osteoprotegerin and Femoral-Fractures

Wnt signaling, a major regulator of bone formation and homeostasis, might be involved in the bone loss of osteoporotic patients and the consequent impaired response to fracture. Therefore we analyzed Wnt-related, osteogenic, and adipogenic genes in bone tissue of elderly postmenopausal women undergoing hip replacement for either femoral fracture or osteoarthritis. Bone specimens derived from the intertrochanteric region of the femurs of 25 women with fracture (F) and 29 with osteoarthritis without fracture (OA) were analyzed. Specific miRNAs were analyzed in bone and in matched blood samples. RUNX2, BGP, and OPG showed lower expression in F than in OA samples, while OSX, OPN, BSP, and RANKL were not different. Inhibitory genes of Wnt pathway were lower in F versus OA.

Topics: Aged; Aged, 80 and over; beta Catenin; Core Binding Factor Alpha 1 Subunit; Female; Femoral Fractures; Humans; MicroRNAs; Osteoarthritis; Osteoprotegerin; Postmenopause; RANK Ligand; Wnt Signaling Pathway

Fracture healing in the elderly is associated with a declined healing potential caused by multiple factors including a delay of vascularization. Erythropoietin (EPO) has been demonstrated to improve vascularization and fracture healing in adult mice. We, therefore, hypothesized that EPO in aged mice also improves fracture healing. For this purpose, EPO was given daily in a femoral fracture model in aged mice and compared to vehicle-treated controls using radiological, biomechanical, histomorphometric and Western blot techniques. Blood analyses revealed significantly higher concentrations of hemoglobin and a higher hematocrit in EPO-treated animals at 14 and 35 days after fracture. Micro-computed tomography (μCT) indicated that the fraction of bone volume/tissue volume within the callus did not differ between the two groups. However, μCT showed a 3-fold increased tissue mineral density (TMD) in the callus of EPO-treated animals compared to controls. The callus TMD of the EPO-treated animals was also 2-fold higher when compared to the TMD of the unfractured contralateral femur. Interestingly, biomechanical analyses revealed a reduced bending stiffness in femurs of EPO-treated animals at day 35. The histomorphometrically analyzed callus size and callus composition did not show significant differences between the study groups. However, Western blot analyses exhibited an increased expression of osteoprotegerin (OPG), but in particular of receptor activator of NF-κB ligand (RANKL) in the callus of the EPO-treated animals. Further histological analyses of the callus tissue showed that this was associated with an increased number of newly formed blood vessels and a higher number of tartrate-resistant acid phosphatase (TRAP)

Topics: Aging; Animals; Biomechanical Phenomena; Bone Remodeling; Bony Callus; Erythropoietin; Female; Femoral Fractures; Fracture Healing; Hemoglobins; Male; Mice; Osteoprotegerin; RANK Ligand; X-Ray Microtomography

As new insights into the complexities of endochondral fracture repair emerge, the temporal role of osteoclast activity remains ambiguous. With numerous antiresorptive agents available to treat bone disease, understanding their impact on bone repair is vital. Further, in light of recent work suggesting osteoclast activity may not be necessary during early endochondral fracture union, we hypothesize instead a pivotal role of matrix metalloproteinase (MMP) secreting cells in driving this process. Although the role of MMPs in fracture healing has been examined, no directly comparative experiments exist. We examined a number of antiresorptive treatments to either block osteoclast activity, including the potent bisphosphonates zoledronic acid (ZA) and clodronate (CLOD), which work via differing mechanisms, or antagonize osteoclastogenesis with recombinant OPG (HuOPG-Fc), comparing these directly to an inhibitor of MMP activity (MMI270). Endochondral ossification to union occurred normally in all antiresorptive groups. In contrast, MMP inhibition greatly impaired endochondral union, significantly delaying cartilage callus removal. MMP inhibition also produced smaller, denser hard calluses. Hard callus remodeling was, as expected, delayed with ZA, CLOD, and OPG treatment at 4 and 6 weeks, resulting in larger, more mineralized calluses at 6 weeks. As a result of reduced hard callus turnover, bone formation was reduced with antiresorptive agents at these time points. These results confirm that the achievement of endochondral fracture union occurs independently of osteoclast activity. Alternatively, MMP secretion by invading cells is obligatory to endochondral union. This study provides new insight into cellular contributions to bone repair and may abate concerns regarding antiresorptive therapies impeding initial fracture union.

Topics: Animals; Bone Density Conservation Agents; Clodronic Acid; Collagenases; Diphosphonates; Femoral Fractures; Fracture Healing; Humans; Hydroxamic Acids; Imidazoles; Male; Matrix Metalloproteinase Inhibitors; Osteoclasts; Osteoprotegerin; Pyrazines; Rats; Rats, Wistar; Recombinant Proteins; Sulfonamides; Time Factors; Zoledronic Acid

Melatonin, the major pineal hormone, is known to regulate distinct physiologic processes. Previous studies have suggested that it supports skeletal growth and bone formation, most probably by inhibiting bone resorption. There is no information, however, whether melatonin affects fracture healing. We therefore studied in a mouse femur fracture model the influence of melatonin on callus formation and biomechanics during fracture healing.. Thirty CD-1 mice received 50 mg/kg body weight melatonin i.p. daily during the entire 2-wk or 5-wk observation period. Controls (n = 30) received equivalent amounts of vehicle. Bone healing was studied by radiological, biomechanical, histomorphometrical, and protein biochemical analyses at 2 and 5 wk after fracture.. Biomechanical analysis at 2 wk after fracture healing showed a significantly lower bending stiffness in melatonin-treated animals compared with controls. A slightly higher amount of cartilage tissue and a significantly larger callus size indicated a delayed remodeling process after melatonin treatment. Western blot analysis showed a significantly reduced expression of receptor activator of nuclear factor-κB ligand (RANKL) and collagen I after melatonin treatment. The reduced expression of RANKL was associated with a diminished number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts within the callus of the newly formed bone.. Because bone resorption is an essential requirement for adequate remodeling during fracture healing, we conclude that melatonin impairs fracture healing by suppressing bone resorption through down-regulation of RANKL-mediated osteoclast activation.

Topics: Animals; Biomechanical Phenomena; Bone Remodeling; Bone Resorption; Collagen Type I; Dose-Response Relationship, Drug; Down-Regulation; Femoral Fractures; Fracture Healing; Melatonin; Mice; Mice, Inbred Strains; Models, Animal; Osteoclasts; Osteoprotegerin; RANK Ligand

As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro-computed tomographic (µCT), and biomechanical measures. EP1(-/-) mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1(-/-) mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1(-/-) mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE(2) receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone Density; Bony Callus; Cartilage; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type II; Collagen Type X; Core Binding Factor Alpha 1 Subunit; Female; Femoral Fractures; Fracture Healing; Gene Expression; Isoenzymes; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoblasts; Osteocalcin; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Sp7 Transcription Factor; Tartrate-Resistant Acid Phosphatase; Time Factors; Torsion, Mechanical; Transcription Factors; X-Ray Microtomography

Several reports indicated that interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF- alpha) play important regulatory roles in bone remodeling and homeostasis. In addition, receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been shown to be important regulators of osteoclastogenesis during bone remodeling, and their expressions were examined during fracture healing in a mouse model of tibial fracture. However, studies linking RANKL, OPG, IL-6 and TNF-alpha in patients with head injury and fracture are lacking.. Within the first few hours of admission to hospital and at 4, 8, and 12 weeks after the injury, we evaluated changes in serum levels of RANKL, OPG, IL-6 and TNF-alpha in 24 male patients with a concomitant head injury and fracture and in 26 male patients with fracture only. These levels were compared with those found in 36 healthy controls.. The RANKL/OPG ratios were found to significantly lower in patients with a concomitant head injury and fracture than in the controls immediately after admission and at 4, 8, and 12 weeks after the injury. In addition, RANKL/OPG ratios were significantly lower in patients with a concomitant head injury and fracture than in those with fracture at 8 and 12 weeks after the injury. The serum IL-6 levels were significantly higher in patients with a concomitant head injury and fracture than in the controls upon admission, and at 4, 8, and 12 weeks after the injury. Moreover, the serum IL-6 levels were significantly higher in patients with a head injury and fracture than in those with just a fracture at 4, 8, and 12 weeks after the injury.. Based on these changes in the profiles of RANKL, OPG, and IL-6 and the RANKL/OPG ratio, altered repair of a fracture can occur in patients with a concomitant head injury and fracture.

Topics: Adult; Comorbidity; Craniocerebral Trauma; Femoral Fractures; Fracture Healing; Fractures, Bone; Glasgow Coma Scale; Hematoma, Subdural; Humans; Humeral Fractures; Interleukin-6; Intracranial Hemorrhage, Traumatic; Male; Middle Aged; Osteoprotegerin; RANK Ligand; Tibial Fractures; Tumor Necrosis Factor-alpha; Young Adult

The osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL)/receptor activator of nuclear factor-kappaB (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB (10(-11) M-10(-7) M) dose-dependently inhibited OPG secretion with an EC(50) of 1.08 x 10(-10) M, and also decreased its expression. This action was blocked by the antagonist CGRP(8-37). Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP (10(-8) M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 x 10(-6) M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect.

Topics: Acid Phosphatase; Aged; Bone Remodeling; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Carrier Proteins; Cathepsin K; Cathepsins; Cell Separation; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Femoral Fractures; Glycoproteins; Humans; Isoenzymes; Leukocytes, Mononuclear; Membrane Glycoproteins; Middle Aged; Osteoblasts; Osteoclasts; Osteoprotegerin; Peptide Fragments; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Calcitonin Gene-Related Peptide; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Tartrate-Resistant Acid Phosphatase