Influence of nanofibers on growth and gene expression of human tendon derived fibroblast

Abstract

Background: Rotator cuff tears are a common and frequent lesion especially in older patients. The mechanisms of tendon repair are not fully understood. Common therapy options for tendon repair include mini-open or arthroscopic surgery. The use of growth factors in experimental studies is mentioned in the literature. Nanofiber scaffolds, which provide several criteria for the healing process, might be a suitable therapy option for operative treatment. The aim of this study was to explore the effects of nanofiber scaffolds on human tendon derived fibroblasts (TDF's), as well as the gene expression and matrix deposition of these fibroblasts.

Methods: Nanofibers composed of PLLA and PLLA/Col-I were seeded with human tendon derived fibroblasts and cultivated over a period of 22 days under growth-inductive conditions, and analyzed during the course of culture, with respect to gene expression of different extra cellular matrix components such as collagens, bigylcan and decorin. Furthermore, we measured cell densities and proliferation by using fluorescence microscopy.

Results: PLLA nanofibers possessed a growth inhibitory effect on TDF's. Furthermore, no meaningful influence on the gene expression of collagen I, collagen III and decorin could be observed, while the expression of collagen X increased during the course of cultivation. On the other hand, PLLA/Col-I blend nanofibers had no negative influence on the growth of TDF's. Furthermore, blending PLLA nanofibers with collagen had a positive effect on the gene expression of collagen I, III, X and decorin. Here, gene expression indicated that focal adherence kinases might be involved.

Conclusion: This study indicates that the use of nanofibers influence expression of genes associated with the extra cellular matrix formation. The composition of the nanofibers plays a critical role. While PLLA/Col-I blend nanofibers enhance the collagen I and III formation, their expression on PLLA nanofibers was more comparable to controls. However, irrespective of the chemical composition of the fibres, the collagen deposition was altered, an effect which might be associated with a decreased expression of biglycanes.

Figure 1

Fiber characterization. SEM analysis of PLLA (A) and PLLA/Col-I nanofibers (B).

Figure 2

Influence of PLLA and PLLA/Col-I nanofibers on growth of TDF's. Area of FDA positive cells cultured over a period of 4 days (A) and relative CCND expression (B). Immunofluorescence microphotographs of FDA staining of TDF's cultured on glass (C), PLLA (D) and PLLA/Col-I (E) nanofiber scaffolds.

Figure 3

Influence of PLLA and PLLA/Col-I nanofibers on gene expression Col-I deposition of TDF's. Time course of collagen-I gene expression of TDF's on nanofibers compared to cover slips control (A). Immunofluorescence microphotographs of Col-I (red) deposition after 22 days of culture cover slip control (B), PLLA nanofibers (C) and PLLA/Col-I nanofibers (D).

Figure 4

Influence of PLLA and PLLA/Col-I nanofibers on gene expression Col-I deposition of TDF. Time course of collagen-III (A) and collagen-X (B) gene expression of TDF's cultured on PLLA and PLLA/Col-I nanofibers as compared to cover slips control.

Figure 5

Influence of PLLA and PLLA/Col-I nanofibers on gene expression proteoglycanes. Time course of decorin (A) and biglycan (B) gene expression of TDF's cultured on PLLA and PLLA/Col-I nanofibers as compared to cover slips control.

Figure 6

Influence of PLLA and PLLA/Col-I nanofibers on gene expression genes involved in integrin signalling. Gene expression of FAK, PYK and PI3K after 4 days of incubation PLLA and PLLA/Col-I as compared to cover slip control.