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, 20 (21)

Transforming Growth Factor Beta 3-Loaded Decellularized Equine Tendon Matrix for Orthopedic Tissue Engineering

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Transforming Growth Factor Beta 3-Loaded Decellularized Equine Tendon Matrix for Orthopedic Tissue Engineering

Susanne Pauline Roth et al. Int J Mol Sci.

Abstract

Transforming growth factor beta 3 (TGFβ3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGFβ3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGFβ3 in an in vitro model. TGFβ3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGFβ3 were not detected in the rinsing fluid of the TGFβ3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGFβ3 to assess its bioactivity. Both scaffold-associated TGFβ3 and TGFβ3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGFβ3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaffold-associated TGFβ3 is preserved, thus TGFβ3 application via absorption in decellularized tendon scaffolds is a feasible approach.

Keywords: horse; multipotent mesenchymal stromal cells (MSC); regeneration; scaffold; surface coating; tendon; tissue engineering; transforming growth factor beta 3 (TGFβ3).

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Score points for scaffold morphology (a), cell distribution (b), and cell integration (c) of MSC-seeded tendon scaffolds normalized to the respective internal controls (w/o TGFβ3; indicated by the horizontal line intersecting the x-axis at 1.0). MSC-seeded tendon scaffolds were either directly loaded with TGFβ3 (scaffold-associated) or TGFβ3 was added as a cell culture medium supplement (dissolved). The morphology of MSC-seeded scaffolds (a) in terms of scaffold contraction was assessed macroscopically. Hematoxylin- and eosin-stained paraffin sections of MSC-seeded scaffolds were microscopically evaluated for cell distribution (b) and cell integration (c) (10× objective). The scoring systems used are given in the Materials and Methods section. The total number of scaffolds within each group was 42 (n = 42). As an exception to this, for the evaluation of cell distribution (b) and cell integration (c), there were altered numbers of scaffolds due to the technical processing (scaffolds directly loaded with TGFβ3 at day 3: n = 41; internal control scaffolds (w/o TGFβ3) in the group of directly loaded scaffolds at day 3: n = 39; scaffolds receiving dissolved TGFβ3 at day 5: n = 41). Bars indicate the normalized median values and error bars the 95% confidence interval; * represents significant differences compared to the corresponding untreated control group (w/o TGFβ3) (p < 0.05).
Figure 2
Figure 2
Microscopic appearance of MSC-seeded tendon scaffolds treated with TGFβ3 (+TGFβ3) and the respective internal control scaffolds (w/o TGFβ3). Representative images of hematoxylin- and eosin-stained paraffin sections of MSC-seeded tendon scaffolds (ad, left) and of corresponding LIVE/DEAD®-stained MSC-seeded tendon scaffolds (ad, right). The panel of LIVE/DEAD®-stained scaffolds shows vital cells in green and cells with defect cellular membranes in red. MSC-seeded tendon scaffolds were either directly loaded with TGFβ3 (scaffold-associated) (a), or TGFβ3 was applied as a standard cell culture medium supplement (dissolved) (c). Respective internal control scaffolds—(b) internal control for scaffolds directly loaded with TGFβ3; (d) internal control for scaffolds that received TGFβ3 as a standard cell culture medium supplement—were not treated with TGFβ3 (w/o TGFβ3). Note the obvious alterations of the scaffold morphology (left panel of hematoxylin- and eosin-stained sections), illustrating the increased cell-mediated scaffold contractions in the presence of TGFβ3 regardless of the route of application ((a) directly applied TGFβ3 and (c) TGFβ3 applied as a cell culture medium supplement). All images shown were taken after 5 days from tendon scaffolds that were seeded with MSC from the same donor horse.
Figure 3
Figure 3
Quantitative image analysis results of LIVE/DEAD®-stained, MSC-seeded tendon scaffolds. Values of the cell shape measurements (a) and of the numbers of viable cells (b) were normalized to the corresponding internal controls (w/o TGFβ3; indicated by the horizontal line intersecting the x-axis at 1.0). Higher values of the cell shape measurement in (a) correspond to more elongated cells. MSC-seeded tendon scaffolds were either directly loaded with TGFβ3 (scaffold-associated) or TGFβ3 was supplemented via the cell culture medium (dissolved). The total number of scaffolds within each group was 42 (n = 42). Bars represent the normalized median values and error bars the 95% confidence interval; one extreme outlier value was removed (scaffold-bound TGFβ3 group in (b), day 3) before plotting the graph; * illustrates significant differences compared to the respective untreated control group (w/o TGFβ3) (p < 0.05).
Figure 4
Figure 4
Gene expression levels of tendon extracellular matrix (ECM) molecules (a) and intracellular tendon markers (b) in tendon scaffold-seeded MSC. Data are given as “fold change” (FC) to the respective internal control (w/o TGFβ3), which is displayed as a horizontal line intersecting the x-axis at zero. Tendon scaffolds seeded with MSC were either directly loaded with TGFβ3 (scaffold-associated) or supplemented with TGFβ3 added to the cell culture medium (dissolved). Part (a) includes the gene expression of collagen 1A2 (Col1A2), collagen 3A1 (Col3A1), decorin (DCN), and tenascin c (TNC), as well as the expression of osteopontin (OPN), which is related to osteogenic differentiation. Part (b) shows the gene expression of scleraxis (SCX), smad8 (SMAD8), and mohawk (MKX). The total number of scaffolds within each group was 42 (n = 42). Bars represent the median fold changes values and error bars the 95% confidence interval; * illustrates significant differences compared to the untreated control (w/o TGFβ3) (p < 0.05).

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