Collagen type I and decorin expression in tenocytes depend on the cell isolation method

Abstract

Background: The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE) might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB) can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED) and cell migration (CM), are investigated on differences in gene expression and cell morphology.

Methods: Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan.

Results: The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC) using ED as isolation method (p < 0.05), however decorin expression was higher in TLCC using CM as isolation method (p < 0.05). Dedifferentiation potential seemed to be similar for both isolation techniques.

Conclusion: In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM) from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.

Figure 1

Yield of cells. Cell yield after both isolation methods and normalized to 1 g of native tendon tissue. Cells were counted using a heamocytometer and trypan blue. The amount of cells after 1 day using ED is 14 times higher than using CM after 50 days.

Figure 2

Migrating tenocytes of the LHB. The tendon slices were put into petri-dishes, incubated (37°C, 5% CO2) in cell culture medium (DMEM/HAM’s F12) and analyzed by light microscopy. An increasing amount of spindle-like cells could be observed at different time points. A = after 8 days, B = after 11 days, C = after 16 days D = after 22 days.

Figure 3

Cell morphology in TLCC. Tenocyte cell cultures at different time of subcultering. ED (enzymatic digestion) CM (cell migration). Cells of the second and third cell passage are shown. Cells show typical morpholpgy for tenocytes and no differences between both methods.

Figure 4

Cell proliferation. Absolute number of cells from passage 1 to 3 (n = 7). Proliferation was almost similar for TLCC of both isolation methods, however yield of cell and proliferation rate seemed to be more variable for ED. Cell number significantly increases from passage 1 to 3 for both groups, ED and CM.

Figure 5

RT-PCR analysis. Cells in the third cell passage were used. cDNA from other musculoskeletal cell types such as f, fibroblasts (passage 6), c, chondrocytes (passage 3) and o, osteoblasts (passage 2) were used for comparison. GAPDH was used for normalization. ED, enzymatic digestion, CM, cell migration. Each band represents one patient/LHB (n = 7).

Figure 6

Quantitative RT-PCR. RT-PCR was performed for collagen type I and decorin. Relative gene expression was estimated against cyclophilin B.