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B77.

Postoperative lumbar spinal stenosis after intertransverse fusion with granules of hydroxyapatite: a case report

In the present case of postoperative lumbar spinal stenosis after non-instrumented intertransverse fusion with granules of hydroxyapatite (HA), bone union was not completed and the patient felt the recurrence of his symptoms within two years. We performed re-decompression with fusion, and in hematoxylin and eosin staining of HA granulation harvested during revision surgery, fibrous tissue with hyaline degeneration surrounded the cavity where the HA had existed. Multinuclear giant cells and lymphocytes infiltrated some parts of the marginal layer of the cavity, and no obvious bony bridge had regenerated from autologous bone. No tartrate-resistant acid phosphate (TRAP) -positive osteoclasts could be seen in the new bone, suggesting that the activity of osteoclasts in the new bone decreased during the seven years after the primary surgery.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3483360258050263

Osteoconductive action of alendronate after implantation of beta tricalcium phosphate in rat adjuvant-induced arthritis

The aim of the study was to determine the effect of alendronate on resorption of β-tricalcium phosphate (β-TCP) and bone formation in rats with adjuvant-induced arthritis (AIA). After preparation of a model of AIA in rats (day 0), alendronate or vehicle was injected intraperitoneally once daily five times in a week. Cylindrical β-TCP was implanted into the rat femoral condyle on day 7. Rats were killed on days 12, 15, and 21, and specimens and serum samples were collected. Specimens were analyzed by tartrate-resistant acid phosphate (TRAP) staining, immunohistochemistry of the ED1 protein, and in situ hybridization with digoxigenin-labeled α1 chain of type I procollagen (COL1A1). Mineralized bone sections were analyzed by Villanueva bone stain. The serum osteocalcin level was measured using an enzyme-linked immunosorbent assay kit. Alendronate decreased the number of TRAP-positive cells attached to β-TCP, the numbers of ED1-positive multinucleated giant cells, and resorption of β-TCP. In AIA rats treated with alendronate, COL1A1 mRNA-positive cells adhered to β-TCP were round or cuboid whereas the cells in untreated AIA rats were fibroblast-like cells. Alendronate increased calcification of newly formed bone whereas it did not restore the bone formation suppressed with inflammation. These results suggest that alendronate has the potential to conduct mature bone after implantation of β-TCP in AIA. Alendronate may help to reduce insufficiency of newly formed bone after implantation of β-TCP in diseases characterized by increased bone resorption such as rheumatoid arthritis.

IL-4 inhibits TNF-α-mediated osteoclast formation by inhibition of RANKL expression in TNF-α-activated stromal cells and direct inhibition of TNF-α-activated osteoclast precursors via a T-cell-independent mechanism in vivo

It has been reported that osteoclastogenesis is induced by tumor necrosis factor (TNF)-α. Interleukin (IL)-4 is the most important cytokine involved in humoral immunity. However, no studies have investigated the effect of IL-4 on TNF-α-mediated osteoclast formation in vivo. In this study, we investigated the effect of IL-4 on TNF-α-mediated osteoclast formation in vivo. TNF-α was administered with and without IL-4 into the supracalvariae of mice. The number of osteoclasts and the levels of mRNA for cathepsin K and tartrate-resistant acid phosphate, both osteoclast markers, in mice administered TNF-α and IL-4 were lower than those in mice administered TNF-α alone. The level of tartrate-resistant acid phosphatase form 5b (TRACP5b) as a marker of bone resorption in mice administered both TNF-α and IL-4 was also lower. We showed that IL-4 inhibited TNF-α-mediated osteoclast formation in osteoclast precursors in vitro. Expression of receptor activator of NF-κB ligand (RANKL) in TNF-α-activated stromal cells was also inhibited. Furthermore, we investigated whether IL-4 had effects on both stromal cells and osteoclast precursors in TNF-α-mediated osteoclast formation in vivo. Using mice whose stromal cells and osteoclast precursors were chimeric for the presence of TNF receptors, IL-4 inhibited TNF-α-mediated osteoclast formation in the presence of TNF-α-responsive stromal cells, and TNF-α-responsive osteoclast precursors in vivo. IL-4 also inhibited TNF-α-induced RANKL expression in the presence of TNF-α-responsive stromal cells in vivo. This event is dependent on p38 inhibition in vitro. Additionally, IL-4 inhibited TNF-α-mediated osteoclast formation in T cell-depleted mice. In summary, we conclude that IL-4 inhibited TNF-α-mediated osteoclast formation by inhibiting expression of RANKL in TNF-α-activated stromal cells, and directly inhibited TNF-α-activated osteoclast precursors in vivo via a T cell-independent mechanism.

Combined Effects of Hydrogen Sulfide and Lipopolysaccharide on Osteoclast Differentiation in Rats

Lipopolysaccharide (LPS) stimulates osteoclast differentiation through toll-like receptors (TLRs) 2 and 4, and hydrogen sulfide (H(2)S) induces osteoclast differentiation. If H(2)S activates TLRs, H(2)S may enhance the effects of LPS on osteoclast differentiation. The purpose of the present study is to examine the combined effects of sodium hydrogen sulfide (NaHS, an H(2)S donor drug) and LPS on osteoclast differentiation and TLR expression in rat periodontal tissue. Twenty-eight male Wistar rats (8 weeks old) were divided into four groups (n = 7 per group): a control (no treatment) group and three experimental groups (NaHS group, LPS group, and a combination [NaHS + LPS] group). At 1 day after topical application of NaHS and/or Porphyromonas gingivalis LPS into the gingival sulcus of first molars, the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclasts in the periodontal tissue was counted. Expression of TLR2 and TLR4 mRNAs and proteins in the gingival was also assessed. The number of TRAP-positive osteoclasts was significantly higher in the combination group than in any other group (P <0.01). The combination group had 11.0-fold higher TLR4 mRNA levels than the control group. TLR4 protein levels were also higher in the combination group than in the NaHS or LPS group. However, the TLR2 mRNA and protein levels were not significantly different in the combination group and the LPS group. In rat periodontal tissue, NaHS and LPS had an additive effect on osteoclast differentiation through activation of the TLR4 pathway but not the TLR2 pathway.

Monocyte proliferation and differentiation to osteoclasts is affected by density of collagen covalently bound to a poly(dimethyl siloxane) culture surface

Osteoclast differentiation is affected by substrate characteristics and environmental conditions; these parameters are therefore of interest for understanding bone remodeling. As a step toward osteoclast mechanotransduction experiments, we aimed to optimize conditions for osteoclast differentiation on extendable poly(dimethylsiloxane) (PDMS) substrates. Because cells attach poorly on PDMS alone, chemical modification by covalent attachment of collagen type I was performed. Effects of collagen surface concentrations on monocyte fusion and osteoclast differentiation were examined. Osteoclasts differentiated on modified PDMS were fewer in number (by ∼50%) than controls on polystyrene physically modified by nonspecific attachment of collagen, and exhibited somewhat different morphologies. Nevertheless, for certain choices of the chemical modification procedures, appropriate differentiation on PDMS was still evident by qRT-PCR analysis for tartrate-resistant acid phosphate (TRAP) and cathepsin K (CTSK) gene expression, positive TRAP staining, fluorescent phalloidin staining showing actin ring formation and bone resorption assays. At relatively high collagen surface densities, monocyte clumps appeared on PDMS suggesting substrate-induced alterations to monocyte fusion. Covalently bound collagen can therefore be used to promote osteoclast differentiation on extendable PDMS substrates. Under appropriate conditions osteoclasts retain similar functionality as on polystyrene, which will enable future studies of osteoclast interactions with microstructured surfaces and mechanostimulation.

Expression of osteoprotegerin and receptor activator of nuclear factor κB ligand in root resorption induced by heavy force in rats

The aim of this study was to investigate the expression patterns of osteoprotegerin (OPG) and the receptor activator of nuclear factor κB ligand (RANKL) in root resorption during orthodontic tooth movement. Forty 12-week-old male SD rats were used with the right maxillary side as the experimental group and the left maxillary side as the control group. After 1N (100g) force was loaded on the right maxillary first molar, the rats were sacrificed on days 0, 1, 4, 8, and 12. Mesial root resorption of the first molar, the number of odontoclasts and osteoclasts, and OPG and RANKL mRNA expression were determined by hematoxylin-eosin and scanning electron microscopy, tartrate-resistant acid phosphate staining, and in situ hybridization, respectively. Serious root resorption was apparent on the pressure side of the mesial root of the right maxillary first molar on days 8 and 12. The number of odontoclasts in the cementum lacuna was elevated on days 8 and 12. OPG expression rose significantly on the tensile side, while RANKL expression increased on the pressure side. The mRNA level of RANKL was significantly elevated on days 4, 8, and 12. Moreover, the RANKL/OPG mRNA ratio was increased on the pressure side, but decreased on the tensile side. Changes in the expression of RANKL mRNA and the RANKL/OPG mRNA ratio are accompanied by a parallel alteration in the number of odontoclasts and tooth resorption, suggesting crucial involvement of RANKL and OPG in tooth resorption.

Effect of diabetes on orthodontic tooth movement in a mouse model

Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading. Type 1 diabetes results in bone remodeling, suggesting that this disease might affect orthodontic tooth movement. The present study investigated the effects of the diabetic state on orthodontic tooth movement. An orthodontic appliance was placed in normoglycemic (NG), streptozotocin-induced diabetes (DB), and insulin-treated DB (IT) C57BL6/J mice. Histomorphometric analysis and quantitative PCR of periodontium were performed. The DB mice exhibited greater orthodontic tooth movement and had a higher number of tartrate-resistant acid phosphate (TRAP) -positive osteoclasts than NG mice. This was associated with increased expression of factors involved in osteoclast activity and recruitment (Rankl, Csf1, Ccl2, Ccl5, and Tnfa) in DB mice. The expression of osteoblastic markers (Runx2, Ocn, Col1, and Alp) was decreased in DB mice. Reversal of the diabetic state by insulin treatment resulted in morphological findings similar to those of NG mice. These results suggest that the diabetic state up-regulates osteoclast migration and activity and down-regulates osteoblast differentiation, resulting in greater orthodontic tooth movement.

마가목 및 현지초 추출물의 골손실 및 연골손상 억제효과

본 연구에서는 마가목 (SC), 현지초 추출물 (GT) 및 이들의 1:1 혼합물 시료 (MIX)가 골손실 및 연골손상 억제에 효과가 있는지 알아보기 위해, 각각의 시료를 조골세포주인 MG-63 세포, 파골세포로의 분화를 유도한 Raw264.7 세포와 연골세포로의 분화를 유도한 ATDC5 세포에 처리하여 세포분화 조절 정도를 확인하였다. 각 세포의 분화 정도는 alkaline phosphatase (ALP) 활성 측정, tartrate-resistant acid phosphatase (TRAP) 염색법 및 alcian-blue 염색법으로 확인하였다. 이들 시료는 MG-63 세포에서 ALP 활성에는 영향을 미치지 않았으나, 마가목 추출물 (SC) 및 마가목과 현지초 추출물의 혼합시료 (MIX)는 농도 의존적으로 파골세포의 분화를 억제하고 연골세포의 분화를 촉진하는 것으로 나타났다. 이상의 결과를 종합하여 볼 때, 마가목과 현지초는 골손실과 연골 손상으로부터 보호할 수 있는 중요한 천연물 소재임을 확인할 수 있었다. 나아가 이들 추출물의 작용기전 및 활성물질 구명에 대한 연구는 추후 더 진행되어야 할 것이다. 【This study was carried out to investigate the effect of Sorbus commixta (SC), Geranium thunbergii (GT) and their mixture (SC:GT=1:1, MIX) on inhibition of bone loss and chondral defect. To examine their activities, we measured the alkaline phosphatase (ALP) activity in human osteoblast-like MG-63 cells and performed tartrate-resistant acid phosphate (TRAP) staining in osteoclast differentiated from Raw264.7 cells. To investigate the influence on chondrocyte differentiation, we performed alcian-blue staining in chondrocyte differentiated from ATDC5 cells. All of SC, GT and MIX did not increase ALP activity in MG-63 cells. However, SC and mixture (SC:GT=1:1, MIX) significantly inhibited osteoclastic differentiation. And they also induced chondrocyte differentiation. These results suggest that SC and GT may have a potential for the treatment of bone loss and chondral defect by suppression of osteoclast differentiation and stimulation of chondrocyte differentiation. Therefore, clarification of their mechanisms and active components will be needed.】

A new osteopetrosis mutant mouse strain (ntl) with odontoma‐like proliferations and lack of tooth roots

A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.

Effect of cyclic mechanical loading on osteoclast recruitment in periodontal tissue

It is well accepted that cyclic mechanical loading induces osteoclastogenesis in periodontal tissue, but its molecular mechanisms are not well understood, in part because of a lack of appropriate models. In this study, we investigated a novel device that allows cyclic mechanical loading to be performed in a well-controlled manner. Furthermore, by employing this model, the effect of cyclic loading on osteoclast recruitment in the periodontal tissue was described. By using a newly developed device, the cyclic loading of 20 n (reference loading corresponding to the fracture hardness of dietary pellets) and two excessive loadings (i.e. 30 and 40 n) were applied to maxillary right molars in rats for up to 7 d, and osteoclast recruitment in the periodontal tissue was evaluated by analyzing relevant marker proteins using immunohistochemistry. Osteoclastogenesis was induced by day 3 within alveolar bone subjected to a compression force of 30 n. With both 30 and 40 n loadings, cells that were positive to for tartrate-resistant acid phosphate, receptor activator of nuclear factor-kappaB ligand and osteoprotegerin were significantly increased in the alveolar bone/periodontal ligament in a time-dependent manner. A new device was developed that allows various levels of cyclic mechanical loading to be exerted. By using this device in rats, early events of osteoclast recruitment in the periodontal tissues were observed with excessive loadings in a time-dependent manner, indicating the usefulness of this model.

Lipopolysaccharides (LPS) induce the differentiation of human monocytes to osteoclasts in a tumour necrosis factor (TNF) α-dependent manner: A link between infection and pathological bone resorption

The degradation of bone is a serious consequence of persistent bacterial infection, including periodontitis, infection-associated non-unions or osteomyelitis. To test the hypothesis that infection and inflammatory conditions promote the differentiation of monocytes to bone-resorbing osteoclasts, highly purified monocytes, or alternatively, cells of the promyeloid cell line U937, differentiated to monocyte-like cells, were cultivated in the presence of lipopolysaccharides (LPS) for up to 30 days. After 2–4 days, a massive aggregation of the cells was observed, after 15–20 days multinuclear cells with the morphological characteristics of osteoclasts became apparent. These cells expressed the osteoclast-typical proteins tartrate-resistant acid phosphate (TRAcP) and cathepsin K. Moreover, these cells formed resorption pits on calcium phosphate coated cover slips or ivory slices. To test whether the differentiation of the monocytes to osteoclast-like cells was mediated by tumour necrosis factor α (TNFα) secreted by the cells in culture, an antibody directed against TNFα was added together with LPS. Differentiation to osteoclast-like cells was inhibited, suggesting a paracrine effect of locally produced TNFα. In conclusion, we propose that local bacterial infections could create a microenvironment that promotes the generation of bone resorbing cells, which, in turn, could contribute to the infection-associated osteolysis.

Wnt / RANKL‐mediated bone growth promoting effects of blueberries in weanling rats

We studied the effects of dietary blueberry supplementation on bone growth in weanling rats. Weanling male and female rats were fed AIN‐93G semi‐purified diets supplemented with 10% whole blueberry powder for 14 and 30 days beginning on PND 21. In both sexes tibial bone mineral density and content and cortical bone thickness were all increased dramatically in blueberry‐fed rats as compared to control casein AIN‐93G fed animals (p &lt; 0.05). The serum bone formation marker alkaline phosphatase (ALP) increased but the resorption marker RatLaps decreased in blueberry‐fed rats. Ex‐vivo osteoclastogenesis and osteoblastogenesis assays and bone histomorphometric studies revealed decreased osteoclastogenesis and increased osteoblastogenesis in blueberry‐fed rats compared to controls (p &lt; 0.05). In contrast to control diets, gene expression from both bone and bone marrow of blueberry‐fed rats showed dramatic decreases in RANKL and tartrate resistant acid phosphate (TRAP) mRNAs but increases in bone formation markers ALP, osteocalcin, RUNX2 and the Wnt signaling related components Wnt5a, LRP5, LRP6 mRNAs. Beta‐catenin protein level was increased in blueberry‐fed rats in bone tissue relative to control rats (p &lt; 0.05). These results indicate significant stimulation of bone growth associated with blueberry consumption in early development. Supported in part by ARS CRIS #6251‐51000‐005‐03S.

Phosphocalcic Metabolism after Biliopancreatic Diversion

Malabsorptive techniques to treat morbid obesity have been followed by alterations in phosphocalcic metabolism. Knowledge of the preoperative situation is important to assess the influence of these techniques on phosphocalcic metabolism and to consider treatments for these alterations. 61 consecutive morbidly obese patients (50 women, 11 men, age 19 to 63 years) having had biliopancreatic diversion (BPD) were studied in a prospective manner. Preoperative and postoperative levels of calcium, phosphorus, 25-hydroxyvitamin D, tartrate resistant acid phosphate, plasma parathormone (PTH), tubular absorption of phosphate, and urinary calcium and pyridinolines were analyzed, as well as the potential risk factors for their alterations. Follow-up of all patients was a minimum of 4 years. Before BPD, 42.3% of patients presented an increase in PTH and 54% a decrease in the 25-OH vitamin D, but the values of calcium and plasma phosphorus maintained at normal level. 81.8% of the patients with an increase in the PTH maintained high levels after BPD, while 60% of those with a normal preoperative PTH also presented hyperparathyroidism 4 years after the intervention. A correlation between the levels of plasma PTH and body mass index was not found. Morbid obesity is accompanied by a high percentage of hyperparathyroidism. BPD produces malabsorption of vitamin D during the first years, favoring the persistence or appearance of hyperparathyroidism. It is important to recognize and treat the secondary hyperparathyroidism. The postoperative period could necessitate more energetic interventions to get more efficient control of the phosphocalcic metabolism.

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor α-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.

Effect of etodolac, a COX-2 inhibitor, on neuropathic pain in a rat model

Etodolac, a cyclooxygenase-2 inhibitor, may alleviate nociceptive pain and inhibit the activation of osteoclasts. The aim of the present study was to determine whether etodolac can alleviate heat-evoked hyperalgesia and investigate its possible protective effects on osteoporosis induced by chronic constriction injury (CCI) in rats. A CCI to the sciatic nerve was performed, after which the rats received etodolac orally in a volume of 2 ml at 0, 1, and 10 mg/kg/day for 1 to 5 weeks following surgery (experiment 1); at 0 and 10 mg/kg/day for 1 day to 5 weeks following surgery (experiment 2); and at 0 mg/kg/day for 1 to 5 weeks, 10 mg/kg/day for 1 to 2 weeks after surgery, or 10 mg/kg/day for 1 to 3 weeks after surgery (experiment 3). Paw withdrawal latency after exposure to heat, bone mineral content (BMC) and bone mineral density (BMD) in the whole tibial bone, and the number of tartrate resistant acid phosphate (TRAP)-positive multinucleated osteoclasts were measured. Etodolac alleviated heat-evoked hyperalgesia in the CCI rats and the increase in number of TRAP-positive multinucleated osteoclasts on the CCI-side was abrogated, however, it did not inhibit the decrease of BMC and BMD on the CCI-side. Our results suggest that etodolac is useful for treatment of neuropathic pain.

Osteoporosis following chronic constriction injury of sciatic nerve in rats.

Osteopathic changes sometimes occur in patients with complex regional pain syndrome (reflex sympathetic dystrophy and causalgia). We aimed to investigate whether such osteopathic changes occurred in rats with chronic constriction injury (CCI) of the sciatic nerve. A CCI of the sciatic nerve was established in a unilateral hind limb in 39 adult Sprague-Dawley rats, which were killed 1, 2, 3, 5, or 7 weeks after the CCI procedure. Bone mineral content (BMC) and bone mineral density (BMD) in extracted tibial bones were measured using a dual-energy X-ray absorptiometer, and the number of osteoclasts in the metaphyseal regions was counted by the use of tartrate-resistant acid phosphate (TRAP) staining. BMC was significantly decreased, compared with that of the contralateral side, 1 to 7 weeks after CCI, and BMD was decreased 2 to 7 weeks after the procedure in the ipsilateral tibial bones, compared with BMD in the contralateral bones. The number of TRAP-positive multinucleated osteoclasts in the ipsilateral bones was significantly increased at 2, 3, and 5 weeks after the CCI, when compared with the number of these osteoclasts in the contralateral bones. The results of the present study demonstrate that osteopathic changes are associated with chronic constrictive injury of the sciatic nerve.