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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

Abstract

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Introduction

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure ....

Protocol

NOTE: All the steps must be conducted using aseptic techniques.

1. Culture and pre-expansion of NHDFs

  1. Rapidly thaw the cryo-vial containing adult cryopreserved normal human dermal fibroblasts (NHDFs, 1 x 106 cells) at 37 °C until they are almost defrosting.
  2. Slowly add 1 mL of prewarmed fibroblast growth medium 2 (ready-to-use kit including basal medium, 2% fetal calf serum (FCS), basic fibroblast growth factor (bFGF) and insulin) supplemen.......

Representative Results

The 3D T/L organoid model was previously established and demonstrated here by implementing commercially purchased NHDF (n=3, 3 organoids per donor, NHDF were used at passages 5-8). The model workflow is summarized in Figure 1. Figure 2 shows representative phase-contrast images of NHDF culture during the pre-expansion in T-75 flasks (Figure 2A) as well as at the beginning and after 5 days of culture in the 2D expansion step in 10 cm.......

Discussion

The results demonstrated in this study provide valuable insights into the establishment and characterization of the NHDF 3D organoid model for studying T/L tissues. The 3-step protocol led to the formation of 3D rod-like organoids that exhibit typical features of T/L niche. This model was previously reported in Kroner-Weigl et al. 20237 and demonstrated in great detail here.

The phase-contrast images presented in Figure 2 showed th.......

Acknowledgements

D.D. and S.M.-D. acknowledge the BMBF Grant "CellWiTaL: Reproducible cell systems for drug research - transfer layer-free laser printing of highly specific single cells in three-dimensional cellular structures" Proposal Nr. 13N15874. D.D. and V.R.A. acknowledge the EU MSCA-COFUND Grant OSTASKILLS "Holistic training of next-generation Osteoarthritis researches" GA Nr. 101034412. All authors acknowledge Mrs. Beate Geyer for technical assistance.

....

Materials

NameCompanyCatalog NumberComments
Ascorbic acid  Sigma-Aldrich, Taufkirchen,Germany  A8960
10 cm adherent cell culture dishSigma-Aldrich, Taufkirchen,Germany CLS430167
10 cm non-adherent petri dish Sigma-Aldrich, Taufkirchen,Germany CLS430591
Cryo-mediumTissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands  4583
Cryomold standard Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands4557
D(+)-Sucrose AppliChem Avantor VWR International GmbH, Darmstadt, GermanyA2211
DMEM high glucose medium Capricorn Scientific, Ebsdorfergrund, Germany DMEM-HA
DMEM low glucoseCapricorn Scientific, Ebsdorfergrund, Germany  DMEM-LPXA
Fetal bovine serum Anprotec, Bruckberg, Germany AC-SM-0027
Fibroblast growth medium 2 PromoCell, Heidelberg, Germany  C-23020
Inverted microscope with high resolution cameraZeissNAZeiss Axio Observer with  Axiocam 506
MEM amino acids Capricorn Scientific, Ebsdorfergrund, Germany  NEAA-B
Metal pins EntoSphinx, Pardubice, Czech Republic 04.31
Normal human dermal fibroblasts  PromoCell, Heidelberg, Germany C-12302
Paraformaldehyde AppliChem, Sigma-Aldrich, Taufkirchen, Germany A3813
Penicillin/streptomycin Gibco, Thermo Fisher Scientific, Darmstadt, Germany15140122
Phosphate buffer saline Sigma-Aldrich, Taufkirchen, Germany P4417
TGFß3 R&D Systems, Wiesbaden, Germany  8420-B3
Trypsin-EDTA 0,05% DPBS Capricorn Scientific, Ebsdorfergrund, Germany  TRY-1B

References

  1. Schneider, M., Angele, P., Järvinen, T. A. H., Docheva, D. Rescue plan for Achilles: Therapeutics steering the fate and functions of stem cells in tendon wound healing. Adv Drug Deliv Rev. 129, 352-375 (2018).
  2. Steinmann, S., Pfeifer, C. G., Brochhausen, C., Docheva, D.

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