Biomedical subjects

Jon E Wergedal

Publications and source records attributed to Jon E Wergedal.

18 recordsLinked to original sources

PubMed2006

Pregnancy-associated plasma protein-A increases osteoblast proliferation in vitro and bone formation in vivo.

Pregnancy-associated plasma protein (PAPP)-A, a protease for IGF binding protein (IGFBP)-2, -4, and -5, may enhance IGF action by increasing its bioavailability. Here we have determined the role and mechanism of action of PAPP-A in the regulation of osteoblast proliferation in vitro and bone metabolism in vivo. Recombinant PAPP-A (100 ng/ml) significantly increased osteoblast proliferation and free IGF-I concentration. These effects were abolished by noncleavable IGFBP-4, suggesting that PAPP-A promotes osteoblast proliferation by increasing IGF bioavailability. To determine whether PAPP-A exerts an anabolic effect on bone in vivo, we developed transgenic mice that overexpress PAPP-A in osteoblasts using the 2.3-kb rat type I collagen promoter. Consistent with the increase in IGFBP-4 proteolysis, free IGF-I concentration was significantly increased in the conditioned medium of cultured osteoblasts derived from transgenic mice compared with the wild-type littermates. Calvarial bone thickness, bone marrow cavity, and skull bone mineral density were significantly increased in transgenic mice. Bone size-related parameters in femur and tibia such as total bone area and periosteal circumference as determined by peripheral quantitated computed tomography and histological analysis were significantly increased in transgenic mice. Bone formation rate and osteoid surface were increased by more than 2-fold, whereas bone resorbing surface was unaffected. These anabolic effects were sustained with aging. These findings provide strong evidence that PAPP-A acts as a potent anabolic factor in the regulation of bone formation. Thus, enhancing IGF bioavailability by PAPP-A can be a powerful strategy in the treatment of certain metabolic diseases such as osteoporosis.

Animals↗

PubMed2006

Identification of genetic loci that regulate bone adaptive response to mechanical loading in C57BL/6J and C3H/HeJ mice intercross.

Strain-dependent differences in bone adaptive responses to loading among inbred mouse strains suggest that genetic background contributes significantly to adaptation to exercise. To explore the genetic regulation of response to loading, we performed a genome-wide search for linkage in a cross between two strains, a good responder, C57BL6/J (B6), and a poor responder, C3H/HeJ (C3H). Using a four-point bending model, the right tibia was loaded by applying 9 N force for 36 cycles for 12 days in 10-week-old female B6xC3H F2 mice. Changes in bone density (BMD) and bone size were evaluated in vivo by pQCT. Measurements from non-loaded left tibia were used as an internal control to calculate loading-induced percent increase in BMD and bone size, thus excluding the possibility of identifying background QTL(s) due to natural allelic variation in mapping strains. A genome-wide scan was performed using 111 microsatellite markers in DNA samples collected from 329 F2 mice. Heritability of bone adaptive response to loading was between 70 and 80%. The mean increase, expressed as percent of unloaded tibia, was 5% for BMD, 9% for periosteal circumference (PC), and 14% for cortical thickness in F2 mice (n = 329). All these phenotypes showed normal distributions. Absence of significant correlation between BMD response to four-point bending and body weight or bone size suggested that the bone adaptive response was independent of bone size. Interval mapping revealed that BMD response to four-point bending was influenced by three significant loci on Chrs 1 (log-of-odds ratio score (LOD) 3.4, 91.8 cM), 3 (LOD 3.6, 50.3 cM), and 8 (LOD 4.2, 60.1 cM) and one suggestive QTL on Chr 9 (LOD 2.5, 33.9 cM). Loading-induced increases in PC and Cth were influenced by four significant loci on Chrs 8 (LOD 3.0, 68.9 cM), 9 (LOD 3.0, 13.1 cM), 17 (LOD 3.0, 39.3 cM), and 18 (LOD 3.0, 0 cM) and two suggestive loci on Chr 9 (LOD 2.2, 24 cM) and 11 (LOD 2.1, 69.9 cM). Pairwise analysis showed the presence of several significant and suggestive interactions between loci on Chrs 1, 3, 8, and 13 for BMD trait. This is the first study that provides evidence for the presence of multiple genetic loci regulating bone anabolic responses to loading in the B6xC3H intercross. Knowledge of the genes underlying these loci could provide novel approaches to improve skeletal mass.

Adaptation, Biological↗

PubMed2006

Regulation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch.

Phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B (NF-kappaB) signaling pathways play a critical role in mediating survival signals. In this study we have investigated how loss of dystrophin (the primary cause of Duchenne muscular dystrophy) modulates the activation of PI3K/Akt and NF-kappaB signaling pathways in skeletal muscle in response to mechanical stimulation. Activation of Akt was significantly higher in diaphragm muscle from dystrophin-deficient mdx mice compared to normal mice at both prenecrotic and necrotic states. Higher activation of Akt was also observed in cultured dystrophin-deficient primary myotubes differentiated in vitro. Application of passive mechanical stretch ex vivo synergistically increased the activation of Akt in diaphragm of mdx mice. Stretch-induced activation of PDK-1 and PI3K were also higher in diaphragm of mdx mice compared to normal mice. Pretreatment of diaphragm with PI3K inhibitor LY294002 blocked the activation of Akt in normal and mdx mice. Higher activation of Akt was associated with increased phosphorylation of its downstream targets glycogen synthase kinase 3beta (GSK3beta), FKHR, and mammalian target of rapamycin (mTOR). Treatment of diaphragm muscle with LY294002 inhibited the stretch-induced activation of IkappaB (IkappaB) kinase (IKK) and NF-kappaB transcription factor in normal and mdx mice. Mechanical stretch also reduced the interaction of HDAC1 with RelA subunit of NF-kappaB in diaphragm muscle. Finally, cellular levels of Bcl-2, cIAP1, and integrin beta1 and activation of integrin linked kinase were higher in diaphragm muscle of mdx mice compared to normal mice. Taken together, our data suggest that loss of dystrophin and/or mechanical stretch results in the up-regulation of P13K/Akt and NF-kappaB signaling pathways in skeletal muscle.

Animals↗

PubMed2006

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.

Animals↗

PubMed2006

Femur mechanical properties in the F2 progeny of an NZB/B1NJ x RF/J cross are regulated predominantly by genetic loci that regulate bone geometry.

UNLABELLED: Genetic analysis of an NZB/B1NJ x RF/J cross has identified QTLs for femur mechanical, geometric, and densitometric phenotypes. Most mechanical QTLs were associated with geometric QTLs, strongly suggesting common genetic regulation. INTRODUCTION: Previous studies have shown that bone architecture and BMD are important factors affecting bone strength, and both are genetically regulated. We conducted genetic analyses for loci regulating femur mechanical properties, geometric properties, and BMD in a cohort of F2 mice derived from intercross matings of (NZB/B1NJ x RF/J)F1 parents. MATERIALS AND METHODS: Femurs were isolated from 662 10-week-old females. Mechanical properties were determined for a femur from each animal by three-point bending. Geometric properties and volumetric BMD (vBMD) were determined by pQCT. Genotype data were obtained by PCR assays for polymorphic markers carried in the genomic DNA of each mouse. Genome-wide scans were carried out for co-segregation of genetic marker data with values from 23 different phenotypes. Quantitative trait loci (QTLs) were identified for mechanical, geometric, and mineral density phenotypes. RESULTS: QTLs for many phenotypes were significantly refined by covariate analyses using body weight and femur length. Major QTLs for mechanical and geometric phenotypes were found on chromosomes 5, 7, 9, 11, and 12. Nine chromosomal locations were identified with mechanical QTLs and 17 locations with one or more geometric QTLs. The significance of five mechanical and nine geometric QTLs was affected by the inclusion of covariates. These changes included both decreases and increases in significance. The QTLs on chromosomes 5 and 12 were decreased by inclusion of the covariates in the analysis, but QTLs on 7 and 11 were unaffected. Mechanical QTLs were almost always associated with geometric QTLs and less commonly (two of six) with vBMD QTLs. CONCLUSIONS: Genetic regulation of mechanical properties in the F(2) mice of this NZB/B1NJ x RF/J cross seems to be caused by genes regulating femur geometry.

Animals↗

PubMed2005

Multiple genetic loci from CAST/EiJ chromosome 1 affect vBMD either positively or negatively in a C57BL/6J background.

UNLABELLED: Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively. INTRODUCTION: BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1). MATERIALS AND METHODS: After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines. RESULTS: Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (-6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = -0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb. CONCLUSIONS: Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.

Alleles↗

PubMed2005

New quantitative trait loci that regulate wound healing in an intercross progeny from DBA/1J and 129 x 1/SvJ inbred strains of mice.

Wound healing/regeneration mouse models are few, and studies performed have mainly utilized crosses between MRL/MPJ (a good healer) and SJL/J (a poor healer) or MRL/lpr (a good healer) and C57BL/6J (a poor healer). Wound healing is a complex trait with many genes involved in the expression of the phenotype. Based on data from previous studies that common and additional quantitative trait loci (QTL) were identified using different crosses of inbred strains of mice for various complex traits, we hypothesized that a new cross would identify common and additional QTL, unique modes of inheritance, and interacting loci, which are responsible for variation in susceptibility to fast wound healing. In this study, we crossed DBA/1J (DBA, a good healer) and 129/SvJ (129, a poor healer) and performed a genome-wide scan using 492 (DBA x 129) F2 mice and 98 markers to identify QTL that regulate wound healing/regeneration. Four QTL on chromosomes 1, 4, 12, and 18 were identified which contributed toward wound healing in F2 mice and accounted for 17.1% of the phenotypic variation in ear punch healing. Surprisingly, locus interactions contributed to 55.7% of the phenotype variation in ear punch healing. In conclusion, we have identified novel QTL and shown that minor interacting loci contribute significantly to wound healing in DBA x 129 mice cross.

Animals↗

PubMed2005

Mechanical loading-induced gene expression and BMD changes are different in two inbred mouse strains.

Our goal is to evaluate skeletal anabolic response to mechanical loading in different age groups of C57B1/6J (B6) and C3H/HeJ (C3H) mice with variable loads using bone size, bone mineral density (BMD), and gene expression changes as end points. Loads of 6-9 N were applied at 2 Hz for 36 cycles for 12 days on the tibia of 10-wk-old female B6 and C3H mice. Effects of a 9-N load on 10-, 16-, and 36-wk-old C3H mice were also studied. Changes in bone parameters were measured using peripheral quantitative computed tomography, and gene expression was determined by real-time PCR. Total volumetric BMD was increased by 5 and 15%, respectively, with 8- and 9-N loads in the B6, but not the C3H, mice. Increases of 20 and 12% in periosteal circumference were reflected by dramatic 44 and 26% increases in total area in B6 and C3H mice, respectively. The bone response to bending showed no difference in the three age groups of B6 and C3H mice. At 2 days, mechanical loading resulted in significant downregulation in expression of bone resorption (BR), but not bone formation (BF) marker genes. At 4 and 8 days of loading, expression of BF marker genes (type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein) was increased two- to threefold and expression of BR marker genes (matrix metalloproteinase-9 and thrombin receptor-activating peptide) was decreased two- to fivefold. Although expression of BF marker genes was upregulated four- to eightfold at 12 days of training, expression of BR marker genes was upregulated seven- to ninefold. Four-point bending caused significantly greater changes in expression of BF and BR marker genes in bones of the B6 than the C3H mice. We conclude that mechanical loading-induced molecular pathways are activated to a greater extent in the B6 than in the C3H mice, resulting in a higher anabolic response in the B6 mice.

Alkaline Phosphatase↗

PubMed2004

Genetic variation in femur extrinsic strength in 29 different inbred strains of mice is dependent on variations in femur cross-sectional geometry and bone density.

The femurs from groups of mice from 29 different inbred strains were characterized to study the genetic variations in bone parameters. For these analyses, we used peripheral quantitative computed tomography to assess bone size and density in addition to three-point bend testing to assess bone mechanical properties. Highly significant differences between inbred strains were found for all size, density, and mechanical parameters measured (P < 0.0001). Correcting femoral cross-sectional geometry values or bone mechanical properties values for body weight or femur length reduced but did not eliminate the variations in bone geometry or bone mechanical properties. Mice of similar body size had as much as a 40% difference in the midshaft total area of the femur. Regression analysis suggested that 50.9% of the variation in maximum load among strains was related to variations in section modulus, i.e., cross-sectional geometry, 21.5% was related to variations in material bone density, and 27.7% to variations in quality. These components were further analyzed to show that 3.9-27.8% of the variation in maximum load was related to adaptation to mechanical stress. These findings indicate that there is a significant genetic variation in the femur cross-sectional area, density, and mechanical properties between inbred mouse strains. These studies identify inbred mouse strains suitable for future studies identifying genes regulating bone geometry and mechanical properties.

Animals↗

PubMed2004

Insulin-like growth factor-binding protein-5 induces a gender-related decrease in bone mineral density in transgenic mice.

IGF-binding protein-5 (IGFBP-5) is abundant in serum and bone during normal skeletal development, but levels decrease in osteoporosis. Studies have shown that IGFBP-5 stimulates markers of bone formation by potentiating IGF actions and by IGF-independent actions. To test the hypothesis that IGFBP-5 promotes the acquisition of bone mineral density (BMD), we generated transgenic (Tg) mice overexpressing Igfbp5 using a cytomegalovirus enhancer and beta-actin promoter (CMV/betaA). Tg animals showed an increase in serum IGFBP-5 concentrations by 7.7- to 3.5-fold at 3-8 wk of age, respectively. Concentrations were 6-49% higher for males compared with females in both wild-type and Tg mice. Surprisingly, BMD decreased in a gender-dependent manner, with Tg male adults affected more severely than Tg females (31.3% vs. 19.2% reduction, respectively, compared with wild-type mice, assessed by dual energy x-ray absorptiometry). Significant gender differences in BMD were confirmed by peripheral quantitative computed tomography. Histomorphometry revealed that although the bone formation rate and mineralizing surface at the periosteum decreased in Tg mice, they increased at the endosteum, suggesting opposing effects of IGFBP-5 on periosteal and endosteal osteoblasts (by altering proliferation or survival). These findings differ from previous observations in Igf1- and Igf2-null animals. In conclusion, IGFBP-5 has a significant influence on BMD acquisition and maintenance that is dependent on gender and age. The phenotype of Igfbp5 mice cannot be explained solely by IGF inhibition; thus, this study provides the first in vivo evidence, by genetic manipulation, for IGF-independent actions of IGFBP-5 in bone function. These findings have implications for the gender-biased progression of osteoporosis.

Absorptiometry, Photon↗

PubMed2004

Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice.

BACKGROUND: Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV-based retroviral vector to express human bone morphogenetic protein 4 (BMP4). METHODS: We assessed two approaches for administering transduced MSCs. beta-Galactosidase (beta-Gal) transduced C57BL/6J mouse MSCs were injected intravenously via tail vein or directly injected into the femoral bone marrow cavity of non-marrow-ablated syngenic recipient mice and bone marrow cavity engraftment was assessed. BMP4- or beta-Gal-transduced cells were injected into the femoral bone marrow cavity and effects on bone were evaluated by X-ray, peripheral quantitative computed tomography (pQCT), and histology. RESULTS: After tail-vein injection less than 20% of recipient mice contained beta-Gal-positive donor cells in femur, humerus or vertebra marrow cavities combined, and in these mice only 0.02-0.29% of injected cells were present in the bone marrow. In contrast, direct intramedullary injection was always successful and an average of 2% of injected cells were present in the injected femur marrow cavity 24 hours after injection. Numbers of donor cells decreased over the next 14 days. Intramedullary injection of BMP4-transduced MSCs induced bone formation. Trabecular bone mineral density (BMD) determined by pQCT increased 20.5% at 14 days and total BMD increased 6.5% at 14 days and 10.4% at 56 days. CONCLUSIONS: The present findings support the feasibility of using ex vivo MSC-based retroviral gene therapy to induce relatively sustained new bone formation, with normal histological appearance, at endosteal bone sites.

Animals↗

PubMed2004

In vivo and in vitro evidence that the high osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is intrinsic to bone cells.

Two inbred mouse strains, C3H/HeJ (C3H) and C57BL/6J (B6), displayed a profound difference in femoral peak bone density. We have previously shown that the difference could be attributed to a greater bone formation rate (BFR) that was due to a higher osteoblastic activity [measured by a mineral apposition rate (MAR)] in the C3H (high density) than B6 (low density) mice. The present study sought to determine (1) whether the BFR/MAR differences between the two mouse strains present in weight-loaded endochondral bones are also seen in less weight-loaded membranous bones and (2) whether the difference in osteoblastic activity was seen in vitro in the absence of systemic factors. To address the first objective, we performed histomorphometric measurements on the weakly loaded membranous bones (i.e., parietal bones of the calvaria) to determine if there were similar differences in MAR and BFR of membranous bones as those of highly loaded, endochondral bones. The parietal bones of adult C3H mice showed similar increases in MAR and BFR as the endochondral bones, compared to B6 mice of same age, suggesting that the differences in the MAR and BFR in the two mouse strains are probably not related to differences in mechanical strain. These findings also suggest that the gene(s) responsible for the difference in MAR between strains may not be a mechanical response gene. With respect to the second objective, we isolated osteoblasts from the parietal bones and determined their differentiation status (i.e., ALP-specific activity) and bone-forming ability (i.e., mineralized nodule formation) in vitro. Consistent with the premise that C3H osteoblasts have an intrinsic, higher differentiation status and bone-forming ability than B6 osteoblasts, osteoblasts isolated from C3H mice as compared with those from B6 mice had a significantly greater ALP-specific activity and a greater ability to form mineralized nodules in vitro in the absence of systemic factors. Because differences in ALP activity, bone-forming ability, cortical bone width, and osteoblastic activity were detected at birth, the different MAR/BFR phenotypes develop at very early life and even perhaps during embryogenesis. In conclusion, we have for the first time provided evidence that the genetic differences responsible for the observed MAR/BFR phenotype in the C3H-B6 strains are intrinsic to osteoblasts and might not depend on responses to mechanical loading and/or alterations in systemic factors.

Animals↗

PubMed2003

Lack of insulin-like growth factor I exaggerates the effect of calcium deficiency on bone accretion in mice.

Recent studies provide evidence that the GH/IGF-I axis plays a critical role in the regulation of bone accretion that occurs during puberty and that the peak bone mineral density (BMD) is dependent on the amount of dietary calcium intake during the active growth phases. To evaluate whether IGF-I deficiency exaggerates the effect of calcium deficiency on bone accretion during active growth phases, IGF-I knockout (KO) and wild-type (WT) mice were fed with low calcium (0.01%) or normal calcium (0.6%) for 2 wk during the pubertal growth phase and were labeled with tetracycline. The low calcium diet caused significant decreases in endosteal bone formation parameters and a much greater increase in the resorbing surface of both the endosteum and periosteum of the tibia of IGF-I KO mice compared with WT mice. Accordingly, femur BMD measured by dual energy x-ray absorptiometry or peripheral quantitative computed tomography increased significantly in IGF-I WT mice fed the low calcium diet, but not in IGF-I KO mice. IGF-I-deficient mice fed the normal calcium diet showed elevated PTH levels, decreased serum 1,25-dihydroxyvitamin D and serum calcium levels at baseline. Serum calcium changes due to calcium deficiency were greater in IGF-I KO mice compared with WT mice. PTH levels were 7-fold higher in IGF-I KO mice fed normal calcium compared with WT mice, which was further elevated in mice fed the low calcium diet. Treatment of IGF-I-deficient lit/lit mice with GH decreased the serum PTH level by 70% (P < 0.01). Based on these and past findings, we conclude that: 1) IGF-I deficiency exaggerates the negative effects of calcium deficiency on bone accretion; and 2) IGF-I deficiency may lead to 1,25-dihydroxyvitamin D deficiency and elevated PTH levels even under normal calcium diet.

Absorptiometry, Photon↗

PubMed2003

In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with bone morphogenetic protein-4.

This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation.

Animals↗

PubMed2003

Patients with Van Buchem disease, an osteosclerotic genetic disease, have elevated bone formation markers, higher bone density, and greater derived polar moment of inertia than normal.

Van Buchem disease is an autosomal recessive disease characterized by overgrowth of the skeleton. In a group of Dutch patients the disease is thought to be due to a 52-kb deletion that results in decreased expression of the SOST gene. To further characterize the disease, the morphology of the metacarpals of six adult subjects and two juveniles with Van Buchem disease were measured on hand x-rays along with nine normal adults and nine adult carriers of the disease. Serum bone formation markers, alkaline phosphatase, type I procollagen peptide, and osteocalcin, and the urinary bone resorption marker, cross-linked N-telopeptide, were determined. Van Buchem patients had increased metacarpal outer diameter, inner diameter, cortical thickness, and bone mineral density. Calculated bone volume and derived polar moment of inertia were markedly elevated (elevations of 158 +/- 33% and 497 +/- 95%, respectively) consistent with increased bone strength. Serum procollagen peptide and osteocalcin were significantly higher in Van Buchem patients. Urinary cross-linked N-telopeptide was significantly elevated in Van Buchem patients. None of these changes was found in Van Buchem carriers. These observations indicate that decreased expression of the SOST gene can lead to increased bone formation and to stronger bones.

Adolescent↗

PubMed2003

A missense mutation in the mouse Col2a1 gene causes spondyloepiphyseal dysplasia congenita, hearing loss, and retinoschisis.

UNLABELLED: A missense mutation in the mouse Col2a1 gene has been discovered, resulting in a mouse phenotype with similarities to human spondyloepiphyseal dysplasia (SED) congenita. In addition, SED patients have been identified with a similar molecular mutation in human COL2A1. This mouse model offers a useful tool for molecular and biological studies of bone development and pathology. INTRODUCTION: A new mouse autosomal recessive mutation has been discovered and named spondyloepiphyseal dysplasia congenita (gene symbol sedc). MATERIALS AND METHODS: Homozygous sedc mice can be identified at birth by their small size and shortened trunk. Adults have shortened noses, dysplastic vertebrae, femora, and tibias, plus retinoschisis and hearing loss. The mutation was mapped to Chr15, and Col2a1 was identified as a candidate gene. RESULTS: Sequence analyses revealed that the affected gene is Col2a1, which has a missense mutation at exon 48 causing an amino acid change of arginine to cysteine at position 1417. Two human patients with spondyloepiphyseal dysplasia (SED) congenita have been reported with the same amino acid substitution at position 789 in the human COL2A1 gene. CONCLUSIONS: Thus, sedc/sedc mice provide a valuable model of human SED congenita with molecular and phenotypic homology. Further biochemical analyses, molecular modeling, and cell culture studies using sedc/sedc mice could provide insight into mechanisms of skeletal development dependent on Col2a1 and its role in fibril formation and cartilage template organization.

Amino Acid Sequence↗

PubMed2002

A time course of bone response to jump exercise in C57BL/6J mice.

Exercise, by way of mechanical loading, provides a physiological stimulus to which bone tissue adapts by increased bone formation. The mechanical stimulus due to physical activity depends on both the magnitude and the duration of the exercise. Earlier studies have demonstrated that jump training for 4 weeks produces a significant bone formation response in C57BL/6J mice. An early time point with significant increase in bone formation response would be helpful in: (1) designing genetic quantitative trait loci (QTL) studies to investigate genes regulating the bone adaptive response to mechanical stimulus; and (2) mechanistic studies to investigate early stimulus to bone tissue. Consequently, we investigated the bone structural response after 2, 3, and 4 weeks of exercise with a loading cycle of ten jumps a day. We used biochemical markers and peripheral quantitative computed tomography (pQCT) of excised femur to measure bone density, bone mineral content (BMC), and area. Four-week-old mice were separated into control ( n = 6) and jump groups ( n = 6), and the latter groups of mice were subjected to jump exercise of 2-week, 3-week, and 4-week duration. Data (pQCT) from a mid-diaphyseal slice were used to compare bone formation parameters between exercise and control groups, and between different time points. There was no statistically significant change in bone response after 2 weeks of jump exercise as compared with the age-matched controls. After 3 weeks of jump exercise, the periosteal circumference, which is the most efficient means of measuring adaptation to exercise, was increased by 3% ( P < 0.05), and total and cortical area were increased by 6% ( P < 0.05) and 11% ( P < 0.01), respectively. Total bone mineral density (BMD) increased by 11% ( P < 0.01). The biggest changes were observed in cortical and total BMC, with the increase in total BMC being 12% ( P < 0.01). Interestingly, the increase in BMC was observed throughout the length of the femur and was not confined to the mid-diaphysis. Consistent with earlier studies, mid-femur bone mass and area remained significantly elevated in the 4-week exercise group when compared with the control group of mice. The levels of the biochemical markers osteocalcin, skeletal alkaline phosphatase, and C-telopeptide were not significantly different between the exercise and control groups, indicating the absence of any systemic response due to the exercise. We conclude that a shorter exercise regimen, of 3 weeks, induced a bone response that was greater than or equal to that of 4 weeks of jump exercise reported earlier.

Alkaline Phosphatase↗

PubMed2002

Expression of the fibroblast growth factor receptor genes in fracture repair.

The spatial and temporal expression domains of the fibroblast growth factor receptor genes were examined in the healing rat femur fracture by in situ hybridization. Fibroblast growth factor receptor gene expression was detected in diverse fracture tissues throughout healing. Fibroblast growth factor receptor 1 and 2 expression was present throughout fracture repair, in the early proliferating periosteal mesenchyme, in the osteoblasts during intramembranous bone formation, and in the chondrocytes and osteoblasts during endochondral bone formation. Fibroblast growth factor receptor 3 expression colocalized with fibroblast growth factor receptor 1 and 2 expression in the chondrocytes and osteoblasts beginning at 10 days of healing, and persisted throughout endochondral bone formation. Fibroblast growth factor receptor 3 recapitulated its expression in fetal skeletal development, suggesting that it has a similar function in the control of endochondral bone growth during fracture repair. Fibroblast growth factor receptor 4 expression was not observed at any time. The extensive colocalized expression of the fibroblast growth factor receptors in healing indicates that fibroblast growth factor regulation of fracture callus maturation is extensive, and accurate identification of the receptor isoforms is necessary to establish the functions of fibroblast growth factor family members in fracture repair.

Animals↗