Tissue development-mimicking extracellular matrices from cultured cells

摘要

Introduction: Extracellular matrix (ECM) provides various signals for manipulating cell functions and maintaining the homeostasis of living organisms. ECM is tissue-specific and dynamically remodelled during the development and aging process. We have established a method to prepare development-mimicking ECM by using cultured cells. By the method, we have prepared "stepwise osteogenesis-mimicking matrices" and "stepwise adipogenesis-mimicking matrices" that respectively replicated the dynamically changing ECM secreted at different stages of osteogenesis or adipogenesis. Although these ECM models can mimic ECM changes during osteogenesis and adipogenesis separately, they cannot simultaneously mimic osteogenesis and adipogenesis to make their complex ECM. Therefore, in this study, ECM mimicking the simultaneous osteogenesis and adipogenesis of MSCs were prepared. Materials and Methods: Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on tissue culture polystyrene (TCPS) plates at a density of 5,000 ce!ls/cm2. To induce cells undergoing simultaneous osteogenesis and adipogenesis, MSCs were cultured in mixture media composed of osteogenic medium (OM) and adipogenic medium (AM) at OM/AM ratio of 95/5 (O95A5), 85/15 (O85A15), 70/30 (O70A30) and 50/50 (O50A50). The cells were cultured for 7,14 and 21 days. The different differentiation stages of MSCs were confirmed by histological staining (alkaline phosphatase and Alizarin red S) and analysis of differentiation process related genes encoding ALP, IBSP and LPL. The cells at different stages of osteogenesis-co-adipogenesis were decellularized to prepare their respective matrices. The main components in the matrices were analysed by immunocytochemical staining of type Ⅰ collagen, biglycan, fibronectin, decorin, laminin α-4 and versican. Results and Discussion: The progress of simultaneous osteogenic and adipogenic differentiation of MSCs was confirmed by histological staining and related gene expression. The results indicated that MSCs cultured in mixture media showed simultaneous osteogenesis and adipogenesis at different stages. MSCs cultured in O85A15 for 1 week showed early osteogenesis and early adipogenesis (EOEA). MSCs cultured in O50A50 for 2 weeks showed early osteogenesis and late adipogenesis (EOLA) stage. MSCs cultured in O95A5 for 3 weeks were at a stage of late osteogenesis and early adipogenesis (LOEA). MSCs cultured in O70A30 for 3 weeks were at a stage of late osteogenesis and late adipogenesis (LOLA). MSCs cultured in basal medium for 1 week were defined as cells at the undlfferentiated stem cell stage (SC). Five different simultaneous osteogenesis- and adipogenesis-mimicking matrices (OEAE, OLAE, OEAL, OLAL and SC) were prepared after decellularization. Immunocytochemical staining showed that the composition of the matrices changed depending on the stepwise stage of osteogenic and adipogenic differentiation. Conclusion: Five types of ECM that mimicked the dynamically changing ECM during simultaneous osteogenesis and adipogenesis of MSCs were prepared by culturing MSCs in mixture media of osteogenic medium and adipogenic medium for different culture period. The stepwise osteogenesis-co-adipogenesis-mimicking ECM had different composition depending on the stage of osteogenesis-co-adipogenesis.