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

Cells were explanted from human adolescent non-degenerative hamstring tendon tissue (n = 5). After the phenotype of the cells was analyzed by immunohistochemical staining and FACS-analysis, cells were cultured for 21 days on osteogenic, adipogenic, or chondrogenic medium. The differentiation potential of the tendon-derived cell population was evaluated by immunohistochemical and histochemical staining and real-time RT-PCR, and was compared with the differentiation potential of human femoral-shaft-derived BMSCs (n = 5).

Human tendon-derived cells were cultured from explants from hamstring tendon tissue of five adolescents (age 12–17 years) undergoing hamstring-tendon release for treatment of knee-contractures (MEC-2006-069). In this clinical condition the tendon is primarily not affected, but is exposed to continuously high tensile strains. After the peritendineum had been carefully removed, the tendon was cut into 3 mm³ sections, transferred into six-well plates (Corning, NY, USA) and cultured in expansion medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum (FCS), 50 μg/ml gentamicin and 1.5 μg/ml fungizone (all Invitrogen, Scotland, UK)). Tissue cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ for ten days, with three medium changes. During this time, fibroblasts migrated out of the tissue and adhered to the bottom of the culture dish. Cells were subcultured and trypsinized at subconfluency and cells from the third to the fifth passage were used for the differentiation experiments.

Human bone marrow stromal cells (BMSCs) were isolated from femoral shaft biopsies of six patients (age 42–72 years) undergoing total hip replacement for treatment of osteoarthritis (MEC-2004-142). BMSCs were isolated from aspirated marrow acccording to procedures described earlier [18]. Briefly, heparinized femoral-shaft marrow aspirate was plated out and after 24 hours, non-adherent cells were removed with 2% FCS in 1×PBS. Adherent cells were subcultured in medium with 10% FCS and trypsinized at subconfluency. Cells from the second to the fourth passage were used for the differentiation experiments. The used serum lot was selected specifically for the maintenance of multipotential cells.

Explants harvested on day 6 of the explantation period were fresh frozen in liquid nitrogen and 6 μm frozen sections were fixated in acetone. Cells in monolayer cultures, passage 1 and 4, were fixated in ice-cold 70% ethanol.

After trypsinisation, cells were seeded in six-well plates and cultured in a modified version of three differentiation media described earlier [18]. Briefly, cells were seeded at 3,000 cells/cm² to induce osteogenic differentiation and then cultured in an osteogenic induction medium containing DMEM plus 10% FCS and freshly added β-glycerophosphate 10 mM (Sigma, St. Louis, USA), dexamethasone 0.1 μM (Sigma) and L-ascorbic acid 2 phosphate 0.5 mM (Sigma). Cells were seeded at 20,000 cells/cm² to induce adipogenic differentiation, and cultured in adipogenic induction medium containing DMEM with 10% FCS, supplemented with dexamethasone 1 μM, indo-methacin 0.2 mM, insulin 0.01 mg/ml, and 3-isobutyl-l-methyl-xanthine 0.5 mM (all from Sigma). To induce chondrogenic differentiation, cells were cultured in 1.2% low viscosity alginate beads at a density of 4 × 10⁶ cells/ml in serum-free chondrogenic induction medium containing DMEM supplemented with TGF-β2 10 ng/ml (R&D Systems, UK), L-ascorbic acid 2 phosphate 25 μg/ml (Sigma), sodium pyruvate 100 μg/ml (Invitrogen), proline 40 μg/ml (Sigma) and ITS+ (diluted 1:100; BD Biosciences, Bedford, MA). All media contained 50 μg/ml gentamicin and 1.5 μg/ml fungizone. Cells were cultured in differentiation media for 21 days, with media changes twice a week. On day 21 of culture, two wells were harvested for RNA extraction and one well was used for histochemical evaluation. One well was cultured for 21 days on expansion medium as control condition for the histochemical stainings.

At harvesting, monolayer cell cultures were suspended in RNA-Bee™ (TEL-TEST, Friendswood, TX, USA). Alginate beads were dissolved in 150 μl of 55 mM sodium citrate in 150 mM sodium chloride per bead (both Fluka, Steinheim, Switzerland) and cell pellets were subsequently suspended in RNA-Bee™. RNA was precipitated with 2-propanol, purified with lithium chloride, and 1 μg total RNA of each sample was reverse-transcribed into cDNA using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). Primers were designed using PrimerExpress 2.0 software (Applied Biosystems, Foster City, CA, USA) to meet Taqman^® or SYBR^®Green requirements and were designed to bind to separate exons to avoid co-amplification of genomic DNA. BLASTN ensured gene specificity of all primers listed in Table 1. As osteogenic markers osterix (SP7), RUNT-related transcription factor 2 (RUNX2), and osteocalcin (BGLAP) were studied, while SOX9, aggrecan (AGC1), collagen 2 (COL2A1), and collagen 10 (COL10A1) were used as chondrogenic markers. Adipogenic markers studied were fatty acid binding protein 4 (FABP4) and peroxisome proliferative activated receptor γ (PPARG). Amplifications were performed as 25 μl reactions using either TaqMan^® Universal PCR MasterMix (ABI, Branchburg, New Jersey, USA) or qPCR™ Mastermix Plus for SYBR^®Green I (Eurogentec, Nederland B.V., Maastricht, The Netherlands) according to the manufacturer's guidelines. Real-Time RT-PCR (QPCR) was done using an ABI PRISM^® 7000 with SDS software version 1.7. Data were normalized to GAPDH which was stably expressed across sample conditions (not shown). Relative expression was calculated according to the 2^-ΔCT formula [25] using averages of duplo samples.

Statistical analysis on averages of duplo samples was performed using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA). Groups on differentiation media were compared with a Kruskall-Wallis H test and post-hoc Mann-Whitney U test. For both tests p < 0.05 was considered to indicate statistically significant differences. The graphs are Box-Whisker plots, with the box representing the middle two quartiles (25–75) and the Whiskers the highest and lowest value. All outlier variables were included in the statistical analyses but excluded in the graphical display.

Histological examination of the adolescent hamstring tendon explants confirmed normal tissue morphology. Specifically, no degenerative lesions, inflammatory cell infiltration, (partial) ruptures, chondroid metaplasia, or calcifications were seen.

During the explant culture period, proliferating cells (Ki-67 positive) were located between the highly organized collagen fibres of the tendon tissue and also in the connective tissue of the endotenon. These cells stained positive for fibroblast-marker D7-FIB. On the other hand, proliferating cells were also seen in the vascular walls, staining negative for D7-FIB but positive for α-SMA, a marker for pericytes and smooth muscle cells (Figure 1).

Cells explanted from the tendon tissue had a characteristic spindle-shaped fibroblastic morphology. Through the first four passages in monolayer culture all tendon-derived cells stained positive for D7-FIB but stained negative for α-SMA (Figure 1). On further characterization by FACS-analysis 99.1 +/- 1.1 % of the tendon-derived fibroblasts were CD34 negative and 72.6 +/- 22.9 % were CD105 positive (average of passage 2 to 5 tendon-derived cells, n = 4). The BMSCs had 99.7 +/- 0.4 % CD34 negative cells and 93.8 +/- 4.6 % CD105 positive cells (average of passage 1 to 5 BMSCs, n = 8).

Light microscopy revealed the presence of vacuoles within approximately one third of the cells in all adipogenic cultures of tendon-derived fibroblasts. Oil Red O staining confirmed that these were lipid vacuoles (Figure 2A). Only cells aggregated into clusters stained positively for lipid vacuoles. Tendon-derived fibroblasts cultured on control medium (Figure 2B), on osteogenic, or on chondrogenic medium (not shown) did not develop any lipid vacuoles. Cellular distribution of Oil Red O positive BMSCs cultured in adipogenic medium was more homogenous with approximately 75% of cells staining positively (results not shown).

In addition to this, culture of tendon-derived fibroblasts in adipogenic medium significantly upregulated expression of FABP4 and PPARG compared to those cultured in osteogenic (both p = 0.009) and chondrogenic medium (both p = 0.025). Similar findings were seen in the BMSC cultures although the difference in PPARG expression between the osteogenic and adipogenic medium condition did not reach statistical significance in the BMSC cultures (Figure 3). In the BMSC cultures PPARG expression was significantly higher in adipogenic medium compared to chondrogenic medium (p = 0.021); FABP4 expression was upregulated in the adipogenic medium compared to osteogenic medium (p = 0.021) and chondrogenic medium (p = 0.021).

Immunohistochemical staining for collagen type 2 was performed on tendon-derived fibroblasts cultured in chondrogenic, adipogenic, osteogenic, and control medium for 21 days. In all chondrogenic medium conditions approximately 5% of the cells stained positive for collagen type 2 (Figure 4A). Tendon-derived fibroblasts cultured in control medium (Figure 4B), as well as adipogenic and osteogenic medium were immunonegative for collagen type 2 (not shown). BMSC cultures showed a similar amount of collagen type 2 staining in chondrogenic medium (not shown).

Culture of tendon-derived fibroblast in chondrogenic medium significantly increased expression of chondrogenic markers COL2A1 and COL10A1 (the latter is considered to be a marker for hypertrophic cartilage formation) compared to the osteogenic condition (p = 0.025 for both genes) and adipogenic condition (p = 0.025 for both genes). Expression of SOX9 and AGC1 in chondrogenic medium compared to osteogenic and adipogenic medium was not significantly different (Figure 5).

BMSC cultures also showed a significantly higher expression of COL2A1 and COL10A1 in chondrogenic medium compared to osteogenic medium (p = 0.014 for both genes) and adipogenic medium (p = 0.028 for COL2A1 and p = 0.009 for COL10A1). SOX9 expression in BMSCs showed the same trend as in the tendon-derived fibroblast cultures, but the differences only reached significance in the BMSC cultures (osteogenic versus adipogenic medium p = 0.014 and osteogenic versus chondrogenic medium p = 0.014). Expression of AGC1 in the BMSCs did not differ significantly between the three medium conditions. Interestingly, BMSCs cultured in osteogenic medium had significantly upregulated COL10A1 compared to the adipogenic condition (p = 0.027). This phenomenon was not seen in the tendon-derived fibroblasts (Figure 5).

Von Kossa staining of tendon-derived fibroblasts in the osteogenic condition showed clustered areas of calcium deposition, whereas the tendon-derived fibroblast cultures in control medium had no calcium deposition (Figure 6). Also, tendon-derived fibroblast cultures in adipogenic and chondrogenic medium remained negative for calcium (results not shown). Similarly, in BMSC cultures, calcium deposition was found only in the osteogenic condition (not shown).

Although we did find expression of osteogenic markers RUNX2, osterix, and osteocalcin, culture of tendon-derived fibroblasts in osteogenic medium did not induce statistically significant upregulation of any of these genes. Similar results were found by QPCR of these markers in BMSCs cultured in osteogenic medium (Figure 7). In the tendon-derived fibroblast cultures SP7 and RUNX2 (both also known to play an important role in chondrogenic differentiation and hypertrophic cartilage formation [26]) were significantly upregulated in the chondrogenic medium compared to the osteogenic (p = 0.025 for both genes) and adipogenic medium (p = 0.025 for both genes)(Figure 7). BMSCs also showed an upregulation of SP7 and RUNX2 in the chondrogenic medium. RUNX2 upregulation was significant (p = 0.016 for the difference in gene expression of RUNX2 between adipogenic and chondrogenic medium in BMCSs), but SP7 upregulation in chondrogenic medium did not reach significance (Figure 7). In summary, chondrogenic medium not only stimulated expression of chondrogenic marker COL2A1, but also of COL10A1, RUNX2, and SP7.