Establishment of a streptozotocin-induced diabetic domestic pig model and a systematic evaluation of pathological changes in the hard and soft tissue over a 12-month period

Authors

Cornelius Von Wilmowsky, Philipp Stockmann, Philipp Metzler, Igor Alexander Harsch, Kerstin Amann, Karl Andreas Schlegel

Abstract

The number of diabetic patients in need of medical treatment is growing steadily. Therefore, a diabetic animal model with high degree of similarities with humans, which is suitable for the systematic evaluation of biomaterials and medical devices, is needed. Twenty domestic pigs were used for the study. Fifteen received Streptozotocin (STZ) to induce diabetes mellitus. Internal parameters were measured and bone as well as soft tissues biopsies were taken after 0, 6 and 12 months and evaluated qualitatively and quantitatively by means of scanning electronic microscopy, light microscopy and microradiography. The results of the clinical internal parameters, determined by the American Diabetes Association for the definition of diabetes mellitus could be fulfilled. Pathological changes of the skin vasculatures were already visible after 6 months with a significant wall thickening in the diabetic group. The bone mineralization was lower in the diabetic group after 6 months and with a significant difference after 12 months. From the present results, it can be concluded that a STZ dosage of 90 mg/kg body weight in the domestic pig is suitable for the induction of an apparent diabetes, leading to histolopathological changes in the hard and soft tissues already after 6 months. The high degree of similarities with humans makes it an interesting diabetic animal model for biomaterial research in a compromised animal model. To cite this article: von Wilmowsky C, Stockmann P, Metzler P, Harsch IA, Amann K, Schlegel KA. Establishment of a streptozotocin-induced diabetic domestic pig model and a systematic evaluation of pathological changes in the hard and soft tissue over a 12-month period.

Link to Article

http://dx.doi.org/10.1111/j.1600-0501.2010.01914.x

The effect of combined delivery of recombinant human bone morphogenetic protein-2...

Title

The effect of combined delivery of recombinant human bone morphogenetic protein-2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium-phosphate-coated implants on osseointegration

Authors

Mustafa Ramazanoglu, Rainer Lutz, Celaletdin Ergun, Cornelius von Wilmowsky, Emeka Nkenke, Karl Andreas Schlegel

Abstract

The delivery of growth factors for enhanced osseointegration depends on the effectiveness of the carrier systems at the bone–implant interface. This study evaluated the effect of solo and dual delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF165) from biomimetically octacalcium phosphate-coated implants on osseointegration. Biomimetic implants, bearing either a single growth factor (BMP or VEGF) or their combination (BMP+VEGF), were established, and compared with acid-etched (AE, control) and biomimetic implants without growth factor (CAP). Implants were placed into frontal skulls of nine domestic pigs. The quality of osseointegration was evaluated using microradiographic and histomorphometric analysis of bone formation inside four defined bone chambers of the experimental implant at 1, 2 and 4 weeks. Biomimetic implants, either with or without growth factor, showed enhanced bone volume density (BVD) values after 2 and 4 weeks. This enhancement was significant for the BMP and BMP+VEGF group compared with the control AE group after 2 weeks (P<0.05). All biomimetic calcium-phosphate (Ca-P) coatings exhibited significantly enhanced bone–implant contact (BIC) rates compared with the uncoated control surface after 2 weeks (P<0.05). However, the combined delivery of BMP-2 and VEGF did not significantly enhance BIC at the final observation period. It was concluded that the combined delivery of BMP-2 and VEGF enhances BVD around implants, but not BIC. Therefore, it may be assumed that changes in the surface characteristics should be considered when designing growth factor-delivering surfaces.

Link to Article

http://dx.doi.org/10.1111/j.1600-0501.2010.02133.x

rhBMP-2 Induces Transient Bone Resorption Followed by Bone Formation in a Nonhuman Primate Core-Defect Model

Authors

Howard J. Seeherman, X. Jian Li, Mary L. Bouxsein, John M. Wozney

Abstract

Bone resorption preceding bone formation has been reported following the administration of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in an absorbable collagen sponge (ACS) in metaphyseal bone. This study characterizes treatment with rhBMP-2/ACS in metaphyseal bone with use of a nonhuman primate core-defect model. Unilateral proximal femoral core defects were treated with 360 µg of rhBMP-2/ACS or ACS alone or were left untreated in seven, five, and five adult male cynomolgus monkeys, respectively. Distal femoral core defects in seven of the above animals were treated with 360 µg of rhBMP-2/ACS in one limb and ACS alone in the contralateral limb. Retention of rhBMP-2 in the proximal part of the femora was determined with use of tracer amounts of 125I-rhBMP-2 imaged with a gamma camera. The distal part of the femora was evaluated with in vivo computed tomography.Computed tomography and histological evaluation were performed on harvested segments in all animals at twenty-four weeks. The histological response in the proximal and distal parts of the femora containing core defects treated with 360 µg of rhBMP-2/ACS in one limb and ACS alone in the contralateral limb was evaluated at one, two, and four weeks in three animals pertime point. Approximately 39.9%, 24.2%, 3.4%, and 0.5% of the rhBMP-2 was retained in the proximal part of the femora at one, seven, fourteen, and twenty-one days, respectively. The mineral density and trabecular volume fraction of the core defects treated with rhBMP-2/ACS, those treated with ACS alone, and untreated core defects in the proximal part of the femora were 81%, 54%, and 20%, respectively, and 94%, 36%, and 31%, respectively, of the corresponding region in the contralateral limbs at twenty-four weeks. The mineral density and trabecular volume fraction of the region surrounding the core defects treated with rhBMP-2/ACS, those treated with ACS alone, and untreated core defects were 112%, 105%, and 104%, respectively, and 117%, 108%, and 107%, respectively, of the corresponding region in the contralateral limbs. Treatment with rhBMP-2/ACS increased the size of the proximal and distal core defects compared with treatment with ACS alone. Histological evaluation of the rhBMP-2/ACS-treated limbs demonstrated that bone resorption was initiated at one week in association withosteoclasts and receptor activator of nuclear factor-B ligand-positive stained spindle-shaped cells and peaked at two weeks. Bone formation was observed at two weeks and was ongoing at twenty-four weeks. Treatment of metaphyseal core defects with rhBMP-2/ACS resulted in bone resorption followed by bone formation in nonhuman primates. Use of rhBMP-2 delivered in a collagen sponge for metaphyseal fracture repair should be avoided when transient bone resorption may lead to loss of fixation or structural support.

Link to Article

http://dx.doi.org/10.2106/JBJS.H.01732

NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis

Authors

Chang Yang, Kathleen McCoy, Jennifer L. Davis, Marc Schmidt-Supprian, Yoshiteru Sasaki, Roberta Faccio, Deborah Veis Novack

Abstract

Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway. Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis. Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0015383

In vivo performance of selective electron beam-melted Ti-6Al-4V structures

Authors

Sabine Ponader, Cornelius von Wilmowsky, Martin Widenmayer, Rainer Lutz, Peter Heinl, Carolin Körner, Robert F. Singer, Emeka Nkenke, Friedrich W. Neukam, Karl A. Schlegel

Abstract

Highly porous titanium structures are widely used for maxillofacial and orthopedic surgery because of their excellent mechanical properties similar to those of human bone and their facilitation of bone ingrowth. In contrast to common methods, the generation of porous titaniumproducts by selective electron beam melting (SEBM), an additive manufacturing technology, overcomes difficulties concerning the extreme chemical affinity of liquid titanium to atmospheric gases which consequently leads to strongly reduced ductility of the metal. The purpose of this study was to assess the suitability of a smooth compact and a porous Ti-6Al-4V structure directly produced by the SEBM process as scaffolds for bone formation. SEBM-processed titanium implants were placed into defects in the frontal skull of 15 domestic pigs. To evaluate the direct contact between bone and implant surfaces and to assess the ingrowth of osseous tissue into the porous structure, microradiographs and histomorphometric analyses were performed 14, 30, and 60 days after surgery. Bone ingrowth increased significantly during the period of this study. After 14 days the most outer regions of the implants were already filled with newly formed bone tissue (around 14%). After 30 days the bone volume inside the implants reached almost 30% and after 60 days abundant bone formation inside the implants attained 46%. During the study only scarce bone–implant contact was found around all implants, which did not exceed 9% around compact specimens and 6% around porous specimens after 60 days. This work demonstrates that highly porous titanium implants with excellent interconnectivity manufactured using the SEBM method are suitable scaffolds for bone ingrowth. This technique is a good candidate for orthopedic and maxillofacial applications.

Link to Article

http://dx.doi.org/10.1002/jbm.a.32337

Inhibition of RANKL increases the anti-tumor effect of the EGFR inhibitor panitumumab in a murine model of bone metastasis

Authors

Jude Canon, Rebecca Bryant, Martine Roudier, Tao Osgood, Jon Jones, Robert Miller, Angela Coxon, Robert Radinsky, William C. Dougall

Abstract

Bone metastases cause severe skeletal complications and are associated with osteoclast-mediated bone destruction. RANKL is essential for osteoclast formation, function, and survival, and is the primary effector of tumor-induced osteoclastogenesis and osteolysis. RANKL inhibition by its soluble decoy receptor osteoprotegerin (OPG) prevents tumor-induced osteolysis and decreases skeletal tumor burden. Because

osteoclast-mediated bone resorption releases growth factors from the bone matrix, the host bone micro-environment induces a vicious cycle of bone destruction and tumor proliferation and survival. A prediction of

this vicious cycle hypothesis is that targeting the host bone microenvironment by osteoclast inhibition would reduce tumor growth and survival and may enhance the anti-tumor effects of targeted therapies. The

epidermal growth factor receptor (EGFR) pathway regulates critical processes such as cell growth and survival, and anti-EGFR therapies can cause tumor cell arrest and apoptosis. We evaluated whether reduction

of osteolysis by RANKL inhibition could enhance the anti-tumor effects of an anti-EGFR antibody (panitumumab) in a novel murine model of human A431 epidermoid carcinoma bone metastasis. Skeletal tumor progression was assessed longitudinally by bioluminescence imaging. RANKL inhibition by OPG-Fc treatment resulted in a reduction in tumor progression in bony sites. OPG-Fc treatment also caused a dose-

dependent reduction in tumor-induced osteolysis, supporting the essential role of RANKL in this process. In combination, RANKL inhibition increased the anti-tumor efficacy of an anti-EGFR antibody, and completely blocked tumor-induced bone breakdown, demonstrating that addition of the indirect anti-tumor effect of RANKL inhibition increases the anti-tumor efficacy of panitumumab, a targeted anti-EGFR antibody.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.03.001