Despite the high incidence of tendon injuries worldwide, an optimal treatment strategy has yet to be defined. A key challenge for tendon repair is the alignment of the repaired matrix into orientations which provide maximal mechanical strength. Using oriented implants for tissue growth combined with either exogenous or endogenous stem cells may provide a solution. Previous research has shown how oriented fiber-like structures within 3D scaffolds can provide a framework for organized extracellular matrix deposition. In this article, we present our data on the remote magnetic alignment of collagen hydrogels which facilitates long-term collagen orientation. Magnetic nanoparticles (MNPs) at varying concentrations can be contained within collagen hydrogels. Our data show how, in response to the magnetic field lines, MNPs align and form string-like structures orientating at 90 degrees from the applied magnetic field from our device. This can be visualized by light and fluorescence microscopy, and it persists for 21 days post-application of the magnetic field. Confocal microscopy demonstrates the anisotropic macroscale structure of MNP-laden collagen gels subjected to a magnetic field, compared to gels without MNP dosing. Matrix fibrillation was compared between non- and biofunctionalized MNP hydrogels, and different gels dosed with varying MNP concentrations. Human adipose stem cells (hASCs) seeded within the magnetically aligned gels were observed to align in parallel to MNP and collagen orientation 7 days post-application of the magnetic field. hASCs seeded in isotropic gels were randomly organized. Tenocyte-likeness of the cells 7 days post-seeding in collagen I scaffolds was confirmed by the positive expression of tenomodulin and scleraxis proteins. To summarize, we have developed a convenient, non-invasive protocol to control the collagen I hydrogel architecture. Through the presence or absence of MNP dosing and a magnetic field, collagen can be remotely aligned or randomly organized, respectively, in situ. Tendon-like cells were observed to organize in parallel to unidirectionally aligned collagen fibers and polydirectionally in non-aligned collagen constructs. In this way, we were able to engineer the constructs emulating a physiologically and pathologically relevant tendon niche. This can be considered as an innovative approach particularly useful in tissue engineering or organ-on-a-chip applications for remotely controlling collagen matrix organization to recapitulate the native tendon.
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.
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