Fetal and adult fibroblasts display intrinsic differences in tendon tissue engineering and regeneration

Abstract

Injured adult tendons do not exhibit optimal healing through a regenerative process, whereas fetal tendons can heal in a regenerative fashion without scar formation. Hence, we compared FFs (mouse fetal fibroblasts) and AFs (mouse adult fibroblasts) as seed cells for the fabrication of scaffold-free engineered tendons. Our results demonstrated that FFs had more potential for tendon tissue engineering, as shown by higher levels of tendon-related gene expression. In the in situ AT injury model, the FFs group also demonstrated much better structural and functional properties after healing, with higher levels of collagen deposition and better microstructure repair. Moreover, fetal fibroblasts could increase the recruitment of fibroblast-like cells and reduce the infiltration of inflammatory cells to the injury site during the regeneration process. Our results suggest that the underlying mechanisms of better regeneration with FFs should be elucidated and be used to enhance adult tendon healing. This may assist in the development of future strategies to treat tendon injuries.

Figure 1. Mechanical properties of repaired tendons at 4 weeks post-surgery.

Samples were carefully attached for mechanical testing on the Instron machine. The stiffness (a), modulus (b), failure force (c) and stress at failure (d) of the FFs repaired tendon were higher than the AFs repaired tendon p < 0.05. ** P < 0.01 between two groups. Abbreviations: mouse fetal fibroblasts (FFs) and mouse adult fibroblasts (AFs).

Figure 2. In vitro construction and characterization of scaffold-free engineered tendon.

(a–b) Morphology of mouse fetal fibroblasts (FFs) and mouse adult fibroblasts (AFs). (c): Cell proliferation of FFs and AFs on day 1, 3, 5, 7 showed high proliferation potential of FFs. (d): quantitative real time PCR confirmed gene expression levels of tendon-related genes expressed by FFs and AFs on day 0, 3, 7 of scaffold-free engineered tendon formation (n = 3, means ± SD). Gene expression levels are normalized to the reference gene GAPDH (y axis); Transcript levels expressed relative to day 0 AF samples. FFs engineered tendon showed high expression of tendon-related genes. * P < 0.05 between two groups. ** P < 0.01 between two groups. (e): Hematoxylin and eosin and Masson-stained sections of in vitro engineered tendons showing bands of collagen fibers. More collagen fibers were formed in the FFs engineered tendon. Scale bar = 200 μm (a) and (b); Scale bar = 20 μm (e).

Figure 3. FFs and AFs contributed to tendon repair.

(a) DiI-stained cells were incorporated within engineered tendons. Then the cells were placed within the tendon defect and sutured to the tendon. (b): Fluorescence detection using a tracking system showed positive orange fluorescent signal existent in the implantation site, indicating positive cell survival after 2 weeks post-implantation. (c): The quantization of relative fluorescence intensity of AFs group and FFs group at 2 weeks post-transplantation. (e–j) and (i–q) denote the fluorescent staining results of the implanted constructs, showing that seed cells survived at the tendon defect and formed tendon-like tissues after 2 and 4 weeks post-surgery. (e), (h), (l) and (o) showed DAPI staining, (f), (i), (m) and (p) showed DiI staining, while (g), (j), (n) and (q) are the merged images of (e), (h), (l), (o) and (f), (i), (m), (p) respectively. (k), (r): The quantization of survived cells/fibroblast ratio of AFs group and FFs group at 2 and 4 weeks post-transplantation respectively. Abb: mouse fetal fibroblasts (FFs) and mouse adult fibroblasts (AFs).

Figure 4. Histology of scaffold-free engineered tendons after 1 week implantation in vivo.

HE (a) and (b) and Masson trichrome (c) and (d) staining of tendon-like tissue showed tissue morphology and ECM deposition. The repaired site was composed mostly of fibroblast and inflammatory cells. Masson trichrome staining showed dense formation of collagen fibers at the repaired site. (e), (f) polarized light microscopy images of tissue-engineered tendon in vivo. The maturity of repaired tissues can be assessed under polarized light microscopy. Dark arrows indicate the axis of tensile load of the tendons. White arrow shows the collagen fibers under polarized light microscopy. Abbreviations: J - tendon-neotendon junction; N - neo-tendon; T - host tendon.

Figure 5. Histology of scaffold-free engineered tendons after 2 weeks implantation in vivo.

HE (a) and (b) and Masson trichrome (c) and (d) staining of tendon-like tissue showed tissue morphology and ECM deposition. HE staining showed a decrease in the number of inflammatory cell infiltration in both FFs-repaired and AFs-repaired groups. Masson trichrome staining indicated extracellular matrix with crimp pattern deposition in the FFs-repaired group. (e), (f) are polarized light microscopy images of tissue-engineered tendon in vivo. Dark arrows indicate the axis of tensile load of the tendons. White arrow shows the collagen fibers under polarized light microscopy. Abbreviations: J - tendon-neotendon junction; N - neo-tendon; T - host tendon.

Figure 6. Histology of scaffold-free engineered tendons after 4 weeks implantation in vivo.

HE (a) and (b) and Masson trichrome (c) and (d) staining of tendon-like tissue showed tissue morphology and ECM deposition. The FFs-repaired tendons displayed more spindle-shaped cells and more arranged bundles of collagen fibers compared to the AFs-repaired group. (e), (f) are polarized light microscopy images of tissue-engineered tendon in vivo. Continuous collagen fibers (bright yellow) were observed over the entire field of vision in the FFs-repaired tendons. Dark arrows indicate the axis of tensile load of the tendons. White arrow shows the collagen fibers under polarized light microscopy. Abbreviations: N - neo-tendon.

Figure 7

The quantity and ratio of fibroblast-like cells and immune cells within the repaired tissue after 1 week (a), 2 weeks (b), 3 weeks (c) and 4 weeks (d) implantation. The FFs-repaired group had more fibroblast-like cells and less immune cells throughout the repair process compared to the AFs-repaired group. (e), (f), (g) and (h): mRNA levels of inflammatory cytokines within FFs and AFs at 2 weeks and 4 weeks post-implantation. Gene expression levels are normalized to the reference gene GAPDH (y axis), Transcript levels expressed relative to 2 w AF samples. The expression levels of inflammatory cytokines within FFs-repaired tendons were significantly down-regulated at 4 weeks post-surgery. HPF: high power field.* p < 0.05 between two groups. ** p < 0.01 between two groups.

Figure 8. Ultrastructure of tissue-engineered tendons at 4 weeks post-implantation.

Histogram and distribution of collagen fibril diameters in the AFs group (a, c) and FFs group (b, d). There were thicker fibrils in the FFs-repaired group and larger diameter of collagen fibrils in the FFs-repaired tendons.