Collagen scaffolds with controlled topography and stiffness and mechanical stimulation direct tissue-specific cell phenotype for tendon regeneration

Tendon and ligament injuries represent a major global cause of disability, frequently requiring surgical intervention to restore function. While tissue grafts remain the clinical gold standard for tendon augmentation, their use is constrained by risks of disease transmission and suboptimal tissue integration. Emerging tissue engineering strategies aim to overcome these limitations through the ex vivo development of tendon substitutes using biomaterial scaffolds and targeted microenvironmental cues; however, further refinement is required to achieve functional regeneration. Collagen type I, the principal structural component of tendons, holds great potential as a scaffold biomaterial, yet its adoption is hindered by sourcing and processing concerns. On the one hand, traditional sources such as bovine and porcine tissues raise concerns related to zoonosis risks and ethical acceptability. On the other hand, collagen manipulation complexity has traditionally limited the architectural and mechanical properties of collagen scaffolds. In this study, we hypothesised that an appropriate collagen type I scaffold with defined architectural and mechanical properties can maintain the phenotype of human tendon cells and induce the deposition of organised tendon-specific ECM in vitro. In the first phase of this study, collagen type I was extracted from caprine skin, digital flexor and digital extensor tendons and compared to that extracted from bovine and porcine Achilles tendons. Biochemical analysis confirmed that caprine collagen type I purity was on pair with that of traditional sources. Subsequently, collagen was either non-crosslinked or crosslinked with 4-arm succinimidyl glutarate and processed into films. Caprine scaffolds displayed macroscopic and microscopic features, including fibril diameter ranges, similar to their bovine and porcine counterparts regardless of the tissue source. Mechanical characterisation revealed that caprine scaffolds were intrinsically stiffer and less susceptible to modulus increases upon crosslinking, suggesting underlying species-specific biochemical differences. With respect to cytocompatibility, caprine tendon-derived scaffolds supported the attachment, proliferation and metabolic activity of fibroblast and macrophages at levels consistent with those observed on bovine and porcine scaffolds. Notably, caprine skin-derived collagen enhanced both fibroblast and macrophage attachment relative to tendon-derived collagen from all species, pointing to tissue-specific functional differences. Immunogenicity analysis revealed all caprine scaffolds induced lower pro-inflammatory responses than Escherichia coli lipopolysaccharides on tissue culture plastic and elicited responses comparable to traditional collagen scaffolds. Minor increases in tumour necrosis factor alpha expression were observed in crosslinked groups, likely reflecting the influence of increased surface stiffness on macrophage behaviour rather than a direct effect of the crosslinker agent, as this was not observed in the indirect cultures. Collectively, these results underscore the potential of caprine tissues as an alternative source of collagen type I for the fabrication of medical devices. In the second phase of this study, collagen scaffolds featuring either planar or grooved (2 x 2 x 2 μm) surface topographies and tuneable mechanical properties were fabricated using soft lithography and chemical crosslinking with different concentrations of succinimidyl glutarate (0.5 mM, 1.0 mM and 1.5 mM). Surface characterisation confirmed the presence of well-defined surface grooves, particularly in crosslinked scaffolds. The crosslinking agent reduced scaffold free amine content and increased Young’s modulus, indicating the formation of covalent bonds. In addition, micro-indentation measurements revealed a concentration-dependent increase in surface stiffness. In vitro experiments using human tendon cells demonstrate that grooved topographies promoted anisotropic cell and extracellular matrix alignment, especially in crosslinked scaffolds, highlighting the importance of collagen crosslinking for pattern stability. Crosslinking also exerted a dose-dependent effect on cell phenotype, with the highest concentration reducing cell proliferation, the lowest concentration inducing the broadest tendon-marker upregulation and all tested concentrations inducing higher tenascin C deposition than the non-crosslinked counterparts, a phenomenon we attributed to crosslinker-mediated surface smoothening and scaffold stiffening, respectively. Scaffolds crosslinked with the lowest crosslinker concentration and subjected to different tensional regimes (no tension, static tension and cyclic tension) showed that, within the parameters utilised in this study (frequency, strain and rest interval), static tension resulted in higher cell proliferation, enhanced cell and extracellular alignment and increased tendon marker upregulation compared to cyclic stimulation in planar, grooved, and both planar and grooved scaffolds, respectively. Collectively, this study advocates the use of combined biophysical cues to maintain physiological cell function. In conclusion, our findings underscore the viability of caprine tissue as a source of collagen type I for the manufacturing of collagen devices and demonstrate that precise modulation of collagen scaffold architecture and mechanical properties directly influences the maintenance of a tendon cell phenotype in vitro. Together, these insights offer a foundation for the design of collagen-based functional tendon substitutes.

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University of Galway

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University of Galway Theses (PhD Theses)