The advent of bioprinting has enabled the creation of precise three‐dimensional (3D) cell cultures suitable for biomimetic in vitro models. In this study, we developed a novel protocol for 3D printing methacrylated collagen (ColMa, or PhotoCol®) combined with tendon stem/progenitor cells (hTSPCs) derived from human tendon explants. Although pure ColMa has not previously been proposed as a printable hydrogel, this paper outlines a robust and highly reproducible pipeline for bioprinting this material. Indeed, we successfully fabricated a 3D bioengineered scaffold and cultured it for 21 days under perfusion conditions with medium supplemented with growth/differentiation factor‐5 (GDF‐5). This bioprinting pipeline and the culture conditions created an exceptionally favorable 3D environment, enabling the cells to proliferate, exhibit tenogenic behaviors, and produce a new collagen type I matrix, thereby remodeling the surrounding environment. Indeed, over the 21‐day culture period under perfusion condition, tenomodulin expression showed a significant upregulation on day 7, with a 2.3‐fold increase, compared to days 14 and 21. Collagen type I gene expression was upregulated nearly 10‐fold by day 14. This trend was further confirmed by western blot analysis, which revealed a statistically significant difference in tenomodulin expression between day 21 and both day 7 and day 14. For type I collagen, significant differences were observed between day 0 and day 21, as well as between day 0 and day 14, with a p‐value of 0.01. These results indicate a progressive over‐expression of type I collagen, reflecting cell differentiation towards a proper tenogenic phenotype. Cytokines, such as IL‐8 and IL‐6, levels peaked at 8566 and 7636 pg/mL, respectively, on day 7, before decreasing to 54 and 46 pg/mL by day 21. Overall, the data suggest that the novel ColMa bioprinting protocol effectively provided a conducive environment for the growth and proper differentiation of hTSPCs, showcasing its potential for studying cell behavior and tenogenic differentiation.

Tendon injuries present significant clinical challenges due to limited intrinsic healing and complex pathological mechanisms. Here, we developed a novel 3D bioprinted methacrylated type I collagen (ColMA) scaffold integrated with Growth Differentiation Factor-5 (GDF-5)-loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticles and dynamically cultured it under perfusion to establish a tenogenic microenvironment in vitro. Pathological human Tendon Stem/Progenitor Cells (hTSPCs) derived from tendinopathic surgical explants were encapsulated to investigate their impaired extracellular matrix (ECM) deposition and associated pro-inflammatory signaling. GDF-5-loaded nanoparticles (average diameter 140 ± 40 nm) were fabricated via microfluidic-assisted nanoprecipitation and homogeneously incorporated within the ColMA synthetic ECM to enable sustained growth factor release. Continuous perfusion culture (1 mL/min) ensured efficient mass transfer and supported cell viability above 70% over 21 days. Pathological hTSPCs exhibited impaired ECM remodeling, characterized by the absence of type I collagen and a 2.56-fold increase in type III collagen at day 7, indicative of a fibrotic-like phenotype. Western blot densitometry demonstrated a 5.31-fold elevation in secreted tenomodulin at day 14, while ECM analysis verified a type III to type I collagen ratio of 4.5. In addition, a markedly pro-inflammatory cytokine profile was observed, with elevated secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) from day 7 onward, consistent with the chronic inflammatory status of cells derived from pathological tendon tissues. This modular 3D platform represents a robust in vitro model for mechanistic studies and the advancement of personalized regenerative strategies targeting chronic tendon disorders.

This study introduces a 3D bioprinted scaffold embedding human Bone Morphogenetic Protein-2 (hBMP-2)-loaded Poly-lactic-co-glycolic Acid nanocarriers (PLGA-NCs) to induce the osteogenic differentiation of human Tendon Stem/Progenitor Cells (TSPCs) harvested from tendon explants. The nanocarriers (120 ± 40 nm) were embedded in a Gelatin Methacryloyl (GelMA) matrix and bioplotted under optimized conditions to preserve cell viability (60%). A 3D dynamic culture system, supported by compartmental modelling, enabled continuous perfusion over 21 days, promoting efficient metabolite exchange and removal of catabolic byproducts. This environment enhanced proliferation and osteogenic differentiation, demonstrated by a 20-fold increase in osteopontin (p < 0.05) and an 8-fold increase in osteocalcin (p < 0.05) expression, along with increased calcium deposition observed via Alizarin Red S staining. In contrast, static monolayer cultures with soluble hBMP-2 (20 ng/mL) showed reduced osteogenic activity. These findings suggest that the engineered scaffold offers a promising strategy for regenerating the bone-tendon interface by leveraging the multipotency of TSPCs in a biomimetic 3D environment.

Tendon tissue engineering requires scaffolds with an appropriate structure that can be cultured under cyclic strain in order to replicate both mechanical cues and biological commitments. This study presents a hybrid scaffold combining 3D-printed auxetic polycaprolactone (PCL) with electrospun gelatin-(3-Glycidyloxypropyl)trimethoxysilane (GPTMS) functionalized with collagen microfibers suitable for the purpose of tenogenic commitment and in vitro modelling. The auxetic core was fabricated via pneumatic melt extrusion at 1000 mbar and printing speed of 200 mm/min, achieving a re-entrant angle of 60.92 ± 2.1° and strut thickness of 350 ± 30 μm with high geometric fidelity. Mechanical characterization revealed that PCL scaffolds exhibited an elastic modulus of 51.97 ± 6.19 MPa and ultimate tensile strain of 6.75 ± 0.59%. Integration of electrospun gelatin-GPTMS without collagen increased the elastic modulus to 66.54 ± 7.16 MPa while reducing ultimate tensile strain to 4.14 ± 1.27%. Incorporation of 1 mg/mL and 2 mg/mL μCollaFibR™ significantly enhanced ultimate tensile strain to 21.70 ± 0.20% and 19.94 ± 0.20%, while decreasing elastic modulus to 47.65 ± 1.33 MPa and 39.83 ± 1.85 MPa. Field Emission-Scanning Electron Microscopy confirmed hierarchical architecture with collagen microfibers (1-5 μm diameter) interspersed throughout the electrospun gelatin matrix. Human tendon stem/progenitor cells seeded at 1 × 10⁵ cells/cm² maintained viability exceeding 85% at day 3 across all formulations, exhibiting elongated morphology and alignment along nanofiber axes. The developed multimaterial platform successfully integrates auxetic mechanics with extracellular matrix-mimetic topographies, offering a biomimetic scaffold structure for subsequent in vitro modelling of tenogenic events.