A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E-2 (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.

Biomimetic bone-like composites fabricated through an automated alternate soaking process.

Hydroxyapatite-gelatin composites have been proposed as suitable scaffolds for bone and dentin tissue regeneration. There is considerable interest in producing these scaffolds using biomimetic methods due to their low energy costs and potential to create composites similar to the tissues they are intended to replace. Here an existing process used to coat a surface with hydroxyapatite under near physiological conditions, the alternate soaking process, is modified and automated using an inexpensive "off the shelf" robotics kit. The process is initially used to precipitate calcium phosphate coatings. Then, in contrast to previous utilizations of the alternate soaking process, gelatin was added directly to the solutions in order to co-precipitate hydroxyapatite-gelatin composites. Samples were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and nanoindentation. Calcium phosphate coatings formed by the alternate soaking process exhibited different calcium to phosphate ratios, with correspondingly distinct structural morphologies. The coatings demonstrated an interconnected structure with measurable mechanical properties, even though they were 95% porous. In contrast, hydroxyapatite-gelatin composite coatings over 2mm thick could be formed with little visible porosity. The hydroxyapatite-gelatin composites demonstrate a composition and mechanical properties similar to those of cortical bone.

A bone remodelling model coupling micro-damage growth and repair by 3D BMU-activity.

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to its specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and micro-damage, accumulated by fatigue or creep, is removed minimizing the risk of fracture. Nevertheless, bone is not always able to repair itself completely. Actually, if bone repairing function is slower than micro-damage accumulation, a type of bone fracture, usually known as "stress fracture", can finally evolve. In this paper, we propose a bone remodelling continuous model able to simulate micro-damage growth and repair in a coupled way and able therefore to predict the occurrence of "stress fractures". The biological bone remodelling process is modelled in terms of equations that describe the activity of basic multicellular units. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation is achieved. In overloading, bone porosity decreases unless the damage rate is so high that causes resorption or "stress fracture".

METHOD FOR ASSESSING EFFECTS OF CIRCUMFERENTIAL FLOW DISTORTION ON COMPRESSOR STABILITY.

This paper describes the development of a new analysis to predict the onset of flow instability for an axial compressor operating in a circumferentially distorted inlet flow. A relatively simple model is used to examine the influence of various distortions in setting this instability point. It is found that the model reproduces known experimental trends for the loss of stability margin with increasing distortion amplitude and with changes in reduced frequency.

Effects of rotating inlet distortion on multistage compressor stability

In multi-spool engines, rotating stall in an upstream compressor will impose a rotating distortion on the downstream compressor, thereby affecting its stability margin. In this paper experiments are described in which this effect was simulated by a rotating screen upstream of several multistage low-speed compressors. The measurements are complemented by, and compared with, a theoretical model of multistage compressor response to speed and direction of rotation of an inlet distortion. For co-rotating distortions (i.e., distortions rotating in the same direction as rotor rotation), experiments show that the compressors exhibited significant loss in stability margin and that they could be divided into two groups according to their response. The first group exhibited a single peak in stall margin degradation when the distortion speed corresponded to roughly 50% of rotor speed. The second group showed two peaks in stall margin degradation corresponding to distortion speeds of approximately 25-35% and 70-75% of rotor speed. These new results demonstrate that multistage compressors can have more than a single resonant response. Detailed measurements suggest that the two types of behavior are linked to differences between the stall inception processes observed for the two groups of compressors and that a direct connection thus exists between the observed forced response and the unsteady flow phenomena at stall onset. For counter-rotational distortions, all the compressors tested showed minimal loss of stability margin. The results imply that counter-rotation of the fan and core compressor, or LP and HP compressors, could be a worthwhile design choice. Calculations based on the two-dimensional theoretical model show excellent agreement for the compressors which had a single peak for stall margin degradation. We take this first-of-a-kind comparison as showing that the model, though simplified, captures the essential fluid dynamic features of the phenomena. Agreement is not good for compressors which had two peaks in the curve of stall margin shift versus distortion rotation speed. The discrepancy is attributed to the three-dimensional and short length scale nature of the stall inception process in these machines; this includes phenomena that have not yet been addressed in any model.

Mechanobiology of bone remodelling

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to the specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and microdamage removed minimizing the risk of fracture. Bone remodelling is controlled by mechanical and metabolical stimuli. In this paper, we introduce a new model of bone remodelling that takes into account both types of influences. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation, while, in overloading, decreases unless the damage rate is so high that causes resorption and "stress fracture". This model has been employed to predict bone adaptation in the proximal femur after total hip replacement proving its consistence and good correspondence with well-known clinical experiences.

Variability of nanoindentation responses of bone and artificial bone-like composites

Nanoindentation is a popular technique for measuring the intrinsic mechanical response of bone and has been used to measure a single-valued elastic modulus. However, bone is a composite material with 20-80 nm hydroxyapatite plates embedded in a collagen matrix, and modern instrumentation allows for measurements at these small length scales. The present study examines the indentation response of bone and artificial gelatin-apatite nanocomposite materials across three orders of magnitude of lengthscale, from nanometers to micrometers, to isolate the composite phase contributions to the overall response. The load-displacement responses were variable and deviated from the quadratic response of homogeneous materials at small depths. The distribution of apparent elastic modulus values narrowed substantially with increasing indentation load. Indentation of particulate nanocomposites was simulated using finite element analysis. Modeling results replicated the convergence in effective modulus seen in the experiments. It appears that the apatite particles are acting as the continuous ("matrix") phase in bone and nanocomposites. Copyright © 2004 by ASME.

Viscoelastic properties of bone as a function of hydration state determined by nanoindentation

Spherical indentation creep testing was used to examine the effect of hydration state on bone mechanical properties. Analysis of creep data was based on the elastic-viscoelastic correspondence principle and utilized a direct solution for the finite loading-rate experimental conditions. The zero-time shear modulus was computed from the creep compliance function and compared to the indentation modulus obtained via conventional indentation analysis, based on an elastic unloading response. The method was validated using a well-known polymer material under three different loading conditions. The method was applied to bone samples prepared with different water content by partial exchange with ethanol, where 70% ethanol was considered as the baseline condition. A hydration increase was associated with a 43% decrease in stiffness, while a hydration decrease resulted in a 20% increase in bone tissue stiffness.

Nanoindentation measurements of bone viscoelasticity as a function of hydration state

Bone is an anisotropic material, and its mechanical properties are determined by its microstructure as well as its composition. Mechanical properties of bone are a consequence of the proportions of, and the interactions between, mineral, collagen and water. Water plays an important role in maintaining the mechanical integrity of the composite, but the manner in which water interacts within the ultrastructure is unclear. Dentine being an isotropic two-dimensional structure presents a homogenous composite to examine the dehydration effects. Nanoindentation methods for determining the viscoelastic properties have recently been developed and are a subject of great interest. Here, one method based on elastic-viscoelastic correspondence for 'ramp and hold' creep testing (Oyen, J. Mater. Res., 2005) has been used to analyze viscoelastic behavior of polymeric and biological materials. The method of 'ramp and hold' allows the shear modulus at time zero to be determined from fitting of the displacement during the maximum load hold. Changes in the viscoelastic properties of bone and dentine were examined as the material was systematically dehydrated in a series of water:solvent mixes. Samples of equine dentine were sectioned and cryo-polished. Shear modulus was obtained by nanoindentation using spherical indenters with a maximum load hold of 120s. Samples were tested in different solvent concentrations sequentially, 70% ethanol to 50% ethanol, 70 % ethanol to 100% ethanol, 70% ethanol to 70% methanol to 100% methanol, and 70% ethanol to 100% acetone, after storage in each condition for 24h. By selectively removing and then replacing water from the composite, insights in to the ultrastructure of the tissue can be gained from the corresponding changes in the experimentally determined moduli, as well as an understanding of the complete reversibility of the dehydration process. © 2006 Materials Research Society.

Effect of water on mechanical properties of mineralized tissue composites

In the current study, the effects of polar solvents on tissue volume and mechanical properties are considered. Area shrinkage measurements are conducted for mineralized bone tissue samples soaked in polar solvents. Area shrinkage is used to calculate approximate linear and volume shrinkage. Results are compared with viscoelastic mechanical parameters for bone in the same solvents (as measured previously) and with both shrinkage measurements and mechanical data for nonmineralized tissues, as taken from the existing literature. As expected, the shrinkage of mineralized tissues is minimal when compared with shrinkage of nonmineralized tissues immersed in the same polar solvents. The mechanical changes in bone are also substantially less than in nonmineralized tissues. The largest stiffness values are found in shrunken bone samples (immersed in acetone and ethanol). The mineral phase in bone thus resists tissue shrinkage that would otherwise occur in the pure soft tissue phase. © 2007 Materials Research Society.

Effects of gelatin on mechanical properties of hydroxyapatite-gelatin nano-composltes

A biomimetic reactor has been developed to synthesize hydroxyapatite- gelatin (HAP-GEL) nanocomposites that mimic ultra-structures of natural bone. We hypothesize that in the reactor, gelatin concentration controls morphology and packing structures of HAP crystals. To test the hypothesis, three types of mechanical tests were conducted, including nanoindentation, compression, and fracture tests. Nanoindentation tests in conjunction with computer modeling were used to assess effects on gelatin-induced microstructures of HAP. The results showed that increasing gelatin content increased both the plane strain modulus and the fracture toughness. The gelatin appeared to shorten the HAP crystal distance, which consolidated the internal structure of the composite and made the material more rigid. The fracture toughness KIC increased partially due to the effect of fiber bridging between gelatin molecules. The highest fracture toughness (1.12 MPa·1/2) was equivalent to that of pure hydroxyapatite. The compressive strength of the HAP-GEL (107.7±26.8 MPa) was, however, less sensitive to microstructural changes and was within the range of natural cortical bone (human 170 MPa, pig: 100 MPa). The compression strength was dominated by void inclusions while the nanoindentation response reflected ultra-structural arrangement of the crystals. The gelatin concentration is likely to modify crystal arrangement as demonstrated in TEM experiments but not void distribution at macroscopic levels. © 2006 Materials Research Society.