Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study

Abstract

Background: Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential.

Methods: Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs).

Results: Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations.

Conclusion: This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

Figure 1

Ki-67, D7-FIB, and α-SMA staining on tendon explants (day 6 of explantation period) and on tendon-derived fibroblasts (TDF) in monolayer culture. Ki-67 positive (proliferating) cells in the explants were located in the tendinous tissue (A, black arrow), in the endotenon (A, white arrow), and in the vascular walls (A, circle). Cells in the tendon tissue and in the endotenon stained positive for fibroblastmarker D7-FIB (B). Cells in the vascular walls remained negative for D7-FIB (B) and instead stained positive for α-SMA, a marker for pericytes and smooth muscle cells (C). All TDFs in monolayer culture stained positive for D7-FIB from passage one (D) to passage four (E) and remained negative for α-SMA from passage one (F) to passage four (G).

Figure 2

Oil Red O staining on tendon-derived fibroblasts cultured for 21 days in adipogenic medium (A) (note that not all cells but merely clusters of cells formed Oil Red O positive lipid vacuoles inside the cell's main body) or in control medium (B). Like cells in control medium, cells cultured in osteogenic or chondrogenic medium were negative (figures not shown).

Figure 3

Expression levels of adipogenic markers in tendon-derived fibroblasts (TDF) and bone marrow-derived stromal cells (BMSC). Cells were cultured for 21 days on osteogenic (N = 5 for TDF, N = 4 for BMSC), adipogenic (N = 5 for TDF, N = 5 for BMSC), or chondrogenic (N = 3 for TDF, N = 5 for BMSC) induction medium. The relative, GAPDH-normalized, expression levels of fatty acid binding protein 4 (FABP4)(A) and peroxisome proliferator activated receptor γ (PPARG)(B) is displayed on the vertical axis. * Indicates a P-value<0.05.

Figure 4

Immunohistochemical staining for collagen type 2 on tendon-derived fibroblasts. 5% of the cells cultured for 21 days in alginate beads in chondrogenic medium stained positive (A). Cells cultured in monolayer in control medium remained negative (B) as did cells in adipogenic or osteogenic media (figures not shown).

Figure 5

Expression levels of chondrogenic markers in tendon-derived fibroblasts (TDF) and bone marrow-derived stromal cells (BMSC). SOX9 (A), aggrecan (AGC1)(B), collagen 2 (COL2A1)(C) and collagen 10 (COL10A1)(D). See figure 3 for reminder of key.

Figure 6

Von Kossa staining on tendon-derived fibroblasts cultured for 21 days in osteogenic (A) or control medium (B). Calcium deposition was seen in osteogenic medium (A), not in control medium (B) or in adipogenic or chondrogenic media (figures not shown).

Figure 7

Expression levels of osteogenic markers in tendon-derived fibroblasts (TDF) and bone marrow-derived stromal cells (BMSC). RUNT-related transcription factor 2 (RUNX2) (A), SP7 (B), and BGLAP (C). See figure 3 for reminder of key.