Applied Compressive Strain Governs Hyaline-like Cartilage versus Fibrocartilage-like ECM Produced within Hydrogel Constructs

Abstract

The goal of cartilage tissue engineering (CTE) is to regenerate new hyaline cartilage in joints and treat osteoarthritis (OA) using cell-impregnated hydrogel constructs. However, the production of an extracellular matrix (ECM) made of fibrocartilage is a potential outcome within hydrogel constructs when in vivo. Unfortunately, this fibrocartilage ECM has inferior biological and mechanical properties when compared to native hyaline cartilage. It was hypothesized that compressive forces stimulate fibrocartilage development by increasing production of collagen type 1 (Col1), an ECM protein found in fibrocartilage. To test the hypothesis, 3-dimensional (3D)-bioprinted hydrogel constructs were fabricated from alginate hydrogel impregnated with ATDC5 cells (a chondrogenic cell line). A bioreactor was used to simulate different in vivo joint movements by varying the magnitude of compressive strains and compare them with a control group that was not loaded. Chondrogenic differentiation of the cells in loaded and unloaded conditions was confirmed by deposition of cartilage specific molecules including glycosaminoglycans (GAGs) and collagen type 2 (Col2). By performing biochemical assays, the production of GAGs and total collagen was also confirmed, and their contents were quantitated in unloaded and loaded conditions. Furthermore, Col1 vs. Col2 depositions were assessed at different compressive strains, and hyaline-like cartilage vs. fibrocartilage-like ECM production was analyzed to investigate how applied compressive strain affects the type of cartilage formed. These assessments showed that fibrocartilage-like ECM production tended to reduce with increasing compressive strain, though its production peaked at a higher compressive strain. According to these results, the magnitude of applied compressive strain governs the production of hyaline-like cartilage vs. fibrocartilage-like ECM and a high compressive strain stimulates fibrocartilage-like ECM formation rather than hyaline cartilage, which needs to be addressed by CTE approaches.

Keywords: Col1; Col2; compressive force; fibrocartilage; hyaline cartilage; hydrogel.

Figure 1

Representative images of (A) an unloaded construct as well as (B) 6%, (C) 12%, and (D) 24% loaded samples. The cleaved strands from the constructs following compression are indicated by arrows (Scale bar = 2 mm).

Figure 2

(A–D) Alcian blue stained sections show deposition of GAGs within different groups at various regions of the sections. (A′–D′) Higher magnification images of the dashed rectangles within the images of (A–D) show blue color related to GAG deposition surrounding the cells which is darker than the background blue color. The arrows and brackets are pointing to the cells depositing GAG (Scale bars: (A–D) = 100 µm, and higher magnification images of (A′–D′) = 25 µm).

Figure 3

(A) Although there was no statistically significant difference in GAG deposition among the groups, the 12% group had the lowest median value, and (B) collagen contents of all groups also tended to be similar with slight variations between them that none were statistically significant. The bar graphs show median and interquartile range (n = 3 for each group, n.s. = not significant).

Figure 4

(A,B) Col1 and Col2 immunofluorescence staining on a same section of pig joint, as a positive control for the antibodies, confirmed that the selected primary antibodies detected Col1 and Col2 deposition at the expected regions, and arrows are pointing Col1 staining in the superficial zone. (C,D) Negative control section of a pig joint double immunofluorescence stained for Col1 and Col2 in the absence of the primary antibodies did not show any positive staining at the cartilage region (scale bars = 500 µm).

Figure 5

(A–D) Staining with DAPI of different groups showed that cell populations seemed to be lower in groups 12% and 24% loaded groups. (E–H) Col2 immunofluorescence staining confirmed chondrogenic differentiation of most of the cells for all the groups. (I–L) Col1 immunofluorescence staining showed its deposition from most of the cells in all groups suggesting that cells were differentiated to fibrochondrocytes (scale bars = 200 µm).

Figure 6

Quantitative results showed all groups seemed to have similar (A) Col2/DAPI quantities, but (B) DAPI pixels tended lower in 12% and 24% loaded groups. Additionally, (C) %hyaline-like cartilage and (D) %fibrocartilage-like ECM tended to be lowest and highest, respectively, in 12% loaded group. The bar graphs are showing median and interquartile range (n = 4 for each group, n.s. = not significant, * p  <  0.05, and ** p  <  0.01).

Figure 7

Overview of experimental design: Hydrogel constructs were fabricated using a 3D-bioplotter and cultured for 10 days. Afterward, they were either cultured in an unloaded condition or subjected to compression using a biodynamic machine. The three different compressive strains used are shown by the strain-time figures.