MECHBIO

EVALUATION AND OPTIMIZATION OF TISSUE ENGINEERING SCAFFOLDS USING COMPUTATIONAL FLUID DYNAMICS
Anderson EJ, Savrin, J, Cooke, M, Dean D, Knothe Tate ML, BMES 2005 ID: 143482  

The design and optimization of tissue engineering scaffolds is in its nascency; discrepancies between the proposed design and actual prototype geometries are an unfortunate artifact of the manufacturing process. This study aims to optimize scaffold geometry to achieve mechanobiologically advantageous permeabilities using computational and experimental techniques. Rapid prototyped, biodegradable scaffolds were manufactured and then imaged using microCT. Computational models were built based on optimal geometries (CAD) and actual manufactured geometries (microCT). Fluid flow and permeability were calculated through each scaffold (using Darcy's Law), as a function of average pore size. These calculations were validated experimentally. In both the theoretical and experimental calculations, a reduction in pore volume was found to have a dramatic affect on permeability. This approach constitutes a novel design tool to investigate the effect of scaffold geometry on intrinsic permeability. Furthermore, it provides a platform to predict and optimize porosity and permeability of scaffolds for specific geometries, thus reducing the number of experimental studies necessary to validate design performance.

MECHANOBIOLOGICAL INFLUENCES IN ENDOGENEOUS BONE TISSUE ENGINEERING
Kreuzer SK, Anderson EJ, Knothe U, Knothe Tate ML, BMES 2005 ID: 143482  

Orthopedic surgeons and tissue engineers face great challenges in regenerating large bone defects subject to cyclic loading. A novel modality for treatment of such defects has been developed utilizing the osteogenic potential of the periosteum and stabilization via intramedullary nailing. Elevation of the periosteal surface and subsequent translation of healthy osteotomized diaphyseal bone into the defect zone allows for the creation of an in situ periosteal tube with associated regenerative and vascular capacity. Additionally, bone graft and the presence of adherent, vascularized bone chips to the periosteal sleeve allow for variation in treatment and the possibility for additional effectiveness. High resolution radiographs and high resolution micro-CT images demonstrate the efficacy of the treatment for regenerating bone in an ovine femur model. Retention of the periosteal sleeve resulted in a significant increase in regenerate volume in all cases throughout the course of the 16 week treatment when compared to treatment without periosteum retention. We have developed a computational finite element of this treatment modality to elucidate which variables exert the greatest influence on tissue regeneration in situ. Predictions of our model are validated based on histological measures, providing insight into the biological mechanisms underlying this novel treatment. Together these analyses promise important implications for both tissue engineering and clinical treatment of bone defects.

IN SITU ASSESSMENT OF OSTEOCYTE VIABILITY & CYTOSKELETAL INTEGRITY IN NATIVE BONE TISSUE AND OSMOTIC STRESS EFFECTS ON THE SAME
Sorkin A , Knothe Tate ML. Trans ORS Annual Meeting, pg 1645, 2005

Osteocytes are the putative mechanosensors in bone; despite their implicit role in bone physiology, in situ study of osteocytes has been limited due to their remote location within the mineralized bone matrix. Nonetheless, native bone remains the ideal environment in which to study the function of osteocytes. Histological methods allow one to study osteocytes in their native milieu, but they do not allow cells to be observed while they remain viable and active. In vivo observation techniques are in development but are currently too technically demanding and expensive to be implemented as a standard laboratory technique. Hence, the purpose of this study was twofold, to adapt and implement, for the first time to our knowledge, two cytochemical methods developed for in vitro cell culture to study osteocyte activity in situ in semi-thin sections of native bone, (ii) to determine what percentage of osteocytes remain viable over time in bone specimens obtained from a recently sacrificed animal, and (iii) to assess the response of osteocytes to osmotic stress while in their native milieu.

DETERMING THE PERMEABILITY OF CORTICAL BONE AT MULTIPLE LENGTH SCALES USING FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING TECHNIQUES
Patel RB, O’Leary, JM, Bhatt SJ; Vasanja, A, Knothe Tate, ML. Trans ORS Annual Meeting, pg 0140, 2005

Orthopaedic surgeons, endocrinologists, tissue engineers and pharmaceutical developers are directing increased attention to decipher the permeability and molecular transport characteristics of bone tissue. Osteocytes, embedded within the mineralized bone matrix, can be at distances up to 250 μm away from the nearest blood supply, which necessitates molecular transport through the porous network of cortical
bone. This transport is not only vital for cell survival but also for cellular signaling and drug delivery between the blood supply and cells. Bone’s hierarchically organized transport network includes haversian capillaries (50-70 μm in diameter), canalicular channels (0.10-1 μm in diameter), pericellular, proteoglycan meshwork-filled fluid spaces (15-50 nm), and matrix microporosity (5-12 nm) through which molecules pass as they are transported between the blood supply and osteocytes [1,2]. It is the nanoporous structure of cortical bone that confers size exclusion properties to the tissue, restricting free diffusion of bioactive molecules and drugs between cells and to/from the blood supply. The ultimate goal of this study was to define the upper permeability limit of large molecular weight molecules in cortical bone. Fluorescence Recovery After Photobleaching (FRAP) was applied to measure diffusive permeability of 300-70,000 Dalton (Da) molecular weight fluorescent probes in fresh, bovine cortical bone. The specific aims of this study
were threefold, (i) to measure the currently unknown diffusion constants of various size macromolecules through the lacunocanalicular network, (ii) to compare differences in permeability in the transverse and longitudinal planes in cortical bone, and (iii) to compare permeability across length scales including the matrix microporosity, cellular syncytium, and “bulk tissue” permeability.

LACUNOCANALICULAR PERMEABILITY MEASUREMENTS IN HEALTHY & OSTEOPOROTIC PATIENTS: AN EXPERIMENTAL FLUID MECHANICS APPROACH USING SCALED PHYSICAL MODELS
Anderson EJ, Knothe Tate ML. Trans ORS Annual Meeting, pg 1128, 2005
Osteocytes are organized in a functional syncytium that provides a biological network for transport and communication across bone tissue. In healthy bone, the cellular syncytium connects cells deep within bone tissue to cells on bone surfaces and near the blood supply. With the onset and progression of osteoporosis, cellular connectivity decreases and efficiency of signal transmission is expected to decrease as well. Interstitial fluid flow within this lacunocanalicular network is postulated to play a key role in mechano-transduction, however, flow regimes within this space are poorly understood due to their remote location and nano-microscale dimensions. This gave us impetus to implement the scaling approach used in fluid mechanics to elucidate flow regimes in very large or small scale systems.

APPLICATION OF STOCHASTIC NETWORK MODELS FOR THE STUDY OF MOLECULAR TRANSPORT PROCESSES IN BONE
Steck R, Knothe Tate ML. Proceedings of 2004 ASME International Mechanical Engineering Congress and Exposition November 2004, Anaheim, California USA, IMECE2004-59746

In order to simulate molecular transport along the different pathways in healthy and diseased bone, we are developing stochastic network models. This method has been previously used in chemical engineering for the simulation and optimization of column chromatography [3]. It is based on the percolation theory and is ideal for the computational simulation of flows and transport within hierarchial, porous networks and allows for taking different transport phenomena into account. In the case of bone, these may include diffusive or convective transport, sources and sinks, as well as cell initiated active transport mechanisms. We have used this method successfully to demonstrate the influence of age related osteocyte density decrease on the bone tissue permeability [4], as well as for a simulation of the molecular sieving characteristics of bone [5]. In the current study, we examined the influence of bone diseases, such as osteoporosis and osteomalacia, on the transport capacity of bone tissue. There is evidence that these diseases affect the state of the osteocyte syncytium, which can be described using parameters such as osteocyte density, connectivity and canalicular tortuosity. While the diseaserelated changes to the lacunocanalicular network have only been described semi-quantitatively (a quantitative study including age matched samples from a big number of donors is currently under way), the aim of this parametric study was to identify the parameters with the biggest influence on the transport of various molecules to and between the osteocytes...[Read more]

MODELING THE EFFECTS OF INTERSTITIAL FLUID FLOW ON A SINGLE OSTEOCYTE AND ITS PROCESSES
Anderson EJ, Kalymoorthy S, Knothe Tate ML. Proceedings of 2004 ASME International Mechanical Engineering Congress and Exposition November, 2004, Anaheim, California USA IMECE2004-61861

In order to understand the manner in which local changes in mechanical environment are translated into cellular activity underlying tissue level bone adaptation, there is a need to explore fluid flow regimes at small scales such as the osteocyte Recent developments provide impetus to model periosteocytic flow using computational fluid dynamics. In building this model, the local effects of fluid flow on the osteocyte cell body and its processes were analyzed. For each model, fluid flow was induced via a pressure gradient, and the CFD calculated, based on the Navier-Stokes equations, the shear stress at the cell-fluid interface and radial stress, acting normal to the cell surface. Based on the model, the osteocyte cell body is exposed primarily to effects of hydrodynamic pressure and the cell processes are exposed primarily to shear and radial stress, with highest stress gradients at sites where the process and the cell body intersect and where two cell processes join at the gap junction. Hence, this model simulates subcellular effects of fluid flow and suggests, for the first time to our knowledge, major differences in modes of loading between...[Read more]

PERFORMANCE EVALUATION OF FOUR CELL FLOW CHAMBERS: HOW WELL IS STRESS CONTROLLED AT A CELLULAR LEVEL?
Anderson EJ, Sorkin AM, Knothe Tate ML. Proceedings of 2004 ASME International Mechanical Engineering Congress and Exposition November 2004, Anaheim, California USA IMECE2004-61837

Fluid structure interactions at the cellular level are poorly understood yet they appear to be universal across tissue types and may hold the key to unraveling mechanisms of mechanotransduction at a cellular and subcellular level. Due to practical difficulties in studying cells in situ during normal physiologic activity, cell perfusion chambers have been developed to simulate physiologic fluid flow in vitro. While this approach has obvious advantages for unraveling cell signaling pathways in mechanotransduction, little is known with regard to how well these in vitro flow profiles emulate actual physiologic flow. The purpose of this computational study was to compare the local stress imparted through fluid flow in four cell perfusion chambers. From the computational models, in each chamber, varying velocity components cause the local shear stress imparted to the cells to vary as a function of location, and in fact only a limited number of cells are exposed to target stress. Due to differences in flow regimes between the four chambers, comparison between experimental data obtained using different perfusion chambers may be inappropriate. [Read more]

The underlying mechanisms of mechanochemical transduction and cellular signaling that modulate processes of growth, repair and adaption of musculoskeletal tissues, in general, and ligament, in particular, are poorly understood. Interstitial fluid flow has been shown to have profound effects on cell activity by direct transmission of shear stresses via fluid flow over cell membranes as well as concentration gradients that are modulated by convective transport processes. The goal of this study was to assess permeability of the ligament matrix to globular protein tracers of different molecular weight in vivo and to document effects of loading on interstitial transport within the ligament. The interosseous ligament of the rat forelimb was chosen as a model system. Cyclic tensile loading of the ligament was achieved via axial end loading of the ulna. The contralateral limb served as an unloaded control. Fluorescent albumins of different molecular weights were injected into the lateral tail vein of the rat before loading commenced. Without loading molecules were poorly transported through the ligament matrix. Load induced convective fluid flow augmented transport of the large macromolecules. In sum, results of the study establish the range of ligament matrix permeability and effects of load induced fluid flow on molecular transport, which further advances our understanding of ligament physiology and chemotransport in musculoskeletal tissues.