Abstract-If musculoskeletal tissues are indeed efficient for their mechanical function, it is most reasonable to assume that this is achieved because the mechanical environment in the tissue influences cell differentiation and expression. Although mechanical stimuli can influence the transport of bio active factors, cell deformation and cytoskeletal strain, the question of whether or not they have the potential to regulate tissue differentiation sequences (for example, during fracture healing or embryogenesis) has not been answered.To assess the feasibility of biophysical stimuli as mediators of tissue differentiation, we analysed intcrfacial tissue formation adjacent to a micromotion device implanted into the condyles of dogs, A biphasic finite element model was used and the mechanical environment in the tissue was characterised in terms of (i) forces opposing implant motion, (ii) relative velocity between constituents, (iii) fluid pressure, (iv) deformation of the tissue and (v) strain in the tissue. It was predicted that, as tissue differentiation progressed, subtle but systematic mechanical changes occur on cells in the interfacial tissue. Specifically, as the forces opposing motion increase, the implant changes from being controlled by the maximum-allowable displacement (motion-control) to being controlled by the maximum-available load (force-control). This causes a decrease in the velocity of the fluid phase relative to the solid phase and a drop in interstitial fluid pressure accompanied by a reduction in peri-prosthetic tissue strains. The variation of biophysical stimuli within the tissue can be plotted as 'mechano-regulatory pathway', which identifies the transition from motion-control to force-control as a branching event in the tissue differentiation sequence. © 1997 Elsevier Science Ltd

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Mechanical forces are essential for normal adult bone function and repair, but the impact of prenatal muscle contractions on bone development remains to be explored in depth in mammalian model systems. In this study, we analyze skeletogenesis in two ‘muscleless’ mouse mutant models in which the formation of skeletal muscle development is disrupted; Myf5nlacZ/nlacZ:MyoD−/− and Pax3Sp/Sp (Splotch). Ossification centers were found to be differentially affected in the muscleless limbs, with significant decreases in bone formation in the scapula, humerus, ulna and femur, but not in the tibia. In the scapula and humerus, the morphologies of ossification centers were abnormal in muscleless limbs. Histology of the humerus revealed a decreased extent of the hypertrophic zone in mutant limbs but no change in the shape of this region. The elbow joint was also found to be clearly affected with a dramatic reduction in the joint line, while no abnormalities were evident in the knee. The humeral deltoid tuberosity was significantly reduced in size in the Myf5nlacZ/nlacZ:MyoD−/− mutants while a change in shape but not in size was found in the humeral tuberosities of the Pax3Sp/Sp mutants. We also examined skeletal development in a ‘reduced muscle’ model, the Myf5nlacZ/+:MyoD−/− mutant, in which skeletal muscle forms but with reduced muscle mass. The reduced muscle phenotype appeared to have an intermediate effect on skeletal development, with reduced bone formation in the scapula and humerus compared to controls, but not in other rudiments. In summary, we have demonstrated that skeletal development is differentially affected by the lack of skeletal muscle, with certain rudiments and joints being more severely affected than others. These findings indicate that the response of skeletal progenitor cells to biophysical stimuli may depend upon their location in the embryonic limb, implying a complex interaction between mechanical forces and location-specific regulatory factors affecting bone and joint development.

Mesenchymal stem cells (MSCs) are multipotent cells capable of developing along the chondrogenic, osteogenic and adipogenic lineages. As such, they have received interest as a potential cell source for tissue engineering strategies. Cartilage is an avascular tissue and thus resides in a microenvironment with reduced oxygen tension. The aim of this study was to examine the effect of a low oxygen environment on MSC differentiation along the chondrogenic route. In MSCs exposed to chondrogenic growth factors, transforming growth factor-beta and dexamethasone, in a hypoxic environment (2% oxygen), the induction of collagen II expression and proteoglygan deposition was significantly greater than that observed when cells were exposed to the chondrogenic growth factors under normoxic (20% oxygen) conditions. The transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is a crucial mediator of the cellular response to hypoxia. Following exposure of MSCs to hypoxia (2% oxygen), HIF-1alpha translocated from the cytosol to the nucleus and bound to its target DNA consensus sequence. Similarly, hypoxia evoked an increase in phosphorylation of both AKT and p38 mitogen activated protein kinase, upstream of HIF-1alpha activation. Furthermore, the PI3 kinase/AKT inhibitor, LY294002, and p38 inhibitor, SB 203580, prevented the hypoxia-mediated stabilisation of HIF-1alpha. To assess the role of HIF-1alpha in the hypoxia-induced increase in chondrogenesis, we employed an siRNA knockdown approach. In cells exposed to HIF-1alpha siRNA, the hypoxia-induced enhancement of chondrogenesis, as evidenced by upregulation of collagen II, sox-9 and proteoglycan deposition, was absent. This provides evidence for HIF-1alpha being a key mediator of the beneficial effect of a low oxygen environment on chondrogenesis.

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40%) while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures.

Adult mesenchymal stem cells have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. Collagen, a normal constituent of bone, provides strength and structural stability and is therefore a potential candidate for use as a substrate on which to engineer bone and cartilage from their respective mesenchymal-derived precursors. In this study, a collagen- glycosaminoglycan scaffold was used to provide a suitable three-dimensional (3-D) environment on which to culture adult rat mesenchymal stem cells and induce differentiation along the osteogenic and chondrogenic lineages. The results demonstrate that adult rat mesenchymal stem cells can undergo osteogenesis when grown on the collagen-glycosaminoglycan scaffold and stimulated with osteogenic factors (dexamethasone, ascorbic acid, beta-glycerophosphate), as evaluated by the temporal induction of the bone-specific proteins, collagen I and osteocalcin, and subsequent matrix mineralization. The osteogenic factors were coupled to activation of the extracellular-regulated protein kinase (ERK), and this kinase was found to play a role in the osteogenic process. As well as supporting osteogenesis, when the cell-seeded scaffold was exposed to chondrogenic factors (dexamethasone and TGF-1beta), collagen II immunoreactivity was increased, providing evidence that the scaffold can also provide a suitable 3-D environment that supports chondrogenesis.

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.