Effect of book-shaped acellular tendon scaffold with bone marrow mesenchymal stem cells sheets on bone-tendon interface healing

Abstract

Background: Tissue engineering has exhibited great effect on treatment for bone-tendon interface (BTI) injury. The aim of this study was to evaluate the effect of a book-shaped acellular tendon scaffold (ATS) with bone marrow mesenchymal stem cells sheets (MSCS) for BTI injury repair.

Methods: ATS was designed based on the shape of "book", decellularization effect was evaluated by Hematoxylin and eosin (H&E), 4', 6-diamidino-2-phenylindole (DAPI) and scanning electron microscopy (SEM), then bone marrow mesenchymal stem cells (MSCs) were cultured on ATS to assess the differentiation inductivity of ATS. A rabbit right partial patellotomy model was established, and MSCS seeded on ATS were implanted into the lesion site. The patella-patellar tendon (PPT) at 2, 4, 8 or 16 weeks post-operation were obtained for histological, biomechanical and immunofluorescence analysis.

Results: H&E, DAPI and SEM results confirmed the efficiency of decellularization of ATS, and their in vitro tenogenic and chondrogenic ability were successfully identified. In vivo results showed increased macrophage polarization toward the M2 phenotype, IL-10 expression, regenerated bone and fibrocartilage at the patella-patellar tendon interface of animals received MSCS modified ATS implantation. In addition, the level of tensile strength was also the highest in MSCS modified ATS implantation group.

Conclusion: This study suggests that ATS combined with MSCS performed therapeutic effects on promoting the regeneration of cartilage layer and enhancing the healing quality of patella-patellar tendon interface.

The translational potential of this article: This study showed the good biocompatibility of the ATS, as well as the great efficacy of ATS with MSCS on tendon to bone healing. The results meant that the novel book-shaped ATS with MSCS may have a great potential for clinical application.

Keywords: Bone marrow mesenchymal stem cell sheets; Book-shaped acellular tendon scaffold; Patella-patellar tendon interface; Regeneration; Tissue engineering.

Figure 1

The diagrammatic drawing of the preparation of MSCS and the combination of MSCS and ATS. ATS = acellular tendon scaffold; MSCS = mesenchymal stem cells sheet.

Figure 2

(A) Macroscopic features of the book-shaped acellular tendon scaffold; (B) H&E staining of the native and acellular book-shaped tendon scaffold; (C) DAPI staining of the native and acellular book-shaped tendon scaffold; (D) SEM observation of the native and acellular book-shaped tendon scaffold. H&E = haematoxylin and eosin; DAPI, 4’,6-diamidino-2-phenylindole; SEM = scanning electron microscope.

Figure 3

(A,B) Analysis of gene expression involving tenogenic differentiation; (C, D) analysis of gene expression involving chondrogenic differentiation. Different letters represent statistical significant difference, p ＜ 0.05. ATS = acellular tendon scaffold; TNMD = Tenomodulin; ACAN = Aggrecan.

Figure 4

The area of new bone of regenerated patella–patellar tendon interface at 8 and 16 weeks postoperatively: (A) Representative H&E staining images; (B) semiquantitative analysis of the area of new bone. Scale bars = 100 μm. ^aSignificantly different from the control group at the same time point postoperatively, p < 0.05; ^bStatistically significant difference between the ATS group and the ATS+ MSCS group at the same time point postoperatively, p < 0.05; ^cStatistical difference between 8 and 16 weeks postoperatively in the same group, p < 0.05. The dotted line shows the osteotomy site. ATS = acellular tendon scaffold; MSCS = mesenchymal stem cells sheet; NB = newly formed bone; RP = remaining patella; TF = tendon fibre.

Figure 5

The regenerated fibrocartilage of regenerated patella–patellar tendon interface at 8 and 16 weeks postoperatively: (A) Representative toluidine blue staining images; (B) semiquantitative analysis of the thickness of the regenerated fibrocartilage layer. Scale bars = 600 μm ^aSignificantly different from the control group at the same time point postoperatively, p < 0.05; ^bStatistical difference between the ATS group and the ATS+ MSCS group at the same time point postoperatively, p < 0.05; ^cStatistical difference between 8 and 16 weeks postoperatively in the same group, p < 0.05. ATS, acellular tendon scaffold; FC, fibrocartilage; MSCS, mesenchymal stem cells sheet; NB = newly formed bone; TF = tendon fibre.

Figure 6

The expression of CD206 at regenerated patella–patellar tendon interface at 2, 4, 8 and 16 weeks postoperatively: (A) IF staining images of representative CD206⁺ macrophages; (B) semiquantitative analysis of CD206+ macrophages. Scale bars = 45 μm. Different letters represent statistical significance, p < 0.05. ATS = acellular tendon scaffold; MSCS = mesenchymal stem cells sheet.