bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential Treatment with Basic Fibroblast Growth Factor and Parathyroid Hormone Restores Lost Cancellous Bone Mass and Strength in the Proximal Tibia of Aged Ovariectomized Rats
Authors: Wronski T. J. , Ratkus A. M. , Thomsen J. S. , Vulcan Q. , Mosekilde Li.
Published: April 2006

This study was designed to determine whether sequential treatment with basic fibroblast growth factor (bFGF) and parathyroid hormone (PTH) can restore lost cancellous bone mass and strength at a severely osteopenic skeletal site in aged ovariectomized (OVX) rats. Female Sprague-Dawley rats were subjected to sham surgery or ovariectomy at 3 months of age and maintained untreated for the first year after surgery. At 15 months of age, groups of baseline control and OVX rats were killed and catheters were inserted in the jugular veins of all remaining rats. Two groups of OVX rats were injected intravenously (iv) daily with bFGF for 14 days at a dose of 200 μg/kg body weight. At the end of bFGF treatment, one group was killed whereas the other group was subjected to 8 weeks of treatment with synthetic human PTH 1-34 [hPTH(1-34)] consisting of subcutaneous (sc) injections 5 days/week at a dose of 80 μg/kg. Another group of OVX rats was treated iv with vehicle for 2 weeks followed by treatment with PTH alone for 8 weeks. Other groups of sham-operated control rats and OVX rats were treated iv and sc with vehicle alone. The right proximal tibia from each rat was processed undecalcified for quantitative bone histomorphometry and the left proximal tibia was subjected to biomechanical testing. Baseline and vehicle-treated OVX rats were severely osteopenic because their tibial cancellous bone volumes were less than 5% compared with mean values of 20.3% and 15.0% in baseline and vehicle-treated control rats, respectively. Treatment of OVX rats for 2 weeks with bFGF alone did not significantly increase tibial cancellous bone volume but induced marked increases in osteoid volume, osteoblast surface, and osteoid surface. Sequential treatment of aged OVX rats with bFGF and PTH increased tibial cancellous bone volume (15.1%) and load to failure to at least the level of vehicle-treated control rats. Tibial cancellous bone volume (10.8%) and load to failure also were significantly increased by treatment with PTH alone, and these variables were not significantly different from those of OVX rats treated with bFGF + PTH. However, tibial ash density was significantly greater in OVX rats treated sequentially with bFGF and PTH compared with OVX rats treated with PTH alone. Our findings suggest that sequential treatment with bFGF and PTH may be useful for restoration of lost cancellous bone in the severely osteopenic, estrogen-deplete skeleton, but it cannot be concluded with certainty that this sequential treatment has a greater bone restorative effect than treatment with PTH alone.
Relation Between Vitamin D Insufficiency, Bone Density, and Bone Metabolism in Healthy Postmenopausal Women
Authors: Mezquita-Raya Pedro , Muñoz-Torres Manuel , De Dios Luna Juan , Luna Victoria , Lopez-Rodriguez Francisca , Torres-Vela Elena , Escobar-Jiménez Fernando
Published: April 2006

Although only few postmenopausal women exhibit biochemical signs of hypovitaminosis D, vitamin D insufficiency has been shown to have adverse effects on bone metabolism and could be an important risk factor for osteoporosis and fracture. We determined serum levels of 25-hydroxyvitamin D [25(OH)D], intact parathyroid hormone (iPTH), bone turnover markers, dietary calcium intake, and bone mineral density (BMD; measured by dual X-ray absorptiometry) in 161 consecutive ambulatory women, healthy except for osteoporosis, referred to a bone metabolic unit. The prevalence of vitamin D insufficiency [25(OH)D ≤ 15 ng/ml] was 39.1%. 25(OH)D was lower in the osteoporotic subjects (15.7 ± 5.3 ng/ml vs. 21.8 ± 9.7 ng/ml; p < 0.001). After controlling for all other variables, lumbar spine (LS) BMD was found to be significantly associated with 25(OH)D, body mass index (BMI), and years after menopause (YSM) (R2 = 0.253; p < 0.001). For femoral neck (FN), significant independent predictors of BMD were YSM, BMI, iPTH, and 25(OH)D (R2 = 0.368; p < 0.001). The probability of meeting osteoporosis densitometric criteria was higher in the vitamin D insufficiency group (odds ratio [OR], 4.17, 1.83-9.48) after adjusting by YSM, BMI, iPTH, and dietary calcium intake. Our study shows that vitamin D insufficiency in an otherwise healthy postmenopausal population is a common risk factor for osteoporosis associated with increased bone remodeling and low bone mass.
Expression Profiles of Receptor Activator of Nuclear Factor κB Ligand, Receptor Activator of Nuclear Factor κB, and Osteoprotegerin Messenger RNA in Aged and Ovariectomized Rat Bones
Authors: Ikeda Tohru , Utsuyama Masanori , Hirokawa Katsuiku
Published: April 2006

The receptor activator of nuclear factor-κB ligand (RANKL; also known as tumor necrosis factor-related activation-induced cytokine [TRANCE], osteoprotegerin ligand [OPGL], and osteoclast differentiation factor [ODF]) is a transmembrane ligand expressed in osteoblasts and bone marrow stromal cells. It binds to RANK, which is expressed in osteoclast progenitor cells, and induces osteoclastogenesis. OPG, a decoy receptor for RANKL, also binds to RANKL, and competitive binding of RANKL with RANK or OPG is thought to regulate bone metabolism. To investigate roles of the RANKL/RANK/OPG system in pathophysiological conditions, the expression of RANKL, RANK, and OPG messenger RNA (mRNA) was analyzed in bones of aged and ovariectomized rats by means of in situ hybridization. In the control 8-week-old male and sham-operated female rat bones, the expression of RANKL mRNA was detected in hypertrophic chondrocytes of the growth plate and some periosteal and endosteal mesenchymal cells. The expression of RANK mRNA was detected in osteoclast-like cells and mononuclear cells in contact with the cortical and trabecular bones. The expression of OPG mRNA was detected in proliferating chondrocytes and osteocytes. In the 2.5-year-old rat bones, the expression of RANKL, RANK, and OPG mRNA tended to decrease except for the endosteal region. In the ovariectomized rat bones, the expression of RANKL, RANK, and OPG mRNA increased, and high expression of OPG mRNA was induced in resting chondrocytes and osteocytes. These results suggest that estrogen deficiency stimulates the RANKL/RANK/OPG system and induces OPG in cells that have been thought to be less important for bone metabolism.
Leptin Is Expressed in and Secreted from Primary Cultures of Human Osteoblasts and Promotes Bone Mineralization
Authors: Reseland Janne E. , Syversen Unni , Bakke Ingunn , Qvigstad Gunnar , Eide Liv G. , Hjertner Øyvind , Gordeladze Jan O. , Drevon Christian A.
Published: April 2006

The adipose hormone leptin and its receptor are important for regulation of food intake and energy metabolism. Leptin also is involved in the growth of different tissues. In this study, we show the expression of leptin in primary cultures of normal human osteoblasts (hOBs) as evidenced by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Release of leptin into the medium also was found. Leptin was not detected in commercially available hOBs (NHOst) or in three different human monoclonal osteosarcoma cell lines. Leptin expression was observed in OBs in the mineralization and/or the osteocyte transition period but not during the matrix maturation period. Furthermore, hOBs and osteosarcoma cell lines expressed the long signal-transducing form of the leptin receptor (OB-Rb) as shown by RT-PCR. We observed no significant changes in leptin or OB-Rb genes in hOBs after incubation with recombinant leptin, indicating no autoregulation of the leptin expression. Incubation of both hOBs entering the mineralization phase and osteosarcoma cell lines with recombinant leptin markedly increased the number of mineralized nodules as shown by alizarin S staining. These findings indicate that leptin may be of importance for osteoblastic cell growth and bone mineralization.
Bone Reinnervation After Fracture: A Study in the Rat
Authors: Li Jian , Ahmad Tashfeen , Spetea Mariana , Ahmed Mahmood , Kreicbergs Andris
Published: April 2006

Reinnervation after tibial fracture in the rat was studied by analyzing the occurrence of growth-associated protein 43 (GAP-43), a marker for regenerating nerve fibers, and protein gene product 9.5 (PGP-9.5), a marker for mature nerve fibers, by immunohistochemistry. At 3 days postfracture, GAP-43-immunoreactive nerve fibers were first observed in the fracture hematoma and periosteum. At 7 days postfracture, abundant sprouting of GAP-43-positive fibers was seen in the callus, hyperplastic periosteum, and edge of fibrocartilage. In the latter region, the nerve fibers were nonvascular, showing dense ramifications and terminal sprouting close to chondroid cells. At 14 days and 21 days postfracture, many GAP-43-positive fibers were still sprouting into the fibrocartilage and new woven bone. Fine varicose GAP-43-positive fibers also were present in the bone marrow. In contrast to GAP-43, PGP-9.5-positive nerve fibers were observed only occasionally at 3 days postfracture but gradually increased in number from day 14 to 21. Our study shows that intense nerve regeneration occurs in early fracture healing partly unrelated to neovascularization. Considering that neuronal mediators have been shown to participate in local bone formation and resorption, the nerve regeneration observed may prove to be essential for delivery of neuronal mediators required for normal callus formation and/or neovascularization.
Selenium Deficiency-Induced Growth Retardation Is Associated with an Impaired Bone Metabolism and Osteopenia
Authors: Moreno-Reyes Rodrigo , Egrise Dominique , Nève Jean , Pasteels Jean-Lambert , Schoutens Andre
Published: April 2006

Although the importance of selenium for bone metabolism is unknown, some clinical conditions such as Kashin-Beck osteoarthropathy have been associated with selenium deficiency. Although selenium deficiency induces growth retardation in rats, it has not been established whether this growth inhibition is associated with changes in bone metabolism. We investigated the effect of selenium deficiency on bone metabolism in growing male rats fed a selenium-deficient diet for two generations (Se−). In Se− rats, erythrocyte glutathione peroxidase activity and plasma selenium concentration were strongly reduced compared with pair-fed selenium-adequate rats (Se+). Weight and tail length were reduced by 31% and 13% in the Se− rats, respectively (p < 0.001). The Se− diet was associated with a 68% reduction of pituitary growth hormone (GH; p = 0.01) and a 50% reduction of plasma insulin-like growth factor I (IGF-I; p < 0.001). Plasma calcium was lower and urinary calcium concentration was greater in Se− rats. This group had a 2-fold increase in parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in plasma. Plasma osteocalcin and urinary deoxypyridoline were reduced by 25% and 57% in the Se− rats (p < 0.001). Selenium deficiency resulted in a 23% and 21% reduction in bone mineral density (BMD) of the femur and tibia (p < 0.001) and this effect persisted after adjustment for weight in a linear regression model. A 43% reduction in trabecular bone volume of the femoral metaphysis (p < 0.001) was found in Se− rats. This experimental study shows that growth retardation induced by selenium deficiency is associated with impaired bone metabolism and osteopenia in second-generation selenium-deficient rats.
Pseudo-(Tumor-Induced) Rickets
Authors: Whyte Michael P. , Podgornik Michelle N. , Wollberg Valerie A. , Eddy Mark C. , McAlister William H.
Published: April 2006

An athletic 8-year-old boy developed severe muscle weakness over 2 years. At the age of 10 years, investigation for possible neuromuscular disease disclosed hypophosphatemia (1.8 mg/dl) and rickets. There was selective renal tubular wasting of inorganic phosphate (Pi) but no history of toxin exposure, familial bone or kidney disease, or biochemical evidence of vitamin D deficiency. Urine amino acid quantitation was unremarkable. Serum 1,25-dihydroxyvitamin D [1,25(OH)2D] concentration was in the lower half of the reference range. Our presumptive diagnosis was tumor-induced rickets; however, physical examination and bone scanning in search of a neoplasm were unrevealing. Soon after 1,25(OH)2D3 and Pi treatment began, muscle strength improved considerably. After 6 months of therapy, radiographic abnormalities were substantially better. During the next 6 years, physical examinations, a second bone scan, whole-body and nasal sinus magnetic resonance imaging, and octreotide scintigraphy were unremarkable. When his physes fused at the age of 16 years, assessment of his course showed excellent control of his rickets requiring decreasing doses of medication. Furthermore, fasting serum Pi levels and tubular maximum phosphorus/glomerular filtration (TmP/GFR) values had increased steadily and normalized after 3 years of treatment. Accordingly, therapy was stopped. Seven months after stopping medication, he continues to feel completely well. Fasting serum Pi levels, TmP/GFR, other biochemical parameters of bone and mineral homeostasis, creatinine clearance, and renal sonography are normal. Neither spontaneous or pharmacologic cure of tumor-induced rickets or osteomalacia nor a patient matching ours has been reported. His disorder, which we call pseudo-(tumor-induced) rickets, should be considered when investigation for oncogenic rickets or osteomalacia discloses no causal lesion. Consequently, prolonged medical therapy and futile searches for a neoplasm may be avoided.
Relationships Between Bone Mineral Density and Incident Vertebral Fracture Risk with Raloxifene Therapy
Authors: Sarkar Somnath , Mitlak Bruce H. , Wong Mayme , Stock John L. , Black Dennis M. , Harper Kristine D.
Published: April 2006

Although low absolute values of bone mineral density (BMD) predict increased fracture risk in osteoporosis, it is not certain how well increases in BMD with antiresorptive therapy predict observed reductions in fracture risk. This work examines the relationships between changes in BMD after 1 year or 3 years of raloxifene or placebo therapy and the risk for new vertebral fractures at 3 years. In the Multiple Outcomes of Raloxifene Evaluation (MORE) trial, 7705 postmenopausal women with osteoporosis were randomized to placebo or raloxifene 60 mg/day or 120 mg/day. Relationships between baseline BMD and changes in BMD from baseline with the risk of new vertebral fractures were analyzed in this cohort using logistic regression models with the raloxifene doses pooled. As has been observed in other populations, women with the lowest baseline lumbar spine or femoral neck BMD in the MORE cohort had the greatest risk for vertebral fractures. Furthermore, for any percentage change, either increase or decrease in femoral neck or lumbar spine BMD at 1 year or 3 years, raloxifene-treated patients had a statistically significantly lower vertebral fracture risk compared with placebo-treated patients. The decrease in fracture risk with raloxifene was similar across the range of percentage change in femoral neck BMD observed at 3 years; patients receiving raloxifene had a 36% lower risk of vertebral fracture compared with those receiving placebo. At any percentage change in femoral neck and lumbar spine BMD observed at 1 year, raloxifene treatment decreased the risks of new vertebral fractures at 3 years by 38% and 41%, respectively. The logistic regression model showed that the percentage changes in BMD with raloxifene treatment accounted for 4% of the observed vertebral fracture risk reduction, and the other 96% of the risk reduction remains unexplained. The present data show that the measured BMD changes observed with raloxifene therapy are poor predictors of vertebral fracture risk reduction with raloxifene therapy.
Identification of the Functional Domain of Osteoclast Inhibitory Peptide-1/hSca
Authors: Koide Masanori , Kurihara Noriyoshi , Maeda Hidefumi , Reddy Sakamuri V.
Published: April 2006

Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-1/human Sca [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1 c-peptide (10-100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c-peptide (10-100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1 and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment.
Disruption of the p53 Gene Results in Preserved Trabecular Bone Mass and Bone Formation After Mechanical Unloading
Authors: Sakai Akinori , Sakata Takeshi , Tanaka Shinya , Okazaki Ryuji , Kunugita Naoki , Norimura Toshiyuki , Nakamura Toshitaka
Published: April 2006

We tested the hypothesis that mechanical unloading facilitates signaling of p53, an important modulator of cell cycling and apoptosis, in bone marrow cells and thereby reduces trabecular bone volume (BV). We performed histomorphometric analyses and bone marrow cell cultures in tail-suspended (TS) p53 null (p53−/−) and wild-type (p53+/+) mice. Eight-week-old male mice were assigned to four groups after 1-week acclimatization: p53+/+ + ground control (GC), p53+/++TS, p53−/−+GC, and p53−/−+TS. Bilateral tibial samples were used for analysis. The histomorphometric parameters of trabecular structure, formation and resorption did not differ between the p53−/−+GC and p53+/++GC groups. Trabecular BV in p53+/++TS mice was significantly reduced to 45% of that in the p53+/++GC group after one week of TS. In contrast, BV in p53−/−+TS mice was preserved at the same level as that in the p53−/−+GC group. The bone formation rate (BFR) was significantly reduced in p53+/++TS but not in p53−/−+TS mice. Unloading significantly increased trabecular osteoclast number (Oc.N) and surface in p53+/++TS mice compared with the p53+/++GC group, but the difference was not significant between p53−/−+TS and p53−/−+GC mice. In bone marrow cell culture, the numbers of alkaline phosphatase-positive (ALP+) colony-forming units fibroblastic (CFU-f) and mineralized nodules were significantly reduced in p53+/++TS, but not p53−/−+TS mice. [3H]thymidine incorporation into bone marrow cells was higher in p53−/− mice than in p53+/+ mice, independent of mechanical loading or unloading. Flow cytometric cell cycle analysis revealed that unloading significantly increased the percentage of hypoploid bone marrow cells in p53+/+ mice relative to that in p53+/++GC mice, but there was no significant difference in ploidy between p53−/−+TS and p53−/−+GC mice. Expression levels of p53 and p21 mRNAs were enhanced after TS in bone marrow cells from p53+/+ mice. Our data show that trabecular bone mass and bone formation were preserved after tail-suspension in p53−/− mice, closely associated with ALP+ CFU-f and mineralized nodule formation in marrow cultures obtained from tibias of p53−/− mice. We speculate that bone loss due to mechanical unloading may be related to facilitation of intracellular p53-p21 signaling.
Sequence Analysis of Measles Virus Nucleocapsid Transcripts in Patients with Paget's Disease
Authors: Friedrichs William E. , Reddy Sakamuri V. , Bruder Jan M. , Cundy Tim , Cornish Jillian , Singer Frederick R. , Roodman G. David
Published: April 2006

It has been debated for almost 30 years whether Paget's disease of bone results from paramyxoviral infection of osteoclasts (OCs). Paramyxoviral-like nuclear inclusions are found in OCs from patients with Paget's disease, and measles virus (MV) or canine distemper virus (CDV) messenger RNA (mRNA) transcripts have been detected by in situ hybridization in bone cells from pagetic lesions. Furthermore, immunocytochemical studies have shown the presence of several paramyxoviral species in OCs from patients with Paget's disease. However, others have been unable to detect paramyxoviral transcripts in bone samples from patients with Paget's disease or marrow cultures from involved sites of patients with Paget's disease. Furthermore, no one has been able to isolate an infectious virus from pagetic bone samples or marrow cells from patients with Paget's disease, and a full-length viral gene has not been sequenced from pagetic samples. In this study, we have obtained the full-length sequence for the MV nucleocapsid (MVNP) gene in bone marrow from an involved site from a patient with Paget's disease and more than 700 base pairs (bps) of MVNP sequence in 3 other patients with Paget's disease. These sequences were undetectable in four normal marrow samples studied simultaneously. The sequences from the patients contained multiple mutations that differed from the Edmonston strain MVNP gene. These findings are consistent with the presence of a chronic MV infection in affected sites from these patients with Paget's disease.
Complex Segregation Analysis of the Radiographic Phalanges Bone Mineral Density and Their Age-Related Changes
Authors: Livshits Gregory , Karasik David , Kobyliansky Eugene
Published: April 2006

The complex segregation analyses performed in our previous studies revealed a significant major gene (MG) effect on the age-adjusted cortical and cancellous bone mineral density (BMD) in two ethnically different populations, Chuvasha and Turkmenians. The aim of the present study was to test the hypothesis of pleiotropic MG control of three components of bone aging, that is, the baseline level of BMD (μgs), the age at onset of the bone mass loss (Tgs), and the rate of this loss over the years (αgs). Nuclear and more complex pedigrees from the same two ethnic samples were assessed for hand phalangeal BMD (Chuvasha, 1208 individuals, and Turkmenians, 643 individuals), and complex segregational analysis incorporating age and sex effects directly into MG penetrance function was carried out. The results of the present analysis clearly confirmed the existence of the putative MG and showed that the proportion of BMD variation attributable to this MG effect within the sex was remarkably similar in both populations and ranged between 34.7% and 35.2%. The most parsimonious model for BMD transmission in Chuvasha pedigrees additionally indicated significant residual correlation between siblings and clear sex differences in the annual rates of bone loss αgs. The latter was more than twice as high in females than that in males (0.086 SD vs. 0.033 SD per year). In Turkmenian pedigrees the most parsimonious model presented obvious evidence of the MG control of BMD baseline levels in both sexes with significantly lower baseline levels and younger age at onset (Tgs) in females. No clear MG effects were inferred on Tgs and/or αgs in either sample, either in males or in females. That is, the present study does not suggest MG × SEX × AGE interaction. We suppose that if the rate of age-related changes in phalangeal BMD is genetically determined, then these are not the same genes as those affecting the BMD baseline levels.
Expansile Skeletal Hyperphosphatasia Is Caused by a 15-Base Pair Tandem Duplication inTNFRSF11AEncoding RANK and Is Allelic to Familial Expansile Osteolysis
Authors: Whyte Michael P. , Hughes Anne E.
Published: April 2006

Expansile skeletal hyperphosphatasia (ESH) is a singular disorder characterized in the year 2000 in a mother and daughter with early-onset deafness, premature loss of teeth, progressive hyperostotic widening of long bones causing painful phalanges in the hands, accelerated bone remodeling, and episodic hypercalcemia likely inherited as a highly penetrant, autosomal dominant trait. Absence of large osteolytic lesions with cortical thinning in major long bones, together with bouts of hypercalcemia, indicated that ESH is not a variant of familial expansile osteolysis (FEO). Here, we investigated the molecular basis of ESH after three families with FEO were reported to have an identical 18-base pair tandem duplication (84dup18) in the signal peptide sequence of the TNFRSF11A gene that encodes receptor activator of nuclear factor-κB (RANK). We find that ESH is caused by a remarkably similar 15-base pair tandem duplication (84dup15) in TNFRSF11A. Hence, ESH and FEO are allelic diseases and ESH, like FEO, probably reflects increased activity in the skeleton of the RANK target, nuclear factor-κB (NF-κB).
Y-Position Collagen II Mutation Disrupts Cartilage Formation and Skeletal Development in a Transgenic Mouse Model of Spondyloepiphyseal Dysplasia
Authors: Gaiser Kelly G. , Maddox B. Kerry , Bann James G. , Boswell Bruce A. , Keene Douglas R. , Garofalo Silvio , Horton William A.
Published: April 2006

Mice were generated by pronuclear injection of a type II collagen transgene harboring an Arg789Cys (R789C) mutation that has been found in patients with spondyloepiphyseal dysplasia (SED). Expression was directed to cartilage by the murine Col2a1 promoter to examine the consequences of mutations involving the Y-position of the collagen helix Gly-X-Y triplet on skeletogenesis. The transgenic mice had very short limbs, short trunk, short snout, and cleft palate; they died at birth. Their growth plates were disorganized and collagen fibrils were sparse in cartilage matrix. When the transgene was expressed in RCS cells, there was no evidence that R789C-bearing collagen chains were incorporated into stable collagen molecules. Molecular modeling of the mutation raised the possibility that it destabilizes the collagen triple helix. Together our results suggest that Y-position mutations, such as R789C, can act in a dominant negative manner to destabilize collagen molecules during assembly, reducing their availability to form fibrils, the deficiency of which profoundly disturbs the template functions of cartilage during skeletogenesis.
Chondrogenic Activity of the Heparan Sulfate Proteoglycan Perlecan Maps to the N-terminal Domain I
Authors: French Margaret M. , Gomes Ronald R. , Timpl Rupert , Höök Magnus , Czymmek Kirk , Farach-Carson Mary C. , Carson Daniel D.
Published: April 2006

C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln Imut) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 μm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.
The Bone Lining Cell: Its Role in Cleaning Howship's Lacunae and Initiating Bone Formation
Authors: Everts V. , Delaissé J. M. , Korper W. , Jansen D. C. , Tigchelaar-Gutter W. , Saftig P. , Beertsen W.
Published: April 2006

In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell “rescues” bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process.
Klotho Gene Polymorphisms Associated With Bone Density of Aged Postmenopausal Women
Authors: Kawano Ken-Ichi , Ogata Naoshi , Chiano Mathias , Molloy Helen , Kleyn Patrick , Spector Tim D. , Uchida Motoyuki , Hosoi Takayuki , Suzuki Takao , Orimo Hajime , Inoue Satoshi , Nabeshima Yoichi , Nakamura Kozo , Kuro-O Makoto , Kawaguchi Hiroshi
Published: April 2006

Because mice deficient in klotho gene expression exhibit multiple aging phenotypes including osteopenia, we explored the possibility that the klotho gene may contribute to age-related bone loss in humans by examining the association between klotho gene polymorphisms and bone density in two genetically distinct racial populations: the white and the Japanese. Screening of single-nucleotide polymorphisms (SNPs) in the human klotho gene identified 11 polymorphisms, and three of them were common in both populations. Associations of the common SNPs with bone density were investigated in populations of 1187 white women and of 215 Japanese postmenopausal women. In the white population, one in the promoter region (G-395A, p = 0.001) and one in exon 4 (C1818T, p = 0.010) and their haplotypes (p < 0.0001) were significantly associated with bone density in aged postmenopausal women (≥65 years), but not in premenopausal or younger postmenopausal women. These associations were also seen in Japanese postmenopausal women. An electrophoretic mobility shift analysis revealed that the G-A substitution in the promoter region affected DNA-protein interaction in cultured human kidney 293 cells. These results indicate that the klotho gene may be involved in the pathophysiology of bone loss with aging in humans.
The Effects of Vitamin D Receptor Polymorphism on Secondary Hyperparathyroidism and Bone Density After Renal Transplantation
Authors: Giannini Sandro , D'Angelo Angela , Nobile Martino , Carraro Gianni , Rigotti Paolo , Silva-Netto Fatima , Pavan Silvia , Marchini Francesco , Zaninotto Martina , Carbonare Luca Dalle , Sartori Leonardo , Crepaldi Gaetano
Published: April 2006

Immunosuppresive treatment and secondary hyperparathyroidism (SHPT) are considered among the most important pathogenetic factors for postrenal transplant bone disease. The aim of this study was to investigate the relationships among vitamin D receptor (VDR) gene polymorphism, parathyroid hormone (PTH) levels, and bone density in renal transplant recipients. We enrolled 69 patients (47 men and 22 women; mean age, 47 ± 11 years) who had undergone kidney transplantation 51 ± 5 months before. All patients underwent an evaluation of the main biochemical parameters of bone metabolism as well as bone densitometry. VDR alleles were typed by a polymerase chain reaction (PCR) assay based on a polymorphic BsmI restriction site. When the patients were categorized according to the VDR genotype (BB, Bb, and bb), serum creatinine, and the cumulative doses of immunosuppressive drugs were similar across the groups. PTH levels higher than 80 pg/ml were found in 53.6% of the patients, with the highest values being detected in the bb VDR genotype (p < 0.05). PTH was significantly correlated to urinary type I collagen cross-linked N-telopeptide (NTx) values. Bone density was low in the whole population; however, spinal bone density was lower in the bb subgroup (p < 0.02). In the whole population, only PTH (p < 0.05) and body mass index (BMI; p < 0.01) were independent predictors of spinal bone density. When grouping the patients by the VDR gene polymorphism, only PTH continued to be an independent predictor of spinal bone density in the bb allele subgroup (R2 adj. = 0.17). We can conclude that the VDR genotype polymorphism affects bone density of renal transplant recipients via its effects on the severity of SHPT.
Continuous Inhibition of MAPK Signaling Promotes the Early Osteoblastic Differentiation and Mineralization of the Extracellular Matrix
Authors: Higuchi Chikahisa , Myoui Akira , Hashimoto Nobuyuki , Kuriyama Kohji , Yoshioka Kiyoko , Yoshikawa Hideki , Itoh Kazuyuki
Published: April 2006

We screened the small molecule compounds that stimulate osteogenesis by themselves or promote bone morphogenetic protein (BMP)-induced bone formation. We found that a specific inhibitor for MAPK/extracellular signal-regulated kinase kinase (MEK)-1, promoted the early osteoblastic differentiation and mineralization of extracellular matrix (ECM) in C2C12 pluripotent mesenchymal cells treated with recombinant human BMP-2 (rhBMP-2) and MC3T3-E1 preosteoblastic cells. ALP activity was synergistically increased by the treatment with a specific MEK-1 inhibitor PD98059 and rhBMP-2 in both cell lines. Twenty-five micromolar PD98059 promoted mineralization of ECM in rhBMP-2-treated C2C12 cells and MC3T3-E1 cells. In contrast, PD98059 reduced osteocalcin (OCN) secretion and its transcriptional level in rhBMP-2-treated C2C12 cells but increased its secretion and mRNA level in MC3T3-E1 cells. Stable expression of a dominant-negative MEK-1 mutant in C2C12 cells represented high ALP activity and low osteocalcin production in the presence of rhBMP-2, while a constitutively active mutant of MEK-1 attenuated both of them. Together, our results indicated that BMP-2-induced mineralization of ECM of pluripotent mesenchymal stem cells and preosteoblastic cells could be controlled by a fine tuning of the MAPK signaling pathway. Further, MEK-1 inhibitors would be useful for the promotion of bone formation, for instance, the treatments for delayed fracture healing or advance of localized osteoporotic change after fracture healing.
Strong Static Magnetic Field Stimulates Bone Formation to a Definite Orientation In Vitro and In Vivo
Authors: Kotani Hiroko , Kawaguchi Hiroshi , Shimoaka Takashi , Iwasaka Masakazu , Ueno Shoogo , Ozawa Hidehiro , Nakamura Kozo , Hoshi Kazuto
Published: April 2006

The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects.
Establishment of a Novel Chondrocytic Cell Line N1511 Derived From p53-Null Mice
Authors: Kamiya Nobuhiro , Jikko Akitoshi , Kimata Koji , Damsky Caroline , Shimizu Katsuji , Watanabe Hideto
Published: April 2006

We established a clonal chondrocytic cell line N1511 derived from rib cartilage of a p53-null mouse. N1511 cells proliferated in polygonal shape and elicited differentiation at confluence when treated with combination of bone morphogenetic protein (BMP) 2 and insulin or parathyroid hormone (PTH) and dexamethasone. BMP-2/insulin-treated cells became refractile without forming cartilaginous nodules and reached terminal differentiation, became positive for alizarin red staining, and developed considerable ALP activity. In contrast, PTH/dexamethasone-treated cells formed Alcian blue-positive nodules but remained negative for alizarin red staining and ALP activity. Northern blot analysis revealed that BMP-2/insulin-treated cells sequentially expressed type II, IX, and X collagens, whereas PTH/dexamethasone-treated cells slowly expressed type II collagen and then type IX, and they did not exhibit type X collagen expression. These results show that BMP-2/insulin treatment induces full differentiation toward hypertrophy, whereas treatment with PTH/dexamethasone slows and limits differentiation. Recovery of p53 expression in N1511 cells by transient transfection inhibited cell proliferation, suggesting that cell proliferation could be regulated with p53 in this cell line. These results indicate that N1511 is the only cell line with known genetic mutation, which undergoes multiple steps of chondrocyte differentiation toward hypertrophy, and because proliferation could be regulated by expression of p53, N1511 could be an excellent model for studies of chondrogenesis, the function of p53, and genetic engineering of cartilage tissue.
Accelerated Chondrogenesis of the Rabbit Cranial Base Growth Plate by Oscillatory Mechanical Stimuli
Authors: Wang Xin , Mao Jeremy J.
Published: April 2006

How mechanical stimuli modulate chondral growth is not well understood. To test a hypothesis that chondral growth is accelerated by oscillatory mechanical stimuli rather than the peak magnitude of mechanical force, we delivered 2-N tensile forces with static (frequency = 0 Hz) and cyclic (f = 1 Hz) profiles noninvasively to the maxillae of growing New Zealand white rabbits for 20 minutes/day over 12 days. Computerized histomorphometry revealed significantly greater maximum height of the cranial base growth plate (GP) treated with cyclic forces (870 ± 130 μm) than static forces (654 ± 29 μm) and sham controls (566 ± 47 μm). In addition, the average total GP area treated with cyclic forces (2.63 ± 0.17 mm2) was significantly greater than static forces (2.12 ± 0.99 mm2) and sham controls (1.65 ± 0.13 mm2). The proliferating zone of GPs treated with cyclic forces (158 ± 38.5 μm) was significantly longer than the corresponding zones of static forces (117 ± 8.6 μm) and sham controls (54 ± 14.9 μm). The average number of chondrocytes in the proliferating zone treated with cyclic forces (1045 ± 127) was significantly greater than static forces (632 ± 85) and sham controls (632 ± 60) in standardized grids. Like natural GPs, the cartilage matrix treated with cyclic and static tensile forces consisted of abundant aggrecan-like proteoglycans. These findings indicate that oscillatory components of mechanical force rather than its peak magnitude are potent anabolic stimulus for chondral growth. A cascade of oscillatory mechanical stimuli is likely capable of engineering chondral growth beyond naturally occurring chondrogenesis.
Annexin VIII Is Differentially Expressed by Chondrocytes in the Mammalian Growth Plate During Endochondral Ossification and in Osteoarthritic Cartilage
Authors: White Alicia H. , Watson Rachel E. B. , Newman Bill , Freemont Anthony J. , Wallis Gillian A.
Published: April 2006

Endochondral ossification is the developmental process that leads to the formation and coordinated longitudinal growth of the majority of the vertebrate skeleton. Central to this process is chondrocyte differentiation occurring in the growth plate that lies at the junction between the epiphyseal cartilage and the bone. To identify novel factors involved in this differentiation process, suppression subtractive hybridization was performed to amplify preferentially cDNAs uniquely expressed in fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes. The subtracted product was used to screen a fetal bovine chondrocyte cDNA library. One of the cDNA clones identified encoded the bovine orthologue of annexin VIII, a protein not previously described in the growth plate. Northern and Western blotting confirmed that annexin VIII was expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. Immunofluorescence localized annexin VIII largely to the chondrocyte cell membrane. In a preliminary study, we examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. Thus annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification and may act as a marker for cells undergoing inappropriate differentiation in OA.
Basic Fibroblast Growth Factor Stimulates Osteoclast Recruitment, Development, and Bone Pit Resorption in Association With Angiogenesis In Vivo on the Chick Chorioallantoic Membrane and Activates Isolated Avian Osteoclast Resorption In Vitro
Authors: Collin-Osdoby Patricia , Rothe Linda , Bekker Simon , Anderson Fred , Huang Yuefang , Osdoby Philip
Published: April 2006

Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory-related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)-1, FGFR-2, FGFR-3, and FGFR-like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo-oxygenase (COX)-2 prostaglandin inhibitor (NS-398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR-1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF-dependent processes may aid in the treatment of inflammatory-related or metastatic bone loss.