A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells

A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells

Wan-Ju Li, Richard Tuli, Chukwuka Okafor, Assia Derfoul, Keith G. Danielson, David J. Hall, Rocky S. Tuan

Summary:

A stem cell is an undifferentiated cell of a multi-cellular organism that has the ability to differentiate into a range of specialized cell types. Adult stem cells are those that are found throughout the body after embryonic development, whereas embryonic stem cells are found in an early-stage embryo. Tissue engineers are interested in adult stem cells because of their multidifferentiative potential of generating several cell types. In this paper, a 3-dimensional nanofibrous scaffold (NFS) was created for cartilage tissue engineering with the use of adult mesenchymal stem cells (MSCs). These cells give rise to chondrocytes, cells that secrete the matrix of cartilage and become embedded in it, as long as the cells are maintained in culture and treated with the transforming growth factor-b (TGF-b). The premise of the experiment is to show that NFS is a more biologically representative method to model cartilage repair than the cell pellet method mentioned below. An in vitro analysis of chondrogenesis was conducted in NFSs with MSCs placed into the scaffold. In order to conduct the experiment, first MSCs were isolated and grown. MSCs were isolated from bone marrow of the femoral neck and grown in a DMEM/bovine serum mixture. Next, 3-dimensional cell pellet cultures and NFSs were created. The cell pellet was formed by placing 250,000 cells in a conical vial, centrifuging the solution and letting the sample sit over night. The NFSs were formed by electrospinning a polymer that resembles an in vivo natural extracellular matrix (Figure 1, A & B). In order to induce chondrogenesis, the cells were supplied with the transforming growth factor and incubated for 21 days. Four samples were analyzed, 2 CP samples (one with TGF-b, one without) and 2 NFS samples (one with TGF-b, one without). Subsequently RNA extraction was performed, followed by RT-PCR for 32 cycles (Figure 3). A sulfated glycosaminoglycan (sGAG) assay was performed to measure the biosynthetic rate of the cells and based on the extracts of the sGAG assay (Figure 4), a cell proliferation assay was conducted to estimate MSC proliferation. Finally, after 21 days of harvesting an immunohistological analysis of CP and NFS was conducted by washing with PBS, staining with a Broad Spectrum Histostain-SP kit and counterstaining with hematoxylin (Figure 6 & 7).

The major goal of the experiment was to compare induced chondrogenesis in a CP culture compared to a 3-dimensional scaffold (NFS). Although the CP method of chondrogenesis is one of the more typical methods used in research, this paper proposes that the NFS method more accurately describes the in vivo setting in the human body. Furthermore, according to figure 2 in the paper, CP produces unorganized collagen-like fibers, whereas NFS cultures showed a smooth, even surface (more biologically representative of the body). Microscopic images were taken of the two techniques to show the cartilage-like tissue growth when TGF-b was present. The NFS model shows zonal architecture similar to native cartilage. According to the literature, this occurs because of increased ECM production in a 3-dimensional setting as opposed to the CP method. In conclusion, the NFS method provides a more realistic comparison to in vivo situations and more appropriately models a tissue engineering-based cartilage repair technique.

Critique:

Overall, the scaffold method seems to be a definite improvement in terms of biological representation for cartilage repair. As we talked about in lecture, a 3-D model more accurately describes an in vivo environment. The NFS method does not require centrifugation and therefore, may more accurately describe cells than cells that were forced into a pellet. The NFS method tries to alleviate this concern by mimicking the 3-dimensional nature of cartilage repair in the body. Furthermore, this method allows the cells to have a high surface area to volume ratio instead of being densely packed into a pellet. The CP culture induces cell-cell interactions, and this action is a necessity to chondrogenesis initiation. The growth seen in the CP , therefore, cannot completely be accredited to the pellet method, and as a result the CP method may be less accurate than NFS. Although a quantitative biochemical assay tells us that the NFS method is equivalent and “sometimes” higher than the CP method, the microstructure resemblance between the 3-dimensional method and an accurate biological description of cartilage growth is very similar (comparison of zonal architecture is accurate) to an in vivo situation. Although the overall methodology behind the proposed scaffold idea seemed to be accurate, it would have been useful to show a comparison between hydrogel and non-hydrogel polymer scaffolds, in order to ensure that the scaffold material did not have a major affect on the cell growth seen. In future works it will be useful to include this data, as well as an image that shows in vivo cartilage repair in comparison to the scaffold method described above.

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