Multiscale computational and experimental approaches to elucidate bone and ligament mechanobiology using the ulna-radius-interosseous membrane construct as a model system.

An in vivo axial loading model of the rat ulna was developed almost two decades ago. As a minimally invasive model, it lends itself particularly well for the study of functional adaptation in bone and the interosseous membrane, a ligament spanning between the radius and ulna. The objective of this paper is to review computational and experimental approaches to elucidate its applicability for the study of multiscale bone and ligament mechanobiology. Specifically, this review describes approaches, including i) measurement of strains on bone tissue surfaces, ii) development of a three-dimensional finite element (FE) mesh of a skeletally mature rat ulna, iii) parametric study of the relative influence of mechanical constants and materials properties on computational model predictions, iv) comparison of experimental and computational strain distribution data, and analysis of the radius and interosseous membrane (IOM) ligament's effect on axial load distribution through the ulna of the rat, and v) the effect of mechanical loading on transport through the IOM using different molecular weight fluorescent tagged dextrans. In the first stage of the study a computational stress analysis was performed after applying a 20 N single static load at the ulnar extremities, corresponding to values of experimental strain gauge measurements. To account for the anisotropy of the bone matrix, transverse isotraopic, elastic material properties were applied. In a parametric study, we analyzed the qualitative effect of different material properties on the global load and displacement behavior of the computational model. In a second stage, the same ulnar model used in the parametric study was extended to account for the interaction between the ulna, radius and IOM. The three-dimensional FE model of the rat forelimb confirms the influence of ulnar curvature on its deformation and underscores the influence of the radius and IOM on strain distribution through the ulna. The mode of strain, {i.e.} compression or tension, and strain distribution along the bone diaphysis correspond to those measured experimentally in vivo. When the radius and, indirectly, the IOM were loaded, the bone deformation shifted distally with respect to the diaphysis. In a final stage, the aforementioned ulnar model was used to study the permeability of fluorescent tagged dextrans with different molecular weights in the presence and absence of ulnar compression. Small molecular weight dextrans (3,000 Da) were distributed throughout the IOM in the absence of as well as after mechanical loading. Interestingly, no gradient in distribution was observed in either case. In contrast, very high molecular weight dextrans (1,000,000 Da) were observed only within vascular and lymphatic spaces in the bone (as well as periosteum) and IOM, both in the absence of and after the application of mechanical loading via end load compression. Between the two extremes, both 10 and 70 kDa tracers were distributed throughout the IOM after application of compressive loading. Loading appears to dissipate the steep gradient of fluorescent 70 kDa tracer observed along the lateral surface of the unloaded IOM and its insertion into the radius and ulna. Hence, this combined computational and experimental analysis of the ulna compression model provides new insight into multiscale mechanobiology of the ulna-radius-interosseous membrane construct and may provide new avenues for elucidation of ligament's remarkable structure-function relationships.

Probing the tissue to subcellular level structure underlying bone's molecular sieving function.

Transport of fluorescent probes between 300 and 2,000,000 Da was studied in mechanically loaded and unloaded ulnae of skeletally mature rats to characterize the permeability of the pericellular space of the lacunocanalicular system (LCS), and the microporosity of the bony matrix. The mineral matrix porosity allowed for penetration of the 300 Da probe but impeded transport of larger probes. The pericellular space of the LCS was permeable up to 10 kDa; above 10 kDa, diffusion was ineffective for transport through the pericellular space. Convective transport via load-induced fluid flow increased penetration of all probes up to 70 kDa. Above this threshold, probes were excluded from bone, both with and without loading. This exploratory study suggests that bone acts as a molecular sieve and that mechanical loading modulates transport of solutes through the pericellular space that links osteocytes deep within the tissue to the blood supply and to osteoblasts and osteoclasts on bone forming and resorbing surfaces. This provides support for the postulate of transport modulated bone remodeling in which osteocytes are influenced by and modulate the local permeability of their surroundings as a means for survival (Knothe Tate et al. 1998, [28]) and has profound implications for osteocyte viability and intercellular communication in bone.

Noninvasive fatigue fracture model of the rat ulna.

Fatigue damage occurs in response to repeated cyclic loading and has been observed in situ in cortical bone of humans and other animals. When microcracks accumulate and coalesce, failure ensues and is referred to as fatigue fracture. Experimental study of fatigue fracture healing is inherently difficult due to the lack of noninvasive models. In this study, we hypothesized that repeated cyclic loading of the rat ulna results in a fatigue fracture. The aim of the study was to develop a noninvasive long bone fatigue fracture model that induces failure through accumulation and coalescence of microdamage and replicates the morphology of a clinical fracture. Using modified end-load bending, right ulnae of adult Sprague-Dawley rats were cyclically loaded in vivo to fatigue failure based on increased bone compliance, which reflects changes in bone stiffness due to microdamage. Preterminal tracer studies with 0.8% Procion Red solution were conducted according to protocols described previously to evaluate perfusion of the vasculature as well as the lacunocanalicular system at different time points during healing. Eighteen of the 20 animals loaded sustained a fatigue fracture of the medial ulna, i.e. through the compressive cortex. In all cases, the fracture was closed and non-displaced. No disruption to the periosteum or intramedullary vasculature was observed. The loading regime did not produce soft tissue trauma; in addition, no haematoma was observed in association with application of load. Healing proceeded via proliferative woven bone formation, followed by consolidation within 42 days postfracture. In sum, a noninvasive long bone fatigue fracture model was developed that lends itself for the study of internal remodeling of periosteal woven bone during fracture healing and has obvious applications for the study of fatigue fracture etiology.

A finite element analysis for the prediction of load-induced fluid flow and mechanochemical transduction in bone.

Interstitial fluid flow through the lacunocanalicular cavities of mechanically loaded bone provides the biophysical basis for a number of postulates regarding mechanotransduction in bone. Recently, the existence of load-induced fluid flow and its influence on molecular transport through bone has been confirmed using tracer methods to visualize fluid flow induced by in vivo four-point-bending of rat tibiae. In this paper, we present a theoretical two-stage approach for the calculation of load-induced flow fields and for the evaluation of their influence on molecular transport in bone loaded in four-point bending, analogous to the aforementioned experimental model. In the first stage, the fluid velocities are calculated using a three-dimensional, poroelastic finite element model. In the second stage, mass transport analysis, this calculated fluid flow serves as a forced convection flow and its contribution to the total transport potential is determined. Based on this combined approach, the overall tracer concentration in the loaded bone is significantly higher than that in the unloaded bone. Furthermore, augmentation of mass transport through convective flow is more pronounced in the tension band of the tissue, as compared to the compression band. In general, augmentation of tracer concentration via convective mechanisms is most pronounced in areas corresponding to lowest fluid velocities, which is indicative of fluid flow direction and areas of increased "dwell time" or accumulation during the loading cycle. This theoretical model, in combination with the corresponding experimental model, provides unique insight into the role of mechanical loads in modulating local flow distributions and concentration gradients within bone tissue.

The role of interstitial fluid flow in the remodeling response to fatigue loading.

Load-induced fluid flow enhances molecular transport through bone tissue and relates to areas of bone resorption and apposition. Remodeling activity is highly coordinated and necessitates a means for cellular communication via intracellular and extracellular means. Osteocytes, osteoblasts, and osteoclasts, which reside in disparate locations within the tissue, communicate intracellularly via the cellular syncytium and extracellularly via the pericellular fluid space of the lacunocanalicular system. Both of these communications systems are physically disrupted by microdamage incurred during fatigue loading of bone. The purpose of this study was to develop an analytical model to understand the role of interstitial fluid flow in the remodeling response to fatigue loading. Adequate transport was assumed a prerequisite for maintenance of cell viability in bone. Diffusive and convective transport were simulated through the lacunocanalicular network in a healthy undamaged state as well as in a damaged state after fatigue loading. The model predicts that fatigue damage impedes transport from the blood supply, depleting the concentration of molecular entities in and downstream from areas of damage. Furthermore, the presence of microcracks alters the distribution of molecular entities between individual lacunae. These effects were confirmed by the results of an in vivo pilot study in which fluorescent, flow-visualizing agents pooled within microcracks and were absent from areas surrounding microcracks, corresponding to areas deprived of fluid flow. Loss of osteocyte viability is coupled to targeting and initiation of new remodeling activity. Taken as a whole, these data suggest a link between interstitial fluid flow, mass transport, maintenance of osteocyte viability, and modulation of remodeling activity.

Investigation of the morphology of the lacunocanalicular system of cortical bone using atomic force microscopy.

Mechanical loading has been implicated as a powerful driving mechanism for interstitial fluid flow through bone. However, little information is available with regard to the morphology of bone fluid spaces, e.g., the canalicular wall, which would be expected to dictate the type of flow regime developing in the lacunocanalicular system under mechanical loads. The purpose of this study was to examine the fine structure of the lacunocanalicular system in cortical bone using atomic force microscopy (AFM), resin casting methods, and selective etching of the specimen surface. A resin-cast replica of the canalicular wall was produced and surface morphology and dimensions were observed using AFM in tapping mode. Material contrast was obtained using surface potential measurements. A striped pattern perpendicular to the canaliculus long axis with a periodicity of 125 nm dominated the structure of the canalicular wall; it is likely that this was caused by the imprint of collagen fibrils arranged in parallel, lining the canaliculus wall. The largest dimension measured for canalicular diameter was on the order of 500 nm. The regular dips and ridges caused by the collagen that lines the wall are a source of roughness which may influence shear stresses imparted by the fluid on the cell surface as well as mixing of solutes within the lacunocanalicular system. In addition, the lacunocanalicular wall lining is likely to affect physicochemical interactions between the fluid and bone matrix. This has important implications for modeling and understanding the microfluid mechanics and rheology of the fluid-filled lacunocanalicular network.

Development and testing of a new self-locking intramedullary nail system: testing of handling aspects and mechanical properties.

A distal interlocking system has been developed which is easy to use, carries out an aligning effect on the distal fracture fragment, reduces the exposure to radiation for the surgeon and the patient, and allows for a decrease in operating time. The goal of this study was to develop and test the handling and mechanical properties of two prototype nails in comparison to a conventional interlocking nail concept (Unreamed Femoral Nail system). It was shown that the prototype designs represent an improvement over this system. Both designs were easy to use. The prototype with the asymmetrically offset interlocking bolts exhibited an exemplary aligning effect on the distal fracture fragment. Both designs showed mechanical stability comparable or superior to that of the standard contralateral control in four-point-bending and axial compression. Given the handling advantages afforded by the new self-locking intramedullary implant system, it would be expected that use of this system would reduce exposure to radiation for the surgeon as well as the patient and allow for a decrease in operating time. This new development may be of particular interest for clinics without access to fluoroscopes in the operating theatre (e.g. in the Third World).

In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation.

Load-induced extravascular fluid flow has been postulated to play a role in mechanotransduction of physiological loads at the cellular level. Furthermore, the displaced fluid serves as a carrier for metabolites, nutrients, mineral precursors and osteotropic agents important for cellular activity. We hypothesise that load-induced fluid flow enhances the transport of these key substances, thus helping to regulate cellular activity associated with processes of functional adaptation and remodelling. To test this hypothesis, molecular tracer methods developed previously by our group were applied in vivo to observe and quantify the effects of load-induced fluid flow under four-point-bending loads. Preterminal tracer transport studies were carried out on 24 skeletally mature Sprague Dawley rats. Mechanical loading enhanced the transport of both small- and larger-molecular-mass tracers within the bony tissue of the tibial mid-diaphysis. Mechanical loading showed a highly significant effect on the number of periosteocytic spaces exhibiting tracer within the cross section of each bone. For all loading rates studied, the concentration of Procion Red tracer was consistently higher in the tibia subjected to pure bending loads than in the unloaded, contralateral tibia. Furthermore, the enhancement of transport was highly site-specific. In bones subjected to pure bending loads, a greater number of periosteocytic spaces exhibited the presence of tracer in the tension band of the cross section than in the compression band; this may reflect the higher strains induced in the tension band compared with the compression band within the mid-diaphysis of the rat tibia. Regardless of loading mode, the mean difference between the loaded side and the unloaded contralateral control side decreased with increasing loading frequency. Whether this reflects the length of exposure to the tracer or specific frequency effects cannot be determined by this set of experiments. These in vivo experimental results corroborate those of previous ex vivo and in vitro studies. Strain-related differences in tracer distribution provide support for the hypothesis that load-induced fluid flow plays a regulatory role in processes associated with functional adaptation.

A finite difference model of load-induced fluid displacements within bone under mechanical loading.

Load-induced fluid flow in the lacunocanalicular network, induced by the mechanical loading of bone, is believed to play an important role in bone modelling, remodelling and adaptation processes. There are strong indications that this fluid flow is responsible for the mechanotransduction from external mechanical loads to the cells responsible for bone apposition or removal. Since direct flow measurements (especially in compact bone, in vivo and in situ) are not yet possible, theoretical modelling offers an alternative approach to determine the fluid flow velocities, displacements and effects of interstitial fluid flow. In this model, the fluid displacements in a middiaphyseal slab of a rat tibia under a cyclic four-point-bending load were calculated by applying Biot's theory of poroelasticity. The resulting differential equations were solved numerically for the fluid displacement vectors using the finite difference method. Thereby, the cross section located in the middle between the two inner points of force application was chosen for examination, such that the problem, although formulated in three dimensions, reduced itself to an essentially planar form. The maximal fluid displacements for the vector components in the cross sectional plane were found in the proximity of the neutral axis of bending. The direction of the displacement vectors was from the lateral aspect, which was in compression in the examined loading situation, towards the medial aspect in tension. In a parameter study it was found that the fluid displacement pattern and the distribution of fluid displacements remained constant for all the examined parameters, while the magnitude was influenced by the model parameters Young's modulus, Poisson's ratio and porosity. This study represents a further step in the examination of load-induced fluid displacements in loaded bone using theoretical models, aiming to understand the relationship between mechanical loading and bone modelling, remodelling and functional adaptation.

An ex vivo model to study transport processes and fluid flow in loaded bone.

Load-induced fluid flow has been postulated to provide a mechanism for the transmission of mechanical signals (e.g. via shear stresses, enhancement of molecular transport, and/or electrical effects) and the subsequent elicitation of a functional adaptation response (e.g. modeling, remodeling, homeostasis) in bone. Although indirect evidence for such fluid flow phenomena can be found in the literature pertaining to strain generated potentials, actual measurement of fluid displacements in cortical bone is inherently difficult. This problem motivated us to develop and introduce an ex vivo perfusion model for the study of transport processes and fluid flow within bone under controlled mechanical loading conditions. To this end, a closed-loop system of perfusion was established in the explanted forelimb of the adult Swiss alpine sheep. Immediately prior to mechanical loading, a bolus of tracer was introduced intraarterially into the system. Thereafter, the forelimb of the left or right side (randomized) was loaded cyclically, via Schanz screws inserted through the metaphyses, producing a peak compressive strain of 0.2% at the middiaphysis of the anterior metacarpal cortex. In paired experiments with perfusion times totalling 2, 4, 8 and 16 min, the concentration of tracer measured at the middiaphysis of the cortex in cross section was significantly higher in the loaded bone than in the unloaded contralateral control. Fluorometric measurements of procion red concentration in the anterior aspect alone showed an enhancement in transport at early stages of loading (8 cycles, 2 min) but no effect in transport after higher number of cycles or increased perfusion times, respectively. This reflects both the small size of the molecular tracer, which would be expected to be transported rapidly by way of diffusive mechanisms alone, as well as the loading mode to which the anterior aspect was exposed. Thus, using our new model it could be shown that load-induced fluid flow represents a powerful mechanism to enhance molecular transport within the lacunocanalicular system of compact bone tissue. Based on these as well as previous studies, it appears that the degree of this effect is dependent on tracer size as well as the mechanical loading mode to which a given area of tissue is exposed.

A novel ex vivo model for investigation of fluid displacements in bone after endoprosthesis implantation.

Tissue perfusion and mass transport in the vicinity of implant surfaces prior to integration or bonding may play a crucial role in modulating cellular activities associated with bone remodeling, in particular, at early stages of the integration process. Furthermore, fluid displacements have been postulated to transduct mechanical stress signals to bone cells via loading-dependent flow of interstitial fluid through the lacunocanalicular network of bone. Thus, an understanding and new possibilities for influencing these processes may be of great importance for implant success. An ex vivo model was developed and validated for investigation of fluid displacements in bone after endoprosthesis implantation. This model serves to explicate the effects of surgical intervention as well as mechanical loading of the implant-bone construct on load-induced fluid flow in the vicinity of the implant. Using this model, we intend to quantify perfusion and extravascular flow dynamics in the vicinity of implants and define optimal conditions for enhancing molecular transport of osteotropic agents from the implant surface to apposing bone as well as from the blood supply to the implant surface. Furthermore, the elucidation of main transport pathways may help in understanding the distribution of wear particles in bone surrounding implant, a process which has been postulated to cause osteolysis and implant loosening.

Experimental elucidation of mechanical load-induced fluid flow and its potential role in bone metabolism and functional adaptation.

Several researchers have developed theories implicating some manifestation of mechanical forces such as stress, strain, and strain energy density for the initiation of cellular processes associated with functional adaptation. The mechanisms underlying dynamic bone growth and repair in response to mechanical stimuli, however, are not fully understood. Load-induced fluid flow has been postulated to provide a mechanism for the transmission of mechanical signals (eg, via shear stresses, enhancement of molecular transport, or electrical effects) and the subsequent elicitation of a functional adaptation response in bone. Although indirect evidence for such fluid flow phenomena can be found in the literature pertaining to strain-generated potentials, experimental studies are inherently difficult. This motivated the authors to develop theoretical as well as ex vivo, in vitro, and in vivo experimental methods for the study of transport processes and fluid flow within bone under well-controlled mechanical loading conditions. By introducing tracer substances such as disulphine blue, procion red, and microperoxidase into the experimental system, transport and fluid flow could be visualized at tissue, cellular, and subcellular levels, respectively. Based on these studies, it could be shown that load-induced fluid flow represents a powerful mechanism to enhance molecular transport within compact bone tissue. Furthermore, the distribution of transport-elucidating tracers is a function of mechanical loading parameters as well as the location within the cross-section of the bone cortex.

In vivo tracer transport through the lacunocanalicular system of rat bone in an environment devoid of mechanical loading.

Although diffusion has been shown to be the major contributing mechanism for molecular transport in the extravascular spaces of organs and soft tissues, it is unlikely that diffusion alone can account for molecular transport in the porous, yet relatively impermeable matrix of bone. Rather, it has been proposed that fluid flow induced by the deformations that bone is subjected to during daily activities may promote molecular transport through convective mixing of fluids or enhancement of molecular transport from the capillaries to the outermost osteocytes within a given osteon. As the relative contribution of diffusive and convective transport in the bone matrix has not yet been elucidated, we conducted experiments to study the primary role of diffusion for molecular transport within bone and to establish a baseline for fluid transport whereby mechanical loading effects are negligible. Procion red and microperoxidase were utilized as short-term (i.e., low MW, transported on the order of minutes) and long-term (i.e., comparatively high MW, transported on the order of hours) molecular tracers, respectively, to elucidate in vivo the pathways and extent of transport in the metacarpus and tibia of 60-day-old (i.e., skeletally immature) and 180-day-old (i.e., skeletally mature) animals. The tracers were introduced intravenously and the animals were maintained in an anesthetized state for the duration of the experiment to prevent physiological loading. In short-term studies, procion red tracer distribution was highly dependent on bone structure, demarcating spaces apposing the vascular pathways in the trabecular bone of immature animals and vascular and extravascular pathways (i.e., specifically, the lacunocanalicular system) within compact bone of mature animals. In longer term studies using microperoxidase, reaction product was concentrated in soft tissues as well as along a subperiosteal and subendosteal band of bone. In contrast, little peroxidase reaction product was observed in the metacarpal and tibial cortices of either immature or mature animals. Based on the results of these studies, diffusive transport mechanisms may suffice to insure an adequate supply of small molecules, such as amino acids, to osteocytes in the midcortex within minutes. In contrast, diffusion alone may not be efficient for transport of larger molecules. Thus, another mechanism of transport, such as convective transport by means of load-induced fluid flow, may be necessary to provide a sufficient supply of larger molecules, such as proteins to osteocytes for the maintenance of metabolic activity, as well as for activation or suppression of modeling processes.