Tendon-to-bone multi-tissue transition exhibits a hierarchical and continuous gradient of matrix composition and alignment, allowing for efficient transmission of mechanical loading between tendon and bone. Upon injury, main problems associated with tendon-to-bone regeneration include disorganized matrix deposition, with a gradual loss of mineral content resulting in poor mechanical properties, limiting tissue integration and the formation of a graded interface. Therefore, we propose to assembly two types of continuous microfibres with distinct topological and compositional features tailored to guide cell alignment and matrix deposition while matching the mechanical requirements of the native tissue.

Wet-spinning was used to produce textured composite microfibres using different flow rates and two polymer blends to replicate the anisotropic architecture of tendon (PCL/Gelatin, 22/9%, w/v) and the isotropic organization together with mineral composition of bone (PCL/Gelatin/Hydroxyapatite, 22/9% w/v and 7.7% w/w HAp). Obtained microfibres morphology, chemical and mechanical properties were evaluated. Biological performance was studied using human adipose-derived stem cells (hASCs). Cytoskeleton alignment, nuclei elongation and matrix mineralization were evaluated. Textile techniques were used to create a 3D fibrous scaffold. Morphological features were analyzed by micro-CT.

PCL/Gelatin fibers produced at 1 mL/h extrusion rate exhibited the highest anisotropic alignment, in opposition to PCL/Gelatin/HAp fibers produced under the same condition. Micro-CT analysis of PCL/Gelatin/HAp fibers demonstrated variations within pore diameter and particles size between the different flow rates. Herein, PCL/Gelatin fibers induced a higher cytoskeleton alignment and nuclei elongation (p < 0.0001) in seeded hASCs. In contrast, significantly higher mineralization was found in PCL/Gelatin/HAp fibres (day 7, p < 0.04; day 14, p < 0.0001) as observed by alizarin red staining and quantification, suggesting the induction of an osteogenic-like phenotype. As proof of concept, textile techniques were used to assemble the two types of fibers and create a 3D scaffold presenting a continuous gradient in HAp content, as well as topological cues. After 14 days of culture with hASCs, a gradient of collagen deposition and matrix mineralization was found (p < 0.014, p < 0.0001). Higher deposition of collagen type II was observed in the tendon and interface parts of the fibrous scaffold and collagen type X in the interface.

Overall, the wet-spinning method was efficiently used to engineer continuous textured composite microfibers. PCL/Gelatin fibers supported cell alignment mimicking tendon one, while PCL/Gelatin/HAp fibers induced mineral deposition and a possible phenotypic change without additional medium supplementation. Textile techniques allowed fibres assemblage and 3D scaffolds fabrication envisioning tendon-to-bone applications.

The selection of a proper material to be used as a scaffold or as a hydrogel to support, hold or encapsulate cells is both a critical and a difficult choice that will determine the success of failure of any tissue engineering and regenerative medicine (TERM) strategy. We believe that the use of natural origin polymers, including a wide range of marine origin materials, is the best option for many different approaches that allow for the regeneration of different tissues. In addition to the selection of appropriate material systems it is of outmost importance the development of processing methodologies that allow for the production of adequate scaffolds/matrices, in many cases incorporating bioactive/differentiation agents in their structures. An adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation, and in many cases the selection of a specific sub-population, methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The development of dynamic ways to culture the cells and of distinct ways to stimulate their differentiation in 3D environments, as well as the use of nano-based systems to induce their differentiation and internalization into cells, is also a key part of some of the strategies that are being developed in our research group. The potential of each combination materials/cells, to be used to develop novel useful regeneration therapies will be discussed. The use of different cells and their interactions with different natural origin degradable scaffolds and smart hydrogels will be described. Several examples of TERM strategies to regenerate different types of musculoskeletal tissues will be presented. Relevance to orthopaedics will be highlighted.

Tendon injuries constitute a major healthcare burden owing to the limited healing ability of these tissues and the poor clinical outcomes of surgical repair treatments. Recent advances in tendon tissue engineering (TTE) strategies, particularly through the use of biotextile technologies, hold great promise toward the generation of artificial living tendon constructs. We have previously developed a braided construct based on suture threads coated with gelMA:alginate hydrogel encapsulating human tendon cells. These cell-laden composite fibers enabled the replication of cell and tissue-level properties simultaneously. Based on this concept, in this study we explored the use of platelet lysate (PL), a pool of supra-physiological concentrations of growth factors (GFs), to generate a hydrogel layer, which is envisioned to act as a depot of therapeutic factors to induce tenogenic differentiation of encapsulated human adipose stem cells (hASCs). For this purpose, commercially available suture threads were first embedded in a thrombin solution and then incubated in PL containing hASCs. Herein, thrombin induces the gelation of PL and consequent hydrogel formation. After coating suture threads with the mixture of PL-ASCs, cells were found to be viable and homogeneously distributed along the fibers. Strikingly, hASCs encapsulated within the PL hydrogel layer around the suture thread were able to sense chemotactic factors present in PL and to establish connections between adjacent independent fibers, suggesting a tremendous potential of PL cell-laden hydrogel fibers as building blocks in the development of living constructs aimed at tendon repair applications.

Tendon injuries are a worldwide problem affecting several age groups and stem cell based therapies hold potential for tendon strategies guiding tendon regeneration.

Tendons rely on mechano-sensing mechanisms that regulate homeostasis and influence regeneration. The mechanosensitive receptors available in cell membranes sense the external stimuli and initiate mechanotransduction processes. Activins are members of the TGF-β superfamily which participate in several tendon biological processes. It is envisioned that the activation of the activin receptor, trigger downstream Smad2/3 pathway thus regulating the transcription of tenogenic genes driving stem cell differentiation.

In this work, we propose to target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), inducing hASCs commitment towards the tenogenic lineage. Since mechanotransduction can be remotely triggered through magnetic actuation combined with magnetic nanoparticles (MNPs), we stimulated hASCs tagged complexes using a vertical oscillating magnetic bioreactor (MICA Biosystems Ltd). Carboxyl functionalised MNPs (Micromod) were coated with anti-ActRIIA antibody (Abcam) by carbodiimide activation. hASCs were then cultured with MNPs-anti-ActRIIA for 14days with or without magnetic exposure (1Hz, 1h/every other day). hASCs cultured alone in αMEM (negative control) or in αMEM supplemented with ActivinA (R&D systems) (positive control of ActRIIA activation) were used as experimental controls. The tenogenic commitment of hASCs was assessed by real time RT-PCR, immunocytochemistry and quantification of collagen and non-collagenous proteins. Moreover, the phosphorylation of Smad2/3 was also evaluated on hASCs incubated for 2, 10, or 30min under magnetic stimulated (1Hz) and non-stimulated conditions.

The increased gene expression of tendon related markers and higher ECM proteins deposition suggests that remote magnetic activation of ActRIIA promotes effectively hASCs tenogenic commitment. Furthermore, the detection of phospho-Smad2/3 proteins by ELISA (Cell Signaling Technology) was significantly more intense after 10min in hASCs under magnetic stimulation and in comparison to the control groups. These outcomes suggest that ActRIIA is a mechanosensitive receptor that can be remotely activated upon magnetic stimulation.

In conclusion, remotely activation of MNPs tagged hASCs has potential for modulating tenogenic differentiation of stem cells envisioning successful cell therapies for tendon regeneration.

Tendon detachment from its bony insertion is one of the most frequent injuries occurring in the musculoskeletal interface, constituting an unmet challenge in orthopaedics. Tendon-to-bone integration occurs at the enthesis, which is characterized by a complex structure organized in a gradient of cells and microenvironments. Hence, the maintenance of a heterotypic cellular niche is critical for tissue functionality and homeostasis. Replicating this unique complexity constitutes a challenge when addressing tendon-to-bone regeneration and interfacial tissue engineering strategies. Currently, mechanisms presiding to tendon-to-bone interface healing are not yet fully understood, particularly the interactions between tendon and bone cells in the orchestration of interfacial repair versus regeneration. Therefore, this study focused on the hypothesis that interactions between human tendon-derived cells (hTDCs) and pre-osteoblasts (pre-OB) can initiate a cascade of events, potentially leading to interfacial regeneration. Thus, hTDCs and pre-OB (pre-differentiated human adipose-derived stem cells) were used. Herein, five different ratios between basal and osteogenic media (100:0,75:25,50:50,25:75,0:100) were assessed to estimate their influence on cell behaviour and identify the ideal parameters for simultaneously supporting tenogenic and osteogenic differentiation before establishing a co-culture. Tenogenic and osteogenic differentiation were assessed through the expression of tendon and bone markers, mineralization (alizarin red, AZ) and alkaline phosphatase (ALP) quantification. Results showed that hTDCs exhibited osteogenic differentiation potential when cultured in the presence of osteogenic media, as demonstrated by an increase in ALP activity and mineralization. Pre-OB expressed osteogenic markers (OCN, OPN) in all media conditions confirming osteogenic commitment, which was simultaneously confirmed by ALP levels and AZ staining. Thus, three different conditions (100:0, 50:50, 0:100) were chosen for further studies in a direct contact co-culture system. Similarly to single cultures, a significant proliferation was observed in all conditions and mineralization was increased as soon as 7 days of culture. Additionally, osteogenic, tenogenic and interface-relevant markers will be assessed to study the effect of co-culture on phenotype maintenance. In summary, the present work addresses major limitations to clinical translation of cell-based therapies aiming at promoting interfacial regeneration. Particularly, we explored the influence of culture media on the maintenance of tenogenic and osteogenic niches, taking a basic and critical step towards the establishment of more complex cell-based systems.

Acknowledgements

Authors thank Fundação para a Ciência e Tecnologia in the framework of FCT-POPH-FSE, SFRH/BD/96593/2013 (RCA) and IF/00593/2015 (MEG); and to FCT/MCTES and the FSE/POCH, PD/59/2013 for PD/BD/128088/2016 (IC).