Pentamidine-loaded gelatin decreases adhesion formation of flexor tendon

Abstract

Background: Prevention of adhesion formation following flexor tendon repair is essential for restoration of normal finger function. Although many medications have been studied in the experimental setting to prevent adhesions, clinical application is limited due to the complexity of application and delivery in clinical translation.

Methods: In this study, optimal dosages of gelatin and pentamidine were validated by gelatin concentration test. Following cell viability, cell migration, live and dead cell, and cell adhesion assay of the Turkey tenocytes, a model of Turkey tendon repair was established to evaluate the effectiveness of the Pentamidine-Gelatin sheet.

Results: Pentamidine carried with gelatin, a Food and drug administration (FDA) approved material for drug delivery, showed good dynamic release, biocompatibility, and degradation. The optimal dose of pentamidine (25ug) was determined in the in vivo study using tenocyte viability, migration, and cell adhesion assays. Further biochemical analyses demonstrated that this positive effect may be due to pentamidine downregulating the Wnt signaling pathway without affecting collagen expression.

Conclusions: We tested a FDA-approved antibiotic, pentamidine, for reducing adhesion formation after flexor tendon repair in both in vitro and in vivo using a novel turkey animal model. Compared with the non-pentamidine treatment group, pentamidine treated turkeys had significantly reduced adhesions and improved digit function after six weeks of tendon healing.

The translational potential of this article: This study for the first time showed that a common clinical drug, pentamidine, has a potential for clinical application to reduce tendon adhesions and improve tendon gliding function without interfering with tendon healing.

Keywords: Adhesion; Flexor tendon repair; Gelatin; Pentamidine; Turkey model.

Graphical abstract

Fig. 1

The cell migration test of pentamidine in turkey tenocytes. (A) The overview of turkey tenocytes treated with different doses of pentamidine (0–50 μg/mL) at 0 h to 48 h in IncuCyte® S3 Live Cell Analysis System. Scale bar: 800 μm. (B) The cell migration test showed the 40 and 50 μg/mL pentamidine groups could significantly reduce the cell migration compare with 0 μg/mL pentamidine groups at 48 h post-seeded.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2

The live/dead and adhesion assay of pentamidine in turkey tenocytes. (A) Tenocytes viability was assessed and visualized by live/dead assay with fluorescent dyes to distinguish live (green) and dead cells (red) at 24 h post-seeded in IncuCyte® S3 Live Cell Analysis System. Scale bar: 200 μm. (B) The quantitative analysis showed high dose of pentamidine (>45 μg/mL) could significantly reduce the cell viability compare with 0 μg/mL pentamidine group. (C) The cell adhesion assay showed 100–200 μg/mL pentamidine could significantly reduce the cell adhesion compare with 0 μg/mL pentamidine at Fibronectin, Collagen I, Collagen IV, and Fibrinogen groups. BSA group was as a negative control. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The overview and SEM of pentamidine-gelatin patch. The scan electron microscopy showed the patch of gelatin only group had a smooth surface without particles. While the gelatin patch with 25 μg/mL pentamidine group had a rough surface with many pentamidine particles clustered together.

Fig. 4

Pentamidine-gelatin patch decreases adhesion formation and WOF after flexor tendon repair. (A) The suture method of tendon repair was modified Pennington technique (Red line) with circumferential running suture (Green line). (B, C) The surgical procedure and casting process of in vivo study. (D) The overview of the surface of FDP tendon. (E) The adhesion score in Pentamidine group was significantly lower than that in Control group and Gelatin group. (F, G) WOF in the Pentamidine group was significantly lower than that in the Control group. (H) No difference was found in frictional force among each group. (I, J) No difference was found in frictional force among each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Expression signatures of differential expression genes in injury tendons and normal tendons. (A) Chromosome distribution showed the numbers of differential expression genes located at different chromosomes. (B) GO analysis of differential expression genes. BP, biological processes; CC, cellular component; GO, gene ontology; (C) PPI network and the core genes of the differential methylation genes. (D) Representative heat map of the 131 Wnt-related differential expression genes.

Fig. 6

Validation of the differential expression genes identified in bioinformation analysis by qRT-PCR. (A) The expression of Wnt-related differential expression genes in turkey tenocytes. (B) The expression of Wnt-related differential expression genes in turkey tissue with different treatments. (C) The expression of collagen-related genes in turkey tissue with different treatments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7

Histology results of healing tendons in different groups at 6 weeks. (A) Representative H&E staining images at 6 weeks after surgery. (B) Representative Masson staining images at 6 weeks after surgery. (C) Representative Sirius red staining images at 6 weeks after surgery. Scale bar: 200 μm. Narrow showed running suture. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Immunohistochemistry results of healing tendons in different groups at 6 weeks. (A) Immunofluorescence staining of COL1A2, COL3A1, Wnt7a, and Wnt11 at 6 weeks after surgery. (B) The quantitative analysis showed there were no significant changes in COL1A2 and COL3A1 among each group. However, the expression of Wnt7A and Wnt11 was significantly decreased in the Gelatin plus 25 μg/mL Pentamidine group compared to Control or Repair with Gelatin group. Scale bar: 100 μm *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.