Dynamic mechanical stimulation for bone tissue engineering.

Mechanical loading is an important regulatory factor in bone homeostasis, and plays an essential role in maintaining the structure and mass of bone throughout a lifetime. Although the exact mechanism is unknown the data presented in this thesis supports the concept that substrate signals influence MSC growth and differentiation. A better understanding of the cellular and molecular responses of bone cells to mechanical stimuli is the key to further improvements to therapeutic approaches in orthopaedics, orthodontics, periodontics, bone repair, bone regeneration, implantology and tissue engineering. However, the mechanisms by which cells transduce mechanical signals are poorly understood. There has also been an increased awareness of the need for improvement and development of 3-D in vitro models of mechanotransduction to mimic the 3-D environment, as found in intact bone tissue and to validate 2-D in vitro results. The aims of the project were (i) to optimize a model system by which bone cells can survive in 3-D static culture and their responses to mechanical stimuli can be examined in vitro, (ii) to test the effects of intermittent mechanical compressive loading on cell growth, matrix maturation and mineralization by osteoblastic cells, (iii) to examine the role of the primary cilia, (iv) to assess the effect of dynamic compressive loading on human mesenchymal stem cells in the 3-D environment. The optimized model system has the potential to be used in in vitro studies of bone in 3-D environments including a better understanding of the mechanically controlled tissue differentiation process and matrix maturation in the engineered bone constructs. It has less complicated equipment and techniques compared to dynamic seeding and culture systems making it easy to use in the laboratory. In addition, cells are not pre stimulated by any mechanical stimuli during seeding and culture which enables the researcher to study selected mechanical stimuli and mechanotransduction in bone tissue constructs. The model can mimic the bone environment providing a better physiological model than cells cultured in 2-D monolayer. Using our 3-D system, several loading regimens were compared and it was shown that intermittent short periods of compressive loading can improve cell growth and/or matrix production by MLO-A5 osteoblastic cells during 3-D static culture. This VI suggests that the cells are responding to the mechanical compression stimulus either by directly sensing the substrate strain or the fluid shear stress induced by flow through the porous scaffold. We also demonstrated that our mechanical loading system has the potential to induce osteogenic differentiation and bone matrix production by human MSCs in the same way as treatment with dexamethasone. Although the exact mechanism is unknown the data presented supports the concept that the dynamic compressive loading influence MSC growth, differentiation and production. In further experiments, we used the optimized 3-D model system to study the effects of mechanical loading on primary cilia, which have recently been shown to be potential mechanosensors in bone. We demonstrated that mature cells lacking a cilium were less responsive, less able to upregulate matrix protein gene expression and did not increase matrix production in response to mechanical stimulation suggesting that the primary cilia are sensors for mechanical forces such as fluid flow and/or strain induced shear stress

Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts