The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.
There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1b, tumor necrosis factor-a, and interferon-g to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE 2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11+ cells compared to unsorted osteo-mESCs and cad-11 -cells. Moreover, the cad-11 + cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.
Hydrogel scaffolds derived from the extracellular matrix (ECM) of mammalian tissues have been successfully used to promote tissue repair in vitro and in vivo. The objective of this study was to evaluate the osteogenic potential of ECM hydrogels prepared from demineralized and decellularized bovine bone in the presence and absence of osteogenic medium. Culture of C2C12 and mouse primary calvarial cells (mPCs) on decellularized bone ECM (bECM) and demineralized bone matrix (DBM) gels resulted in increased expression of osteogenic gene markers, including a 3.6- and 13.4-fold increase in osteopontin and 15.7- and 27.1-fold increase in osteocalcin when mPCs were cultured upon bECM with basal and osteogenic media, respectively. bECM hydrogels stimulated the osteogenic differentiation of C2C12 and mPCs even in the absence of osteogenic medium. These results suggest that bECM hydrogel scaffolds may have great utility in future clinical applications for bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 900-908, 2018.
It is possible to influence osteoblast activity by the application of mechanical forces. There is potential in using these forces for tissue engineering applications in that cell matrix production may be upregulated, resulting in a functional tissue engineered construct created in a shorter culture time. We have been developing a novel technique for applying mechanical forces directly to the cell with the use of magnetic particles. Particles attached to the cell membrane can be manipulated using an external magnetic field thus applying forces in the piconewton range. We have previously demonstrated that primary human osteoblasts respond to this type of stimulus by upregulating bone related gene expression and producing mineralized matrix at early time points. In this paper we discuss the optimization of this technique by presenting data on the effects of this type of force on osteoblast proliferation, phagocytosis and also the potential use of this technique in developing 3D tissue engineered constructs.
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