Growth factor priming differentially modulates components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells

Abstract

To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB) in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC)-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes) and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs). However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.

Figure 1. Schematic of experiments.

Cells isolated from canine cartilage and synovium were cultured in 2D with (primed, -P) or without (unprimed, -U) a growth factor cocktail (see Materials and Methods). Surface marker expression by flow cytometry and label-free proteomic profiling were assessed in chondrocytes and SDSCs at passage 2 (P2). Cells were subsequently evaluated for chondrogenic capacity in 3D pellet culture at passage 2 (P2).

Figure 2. Morphological differences between unprimed and primed cells.

Phase contrast micrographs (20×) of canine chondrocytes and SDSCs at passage 4 cultured unprimed (left side) or primed (right side). Scale bar = 50 µm. Initial plating density was 12.5-fold greater for chondrocytes than SDSCs.

Figure 3. Doubling times of canine chondrocytes and SDSCs.

Doubling times of canine (A) chondrocytes (unprimed (C-U), primed (C-P)) and (B) SDSCs (unprimed (S-U), primed (S-P)) from P1 to P4 (n = 5). Statistically significant differences relative to unprimed cells are represented as **p<0.01 and ***p<0.001.

Figure 4. Examples of differential protein expression in canine chondrocytes and SDSCs.

A) Example of differential expression of collagen alpha-1(II) chain. The plot represents one isotopic signal from the mass spectrum of peptide GFTGLQGLPGPPGPSGDQGASGPAGPSGPR at 1337.1 m/z and 45.6 min retention time. Unprimed chondrocytes (C-U) cells exhibit strong accumulation of this protein, compared to primed chondrocytes (C-P) samples or either S-U or S-P samples B) Example of differential expression of aminopeptidase N (CD13). Plot represents one isotopic signal from the mass spectrum of peptide ESALLYDPQSSSIGNK at 854.9 m/z and 46.3 min retention time. Primed SDSCs (S-P cells) exhibit strong accumulation of this protein in response to growth factor priming; in comparison, neither C-P nor unprimed SDSCs (S-U cells) show a significant signal.

Figure 5. Biochemical properties of canine chondrocyte- and SDSC-pellet culture.

(A, C) Glycosaminoglycan (GAG) content (g) normalized to DNA (g). After 28 days in culture, primed cells produced significantly more GAG/DNA. (B, D) Collagen content (g) normalized to DNA (g). Primed SDSCs (S-P cells) produced more collagen after 14 or 28 days. Results are shown as mean ± SD (n = 5). Statistically significant differences relative to unprimed cells are represented as *p<0.05, **p<0.01 and ***p<0.001.