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2.
J Orthop Res ; 34(10): 1716-1724, 2016 10.
Article in English | MEDLINE | ID: mdl-26867804

ABSTRACT

The majority of Osteosarcoma (OS) patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. These protocols include distraction osteogenesis (DO), which is characterized by direct new bone formation. Cisplatin (CDP) is extensively used for OS chemotherapy and recent studies, using a mouse DO model, have demonstrated that CDP has profound negative effects on bone repair. Recent oncological therapeutic strategies are based on the use of standard cytotoxic drugs plus an assortment of biologic agents. Here we demonstrate that the previously reported CDP-associated inhibition of bone repair can be modulated by the administration of a small molecule p53 inducer (nutlin-3). The effects of nutlin-3 on CDP osteotoxicity were studied using both pre- and post-operative treatment models. In both cases the addition of nutlin-3, bracketing CDP exposure, demonstrated robust and significant bone sparing activity (p < 0.01-0.001). In addition the combination of nutlin-3 and CDP induced equivalent OS tumor killing in a xenograft model. Collectively, these results demonstrate that the induction of p53 peri-operatively protects bone healing from the toxic effects of CDP, while maintaining OS toxicity. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1716-1724, 2016.


Subject(s)
Antineoplastic Agents/therapeutic use , Bone Regeneration/drug effects , Cisplatin/therapeutic use , Imidazoles/therapeutic use , Osteosarcoma/drug therapy , Piperazines/therapeutic use , Animals , Female , Humans , Imidazoles/pharmacology , Male , Mice, Inbred C57BL , Mice, Nude , Osteogenesis, Distraction , Osteosarcoma/surgery , Piperazines/pharmacology , Random Allocation , Xenograft Model Antitumor Assays
3.
Bone ; 82: 101-7, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26211996

ABSTRACT

Persons with type 1 and type 2 diabetes have increased fracture risk, attributed to deficits in the microarchitecture and strength of diabetic bone, thought to be mediated, in part, by the consequences of chronic hyperglycemia. Therefore, to examine the effects of a glucose-lowering SGLT2 inhibitor on blood glucose (BG) and bone homeostasis in a model of diabetic bone disease, male DBA/2J mice with or without streptozotocin (STZ)-induced hyperglycemia were fed chow containing the SGLT2 inhibitor, canagliflozin (CANA), or chow without drug, for 10weeks of therapy. Thereafter, serum bone biomarkers were measured, fracture resistance of cortical bone was assessed by µCT analysis and a three-point bending test of the femur, and vertebral bone strength was determined by compression testing. In the femur metaphysis and L6 vertebra, long-term diabetes (DM) induced deficits in trabecular bone microarchitecture. In the femur diaphysis, a decrease in cortical bone area, cortical thickness and minimal moment of inertia occurred in DM (p<0.0001, for all) while cortical porosity was increased (p<0.0001). These DM changes were associated with reduced fracture resistance (decreased material strength and toughness; decreased structural strength and rigidity; p<0.001 for all). Significant increases in PTH (p<0.0001), RatLAPs (p=0.0002), and urine calcium concentration (p<0.0001) were also seen in DM. Canagliflozin treatment improved BG in DM mice by ~35%, but did not improve microarchitectural parameters. Instead, in canagliflozin-treated diabetic mice, a further increase in RatLAPs was evident, possibly suggesting a drug-related intensification of bone resorption. Additionally, detrimental metaphyseal changes were noted in canagliflozin-treated control mice. Hence, diabetic bone disease was not favorably affected by canagliflozin treatment, perhaps due to insufficient glycemic improvement. Instead, in control mice, long-term exposure to SGLT2 inhibition was associated with adverse effects on the trabecular compartment of bone.


Subject(s)
Blood Glucose/drug effects , Bone Diseases/drug therapy , Canagliflozin/therapeutic use , Diabetes Mellitus, Experimental/drug therapy , Sodium-Glucose Transporter 2 Inhibitors , Animals , Blood Glucose/metabolism , Bone Diseases/metabolism , Bone Diseases/prevention & control , Canagliflozin/pharmacology , Diabetes Mellitus, Experimental/metabolism , Male , Mice , Mice, Inbred DBA , Sodium-Glucose Transporter 2/metabolism
4.
Bone Rep ; 1: 16-19, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25685827

ABSTRACT

In type 1 diabetes, diabetic bone disease (DBD) is characterized by decreased bone mineral density, a state of low bone turnover and an increased risk of fracture. Animal models of DBD demonstrate that acquired alterations in trabecular and cortical bone microarchitecture contribute to decreased bone strength in diabetes. With anti-collagenolytic and anti-inflammatory properties, tetracycline derivatives may prevent diabetes-related decreases in bone strength. To determine if doxycycline, a tetracycline class antibiotic, can prevent the development of DBD in a model of long-term diabetes, male DBA/2J mice, with or without diabetes, were treated with doxycycline-containing chow for 10 weeks (dose range, 28-92 mg/kg/day). Long-term doxycycline exposure was not deleterious to the microarchitecture or biomechanical properties of healthy bone in male DBA/2J mice. Doxycycline treatment also did not prevent or alleviate the deleterious changes in trabecular microarchitecture, cortical structure, and biomechanical properties of bone induced by chronic diabetes.

5.
J Orthop Res ; 32(3): 464-70, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24259375

ABSTRACT

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. Surgical reconstructions after tumor resection include structural allografts, non-cemented endoprostheses, and distraction osteogenesis (DO), which require direct bone formation. Although cisplatin (CDP) is extensively used for OS chemotherapy, the effects on bone regeneration are not well studied. The effects of CDP on direct bone formation in DO were compared using two dosing regimens and both C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice, as CDP toxicity is associated with elevated TNF levels. Detailed evaluation of the five-dose CDP regimen (2 mg/kg/day), demonstrated significant decreases in new bone formation in the DO gaps of CDP treated versus vehicle treated mice (p < 0.001). Further, no significant inhibitory effects from the five-dose CDP regimen were observed in TNFR1KO mice. The two-dose regimen significantly inhibited new bone formation in B6 mice. These results demonstrate that CDP has profound short term negative effects on the process of bone repair in DO. These data provide the mechanistic basis for modeling peri-operative chemotherapy doses and schedules and may provide new opportunities to identify molecules that spare normal cells from the inhibitory effects of CDP.


Subject(s)
Antineoplastic Agents/toxicity , Bone Regeneration/drug effects , Cisplatin/toxicity , Osteogenesis, Distraction , Receptors, Tumor Necrosis Factor, Type I/metabolism , Animals , Antineoplastic Agents/administration & dosage , Cisplatin/administration & dosage , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Tumor Necrosis Factor-alpha/metabolism
6.
Bone ; 57(1): 36-40, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23886838

ABSTRACT

OBJECTIVE: Using a streptozotocin (STZ)-induced mouse model of type 1 diabetes (T1D), we have previously demonstrated that long-term diabetes inhibits regenerative bone formation during tibial distraction osteogenesis (DO) and perturbs skeletal integrity by decreasing cortical thickness, bone mineral density and bone's resistance to fracture. Because long-standing T1D is also associated with a deficiency of insulin-like growth factor I (IGF-I), we examined the effects of systemic IGF-I treatment on skeletal microarchitecture and strength, as well as on bone formation in diabetic mice. RESEARCH DESIGN AND METHODS: Streptozotocin-induced diabetic or control mice were treated with recombinant human IGF-I (rhIGF-I, 1.5mg/kg/day as subcutaneous infusion) or vehicle throughout a 14day DO procedure. Thereafter, trunk blood was assayed for glucose, insulin, rhIGF-I, mouse IGF-I and leptin. Bone formation in distracted tibiae was quantified. Effects on cortical bone strength and trabecular bone architecture were assessed by µCT analysis and three-point bend testing of contralateral femurs. RESULTS: New bone formation during DO was reduced in diabetic mice but significantly improved with rhIGF-I treatment. The contralateral femurs of diabetic mice demonstrated significant reductions in trabecular thickness, yield strength and peak force of cortical bone, which were improved with rhIGF-I treatment. rhIGF-I also reduced intracortical porosity in control mice. However, treatment with rhIGF-I did not normalize serum glucose, or correct concurrent deficiencies of insulin or leptin seen in diabetes. CONCLUSIONS: These findings demonstrate that despite persistent hyperglycemia, rhIGF-I promoted new bone formation and improved biomechanical properties of bone in a model of T1D, suggesting that it may be useful as a fracture preventative in this disease.


Subject(s)
Diabetes Mellitus, Type 1/drug therapy , Insulin-Like Growth Factor I/therapeutic use , Animals , Hyperglycemia/drug therapy , Mice , Osteogenesis/drug effects
7.
Alcohol ; 46(2): 133-8, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21908154

ABSTRACT

Distraction osteogenesis (DO) is an orthopedic protocol, which induces direct new bone formation as a result of the stimulating effects of mechanical distraction. Chronic ethanol exposure has been demonstrated to inhibit bone formation in rodent models of DO. Further, it has been demonstrated that (1) tumor necrosis factor-α (TNF) blockers are protective against ethanol exposure and (2) recombinant mouse TNF (rmTNF) inhibits direct bone formation in ethanol naïve mice through TNF receptor 1 (TNFR1). These results suggest that the inhibitory effects are significantly mediated by TNF signaling. Therefore, we hypothesized that direct new bone formation in TNFR1 knockout (KO) mice would be protected from ethanol exposure. We used a unique model of mouse DO combined with liquid/chow diets to compare the effects of ethanol on both a strain of TNFR1 knockout (TNFR1 KO) mice and on mice of their C57BL/6 (B6) control strain. In the B6 study, and in concordance with previous work, both radiological and histological analyses of direct bone formation in the distraction gaps demonstrated significant osteoinhibition due to ethanol compared with chow- or pair-fed mice. In the TNFR1 KO study and in support of the hypothesis, both radiological and histological analyses of distraction gap bone formation demonstrated no significant differences between the ethanol, chow fed, or pair fed. We conclude that exogenous rmTNF and ethanol-induced endogenous TNF act to inhibit new bone formation during DO by signaling primarily through TNFR1.


Subject(s)
Ethanol/pharmacology , Osteogenesis, Distraction/methods , Osteogenesis/drug effects , Tumor Necrosis Factor-alpha/physiology , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Tumor Necrosis Factor, Type I/deficiency , Signal Transduction/drug effects , Tibia/drug effects , Tibia/growth & development , Tibial Fractures/surgery
8.
Exp Diabetes Res ; 2011: 269378, 2011.
Article in English | MEDLINE | ID: mdl-21747824

ABSTRACT

Microalbuminuria in humans with Type 1 diabetes (T1D) is associated with increased urinary excretion of megalin, as well as many megalin ligands, including vitamin-D-binding protein (VDBP). We examined the DBA/2J diabetic mouse, nephropathy prone model, to determine if megalin and VDBP excretion coincide with the development of diabetic nephropathy. Megalin, VDBP, and 25-hydroxy-vitamin D (25-OHD) were measured in urine, and genes involved in vitamin D metabolism were assessed in renal tissues from diabetic and control mice at 10, 15, and 18 weeks following the onset of diabetes. Megalin, VDBP, and 25-OHD were increased in the urine of diabetic mice. 1-α hydroxylase (CYP27B1) mRNA in the kidney was persistently increased in diabetic mice, as were several vitamin D-target genes. These studies show that intrarenal vitamin D handling is altered in the diabetic kidney, and they suggest that in T1D, urinary losses of VDBP may portend risk for intrarenal and extrarenal vitamin D deficiencies.


Subject(s)
Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/metabolism , Diabetic Nephropathies/etiology , Diabetic Nephropathies/metabolism , Endocytosis , Vitamin D/metabolism , Animals , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/chemically induced , Diabetes Mellitus, Type 1/genetics , Diabetic Nephropathies/pathology , Disease Models, Animal , Disease Susceptibility , Endocytosis/physiology , Kidney/metabolism , Kidney/pathology , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Mice , Mice, Inbred DBA , Signal Transduction/physiology , Streptozocin
9.
Bone ; 48(4): 733-40, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21185416

ABSTRACT

Type 1 diabetes (T1DM) increases the likelihood of a fracture. Despite serious complications in the healing of fractures among those with diabetes, the underlying causes are not delineated for the effect of diabetes on the fracture resistance of bone. Therefore, in a mouse model of T1DM, we have investigated the possibility that a prolonged state of diabetes perturbs the relationship between bone strength and structure (i.e., affects tissue properties). At 10, 15, and 18 weeks following injection of streptozotocin to induce diabetes, diabetic male mice and age-matched controls were examined for measures of skeletal integrity. We assessed 1) the moment of inertia (I(MIN)) of the cortical bone within diaphysis, trabecular bone architecture of the metaphysis, and mineralization density of the tissue (TMD) for each compartment of the femur by micro-computed tomography and 2) biomechanical properties by three-point bending test (femur) and nanoindentation (tibia). In the metaphysis, a significant decrease in trabecular bone volume fraction and trabecular TMD was apparent after 10 weeks of diabetes. For cortical bone, type 1 diabetes was associated with decreased cortical TMD, I(MIN), rigidity, and peak moment as well as a lack of normal age-related increases in the biomechanical properties. However, there were only modest differences in material properties between diabetic and normal mice at both whole bone and tissue-levels. As the duration of diabetes increased, bone toughness decreased relative to control. If the sole effect of diabetes on bone strength was due to a reduction in bone size, then I(MIN) would be the only significant variable explaining the variance in the maximum moment. However, general linear modeling found that the relationship between peak moment and I(MIN) depended on whether the bone was from a diabetic mouse and the duration of diabetes. Thus, these findings suggest that the elevated fracture risk among diabetics is impacted by complex changes in tissue properties that ultimately reduce the fracture resistance of bone.


Subject(s)
Bone and Bones/physiopathology , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Animals , Bone Density , Diabetes Mellitus, Experimental/chemically induced , Male , Mice , Mice, Inbred DBA , Streptozocin , Tomography, X-Ray Computed
10.
Bone ; 46(2): 410-7, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19772956

ABSTRACT

Distraction osteogenesis (DO) is a process which induces direct new bone formation as a result of mechanical distraction. Tumor necrosis factor-alpha (TNF) is a cytokine that can modulate osteoblastogenesis. The direct effects of TNF on direct bone formation in rodents are hypothetically mediated through TNF receptor 1 and/or 2 (TNFR1/2) signaling. We utilized a unique model of mouse DO to assess the effects of 1) TNFR homozygous null gene alterations on direct bone formation and 2) rmTNF on wild type (WT), TNFR1(-/-) (R1KO), and TNR2(-/-) (R2KO) mice. Radiological and histological analyses of direct bone formation in the distraction gaps demonstrated no significant differences between the WT, R1KO, R2KO, or TNFR1(-/-) and R2(-/-) (R1 and 2KO) mice. R1 and 2KO mice had elevated levels of serum TNF but demonstrated no inhibition of new bone formation. Systemic administration by osmotic pump of rmTNF during DO (10 microg/kg/day) resulted in significant inhibition of gap bone formation measures in WT and R2KO mice, but not in R1KO mice. We conclude that exogenous rmTNF and/or endogenous TNF act to inhibit new bone formation during DO by signaling primarily through TNFR1.


Subject(s)
Osteogenesis, Distraction , Osteogenesis/physiology , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type I/deficiency , Tumor Necrosis Factor-alpha/pharmacology , Animals , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Bone and Bones/pathology , Male , Mice , Osteogenesis/drug effects , Radiography , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/pharmacology , Staining and Labeling , Tumor Necrosis Factor-alpha/blood
11.
J Bone Miner Res ; 25(1): 114-23, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19580462

ABSTRACT

Skeletal changes accompanying aging are associated with both increased risk of fractures and impaired fracture healing, which, in turn, is due to compromised bone regeneration potential. These changes are associated with increased serum levels of selected proinflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-alpha). We have used a unique model of bone regeneration to demonstrate (1) that aged-related deficits in direct bone formation can be restored to young mice by treatment with TNF blockers and (2) that the cyclin-dependent kinase inhibitor p21 is a candidate for mediation of the osteoinhibitory effects of TNF. It has been hypothesized recently that TNF antagonists may represent novel anabolic agents, and we believe that the data presented here represent a successful test of this hypothesis.


Subject(s)
Aging/drug effects , Osteoblasts/cytology , Osteoblasts/drug effects , Osteogenesis/drug effects , Regeneration/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Aging/blood , Animals , Blotting, Western , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cytokines/blood , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Radiography , Receptors, Tumor Necrosis Factor, Type I/pharmacology , Receptors, Tumor Necrosis Factor, Type II/pharmacology , Recombinant Proteins/pharmacology , Solubility/drug effects , Tibia/diagnostic imaging , Tibia/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Wound Healing/drug effects
12.
Endocrinology ; 149(4): 1697-704, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18162513

ABSTRACT

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.


Subject(s)
Core Binding Factor Alpha 1 Subunit/genetics , Diabetes Mellitus, Type 1/physiopathology , Osteogenesis/physiology , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Core Binding Factor Alpha 1 Subunit/physiology , Down-Regulation , Female , Insulin/pharmacology , Matrix Metalloproteinase 9/genetics , Mice , Osteogenesis, Distraction , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/genetics
13.
Acta Orthop ; 78(1): 46-55, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17453392

ABSTRACT

BACKGROUND: There is evidence to suggest that impairment in bone formation and/or turnover is associated with the metabolic abnormalities characteristic of type-2 diabetes mellitus. However, bone regeneration/repair in type-2 diabetes has not been modeled. Using Zucker Diabetic Fatty (ZDF) rats (a model of type-2 diabetes) for tibial distraction osteogenesis (DO), we hypothesized that bone formation within the distraction gap would be impaired. ANIMALS AND METHODS: Rats were examined for body weight, glycosuria, and glycosemia to confirm the diabetic condition during the study. The rats received placement of the external fixators and osteotomies on the left tibia. Distraction was initiated the following day at 0.2 mm twice a day and continued for 14 days. The lengthened tibiae were harvested and distraction gaps were examined radiographically and histologically. RESULTS: We found significant reduction in new bone formation in the distraction gaps of the ZDF rats, both radiographically and histologically, compared to lean rats. We found a decrease in a marker of cellular proliferation in the distraction gaps and increased adipose volume in adjacent bone marrow of the ZDF rats. INTERPRETATION: Our findings suggest that this model might be used to study the contributions of leptin resistance, insulin resistance and/or hyperglycemia to impaired osteoblastogenesis in vivo.


Subject(s)
Bone Regeneration/physiology , Bone and Bones/metabolism , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Osteogenesis/physiology , Animals , Bone Density , Bone and Bones/cytology , Bone and Bones/pathology , Cell Proliferation , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Immunohistochemistry , Models, Biological , Rats , Rats, Zucker , Tibia/cytology , Tibia/metabolism , Tibia/pathology
14.
Toxicol Appl Pharmacol ; 220(3): 302-10, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17391719

ABSTRACT

Tumor necrosis factor-alpha (TNF-alpha) is an inflammatory cytokine that modulates osteoblastogenesis. In addition, the demonstrated inhibitory effects of chronic ethanol exposure on direct bone formation in rats are hypothetically mediated by TNF-alpha signaling. The effects in mice are unreported. Therefore, we hypothesized that in mice (1) administration of a soluble TNF receptor 1 derivative (sTNF-R1) would protect direct bone formation during chronic ethanol exposure, and (2) administration of recombinant mouse TNF-alpha (rmTNF-alpha) to ethanol naïve mice would inhibit direct bone formation. We utilized a unique model of limb lengthening (distraction osteogenesis, DO) combined with liquid diets to measure chronic ethanol's effects on direct bone formation. Chronic ethanol exposure resulted in increased marrow TNF, IL-1, and CYP 2E1 RNA levels in ethanol-treated vs. control mice, while no significant weight differences were noted. Systemic administration of sTNF-R1 during DO (8.0 mg/kg/2 days) to chronic ethanol-exposed mice resulted in enhanced direct bone formation as measured radiologically and histologically. Systemic rmTNF-alpha (10 microg/kg/day) administration decreased direct bone formation measures, while no significant weight differences were noted. We conclude that chronic ethanol-associated inhibition of direct bone formation is mediated to a significant extent by the TNF signaling axis in a mouse model.


Subject(s)
Ethanol/pharmacology , Osteogenesis, Distraction/methods , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacology , Animals , Bone Marrow/drug effects , Bone Marrow/metabolism , Central Nervous System Depressants/administration & dosage , Central Nervous System Depressants/pharmacology , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , Ethanol/administration & dosage , Ethanol/blood , Interleukin-6/genetics , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Osteogenesis/drug effects , Osteogenesis/physiology , Osteotomy/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction/methods , TNF Receptor-Associated Factor 1/metabolism , TNF Receptor-Associated Factor 1/pharmacology , Tibia/surgery , Tumor Necrosis Factor-alpha/genetics
15.
Alcohol ; 39(3): 159-67, 2006 Jul.
Article in English | MEDLINE | ID: mdl-17127135

ABSTRACT

Excessive alcohol consumption has been reported to interfere with human bone homeostasis and repair in multiple ways. Previous studies have demonstrated that chronic ethanol exposure in the rat via an intragastric dietary delivery system inhibits direct bone formation during distraction osteogenesis (DO, limb lengthening). The opportunity to extend the rat ethanol studies to mice is now possible due to the development of mouse models of DO. This study employed a novel combination of liquid ethanol diet delivery and a murine DO model to test the hypothesis that chronic ethanol exposure would result in deficits in direct bone formation during DO in contrast to the pair-fed controls. Twenty-eight 12-month-old C57BL/6 male mice were acclimated to the Lieber-DeCarli liquid control diet #710027 (Dyets Inc.) over a 1-week period. The mice were separated into two diet groups (n=14/group): pair-fed control and ethanol (diet #710260). After being on diet for 82 days, all mice underwent placement of an external fixator and osteotomy on the left tibia. Following a 6-day latency period, distraction began at a rate of 0.075 mm twice a day (b.i.d.) for 14 days. The weight changes were equivalent for both groups. The hypothesis that chronic ethanol exposure would inhibit direct bone formation and produce skeletal toxicity was supported by radiographic (P=.011) and histologic (P=.002) analyses of the % new bone formation in the DO gaps, by peripheral quantitative computed tomography analysis of the total volumetric bone mineral density of the contralateral proximal tibias (P<.001) and contralateral femoral necks (P=.012), by three-point bending on the contralateral tibias (P<.001 energy to break), by pin site bone formation measures (P<.001), and by ethanol-associated increased adipocyte area (adjacent to the gap) percentages (P<.002). We conclude that this model can be used to study the mechanisms underlying inhibition of bone formation by chronic ethanol exposure and to test preclinical interventions.


Subject(s)
Ethanol/pharmacology , Osteogenesis, Distraction , Osteogenesis/drug effects , Tibia/drug effects , Absorptiometry, Photon , Adipocytes/drug effects , Animals , Male , Mice , Mice, Inbred C57BL , Models, Animal , Tibia/diagnostic imaging , Tibia/growth & development
16.
J Bone Miner Res ; 21(9): 1359-66, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16939394

ABSTRACT

UNLABELLED: DO was used in an aged mouse model to determine if systemically and/or locally administered rhIGF-I improved osteoblastogenesis and new bone formation. Local and systemic rhIGF-I treatment increased new bone formation. However, only systemic delivery produced measurable concentrations of rhIGF-I in the circulation. INTRODUCTION: Human and rodent research supports a primary role for IGF-I in bone formation. Significant roles for both endocrine and paracrine/autocrine IGF-I have been suggested for normal osteoblastogenesis and bone formation. We have assessed, using a mouse model of distraction osteogenesis (DO), the impact of continuous administration of recombinant human (rh)IGF-I, delivered either locally to the distraction site or absorbed systemically, on bone formation in an aged mouse model. MATERIALS AND METHODS: DO was performed in aged mice (18-month-old C57BL/6 male mice), which were distracted at 0.15 mm daily. At the time of osteotomy, miniosmotic pumps were inserted subcutaneously to (1) deliver vehicle or rhIGF-I subcutaneously for systemic delivery or (2) deliver vehicle or rhIGF-I directly to the newly forming bone through infusion tubing routed subcutaneously from the pump to the distraction site. Serum concentrations of mouse IGF-I, human IGF-I, and osteocalcin were determined at the end of the study. RESULTS: New bone formation observed in DO gaps showed a significant increase in new bone formation in rhIGF-I-treated mice, irrespective of delivery route. However, detectable levels of human IGF-I were found only in the serum of animals receiving rhIGF-I systemically. Osteocalcin levels did not differ between controls and rhIGF-I-treated groups. CONCLUSIONS: Locally and systemically delivered rhIGF-I both produce significant increases in new bone formed in an aged mouse model in which new bone formation is normally markedly impaired, suggesting that rhIGF-I may improve senile osteoporosis. Because systemic administration of IGF-I can result in untoward side effects, including an increased risk for cancer, the findings that locally delivered IGF-I improves bone regeneration without increasing circulating IGF-I levels suggests that this delivery route may be preferable in an at-risk, aged population.


Subject(s)
Aging/drug effects , Injections, Intralesional/methods , Insulin-Like Growth Factor I/administration & dosage , Osteogenesis/drug effects , Recombinant Proteins/administration & dosage , Animals , Electron Probe Microanalysis , Humans , Injections, Intralesional/instrumentation , Male , Mice , Mice, Inbred C57BL , Models, Animal , Models, Biological , Osteotomy/rehabilitation , Radiography , Tibia/cytology , Tibia/diagnostic imaging
17.
Diabetes ; 54(10): 2875-81, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16186388

ABSTRACT

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (microCT). Contralateral tibiae were analyzed using microCT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < or = 0.001) and radiographically (P < or = 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < or = 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration.


Subject(s)
Diabetes Mellitus, Type 1/physiopathology , Osteogenesis/physiology , Animals , Bone and Bones/chemistry , Collagen/blood , Collagen Type I , Diabetes Mellitus, Type 1/drug therapy , Female , Immunohistochemistry , Insulin/blood , Insulin/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Osteocalcin/blood , Osteogenesis/drug effects , Osteogenesis, Distraction , Peptides/blood , Receptor, Insulin/analysis , Tibia/chemistry , Tomography, X-Ray Computed
18.
Alcohol Clin Exp Res ; 29(8): 1466-72, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16131855

ABSTRACT

BACKGROUND: Chronic ethanol exposure inhibits the rapid bone formation demonstrated during limb lengthening by distraction osteogenesis (DO). This inhibition is attenuated by simultaneous administration of antagonists to the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. The individual effects on inhibition of osteogenesis by these cytokines were tested. We hypothesized that administration of individual antagonists to these cytokines [IL-1 receptor antagonist (IL-1ra) or polyethylene glycol-conjugated soluble TNF receptor type 1 (sTNFR1)] would enhance DO and that the individual administration of each cytokine [recombinant rat (rr) IL-1 or recombinant rat (rr) TNF] would inhibit DO. METHODS: Rats were either infused with a liquid diet with or without ethanol (antagonist studies) or given rat chow (recombinant studies) and underwent tibial fractures stabilized with external fixators for DO. The bioactive substances were administered by systemic (antagonist studies) or local (recombinant) diffusion. RESULTS: A comparison of histologic sections from these distracted tibias demonstrated a protective effect on bone formation by sTNFR1 (p<0.05), unexpectedly, an IL-1ra-related decrease in bone formation (p<0.02), significant decreases in bone formation with rrTNF compared with the vehicle controls (p<0.02), and no significant changes in bone formation with rrIL-1. The cellular responses (fibroblastic and inflammatory cells) were unique for each recombinant cytokine administered. CONCLUSIONS: These results suggest that the osteoinhibitory effects of chronic ethanol exposure are mediated in part by the TNF signaling axis.


Subject(s)
Alcoholism/physiopathology , Bone Regeneration/drug effects , Ethanol/toxicity , Interleukin-1/physiology , Osteogenesis, Distraction , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/physiology , Animals , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology
19.
Toxicol Sci ; 82(2): 656-60, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15470231

ABSTRACT

We tested the hypothesis that combined administration of IL-1 and TNF antagonists would protect fracture healing from inhibition by chronic ethanol exposure. Adult male rats were fed a liquid diet +/- ethanol (CON and ETOH) by intragastric infusion for three weeks prior to and three weeks after creation of an externally fixated tibial fracture. Beginning the day of fracture, one-half of each dietary group received 2.0 mg/kg/day IL-1ra and 2.0 mg/kg/2-days sTNFR1 (CON + ANTAG and ETOH + ANTAG), while all other animals received vehicle alone (CON + VEH and ETOH + VEH). Scoring of ex vivo radiographs and analysis by pQCT revealed a significantly lower incidence of bridging and reduced total mineral content in the ETOH + VEH group compared to all other groups. These results support, for the first time, the hypothesis that IL-1 and TNF antagonists are capable of protecting fracture healing from the inhibition associated with chronic ethanol consumption.


Subject(s)
Central Nervous System Depressants/toxicity , Ethanol/antagonists & inhibitors , Ethanol/toxicity , Fracture Healing/drug effects , Interleukin-1/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Bony Callus/drug effects , Male , Osteogenesis/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Tumor Necrosis Factor, Type I/drug effects , Signal Transduction/drug effects , Tomography, X-Ray Computed , Weight Gain/drug effects
20.
Cytokine ; 23(6): 179-89, 2003 Sep 21.
Article in English | MEDLINE | ID: mdl-12967643

ABSTRACT

Chronic alcohol consumption is a risk factor for osteoporosis and inhibits osseous repair and regeneration. We investigated the hypothesis that chronic ethanol exposure induces the expression of TNF-alpha and/or IL-1beta and inhibits proliferation during distraction osteogenesis (DO). Following six weeks of liquid diet infusion (+/-ethanol) and 14 days of DO, the expression of TNF-alpha and IL-1beta in the distraction gap and contralateral femoral marrow of adult male rats was examined by immunohistochemistry and RT-PCR, respectively. In the bone marrow, the expression of both TNF-alpha and IL-1beta mRNA was significantly increased by ethanol (p<0.04 for both). In the DO gap, ethanol exposure increased the expression of TNF-alpha in both the fibrous interzone and primary matrix front (PMF), while IL-1beta expression was not significantly affected in either region. A negative correlation was found between the percentage of PCNA+ and TNF+ cells in the PMF (p<0.015, R(2)=0.655). Incubation of MC3T3-E1 cells with ethanol for 24 or 48 h produced a time and dose dependent two- to fourfold increase in TNF-alpha transcripts as measured by RT-PCR, demonstrating that ethanol can directly induce TNF-alpha expression in osteoblast-like cells. These results support the hypothesis that attenuation of bone formation by ethanol may be mediated, in part, by local increases in TNF-alpha during osteogenesis.


Subject(s)
Alcoholism/metabolism , Ethanol/administration & dosage , Ethanol/pharmacology , Osteogenesis, Distraction , Tumor Necrosis Factor-alpha/metabolism , Animals , Cell Division/drug effects , Cell Line , Femur/drug effects , Femur/metabolism , Immunohistochemistry , Interleukin-1/biosynthesis , Interleukin-1/genetics , Male , Proliferating Cell Nuclear Antigen/biosynthesis , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Tibia/drug effects , Tibia/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
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