Determining the most important cellular characteristics for fracture healing using design of experiments methods

Computational models are employed as tools to investigate possible mechanoregulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The objective of this study was to determine the most important cellular characteristics of a mechanoregulatory model describing both cell phenotype-specific and mechanobiological processes that are active during bone healing using a statistical approach. The computational model included an adaptive two-dimensional finite element model of a fractured long bone. Three different outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at early, mid and late stages of healing and (3) the total time until complete healing. For the statistical analysis, first a resolution IV fractional factorial design (L₆₄) was used to identify the most significant factors. Thereafter, a three-level Taguchi orthogonal array (L₂₇) was employed to study the curvature (non-linearity) of the 10 identified most important parameters. The results show that the ability of the model to predict the sequences of normal fracture healing was predominantly influenced by the rate of matrix production of bone, followed by cartilage degradation (replacement). The amount of bone formation at early stages was solely dependent on matrix production of bone and the proliferation rate of osteoblasts. However, the amount of bone formation at mid and late phases had the rate of matrix production of cartilage as the most influential parameter. The time to complete healing was primarily dependent on the rate of cartilage degradation during endochondral ossification, followed by the rate of cartilage formation. The analyses of the curvature revealed a linear response for parameters related to bone, where higher rates of formation were more beneficial to healing. In contrast, parameters related to fibrous tissue and cartilage showed optimum levels. Some fibrous connective tissue- and cartilage formation was beneficial to bone healing, but too much of either tissue delayed bone formation. The identified significant parameters and processes are further confirmed by in vivo animal experiments in the literature. This study illustrates the potential of design of experiments methods for evaluating computational mechanobiological model parameters and suggests that further experiments should preferably focus at establishing values of parameters related to cartilage formation and degradation.     

Micro-finite element simulation of trabecular-bone post-yield behaviour--effects of material model, element size and type

Micro-finite element (micro-FE) analysis became a standard tool for the evaluation of trabecular bone mechanical properties. The accuracy of micro-FE models for linear analyses is well established. However, the accuracy of recently developed nonlinear micro-FE models for simulations of trabecular bone failure is not known. In this study, a trabecular bone specimen was compressed beyond the apparent yield point. The experiment was simulated using different micro-FE meshes with different element sizes and types, and material models based on cortical bone. The results from the simulations were compared with experimental results to study the effects of the different element and material models. It was found that a decrease in element size from 80 to 40 mum had little effect on predicted post-yield behaviour. Element type and material model had significant effects. Nevertheless, none of the established material models for cortical bone were able to predict the typical descent in the load-displacement curve seen during compression of trabecular bone.     

Hip-joint and abductor-muscle forces adequately represent in vivo loading of a cemented total hip reconstruction.

Using finite element analyses, we investigated which muscle groups acting around the hip-joint most prominently affected the load distributions in cemented total hip reconstructions with a bonded and debonded femoral stem. The purpose was to determine which muscle groups should be included in pre-clinical tests, predicting bone adaptation and mechanical failure of cemented reconstructions, ensuring an adequate representation of in vivo loading of the reconstruction. Loads were applied as occurring during heel-strike, mid-stance and push-off phases of gait. The stress/strain distributions within the reconstruction, produced by the hip-joint contact force, were compared to ones produced after sequentially including the abductors, the iliotibial tract and the adductors and vastii. Inclusion of the abductors had the most pronounced effect. They neutralized lateral bending of the reconstruction at heel-strike and increased medial bending at mid-stance and push-off. Bone strains and stem stresses were changed accordingly. Peak tensile cement stresses were reduced during all gait phases by amounts up to 50% around a bonded stem and 11% around a debonded one. Additional inclusion of the iliotibial tract, the adductors and the vastii produced relatively small effects during all gait phases. Their most prominent effect was a slight reduction of bone strains at the level of the stem tip during heel-strike. These results suggest that a loading configuration including the hip-joint contact force and the abductor forces can adequately reproduce in vivo loading of cemented total hip reconstructions in pre-clinical tests.     

<Emphasis Type="Italic">Usnea antarctica</Emphasis>, an important Antarctic lichen,is vulnerable to aspects of regional environmental change

Studies of cryptogam responses to climate change in the polar regions are scarce because these slow-growing organisms require long-term monitoring studies. Here, we analyse the response of a lichen and moss community to 10 years of passive environmental manipulation using open-top chambers (OTCs) in the maritime Antarctic region. Cover of the dominant lichen Usnea antarctica declined by 71 % in the OTCs. However, less dominant lichen species showed no significant responses except for an increase in Ochrolechia frigida, which typically covered dying lichen and moss vegetation. There were no detectable responses in the moss or associated micro-arthropod communities to the influence of the OTCs. Based on calculated respiration rates, we hypothesise that the decline of U. antarctica was most likely caused by increased net winter respiration rates (11 %), driven by the higher temperatures and lower light levels experienced inside the OTCs as a result of greater snow accumulation. During summer, U. antarctica appears unable to compensate for this increased carbon loss, leading to a negative carbon balance on an annual basis, and the lichen therefore appears to be vulnerable to such climate change simulations. These findings indicate that U. antarctica dominated fell-fields may change dramatically if current environmental change trends continue in the maritime Antarctic, especially if associated with increases in winter snow depth or duration.     

Potential of endozoochorous seed dispersal by sheep in calcareous grasslands: correlations with seed traits

Questions: What is the potential of sheep to serve as seed dispersers via ingestion and defecation in calcareous grasslands? Is the presence of viable seeds from dung correlated with specific seed traits? Location: Calcareous grasslands, South Limburg, the Netherlands/Belgium. Methods: Dung samples (n=24) from sheep were collected between September 2006 and November 2007 from five sites with Mesobromion plant communities, and communities of Nardo‐Galion saxatilis. Germinability and identity of seeds in the dung samples were ascertained from germination of seedlings under glasshouse conditions. Seed traits of species with viable seeds in dung were compared with those present in the local species pool. Results: Seventy‐two plant species from 23 plant families had viable seeds in sheep dung. The plant families encountered most frequently were Gramineae and Compositae. The most abundant and frequently recorded plant species in dung samples was Urtica dioica, accounting for >80% of the total number of seeds. Mean seed density in sheep dung was 0.8 seeds g^?1 dry matter. Seeds with low seed mass and a high seed longevity index were over‐represented in dung. Viable seeds >2.5 mg were infrequent in the dung samples. Conclusions: We conclude that sheep are potentially important dispersers of plant species in Dutch calcareous grasslands. Although smaller seeds were relatively abundant in sheep dung, it cannot be excluded that this was mainly caused by differences in seed abundance.     

How morphology predicts mechanical properties of trabecular structures depends on intra-specimen trabecular thickness variations

Two observations underlie this work. First, that the architecture of trabecular bone can accurately predict the mechanical stiffness characteristics of bone specimens when considering the combination of volume fraction and fabric, which is a measure of architectural anisotropy. Second, that the same morphological measures could not accurately predict the mechanical properties of porous structures in general. We hypothesize that this discrepancy can be explained by the special nature of trabecular bone as a structure in remodeling equilibrium relative to the external loads. We tested this hypothesis using a generic model of trabecular bone. Five series of 153 different architectures were created with this model. Each architecture was subjected to morphological analysis, and four different fabric measures were calculated to evaluate their effectiveness in characterizing the architecture. Relationships were determined relating morphology to the elastic constants. The quality of these relationships was tested by correlating the predicted elastic constants with those determined from finite element analysis. We found that the four fabric measures used could estimate the mechanical properties almost equally well. So the suggestion that fabric measures based on trabecular bone volume better represent the architecture than mean intercept length could not be affirmed. We conclude that for structures with equally sized elliptical voids the mechanical properties can be predicted well only if trabecular thickness variations within each structure are limited. These structures closely resemble previously developed models of trabecular bone. Furthermore, they are stiff in the principal fabric direction, hence, according to Cowin (J. Biomech. Eng. (108) (1986) 83), they are in remodeling equilibrium. These structures are also stiff over a large range of loading orientations, hence, are relatively insensitive to deviations in direction of loading.     

A sclerostin-based theory for strain-induced bone formation

Bone formation responds to mechanical loading, which is believed to be mediated by osteocytes. Previous theories assumed that loading stimulates osteocytes to secrete signals that stimulate bone formation. In computer simulations this 'stimulatory' theory successfully produced load-aligned trabecular structures. In recent years, however, it was discovered that osteocytes inhibit bone formation via the protein sclerostin. To reconcile this with strain-induced bone formation, one must assume that sclerostin secretion decreases with mechanical loading. This leads to a new 'inhibitory' theory in which loading inhibits osteocytes from inhibiting bone formation. Here we used computer simulations to show that a sclerostin-based model is able to produce a load-aligned trabecular architecture. An important difference appeared when we compared the response of the stimulatory and inhibitory models to loss of osteocytes, and found that the inhibitory pathway prevents the loss of trabeculae that is seen with the stimulatory model. Further, we demonstrated with combined stimulatory/inhibitory models that the two pathways can work side-by-side to achieve a load-adapted bone architecture.