Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy

Abstract

Objective: This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy.

Method: In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-β (TGFβ) following 21 days of culture in either growth or chondrogenic media.

Results: In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFβ. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets.

Conclusion: The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.

Figure 1. hMSC pellets cultured with TGF-β express aggrecan, but also the hypertrophic gene MMP13

(*, p<0.001). hMSC pellets cultured in chondrogenic media for 21 days with TGFβ (5ng/mL) express more aggrecan and MMP13 mRNA than comparable pellets grown in the absence of TGFβ, N=5 (A, C). The gene expression data is confirmed by protein analyses: aggrecan immunohistochemistry reveals greater staining in hMSC pellets cultured with TGFβ compared to those without TGFβ, N≥3 (B), and more MMP13 was detected in media of hMSC pellets cultured with TGFβ than those cultured without TGFβ, N=3 (D). Error bars represent 95% confidence intervals.

Figure 2. BCPs produce significantly more GAG than hMSC pellets

(+, p = 0.0015 compared to hMSC; *, p = 0.0121 compared to hMSC). BCPs constructed with cell populations labeled with DiI (red) or DiO (green), N≥3 (A), show bilaminar structure as portrayed in a schematic (B). BCPs are intended to facilitate inductive interactions (arrow) between an outer layer of male juvenile chondrocytes (XY, orange), and a core of female hMSC (XX, green). Following 21 days of culture in growth media, BCPs containing 75% hMSC and 25% JC produce as much proteoglycan as pellets containing 100% JC as determined by DMMB analysis N=10 (C). Error bars represent 95% confidence intervals.

Figure 3. BCP co-culture is sufficient to induce chondrogenic differentiation of hMSC

In situ hybridization (A) and immunohistochemistry (B) reveal that JC and BCP pellets express qualitatively more aggrecan mRNA and protein than hMSC pellets. FISH detection of male JC (C) and female hMSC (D), respectively, in boxed regions of (B). N≥3 for all.

Figure 4. Unlike TGF-β, BCP co-culture induces chondrogenic differentiation of hMSC without hypertrophy

(*, p<0.001; +, p = 0.0021). Following 21 days of culture in growth media, BCPs exhibit enhanced chondrogenic differentiation relative to TGF-β treated hMSC pellets, as shown by increased mRNA expression for anabolic chondrocyte marker genes Sox9, collagen 2A1, and aggrecan (A) and reduced mRNA expression of hypertrophic genes Runx2, Col 10A1 and MMP13 (C) as assessed by quantitative RT-PCR. Data are normalized to β-2-macroglobulin expression and show averages of 5 biological replicates. The gene expression data is confirmed by protein analyses: Western blotting confirms the upregulation of Sox9 in the BCPs compared to the TGFβ-treated hMSCs, N≥3 (B), and an enzyme activity assay confirms the upregulation of MMP13 in the TGFβ treated hMSCs compared to the BCPs N=3 (D). Error bars represent 95% confidence intervals.

Figure 5. BCP co-culture performs similarly in chondrogenic media compared to culture in growth

media (*, p<0.001 compared to hMSC; +, p <0.001 compared to TGFβ treated hMSC; ◆, p = 0.0038 compared to JC; x, p = 0.045 compared to hMSC). In chondrogenic media, BCP and JC pellets produce more proteoglycan compared to hMSC in the presence or absence of TGFβ as assessed by DMMB, N=8 (A), a result that is consistent with Aggrecan IHC N≥3 (B). JC pellets showed increased expression of chondrogenic genes relative to BCPs and hMSC+TGFβ pellets. hMSC+TGFβ pellets had greater expression of hypertrophic genes than BCP pellets N=5 (C). Error bars represent 95% confidence intervals.

Figure 6. BCPs produce more GAG than hMSC or JC pellets when subjected to conditions that mimic the hypoxic, proinflammatory environment found in cartilage

defects (*, p <.001). A DMMB assay revealed a significant advantage for BCP proteoglycan production relative to JC or hMSC pellets that were cultured with TNF-α (10 ng/mL) and IL1-β cytokines (10 ng/mL), and/or at 2% O₂ for 21 days, N=10. Results were normalized to DNA content, N=10 (B), which showed no statistical differences between the cell types (hMSC, JC and BCP) within the conditions: TNFα/IL1β, Hypoxia or Hypoxia + TNFα/IL1β).