A preliminary biomechanical study of cyclic preconditioning effects on canine cadaveric whole femurs.

Biomechanical preconditioning of biological specimens by cyclic loading is routinely done presumably to stabilize properties prior to the main phase of a study. However, no prior studies have actually measured these effects for whole bone of any kind. The aim of this study, therefore, was to quantify these effects for whole bones. Fourteen matched pairs of fresh-frozen intact cadaveric canine femurs were sinusoidally loaded in 4-point bending from 50 N to 300 N at 1 Hz for 25 cycles. All femurs were tested in both anteroposterior (AP) and mediolateral (ML) bending planes. Bending stiffness (i.e., slope of the force-vs-displacement curve) and linearity R(2) (i.e., coefficient of determination) of each loading cycle were measured and compared statistically to determine the effect of limb side, cycle number, and bending plane. Stiffnesses rose from 809.7 to 867.7 N/mm (AP, left), 847.3 to 915.6 N/mm (AP, right), 829.2 to 892.5 N/mm (AP, combined), 538.7 to 580.4 N/mm (ML, left), 568.9 to 613.8 N/mm (ML, right), and 553.8 to 597.1 N/mm (ML, combined). Linearity R(2) rose from 0.96 to 0.99 (AP, left), 0.97 to 0.99 (AP, right), 0.96 to 0.99 (AP, combined), 0.95 to 0.98 (ML, left), 0.94 to 0.98 (ML, right), and 0.95 to 0.98 (ML, combined). Stiffness and linearity R(2) versus cycle number were well-described by exponential curves whose values leveled off, respectively, starting at 12 and 5 cycles. For stiffness, there were no statistical differences for left versus right femurs (p = 0.166), but there were effects due to cycle number (p < 0.0001) and AP versus ML bending plane (p < 0.0001). Similarly, for linearity, no statistical differences were noted due to limb side (p = 0.533), but there were effects due to cycle number (p < 0.0001) and AP versus ML bending plane (p = 0.006). A minimum of 12 preconditioning cycles was needed to fully stabilize both the stiffness and linearity of the canine femurs. This is the first study to measure the effects of mechanical preconditioning on whole bones, having some practical implications on research practices.

Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer.

PURPOSE: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. EXPERIMENTAL DESIGN: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. RESULTS: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD=54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 microg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. CONCLUSIONS: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

A fully human monoclonal antibody to the insulin-like growth factor I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo.

The insulin-like growth factor I receptor (IGF-IR) is overexpressed in many diverse tumor types and is a critical signaling molecule for tumor cell proliferation and survival. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. Through screening of a Fab phage display library, we have generated a fully human antibody (A12) that binds to the IGF-IR with high affinity (4.11 x 10(-11) M) and inhibits ligand binding with an IC(50) of 0.6-1 nM. Antibody-mediated blockade of ligand binding to the IGF-IR inhibited downstream signaling of the two major insulin-like growth factor (IGF) pathways, mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt, in MCF7 human breast cancer cells. As a result, the mitogenic and proliferative potential of IGF-I and IGF-II were significantly reduced. A12 did not block insulin binding to the insulin receptor but could block binding to atypical IGF-IR in MCF7 cells. In addition, A12 was shown to induce IGF-IR internalization and degradation on specific binding to tumor cells, resulting in a significant reduction in cell surface receptor density. In xenograft tumor models in vivo, IGF-IR blockade by A12 was shown to occur rapidly, resulting in significant growth inhibition of breast, renal, and pancreatic tumors. Histological analysis of tumor sections demonstrated a marked increase in apoptotic tumor cells in antibody-treated animals. These results demonstrate that A12 possesses strong antitumor activity in vitro and in vivo and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on IGF-IR signaling for growth and survival.

The effect of muscle contusion on cortical bone and muscle perfusion following reamed, intramedullary nailing: a novel canine tibia fracture model.

BACKGROUND: Management of tibial fractures associated with soft tissue injury remains controversial. Previous studies have assessed perfusion of the fractured tibia and surrounding soft tissues in the setting of a normal soft tissue envelope. The purpose of this study was to determine the effects of muscle contusion on blood flow to the tibial cortex and muscle during reamed, intramedullary nailing of a tibial fracture. METHODS: Eleven adult canines were distributed into two groups, Contusion or No-Contusion. The left tibia of each canine underwent segmental osteotomy followed by limited reaming and locked intramedullary nailing. Six of the 11 canines had the anterior muscle compartment contused in a standardized fashion. Laser doppler flowmetry was used to measure cortical bone and muscle perfusion during the index procedure and at 11 weeks post-operatively. RESULTS: Following a standardized contusion, muscle perfusion in the Contusion group was higher compared to the No-Contusion group at post-osteotomy and post-reaming (p < 0.05). Bone perfusion decreased to a larger extent in the Contusion group compared to the No-Contusion group following osteotomy (p < 0.05), and the difference in bone perfusion between the two groups remained significant throughout the entire procedure (p < 0.05). At 11 weeks, muscle perfusion was similar in both groups (p > 0.05). There was a sustained decrease in overall bone perfusion in the Contusion group at 11 weeks, compared to the No-Contusion group (p < 0.05). CONCLUSIONS: Injury to the soft tissue envelope may have some deleterious effects on intraosseous circulation. This could have some influence on the fixation method for tibia fractures linked with significant soft tissue injury.

The effect of cement mixing time on the biomechanics of cement augmented plated fractures in canine femora.

OBJECTIVE: The goal of this study was to determine the effect of cement mixing time and, hence, cement viscosity on the biomechanical behavior of femoral fracture fixation. DESIGN: Cadaveric plated canine femoral fracture model, comparing treatments in matched pairs. SETTING: Orthopaedic biomechanics laboratory. INTERVENTION: Cement was inserted both as a liquid and as a paste in standard and oversized screw holes to augment fixation with plates and screws. MAIN OUTCOME MEASUREMENTS: Standard 4-point bending tests were performed to obtain stiffness and failure load values. RESULTS: Liquid cement had a 1.38 times increase in stiffness and a failure load 1.84 times greater compared with paste cement, regardless of hole size with a gap at the fracture site (P < 0.05). Liquid cement had a force to failure of 1.77 and 1.91 times in the standard-sized and oversized holes, respectively, when compared with paste cement (P < 0.05). CONCLUSIONS: When the cement was inserted in a liquid state in a plated femoral diaphyseal fracture with a gap, screw purchase augmentation achieved greater bending stiffness and resisted a greater failure load.

Interobserver reliability of the young-burgess and tile classification systems for fractures of the pelvic ring.

OBJECTIVES: The purpose of this study was to measure interobserver reliability of 2 classification systems of pelvic ring fractures and to determine whether computed tomography (CT) improves reliability. The reliability of several radiographic findings was also tested. METHODS: Thirty patients taken from a database at a Level I trauma facility were reviewed. For each patient, 3 radiographs (AP pelvis, inlet, and outlet) and CT scans were available. Six different reviewers (pelvic and acetabular specialist, orthopaedic traumatologist, or orthopaedic trainee) classified the injury according to Young-Burgess and Tile classification systems after reviewing plain radiographs and then after CT scans. The Kappa coefficient was used to determine interobserver reliability of these classification systems before and after CT scan. RESULTS: For plain radiographs, overall Kappa values for the Young-Burgess and Tile classification systems were 0.72 and 0.30, respectively. For CT scan and plain radiographs, the overall Kappa values for the Young-Burgess and Tile classification systems were 0.63 and 0.33, respectively. The pelvis/acetabular surgeons demonstrated the highest level of agreement using both classification systems. For individual questions, the addition of CT did significantly improve reviewer interpretation of fracture stability. The pre-CT and post-CT Kappa values for fracture stability were 0.59 and 0.93, respectively. CONCLUSIONS: The CT scan can improve the reliability of assessment of pelvic stability because of its ability to identify anatomical features of injury. The Young-Burgess system may be optimal for the learning surgeon. The Tile classification system is more beneficial for specialists in pelvic and acetabular surgery.

A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity.

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.

Cloning, expression, and purification of mouse heparanase.

Heparanase is an endoglucuronidase that plays an important role in tumor invasion and metastasis. A full-length heparanase gene was cloned from a mouse embryo cDNA library and determined to encode a protein of 535 amino acids that is 77% identical to human heparanase. The full-length mouse gene was stably expressed in NS0 myeloma cells. The recombinant mouse heparanase protein was purified to homogeneity from cell lysates by a combination of Con-A affinity chromatography, heparin affinity chromatography, and size exclusion chromatography. The purified protein consisted of a non-covalent heterodimer of 50- and 8-kDa polypeptides, similar to the human homolog. The protein was enzymatically active in assays using radiolabeled ECM and heparan sulfate as substrates. The maximum heparanase activity was observed at acidic conditions; however, significant activity was also detected at neutral pH. The enzymatic activity of mouse heparanase was blocked by known heparanase inhibitors.