Xuejun Xin, Mohammad Hussain, Jeremy J. Mao. Biomaterials 28 (2007) 316–325
The development of artificial extracellular matricies (ECMs) has become an active area of research in recent years. Currently, randomly oriented nanofiber sheets produced via electrospinning are being explored as possible synthetic ECMs for tissue engineering. In this paper, electrospun poly(D,L-lactide-co-glycolide) (PLGA) nanofibers are used to study the growth and differentiation of human mesenchymal stem cells (hMSCs), as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives.
After PLGA fibers were electrospun and sterilized, the hMSCs, hMSC-Obs, and hMSC-Chs were seeded and incubated for 1-14 days. After incubation, the cell and nanofiber constructs were treated to observe their morphology, to determine their viability and proliferation, and to show whether or not the seeded hMSCs differentiated.
To visualize cellular attachment and morphology on the nanofiber sheets, the constructs were sputter coated with platinum and observed under SEM. SEM micrographs indicated that the cells had increased attachment to the nanofibers over time because they transitioned from round to elongated shapes along the direction of the nanofibers.
The cell-seeded scaffolds were analyzed using 3 different techniques to determine their viability. To extract the total amount of DNA, the constructs were sonicated and tagged with fluorescent molecules. A fluorometer was used to quantify the amount of DNA, which indicated that the amount of DNA increased over time. The constructs were treated with a live/dead cell assay on their surfaces and observed. This assay showed that most of the cells were alive after incubation. Additionally, the constructs were labeled with bromodeoxyuridine (BrdU). Results from the BrdU labeling revealed that a significant portion of the cells continued to undergo mitosis after being seeded onto the PLGA nanofiber sheets.
Cellular differentiation of the hMSC on the PLGA nanofiber sheets was determined using histology (hemotoxylin and eosin staining) and immunochemistry/confocal microscopy. In the presence of glycosaminoglycans and mineral deposition, the hMSCs showed signs of differentiation into chondrogenic and osteogenic cells, respectively.
This paper is particularly interesting because my group is considering using electrospun fibers as substrates for growing cells. The work in this paper demonstrated both successful growth of cells on synthetic electrospun ECMs, as well as several of the techniques we have covered in lab. Unlike the previous tissue engineering work that has been done in the electrospinning field, we are planning on using fibers that are spun using a new type of electrospinning technology, near-field electrospinning (NFES). NFES has a higher level of control than conventional electrospinning, allowing patterns of nanofibers to be quickly and cheaply drawn on a substrate. It will be fascinating to see if we can force cells to grow in patterns with this technology.
4 comments:
I don't think that the authors are necessarily proving that this technique can lead to increased differentiation in comparison to other methods--I think they are just trying to demonstrate that it is possible. However, I can imagine that if these nanofibers could be ordered in 3D space in a desired way, seeded cells could exhibit increased differentiation because of the nanofiber guidance.
just out of curiosity, what gives NFES the higher level of control than conventional electrospinning?
Many factors enable NFES to be much more controllable than conventional electrospinning. Please take a look at the following paper:
http://www.me.berkeley.edu/~chieh/papers/Nanolett06_NFES.pdf
What different information comes out between measuring both amount of DNA increase and BrdU incorporation? It seems both are measures of DNA synthesis.
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