Antitumoral activity and osteogenic potential of mesenchymal stem cells expressing the urokinase-type plasminogen antagonist amino-terminal fragment in a murine model of osteolytic tumor.

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

Post-processing technique for improved assessment of hard tissues in the submicrometer domain using local synchrotron radiation-based computed tomography.

During the last two decades micro-computed tomography has become the method of choice for the non-destructive assessment and quantitative morphometry of hard tissues in three dimensions. With the advent of third-generation synchrotron radiation sources, micro-computed tomography in the micrometer range has become feasible and has been employed to analyze local bone tissue properties. However, owing to limitations regarding the tradeoff between object size and spatial resolution, non-destructive conventional global computed tomography of hard tissues, such as bone, remains unachievable in the submicrometer domain so far. Here, we report on a post-processing technique for the assessment of hard tissues using local synchrotron radiation-based computed tomography, which overcomes this experimental limitation.

Tissue modulus calculated from beam theory is biased by bone size and geometry: implications for the use of three-point bending tests to determine bone tissue modulus.

Current practice to determine bone tissue modulus of murine cortical bone is to estimate it from three-point bending tests, using Euler-Bernoulli beam theory. However, murine femora are not perfect beams; hence, results can be inaccurate. Our aim was to assess the accuracy of beam theory, which we tested for two commonly used inbred strains of mice, C57BL/6 (B6) and C3H/He (C3H). We measured the three-dimensional structure of male and female B6 and C3H femora (N=20/group) by means of micro-computed tomography. For each femur five micro-finite element (micro-FE) models were created that simulated three-point bending tests with varying distances between the supports. Tissue modulus was calculated from beam theory using micro-FE results. The accuracy of beam theory was assessed by comparing the beam theory-derived moduli with the modulus as used in the micro-FE analyses. An additional set of fresh-frozen femora (10 B6 and 12 C3H) was biomechanically tested and subjected to the same micro-FE analyses. These combined experimental-computational analyses enabled an unbiased assessment of specimen-specific tissue modulus. We found that by using beam theory, tissue modulus was underestimated for all femora. Femoral geometry and size had strong effects on beam theory-derived tissue moduli. Owing to their relatively thin cortex, underestimation was markedly higher for B6 than for C3H. Underestimation was dependent on support width in a strain-specific manner. From our combined experimental-computational approach we calculated tissue moduli of 12.0+/-1.3 GPa and 13.4+/-2.1 GPa for B6 and C3H, respectively. We conclude that tissue moduli in murine femora are strongly underestimated when calculated from beam theory. Using image-based micro-FE analyses we could precisely quantify this underestimation. We showed that previously reported murine inbred strain-specific differences in tissue modulus are largely an effect of geometric differences, not accounted for by beam theory. We suggest a re-evaluation of the tissue properties obtained from three-point bending tests, especially in mouse genetics.

Femoral stiffness and strength critically depend on loading angle: a parametric study in a mouse-inbred strain.

Biomechanical tests of human femora have shown that small variations of the loading direction result in significant changes in measured bone mechanical properties. However, the heterogeneity in geometrical and bone tissue properties does not make human bones well suited to reproducibly assess the effects of loading direction on stiffness and strength. To precisely quantify the influence of loading direction on stiffness and strength of femora loaded at the femoral head, we tested femora from C57BL/6 inbred mice. We developed an image-based alignment protocol and investigated the loading direction influence on proximal femur stiffness and strength. An aluminum femoral phantom and C57BL/6 femora were tested under compression with different loading directions. Both tests, with the aluminum phantom and the murine bones, showed and quantified the linear dependence of stiffness on loading direction: a 5 degrees change in loading direction resulted in almost 30% change in stiffness. Murine bone testing also revealed and quantified the variation in strength due to loading direction: 5 degrees change in loading direction resulted in 8.5% change in strength. In conclusion, this study quantified, for the first time, the influence of misalignment on bone stiffness and strength for femoral head loading. We showed the extreme sensitivity of this site regarding loading direction.

Ultrastructural properties in cortical bone vary greatly in two inbred strains of mice as assessed by synchrotron light based micro- and nano-CT.

UNLABELLED: Nondestructive SR-based microCT and nano-CT methods have been designed for 3D quantification and morphometric analysis of ultrastructural phenotypes within murine cortical bone, namely the canal network and the osteocyte lacunar system. Results in two different mouse strains, C57BL/6J-Ghrhr(lit)/J and C3.B6-Ghrhr(lit)/J, showed that the cannular and lacunar morphometry and their bone mechanics were fundamentally different. INTRODUCTION: To describe the different aspects of bone quality, we followed a hierarchical approach and assessed bone tissue properties in different regimens of spatial resolution, beginning at the organ level and going down to cellular dimensions. For these purposes, we developed different synchrotron radiation (SR)-based CT methods to assess ultrastructural phenotypes of murine bone. MATERIALS AND METHODS: The femoral mid-diaphyses of 12 C57BL/6J-Ghrhr(lit)/J (B6-lit/lit) and 12 homozygous mutants C3.B6-Ghrhr(lit)/J (C3.B6-lit/lit) were measured with global SR microCT and local SR nano-CT (nCT) at nominal resolutions ranging from 3.5 microm to 700 nm, respectively. For volumetric quantification, morphometric indices were determined for the cortical bone, the canal network, and the osteocyte lacunar system using negative imaging. Moreover, the biomechanics of B6-lit/lit and C3.B6-lit/lit mice was determined by three-point bending. RESULTS: The femoral mid-diaphysis of C3.B6-lit/lit was larger compared with B6-lit/lit mice. On an ultrastructural level, the cannular indices for C3.B6-lit/lit were generally bigger in comparison with B6-lit/lit mice. Accordingly, we derived and showed a scaling rule, saying that overall cannular indices scaled with bone size, whereas indices describing basic elements of cannular and lacunar morphometry did not. Although in C3.B6-lit/lit, the mean canal volume was larger than in B6-lit/lit, canal number density was proportionally smaller in C3.B6-lit/lit, so that lacuna volume density was found to be constant and therefore independent of mouse strain and sex. The mechanical properties in C3.B6-lit/lit were generally improved compared with B6-lit/lit specimens. For C3.B6-lit/lit, we observed a sex specificity of the mechanical parameters, which could not be explained by bone morphometry on an organ level. However, there is evidence that for C3.B6-lit/lit, the larger cortical bone mass is counterbalanced or even outweighed by the larger canal network in the female mice. CONCLUSIONS: We established a strategy to subdivide murine intracortical porosity into ultrastructural phenotypes, namely the canal network and the osteocyte lacunar system. Nondestructive global and local SR-based CT methods have been designed for 3D quantification and subsequent morphometric analysis of these phenotypes. Results in the two different mouse strains C57BL/6J-Ghrhr(lit)/J and C3.B6-Ghrhr(lit)/J showed that the cannular and lacunar morphometry and the biomechanical properties were fundamentally different.

Micro-CT combined with bioluminescence imaging: a dynamic approach to detect early tumor-bone interaction in a tumor osteolysis murine model.

Prostate cancer (CaP) cells possess high affinity for bone marrow and predilection to induce bone metastasis. Although the end result of metastasis is predominantly osteoblastic, most patients present mixed lesions with osteolytic component which could initiate and precede bone formation. A precise characterization of tumor-induced bone resorption is thus necessary for early evaluation of therapeutic efficiency. Herein, we investigate the advantage of combining micro-computed tomography (microCT) and in vivo bioluminescence imaging (BLI) to determine the kinetics of the intraosseous CaP growth and bone lesions appearance in an experimental murine model. To mimic established osteolytic bone metastasis, the left tibiae of SCID mice were injected with the human CaP cell line PC-3 expressing luciferase (PC-3 Luc). Noninvasive monitoring of tumor progression was followed weekly by BLI during 4 weeks and bone morphometric parameters were quantified by microCT. Data were compared with conventional radiological and histological analyses. While BLI monitoring in vivo revealed an exponential growth of PC-3 Luc after 2 weeks, a decrease of bone density and bone mineral content was evidenced by microCT as early as 7 days post-injection, reaching significant values at day 21 (30% and 25% loss, respectively), compared with mock-injected controls. Enhanced osteoclast TRAP activity was observed during the first two weeks, highlighting an active interaction between low proliferative PC-3 cells and osteoclasts at the early stage of tumor establishment in bone. Tumor growth detected by BLI was tightly correlated to the osteolysis assessed by microCT (p<0.05). Our results show that the combination of microCT and BLI applied to this tumor osteolysis murine model allows early measurement of intraosseous tumor growth and bone destruction, as well as correlation between both processes kinetics. This model will help to assess new therapeutic approaches targeting intraosseous tumor growth or tumor/osteoclast crosstalk.

The importance of the intracortical canal network for murine bone mechanics.

As shown by recent data bone strength estimation can greatly be improved by including microarchitectural parameters in the analysis. Our previous results showed that intracortical canals (the living space of the vasculature and/or remodeling units) are a major contributor to cortical tissue porosity, and therefore, can be linked to mechanical bone properties. Consequently, the goal of this study was to investigate the importance of the intracortical canal network for murine bone mechanics. To study intracortical canals within murine femoral bone, we used a mouse model, including two mouse strains, C57BL/6J-Ghrhr(lit)/J (B6-lit/+) and C3.B6-Ghrhr(lit)/J (C3.B6-lit/+) representing low and high bone mass, respectively. The intracortical canal network was assessed by synchrotron radiation-based micro-computed tomography and the mechanical bone properties were derived from three-point bending experiments. Multiple linear regression models were built to explain the variation in ultimate force, work to fracture, and stiffness in terms of the morphometric parameters. The power to explain the variation in bone mechanics was increased significantly for most mechanical measures when including morphometric parameters of intracortical canals in addition to macroscopic morphometric measures. Specifically, we could derive generalized (mouse strain-independent) models for ultimate force, where the incorporation of intracortical canals in addition to macroscopic bone measures improved the explained variation in ultimate force considerably, which was confirmed by an increase in adjusted R(2) of 73% and 8% for B6-lit/+ and C3.B6-lit/+, respectively. Further, we observed that the heterogeneity of the morphometric measures for the individual canal branches play an important role for explaining the variation in ultimate force. Finally, the current study provides strong evidence that work to fracture of murine bone, which is triggered critically by microcracks, is affected by intracortical canals. In summary, the study suggests that the intracortical canal network is important for bone mechanics.

Endosseous implant anchorage is critically dependent on mechanostructural determinants of peri-implant bone trabeculae.

Low bone mass is highly prevalent among patients receiving endosseous implants. In turn, the implantation prognosis in low-density skeletal sites is poor. However, little is known about the mechanostructural determinants of implant anchorage. Using metabolic manipulations that lead to low bone density and to its rescue, we show here that anchorage is critically dependent on the peri-implant bone (PIB). Titanium implants were inserted horizontally into the proximal tibial metaphysis of adult rats 6 weeks after orchiectomy (ORX) or sham ORX. Systemic intermittent administration of human parathyroid hormone (1-34) [iahPTH(1-34)] or vehicle commenced immediately thereafter for 6 weeks. The bone-implant apparatus was then subjected to image-guided failure assessment, which assesses biomechanical properties and microstructural deformation concomitantly. Anchorage failure occurred mainly in PIB trabeculae, 0.5 to 1.0 mm away from the implant. Mechanically, the anchorage performed poorly in ORX-induced low-density bone, attributable mainly to decreased trabecular number. iahPTH(1-34) rescued the PIB density and implant mechanical function by augmenting trabecular thickness (Tb.Th). However, implant biomechanical properties in low-density bone were relatively insensitive to implant surface treatment that affected only the osseointegration (%bone-implant contact). These results support a model wherein anchorage failure involves buckling of the weakest trabecular struts followed by sequential failure of the stronger trabeculae. Treatment with iahPTH(1-34) induced thicker struts, which were able to delay and even prevent failure of individual elements, thus implicating trabecular thickness as a prime target for enhancing implant anchorage by systemic bone anabolic therapy.

Bone morphometry strongly predicts cortical bone stiffness and strength, but not toughness, in inbred mouse models of high and low bone mass.

Inbred strains of mice make useful models to study bone properties. Our aim was to compare bone competence and cortical morphometric parameters of two inbred strains to better determine the role of bone structure and geometry in the process of bone failure. Morphometric analysis was performed on 20 murine femora with a low bone mass (C57BL/6J; B6) and 20 murine femora with a high bone mass (C3H/HeJ; C3H) using desktop microCT. The bones were tested under three-point bending to measure their mechanical properties. Results showed that the C3H strain is a more reproducible model regarding bone morphometric and mechanical phenotypes than the B6 strain. Bone strength, stiffness, yield force, yield displacement, and toughness, as well as morphometric traits, were all significantly different between the two strains, whereas postyield displacement was not. It was found that bone volume, cortical thickness, and cross-sectional area predicted almost 80% (p < 0.05) of bone stiffness, strength, and yield force. Nevertheless, cortical bone postyield properties such as bone toughness could not be explained by morphometry, but postyield whitening was observed in that phase. In conclusion, we found that morphometric parameters are strong predictors of preyield but not postyield properties. The lack of morphometric influence on bone competence in the postyield phase in combination with the observed postyield whitening confirmed the important contribution of ultrastructure and microdamage in the process of overall bone failure behavior, especially in the postyield phase.

Differential effects of bone structural and material properties on bone competence in C57BL/6 and C3H/He inbred strains of mice.

The femoral neck is a relevant and sensitive site for studying the degree of osteopenia. Engineering principles predict that bone structural parameters, like cross-sectional geometry, are important determinants of bone mechanical parameters. Mechanical parameters are also directly affected by the material properties of the bone tissue. However, the relative importance of structural and material properties is still unknown. The aim of this study was to compare bone competence and structural parameters between a murine strain showing a low bone mass phenotype, C57BL/6 (B6), and another one showing a high bone mass phenotype, C3H/He (C3H), in order to better determine the role of bone structure and geometry in bone failure behavior. Murine femora of 12- and 16-week-old B6 and 12- and 16-week-old C3H inbred strains were mechanically tested under axial loading of the femoral head. In order to assess the structural properties, we performed three-dimensional morphometric analyses in five different compartments of the mouse femur using micro-computed tomography. The mechanical tests revealed that B6 femora became stiffer, stronger, and tougher at 12-16 weeks, while bone brittleness stayed constant. C3H bone stiffness increased, but strength remained constant, work to failure decreased, and bone became more brittle. These age effects indicated that B6 did not reach peak bone properties at 16 weeks of age and C3H did reach maximal skeletal biomechanical properties before 16 weeks of age. Our investigations showed that 83% of the strength of the femoral neck in the B6 strain was explained by cortical thickness at this location; in contrast, in C3H none of the mechanical properties of the femoral neck was explained by bone structural parameters. The relative contributions of bone structural and material properties on bone strength are different in B6 and C3H. We hypothesize that these different contributions are related to differences at the ultrastructural level of bone that affect bone failure.