Mechanical and physical guidance of osteogenic differentiation and matrix production

Summary Tissue engineering and regenerative medicine strategies until now have mostly relied on static culture using chemical stimulation to induce cell differentiation. However, these strategies neglect the dynamic environment in which cells reside in the body where they are surrounded by a chemically and physically well-defined threedimensional (3D) topography. Not only does this environment control cellular differentiation, but its structure also determines the mechanical function of that tissue. Alongside physical cues, external mechanical forces play an essential role in the homeostasis of many tissues, particularly bone. In order to develop tissue engineered constructs that are suitable for implantation, it may be important to incorporate these essential cues into pre-culture methods and in order to do this, a better understanding of the cellular responses is required. The main aim of this research was to understand how physical and mechanical cues affect cell behaviour, differentiation and matrix production, with particular emphasis on osteogenesis and collagen organisation. In order to achieve this, electrospun scaffolds were fabricated with controllable fibre orientation for studies involving fibroblast matrix organisation, and the affect on the differentiation of osteoprogenitor cells. Short bouts of tensile loading were conducted using a previously established bioreactor model for conditioning collagen-producing cells. A simple rocking platform method for subjecting cells to fluid-flow was also investigated for its potential to enhance osteogenesis and collagen organisation. This system was further used to study the role of the primary cilium for the mechanotransduction of bone cells. The overall goal was to understand how to manipulate cell differentiation and matrix production in order to develop a more suitable construct with correct tissue structure in a rapid manner. Monitoring of the major structural matrix protein collagen was achieved using the minimally-invasive technique of second harmonic generation, which was optimised. Electrospun scaffolds with a random architecture caused cells to deposit matrix in a similar random manner, however highly aligned scaffolds caused deposited collagen to orientate in the fibre direction giving superior tensile properties. Further to this, random fibres appeared to be more favourable for the differentiation of osteoprogenitor cells than highly aligned substrates. 9 Short bouts of tensile stimulation of collagen producing cells on 3D substrates caused an increase in collagen deposition. Another stimulation method, a simple rocking platform, created oscillatory fluid shear stress (FSS) suitable for stimulation of osteogenic cells and enhanced collagen organisation. Further to this, human dermal fibroblasts could be induced to form a mineralised matrix when cultured in osteogenic media, which was further enhanced with FSS. It was also demonstrated that this simple rocking system could be used to test a wide variety of loading parameters. Finally, rocking was used to examine the role of the primary cilium in the load-induced mineral deposition response of bone cells. When mature bone cells were subjected to FSS, primary cilia shortened in length and removal of primary cilia resulted in loss of the load-induced matrix response suggesting that primary cilia are mechanosensors in bone cells.