Increased bone formation in mice lacking apolipoprotein E.

ApoE is a plasma protein that plays a major role in lipoprotein metabolism. Here we describe that ApoE expression is strongly induced on mineralization of primary osteoblast cultures. ApoE-deficient mice display an increased bone formation rate compared with wildtype controls, thereby showing that ApoE has a physiologic function in bone remodeling. INTRODUCTION: Apolipoprotein E (ApoE) is a protein component of lipoproteins and facilitates their clearance from the circulation. This is confirmed by the phenotype of ApoE-deficient mice that have high plasma cholesterol levels and spontaneously develop atherosclerotic lesions. The bone phenotype of these mice has not been analyzed to date, although an association between certain ApoE alleles and BMD has been reported. MATERIALS AND METHODS: Primary osteoblasts were isolated from newborn mouse calvariae and mineralized ex vivo. A genome-wide expression analysis was performed during the course of differentiation using the Affymetrix gene chip system. Bones from ApoE-deficient mice and wildtype controls were analyzed using radiography, micro CT imaging, and undecalcified histology. Cellular activities were assessed using dynamic histomorphometry and by measuring urinary collagen degradation products. Lipoprotein uptake assays were performed with (125)I-labeled triglyceride-rich lipoprotein-remnants (TRL-R) using primary osteoblasts from wildtype and ApoE-deficient mice. Serum concentrations of osteocalcin were determined by radioimmunoassay after hydroxyapatite chromatography. RESULTS: ApoE expression is strongly induced on mineralization of primary osteoblast cultures ex vivo. Mice lacking ApoE display a high bone mass phenotype that is caused by an increased bone formation rate, whereas bone resorption is not affected. This phenotype may be explained by a decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin in the serum of ApoE-deficient mice. CONCLUSION: The specific induction of ApoE gene expression during osteoblast differentiation along with the increased bone formation rate observed in ApoE-deficient mice shows that ApoE has a physiologic role as a regulator of osteoblast function.     

Increased trabecular bone formation in mice lacking the growth factor midkine

Midkine (Mdk) and pleiotrophin (Ptn) comprise a family of heparin‐binding growth factors known primarily for their effects on neuronal cells. Since transgenic mice overexpressing Ptn have been reported to display increased bone density, we have previously analyzed Ptn‐deficient mice but failed to detect any abnormality of skeletal development and remodeling. Together with the finding that Mdk expression increases in the course of primary osteoblast differentiation, we reasoned that Mdk, rather than Ptn, could play a physiologic role in bone formation. Here, we show that Mdk‐deficient mice display an increased trabecular bone volume at 12 and 18 months of age, accompanied by cortical porosity. Histomorphometric quantification demonstrated an increased bone‐formation rate compared with wild‐type littermates, whereas bone resorption was differentially affected in trabecular and cortical bone of Mdk‐deficient mice. To understand the effect of Mdk on bone formation at the molecular level, we performed a genome‐wide expression analysis of primary osteoblasts and identified Ank and Enpp1 as Mdk‐induced genes whose decreased expression in Mdk‐deficient osteoblasts may explain, at least in part, the observed skeletal phenotype. Finally, we performed ovariectomy and observed bone loss only in wild‐type but not in Mdk‐deficient animals. Taken together, our data demonstrate that Mdk deficiency, at least in mice, results in an increased trabecular bone formation, thereby raising the possibility that Mdk‐specific antagonists might prove beneficial in osteoporosis therapy. © 2010 American Society for Bone and Mineral Research     

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)-null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased.     

Decreased bone formation and osteopenia in mice lacking alpha-calcitonin gene-related peptide.

We recently described an unexpected high bone mass phenotype in mice lacking the Calca gene that encodes CT and alphaCGRP. Here we show that mice specifically lacking alphaCGRP expression display an osteopenia caused by a decreased bone formation. These results show that alphaCGRP is a physiological activator of bone formation and that the high bone mass phenotype of the Calca-deficient mice is caused by the absence of CT. INTRODUCTION: Calcitonin (CT) and alpha-calcitonin gene-related peptide (alphaCGRP) are two polypeptides without completely defined physiologic functions that are both derived from the Calca gene by alternative splicing. We have recently described an unexpected high bone mass phenotype in mice carrying a targeted deletion of the Calca gene. To uncover whether this phenotype is caused by the absence of CT or by the absence of alphaCGRP, we analyzed a mouse model, where the production of alphaCGRP is selectively abolished. MATERIALS AND METHODS: Bones from Calca(-/-) mice, alphaCGRP(-/-) mice, and their corresponding wildtype controls were analyzed using radiography, muCT imaging, and undecalcified histology. Cellular activities were assessed using dynamic histomorphometry and by measuring the urinary collagen degradation products. CT expression was determined using radioimmunoassay and RT-PCR. Immunohistochemistry was performed using an anti-CGRP antibody on decalcified bone sections. RESULTS: Unlike the Calca-deficient mice, the alphaCGRP-deficient mice do not display a high bone mass phenotype. In contrast, they develop an osteopenia that is caused by a reduced bone formation rate. Serum levels and thyroid expression of CT are not elevated in alphaCGRP-deficient mice. While CGRP expression is detectable in neuronal cell close to trabecular bone structures, the components of the CGRP receptor are expressed in differentiated osteoblast cultures. CONCLUSION: The discrepancy between the bone phenotypes of Calca(-/-) mice and alphaCGRP(-/-) mice show that the high bone mass phenotype of the Calca(-/-) mice is caused by the absence of CT. The osteopenia observed in the alphaCGRP(-/-) mice that have normal levels of CT further show that alphaCGRP is a physiologic activator of bone formation.     

Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone.

The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were investigated in cultures of fetal mouse calvariae with the use of either a chromogenic substrate or a zymographic assay. PA was detected essentially in the tissue extracts of the explanted bones, with only 1-2% of the total activity released in the surrounding culture media. From their electrophoretic behavior compared to PAs of other mouse tissues and from their response to a specific antibody raised against the tissue type PA (tPA), two major molecular species, of 70 and 48 kD were identified as tPA and urokinase (uPA), respectively, a third minor species of 105 kD being likely to correspond to complexes between tPA and an inhibitor; the culture fluids, moreover, contained enzymatically active degradation products of uPA of 42 and 29 kD. The PA activity of the bone extracts was only minimally affected by the addition of fibrinogen fragments to the chromogenic assays. PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-resorbing agents, 1,25-dihydroxyvitamin D3 and prostaglandin E2, had similar effects. Densitometric scanning of the zymograms of the bone extracts indicated that PTH stimulated only the production of tPA and had no effect on that of uPA. However, PTH also enhanced the release of uPA (both the 48 kD and the 29 kD forms) from the bones into the media. Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of tPA, but it prevented the PTH-induced accumulation of 29 kD uPA in the culture fluids. Thus these studies support the view that tPA and possibly also uPA may have a role in the physiology of bone; the nature of this role remains to be elucidated, however.     

Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor 1

Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment.     

Use of plasminogen activators in venous thrombosis

A major advance in the treatment of thrombosis has been the development of thrombolytic agents. Streptokinase and urokinase have been the standard agents available for many years, but in recent years the most exciting change in the field has been the development of a new generation of plasminogen activators, the principal one being tissue plasminogen activator.The first generation of plasminogen activators—streptokinase and urokinase—do not have fibrin specificity and predictably induce plasma proteolysis when administered systemically in doses which introduce thrombolysis. The second generation of plasminogen activators are much more fibrin-specific and offer a promise of fewer complications.In a number of major randomized studies, these thrombolytic agents have proved effective clinically. The major complication of thrombolytic therapy, however, is hemorrhage. The risk of hemorrhage increases with the length of infusion and occurs most often from sites of vascular invasion such as needle punctures or cutdown sites from surgical wounds. This can be treated by applying pressure over the wound and discontinuing the thrombolytic agent whose half-life is measured in hours. It is believed that as more experience is acquired with the second-generation plasminogen activators, better control of these drugs will result in fewer complications and more effective and wider application of therapy.

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Resumen Uno de los mayores avances en el tratamiento de la trombosis ha sido el desarrollo de los agentes trombolíticos. La estreptoquinasa y la uroquinasa han sido agentes estándar disponibles desde hace años; el cambio reciente más dramático ocurrido en este campo es el desarrollo de una nueva generación de activadores de plasminógeno, cuyo principio activo es el activador de plasminógeno tisular.La primera generación de activadores de plasminógeno—estreptoquinasa y uroquinasa—no poseen especificidad por la fibrina y predeciblemente inducen la proteolisis plasmática cuando se administran sistémicamente en dosis suficientes para inducir trombolisis. La segunda generación esta constituida por agentes de mucha mayor especificidad por la fibrina y ofrecen la perspectiva de menos complicaciones.Estos agentes trombolíticos han probado su efectividad clínica en un número de estudios randomizados. La mayor complicación de la terapia trombolítica es la hemorragia. El riesgo de hemorragia aumenta con la duración de la infusión y ocurre más frecuentemente en los lugares de invasión vascular, tales como sitios de venopunción o de cateterismo venoso. Este tipo de sangrado puede ser controlado mediante presión local e interrupción de la infusión del agente trombolítico, cuya vida media es apenas de unas horas. Creemos que en la medida que se adquiera mayor experiencia con la segunda generación de los activadores de plasminógeno, se logrará un mejor control de estas drogas con menores complicaciones, mayor efectividad y más amplia aplicación terapéutica.

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Résumé Un des progrès majeurs dans le traitement de la thrombose a été le développement des agents thrombolytiques. La streptokinase et l'urokinase sont devenues classiques; ces dernières années, l'évènement le plus marquant a été le développement d'une nouvelle génération d'activateurs de plasminogène, le principal étant l'activateur de plasminogène tissulaire.La première génération des activateurs de plasminogène—la streptokinase et l'urokinase—n'a pas de spécificité pour la fibrine, et induit une protéolyse plasmatique lorsqu'on les administre aux doses thrombolytiques par voie sytémique. La deuxième génération des activateurs est beaucoup plus spécifique pour la fibrine et offre la possibilité d'un taux de complication moindre.Dans bon nombre d'études randomisées majeures, ces agents thrombolytiques ont prouvé leur efficacité clinique. La complication majeure de la thérapeutique thrombolytique, cependant, est l'hémorragie. Le risque d'hémorragie augmente avec la durée de la perfusion et se produit le plus souvent aux sites de l'agression vasculaire comme piqûre d'aiguille ou plaies chirurgicales. Le traitement comporte la compression directe et l'arrêt des agents thrombolytiques dont la demi-vie se mesure en heures. Avec l'expérience, des activateurs de plasminogène de deuxième génération, leur meilleur contrôle donnera un taux de complication moindre et élargira leur champ d'application et leur efficacité.

     

纤维蛋白对肾小球内皮细胞表达纤溶酶原激活物及纤溶酶原激活物抑制物的作用

目的：观察体外培养人肾小球内皮细胞（GEC）表面原位形成的纤维蛋白对GEC表达纤溶酶原激活物及纤溶酶原激活物抑制物（PA／PAI）的影响。方法：应用逆转录聚合酶链反应（RT－PCR）,酶谱分析法与反向酶谱法分别在基因转录水平与蛋白质活性水平上检测纤维蛋白对GEC表达tPA,uPA gn PAI-1r 作用,纤维蛋白平板法检测纤维蛋白对GEC PA／PAI系统的综合效应,结果：纤维蛋白能够明显促进tPA,uPA与PAI－1的mRNA表达上调,无血清RPMI 1640培养下的GEC几乎检测不到PAI知性,但可检测到PAI－1的活性。纤维蛋白能够浓度依赖性刺激GEC tPA与uPA活性增加以及PAI01的活性增加,呈浓度依赖性与时间依赖性,相同剂量的纤维蛋白原与纤维蛋白的作用相似,放线菌酮与放线菌素D均可抑制纤维蛋白上调GEC表达tPA,uPA与PAI的作用,纤维蛋白平板法显示,纤维蛋白对GEC PA／PAI系统的综合效应是以升高PA活性为主,其活性能够被抑肽酶完全阻断。结论：肾脏局部毛细血[管内沉积的纤维蛋白可能通过对GEC PA／PAI系统的调节发挥其病理作用。     

Increased bone resorption precedes increased bone formation in the ovariectomized rat

This study describes an increase in biochemical and histomorphometric markers of bone resorption prior to increased bone formation and trabecular bone loss in the ovariectomized rat. Six-month-old, female Sprague Dawley rats were either sham operated or ovariectomized (Ovx) and killed at 0, 6, 9, 15, 18, 21, and 42 days postOperation when femora were collected and trabecular bone volume (BV/TV) was determined from von Kossa silver-stained sections using the Quantimet 520 image analysis system in the distal region. A number of these sections were also examined unstained for fluorochrome labels, and stained for acid phosphatase to detect osteoclast-like cells (ACP surface). At 18 days postoperation, lumbar vertebrae were examined. Blood and urine specimens were analyzed for bone-related biochemical variables. ACP surface was significantly greater in Ovx rats compared with sham at 6 days postoperation (mean ACP surface (%TS) ± SEM: sham 36.4 ± 1.9; Ovx 40.3 ± 1.2,P < 0.05) as was urinary hydroxyproline excretion. Serum osteocalcin and alkaline phosphatase activity were not elevated in Ovx rats compared with Sham until 9 days postoperation. Mineral apposition rate (MAR) was increased at 12 days after ovariectomy (mean MAR (Μm/day) ± SEM: sham 0.85 ± 0.06; Ovx 1.23 ± 0.06,P < 0.05). Trabecular bone volume (BV/TV) at a specific site in the metaphyseal-diaphyseal core area was significantly lower at 15 days postoperation (mean (%) ± SEM: Sham 7.40 ± 1.23, Ovx 4.25 0 0.65,P < 0.05). There was no difference in lumbar vertebral BV/TV between the two groups at 18 days postoperation, however, ACP surface was elevated in the Ovx rats (P < 0.05). A systemic increase in bone resorption at 6 days postovariectomy precedes increased formation whereas the length of time required for the dissolution of trabeculae postoperation is determined locally.     

Subchondral bone formation in arthrosis. Polychrome labeling studies in mice

We used polychrome sequential labeling to study the dynamics of subchrondral bone sclerosis during developing arthrosis in knee joints of male STR/1N mice. This technique, using four different colored vital markers, gives detailed information about the site and time of new bone formation in the subchondral bone. In arthrotic joints, we found fluorescent bands arranged excentrically around the marrow cavities always pointing towards the cartilage lesions. The linear separation between the first label and the anatomic surface of the bone marrow cavity varied considerably between the experimental groups. In arthrotic joints, we found bone growth rates 3-4 times greater than in control joints. We also found that the degenerative process in cartilage and subchondral bone was a local phenomenon, because in areas with normal cartilage next to the sclerotic lesions appositional bone growth rates were unaffected.     

PTH stimulates bone formation in mice deficient in Lrp5.

Lrp5 deficiency decreases bone formation and results in low bone mass. This study evaluated the bone anabolic response to intermittent PTH treatment in Lrp5-deficient mice. Our results indicate that Lrp5 is not essential for the stimulatory effect of PTH on cancellous and cortical bone formation. INTRODUCTION: Low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor in canonical Wnt signaling, increases osteoblast proliferation, differentiation, and function. The purpose of this study was to use Lrp5-deficient mice to evaluate the potential role of this gene in mediating the bone anabolic effects of PTH. MATERIALS AND METHODS: Adult wildtype (WT, 23 male and 25 female) and Lrp5 knockout (KO, 27 male and 26 female) mice were treated subcutaneously with either vehicle or 80 microg/kg human PTH(1-34) on alternate days for 6 weeks. Femoral BMC and BMD were determined using DXA. Lumbar vertebrae were processed for quantitative bone histomorphometry. Bone architecture was evaluated by microCT. Data were analyzed using a multiway ANOVA. RESULTS: Cancellous and cortical bone mass were decreased with Lrp5 deficiency. Compared with WT mice, cancellous bone volume in the distal femur and the lumbar vertebra in Lrp5 KO mice was 54% and 38% lower, respectively (p<0.0001), whereas femoral cortical thickness was 11% lower in the KO mice (p<0.0001). The decrease in cancellous bone volume in the lumbar vertebrae was associated with a 45% decrease in osteoblast surface (p<0.0001) and a comparable decrease in bone formation rate (p<0.0001). Osteoclast surface, an index of bone resorption, was 24% lower in Lrp5 KO compared with WT mice (p<0.007). Treatment of mice with PTH for 6 weeks resulted in a 59% increase in osteoblast surface (p<0.0001) and a 19% increase in osteoclast surface (p=0.053) in both genotypes, but did not augment cancellous bone volume in either genotype. Femur cortical thickness was 11% higher in PTH-treated mice in comparison with vehicle-treated mice (p<0.0001), regardless of genotype. CONCLUSIONS: Whereas disruption of Lrp5 results in decreased bone mass because of decreased bone formation, Lrp5 does not seem to be essential for the stimulatory effects of PTH on cancellous and cortical bone formation.     

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency.

Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.     

Increased bone resorption and decreased bone formation in Chinese patients with hip fracture

Biochemical markers of bone formation (bone-specific alkaline phosphatase and osteocalcin) and bone resorption (hydroxyproline excretion and bone isoenzyme of acid phosphatase) were measured in 30 patients (15 M and 15 F) with hip fracture and 30 healthy subjects matched for age and sex. Bone isoenzyme of tartrate-resistant acid phosphatase (TRACP) was measured by a recently developed specific immunoassay. Serum osteocalcin concentration and bone-specific alkaline phosphatase activity were significantly lower and serum TRACP concentration and urinary hydroxyproline excretion were elevated in patients compared with healthy subjects. We suggest that there is reduced bone formation and increased bone resorption in patients with hip fracture.     

Regulating DeltaFosB expression in adult Tet-Off-DeltaFosB transgenic mice alters bone formation and bone mass.

The DeltaFosB isoforms are naturally occurring AP-1 family members that increase bone volume via a cell-autonomous effect on osteoblastic bone formation. Mice overexpressing DeltaFosB demonstrate a very high level of bone formation, resulting in a progressive osteosclerosis. Despite the linkage of bone formation and resorption in physiological systems, no alteration in bone resorption was detected in mice overexpressing DeltaFosB. To determine whether altering DeltaFosB expression can regulate bone formation independently of bone resorption in adult mice, we used the Tet-Off-inducible transgene system to induce or block transgenic DeltaFosB overexpression and thereby regulate bone formation in vivo. Overexpression of DeltaFosB after skeletal maturity increased trabecular bone volume by increasing bone formation, again without altering bone resorption, indicating that developmental DeltaFosB overexpression is not required for the osteosclerotic phenotype. Similarly, switching off DeltaFosB overexpression after osteosclerosis had developed led to a marked decrease in bone formation and loss of bone mass such that trabecular bone volume approached normal levels. Despite this dramatic reduction, no alteration in bone resorption was detected. These results clearly demonstrate that DeltaFosB regulates bone formation and bone mass in adult mice with no effect on bone resorption.     

Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene

Humans with inherited sclerostin deficiency have high bone mass. Targeted deletion of the sclerostin gene in mice (SOST-KO) causes increases in bone formation, bone mass and bone strength. Inhibition of sclerostin by a monoclonal antibody increases bone formation and enhances fracture healing in rodent and primate models. In this study, we describe the temporal progression of femoral fracture healing in SOST-KO mice compared with wild type (WT) control mice to further characterize the role of sclerostin in fracture healing. Sixty-seven male 9-10 week-old SOST-KO (N=37) and WT (N=30) mice underwent a closed femoral fracture. Weekly radiography was used to monitor the progress of healing. Histologic sections were used to characterize callus composition, evaluate callus bridging, and quantify lamellar bone formation on days 14 and 28. Densitometry and biomechanical testing were utilized to characterize bone mass and strength at the fractured and contralateral femurs on day 45. A significant improvement in time to radiographic healing (no discernible fracture line) was observed in SOST-KO mice, which corresponded to an increase in histologic bony bridging at 14 days (38% versus 0% in WT). Both genotypes appeared to be nearly fully bridged at 28 days post-fracture. The increased bridging at 14 days was associated with 97% greater bone area and 40% lower cartilage area in the callus of SOST-KO mice as compared to WT mice. Bone formation-related endpoints were higher in SOST-KO mice at both 14 and 28 days. At 45 days post-fracture, peak load and bone mass were significantly greater in the fractured femurs of SOST-KO mice as compared to WT mice. In conclusion, fractures in mice lacking sclerostin showed accelerated bridging, greater callus maturation, and increased bone formation and strength in the callus.     

Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice

BACKGROUND: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. METHODS: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. RESULTS: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. CONCLUSIONS: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.     

Temporal activity of plasminogen activators and matrix metalloproteinases during cutaneous wound repair.

BACKGROUND: Response to tissue injury begins with the deposition of a fibrin-rich clot or the provisional matrix. The provisional matrix consists of plasma-borne matrix molecules that serve as scaffolding for the ensuing migration of cells. During wound repair multiple cell types must migrate through the clot-matrix scaffolding. The migration of these cells through the matrix is dependent on the activity of the fibrinolytic and proteolytic systems, which include the plasminogen activator (PA) system and matrix metalloproteinases (MMP). The aim of this study was to better understand the temporal activity of these enzymes during normal wound repair. METHODS: We used the murine excisional wound model and extracted proteins under nonreducing conditions. With use of gelatin and casein zymography, we determined the activity of the MMPs during the course of wound repair. In addition, we quantified the activity of MMP-2 and MMP-9 by a standardized assay. Plasminogen zymograms were used to detect urokinase PA and tissue PA activity. Western blots were used to detect the natural inhibitor of PAs, plasminogen activator inhibitor type 1. RESULTS: Our results demonstrate the temporal activity of MMP-2, MMP-3, MMP-7, and MMP-9 during the course of normal dermal repair. The activity of urokinase PA and tissue PA were also determined; it preceded the activity of the MMPs. CONCLUSIONS: We demonstrate the temporal activity of the 2 protease families, MMPs and PAs, in the normal process of cutaneous wound healing.     

Dissociation of angiogenesis and osteoclastogenesis during endochondral bone formation in neonatal mice.

Invasion of the mineralized matrix by endothelial cells and osteoclasts is a key event in endochondral bone formation. To examine the putative role of osteoclast activity in the angiogenic process, we used two in vivo models of suppressed bone resorption: mice treated with the bisphosphonate clodronate and in osteoclast-deficient, osteopetrotic mice. Angiogenesis was assessed in caudal vertebrae of these neonatal mice. This model enables us to study the interaction between osteoclasts and endothelial cells during endochondral bone formation. In control conditions, sinusoid-like structures were detected in the vicinity of tartrate resistance acid phosphatase positive (TRAcP+) osteoclasts. Treatment with clodronate completely abolished osteoclastic bone resorption, whereas angiogenesis remained unaffected. In line with these observations, in the osteopetrotic mouse mutants c-fos knockout mice and op/op mice, capillaries invaded the calcified cartilage in the absence of osteoclasts. In conclusion, our data strongly suggest that during endochondral bone formation, vascular invasion can occur in the absence of osteo(chondro)clastic resorption. In addition, bisphosphonates show no apparent effect on angiogenesis in this in vivo model. These findings may have important clinical implications in the management of skeletal disorders such as metastatic bone disease, in which both osteoclastic bone resorption and angiogenesis contribute to tumor growth. On the other hand, our results confirm that bisphosphonates can be used safely in the treatment of disorders that affect the growing skeleton, such as in juvenile osteoporosis.     

Abnormal expression of plasminogen activators in aortic aneurysmal and occlusive disease

Purpose and Methods : Aortic aneurysms are characterized by the destruction of the extracellular matrix of the media, whereas occlusive disease involves excess matrix accumulation within the intima. Plasmin degrades extracellular matrix directly and indirectly by activation of latent metalloenzymes. To determine the expression of tissue- and urokinase-type plasminogen activators, immunoassay, fibrin autography, Northern analysis, and immunohistochemistry were performed on specimens of aneurysmal (n = 12), occlusive (n = 8), and healthy (n = 6) aorta.Results: Immunoassay of tissue-type plasminogen activator revealed 8.7 ± 0.9 ng tissue-type plasminogen activator/mg extracted protein in aneurysmal aorta, 5.7 ± 0.3 ng/mg in normal aorta, and 2.5 ± 0.3 ng/mg in occlusive aorta (p < 0.05 for comparisons between all groups). No urokinase-type plasminogen activator antigen was detected by urokinase-type plasminogen activator immunoassay. Fibrin autography exhibited lytic activity at 64 kDa and 54 kDa attributable to tissue-type plasminogen activator and urokinase-type plasminogen activator. The vast majority of fibrinolysis was secondary to free tissue-type plasminogen activator and was greatest in aneurysmal disease and least in occlusive disease. There was only a small amount of lysis secondary to urokinase-type plasminogen activator. Expression of tissue-type plasminogen activator and urokinase-type plasminogen activators mRNA was comparable in aneurysmal and occlusive aortas. In contrast to occlusive disease, aneurysms had an inflammatory cell infiltrate characterized by the expression of urokinase-type plasminogen activator by specific mononuclear cells. Tissue-type plasminogen activator expression was evident in the intima of normal and diseased aorta and in the media of diseased aorta.Conclusion : Differential expression of plasminogen activators within the arterial wall may contribute to the unique pathogenesis of aneurysmal and occlusive aortic disease. (J VASC SURG 1994;19:865-72)