Histological evaluation of tendon formation using a scaffold-free three-dimensional-bioprinted construct of human dermal fibroblasts under in vitro static tensile culture

Abstract

Introduction: Tendon tissue engineering requires scaffold-free techniques for safe and long-term clinical applications and to explore alternative cell sources to tenocytes. Therefore, we histologically assessed tendon formation in a scaffold-free Bio-three-dimensional (3D) construct developed from normal human dermal fibroblasts (NHDFs) using our Bio-3D printer system under tensile culture in vitro.

Methods: Scaffold-free ring-like tissues were constructed from 120 multicellular spheroids comprising NHDFs using a bio-3D printer. Ring-like tissues were cultured in vitro under static tensile-loading with or without in-house tensile devices (tension-loaded and tension-free groups), with increases in tensile strength applied weekly to the tensile-loaded group. After a 4 or 8-week culture on the device, we evaluated histological findings according to tendon-maturing score and immunohistological findings of the middle portion of the tissues for both groups (n = 4, respectively).

Results: Histology of the tension-loaded group revealed longitudinally aligned collagen fibers with increased collagen deposition and spindle-shaped cells with prolonged culture. By contrast, the tension-free group showed no organized cell arrangement or collagen fiber structure. Additionally, the tension-loaded group showed a significantly improved tendon-maturing score as compared with that for the tension-free group at week 8. Moreover, immunohistochemistry revealed tenascin C distribution with a parallel arrangement in the tensile-loading direction at week 8 in the tension-loaded group, which exhibited stronger scleraxis-staining intensity than that observed in the tension-free group at weeks 4 and 8.

Conclusions: The NHDF-generated scaffold-free Bio-3D construct underwent remodeling and formed tendon-like structures under tensile culture in vitro.

Keywords: 3D, three-dimensional; ECM, extracellular matrix; ESCs, embryonic stem cells; H&E, hematoxylin and eosin; HDF, human dermal fibroblast; Human dermal fibroblast; In vitro study; MCSs, multicellular spheroids; Multicellular spheroid; NHDFs, normal HDFs; Scaffold-free; TDSCs, tendon-derived stem cells; TGF, transforming growth factor; Tendon formation; Tensile culture.

Fig. 1

Materials for and process of manufacturing three-dimensional (3D) Bio-printed ring-like tissue and human neo-tendon tissue under static tensile loading. (a) Image of multicellular spheroid (MCS) produced from normal human dermal fibroblasts (NHDFs). Scale bar = 100 μm. (b) Predesigned 3D ring-like structure. Green spheres represent MCSs. (c) Image of the Bio-3D printer (Regenova). (d) Image of the Kenzan needle-array after skewering MCSs. (e) Image of ring-like tissue after removal from the needle array. (f) Image of the in-house uniaxial tensile device. (g) Image of the tensile device without tissues (upper) and with tissues (lower) at the initial strength point. A: main, B: middle, and C: spring-anchor base. Image of ring-like tissues in the (h) tension-free and (i) tension-loaded groups.

Fig. 2

Histological analysis of the tension-free group. Images showing changes in construct shape with prolonged culture (a) and gross view at weeks 4 (b) and 8 (c). Photomicrographs showing hematoxylin and eosin (H&E) staining (d and g) and Masson's trichrome staining (e, f, h, and i) of in vitro-cultured tissue without tension-loading at weeks 4 (b and d–f) and 8 (c and g–i). Scale bars = 2 mm (a), 100 μm (d, e, h, and i), and 50 μm (f and i).

Fig. 3

Histological analysis of the tension-loaded group. Images showing changes in construct shape with prolonged culture (a) and gross view at weeks 4 (b) and 8 (c). Photomicrographs showing hematoxylin and eosin (H&E) staining (d and g) and Masson's trichrome staining (e, f, h, and i) of in vitro-cultured tissue with tension loading at weeks 4 (b and d–f) and 8 (c and g–i). Scale bars = 2 mm (a), 100 μm (d, e, g, and h), 50 μm (f) and 20 μm (i).

Fig. 4

Histological scoring. Histological scoring of tension-free and tension-loaded groups at weeks 4 and 8. A perfect score on this scale is 20 points. *P < 0.0001; **P < 0.0001; and ***P < 0.0001 (n = 4).

Fig. 5

Immunohistochemical analysis of tenascin. Photomicrographs of immunohistochemical staining for tenascin C (a–d) and the magnification (f–i). (e and j) Negative controls for immunostaining. Scale bars = 50 μm (a–e) and 20 μm (f–j).

Fig. 6

Immunohistochemical analysis of scleraxis. Photomicrographs of immunohistochemical staining for scleraxis (a–d) and the magnification (f–i). (e and j) Negative controls for immunostaining. Scale bars = 50 μm (a–e) and 10 μm (f–j).