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.

Development of a novel method for surgical implant design optimization through noninvasive assessment of local bone properties.

A method was developed to improve the design of locking implants by finding the optimal paths for the anchoring elements, based on a high resolution pQCT assessment of local bone mineral density (BMD) distribution and bone micro-architecture (BMA). The method consists of three steps: (1) partial fixation of the implant to the bone and creation of a reference system, (2) implant removal and pQCT scan of the bone, and (3) determination of BMD and BMA of all implant-anchoring locations along the actual and alternative directions. Using a PHILOS plate, the method uncertainty was tested on an artificial humerus bone model. A cadaveric humerus was used to quantify how the uncertainty of the method affects the assessment of bone parameters. BMD and BMA were determined along four possible alternative screw paths as possible criteria for implant optimization. The method is biased by a 0.87 ± 0.12 mm systematic uncertainty and by a 0.44 ± 0.09 mm random uncertainty in locating the virtual screw position. This study shows that this method can be used to find alternative directions for the anchoring elements, which may possess better bone properties. This modification will thus produce an optimized implant design.

Inhibition of angiotensin-converting enzyme stimulates fracture healing and periosteal callus formation - role of a local renin-angiotensin system.

BACKGROUND AND PURPOSE: The renin-angiotensin system (RAS) regulates blood pressure and electrolyte homeostasis. In addition, 'local' tissue-specific RAS have been identified, regulating regeneration, cell growth, apoptosis, inflammation and angiogenesis. Although components of the RAS are expressed in osteoblasts and osteoclasts, a local RAS in bone has not yet been described and there is no information on whether the RAS is involved in fracture healing. Therefore, we studied the expression and function of the key RAS component, angiotensin-converting enzyme (ACE), during fracture healing. EXPERIMENTAL APPROACH: In a murine femur fracture model, animals were treated with the ACE inhibitor perindopril or vehicle only. Fracture healing was analysed after 2, 5 and 10 weeks using X-ray, micro-CT, histomorphometry, immunohistochemistry, Western blotting and biomechanical testing. KEY RESULTS: ACE was expressed in osteoblasts and hypertrophic chondrocytes in the periosteal callus during fracture healing, accompanied by expression of the angiotensin type-1 and type-2 receptors. Perindopril treatment reduced blood pressure and bone mineral density in unfractured femora. However, it improved periosteal callus formation, bone bridging of the fracture gap and torsional stiffness. ACE inhibition did not affect cell proliferation, but reduced apoptotic cell death. After 10 week treatment, a smaller callus diameter and bone volume after perindopril treatment indicated an advanced stage of bone remodelling. CONCLUSIONS: Our study provides evidence for a local RAS in bone that influenced the process of fracture healing. We show for the first time that inhibition of ACE is capable of accelerating bone healing and remodelling.

Nanohydroxyapatite/poly(ester urethane) scaffold for bone tissue engineering.

Biodegradable viscoelastic poly(ester urethane)-based scaffolds show great promise for tissue engineering. In this study, the preparation of hydroxyapatite nanoparticles (nHA)/poly(ester urethane) composite scaffolds using a salt-leaching-phase inverse process is reported. The dispersion of nHA microaggregates in the polymer matrix were imaged by microcomputed X-ray tomography, allowing a study of the effect of the nHA mass fraction and process parameters on the inorganic phase dispersion, and ultimately the optimization of the preparation method. How the composite scaffold's geometry and mechanical properties change with the nHA mass fraction and the process parameters were assessed. Increasing the amount of nHA particles in the composite scaffold decreased the porosity, increased the wall thickness and consequently decreased the pore size. The Young's modulus of the poly(ester urethane) scaffold was improved by 50% by addition of 10 wt.% nHA (from 0.95+/-0.5 to 1.26+/-0.4 MPa), while conserving poly(ester urethane) viscoelastic properties and without significant changes in the scaffold macrostructure. Moreover, the process permitted the inclusion of nHA particles not only in the poly(ester urethane) matrix, but also at the surface of the scaffold pores, as shown by scanning electron microscopy. nHA/poly(ester urethane) composite scaffolds have great potential as osteoconductive constructs for bone tissue engineering.

Longitudinal as well as age-matched assessments of bone changes in the mature ovariectomized rat model.

In the past, bone loss in the ovariectomized (OVX) osteoporotic rat model has been monitored using in vitro micro-computed tomography (micro-CT) to assess bone structure (bone volume/total volume, BV/TV). The purpose of this study was to assess the importance of baseline control and sham groups in 12-16-week-old, reproductively mature rats. Measurements were carried out in a longitudinal and age-matched fashion using newer in vivo peripheral quantitative computed tomography (pQCT), which measures apparent bone mineral density (BMD). BMD was measured at the distal femoral metaphysis of 12-week-old female Wistar rats with pQCT. Subsequently, animals were either OVX or sham operated, and pQCT measurements were repeated four weeks later. Then, all rats were euthanized and in vitro BMD and BV/TV were obtained by micro-CT imaging. Results from three consistently differentiated regions of interest showed that there was significant bone loss and growth during the four weeks in the OVX and sham group, respectively. Taking this into account, i.e. a posteriori superimposing growth to loss, no differences resulted between BMD values measured in a longitudinal fashion with pQCT and that measured in comparison with an age-matched sham group with micro-CT and pQCT. In addition, there was a strong linear correlation between BMD measured with pQCT and BV/TV obtained from micro-CT. In conclusion, this outcome provides new insights into individual bone changes due to OVX and growth in Wistar rats during the age period of 12-16 weeks, which is often applied in osteoporosis research as the 'mature' rat model. Data can be used as baseline information upon which future in vivo study designs with this rat model can refer to reduce and minimize animal use.

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.

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.