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

Tianchi Ma,Ignacio Sallent,Jennifer L Puetzer,Amy Gelmi,Molly M Stevens

doi:10.1016/j.biomaterials.2020.120527

Tianchi Ma, Ignacio Sallent + Show 3 more

Open Access

https://doi.org/10.1016/j.biomaterials.2020.120527

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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 fibers organize similar to bovine juvenile 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 juvenile 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.

Highlights

Collagen fibers are the primary source of strength in connective tissues throughout the body, musculoskeletal tissues such as tendons, ligaments, and menisci
Tenocytes were isolated from deep flexor tendons, and ligament fibroblasts were isolated from the anterior cruciate ligament (ACL) with 0.2% w/v collagenase digestion overnight [11]
Confocal reflectance revealed that all unclamped scaffolds remained unorganized throughout culture, while clamped constructs developed aligned collagen fibrils by 2 weeks which developed into fibers (20-40 μm in diameters) similar to their respective native tissue by 6 weeks

Summary

Introduction

Collagen fibers are the primary source of strength in connective tissues throughout the body, musculoskeletal tissues such as tendons, ligaments, and menisci These tissues are characterized by large hierarchically organized type I collagen fibers which assemble from tropocollagen molecules into fibrils (10 - 300 nm, up to 1 μm diameter), fibers ( >10 μm diameter) and fascicles (100s μm to mm diameter, Figure 1) [1,2,3]. Due to limited blood supply and demanding load environments, these tissues are characterized by little to no healing Injuries to these tissues result in over 1.4 million surgeries a year in the US (based on 1,000,000 meniscus, 130,000 ACL, 275,000 rotator cuff repairs per year) [4,6,7,8]. Engineered replacements are a promising alternative [4,6,7]; these replacements often fail to produce sufficient collagen, let alone the organized collagen fibers essential to long term mechanical success and often do not translate to the clinic

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