The aim of this study was to synthesize functional in vitro and in vivo 3-dimensional (3D) constructs using a mix of human mesenchymal populations and articular chondrocytes encapsulated in biomineralized polysaccharide templates. Single-cell-type populations or mixtures of both cell types were encapsulated in alginate/chitosan and cultured within a rotating-bioreactor, perfused bioreactor system, or static conditions for 28 days. Within single cell-type populations, type II collagen immunopositive cells were present within lacunae in rotating-bioreactor capsules, with an increased proportion of metabolically active cells compared with perfused and static constructs. Biochemical analysis indicated significantly increased ( p < 0.05) DNA and protein in rotating-bioreactor conditions compared with perfused or static. However, in coculture samples, DNA and protein was significantly increased in static cultures owing to the formation of large regions of partially mineralized osteoid. This osteoid was found only in static cultures and when the ratio of human bone marrow cells to chondrocytes was 2:1 or, to a lesser extent, 5:1 ratio capsules. Subcutaneous implantation of capsules into immunocompromised mice also showed optimal osteoid formation when the ratio was 2:1. The current studies demonstrate the pivotal role of robust 3D biomimetic microenvironments and indicate the potential to harness the interactions between different cell types to create specific tissues.
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