Fracture characterization in cancellousbone specimens via surface difference evaluation of 3D registered pre- and post-compression micro-CT scans
M. Prot a , G. Dubois a , T. J. Cloete b , D. Saletti c and S. Laporte a
a lBm/institut de Biomécanique humaine georges Charpark, arts et métiers paristech, paris, France; b Blast impact and survivability research unit
Automobile accidents and sporting injuries may lead to osseous fractures. To reduce the number of road accidents and their societal costs, governments have partnered with car manufacturers to develop an overall road safety. To achieve this, researchers are working on improving the design of automotive structures. However, researchers must first quantify the risk of injury incurred during an impact. Indeed, in France osteoporosis is responsible of approximately 150 000 fractures per year. A better understanding of this fracture mechanism will aid in the design of protective features that will guard against fracture under these loading conditions. Bone is generally divided into two micro-structural types: cortical and cancellousbone. Cortical bone is a compact bone, denser than cancellousbone and accounts for 80 % of the skeletal mass in the human body. Cancellousbone, also called trabecular or spongy bone has a porous structure that protects the bone marrow, acts as a core material to support the shape of thin layers of cortical bone and assist in transferring joint forces to the thick load bearing cortical bone layers.
a b s t r a c t
Previous studies show that in vivo assessment of fracture risk can be achieved by identifying the relationships between microarchitecture description from clinical imaging and mechanical properties. This study demonstrates that results obtained at low strain rates can be extrapolated to loadings with an order of magnitude similar to trauma such as car crashes. Cancellous bovine bone specimens were compressed under dynamic loadings (with and without conﬁnement) and the mechanical response properties were identiﬁed, such as Young's modulus, ultimate stress, ultimate strain, and ultimate strain energy. Specimens were previously scanned with pQCT, and architectural and structural microstructure properties were identiﬁed, such as parameters of geometry, topology, connectivity and anisotropy. The usefulness of micro-architecture description studied was in agreement with statistics laws. Finally, the differences between dynamic con ﬁned and non-conﬁned tests were assessed by the bone marrow inﬂuence and the cancellousbone response to different boundary conditions. Results indicate that architectural parameters, such as the bone volume fraction (BV/TV), are as strong determinants of mechanical response parameters as ultimate stress at high strain rates (p-valueo0.001). This study reveals that cancellousbone response at high strain rates, under different boundary conditions, can be predicted from the architectural parameters, and that these relations with mechanical properties can be used to make fracture risk prediction at a determined magnitude.
What is the recommended size of a Volume of Interest for cancellousbone? A skeleton-based study
G. Dubois a , M. Prot a , S. Laporte a and T.J. Cloete b
a lBm/institut de Biomécanique humaine georges Charpark, arts et métiers paristech, paris, France; b Blast impact and survivability research unit (Bisru), department of mechanical engineering, university of Cape town (uCt), rondebosch, south africa
2.2. Specimen preparation
2.2.1. Bone model specimen: Polyurethane foam
Sawbone Ò models #1522 02 representing a validated alterna tive for testing human cancellousbone were used as preliminary controls for static and dynamic tests ( Thompson et al. 2003; Elfar et al. 2014 ). Four straight beams of 4 mm in width (b) and 40 mm in length were cut from a polyurethane sheet of 3 mm thickness (h) with an oscillating saw (IsoMet 1000, Buehler Ò ) because sheet lightness was not adapted to the water jet machine used for canine femurs. For the three points bending test, the sam ple span was 30 mm. Manufacturer data were E s = 123 MPa in com pression and E s = 173 MPa in traction. The homogenous porosity was / = 80% and density was q = 240 kg/m 3 .
Results at low strain rates were found to be consistent with the literature. The high strain rate results show the same trend as that observed by (Shim 2005) in human cervical cancellousbone. Given the novel nature of the intermediate strain rate results for cancellousbone presented in this study, no comparison with the literature could be made. However, the results are consistent with lower and higher strain rate behavior. It appears that fracture occurs earlier at intermediate strain rates (0.03 strain versus 0.04 for lower and higher strain rate). Note that the intermediate strain rate data was obtained using two different techniques and, therefore, the distinct intermediate strain rate behaviour is not an artifact of any particular testing method.
It is very diﬃcult to extrapolate the behaviour of small cancellousbone samples to that of massive allografts. Moreover, the control material had not been chosen to
model the clinical situation. However, at the trabe- cular level, interesting information could be obtained about the behaviour of the healing tissue in relation to the implant. Due to its unique diﬀusion property, supercritical fluid can be adapted to treat a large bone volume. The eﬃcient volume of delipidation can be im- proved by increasing the time spent within the SC.CO ". The eﬃcient bone volume for bone marrow deproteina- tion can, as has previously been described be greatly enhanced by dissolution of protease in the SC.CO " . It is therefore likely that the treatment of large bone volumes by SCF technology could facilitate their sub- sequent integration.
A better understanding of the bone fracture mechanisms could help to improve the design of the safety systems. If the strength of bone mainly depends on the cortical bone, the rupture of the cancellousbone, hosting the bone marrow, could lead to vascular problems or local irreversible damage. The study of the mechanical behavior of the cancellousbone is mostly focused on quasi-static compressive tests , for different boundary conditions or with or without bone marrow . Recent experimental works have shown a strain-rate dependency of the cancellousbone mechanical behavior  and the influence of the bone marrow for high strain rate loading . Even if studies have demonstrated the link between cancellousbone microarchitecture parameters, bone mineral density and mechanical parameters , cancellousbone fracture via architecture characterization has not been fully assessed, especially under high strain rate loading.
Universite´ de Lyon, Lyon, France; IFSTTAR, LBMC, Bron; Universite´ Lyon 1, Villeurbanne, France
The manuscript was received on 18 May 2011 and was accepted after revision for publication on 23 June 2011. DOI: 10.1177/0954411911416858
Abstract: For various applications, precision of the Young’s modulus of cancellousbone speci- mens is needed. However, measurement variability is rarely given. The aim of this study was to assess the Young’s modulus repeatability using a uniaxial cyclic compression protocol on embedded specimens of human cancellousbone. Twelve specimens from 12 human calcanei were considered. The specimens were first defatted and then 1 or 2 mm at the ends were embedded in an epoxy resin. The compression experiment consists in applying 20 compres- sive cycles between 0.2 per cent and 0.6 per cent strain with a 2 Hz loading frequency. The coefficient of variation of the current protocol was found to be 1.2 per cent. This protocol showed variability similar to the end-cap technique (considered as a reference). It can be applied on porous specimen (especially human bone) and requires minimal bone length to limit end-artifact variability. The current method could be applied in association with nonin- vasive measurements (such as ultrasound) with full compatibility. This possibility opens the way for bone damage follow-up based on Young’s modulus monitoring.
From the measured displacements, macroscopic and meso- scopic strain ﬁelds were evaluated. The latter show that the local strain ﬁeld is not uniform in the present experiment. This phenomenon is due to the coarseness of the studied microstruc- ture. Furthermore, the variation of the macroscopic principal strains along the height of the sample shows that a classical identiﬁcation procedure to evaluate Poisson’s ratio would lead to erroneous results. More advanced identiﬁcation tools are needed to determine the elastic parameters at a microscopic (at the level of trabeculae), mesoscopic (at the level of the elements considered herein) or even macroscopic scale (i.e., the studied sample). This analysis shows that the representative volume element is signiﬁcantly larger than the volume tested herein. Such metho- dology opens the way for in-depth validation of micro-ﬁnite- element models of cancellousbone.
Materials and Methods Crystal Isolation
Frozen bovine tibias and femurs from animals 1–3 months or 4–5 years old were obtained from Research 87 (Boston, MA). The bones were dissected out and cleaned free of soft tissues. The midshafts (diaphyses) were separated from the metaphyses and then rinsed with ethanol and ether. Cortical bone samples were prepared from midshaft bone sections. Cancellousbone samples were obtained from metaphyseal bone. Bones from several animals of the same age or bone type were combined together to create the samples ana- lyzed due to the length of time required to process large samples by the low-temperature, nonaqueous methods [ 34 ]. Bones (from both hind limbs) from 15 animals were used for the 1–3 month group and from five animals for the 4– 5 year group. As such, the parameters measured reflect average, pooled values. Replicate analysis of samples was done to determine means and standard deviations. Bone chips were obtained by hammering or using an industrial blender with ethanol. The chips were further ground in a liquid nitrogen-cooled SPEX 6700 freezer/mill (SPEX CertiPrep, Metuchen, NJ) to obtain a starting material with a particle size less than 200 mesh. The samples were first defatted in 2:1 (v:v) chloroform and methanol (1 g powder to 10 mL liquid). Following the procedure of Kim et al. [ 34 ], defatted bone powder was plasma-ashed in an oxygen plasma for 40 days to degrade the organic matrix compo- nents. The samples were stirred every fifth day to assure uniform ashing. It has been shown that these techniques, when used in moderation, do not affect the structure or composition of calcium phosphate phases including brushite, octacalcium phosphate, and poorly crystalline carbonated apatite [ 34 ].
Indeed, oestrogens exhibit a broad range of physiological activities that are important in the homeostasic regu- lation of many cells, including bone cells. In women the
menopause initiates an accelerated phase of predominantly cancellousbone loss that declines rapidly over 5–10 years to become asymptotic, with a subsequent slow phase that continues indefinitely. Oestrogen deficiency affects remo- delling in several ways: it increases the activation fre- quency of bone modelling units, which leads to higher bone turnover; it induces a remodelling imbalance by prolonging the resorption phase and shortening the form- ation phase. As a consequence, the volume of the resorp- tion cavity increases beyond the capacity of the osteoblasts
 . These defects require additional interventions with a major impact on patient life quality and with socio-economic burden [2,3] . To manage this problem, different therapeutic strategies have been developed. Autologous cancellousbone graft is still until today the gold standard for regeneration of bone defects exceeding 4 cm  . Although this bone transplantation approach has shown its clinical efficacy, it presents several drawbacks (i.e. limited bone stock, additional surgical site with consequent pain and risk of infections)  . Other approaches have been developed for clinical use. Allogeneic bone graft (i.e. surgical waste, post-mortem donors) is an alternative to autograft  . Bone xenograft has also been used, such as bovine bone whose structure is close to humans  . However, these strategies may transfer pathogens  or lead to immunological reactions  . To overcome bone graft disadvan- tages, several biocompatible scaffolds have been developed [9– 11] . Synthetic bioceramics of hydroxyapatite (HA); tricalcium phosphate (TCP) or their mixture biphasic calcium phosphate (BCP) are the most used in skeletal engineering because of their osteoconductive properties and the easy management of their architecture  . Alternative natural scaffolds including secure amorphous human bone [9,10] , animal derived bone matrices  or the coral exoskeleton  offer a similar structure to can- cellous bone and thus mimic the physiological bone tissue environ- ment  . However, when used alone these scaffolds lack osteoinductive properties and serve only to guide bone tissue  . To enhance new bone formation, growth factors (FGF-2, PDGFs) or differentiating factors (BMPs) and more recently stem cells have been associated to these scaffolds [3,14,15] . An approach involving human bone marrow mesenchymal stromal cells (hBMSC) associ- ated with biomaterials emerges as an innovative strategy to repair delayed unions  . Mesenchymal stromal
I. Papet et al.: Bone quality during spontaneous inflammatory bowel disease
greater for HLA-B27 rats than for controls (Table 1). Concerning bone parameters, femoral BMC was 22% lower in HLA-B27 than in control rats (Table 2). T-BMD, D-DMB and M-BMD were 15, 12 and 15% lower in HLA- B27 rats. The difference observed in D-DBM tended to be lower than that for M-BMD (P=0.057, for interaction). Femoral failure load was 12% lower in HLA-B27 than in control rats. Plasma osteocalcin concentration did not differ between the two groups of animals, while urinary excretion of deoxypyridinoline was 34% greater in the HLA-B27 than in the control rats (Table 3). The bone remodelling index, calculated as serum osteocalcin divided by creatinine-cor- rected deoxypyridinoline, was 41% lower in HLA than in control rats. Protein concentration and fractional synthesis rate measured in the tibial epiphysis were not significantly different between the two groups of rats (Table 4).
It is essential to understand in each clinical setting the precise mechanism of repair in order to optimize the procedure of administration (route, schedule, dose, pretreatment with cytokines of chemokines…). It is also critical to standardize amplification procedures such that cells with similar properties are delivered to each patient, and to make possible the comparison of clinical results. The multiple characteristics of the MSCs accounts for the versatility of the mechanisms of injured tissue repair (Phinney and Prockop, 2007). Unlike HSCs, it is only in a few situations that regeneration by repopulation and differentiation is taking place, e.g. MSCs implanted in bone or cartilage lesions may repair by direct differentiation into osteoblasts or chondrocytes. In other disease models, including vascular diseases, cardiac infarcts or immune diseases, benefit from MSC injection might result from the secretion of cytokines that would induce the proliferation and/or differentiation of nearby differentiated cells or resident stem cells. Alternatively, because of the known immunomodulatory properties of MSCs, MSC-dependent repair might be secondary to impairment of migration and proliferation or increased apoptosis of inflammatory cells. Finally, it remains possible that reprogrammation occurs in certain situations, following fusion with local cells, or due to a specific cytokinic context of the diseased/injured tissue.
and reported that the girls playing the high-impact sport of soccer exhibited greater cortical thickness than swimmers due to increased bone section (width). The authors suggested that the “unloading” environment of the swimming pool may have a deleterious effect on bone geometry. We observed no alteration in the bone geometry of swimmers as compared with age-paired controls. The discrepancy between our data and Ferry's may be related to the training volume (mean 14 h/ wk vs. 10 h/wk), the type of training (associated with or without resistance training), the age of the participants, and the number of years of practice. It is possible that the normal/ low aBMD associated with normal bone geometry conferred an advantage in terms of buoyancy for these athletes [39,40] . The HSA results seem particularly pertinent in sport because they reinforce those obtained by direct evaluation of bone geometry such as pQCT at the tibial cortical site in preme- narcheal rhythmic gymnasts  . Independently, the modifi- cation in bone geometry and strength as evaluated by pQCT was also observed in artistic gymnasts at the forearm  .
1 INSERM, UMR 957, Nantes, France; 2 Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopa-
thologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, EA3822, Nantes, France
Received September 15, 2011; accepted September 30, 2011; Epub October 9, 2011; Published January 1, 2012 Abstract: Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) has been reported to specifi- cally induce malignant cell death being relatively nontoxic to normal cells. Since its identification 15 years ago, the antitumor activity and therapeutic value of TRAIL have been extensively studied. Five receptors quickly emerged, two of them being able to induce programmed cell death in tumor cells. This review takes a comprehensive look at this ligand and its receptors, and its potential role in primary bone tumors (osteosarcoma and Ewing’s sarcoma) therapy. The main limit of clinical use of TRAIL being the innate or acquired resistance mechanisms, different possibilities to sensitize resistant cells are discussed in this review, together with the impact of bone microenvironment in the regu- lation of TRAIL activity.
THYROID AND BONE : Actions of T3 :
- During bone formation : - stimulation of osteoblast proliferation, differentiation and apopotosis
- ↑ the expression of osteocalcin, type 1 collagen, alkalin phosphatase, IGF1,… - During bone resorption : - ↑ expression of IL6 and PGE2 (differenting factors of osteoclast lineage)
The representativity of OCP-citrate crystals as a model for intercrystalline interactions may appear in some ways as the continuation of Brown’s hypothesis on the relationship between a crystalline OCP structure and a surface of nanocrystalline apatite. The proposed model is in fact one monolithic block containing apatite domains and OCP-citrate hydrated layer. In this case, there are no separate nanocrystals, and citrate cannot be depicted as a bridge between preformed nanocrystals. In the proposed model, citrate appears simply as an element of a single continuous structural unit. The authors refer to a pub- lication, indicating that bone crystals form a continuum; however, this is rather misleading as the use of sodium hypochlorite to remove the organic matrix of bone, especially during excessive contact time, significantly alters biological apatite crystals. 9 Bone crystals can be isolated using gentler procedures, 10 and they appear as individualised entities. In addition, the effect of citrate ions on apatite crystals is not clear, and it has been reported in different papers 11 that these molecules do not act as agglomerating agents, but, on the contrary, as dispersing ones, maintaining a high negative zeta potential at the apatite crystals surface and preventing them from getting into close contact. Returning to the proposed