Stiffness of cell micro-environment guides long term cell growth in cell seeded collagen microspheres

Mesenchymal stem cells are widely implicated as a cell source for tissue engineering of skeletal tissue in cell-based therapy. Physical and mechanical cues are potent controlling factors in cell differentiation and can be implemented as a guide to study cellular response, matrix production and tissue regeneration. Microspheres were produced by gelation of bovine collagen type I with concentration of 2 mg/mL and 1,000-2,000 cells per droplet. Short and long term cell viability of human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) and MG-63 osteoblastic cells as well as collagen microstructure and contraction were monitored during 28 days post encapsulation (pc). Results indicated that collagen concentration, hence mechanical properties of cell’s extracellular micro-environment are important in cell proliferation and differentiation. Contraction of cell-embedded microspheres was found to be vital in cell adaptation and the remodelling of their new environment. It was also found that collagen concentration of 2 mg/mL supports proliferation of hES-MPs while higher collagen concentration promoted the viability of MG-63s. Results of hES-MPs characterization in 3D soft environment and mechanically stimulated hES-MPs collagen microspheres can be used in cells/therapeutic carriers, implants in bone and cartilage healing applications. The microspheres developed in this study can also be used as a tool to build more optimised construct to transfer mechanically stimulated stem cells to the specific area of a defective bone which would add significant benefit to the field of bone regeneration

Effect of mechanical loading on osteogenesis of human embryonic stem cell-derived mesenchymal progenitors within collagen microspheres

Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells. However, mesenchymal stem cell fate determined by the effect of physical and mechanical cues is not yet fully understood. Understanding how mechanical cues in the microenvironment orchestrate stem cell differentiation provides valuable insight that can be used to improve current techniques in cell therapy. This study investigates the osteogenic effect of mechanical stimulations on soft cellular microspheres loaded with human embryonic stem cellderived mesenchymal progenitors (hES-MPs) when subjected to dynamic loading and in the absence of chemical stimulation. Microspheres were produced by gelation of bovine collagen type I with 1000 to 2000 hES-MP cells seeded per droplet. Four loading conditions were studied: (1) 10% constant strain was applied by a Bose biodynamic bioreactor for 15 min/day or 40 min/day for 5 or 10 days respectively; (2) 10% adjusted strain was applied (subtraction of polydimethylsiloxane (PDMS) plastic elongation from global strain) using Bose biodynamic bioreactor for the same 4 duration/conditions as in the constant strain protocol. The results indicate that applying mechanical stimulation to hES-MPs/collagen microspheres induced osteogenic differentiation of cells when the loading protocol was adjusted. Alkaline phosphatase activity of samples in the adjusted loading protocol increased significantly on day 14 whilst, the deposited minerals, matrix reorganisation and alignment of collagen fibres enhanced from day 21 post encapsulation onward. Application of cyclic loading to 3D culture of hES-MP cells can be used as a model to regulate mechanostimulation and linage differentiation in vitro