Differences between the cell populations from the peritenon and the tendon core with regard to their potential implication in tendon repair

Abstract

The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency assays, and real-time quantitative polymerase chain reaction analyses of markers of osteogenic and adipogenic differentiation after culture in induction media were performed. Finally, cellular capacity for differentiation towards a myofibroblastic phenotype was assessed. Our results demonstrate that both tendon- and peritenon-derived cell populations are capable of adipogenic and osteogenic differentiation, with higher expression of progenitor cell markers in peritenon cells. Cells from the peritenon also migrated faster, replicate more quickly, and show higher differentiation potential toward a myofibroblastic phenotype when compared to cells from the tendon core. Based on these data, we suggest that cells from the peritenon have substantial potential to influence tendon-healing outcome, warranting further scrutiny of their role.

Figure 1. Gene expression levels of tenogenic markers, matrix remodeling markers, and western blots on tenogenic markers to confirm gene expression results.

Gene expression of (A) SCX, (B) TNMD, (C) COL1A1, (D) COL3A1, (E) MMP1, and (F) MMP3 in tendon core cell population and peritenon cell population cultured in expansion medium. The cells were extracted from eight different horses. Using the 2^−ΔΔCT method, the data are presented both in terms of “relative expression” normalized to three housekeeping genes, as well as the fold change in gene expression normalized relative to the tendon core expression. The respective p values are as follows:(A) SCX (24-fold; p = 0.01), (B) TNMD low expression and not significantly different, (C) no difference for COL1A1 (p = 0.48) (D) Col3A1 (2.1-fold; p = 0.02), (E) MMP1 (330-fold; p = 0.02), (F) MMP3 (13-fold; p = 0.02. (G) Western blots on tenogenic markers to confirm the gene expression results. The graphs display the relative band signal strength in Western blots analyzed by optical densitometry and related to loading control vimentin. Cells from the tendon core population demonstrated a higher level of scleraxis protein expression compared to cells from the peritenon when cultured in expansion medium for a week (p<0.05). The difference between the two cell populations for collagen protein expression was not significant.

Figure 2. Gene expression levels of progenitor cell markers, indicates higher levels of progenitor cell markers in peritenon cell populations.

(A) CD45 (31-fold; p = 0.03), (B) CD90 (1.9-fold; p = 0.02) (C) CD105 (2.4-fold; p = 0.02), and (D) Oct-4 (2.6-fold; p = 0.02).

Figure 3. Migration and replication rate.

(A) Average migration speed of the cell populations from the core of the tendon and from the peritenon, obtained with a scratch assay monitored over 8 h. The peritenon cell population was 1.2 times faster than the tendon core population (p  = 0.04). The cells were from twelve different horses. (B) Replication rate: Starting with equivalent numbers of cells, we calculated the number of cells after 7 days in culture in expansion medium. There were 1.33 times more cells in the flasks containing cells isolated from the peritenon compared to the flasks containing cells isolated from the tendon core (p = 0.026). The cells were from four different horses.

Figure 4. Clonogenicity of the two cell populations.

Representative wells with colonies from the tendon core population (A) and from the peritenon population (B) were evaluated (n = 14; the cells were from seven different horses; scale bar  = 0.5 cm). The cells isolated from the tendon core demonstrated 2.6 times more colonies compared to the peritenon cell population (C; p = 0.02). The colonies were 2.5 times larger in the peritenon cell population than in the cells isolated from the tendon core (D; p = 0.018).

Figure 5. Potential for osteogenic and adipogenic differntiation of cells from the tendon and peritenon after 21 day culture in induction medium.

Osteogenic (Alizarin Red; A–D) and adipogenic (Oil red O; E–H) differentiation of cells from the core of the tendon and from the tendon core (A,B; E,F) and from the peritenon (C,D; G,H). Larger calcification nodules (black arrows) were observed in the peritenon cell population (C) compared to the cells isolated from the tendon core (A) in osteogenic medium. Controls lacked nodule formation (B,D). Lipid vacuoles (black arrows) inside the cells of both populations when cultured in adipogenic medium (E,G), but not in the control condition with expansion medium (F,H). Scale bar  = 100 μm.

Figure 6. Gene expression of markers for osteogenic and adipogenic induction.

Fold changes were calculated by dividing the relative expression of the gene of interest in the differentiation medium by the relative expression in the control expansion medium. No significant differences were found for osteogenic markers, either between induction and control media or between cell types (A,B). With respect to the adipogenic markers (C,D), both markers were substantially upregulated in induction medium. No significant difference was found between cell populations for FABP4 (C), while PPARG (D) was significantly upregulated (3.8-fold) in the cells isolated from the tendon core compared to the cell population from the peritenon (p<0.03); The cells were extracted from eight horses).

Figure 7. Cells at the margins of a tendon explant, but not those inside the more densely packed, well-organized intact matrix, show propensity for differentiation to non-tendinous phenotypes.

Arrows indicate cells that are stained for markers of differentiation in adipogenic medium (Oil red O staining; row A) and osteogenic induction medium (Alizarin red S staining (row B). Images correspond to the edge of the sample, the middle of the explant section and in the endotenon (columns from left to right). Differentiated cells (black arrows) appear in the endotenon and peritenon, but not in the tendon core. Explants were analyzed from six different horses.

Figure 8. Comparison of the propensity to differentiate into myofibroblasts of the cell populations from the peritenon and from the core of the tendon and the effects of ligands on the differentiation propensity of cells isolated from the peritenon and cells isolated from the tendon core.

(A–C) Expression of myofibroblast marker α-SMA was assessed by Western blotting together with loading control vimentin. (B–D) Band signal strength in Western blots was analyzed by optical densitometry and related to loading control vimentin. (A–B)Following addition of TGF-β1, the level of α-SMA was 2.4 times increased in the cell population from the tendon core and 3.6 times increased in the cell population from the peritenon. The cell population from the peritenon showed a 1.2-fold increased expression of α-SMA compared to the cell population from the tendon core when cultured with TGF-β1. (C–D) The cell population from the peritenon showed a 1.7-fold increase of α-SMA expression on 100 kPa PDMS dishes coated with collagen compared to the cell population of the tendon core and a 4-fold increase of α-SMA expression on same dishes coated with fibronectin (for both experiments, the cells were extracted from three different horses).