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. 2023 Jan 13:2023:2915826.
doi: 10.1155/2023/2915826. eCollection 2023.

Micropattern Silk Fibroin Film Facilitates Tendon Repair In Vivo and Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells through the α 2 β 1/FAK/PI3K/AKT Signaling Pathway In Vitro

Kang Lu  1 , Hong Tang  2 , Yang Wang  1 , Liyuan Wang  1 , Mei Zhou  2 , Gang He  2 , Hao Lu  3 , Chuyue Tang  2 , Wan Chen  2 , Xiaoqing Ma  4 , Kanglai Tang  2 , Zhongliang Deng  1
Affiliations

Micropattern Silk Fibroin Film Facilitates Tendon Repair In Vivo and Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells through the α 2 β 1/FAK/PI3K/AKT Signaling Pathway In Vitro

Kang Lu et al. Stem Cells Int. .

Abstract

Background: Tendon injuries are common clinical disorders. Due to the limited regeneration ability of tendons, tissue engineering technology is often used as an adjuvant treatment. This study explored the molecular pathways underlying micropattern SF film-regulated TSPC propensity and their repairing effects to highlight the application value of micropattern SF films.

Methods: First, we characterized the physical properties of the micropattern SF films and explored their repairing effects on the injured tendons in vivo. Then, we seeded TSPCs on SF films in vitro and determined the micropattern SF film-induced gene expression and activation of signaling pathways in TSPCs through high-throughput RNA sequencing and proteomics assays.

Results: The results of in vivo studies suggested that micropattern SF films can promote remodeling of the injured tendon. In addition, immunohistochemistry (IHC) results showed that tendon marker genes were significantly increased in the micropattern SF film repair group. Transcriptomic and proteomic analyses demonstrated that micropattern SF film-induced genes and proteins in TSPCs were mainly enriched in the focal adhesion kinase (FAK)/actin and phosphoinositide 3-kinase (PI3K)/AKT pathways. Western blot analysis showed that the expression of integrins α2β1, tenascin-C (TNC), and tenomodulin (TNMD) and the phosphorylation of AKT were significantly increased in the micropattern SF film group, which could be abrogated by applying PI3K/AKT inhibitors.

Conclusion: Micropattern SF films modified by water annealing can promote remodeling of the injured tendon in vivo and regulate the tendon differentiation of TSPCs through the α2β1/FAK/PI3K/AKT signaling pathway in vitro. Therefore, they have great medical value in tendon repair.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The Achilles tendon defect model and macroscopic repair assessment. (a) Schematic diagram of the anatomy of the rat Achilles tendon and comparison of postoperative surgical areas in different repair groups. Group N: normal tendon (control group); Group D: defect suture group; Group R: allogeneic replacement repair group; Group S: smooth SF film repair group; Group G: micropattern SF film repair group. (b) MRI comparison of tendon repair after 4 and 8 weeks. The red arrow indicates the repaired area, and the repair signal intensity in group G was significantly higher than that in other groups at 8 weeks. (c) Comparison of the width and thickness of the Achilles tendon repair samples. The width and thickness of Group G were significantly lower than those of the other repair groups.
Figure 2
Figure 2
SEM, FTIR, and AFM characterization of SF films. (a) SEM. Group S: SF films without micropatterns; Group G: SF film with micropatterns. The surface of SF film material was clear and stable. (b) FTIR. The β-sheet content in micropattern SF film after water annealing was significantly increased. (c) AFM. The nanoscale roughness on the surface of the micropattern SF film after water annealing treatment was significantly increased.
Figure 3
Figure 3
Comparison of histological evaluation of different repair groups. (a) H&E staining. (b) Histological score. The repair score of Group G was higher than that in the other repair groups. (c) Results of IHC. (d) Statistics of IHC results. The tenogenic markers in Group G were significantly higher than those in the other repair groups.
Figure 4
Figure 4
Results of bioinformatics analysis. (a) The quadrant diagram, Venn diagram (Venn), and heatmap of combined analysis of transcriptomics and proteomics. (b) The transcriptome sequencing results, including GO and KEGG enrichment analyses. (c) The proteomic results, including GO and KEGG enrichment analyses. (d) Signaling pathway enrichment analysis of combined transcriptomic and proteomic sequencing results.
Figure 5
Figure 5
Validation of key proteins in signaling pathways. (a) Immunofluorescence results of α2β1. The expression level of Group G was significantly higher than that of group N. (b) Western blot analysis of integrin α2β1. α2β1 was significantly increased in Group G. (c) Western blot analysis of the PI3K/AKT signaling pathway. The TNC, TNMD, and phosphorylated PI3K/AKT (p-PI3K, p-AKT308, and p-AKT473) were significantly increased in Group G. After adding PI3K/AKT pathway inhibitors, the expression of TNC and TNMD was inhibited simultaneously with phosphorylated PI3K and AKT. (d) Schematic diagram of the signaling pathway of micropattern SF films regulating TSPCs.
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