iPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration

Abstract

Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional (3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSC^SCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSC^SCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSC^SCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence. iMSC^SCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSC^SCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSC^SCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSC^SCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSC^SCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.

Keywords: Achilles tendon; iPSC; microgrooves; scaffold; scleraxis.

Figure 1

Microgrooved appear to increase cellular alignment on cells seeded on scaffold. (A) Graphical depiction of 3D printing scaffold. (B) Scaffold micro-topography imaged via scanning electron microscope (C) Scaffold filament diameters. (D) Young’s Modulus of microgrooved and non-patterned scaffolds compared to native rat tendon. (E) Microgrooved and non-patterned scaffolds seeded with iMSC^SCX+ stained with Phalloidin (green), TNMD (magenta), and DAPI (blue).

Figure 2

Microgrooved scaffolds induced greater cellular alignment and upregulation of tenogenic markers in iMSC^SCX+ in vitro. (A) Quantitative reverse transcription PCR results show significant upregulation of tenogenic markers in the microgrooved scaffolds compared to the control groups in vitro, n = 3/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Immunofluorescence staining of the scaffolds show expression of tenogenic markers on the protein level and increased cell alignment and linearity in the microgrooved scaffolds.

Figure 3

Induction of tendon defect and repair. The Achilles tendon was exposed and a 0.5 cm length resected from the mid-portion of the belly of the tendon. For control, the defect was sutured leaving some gap between the two ends. The 5 mm long polycaprolactone scaffold was implanted and sutured to the proximal and distal ends of the Achilles tendon. (A) Non-injured Achilles tendon. Tendon defects were repaired with (B) Suture only, (C) Scaffold, or Scaffold + iMSC^SCX+, (D) and the wound sutured shut. (E, F) At sacrifice, the tendon was explanted for biomechanical tests and histological analysis.

Figure 4

Gait analysis demonstrated increased functional gait properties in the Scaffold + iMSC^SCX+ group. Gait was assessed pre and postoperatively. (A) Measurements of the prints. (B) The Scaffold + iMSC^SCX+ showed significant improvement compared to the Suture only and Scaffold only groups indicating better functional recovery; n = 9–10, *p < 0.05, **p < 0.01.

Figure 5

Histology revealed increased regular tissue formation and organization in the Scaffold + iMSC^SCX+ group. Histological staining with hematoxylin and eosin (top) and Masson’s Trichrome (bottom) of the harvested tendons.

Figure 6

Immunofluorescence seemed to show greater cellular alignment and decreased scar and inflammation marker in the Scaffold + iMSC^SCX+ group. (A–C) Immunofluorescence staining for tendon markers, (D–F) scar markers and immune markers. Scale bar −50 μm.

Figure 7

Biomechanical testing showed increased in biomechanical properties of the Scaffold + iMSC^SCX+. (A–D) Tendons were explanted, mounted, and loaded to failure using MTS mschine. (E) Biomechanical tests show improved biomechanical properties in the Scaffold + iMSC ^SCX+ group compared to the Suture only and Scaffold only groups. n = 9–10, *p < 0.05, **p < 0.01.