Enhancing tendon-bone integration and healing with advanced multi-layer nanofiber-reinforced 3D scaffolds for acellular tendon complexes

Abstract

Advancements in tissue engineering are crucial for successfully healing tendon-bone connections, especially in situations like anterior cruciate ligament (ACL) restoration. This study presents a new and innovative three-dimensional scaffold, reinforced with nanofibers, that is specifically intended for acellular tendon complexes. The scaffold consists of a distinct layered arrangement comprising an acellular tendon core, a middle layer of polyurethane/type I collagen (PU/Col I) yarn, and an outside layer of poly (L-lactic acid)/bioactive glass (PLLA/BG) nanofiber membrane. Every layer is designed to fulfill specific yet harmonious purposes. The acellular tendon core is a solid structural base and a favorable environment for tendon cell functions, resulting in considerable tensile strength. The central PU/Col I yarn layer is vital in promoting the tendinogenic differentiation of stem cells derived from tendons and increasing the expression of critical tendinogenic factors. The external PLLA/BG nanofiber membrane fosters the process of bone marrow mesenchymal stem cells differentiating into bone cells and enhances the expression of markers associated with bone formation. Our scaffold's biocompatibility and multi-functional design were confirmed through extensive in vivo evaluations, such as histological staining and biomechanical analyses. These assessments combined showed notable enhancements in ACL repair and healing. This study emphasizes the promise of multi-layered nanofiber scaffolds in orthopedic tissue engineering and also introduces new possibilities for the creation of improved materials for regenerating the tendon-bone interface.

Keywords: Acellular tendon complexes; Anterior cruciate ligament; Bioactive glass; Nanofiber reinforcement; Tendon-bone healing.

Graphical abstract

Scheme 1

Schematic illustration of the fabrication process and potential application of multi-layer nanofiber-reinforced three-dimensional scaffold for acellular tendon complex.

Fig. 1

Characterization of decellularized tendons and normal tendons. (a) Decellularized tendon H&E staining. (b) Normal tendon H&E staining. (c) Comparison of DNA content between decellularized tendons and normal tendons, (***P < 0.001). (d) The results of agarose gel electrophoresis.

Fig. 2

Characterization of PU/Col I yarn. (a) The SEM images of PU/Col I yarn. (b) Fluorescence micrographs showing the morphologies of TDSCs after co-culturing with the PU/Col I yarn for 3 days. The cells demonstrate a morphology characterized by elongation. (c) The contact angle of PU yarn and PU/Col I yarn. The contact angle of the PU yarn remained consistently stable at 123° ± 7°, while the angle of PU/Col I yarn was significantly decreased to 36° ± 6° within 10 s. (d) The proliferation of TDSCs on the different samples tested by CCK-8 assay on days 1, 3, 5, and 7. The addition of Col I enhances the cell proliferation of yarn. (e, f) qRT-PCR results detecting the tendinogenesis-related genes (SCX, TNMD, and COL I) expression of TDSCs cultured with different samples. The addition of Col I enhances the expression of TNMD and COL I. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3

Characterization of PLLA/BG nanofiber membrane. (a) The SEM and TEM images of PLLA/BG nanofiber membrane. (b) Fluorescence micrographs showing the morphologies of BMSCs after co-culturing with the PLLA/BG nanofiber membrane for 3 days. The cells were spread on the membrane. (c) The proliferation of BMSCs on the different samples tested by CCK-8 assay on days 1, 3, and 5. The proliferation of BMSCs seeded on the membrane with a 2 % concentration of BG is better than that on the membranes with 1 % and 3 % concentration of BG on days 3 and 5. The membrane with a 2 % concentration of BG exhibits the most pronounced enhancement in cell proliferation. (d) Quantitative analysis of APL shows the expression of ALP in the different groups. The 2 % concentration of BG demonstrates a superior effect in promoting ALP expression compared to concentrations of 0 % and 1 % BG. (e, f) qRT-PCR results detecting the osteogenic gene (ALP, RUNX2, and OPN) expression of BMSCs cultured with different PLLA/BG nanofiber membranes containing different BG concentrations. The 2 % concentration of BG demonstrates remarkable efficacy in promoting the expression of osteogenic genes in BMSCs. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 4

The SEM images of multi-layer nanofiber-reinforced three-dimensional scaffold for acellular tendon complex. (a) The SEM image of a cross section of tendon complexus. (b) The SEM image of cross section of the PLLA/BG nanofiber membrane. (c) The SEM image of a cross section of PU/Col I yarn. (d) The SEM image of a cross section of the acellular tendon.

Fig. 5

The images and analysis of Micro-CT test in different groups at 4 and 8 weeks. (a) The images of Micro-CT test in different groups at 4 and 8 weeks. (b, c) Bone regeneration of bone tunnel in tibia and femur. *P < 0.05 relative to the Control and ATendon groups. They show that the Complexus group has a significant difference with the Control group and ATendon group in bone regeneration of the tibia and femur at 4 weeks, but there is no significant difference in BV/TV results of tibia and femur at 8 weeks. (*P < 0.05).

Fig. 6

Biomechanical tests of the scaffolds at 4- and 8-week post in-vivo implantation. (a, b) Photographs showing the biomechanical test procedure of the samples explanted at 4 or 8 weeks. (c) Ultimate failure load of the different samples. (d) Young's modulus of the different samples. (*P < 0.05, **P < 0.01).

Fig. 7

H&E, Masson and immunofluorescence staining of the samples. (a) H&E and Masson staining of the samples. The Control group exhibited the formation of a robust osseous tunnel, while both the decellularized tendon group and Complexus group demonstrated successful integration of the graft with the surrounding bone tissue. (b) The modified tendon-to-bone maturity score of H&E and Masson staining. ***P < 0.001 as compared with the ATendon and Conplexus groups. The Complexus group resulted in a more mature attachment at 8 weeks of healing compared with ATendon group. (c) Immunofluorescence staining for OCN of the samples at 8 weeks. (d) Semi-quantitative analysis of immunofluorescence staining for OCN. ***P < 0.001 relative to the Control and Complexus groups. The Complexus group and Control group express more OCN protein than the ATendon group. (B: bone; I: interface; T: tendon; **P < 0.01, ***P < 0.001).

Fig. 8

Immunohistochemical and immunofluorescent staining of the samples and semi-quantitative analysis. (a) Immunohistochemistry staining of samples for type I collagen (COL I). (b) Semi-quantitative analysis of Immunohistochemistry staining for COL I. The Complexus group expresses more COL I than the ATendon group and Control group. (c) Immunofluorescence staining for TNC of the samples at 8 weeks. (d) Semi-quantitative analysis of immunofluorescence staining for TNC. ***P < 0.001 as compared with the ATendon and Conplexus groups. The Complexus group expresses more TNC protein than the ATendon group and Control group. (*P < 0.05, **P < 0.01, ***P < 0.001).