Integrating electrospun aligned fiber scaffolds with bovine serum albumin-basic fibroblast growth factor nanoparticles to promote tendon regeneration

Abstract

Background: Electrospun nanofiber scaffolds have been widely used in tissue engineering because they can mimic extracellular matrix-like structures and offer advantages including high porosity, large specific surface area, and customizable structure. In this study, we prepared scaffolds composed of aligned and random electrospun polycaprolactone (PCL) nanofibers capable of delivering basic fibroblast growth factor (bFGF) in a sustained manner for repairing damaged tendons.

Results: Aligned and random PCL fiber scaffolds containing bFGF-loaded bovine serum albumin (BSA) nanoparticles (BSA-bFGF NPs, diameter 146 ± 32 nm) were fabricated, respectively. To validate the viability of bFGF-loaded aligned PCL nanofiber scaffold (aPCL + bFGF group) in tendon tissue engineering, we assessed the in vitro differentiation of human amniotic mesenchymal stem cells (hAMSCs) towards a tenogenic lineage and the in vivo regeneration of tendons using a rat Achilles tendon defect model. The encapsulated bFGF could be delivered in a sustained manner in vitro. The aPCL + bFGF scaffold promoted the in vitro differentiation of human amniotic mesenchymal stem cells (hAMSCs) towards a tenogenic lineage. In the repair of a rat Achilles tendon defect model, the aPCL + bFGF group showed a better repair effect. The scaffold offers a promising substrate for the regeneration of tendon tissue.

Conclusions: The aligned and random PCL fiber scaffolds containing bFGF nanoparticles were successfully prepared, and their physical and chemical properties were characterized. The aPCL + bFGF scaffold could promote the expression of the related genes and proteins of tendon-forming, facilitating tendon differentiation. In the rat Achilles tendon defect experiments, the aPCL + bFGF exhibited excellent tendon regeneration effects.

Keywords: Drug release; Electrospun nanofiber; Fiber orientation; Tendon repair; Tissue engineering.

Fig. 1

Schematic illustration indicating the fabrication of the tissue engineered electrospun nanofiber scaffold for repairing tendon injury. A Schematic illustration of the preparation of chitosan-stabilized bFGF-loaded electrospun PCL nanofiber scaffold. B Schematic illustration indicating aligned PCL nanofiber scaffold seeded with hAMSCs was transplanted to repair injured Achilles tendon

Fig. 2

Morphological analysis of nanoparticles and nanofibers. A and D are TEM images of BSA NPs and BSA-bFGF NPs, respectively, and B and E are the particle size distribution charts of corresponding nanoparticles measured by DLS; C and F are TEM images of nanofibers, containing BSA NPs and BSA-bFGF NPs, respectively

Fig. 3

The characterization of morphology, diameter distribution, and orientation distribution of the nanofiber scaffolds by SEM. The SEM micrographs of A, E aPCL + bFGF group, B, F rPCL + bFGF group, C, G aPCL group, and D, H rPCL group at different magnifications. I–L The fiber diameter distribution and M–P the fiber orientation distribution corresponding to (A–D), respectively

Fig. 4

Biocompatibility, cumulative drug release profile, and mechanical properties of the electrospun nanofiber scaffolds. A Proliferation activity of hAMSCs incubated on the different types of nanofiber scaffolds detected by CCK-8 assay. B Cumulative release curves of bFGF-loaded nanofiber scaffolds. C Stress–strain curves of the different types of nanofiber scaffolds

Fig. 5

Cell morphology and mRNA and protein expression of tendon differentiation of hAMSCs in vitro. A Fluorescence micrographs of live and dead cells staining in each group (scale bar = 100 μm). B mRNA expression levels of tendon-related genes after incubating hAMSCs on different scaffolds for 7 and 14 days. C After culturing hAMSCs on different scaffolds for 14 days, Western blotting was used to detect the expression of SCX and TNMD, and the quantitative analysis of the expression of SCX and TNMD according to the images and normalized to β-actin. The data are expressed as mean ± SD, *p < 0.05

Fig. 6

Surgical procedures and macroscopic evaluation of regenerated tendon. A Surgical procedures of Achilles tendon defect model (a) exposed Achilles tendon, (b) defect caused by no suture, (c) Achilles tendon suture, (d) PCL-hAMSCs construct implantation, and (e) skin suture. B, C The general shape and anatomical images of the regenerated Achilles tendon at 4 and 8 weeks after surgery. D Quantitative analysis of histological results, including the macroscopical scores, thickness, and weight of tendon

Fig. 7

Sagittal T2-weighted MRI scan results of regenerated Achilles tendon and related quantitative analysis. A MRI scan results in each group at day 3, week 4 and 8. Regenerated Achilles tendon structure (red arrow indicates tendon area and blue arrow indicates implant construct). B Quantitative measurement of high- or intermediate-intensity signal area (AHS) and total longitudinal area (AT) within the Achilles tendon region. C The AHS/AT ratio analysis and the ratio of the intensity of intra-tendon lesions based on the background and the results were normalized to the level of the control group; mean anteroposterior analysis. The data were expressed as mean ± SD, *p < 0.05

Fig. 8

The immunofluorescence results of regenerated tendons in each group 8 weeks after surgery. A Immunofluorescence micrographs indicating the expression levels of Col-I, COL-III, FN1, TNC and TNC in different groups. The red fluorescence showed the expression of the protein, and the blue showed the nucleus (scale bar = 100 μm). B Quantification of the expressions of the corresponding proteins in each group by measuring the integrated optical density (IOD); C Protein expression level and quantification of SCX and TNMD at 8 weeks after surgery, ∗ p < 0.05

Fig. 9

Representative histological staining of normal control tendons and regenerated tendons. A Results of hematoxylin–eosin (HE), Masson's trichrome, Sirius red staining and polarized light observation. (Scale bar = 50 μm). B Histological scores were detected by quantitative analysis of regenerated Achilles tendon 8 weeks after surgery; Quantification of collagen fibers in Masson staining and in polarized light observation of Sirius red staining, *p < 0.05