Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human and equine athletes, due to the cumulative degenerative damage. These injuries present significant challenges as the healing process often results in the formation of inferior scar tissue. The poor success with conventional therapy supports the need to search for novel treatments to restore functionality and regenerate tissue as close to native tendon as possible. Mesenchymal stem cell (MSC)-based strategies represent promising therapeutic tools for tendon repair in both human and veterinary medicine. The translation of tissue engineering strategies from basic research findings, however, into clinical use has been hampered by the limited understanding of the multifaceted MSC mechanisms of action. In vitro models serve as important biological tools to study cell behavior, bypassing the confounding factors associated with in vivo experiments. Controllable and reproducible in vitro conditions should be provided to study the MSC healing mechanisms in tendon injuries. Unfortunately, no physiologically representative tendinopathy models exist to date. A major shortcoming of most currently available in vitro tendon models is the lack of extracellular tendon matrix and vascular supply. These models often make use of synthetic biomaterials, which do not reflect the natural tendon composition. Alternatively, decellularized tendon has been applied, but it is challenging to obtain reproducible results due to its variable composition, less efficient cell seeding approaches and lack of cell encapsulation and vascularization. The current review will overview pros and cons associated with the use of different biomaterials and technologies enabling scaffold production. In addition, the characteristics of the ideal, state-of-the-art tendinopathy model will be discussed. Briefly, a representative in vitro tendinopathy model should be vascularized and mimic the hierarchical structure of the tendon matrix with elongated cells being organized in a parallel fashion and subjected to uniaxial stretching. Incorporation of mechanical stimulation, preferably uniaxial stretching may be a key element in order to obtain appropriate matrix alignment and create a pathophysiological model. Together, a thorough discussion on the current status and future directions for tendon models will enhance fundamental MSC research, accelerating translation of MSC therapies for tendon injuries from bench to bedside.

(1) Background: Tendinopathy is a common injury in both human and equine athletes. Representative in vitro models are mandatory to facilitate translation of fundamental research into successful clinical treatments. Natural biomaterials like gelatin provide favorable cell binding characteristics and are easily modifiable. In this study, methacrylated gelatin (gel-MA) and norbornene-functionalized gelatin (gel-NB), crosslinked with 1,4-dithiotreitol (DTT) or thiolated gelatin (gel-SH) were compared. (2) Methods: The physicochemical properties (¹H-NMR spectroscopy, gel fraction, swelling ratio, and storage modulus) and equine tenocyte characteristics (proliferation, viability, and morphology) of four different hydrogels (gel-MA, gel-NB85/DTT, gel-NB55/DTT, and gel-NB85/SH75) were evaluated. Cellular functionality was analyzed using fluorescence microscopy (viability assay and focal adhesion staining). (3) Results: The thiol-ene based hydrogels showed a significantly lower gel fraction/storage modulus and a higher swelling ratio compared to gel-MA. Significantly less tenocytes were observed on gel-MA discs at 14 days compared to gel-NB85/DTT, gel-NB55/DTT and gel-NB85/SH75. At 7 and 14 days, the characteristic elongated morphology of tenocytes was significantly more pronounced on gel-NB85/DTT and gel-NB55/DTT in contrast to TCP and gel-MA. (4) Conclusions: Thiol-ene crosslinked gelatins exploiting DTT as a crosslinker are the preferred biomaterials to support the culture of tenocytes. Follow-up experiments will evaluate these biomaterials in more complex models.

This study investigates photo-crosslinkable gelatin-based hydrogels - thiolated gelatin (GelSH) and gelatin norbornene (GelNB) - for volumetric additive manufacturing (VAM). GelSH was synthesized with degrees of thiol substitution (DS) of 39%, 54%, and 63%, and GelNB with a DS of 60% (with respect to primary amine content). These were combined into GelNB-GelSH photo-resins at 5, 7.5, and 10% (w/v) and crosslinked via thiol-ene chemistry. Physico-chemical analysis showed that increasing DS and polymer concentration reduced swelling and increased moduli. VAM enabled the fabrication of high-resolution 3D hydrogel constructs from optimized formulations, demonstrating the ability to encapsulate mesenchymal stromal cells (MSCs) within a mechanically tunable, cell-supportive hydrogel environment. Film-cast hydrogels, also with embedded MSCs, served as comparative controls. VAM-printed constructs exhibited significantly higher alkaline phosphatase activity and calcium deposition, indicating enhanced osteogenesis. In contrast, chondrogenic and adipogenic differentiation were more pronounced in film-cast samples, due to their lower crosslinking density and stiffness. These findings emphasize the importance of matrix mechanics in guiding stem cell differentiation and demonstrate the potential of VAM for producing complex, functional scaffolds for tissue engineering. This work supports further development of tunable gelatin-based bioresins for applications requiring lineage-specific differentiation, including those targeting softer tissue types. Highlights: Volumetric additive manufacturing (VAM) enables high-fidelity 3D hydrogel scaffolds. GelNB-GelSH bioresins support MSC encapsulation and differentiation. VAM scaffolds enhance osteogenesis, film-cast gels favor chondro- and adipogenesis. Mechanical properties and crosslinking density regulate stem cell fate in hydrogels. This study advances bioresin development for multi-lineage tissue engineering. Abstract Figure: