Timo Damm

Osteosynthesis of a cranio-osteoplasty with a biodegradable magnesium plate system in miniature pigs

Biodegradable magnesium alloys are a new class of implant material suitable for bone surgery. The aim of this study was to investigate plates and screws made of magnesium for osteosynthesis in comparison to titanium in a cranial fracture model. Implants were used for internal fixation of a cranio-osteoplasty in nine minipigs. Computed tomography was conducted repeatedly after surgery. The implants and the adjacent tissues were harvested 10, 20 and 30weeks after surgery and investigated by micro-computed tomography and histological analysis. The surgical procedure and the inserted osteosynthesis material were well tolerated by the animals, and the bone healing of the osteoplasty was undisturbed at all times. The adjacent bone showed formation of lacunas in the magnesium group, resulting in a lower bone-to-implant contact ratio than that of titanium (72 vs. 94% at week 30), but this did not lead to clinical side effects. Radiological measurements showed no reduction in osteosynthesis material volume, but indicated signs of degradation: distinct volumes within the magnesium osteosynthesis group had lower density in micro-computed tomography, and these volumes increased up to 9% at week 30. The histological preparations showed areas of translucency and porosity inside the magnesium, but the outer shape of the osteosynthesis material remained unchanged. No fracture or loosening of the osteosynthesis devices appeared. Soft tissue probes confirmed sufficient biocompatibility. Given their biodegradable capacity, biocompatibility, mechanical strength and visibility on radiographs, osteosynthesis plates made of magnesium alloys are suitable for internal fixation procedures. STATEMENT OF SIGNIFICANCE To the best of our knowledge this is the first study that used biodegradable magnesium implants for osteosynthesis in a cranial fracture model. The cranio-osteoplasty in miniature pigs allowed in vivo application of plate and screw osteosynthesis of standard-sized implants and the implementation of surgical procedures similar to those conducted on human beings. The osteosynthesis configuration, size, and mechanical properties of the magnesium implants within this study were comparable to those of titanium-based osteosynthesis materials. The results clearly show that bone healing was undisturbed in all cases and that the biocompatibility to hard- and soft tissue was sufficient. Magnesium implants might help to avoid long-term complications and secondary removal procedures due to their biodegradable properties.

Assessment of Bone Fragility in Patients With Multiple Myeloma Using QCT-Based Finite Element Modeling.

Multiple myeloma (MM) is a malignant plasma cell disease associated with severe bone destruction. Surgical intervention is often required to prevent vertebral body collapse and resulting neurological complications; however, its necessity is determined by measuring lesion size or number, without considering bone biomechanics. Finite element (FE) modeling, which simulates the physiological loading, may improve the prediction of fragility. To test this, we developed a quantitative computed tomography (QCT)‐based FE model of the vertebra and applied it to a dataset of MM patients with and without prevalent fracture. FE models were generated from vertebral QCT scans of the T12 (T11 if T12 was fractured) of 104 MM patients, 45 with fracture and 59 without, using a low‐dose scan protocol (1.5 mm slice thickness, 4.0 to 6.5 mSv effective dose). A calibration phantom enabled the conversion of the CT Hounsfield units to FE material properties. Compressive loading of the vertebral body was simulated and the stiffness, yield load, and work to yield determined. To compare the parameters between fracture and nonfracture groups, t tests were used, and standardized odds ratios (sOR, normalized to standard deviation) and 95% confidence intervals were calculated. FE parameters were compared to mineral and structural parameters using linear regression. Patients with fracture showed lower vertebral stiffness (–15.2%; p = 0.010; sOR = 1.73; 95% CI, 1.11 to 2.70), yield force (–21.5%; p = 0.002; sOR = 2.09; 95% CI, 1.27 to 3.43), and work to yield (–27.4%; p = 0.001; sOR = 2.28; 95% CI, 1.33 to 3.92) compared to nonfracture patients. All parameters correlated significantly with vBMD (stiffness: R2 = 0.57, yield force: R2 = 0.59, work to yield: R2 = 0.50, p < 0.001), BV/TV (stiffness: R2 = 0.56, yield force: R2 = 0.58, work to yield: R2 = 0.49, p < 0.001), and Tb.Sp (stiffness: R2 = 0.51, yield force: R2 = 0.53, work to yield: R2 = 0.45, p < 0.001). FE modeling identified MM patients with compromised mechanical integrity of the vertebra. Higher sOR values were obtained for the biomechanical compared to structural or mineral measures, suggesting that FE modeling improves fragility assessment in these patients.

Evaluation of the degradation behavior of resorbable metal implants for in vivo osteosynthesis by synchrotron radiation based x-ray tomography and histology

Magnesium(Mg)-alloys are promising candidates as temporary implants for orthopedic and cranio-facial applications. They can sustain tissues during healing, thanks to favorable mechanical properties, and then they slowly degrade into biocompatible products, avoiding the need of a second surgery for implant removal. They have the potential to benefit a vast number of patients, especially children and elderly patients. However, to be able to tailor their degradation to match the speed of tissue regeneration it is crucial to understand how they actually degrade in the living organism. We utilized high-resolution synchrotron-based tomography at the beamline P05 operated by HZG at the storage ring PETRA III at DESY to study the degradation of 3 novel Mg-alloys in rat bone and the consequent bone response. On threedimensional reconstructions of the bone-implant explants we were able to follow the dynamic transformation that the materials underwent at different healing times and on the basis of absorption coefficients we could distinguish and quantify the amount of remaining implants, the corrosion layers and the new bone. This was a great advantage compared to laboratory CT, for which the limitation in contrast and in resolution made impossible to discriminate between original alloy, degradation products and bone, leading to inaccurate determination of the materials degradation rates. The same samples imaged by tomography were used for non-decalcified histology. The combination of histological and tomographical images provided new insight on the nature of the bone-to-implant interface and of the degradation products, which appeared to have great similarities to the host bone.

Bone‐marrow densitometry: Assessment of marrow space of human vertebrae by single energy high resolution‐quantitative computed tomography

PURPOSE Accurate noninvasive assessment of vertebral bone marrow fat fraction is important for diagnostic assessment of a variety of disorders and therapies known to affect marrow composition. Moreover, it provides a means to correct fat-induced bias of single energy quantitative computed tomography (QCT) based bone mineral density (BMD) measurements. The authors developed new segmentation and calibration methods to obtain quantitative surrogate measures of marrow-fat density in the axial skeleton. METHODS The authors developed and tested two high resolution-QCT (HR-QCT) based methods which permit segmentation of bone voids in between trabeculae hypothesizing that they are representative of bone marrow space. The methods permit calculation of marrow content in units of mineral equivalent marrow density (MeMD). The first method is based on global thresholding and peeling (GTP) to define a volume of interest away from the transition between trabecular bone and marrow. The second method, morphological filtering (MF), uses spherical elements of different radii (0.1-1.2 mm) and automatically places them in between trabeculae to identify regions with large trabecular interspace, the bone-void space. To determine their performance, data were compared ex vivo to high-resolution peripheral CT (HR-pQCT) images as the gold-standard. The performance of the methods was tested on a set of excised human vertebrae with intact bone marrow tissue representative of an elderly population with low BMD. RESULTS 86% (GTP) and 87% (MF) of the voxels identified as true marrow space on HR-pQCT images were correctly identified on HR-QCT images and thus these volumes of interest can be considered to be representative of true marrow space. Within this volume, MeMD was estimated with residual errors of 4.8 mg/cm(3) corresponding to accuracy errors in fat fraction on the order of 5% both for GTP and MF methods. CONCLUSIONS The GTP and MF methods on HR-QCT images permit noninvasive localization and densitometric assessment of marrow fat with residual accuracy errors sufficient to study disorders and therapies known to affect bone marrow composition. Additionally, the methods can be used to correct BMD for fat induced bias. Application and testing in vivo and in longitudinal studies are warranted to determine the clinical performance and value of these methods.

Tracking the Progression of Osteolytic and Osteosclerotic Lesions in Mice Using Serial In Vivo μCT: Applications to the Assessment of Bisphosphonate Treatment Efficacy

The metastasis of tumor cells to bone can lead to osteolytic and osteosclerotic lesions, which cause severe, highly‐localized bone destruction and abnormal bone apposition, respectively. Accurate quantification of lesion progression is critical to understand underlying mechanisms and assess treatment efficacy; however, standard structural parameters may be insensitive to local changes. We developed methods to quantify osteolytic and osteosclerotic lesions using micro–computed tomography (μCT) within in vivo mouse datasets. Two Balb/c nude datasets were used: (i) bone‐homing MDA‐MB‐231 (osteolytic) cells injected into the left ventricle, treatment with alendronate or vehicle, and weekly μCT (proximal tibia) for 4 weeks, and (ii) MCF7 (osteosclerotic) cells injected into the right tibia and weekly μCT over 12 weeks. After registering images to baseline, osteolytic lesion volume was determined by summing all baseline bone voxels at distances greater than a threshold (150 μm) from the nearest follow‐up. Osteosclerotic lesions were determined by measuring the distance from each follow‐up surface voxel to the nearest baseline surface and calculating the standard deviation of distance values (SDDT) of the surrounding voxels. Bone mineral density (BMD), bone volume density (BV/TV), and separation (Sp) were determined for comparison. Osteolytic lesions were observed 1 week after tumor cell injection; however, no corresponding BV/TV losses or Sp increases were observed, indicating that standard parameters were unable to detect early metastatic changes. Lesion volume was smaller in the alendronate versus control group (15.0%, p = 0.004 and 18.6%, p = 0.002 of control lesion volume at weeks 3 and 4, respectively). In the osteosclerotic dataset, increased SDDT was observed following injection, providing a potential new measure of osteosclerotic bone apposition. These data show that quantification of local structural change with serial μCT may overcome the limitations of standard mineral and microstructural parameters, and successfully separates metastatic and normal bone turnover.

Improved accuracy in the assessment of vertebral cortical thickness by quantitative computed tomography using the Iterative Convolution Optimization (ICON) method

Vertebral whole bone strength is substantially affected by cortical bone properties. Disease and therapy may affect cancellous and cortical bone differently. Unlike Dual X-ray Absorptiometry (DXA), Quantitative Computed Tomography (QCT) permits selective assessment of cortical and cancellous bone, but image quality limits the accuracy. We present an image processing method specifically adopted to thin cortices that substantially improves accuracy. Ten human vertebrae embedded in epoxy resin were imaged using clinical QCT and High-Resolution QCT (HR-QCT) protocols, both acquired on a clinical whole body CT scanner, whereas high resolution peripheral QCT (HR-pQCT) was used as gold standard. Microstructural variables and BMD were calculated using in-house software StructuralInsight for QCT and HR-QCT and the manufacturers μCT evaluation software for HR-pQCT. An adjusted measure, a deconvolved cortical thickness (dcCt.Th), corrected for partial volume effects, was derived applying the new Iterative Convolution OptimizatioN (ICON) method. Direct measurements of cortical thickness (Ct.Th) showed substantial overestimation with mean ± standard deviation of 1.8 ± 0.5 mm for QCT and 1.5 ± 0.3 mm for HR-QCT compared to 0.37 ± 0.07 mm using HR-pQCT. Correlations of both QCT (r2 = 0.05, p > 0.5.) and HR-QCT (r2 = 0.38, p = 0.060) with the gold standard HR-pQCT were not significant. Also QCT-based BMD and BMC as well as HR-QCT-based BMD did not show a significant correlation with the gold standard approach. Only HR-QCT-based BMC showed a modest correlation (r2 = 0.59, p = 0.01) After applying ICON corrections, dcCt.Th resulted in 0.52 ± 0.09 mm for QCT and 0.43 ± 0.07 mm for HR-QCT, both significantly correlated to HR-pQCT (r2 = 0.75, p = 0.0012 and r2 = 0.93, p < 0.0001, respectively). The average overestimation bias of Ct.Th was reduced from (402 ± 157)% to (45 ± 17)% for QCT and from (330 ± 69)% to (19 ± 8)% for HR-QCT. Due to inaccurate segmentation uncorrected QCT-based Ct.Th measures as well as BMD and BMC showed no correlation to HR-pQCT and thus such bias cortical data can be misleading. The application of ICON reduced random overestimation bias to about 50 μm and 20 μm for QCT and HR-QCT, respectively, leading to a recovery of a significant correlation with the reference data of HR-pQCT. This reveals the potential for fairly accurate assessment of cortical thickness, allowing to better characterize cortical mechanical competence. These results warrant testing of the performance in vivo.

Longitudinal micro-computed tomography monitoring of progressive liver regeneration in a mouse model of partial hepatectomy.

The partial hepatectomy (PH) model is widely used to study liver regeneration. Currently, the extent of regeneration is analyzed by measuring the weight of the liver post-mortem or by magnetic resonance imaging. In this study we aimed to determine whether liver volume gain can be accurately measured using micro-computed tomography (microCT). Approximately 42% of the liver was removed by ligation in C57BL/6 N mice. Mice were divided into two study groups. In group 1 conventional characterization of liver hyperplasia was performed by weighing the liver post-mortem. In group 2, liver volume gain was determined by microCT volume estimation. MicroCT results showed equivalent regeneration rates compared with the conventional method without the need to mathematically determine initial liver weights before PH. This parameter is strongly influenced by the age, strain and sex of the mice. In addition non-invasive microCT determination of volume gain over multiple time-points using the same animal reduces the number of animals needing to be used (in line with the 3R principle of replacement, reduction and refinement).

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4β1 Integrin: OFN FIBRONECTIN INHIBITS OSTEOBLAST DIFFERENTIATION

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model.

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through alpha 4 beta 1 Integrin

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model.