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 (CT). Contralateral tibiae were analyzed using CT. 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 < 0.001) and radiographically (P < 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by CT 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. Diabetes 54:2875-2881, 2005 T ype 1 diabetes is associated with several disorders of skeletal health, including decreased bone density, an increased risk for osteoporosis (1-6), and fragility fracture (7-9), as well as poor bone healing and regeneration characteristics (10), conditions which all rely, in part, upon an intramembranous component to bone formation. Increasing evidence suggests that skeletal abnormalities in type 1 diabetes may, in part, result from the detrimental effects of type 1 diabetes on bone formation. For example, decreased expression of transcription factors that regulate osteoblast differentiation have been demonstrated in animal models of type 1 diabetes (11). Numerous reports of bone histology in diabetic animals demonstrate decreased osteoblast number, osteoid volume, and mineral apposition rates (rev. in 12). In diabetic rats, plasma osteocalcin concentrations, a marker of osteoblast activity, acutely decline beginning on the 1st day of glucosuria (13). Similarly, serum concentrations of osteocalcin in children with newly diagnosed type 1 diabetes are significantly lower at the onset of disease (14). Serum markers correlated with bone formation (IGF-I, alkaline phosphatase, and osteocalcin) also are significantly lower in diabetic patients with osteopenia compared with those without osteopenia (2), and studies have demonstrated that lower bone mineral density (BMD) in type 1 diabetes is correlated with decreased markers of bone formation and more exaggerated dysregulation of the IGF system (15).The present study was designed to test the hypothesis that type 1 diabetes specifically impedes intramembranous bone formation by using a model of tibial distraction osteogenesis uniquely modified for use ...

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.

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 α (TNF-α). 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. © 2010 American Society for Bone and Mineral Research

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.

Distraction Osteogenesis (DO) is a process which induces direct new bone formation as a result of mechanical distraction. Tumor necrosis factor-α (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 -/-& R2 -/-(R1&2KO) mice. R1&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 ug/ 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.

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.

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.

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 (2mg/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 5 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.