The drastic rise in the worldÂ’s population coupled to an ever increasing aging population poses a considerable challenge to the orthopaedic community to maintain healthy activity levels. The field of Tissue Engineering and Regenerative Medicine aims to tackle these challenges by implementing more biomimetic strategies to improve upon current treatments. The success of new therapeutic developments in musculoskeletal tissue engineering relies on our ability to study and understand the complex biological interactions between cells, materials, and native tissues so that we may subsequently guide neotissue formation. This thesis is focused on the development of novel, welldefined, and reproducible in-vitro tissue culture models to explore, characterise, and control cellular behaviour and differentiation for osteochondral regeneration. In particular, these models utilised combinations of polymeric biomaterials, differentiated osteoblasts, human periosteal stem cells, and physico-chemical cell signalling cues. In a commercial venture with PolyNovo Ltd (Melbourne, Australia), a novel two-component injectable polymer platform was synthesized and evaluated for uses as a biomaterial construct in orthopaedic applications. The second aspect of this thesis focuses on the harvest, isolation, expansion, and extensive characterisation of human periosteal cells in-vitro. The periosteum is a bi-layered membrane that covers the outside of cortical bone and has been recently identified as a potential stem cell source; with the ability to form osteogenic, chondrogenic, adipogenic, and myogenic tissue types. To detail the heterogeneous cellular features and behaviours of human periosteal cells in-vitro, cells were isolated from surgical explants, expanding in monolayer in the absence of differentiation supplements, and characterised for changes in morphology, growth rate, cell-cycle, gene expression, and phenotype. Additionally, enrichment techniques were designed to preferentially isolate distinct progenitor cell types identified in periosteal cell cultures. Most interestingly, a novel cell-sorting platform utilising droplet microfluidic approaches, was developed and evaluated for its ability to identify and separate periosteal progenitor cells. In the third part of this thesis, a 3-dimensional agarose culture model was created to control and monitor lineage specific human periosteal cell differentiation in various biomechanical and biochemical environments. The work presented herein further demonstrates the potential of human periosteal cells for osteochondral repair and more importantly provides critical information regarding human periosteal cell expansion, phenotype, and differentiation.