Driving Hierarchical Collagen Fiber Formation for Functional Tendon, Ligament, and Meniscus Replacement

Abstract

Hierarchical collagen fibers are the primary source of strength in musculoskeletal tendons, ligaments, and menisci. It has remained a challenge to develop these large fibers in engineered replacements or in vivo after injury. The objective of this study was to investigate the ability of restrained cell-seeded high density collagen gels to drive hierarchical fiber formation for multiple musculoskeletal tissues. We found boundary conditions applied to high density collagen gels were capable of driving tenocytes, ligament fibroblasts, and meniscal fibrochondrocytes to develop native-sized hierarchical collagen fibers 20-40 µm in diameter. The collagen fibers organize similar to native collagen with native fibril banding patterns and hierarchical fiber bundles 50-350 µm in diameter by 6 weeks. Mirroring fiber organization, tensile properties of restrained samples improved significantly with time, reaching ∼1 MPa. Additionally, tendon, ligament, and meniscal cells produced significantly different sized fibers, different degrees of crimp, and different GAG concentrations, which corresponded with respective native tissue. To our knowledge, these are some of the largest, most organized fibers produced to date in vitro. Further, cells produced tissue specific hierarchical fibers, suggesting this system is a promising tool to better understand cellular regulation of fiber formation to better stimulate it in vivo after injury.

Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels

“…Since tendons are composed of 60 – 85% (dry weight) collagens, of which 95% is type I [22,23], in vitro reconstituted collagen type I hydrogels are commonly investigated as a 3D tendon scaffold. However, extracted self-assembled collagen hydrogels, exhibit poor mechanical properties and random fibrillar orientations [24,25]. These properties are not ideal as tendon scaffolds as they do not reflect the structure and anisotropy of the load-bearing aligned tissue and are mechanically weak.…”

Biofabrication of development-inspired scaffolds for regeneration of the annulus fibrosus macro- and microarchitecture

“…Alternatively, 3D matrices composed of fibrous collagen (51)(52)(53)(54) or fibrin (55) have been studied in combination with mechanical stretch to achieve oriented collagen matrix deposition. Overall, however, the de novo tissues still lack the biomechanical performance and/or nanometer-scale organization of native collagen (51,56). Notably, current scaffold-based approaches are characterized by the random spatial arrangement of elongated cells relative to one another.…”

“…However, a major disadvantage hampering clinical translation of this approach is the requirement of a post-processing step, such as folding (48), stacking (21), or rolling (49) to generate 3D structures, and a tendency to exhibit limited porosity for cell viability and matrix deposition (50). Alternatively, 3D matrices composed of fibrous collagen (51)(52)(53)(54) or fibrin (55) have been studied in combination with mechanical stretch to achieve oriented collagen matrix deposition. Overall, however, the de novo tissues still lack the biomechanical performance and/or nanometer-scale organization of native collagen (51,56).…”

Construction of a liquid-liquid phase separation system from the gel-sol transition of elongated protein microgels in a crowding agent

“…Phase transitions are common guiding principles for the processing of soft materials for biological and medical studies (1)(2)(3)(4). To date, sol-gel transitions have been substantially studied and utilised for the fabrication of solid-state materials like soft hydrogels for diverse purposes such as tissue engineering and pathology studies (1)(2)(3)(4)(5)(6)(7)(8).…”

“…Phase transitions are common guiding principles for the processing of soft materials for biological and medical studies (1)(2)(3)(4). To date, sol-gel transitions have been substantially studied and utilised for the fabrication of solid-state materials like soft hydrogels for diverse purposes such as tissue engineering and pathology studies (1)(2)(3)(4)(5)(6)(7)(8). Recently, there is an increasing interest in liquid proteinaceous materials, as they are found to be highly related to diverse biological functions or malfunctions for healthcare and diseases (9)(10)(11)(12).…”