Development of channelled hydrogel structures to engineer complex tissues

摘要

Introduction: Mesenchymal stem cells (MSC's) have shown great promise as tools for tissue engineering due to their multipotency. A popular culture method involves the use of 3D scaffolds to mimic mechanical properties of extracellular matrix in vitro. Difficulties in directing multiple tissue development from a population of MSC's within a single structure still persist. In this study, we have developed a method of incorporating multiple channels within a hydrogel structure to deliver osteogenic and chondrogenic differentiation cues to specified areas of a hydrogel to promote region specific differentiation of a MSC's population within a single structure. Materials and Methods: Rat bone marrow stromal stem cells (rBMSC's) were mixed with a 0.5% w/w agarose solution at a cell density of 5×105 cells/ml (passage 4). The cell loaded agarose was then cast into a gel over rods that were removed post-gelation to create a cell-loaded scaffold that contained two channels approximately 0.5 mm in diameter. The cells were then cultured for 6 weeks in supplemented aMEM added to the surface of the gel, with fresh media added every 3 days. Following an initial 24 h culture the channels were used to deliver differentiation supplements. Chondrogenic media was injected into one channel and osteogenic media was injected into another. After 24 hours exposed to the differentiation media, supplemented aMEM was injected into the channels and cultured for a further 24 h before once again injecting the differentiation media into the channels. This process was repeated for 6 weeks before cultures were fixed and stained with alizarin red and alcian blue. Results and Discussion: Mistological analysis reveals evidence of matrix depositions associated with both osteogenic and chondrogenic differentiation (Fig. 1). Alizarin red staining of cells near the osteogenic channel revealed potential mineral depositions associated with osteogenesis. In regions nearer the chondrogenic channel staining with alcian blue showed potential glycosaminoglycan deposition alluding to chondrogenic differentiation. This suggests that by using a channeled structure and applying time constraints, delivery of differentiation supplements can be controlled such that multiple cell types can be cultured within a single structure. Conclusion: Controlled delivery of differentiation cues to a population of rBMSC's shows promise as a method for culturing multiple cell types within a single structure. Biochemical analysis of cultures is required however, in order to both confirm and quantify potential matrix depositions.