The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells

It is becoming increasingly clear that mesenchymal stem cell (MSC) differentiation is regulated by mechanical signals. Mechanical forces generated intrinsically within the cell in response to its extracellular environment, and extrinsic mechanical signals imposed upon the cell by the extracellular environment, play a central role in determining MSC fate. This article reviews chondrogenesis and osteogenesis during skeletogenesis, and then considers the role of mechanics in regulating limb development and regenerative events such as fracture repair. However, observing skeletal changes under altered loading conditions can only partially explain the role of mechanics in controlling MSC differentiation. Increasingly, understanding how epigenetic factors, such as the mechanical environment, regulate stem cell fate is undertaken using tightly controlled in vitro models. Factors such as bioengineered surfaces, substrates, and bioreactor systems are used to control the mechanical forces imposed upon, and generated within, MSCs. From these studies, a clearer picture of how osteogenesis and chondrogenesis of MSCs is regulated by mechanical signals is beginning to emerge. Understanding the response of MSCs to such regulatory factors is a key step towards understanding their role in development, disease and regeneration. Birth Defects Research (Part C) 90:75–85, 2010. © 2010 Wiley‐Liss, Inc.     

Sympathetic nervous system and bone adaptive response to its mechanical environment

While bone adaptive response to its mechanical environment was considered to be controlled locally by cytokines and systemic hormones, some recent work suggests that it could also be neuronally regulated. Bone is indeed very densely innervated and many experimental and clinical studies have previously shown the involvement of the nervous system in the control of bone metabolism. The demonstration that the central nervous system regulates bone mass via the sympathetic nervous system (SNS) has prompted recent studies aimed to investigate the role of the SNS in the bone mechano-adaptive response. This review will focus on this work and summarize the evidence for a contribution of the beta-adrenergic signalling in the response of bone cells to mechanical loading. The apparent conflicting results obtained in diverse experimental models of loading and unloading, at different skeletal sites, and in relation to various hormonal levels, will be discussed. While those studies do not support a major influence of the SNS on the bone mechano-adaptive response, there is nevertheless strong evidence that the SNS is part of a complex system which contributes to the metabolic regulation of bone.     

Understanding site-specific residual strain and architecture in bovine cortical bone

Living bone is considered as adaptive material to the mechanical functions, which continually undergoes change in its histological arrangement with respect to external prolonged loading. Such remodeling phenomena within bone depend on the degree of stimuli caused by the mechanical loading being experienced, and therefore, are specific to the sites. In the attempts of understanding strain adaptive phenomena within bones, different theoretical models have been proposed. Also, the existing literatures mostly follow the measurement of surface strains using strain gauges to experimentally quantify the strains experienced in the functional environment. In this work, we propose a novel idea of understanding site-specific functional adaptation to the prolonged load in bone on the basis of inherited residual strains and structural organization. We quantified the residual strains and amount of apatite crystals distribution, i.e., the degree of orientation, using X-ray diffraction procedures. The sites of naturally existing hole in bone, called foramen, are considered from bovine femur and metacarpal samples. Significant values of residual strains are found to exist in the specimens. Trends of residual strains noted in the specimens are mostly consistent with the degree of orientation of the crystallites. These features explain the response behavior of bone to the mechanical loading history near the foramen sites. Preferential orientation of crystals mapped around a femoral foramen specimen showed furnished tailored arrangement of the crystals around the hole. Effect of external loading at the femoral foramen site is also explained by the tensile loading experiment.     

Toward an identification of mechanical parameters initiating periosteal remodeling: a combined experimental and analytic approach

The ability of bone to adapt to its mechanical environment is well recognized, although the specific mechanical parameters initiating or maintaining the adaptive responses have yet to be identified. Recently introduced mathematical models offer the potential to aid in the identification of such parameters, although these models have not been well validated experimentally or clinically. We formulated a complementary experimental/analytic approach, using an animal model with a well-controlled mechanical environment combined with finite element modeling (FEM). We selected the functionally isolated turkey ulna, since the loading could be completely characterized and the periosteal adaptive responses subsequently monitored and quantified after four and eight weeks of loading. Known loads input into a three-dimensional, linearly elastic FEM of the ulna then permitted full-field mechanical characterization of the ulna. The FEM was validated against a normal strain-gaged turkey ulna, loaded in vivo in an identical fashion to the experimental ulnae. Twenty-four candidate mechanical parameters were then compared to the quantified adaptive responses, using statistical techniques. The data supported strain energy density, longitudinal shear stress, and tensile principal stress/strain as the mechanical parameters most likely related to the initiation of the remodeling response. Model predictions can now suggest new experiments, against which the predictions can be supported or falsified.     

Wolff’s law in action: a mechanism for early knee osteoarthritis

There is growing interest in the role of bone in knee osteoarthritis. Bone is a dynamic organ, tightly regulated by a multitude of homeostatic controls, including genetic and environmental factors. One such key environmental regulator of periarticular bone is mechanical stimulation, which, according to Wolff’s law, is a key determinant of bone properties. Wolff’s law theorizes that repetitive loading of bone will cause adaptive responses enabling the bone to better cope with these loads. Despite being an adaptive response of bone, the remodeling process may inadvertently trigger maladaptive responses in other articular structures. Accumulating evidence at the knee suggests that expanding articular bone surface area is driven by mechanical stimulation and is a strong predictor of articular cartilage loss. Similarly, fractal analysis of bone architecture provides further clues that bone adaptation may have untoward consequences for joint health. This review hypothesizes that adaptations of periarticular bone in response to mechanical stimulation cause maladaptive responses in other articular structures that mediate the development of knee osteoarthritis. A potential disease paradigm to account for such a hypothesis is also proposed, and novel therapeutic targets that may have a bone-modifying effect, and therefore potentially a disease-modifying effect, are also explored.     

Emerging evidence that adaptive bone formation inhibition by non-steroidal anti-inflammatory drugs increases stress fracture risk

There is mounting evidence suggesting that the commonly used analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), may inhibit new bone formation with physical training and increase risk of stress fractures in physically active populations. Stress fractures are thought to occur when bones are subjected to repetitive mechanical loading, which can lead to a cycle of tissue microdamage, repair, and continued mechanical loading until fracture. Adaptive bone formation, particularly on the periosteal surface of long bones, is a concurrent adaptive response of bone to heightened mechanical loading that can improve the fatigue resistance of the skeletal structure, and therefore may play a critical role in offsetting the risk of stress fracture. Reports from animal studies suggest that NSAID administration may suppress this important adaptive response to mechanical loading. These observations have implications for populations such as endurance athletes and military recruits who are at risk of stress fracture and whose use of NSAIDs is widespread. However, results from human trials evaluating exercise and bone adaptation with NSAID consumption have been less conclusive. In this review, we identify knowledge gaps that must be addressed to further support NSAID-related guidelines intended for at-risk populations and individuals.     

Influence of cortical canal architecture on lacunocanalicular pore pressure and fluid flow

Bone is a dynamic tissue that undergoes structural modification in response to its mechanical environment, but how bone cells sense and respond to loading conditions remains incompletely understood. Current theories focus on strain-induced fluid flow for the primary means of mechanotransduction. To examine the influence of age-related cortical rarefaction on lacunocanalicular fluid characteristics, coupled fluid flow and mechanical computational models of bone specimens representing young, mid-age and aged samples were derived artificially from the same original micro-computed tomography image data. Simulated mechanical loading was applied to the bone models to induce pressure-driven interstitial fluid flow. Results demonstrated a decrease in pore pressure and fluid velocity magnitudes with age as a result of increased cortical porosity. Mean canal separation, as opposed to canal size, was implicated as a primary factor affecting age-related fluid dynamics. Future investigations through refinement of the model may implicate fluid stasis or inadequate nutrient transport experienced by osteocytes as a key factor in the initiation of cortical remodelling events.     

Influence of cortical canal architecture on lacunocanalicular pore pressure and fluid flow

Bone is a dynamic tissue that undergoes structural modification in response to its mechanical environment, but how bone cells sense and respond to loading conditions remains incompletely understood. Current theories focus on strain-induced fluid flow for the primary means of mechanotransduction. To examine the influence of age-related cortical rarefaction on lacunocanalicular fluid characteristics, coupled fluid flow and mechanical computational models of bone specimens representing young, mid-age and aged samples were derived artificially from the same original micro-computed tomography image data. Simulated mechanical loading was applied to the bone models to induce pressure-driven interstitial fluid flow. Results demonstrated a decrease in pore pressure and fluid velocity magnitudes with age as a result of increased cortical porosity. Mean canal separation, as opposed to canal size, was implicated as a primary factor affecting age-related fluid dynamics. Future investigations through refinement of the model may implicate fluid stasis or inadequate nutrient transport experienced by osteocytes as a key factor in the initiation of cortical remodelling events.     

An integrated proteomics analysis of bone tissues in response to mechanical stimulation

Bone cells can sense physical forces and convert mechanical stimulation conditions into biochemical signals that lead to expression of mechanically sensitive genes and proteins. However, it is still poorly understood how genes and proteins in bone cells are orchestrated to respond to mechanical stimulations. In this research, we applied integrated proteomics, statistical, and network biology techniques to study proteome-level changes to bone tissue cells in response to two different conditions, normal loading and fatigue loading. We harvested ulna midshafts and isolated proteins from the control, loaded, and fatigue loaded Rats. Using a label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) experimental proteomics technique, we derived a comprehensive list of 1,058 proteins that are differentially expressed among normal loading, fatigue loading, and controls. By carefully developing protein selection filters and statistical models, we were able to identify 42 proteins representing 21 Rat genes that were significantly associated with bone cells' response to quantitative changes between normal loading and fatigue loading conditions. We further applied network biology techniques by building a fatigue loading activated protein-protein interaction subnetwork involving 9 of the human-homolog counterpart of the 21 rat genes in a large connected network component. Our study shows that the combination of decreased anti-apoptotic factor, Raf1, and increased pro-apoptotic factor, PDCD8, results in significant increase in the number of apoptotic osteocytes following fatigue loading. We believe controlling osteoblast differentiation/proliferation and osteocyte apoptosis could be promising directions for developing future therapeutic solutions for related bone diseases.     

Candidates for the mechanosensory system in bone

Some potential mechanisms by which bone cells sense mechanical loads are described and hypotheses concerning the functioning of these mechanisms are explored. It is well known that bone tissue adapts its structure to its mechanical load environment. Recent research has illuminated the biological response of bone to mechanical loading at the cellular level, but the precise mechanosensory system that signals bone cells to deposit or resorb tissue has not been identified. The purpose of this paper is to describe the current status of this research and to suggest some possible mechanosensory systems by which bone cells might sense environmental loads.     

Directional dependence of osteoblastic calcium response to mechanical stimuli

In adaptive bone remodeling, mechanical signals such as stress/strain caused by loading/deformation are believed to play important roles as regulators of the process in which osteoclastic resorption and osteoblastic formation are coordinated under a local mechanical environment. The mechanism by which cells sense and transduce mechanical signals to the intracellular biochemical signaling cascade is still unclear, however to address this issue, the present study investigated the characteristic response of a single osteoblastic cell, MC3T3-E1, to a well-defined mechanical stimulus and the involvement of the cytoskeletal actin fiber structure in the mechanotransduction pathway. First, by mechanically perturbing to a single cell using a microneedle, a change in the intracellular calcium ion concentration [Ca²⁺]_i was observed as a primal signaling response to a mechanical stimulus, and the threshold value of the perturbation as the mechanical stimulus was evaluated quantitatively. Second, to study directional dependence of the response to the mechanical stimulus, the effect of actin fiber orientation on the threshold value of the calcium response was investigated at various magnitudes and directions of the stimulus. It was found that the osteoblastic response to the perturbation exhibited a directional dependence. That is, the sensitivity of osteoblastic cells to a mechanical stimulus depends on the angle of the applied deformation with respect to the cytoskeletal actin fiber orientation. This finding is phenomenological evidence that cytoskeletal actin fiber structures are involved in the mechanotransduction mechanism, which may be related to cell polarization behaviors such as cellular alignment caused by mechanical stimulation.     

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.     

The effects of total hip arthroplasty on the structural and biomechanical properties of adult bone

The responsiveness of bone to mechanical stimuli changes throughout life, with adaptive potential generally declining after skeletal maturity is reached. This has led some to question the importance of bone functional adaptation in the determination of the structural and material properties of the adult skeleton. A better understanding of age-specific differences in bone response to mechanical loads is essential to interpretations of long bone adaptation. The purpose of this study is to examine how the altered mechanical loading environment and cortical bone loss associated with total hip arthroplasty affects the structural and biomechanical properties of adult bone at the mid-shaft femur. Femoral cross sections from seven individuals who had undergone unilateral total hip arthroplasty were analyzed, with intact, contralateral femora serving as an approximate internal control. A comparative sample of individuals without hip prostheses was also included in the analysis. Results showed a decrease in cortical area in femora with prostheses, primarily through bone loss at the endosteal envelope; however, an increase in total cross-sectional area and maintenance of the parameters of bone strength, I(x), I(y), and J, were observed. No detectable differences were found between femora of individuals without prostheses. We interpret these findings as an adaptive response to increased strains caused by loading a bone previously diminished in mass due to insertion of femoral prosthesis. These results suggest that bone accrued through periosteal apposition may serve as an important means by which adult bone can functional adapt to changes in mechanical loading despite limitations associated with senescence.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Activity intensity,assistive devices and joint replacement influence predicted remodelling in the proximal femur

Bone morphology and density changes are commonly observed following joint replacement, may contribute to the risks of implant loosening and periprosthetic fracture and reduce the available bone stock for revision surgery. This study was presented in the ‘Bone and Cartilage Mechanobiology across the scales’ WCCM symposium to review the development of remodelling prediction methods and to demonstrate simulation of adaptive bone remodelling around hip replacement femoral components, incorporating intrinsic (prosthesis) and extrinsic (activity and loading) factors. An iterative bone remodelling process was applied to finite element models of a femur implanted with a cementless total hip replacement (THR) and a hip resurfacing implant. Previously developed for a cemented THR implant, this modified process enabled the influence of pre- to post-operative changes in patient activity and joint loading to be evaluated. A control algorithm used identical pre- and post-operative conditions, and the predicted extents and temporal trends of remodelling were measured by generating virtual X-rays and DXA scans. The modified process improved qualitative and quantitative remodelling predictions for both the cementless THR and resurfacing implants, but demonstrated the sensitivity to DXA scan region definition and appropriate implant–bone position and sizing. Predicted remodelling in the intact femur in response to changed activity and loading demonstrated that in this simplified model, although the influence of the extrinsic effects were important, the mechanics of implantation were dominant. This study supports the application of predictive bone remodelling as one element in the range of physical and computational studies, which should be conducted in the preclinical evaluation of new prostheses.     

Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone

Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading.     

Functional strain in bone tissue as an objective, and controlling stimulus for adaptive bone remodelling

The skeleton consists of a series of elements with a variety of functions. In locations where shape or protection are of prime importance the bone's architecture is achieved during growth under direct genetic control. In locations where resistance to repetitive loading is important only the general form of the bone will be achieved as a result of growth alone, the remaining characteristics result from functional adaptation. This mechanism ensures that bone architecture is modelled and remodelled until prevailing strains match those genetically prescribed for that location. For this match to be established, and subsequently maintained, bone cells must be able to 'assess' feedback derived directly or indirectly from the functional strains produced within the tissue. These strains are therefore the objective of functionally adaptive remodelling, and the stimulus for its control. Evans was the first person to refer to the recording of functional strains from gauges attached to bone in vivo. This technique has allowed quantitative investigations on bone's normal functional strain environment, and its adaptive response to changes in its state of strain. Recent investigations have extended to the immediate effects of dynamic strains on the structure of the bone matrix, and the biochemical behaviour of the resident bone cells. Such studies should reveal the mechanism by which strains within the matrix are transduced into the biochemical signals by which adaptive remodelling is controlled.     

Computational model for the cell-mechanical response of the osteocyte cytoskeleton based on self-stabilizing tensegrity structures

The mechanism by which mechanical stimulation on osteocytes results in biochemical signals that initiate the remodeling process inside living bone tissue is largely unknown. Even the type of stimulation acting on these cells is not yet clearly identified. However, the cytoskeleton of osteocytes is suggested to play a major role in the mechanosensory process due to the direct connection to the nucleus. In this paper, a computational approach to model and simulate the cell structure of osteocytes based on self-stabilizing tensegrity structures is suggested. The computational model of the cell consists of the major components with respect to mechanical aspects: the integrins that connect the cell with the extracellular bone matrix, and different types of protein fibers (microtubules and intermediate filaments) that form the cytoskeleton, the membrane-cytoskeleton (microfilaments), the nucleus and the centrosome. The proposed geometrical cell models represent the cell in its physiological environment which is necessary in order to give a statement on the cell behavior in vivo. Studies on the mechanical response of osteocytes after physiological loading and in particular the mechanical response of the nucleus show that the load acting on the nucleus is rising with increasing deformation applied to the integrins.     

Trabecular bone response to mechanical loading in ovariectomized Sprague-Dawley rats depends on baseline bone quantity

Mechanical loading is one of the determining factors for bone modulation, and is therefore frequently used to treat or prevent bone loss; however, there appears to be no data on the effects of baseline bone quantity on this response. This study aimed to verify whether baseline bone quantity affects osteoporotic trabecular bone adaptive response to mechanical stimulation. Twenty-four female Sprague-Dawley (SD) rats were ovariectomized (OVX). After 3 weeks of OVX, rats were divided into a high bone quantity and a low bone quantity group, and rats in each group were then subdivided into 4 groups that were exposed to different loading strategies. In the loading groups, tibiae were stimulated through axial loading at 2000με of strain, for 1500 cycles each of 75s, 150s, or 250s. The sham treatment groups received no loading. Changes in BV/TV for trabecular bone in the tibia were measured at the baseline (before loading), and at 3 weeks and 6 weeks after loading. BV/TVs in loading groups of the low baseline bone quantity group were significantly increased at 6 weeks, compared with those in the no-loading groups (p<0.05), while those in the high quantity groups were not increased (p>0.05). A significant negative correlation was observed between baseline BV/TV and its relative variations at 3 weeks or 6 weeks (p<0.05). These results indicate that adaptive responses of osteoporotic trabecular bone to mechanical loading depend on baseline bone quantity.