Interaction between osteoarthritic chondrocytes and adipose-derived stem cells is dependent on cell distribution in three-dimension and transforming growth factor-β3 induction

Abstract

Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-β3 supplementation on ADSC-OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-β3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC-ADSC interactions in different co-culture models. In the absence of TGF-β3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-β3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.

FIG. 1.

Schematics of the co-culture models. Three different in vitro co-culture models were used to examine the effects of paracrine factor concentration and intercellular distance on the interactions between adipose-derived stem cells (ADSCs) and osteoarthritic chondrocytes (OACs). (A) In the conditioned medium (CM-) co-culture, each cell type was cultured with supplementation of CM from the other cell type. (B) In the bi-layered (Bi) co-culture, ADSCs and OACs were encapsulated in two separate hydrogel layers with an acellular interface (∼250 μm) separating them. (C) In the mixed cell co-culture, ADSCs and OACs were mixed together in three dimensional (3D) at three different cell ratios (OAC:ADSC: 50C:50A, 25C:75A, and 10C:90A). In all the co-culture models, OACs and ADSCs were encapsulated 3D biomimetic hydrogels and cultured for 21 days in vitro, and two media conditions were examined: standard chondrogenic medium with or without transforming growth factor (TGF)-β3 supplementation. Color images available online at www.liebertpub.com/tea

FIG. 2.

Effects of the media condition and CM or bi-layered co-culture on gene expression. Gene expression of (A) aggrecan (Agg), (B) collagen type II (COL2), (C) collagen type I (COL1), and (D) collagen type X (COLX) in control culture (C, OACs, A, ADSC), CM, or bi-layered co-culture (Bi) after 21 days with or without TGF-β3 supplementation. Fold changes relative to OAC control without TGF-β supplementation. Bi-layered co-culture group included both OACs and ADSCs (equal initial cell number). Data presented as mean±standard deviation (SD, n=3 samples/group); * represents significance versus−TGF-β control and ^ represents significance versus−CM control of the same cell type; +and # represent significance of the bi-layered co-culture group versus OAC and ADSC under the same media condition, respectively; *p<0.05, **p<0.01, and ***p<0.001.

FIG. 3.

Effects of media condition and CM or bi-layered co-culture on cartilage matrix production. After 21 days of culture with or without TGF-β3 supplementation, (A) DNA, (B) sGAG, and (C) collagen content per wet weight were evaluated. Data presented as mean±SD (n=3 samples/group). C, OACs; A, ADSC, CM, conditioned medium; Bi, bi-layered co-culture. * Represents significance versus−TGF-β control and ^ represents significance versus−CM control of the same cell type; +and # represent significance of the bi-layered co-culture group versus OAC and ADSC under the same media condition, respectively; *p<0.05, **p<0.01, and ***p<0.001. sGAG, sulfated glycosaminoglycan.

FIG. 4.

Effects of media condition and mixed co-culture on gene expression. Cartilage-specific gene expression of (A) Agg, (B) COL2, (C) COL1, and (D) COLX after 21 days in mixed co-culture at various cell ratios (OAC:ADSC: 50C:50A, 25C:75A, and 10C:90A) with or without TGF-β supplementation. Fold changes relative to OAC control without TGF-β supplementation at day 21. Data presented as mean±(n=3 samples/group). * and ^ represent significance versus OAC and ADSC control under the same media condition, respectively; *p<0.05, **p<0.01, and ***p<0.001.

FIG. 5.

Effects of media condition and mixed co-culture on cartilage matrix production. (A) DNA, (B) sGAG, and (C) collagen content per wet weight in mixed co-culture at day 21 with or without TGF-β3 supplementation. Data presented as mean±SD (n=3 samples/group). * and ^ represent significance versus OAC and ADSC control under the same media condition, respectively; *p<0.05, **p<0.01, and ***p<0.001.

FIG. 6.

Cartilage formation in mixed co-culture. Immunostaining of collagen type II (top row), collagen type I (middle row), and collagen type X (bottom row after 21 days of culture in control and mixed co-culture groups in standard chondrogenic media (A) without or (B) with TGF-β3 supplementation. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 7.

Cell labeling and co-staining of collagen type II in mixed co-culture. To identify the relative contribution of each cell type to collagen type II deposition in mixed co-culture, OACs (red) and ADSCs (green) were fluorescently labeled before encapsulation in hydrogels and co-stained with collagen type II (blue) after 21 days of mixed co-culture without (A) or with (B) TGF-β3 supplementation. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 8.

Catabolic marker expression in mixed co-culture with TGF-β3 supplementation. Catabolic gene expression, including (A) matrix metalloproteinase (MMP)-3, (B) MMP-13, and (C) ADAMTS-5, after 21 days of CM, bi-layered, or mixed co-culture in standard chondrogenic medium with TGF-β3 supplementation. * and ^ represent significance versus OAC and ADSC control, respectively; *p<0.05, **p<0.01, and ***p<0.001. CM-ADSC, CM-treated ADSCs; CM-OAC, CM-treated OACs.