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The anterior cruciate ligament (ACL) connects to bone via structurally complex insertions known as entheses that translate load from elastic ligament and stiff bone via gradients in organization, composition, and cell phenotype [1]. These gradients are not recreated in graft repair or engineered replacements, yielding limited repair options and high failure rates [2]. Previously, we developed a culture system that uses a tensile-compressive interface to guide ligament fibroblasts to develop early postnatal-like entheses by 6 weeks [3]; however, cells used were isolated from the entirety of the neonatal bovine ACL from bone to bone and likely contained multiple cell phenotypes and progenitor cells [3]. This study explored how ligament fibroblasts from ACL mid-substance and fibrochondrocytes from ACL entheses respond to mechanical cues in our system to assess if cells localize to specific tissue regions or remain mixed and differentiate in response to the local mechanical environment over 6 weeks. Confocal microscopy revealed at 0 weeks all regions begin unorganized with an even distribution of ligament (green) and enthesis (violet) cells for 50/50 ligament/enthesis co-culture (Fig 1). By 6 weeks, 50/50 co-culture resulted in early postnatal-like organization [1,3] with mixed cell distribution; however, ligament cells appear to undergo zonal morphological changes with elongated cells in the middle and larger rounded cells under the clamp. Zonal cell morphology, biochemical, and mechanical analysis of 100% ligament, 100% enthesis, and 50/50 co-culture are ongoing.

References: [1] Lu+ 2013, [2] Patel+ 2018, [3] Brown+ 2022.

Publication Date


Subject Major(s)

Biomedical Engineering


anterior cruciate ligament, enthesis, tensile-compressive, fibrochondrocytes, fibroblasts, cell localization, cell differentiation


Molecular, Cellular, and Tissue Engineering | Musculoskeletal System

Current Academic Year


Faculty Advisor/Mentor

Dr. Jennifer L. Puetzer

Faculty Advisor/Mentor

Michael E. Brown


© The Author(s)

Exploring Cell Differentiation Vs. Localization in Engineered Ligament-to-Bone Entheses