Tendons display poor intrinsic healing properties and are difficult to treat[1]. Prior in vitro studies[2] have shown that, by targeting the Activin A receptor with magnetic nanoparticles (MNPs), it is possible to remotely induce the tenogenic differentiation of human adipose stem cells (hASCs). In this study, we investigated the tenogenic regenerative potential of remotely-activated MNPs-labelled hASCs in an in vivo rat model. We consider the potential for magnetic controlled nanoparticle mediated tendon repair strategies.

hASCs were labelled with 250 nm MNPs functionalized with anti-Activin Receptor IIA antibody. Using a rapid curing fibrin gel as delivery method, the MNPs-labelled cells were delivered into a Ø2 mm rat patellar tendon defect. The receptor was then remotely stimulated by exposing the rats to a variable magnetic gradient (1.28T), using a customised magnetic box. The stimulation was performed 1 hour/day, 3 days/week up to 8 weeks. Tenogenesis, iron deposition and collagen alignment were assessed by histological staining and IHC. Inflammation mediators levels were assessed by ELISA and IHC. The presence of human cells in tendons after 4 and 8 weeks was assessed by FISH analysis.

Histological staining showed a more organised collagen arrangement in animals treated with MNPs-labelled cells compared to the controls. IHC showed positive expression of tenomodulin and scleraxis in the experimental groups. Immunostaining for CD45 and CD163 did not detect leukocytes locally, which is consistent with the non-significant levels of the inflammatory cytokines analysis performed on plasma. While no iron deposition was detected in the main organs or in plasma, the FISH analysis showed the presence of human donor cells in rat tendons even after 8 weeks from surgery.

Our approach demonstrates in vivo proof of concept for remote control stem cell tendon repair which could ultimately provide injectable solutions for future treatment.

We are grateful for ERC Advanced Grant support ERC No.789119, ERC CoG MagTendon No.772817 and FCT grant 2020.01157.CEECIND.

Introduction: Tendon tissue engineering entails the generation of a highly ordered collagen matrix with several organization scales that confer the tendon its mechanical functionality. Endogenous production of proteoglycans account for the typical microscopic organization in bundles of the tendon extracellular matrix, as they prevent lateral fusion of collagen fibril by binding the shaft of the fibres and promoting tip to tip fusion. The approach developed in this study is to rely on this molecular endogenous production and to induce a supramolecular uniaxial alignment of collagen fibres bundles with the help of specially designed scaffolds under continuous fluid shear stress.

Methods: Microchannel chitosan scaffolds were produced by casting 2% chitosan gel on a mould equipped with stainless steel needles array that was imaged by optical coherence tomography with a resolution at ~10microns. From OCT measurements, regularly spaced microchannels with clearly delimited boundaries are obtained inside a microporous core of chitosan. By varying the number and the diameter of needles (from 250 μm (microns)to 500 μm (microns)) different types of microstructure have been produced. Microchannels scaffolds were seeded with primary tenocytes explanted from pig tendons and cultured in static culture, as nonstimulated group, and in a perfusion bioreactor.

Results: There was a general increase in the channels occupation ratio for the group stimulated by perfusion, and inversely proportional to the microchannel diameter. Tenocytes were able to proliferate and to produce collagen extracellular matrix from the inner surface of the microchannel up to the whole channel volume.

Conclusion: The proposed microstructure was appropriate for tendon engineering and its channel structure is adequate for direct OCT monitoring.