Development of annulus fibrosus tissue construct with hydrogel coils containing pre-conditioned mesenchymal stem cell

doi:10.1016/j.jmst.2020.03.051

Abstract

Abstract:

Low back pain associated with degenerative disc diseases has been a major health concern that brings suffering to the patients physically and economically. Many existing therapeutic strategies provide short-term relief of symptoms rather than treatment of the underlying cause. Development of an engineered tissue for disc regeneration is still in its infancy due to the limited autologous healthy disc cell source from the patients. It is also challenging to mimic the complexity of micro-architecture in the native disc tissue that determine their unique structural properties. To date, simple tissue models that mimic the annulus fibrosus (AF) micro-environment for understanding the potential of mesenchymal stem cells (MSCs) in AF tissue engineering are still lacking. In this study, the assembly of a coiled hydrogel microfiber has shown its capability to encapsulate MSCs and create an engineered tissue model that mimics the multiple lamellae of native AF. Using this model, we investigated the potential of MSCs that were previously induced by ascorbic acid (AA). Compared to non-induced MSCs, AA-induced MSCs exhibited significant increase in AF-associated biomarkers during later development in the engineered AF tissue model and also encouraged collagen accumulation through the down-regulated catabolic gene MMP1 and upregulated anti-catabolic gene TIMP1. Furthermore, AA-induced MSCs exhibited a Col2/Col1 ratio closer to that of a native AF tissue. These results suggested that AA-induced MSCs could be a potential cell source for AF tissue engineering and this established tissue model may provide a simple tool for successful AF tissue engineering strategies in the future.

Yon Jin Chuah, Yingnan Wu, Mei Ling Shirlynn Cheong, Yan Qing Chia, Ching Ann Tee, Hwan Tak Hee, Chenjie Xu, Yuejun Kang, Dong-An Wang. Development of annulus fibrosus tissue construct with hydrogel coils containing pre-conditioned mesenchymal stem cell[J]. J. Mater. Sci. Technol., 2021, 63: 27-34.

Figures/Tables 8

Fig. 1. Experimental procedure of creating a coiled hydrogel microfiber that is stabilized with calcium alginate solution.

Fig. 2. (a) Fluorescence staining of F-actin (in red) and nucleus (in blue) of MSCs after 7 days of cell expansion. Scale bar: 100 μm. (b) PrestoBlue assay for MSC proliferation (n = 4) on day 4 and day 7. (c) Gene expression of proliferative marker Ki67 in MSCs after 7 days culture. Data are shown as mean ± SD (n = 4). *p-value < 0.022 between two groups (Student’s t-test).

Fig. 3. Gene expression of AF-associated markers including (a) collagen type I (Col1), collagen type II (Col2), aggrecan, and Sox9 and (b) catabolic marker MMP1 and anti-catabolic marker TIMP1 in MSC populations after 1 week of cell expansion. (c) Quantification of total secreted collagen (normalized based on the data for non-induced MSCs) and (d) concentrated collagen collected from a 1.9 cm2 well after 1 week of cell expansion. -AA refers to non-induced and + AA refers to AA-induced. All data are shown as mean ± SD (n = 4). *p-value < 0.041 between two groups (Student’s t-test).

Fig. 4. (a) Image of a stabilized coiled hydrogel microfiber. Scale bar: 5 mm. (b) Phase contrast microscopic image showing the coiled hydrogel microfiber. Scale bar: 500 μm.

Fig. 5. Fluorescence staining of F-actin (in red) and nucleus (in green) of MSCs distributed in the coiled microfibers after 3 days of culture. Scale bar: 200 μm.

Fig. 6. Gene expression of AF-associated markers including (a) collagen type I (Col1), collagen type II (Col2), aggrecan, Sox9 and (b) catabolic marker MMP1 and anti-catabolic marker TIMP1 in the engineered AF tissue constructs after 3 weeks of chondrogenic induction. All data are shown as mean ± SD (n = 4). *p-value < 0.0039 between two groups (Student’s t-test).

Fig. 7. Immunofluorescence staining of collagen type I, collagen type II, and aggrecan within the MSC based engineered AF tissue constructs after 4 weeks of chondrogenic induction. Scale bar: 100 μm.

Fig. 8. Collagen type II to collagen type I gene expression ratio (Col2/Col1) as the phenotypic signature of engineered AF tissue constructs. *p-value < 0.0001 between two groups (Student’s t-test).

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