Equiaxial Strain Modulates Adipose-derived Stem Cell Differentiation within 3D Biphasic Scaffolds towards Annulus Fibrosus

Abstract

Recurrence of intervertebral disc (IVD) herniation is the most important factor leading to chronic low back pain and subsequent disability after discectomy. Efficacious annulus fibrosus (AF) repair strategy that delivers cells and biologics to IVD injury site is needed to limit the progression of disc degeneration and promote disc self-regeneration capacities after discectomy procedures. In this study, a biphasic mechanically-conditioned scaffold encapsulated with human adipose-derived stem cells (ASCs) is studied as a potential treatment strategy for AF defects. Equiaxial strains and frequencies were applied to ASCs-encapsulated scaffolds to identify the optimal loading modality to induce AF differentiation. Equiaxial loading resulted in 2-4 folds increase in secretion of extracellular matrix proteins and the reorganization of the matrix fibers and elongations of the cells along the load direction. Further, the equiaxial load induced region-specific differentiation of ASCs within the inner and outer regions of the biphasic scaffolds. Gene expression of AF markers was upregulated with 5-30 folds within the equiaxially loaded biphasic scaffolds compared to unstrained samples. The results suggest that there is a specific value of equiaxial strain favorable to differentiate ASCs towards AF lineage and that ASCs-embedded biphasic scaffold can potentially be utilized to repair the AF defects.

Figure 1

Effect of varying magnitudes and frequencies of equiaxial strain on the gene expression profiles of the ECM proteins and AF tissue markers. The gene expression profiles for the ECM proteins as well as AF markers were upregulated by equiaxial mechanical loading. Data represent the mean fold change (n = 8), and the error bars represents the standard deviation. *Indicated significant difference with respect to 0% strain group (control) with p < 0.05. ^§Represented significant difference between 0.1 Hz and 1 Hz (at the same strain magnitude), ^†Represented significant difference between 3% and 6% strain groups (at the same frequency), ^‡represented significant difference between 3% and 12% strain groups (at the same frequency) while ^#represented significant difference between 6% and 12% strain groups (at the same frequency), each with p < 0.05.

Figure 2

(A) Effect of varying magnitudes and frequencies of equiaxial strain on the osteogenic markers expression profiles. Loaded samples showed mixed osteogenic response with 3% and 6% groups loaded at 1 Hz showing no osteogenic expression. Data represent the mean fold change (n = 8), and the error bars represents the standard deviation. *Indicated significant difference with respect to 0% strain group (control). (B) Effect of varying magnitudes and frequencies of equiaxial strain on the gene expression profiles of catabolic proteins (MMP-2 and MMP-13) and TIMP-1 that are associated with the AF tissue. Loaded samples showed elevated anabolic response compared to control. *Indicates p < 0.05. ^§Represented significant difference between 0.1 Hz and 1 Hz (at the same strain magnitude), ^†represented significant difference between 3% and 6% strain groups (at the same frequency), ^‡represented significant difference between 3% and 12% strain groups (at the same frequency) while ^#represented significant difference between 6% and 12% strain groups (at the same frequency), each with p < 0.05.

Figure 3

Effect of varying magnitudes and frequencies of equiaxial strain on the amount of soluble secreted EMC proteins in the culture media. (A) Normalized secreted sGAG in the cell culture media. Samples loaded at 3% (0.1 Hz) and samples loaded with 3%, 6% at 1 Hz showed higher normalized soluble GAG content in the culture media (B) Normalized soluble collagens in the cell culture media. Loaded samples showed higher amounts of soluble collagens in the culture media. Data represents the average (n = 8), and the error bars represents the standard deviation. *Represents statistical difference from the control (unstrained group) with p < 0.05.

Figure 4

Effect of equiaxial strain on matrix organization and the cells morphology of ASC-encapsulated 3D collagen scaffolds. (A) Histological visualization using Mason’s Trichrome staining. Mason’s trichrome stains the collagen fibers with blue color, the cytoplasm of the cell is stained with red, and the nucleus is stained with purple color. Loaded ASCs-encapsulated collagen (6% strain and 1 Hz frequency) showed more aligned fibers and elongated cells compared to control. Scale bar represents 100 µm. Black arrows point to the aligned and elongated cells while yellow arrows point to the round and randomly distributed cells (B) Representative directionality histograms for the loaded and control samples. (C) The average amount of directionality obtained from various field views (n = 4) through imageJ. Loaded samples showed higher directionality compared to control. *Represents statistical difference (p < 0.05) from the control group. Error bars represent the standard deviation.

Figure 5

Effect of equiaxial strain on the amount of soluble secreted sGAG (A) and Collagens (B) in the culture media. Loaded ASCs-collagen-PNCOL biphasic scaffolds showed higher secreted ECM proteins amounts in the culture media. Data represents the average (n = 8), and the error bars represents the standard deviation. *Represents statistical difference from the control (unstrained group) with P < 0.05. (C) Histological visualization of the secreted GAG within the biphasic scaffolds using Alcian Blue staining. Alcian Blue dye stains GAGs with blue color. Mechanically loaded samples showed higher amounts of secreted GAG within the scaffolds. Scale bar represents 100 µm.

Figure 6

Effect of equiaxial strain on matrix organization and the cells morphology of ASC-encapsulated collagen-PNCOL biphasic scaffolds. (A) Histological visualization using Mason’s Trichrome staining demonstrated that loaded samples had higher aligned fibers and elongated cells compared to control samples. The black arrows point to the interface between collagen (inner) and PNCOL (outer) regions of the biphasic scaffold. Scale bar represents 100 µm. (B) The average amount of directionality obtained from various field views (n = 4) through imageJ. Loaded samples showed higher directionality compared to control. *Represents statistical difference (p < 0.05) from the control group. Error bars represent the standard deviation.

Figure 7

Effect of equiaxial strain on the gene expression profiles of the native AF ECM marker proteins. Loaded samples exhibited higher gene expression compared to control group. Data represent the mean fold change (n = 8), and the error bars represents the standard deviation. *Represents statistical difference from the control (unstrained group) with 0.05 P-value. ^†Represented a significant difference between the inner and outer regions with a 0.05 P-value.

Figure 8

Effect of equiaxial strain on the gene expression profiles of the native AF tissue markers. Loaded samples exhibited higher gene expression compared to control group. Data represent the mean fold change (n = 8), and the error bars represents the standard deviation. *Represents statistical difference from the control (unstrained group) with 0.05 P-value. ^†Represented a significant difference between the inner and outer regions with a 0.05 P-value.

Figure 9

Representative visualization photograph of the biphasic scaffolds after 7 days of mechanical loading in culture conditions and a schematic view of the biphasic scaffolds. Collagen (inner)-PNCOL (outer) layers of the scaffold are depicted. The scale bar represents 4 mm.