Tendon and ligament (T/L) function is intrinsically related with their unique hierarchically and anisotropically organized extracellular matrix. Their natural healing capacity is, however, limited. Here, continuous and aligned electrospun nanofiber threads (CANT) based on synthetic/natural polymer blends mechanically reinforced with cellulose nanocrystals are produced to replicate the nanoscale collagen fibrils grouped into microscale collagen fibers that compose the native T/L. CANT are then incrementally assembled into 3D hierarchical scaffolds, resulting in woven constructions, which simultaneously mimic T/L nano-to-macro architecture, nanotopography, and nonlinear biomechanical behavior. Biological performance is assessed using human-tendon-derived cells (hTDCs) and human adipose stem cells (hASCs). Scaffolds nanotopography and microstructure induce a high cytoskeleton elongation and anisotropic organization typical of tendon tissues. Moreover, the expression of tendon-related markers (Collagen types I and III, Tenascin-C, and Scleraxis) by both cell types, and the similarities observed on their expression patterns over time suggest that the developed scaffolds not only prevent the phenotypic drift of hTDCs, but also trigger tenogenic differentiation of hASCs. Overall, these results demonstrate a feasible approach for the scalable production of 3D hierarchical scaffolds that exhibit key structural and biomechanical properties, which can be advantageously explored in acellular and cellular T/L TE strategies.

Tendon tissues have limited healing capacity. The incidence of tendon injuries and the unsatisfactory functional outcomes of tendon repair are driving the search for alternative therapeutic approaches envisioning tendon regeneration. Cellular therapies aim at delivering adequate, regeneration-competent cell types to the injured tendon and toward ultimately promoting its reconstruction and recovery of functionality. Mesenchymal stem cells (MSCs) either obtained from tendons or from non-tendon sources, like bone marrow (BM-MSCs) or adipose tissue (ASCs), have been receiving increasing attention over the years toward enhancing tendon healing. Evidences from in vitro and in vivo studies suggest MSCs can contribute to accelerate and improve the quality of tendon healing. Nonetheless, the exact mechanisms underlying these repair events are yet to be fully elucidated. This review provides an overview of the main challenges in the field of cell-based regenerative therapies, discussing the role of MSCs in boosting tendon regeneration, particularly through their capacity to enhance the tenogenic properties of tendon resident cells.

Tendon‐to‐bone interfaces exhibit a hierarchical multitissue transition. To replicate the progression from mineralized to nonmineralized tissue, a novel 3D fibrous scaffold is fabricated with spatial control over mineral distribution and cellular alignment. For this purpose, wet‐spun continuous microfibers are produced using polycaprolactone (PCL)/ gelatin and PCL/gelatin/hydroxyapatite nano‐to‐microparticles (HAp). Higher extrusion rates result in aligned PCL/gelatin microfibers while, in the case of PCL/gelatin/HAp, the presence of minerals leads to a less organized structure. Biological performance using human adipose‐derived stem cells (hASCs) demonstrates that topography of PCL/gelatin microfibers can induce cytoskeleton elongation, resembling native tenogenic organization. Matrix mineralization on PCL/gelatin/HAp wet‐spun composite microfibers suggest the production of an osteogenic‐like matrix, without external addition of osteogenic medium supplementation. As proof of concept, a 3D gradient structure is produced by assembling PCL/gelatin and PCL/gelatin/HAp microfibers, resulting in a fibrous scaffold with a continuous topographical and compositional gradient. Overall, the feasibility of wet‐spinning for the generation of continuously aligned and textured microfibers is demonsrated, which can be further assembled into more complex 3D gradient structures to mimic characteristic features of tendon‐to‐bone interfaces.

Vascularisation is considered to be one of the greatest challenges in tissue engineering. Different strategies exist but cell-based approaches have emerged as a promising therapy to achieve successful vascularisation. The use of endothelial cells to engineer vascularised tissues has been extensively investigated. This field of research has evolved with the discovery of endothelial progenitor cells, a subpopulation with a high regenerative potential. However, the survival of endothelial cell populations alone seems to be impaired. To overcome this problem, co-culture systems, involving supporting cells, like mural cells, fibroblasts, or more tissue-specific cells have been developed. Endothelial cells benefit from the extracellular matrix components and growth factors produced by the supporting cells, which results in neovessel stabilisation and maturation. The use of endothelial progenitor cells in co-culture systems appears to be a promising strategy to promote vascularisation in approaches of increasing complexity. Herein, the authors provide an overview of the cellular strategies that can be used for increasing vascularisation in tissue engineering and regeneration.

Cell-based approaches have emerged as a promising therapy to achieve successful vascularization in tissue engineering. Since fibroblasts activation and migration is required for physiological events relying on angiogenesis, we hypothesize herein that different fibroblasts exhibit distinct capacity to promote capillary-like structures assembly, by mature and progenitor endothelial cells (ECs). Outgrowth endothelial cells (OECs) were isolated from human umbilical cord blood samples and characterized by immunofluorescence and imaging flow cytometry for endothelial markers. Coculture systems were established using either human umbilical vein ECs (HUVECs) or OECs with fibroblasts, being evaluated at 7, 14, and 21 days of culture. Two types of human dermal fibroblasts (HDF) were used, namely neonatal human foreskin fibroblasts-1 (HFF-1) and juvenile HDF. OECs expressed EC markers and formed capillary-like structures. HFF-1 exhibited higher expression of transglutaminase-2, while HDF exhibited a higher expression of a-smooth muscle actin (a-SMA) and podoplanin, which were not observed for HFF-1. Formation of capillary-like structures was only observed in cocultures with HDF and not with HFF-1. No significant differences were found between HDF and OECs or HUVECs cocultures. These findings suggest that HDF is a preferential cell source for promoting vascularization, either using mature or progenitor ECs, probably due to their higher expression of a-SMA and podoplanin, and increased synthesis of extracellular matrix. This work opens new research possibilities regarding the use of specific fibroblast populations cocultured with ECs, as efficient partners for vascular development in regenerative medicine strategies.