Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold
Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold
H. Fan, H. Liu, S.L. Toh, and J.C.H. Goh, Biomaterials 29 (2008), pp. 1017-1027.
In this paper, MSCs were co-cultured with anterior cruciate ligament (ACL) fibroblasts to determine whether soluble signals released by the fibroblasts enhanced the differentiation of the MSCs into fibroblasts. The motivation behind this research is to be able to use such a co-culture system to create a tissue-engineered ligament in vitro to remedy ACL injuries.
MSCs were harvested from rabbit bone marrow and seeded onto silk cable-reinforced gelatin/silk fibroin scaffolds. After incubation, the constructs were transferred into co-culture systems with fibroblasts (isolated from the ACL of the same rabbits) or into non-co-culture systems (control). Over a two-week culture period, observations for the following were made:
1) Cell proliferation (by quantifying DNA content with Hoechst Dye and a fluorescent plate reader)
2) Cell metabolism (by alamar blue assay)
3) Cell viability (by FDA/PI staining)
4) Cell morphology (by SEM)
5) Collagen production (by Sircol collagen dye binding assay)
6) Gene expression of collagen I, collagen III, and tenascin-C (by quantitative RT-PCR)
In addition, histological slides were prepared as well as a Western blot for collagen I, collagen III, and tenascin-C.
It was found that the MSCs in the co-cultures developed fibroblast-like morphology. Also, the mRNA and protein levels of collagen I, collagen III, and tenascin-C were significantly higher for the MSCs in the co-cultures than for the non-co-cultures. These results support the hypothesis that the 3-D co-culture of MSCs with ACL fibroblasts facilitated the differentiation of the MSCs.
I chose this paper because it discusses co-culturing as an alternative to the usual biochemical and mechanical stimulation of MSC differentiation. Co-culturing is certainly more efficient and cost-effective than having to figure out and to obtain the proper growth factors to induce differentiation.
Overall, I liked this paper because the authors fairly thoroughly characterized the behavior of the MSCs. Their protocol was clearly detailed and easy to understand (especially since we've already discussed each of these techniques in this class). However, I do wish that they had gone one step further and characterized some of the mechanical properties of their tissue-engineered constructs.
6 comments:
Co-culturing is certainly more efficient and cost-effective than having to figure out and to obtain the proper growth factors to induce differentiation.
Excellent point!
As Terry said, co-culturing is quite the phenomenon these days. The paper Jackie posted explored co-culturing osteoblasts and endothelial cells and the paper I posted explored co-culturing cardiomyocytes with endothelial cells and fibroblasts. It seems like the results of all 3 papers are quite promising, especially because they are 3-d, clinically relevant engineered tissues. One problem I do have in these papers is the lack of an explanation of how the co-culturing, the interactions of cells, growth factors, and other signals lead to these improved changes, but I tell myself that it is not an easy task and that there is probably a lot of research being done on it as I type. Anyways, I thought the paper was excellent; there were numerous relevant experiments with the numbers and statistics to back it up, as well as graphs to explain the trends or lack there of (unlike some papers that show the raw data). Thanks Ginger!
It seems like co-culturing is the closest step to being able to grow tissue in the future. It would be nice to determine the minimum ACL to MSC ratio for enhanced differentiation for even further efficiency. Great paper! thanks Ginger!
Are ACL injuries characterized by loss of ACL fibroblasts? And are MSCs relatively abundant near ACL injuries? Can normal bone marrow stem cells migrate and differentiate into ACL fibroblasts?
Where are these MSC from? Are they from embroys? Also, it would be interesting to see if these types of scaffolds hold up when placed in animal/human bodies. This type of co-culturing seems to be the treatment of the future since the environment the cells are grown in is much like that in the body.
In response to Sisi's comments:
As far as I can tell, ACL injuries are not so much about fibroblast loss as they are about the inability of the torn ECM to heal itself. I guess the reason they want to induce MSCs to become fibroblasts is because they need cells to seed onto a scaffold (cellular implants are generally better than acellular scaffolds).
I don't think MSCs are abundant near the ACL. In fact, from what I've learned, MSCs are pretty rare in general. (One way to obtain them is to isolate them from bone marrow using cell sorting. Bone marrow itself in the adult is not wildly abundant either.) I don't think they migrate to the injury site because, again, the ACL can't repair itself. It's not like other parts of the body that can undergo wound healing, making injuries a major concern and a popular research topic.
In response to Amanda's comments:
The MSCs from this experiment were harvested from bone marrow. They are definitely not embattled with the ethical problems of embryonic stem cells. Thanks for mentioning embryos, because it brings up a great point: MSCs are difficult to isolate. Maybe someone could try these experiments with umbilical-cord stem cells.
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