Gene-Loaded Nanoparticle-Coated Sutures Provide Effective Gene Delivery to Enhance Tendon Healing

Abstract

How to accelerate tendon healing remains a clinical challenge. In this study, a suture carrying nanoparticle/pEGFP-basic fibroblast growth factor (bFGF) and pEGFP-vascular endothelial growth factor A (VEGFA) complexes was developed to transfer the growth factor genes into injured tendon tissues to promote healing. Polydopamine-modified sutures can uniformly and tightly absorb nanoparticle/plasmid complexes. After tendon tissues were sutured, the nanoparticle/plasmid complexes still existed on the suture surface. Further, we found that the nanoparticle/plasmid complexes delivered into tendon tissues could diffuse from sutures to tendon tissues and effectively transfect genes into tendon cells, significantly increasing the expression of growth factors in tendon tissues. Finally, biomechanical tests showed that nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated sutures could significantly increase the ultimate strengths of repaired tendons, especially at 4 weeks after operation. Two kinds of nanoparticle/plasmid complex-coated sutures significantly increased flexor tendon healing strength by 3.7 times for Ethilon and 5.8 times for PDS II, respectively, compared with the corresponding unmodified sutures. In the flexor tendon injury model, at 6 weeks after surgery, compared with the control suture, the nanoparticle/plasmid complex-coated sutures can significantly increase the gliding excursions of the tendon and inhibit the formation of adhesion. These results indicate that this nanoparticle/plasmid complex-coated suture is a promising tool for the treatment of injured tendons.

Keywords: gene delivery; growth factors; nanoparticle; surgical suture; tendon repair.

Graphical abstract

Figure 1

Schematic Diagram of the Process of Preparing the Nanoparticle/Plasmid Complex-Coated Sutures for Repairing the Tendon (A) The preparation process of nanoparticle/plasmid complexes. (B) The preparation process of nanoparticle/plasmid complex-coated sutures. (C) The mechanism of the nanoparticle/plasmid complex-coated sutures for promoting tendon healing.

Figure 2

Characteristics of Nanoparticle/Plasmid Complexes (A) Typical scanning electron microscopy images of the nanoparticle/plasmid complexes. (B) Typical TEM images of the nanoparticle/plasmid complexes. (C) Hydrodynamic diameter distributions of the nanoparticle/plasmid complexes. (D) Gel retardation assay of nanoparticle/plasmid complexes at different N/P ratios. Typical fluorescent images of EGFP expression (E) and corresponding bright images (F) in chicken tenocytes transfected with naked plasmid. Typical fluorescent images of EGFP expression (H) and corresponding bright images (I) in chicken tenocytes transfected with nanoparticle/plasmid complexes. Typical fluorescent images of EGFP expression (K) and corresponding bright images (M) in rat tenocytes transfected with naked plasmid. Typical fluorescent images of EGFP expression (N) and corresponding bright images (O) in rat tenocytes transfected with nanoparticle/plasmid complexes. The scale bar represents 200 μm. Typical flow cytometry analysis of chicken tenocytes (G) or rat tenocytes (M) treated with naked plasmid. Typical flow cytometry analysis of chicken tenocytes (J) or rat tenocytes (P) treated with nanoparticle/plasmid complexes.

Figure 3

Typical Scanning Electron Microscopy Images of Sutures (A and B) Scanning electron microscopy images of two kinds of sutures, Ethilon (A) and PDS II (B), under different conditions (unmodified sutures, polydopamine-modified sutures, nanoparticle/plasmid complex-coated sutures, and stitched nanoparticle/plasmid complex-coated sutures).

Figure 4

Characteristics of Nanoparticle/Plasmid Complex-Coated Sutures (A and B) In vitro release profiles of pEGFP-bFGF and pEGFP-VEGFA from nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon (A) and PDS II (B) sutures in PBS (pH 7.4). (C–F) In vitro cytotoxicity of nanoparticle/pEGFP-N1 complex-coated Ethilon (C and E) and PDS II (D and F) sutures against tenocytes of chicken at 48 and 72 h after treatment. Compared with the control group, polydopamine-modified sutures could significantly increase cell viability (*p < 0.05), whereas polydopamine-modified sutures loaded with nanoparticle/plasmid complexes had no significant effect on cell viability.

Figure 5

Gene Delivery of Nanoparticle/Plasmid Complex-Coated Sutures Representative EGFP expression images of two kinds of sutures, Ethilon (A) and PDS II (B), in the tendon tissues treated with unmodified sutures or nanoparticle/plasmid complex-coated sutures (NP/plasmid sutures) at 1 week after surgery. Scale bars represent 50 μm.

Figure 6

Effects of Nanoparticle/Plasmid Complex-Coated Sutures on bFGF and VEGFA Protein Expression (A and C) The typical bFGF and VEGFA protein bands of western blotting assay in the injured tendons at 2 and 3 weeks after treatment with nanoparticle/plasmid complex-coated Ethilon (A) and PDS II (C) sutures. (B and D) The expression levels of bFGF and VEGFA protein in the nanoparticle/plasmid complex-coated Ethilon (B) and PDS II (D) sutures group were significantly higher than that in corresponding unmodified sutures group. Statistically significant differences are as indicated (p value).

Figure 7

Effects of Nanoparticle/Plasmid Complex-Coated Sutures on the Functional Recovery and Healing Strengths of Repaired Tendons The tendon gliding excursions (A), work of toe flexion (B), and adhesion scores (C) in the unmodified Ehilon sutures group or nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon sutures (NP/plasmid sutures) group at 6 weeks after treatment. The tendon gliding excursions (D), work of toe flexion (E), and adhesion scores (F) in the unmodified sutures PDS II group or nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated or PDS II sutures (NP/plasmid sutures) group at 6 weeks after treatment. (G and I) The ultimate strengths of the repaired tendons treated with the unmodified sutures or nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon (G) or PDS II (I) sutures at weeks 1, 2, 4, and 6 after surgery in the chicken flexor tendon injury model. (H and J) The ultimate strengths of the repaired tendons treated with the unmodified sutures or nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon (H) or PDS II (J) sutures at weeks 1, 2, and 3 after operation in the rat Achilles tendon injury model. Statistically significant differences are as indicated (p value).

Figure 8

Effects of Nanoparticle/Plasmid Complex-Coated Sutures on Adhesion Information and Tissue Reactions Typical images of adhesion morphology in the unmodified sutures group or nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon (A) or PDS II (B) sutures (NP/plasmid sutures) group at 6 weeks after treatment. Histology of tendons treated with nanoparticle/pEGFP-bFGF and pEGFP-VEGFA complex-coated Ethilon (C) or PDS II (D) sutures. Scale bars represent 100 μm.