Tendons are dense connective tissues with a hierarchical polarized structure that respond to and adapt to the transmission of muscle contraction forces to the skeleton, enabling motion and maintaining posture. Tendon injuries, also known as tendinopathies, are becoming more common as populations age and participation in sports/leisure activities increases. The tendon has a poor ability to self-heal and regenerate given its intrinsic, constrained vascular supply and exposure to frequent, severe loading. There is a lack of understanding of the underlying pathophysiology, and it is not surprising that disorder-targeted medicines have only been partially effective at best. Recent tissue engineering approaches have emerged as a potential tool to drive tendon regeneration and healing. In this review, we investigated the physiochemical factors involved in tendon ontogeny and discussed their potential application in vitro to reproduce functional and self-renewing tendon tissue. We sought to understand whether stem cells are capable of forming tendons, how they can be directed towards the tenogenic lineage, and how their growth is regulated and monitored during the entire differentiation path. Finally, we showed recent developments in tendon tissue engineering, specifically the use of mesenchymal stem cells (MSCs), which can differentiate into tendon cells, as well as the potential role of extracellular vesicles (EVs) in tendon regeneration and their potential for use in accelerating the healing response after injury.

Tendon tissue engineering draws on regenerative medicine principles, offering innovative solutions to address the challenges posed by tendon injuries and degenerative conditions. Tendons’ inherent limited regenerative capacity often hinders complete recovery from injuries, leading to chronic conditions and impaired functionality. Autologous mesenchymal/stromal stem cells (MSCs) and tendon‐derived stem cells (TSCs), combined with growth factors (GFs) like GDF‐5, GDF‐6 and GDF‐7, are emerging as potential therapies for tendinopathy. These GFs are crucial for tendon development and promoting tenogenic differentiation, though the exact pathways they activate remain unclear. For this reason, directly comparing all three pathways to assess their impact on both MSCs and TSCs is essential. This study examined the effects of GDF‐5, GDF‐6 and GDF‐7 on tenogenic differentiation in MSCs and TSCs, with a focus on how oxygen levels (21% O ₂ vs. physoxia at 2% O ₂ ) influence this process. The expression profiles of key tenogenic genes (Scleraxis [Scx], Tenomodulin [Tnmd], Thrombospondin‐4 [Thromb‐4] and Tenascin‐C [Tnc‐C]) were explored by quantitative reverse transcription PCR (RT‐qPCR) following supplementation with individual GFs. Transcriptional analysis was complemented by Tnmd immunofluorescence (IF) and image analysis to identify optimal differentiation parameters. The study highlighted GDF‐7 as a powerful inducer of tenocyte‐like cell differentiation in MSCs, showcasing sustained expression of tenogenic genes over time in 21% O ₂ . Moreover, TSCs in physoxia differentiate into tenocytes without an additional GF requirement. In conclusion, the study lays a foundation for understanding the complex interplay of GFs, oxygen levels and cellular responses in the quest for tendon regeneration. In doing so, it establishes that different cell types have differing biochemical requirements for induction of tenogenic differentiation. While offering promising avenues for tissue engineering platforms, it underscores the need for further research to fully harness the potential of MSCs and TSCs in vivo for tendon regeneration.

Tendon injuries caused by overuse or age-related deterioration are frequent. Incomplete knowledge of somatic tendon cell biology and their progenitors has hindered interventions for the effective repair of injured tendons. Here, we sought to compare and contrast distinct tendon-derived cell populations: type I and II tendon stem cells (TSCs) and tenocytes (TNCs). Porcine type I and II TSCs were isolated via the enzymatic digestion of distinct membranes (paratenon and endotenon, respectively), while tenocytes were isolated through an explant method. Resultant cell populations were characterized by morphology, differentiation, molecular, flow cytometry, and immunofluorescence analysis. Cells were isolated, cultured, and evaluated in two alternate oxygen concentrations (physiological (2%) and air (21%)) to determine the role of oxygen in cell biology determination within this relatively avascular tissue. The different cell populations demonstrated distinct proliferative potential, morphology, and transcript levels (both for tenogenic and stem cell markers). In contrast, all tendon-derived cell populations displayed multipotent differentiation potential and immunophenotypes (positive for CD90 and CD44). Type II TSCs emerged as the most promising tendon-derived cell population for expansion, given their enhanced proliferative potential, multipotency, and maintenance of a tenogenic profile at early and late passage. Moreover, in all cases, physoxia promoted the enhanced proliferation and maintenance of a tenogenic profile. These observations help shed light on the biological mechanisms of tendon cells, with the potential to aid in the development of novel therapeutic approaches for tendon disorders.

Tendon injuries significantly impact quality of life, prompting the exploration of innovative solutions beyond conventional surgery. Extracellular Vesicles (EVs) have emerged as a promising strategy to enhance tendon regeneration. In this study, human Tendon Stem/Progenitor Cells (TSPCs) were isolated from surgical biopsies and cultured in a Growth-Differentiation Factor-5-supplemented medium to promote tenogenic differentiation under static and dynamic conditions using a custom-made perfusion bioreactor. Once at 80% confluence, cells were transitioned to a serum-free medium for conditioned media collection. Ultracentrifugation revealed the presence of vesicles with a 10⁶ particles/mL concentration and sub-200nm diameter size. Dynamic culture yielded a 3-fold increase in EV protein content compared to static culture, as confirmed by Western-blot analysis. Differences in surface marker expression were also shown by flow cytometric analysis. Data suggest that we efficiently developed a protocol for extracting EVs from human TSPCs, particularly under dynamic conditions. This approach enhances EV protein content, offering potential therapeutic benefits for tendon regeneration. However, further research is needed to fully understand the role of EVs in tendon regeneration.

Cancer is one of the leading causes of death in the world and protein therapeutics play an important role in combating this disease. Novel nanocarriers are needed for optimal delivery, enhanced therapeutic effect, and protection of proteins. Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles are commonly used, since they are non-toxic, biodegradable, and allow for the sustained release of the active pharmaceutical ingredient (API). Accurate quantification of the therapeutic inside these nanocarriers is essential for further development and precise in vivo experiments, especially for determining the correct therapeutic dose. Bicinchoninic acid (BCA) assay is one of the most popular methods of protein quantification, known for its low sensitivity to common surfactants. However, large discrepancies between published results are often observed, with determined protein encapsulation efficiencies (EE) varying from 20 to 80%. We investigate the interference of excipients or the combination of excipients, on accurate EE determination of PLGA nanoparticles using the micro BCA assay. The EE was determined using multiple methods: by measuring the un-encapsulated protein (indirect approach) and directly by extracting the protein using sodium hydroxide and dimethyl sulfoxide. We show differences between the methods, highlight the most common pitfalls, and show the importance of using correct standards in assessing EE.

Among the most promising technologies for sustained drug delivery systems are core-shell nanofibres prepared by electrospinning. However, the most common method for the production of those, coaxial electrospinning, suffers from extremely low flow rates limiting the practical applications of such fibres. Emulsion electrospinning, on the other hand, enables the use of the high-throughput needle-less electrospinning devices for the production of the core-shell nanofibres with active pharmaceutical ingredients embedded and protected in their core. The development of such drug delivery systems for the applications in bone regeneration is further challenged by the need for the inorganic additives meant to stimulate the regeneration of the bone. The main objective of this work is to develop a high-throughput electrospinning method for the production of hybrid (organic-inorganic) and bioactive scaffolds needed for bone regeneration. We demonstrate the importance of the formulation, e.g., presence of surfactants, on the stability of the emulsion, and thus on electrospinning. Our work is an important step forward towards the high-throughput production of complex multi-material scaffolds for sustained drug delivery.