Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels

Abstract

Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

Figure 1. Schematic representation of the experimental design.

To examine the effects of intercellular distance and cell distribution on the interactions between adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (NChons), three different in vitro co-culture models were used: (A) In the condition medium (CM-) model, each cell type was cultured alone with supplementation of conditioned medium (CM) from the other cell type. (B) In the bi-layered (bi-) co-culture, ADSCs and NChons were confined in separate hydrogel layers with no direct cell-cell contact, and soluble paracrine signals were allowed to diffuse into the adjacent layer. (C) In the mixed cell co-culture model, ADSCs and NChons were mixed together in 3D at 4 different cell ratios (NChon:ADSC: 75C:25A, 50C:50A, 25C:75A, and 10C:90A). The initial cell density was maintained constant at 15 million/ml in all co-culture models. (D) The concentration of paracrine factors decays rapidly with distance from the secreting cell. In all the co-culture models, human adult ADSCs and bovine NChons were encapsulated in 3D biomimetic hydrogels and cultured in vitro for 21 days in chondrogenic medium supplemented with TGF-β3 (10 ng/ml).

Figure 2. Quantitative gene expression showed markedly enhanced ADSC chondrogenesis in mixed co-culture with all ratios, but not in conditioned medium or bi-layered co-culture.

To distinguish the fate of each cell type, species-specific primers were used to identify the gene expression of human ADSCs and bovine NChons in the xenogenic co-culture. Human-specific (A–C) and bovine-specific (D–F) gene expression of (A, D) aggrecan (Agg), (B, E) type II collagen (COL II), and (C, F) type I collagen (COL I) after 21 days of in vitro co-culture in chondrogenic medium supplemented with TGF-β3. Fold changes relative to (A–C) ADSC and (D–F) NChon control at day 21. Data presented as mean +/− SD (n = 3 samples/group). Asterisks (*) indicate statistical significance between control (ADSC or NChon) and co-culture groups, with *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Biochemical assays and mechanical testing showed that enhanced cell proliferation, cartilage matrix production, and mechanical property in the mixed co-culture, but not in conditioned medium or bi-layered co-culture.

Such synergy increased as ratio of ADSC increased in the mixed co-culture. (A) DNA, (B) glycosaminoglycan (GAG), and (C) collagen per wet weight (w.w.) after 21 days of culture. (D) Compressive tangent moduli of tissue-engineered constructs at day 21. Data was presented as mean +/− SD (n = 4 samples/group). To quantify the synergy between ADSCs and NChons during mixed co-culture, interaction index was calculated as the measured matrix content at various cell ratios normalized by the expected matrix content given no interaction between the two cell types. Interaction index for (E) DNA/w.w., (F) GAG/w.w., and (G) collagen/w.w. increased with an increase in ADSC ratio in the mixed cell co-culture. (*) indicates statistical significance against NChon control and (∧) indicates statistical significance against ADSC control, with *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. The morphology of newly formed cartilage nodules and cell distribution over 21 days.

(A) Newly formed cartilage nodules were visualized by immunostaining of type II collagen at day 21. Scale bars, 100 μm. (B) To determine the distribution of the two cell types in the mixed cell culture, ADSCs were membrane-labeled (red) prior to encapsulation in the hydrogels; co-staining with type II collagen (green) revealed that ADSCs always resided outside the neocartilage nodules. Scale bars, 100 μm. (C–F) Quantification of type II collagen immunostaining images, including cartilage nodule size at different ratios of ADSCs at (C) day 7, (D) day 14, and (E) day 21 (horizontal bars indicate average size), as well as the (F) total percentage of area occupied by cartilage nodules at different cell ratios at day 7, 14, and 21. Both the cartilage nodule size as well as the total area of hydrogel being replaced by cartilage nodules increased with an increase in ADSC ratio in the mixed co-culture.

Figure 5. Large cartilage nodule formation was localized at the interface of the two cells and only occurred in the absence an acellular intermediate hydrogel layer.

Newly formed cartilage nodules were visualized by immunostaining of type II collagen at day 21 in bi-layered co-culture (A) in the absence of an intermediate acellular hydrogel layer and (B) with a 250 μm acellular layer. Scale bars, 200 μm. Dotted lines in (A) indicate the localization of large cartilage nodules at the bi-layer interface, and in (B), the acellular intermediate layer.