bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Engineering of Human Autogenous Cartilage
Authors: Anderer Ursula , Dr. Libera Jeanette 
Published: April 2006

Abstract
A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro-engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three-dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to human serum.
Autosomal Dominant Hypocalcemia Caused by a Novel Mutation in the Loop 2 Region of the Human Calcium Receptor Extracellular Domain
Authors: Hu Jianxin , Mora Stefano , Colussi Giacomo , Proverbio Maria Carla , Jones Kendra A. , Bolzoni Laura , De Ferrari Maria E. , Civati Giovanni , Spiegel Allen M. 
Published: April 2006

Abstract
We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was comparable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified in subjects with ADH. Functional characterization by the extracellular Ca2+ ion ([Ca2+]0)-stimulated phosphoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was left-shifted in Ca2+ sensitivity. This biochemical gain-of-function is comparable with that seen in other missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions (R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent N124R caused a left shift in Ca2+ sensitivity. Thus, a specific substitution, not merely a mutation of the N124 residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We present a hypothesis to explain receptor activation by mutations in this region based on the recently described three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1).
Colocalization of Intracellular Osteopontin With CD44 Is Associated With Migration, Cell Fusion, and Resorption in Osteoclasts
Authors: Suzuki K. , Zhu B. , Rittling S. R. , Denhardt D. T. , Goldberg H. A. , Mcculloch C. A. G. , Sodek J. 
Published: April 2006

Abstract
Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and β1- and β3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.
Prevention of Bone Loss in Ovariectomized Rats by Combined Treatment With Risedronate and 1α,25-Dihydroxyvitamin D3
Authors: Erben Reinhold G. , Mosekilde Lis , Thomsen Jesper S. , Weber Karin , Stahr Kerstin , Leyshon Alyson , Smith Susan Y. , Phipps Roger 
Published: April 2006

Abstract
Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1α,25-dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in rats. One hundred ten female 4-month-old Sprague-Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 μg or 0.1 μg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency-induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.
Is Leptin the Link Between Fat and Bone Mass?
Authors: Thomas Thierry , Burguera Bartolome 
Published: April 2006

AbstractRecently, leptin has emerged as a potential candidate responsible for protective effects of fat on bone tissue. However, it remains difficult to draw a clear picture of leptin effects on bone metabolism because published data are sometimes conflicting or apparently contradictory. Beyond differences in models or experimental procedures, it is tempting to hypothesize that leptin exerts dual effects depending on bone tissue, skeletal maturity, and/or signaling pathway. Early in life, leptin could stimulate bone growth and bone size through direct angiogenic and osteogenic effects on stromal precursor cells. Later, it may decrease bone remodeling in the mature skeleton, when trabecular bone turnover is high, by stimulating osteoprotegerin (OPG) expression. Leptin negative effects on bone formation effected through central nervous system pathway could counterbalance these peripheral and positive effects, the latter being predominant when the blood‐brain barrier permeability decreases or the serum leptin level rises above a certain threshold. Thus, the sex‐dependent specificity of the relationship between leptin and bone mineral density (BMD) in human studies could be, at least in part, caused by serum leptin levels that are two‐ to threefold higher in women than in men, independent of adiposity. Although these hypotheses remain highly speculative and require further investigations, existing studies consistently support the role of leptin as a link between fat and bone.
Expression of a 1,25‐Dihydroxyvitamin D3 Membrane‐Associated Rapid‐Response Steroid Binding Protein During Human Tooth and Bone Development and Biomineralization
Authors: Mesbah Mohand , Nemere Ilka , Papagerakis Petros , Nefussi Jean‐Raphael , Orestes‐Cardoso Silvana , Nessmann Catherine , Berdal Ariane 
Published: April 2006

AbstractThe calciotropic hormone 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] has been established to control skeletal tissue formation and biomineralization via the regulation of gene expression. This action involves the well‐characterized nuclear 1,25(OH)2D3 receptor. However, it has been recognized that several cellular responses to 1,25(OH)2D3 may not to be related to the exclusive nuclear receptor. Indeed, this secosteroid is able to generate rapid responses that have been proposed to be mediated by interactions of the ligand, which is a putative cell membrane‐associated rapid‐response steroid (MARRS) binding protein for 1,25(OH)2D3 [1,25D3‐MARRS]. The nongenomic pathway of 1,25(OH)2D3 was studied here in detail by immunolocalization of the 1,25D3‐MARRS during the specific context of human prenatal development. Western blotting with proteins extracted from 4 week‐ to 27‐week‐old embryos was performed, evidencing a 65‐kDa molecular species recognized by antibody Ab 099 generated against synthetic peptides corresponding to the N terminus of the 1,25D3‐MARRS from chick intestinal basolateral membranes. Based on this biochemical conservation of protein in the human species, the temporospatial expression patterns were established in the craniofacial skeleton at the same ages. Comparative analysis was performed in teeth and bones from early morphogenesis to terminal cell differentiation and extracellular biomineralization. The data show the potential implication of 1,25D3‐MARRS in the heterogeneous cell population including ameloblasts, odontoblasts, osteoblasts, and osteoclasts. The epithelial‐mesenchymal cascade related to odontogenesis was coincident with a sequence of up‐ and down‐regulation of immunoreactive 1,25D3‐MARRS. Biomineralization was associated with a striking up‐regulation in the adjoining secretory cells in all tissues. Finally, osteoclasts appeared also to express the 1,25D3‐MARRS during these early phases of bone modeling. Previously obtained data of the nuclear vitamin D receptor (VDR) expression and this study on 1,25D3‐MARRS suggest the existence of cross‐talk between the genomic and nongenomic pathways during human development.
Radius Bone Strength in Bending, Compression, and Falling and Its Correlation With Clinical Densitometry at Multiple Sites
Authors: Lochmüller Eva‐Maria , Lill Christoph A. , Kuhn Volker , Schneider Erich , Eckstein Felix 
Published: April 2006

AbstractThis study comprehensively analyzes the ability of site‐specific and nonsite‐specific clinical densitometric techniques for predicting mechanical strength of the distal radius in different loading configurations. DXA of the distal forearm, spine, femur, and total body and peripheral quantitative computed tomography (pQCT) measurements of the distal radius (4, 20, and 33%) were obtained in situ (with soft tissues) in 129 cadavers, aged 80.16 ± 9.8 years. Spinal QCT and calcaneal quantitative ultrasound (QUS) were performed ex situ in degassed specimens. The left radius was tested in three‐point bending and axial compression, and the right forearm was tested in a fall configuration, respectively. Correlation coefficients with radius DXA were r = 0.89, 0.84, and 0.70 for failure in three‐point bending, axial compression, and the fall simulation, respectively. The correlation with pQCT (r = 0.75 for multiple regression models with the fall) was not significantly higher than for DXA. Nonsite‐specific measurements and calcaneal QUS displayed significantly (p < 0.01) lower correlation coefficients, and QUS did only contribute to the prediction of axial failure stress but not of failure load. We conclude that a combination of pQCT parameters involves only marginal improvement in predicting mechanical strength of the distal radius, nonsite‐specific measurements are less accurate for this purpose, and QUS adds only little independent information to site‐specific bone mass. Therefore, the noninvasive diagnosis of loss of strength at the distal radius should rely on site‐specific measurements with DXA or pQCT and may be the earliest chance to detect individuals at risk of osteoporotic fracture.
A New Method for In Vivo Analysis of Parathyroid Hormone–Calcium Set Point in Mice
Authors: Imanishi Yasuo , Hall Charles , Sablosky Marilyn , Brown Edward M. , Arnold Andrew 
Published: April 2006

AbstractAlthough methods for measuring the parathyroid hormone (PTH)‐calcium set point in vivo in humans and large animals exist, translating such methods to the increasingly important mouse model poses considerable challenges. We also found that manipulation of dietary calcium does not yield sufficiently high or low serum calcium levels to achieve the minimum and maximum PTH levels needed for set point estimations. Therefore, we developed a new method for in vivo evaluation of the relative set point in mice. Intraperitoneal injection of calcium gluconate caused progressive increases in serum calcium over 120 minutes, with corresponding decreases in serum PTH levels. Intraperitoneal injection of Na2‐EGTA caused a nadir of serum calcium at 30 minutes and a corresponding peak value of serum PTH. The lowest and highest serum calcium concentrations achieved were 6.7 mg/dl and 10.0 mg/dl, respectively. Linear regression analyses indicated high correlation coefficients (serum calcium vs. serum PTH; r = 0.969). To obtain the additional data points needed for set point estimation, blood was collected 30 minutes after intraperitoneal injection by tail nicking from three mice once a day, on 8 consecutive days, using multiple doses of calcium gluconate or Na2‐EGTA. The lowest and highest serum calcium concentrations achieved were 5.0 mg/dl and 11.4 mg/dl. Maximum and minimum PTH levels were indeed observed, and a sigmoidal curve with a set point of 7.8 mg/dl was readily generated by the four‐parameter model that was fit using a nonlinear mixed effects statistical approach. This protocol for in vivo set point analysis should be applicable in the future study of multiple genetically engineered and pharmacologically treated mouse models.
Differential Regulation of Chemokine Secretion in Tuberculous and Staphylococcal Osteomyelitis
Authors: Wright Kathleen M. , Friedland Jon S. 
Published: April 2006

AbstractBone infection or osteomyelitis is characterized by uncontrolled inflammation and destructive bone loss although little is known about immunopathogenesis of infection. We investigated control of chemokine secretion from osteoblasts infected with either Mycobacterium tuberculosis, which normally elicits a granulomatous host response, or Staphylococcus aureus, which drives a host response dominated by neutrophil influx. We show that M. tuberculosis infection of cultured and primary osteoblasts induces extensive secretion of the chemokines interleukin (IL)‐8, inducible protein (IP) 10, RANTES, and monocyte chemoattractant protein (MCP) 1 within 72 h (1630 ± 280 pg/ml per 4 × 105 cells, 74,130 ± 8480 pg/ml per 4 × 105 cells, 18,330 ± 3040 pg/ml per 4 × 105 cells, and 13,8670 ± 13,340 pg/ml per 4 × 105 cells, respectively, for MG‐63 osteoblasts). S. aureus infection also results in secretion of these chemokines but secretion is delayed and of lesser magnitude (210 ± 10 pg/ml per 4 × 105 cells, 11,570 ± 1240 pg/ml per 4 × 105 cells, 930 ± 34 pg/ml per 4 × 105 cells, and 13,770 ± 720 pg/ml per 4 × 105 cells for IL‐8, IP‐10, RANTES, and MCP‐1, respectively). The minimal up‐regulation of secretion of the neutrophil attractant IL‐8 in staphylococcal infection is both striking and unexpected. In both infections, chemokine secretion was dependent on the presence of live organisms. Differences in kinetics and magnitude of chemokine secretion are associated with distinct patterns of mRNA expression, as assessed by ribonuclease protection assay (RPA) and reverse‐transcription polymerase chain reaction (RT‐PCR). In addition, nuclear localization of the transcription factor activator protein (AP) 1 in M. tuberculosis‐infected osteoblasts also is distinct as compared with S. aureus‐infected cells. In summary, this study shows that osteoblasts have an important pathogen‐specific role in control of chemokine gene expression and secretion during the human immune response to osteomyelitis.
Idiopathic Hyperphosphatasia and TNFRSF11B Mutations: Relationships Between Phenotype and Genotype
Authors: Chong Belinda , Hegde Madhuri , Fawkner Matthew , Simonet Scott , Cassinelli Hamilton , Coker Mahmut , Kanis John , Seidel Joerg , Tau Cristina , Tüysüz Beyhan , Yüksel Bilgin , Love Donald , Cundy Tim 
Published: April 2006

Abstract
Homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin, were found in affected members from six of nine families with idiopathic hyperphosphatasia. The severity of the phenotype was related to the predicted effects of the mutations on osteoprotegerin function.
Introduction: Idiopathic hyperphosphatasia (IH) is a rare high bone turnover congenital bone disease in which affected children are normal at birth but develop progressive long bone deformities, fractures, vertebral collapse, skull enlargement, and deafness. There is, however, considerable phenotypic variation from presentation in infancy with severe progressive deformity through to presentation in late childhood with minimal deformity. Two recent reports have linked idiopathic hyperphosphatasia with deletion of, or mutation in, the TNFRSF11B gene that encodes osteoprotegerin (OPG), an important paracrine modulator of RANKL-mediated bone resorption.
Materials and Methods: We studied subjects with a clinical diagnosis of IH and unaffected family members from nine unrelated families. Clinical, biochemical, and radiographic data were collected, and genomic DNA examined for mutations in TNFRSF11B. The relationship between the mutations, their predicted effects on OPG function, and the phenotype were then examined.
Results: Of the nine families studied, affected subjects from six were homozygous for novel mutations in TNFRSF11B. Their parents were heterozygous, consistent with autosomal recessive inheritance. Four of the six mutations occurred in the cysteine-rich ligand-binding domain and are predicted to disrupt binding of OPG to RANKL. Missense mutations in the cysteine residues, predicted to cause major disruption to the ligand-binding region, were associated with a severe phenotype (deformity developing before 18 months age and severe disability), as was a large deletion mutation. Non-cysteine missense mutations in the ligand-binding domain were associated with an intermediate phenotype (deformity recognized around the age of 5 years and an increased rate of long bone fracture). An insertion/deletion mutation at the C-terminal end of the protein was associated with the mildest phenotype.
Conclusion: Mutations in TNFRSF11B account for the majority of, but not all, cases of IH, and there are distinct genotype-phenotype relationships.
12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by Suppressing RANKL-Induced NF-κB Activation
Authors: Wang Cathy , Steer James H , Joyce David A , Yip Kirk HM , Zheng Ming H , XU Jiake 
Published: April 2006

Abstract
The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF-κB nuclear translocation, NF-κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-κβ activation.
Introduction: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling.
Materials and Methods: RANKL-induced RAW264.7 cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-κB nuclear translocation, NF-κB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-κβ, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways.
Results: We found that TPA inhibited RANKL-induced RAW264.7 cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-κβ activation in RAW264.7 cells, but it suppresses the RANKL-induced NF-κβ activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-κβ activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-κβ complexes consisted of C-Rel, NF-κB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW264.7 cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation.
Conclusion: Given that NF-κB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-κβ during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.