Comparative transcriptional analysis of three human ligaments with distinct biomechanical properties

Abstract

One major aim of regenerative medicine targeting the musculoskeletal system is to provide complementary and/or alternative therapeutic approaches to current surgical therapies, often involving the removal and prosthetic substitution of damaged tissues such as ligaments. For these approaches to be successful, detailed information regarding the cellular and molecular composition of different musculoskeletal tissues is required. Ligaments have often been considered homogeneous tissues with common biomechanical properties. However, advances in tissue engineering research have highlighted the functional relevance of the organisational and compositional differences between ligament types, especially in those with higher risks of injury. The aim of this study was to provide information concerning the relative expression levels of a subset of key genes (including extracellular matrix components, transcription factors and growth factors) that confer functional identity to ligaments. We compared the transcriptomes of three representative human ligaments subjected to different biomechanical demands: the anterior cruciate ligament (ACL); the ligamentum teres of the hip (LT); and the iliofemoral ligament (IL). We revealed significant differences in the expression of type I collagen, elastin, fibromodulin, biglycan, transforming growth factor β1, transforming growth interacting factor 1, hypoxia-inducible factor 1-alpha and transforming growth factor β-induced gene between the IL and the other two ligaments. Thus, considerable molecular heterogeneity can exist between anatomically distinct ligaments with differing biomechanical demands. However, the LT and ACL were found to show remarkable molecular homology, suggesting common functional properties. This finding provides experimental support for the proposed role of the LT as a hip joint stabiliser in humans.

Keywords: Sox9; biglycan; collagen; decorin; transforming-growth-factor-beta.

Fig. 1

Comparative analysis of relative gene expression levels of fibrillar components of the ECM in the iliofemoral ligament (IL), the ligamentum teres of the hip (LT) and the anterior cruciate ligament (ACL), as measured by Q-PCR. Note that the expression level profiles for fibrillar components of the ECM are comparable between the LT and ACL but different in the IL. The expression level in the IL was considered as the baseline for each gene (calibrator). n = 6 LT, 4 ACL and 4 IL. *P < 0.05, **P < 0.01 or ***P < 0.001 in ACL or LT vs. LT. COL, collagen; ELN, elastin. Each bar represents the mean value ± SEM.

Fig. 2

Comparative analysis of relative gene expression levels of characteristic components of the ECM of ligaments in the iliofemoral ligament (IL), the ligamentum teres of the hip (LT) and the anterior cruciate ligament (ACL), as measured by Q-PCR. Note that the expression levels of the canonical ligament PGs DCN, BGN and FMOD are always higher in the ACL and/or LT than in the IL. The same pattern is observed for BIGH3, an important component of the ECM. The expression level in the IL was considered the baseline level for each gene (calibrator). BGN, Biglycan; BIGH3, Transforming growth factor β induced gene; DCN, Decorin; FMOD; Fibromodulin. n = 6 LT, 4 ACL and 4 IL. *P < 0.05, **P < 0.01 or ***P < 0.001 in ACL or LT vs. LT. ⁺P < 0.05 or ⁺⁺⁺P < 0.001 in ACL vs. LT. Each bar represents the mean value ± SEM.

Fig. 3

Comparative analysis of relative gene expression levels of growth and transcription factor genes in the iliofemoral ligament (IL), the ligamentum teres of the hip (LT) and the anterior cruciate ligament (ACL) as evaluated by Q-PCR. Note that the expression levels of the growth and transcription factors illustrated in the figure are similar in the ACL and LT but distinct in the IL. The expression level in the IL was considered the baseline for each gene (calibrator). BMP12, Bone morphogenetic protein 12; HIF1A, Hypoxia inducible factor 1α; SOX9, SRY (sex-determining region Y)-box 9 gen; TGFβ1, Transforming growth factor β1; TGiF1, Transforming growth interacting factor 1. n = 6 LT, 4 ACL and 4 IL. * P < 0.05, ** P < 0.01 or *** P < 0.001 in ACL or LT vs. LT. ⁺ P < 0.05 in ACL vs. LT. Each bar value represents the mean ± SEM.

Fig. 4

Representative Western blots illustrating DECORIN, TGFβ1 precursor and SOX9 expression in the iliofemoral ligament (IL), the ligamentum teres (LT) and the anterior cruciate ligament (ACL). Note that the findings at the protein level reproduce to some extent those for gene expression. GAPDH was used as the loading control in all the experiments. n = 4 LT, 4 ACL and 4 IL.