Sex matters in the establishment of murine tendon composition and material properties during growth.

The existence of sex-based differences in tendon and ligament injury rates has led investigators to test the hypothesis that sex plays a significant role in modulating tendon and ligament composition and material properties. To date, no studies have attempted to characterize how such differences develop during the course of normal tissue maturation and growth. Thus, the primary aim of the present study was to use a murine model to test the hypothesis that sex-based differences in the normal age-related development of tendon composition and material properties exist by assessing these parameters in the Achilles and tail tendons from 4-, 6-, 9-, 12-, and 15-week-old male and female C57Bl/6J mice. Despite significantly lower levels of total collagen content in females subsequent to sexual maturity (p<0.0001), as well as a significant effect of sex on glycosaminoglycan content (p<0.0001), Achilles tendon elastic modulus was not compromised in females. Female Achilles tendons did exhibit a significantly higher failure strain (p=0.0201) and strain energy density (p=0.0004) than did males, as well as a trend toward higher ultimate strength (p=0.0556). In contrast to the high load-bearing environment of the Achilles tendon site, sex did not have a statistically significant effect on any compositional or material property in the low load-bearing tendon fascicles of the tail. These data support recent studies by others, which suggest that male and female tendons have a differential adaptational response to their local mechanical loading environment.

Sexual dimorphism in the effect of GDF-6 deficiency on murine tendon.

Three members of the growth/differentiation factor (GDF) subfamily of bone morphogenetic proteins (BMPs), GDFs-5, -6, and -7, have demonstrated the potential to augment tendon and ligament repair. To gain further insight into the in vivo role of these molecules, previous studies have characterized intact and healing tendons in mice with functional null mutations in GDF-5 and -7. The primary goal of the present study was to perform a detailed characterization of the intact tendon phenotype in 4- and 16-week-old male and female GDF6-/- mice and their +/+ littermates. The results demonstrate that GDF6 deficiency was associated with an altered tendon phenotype that persisted into adulthood. Among males, GDF6-/- tail tendon fascicles had significantly less collagen and glycosaminoglycan content, and these compositional differences were associated with compromised material properties. The effect of GDF6 deficiency on tendon was sexually dimorphic, however, for among female GDF6-/- mice, neither differences in tendon composition nor in material properties were detected. The tendon phenotype that was observed in males appeared to be stronger in the tail site than in the Achilles tendon site, where some compositional differences were present, but no material property differences were detected. These data support existing in vitro studies, which suggest a potential role for BMP-13 (the human homologue to GDF-6) in tendon matrix modeling and/or remodeling.

Identification of a tendon phenotype in GDF6 deficient mice.

Increasing evidence suggests that the growth/differentiation factors, GDFs 5, 6, and 7 in particular, may play a role in tendon and ligament biology. Mice with genetic mutations in Gdf5 have altered tendon composition and mechanical behavior, whereas animals with functional null mutations in Gdf7 have a more subtle tendon phenotype. The present study demonstrates for the first time that a null mutation in Gdf6 is associated with substantially lower levels of tail tendon collagen content (-33%) in 4-week-old male mice, which has direct functional consequences for the mechanical integrity of the tissue (45-50% reduction in material properties). These data support a role for GDF6 in tendon matrix modeling.

Effect of GDF-7 deficiency on tail tendon phenotype in mice.

The subfamily of growth/differentiation factors (GDFs) known as GDFs 5, 6, and 7 appears to be involved in tendon maintenance and repair, although the precise nature of this role has yet to be elucidated. The aim of the present study was to examine the role of GDF-7 in tendon maintenance by studying tail tendon fascicle gene expression, composition, and material property strain rate dependency in 16-week-old male and female GDF-7 deficient mice. GDF-7 deficiency did not affect the biochemical composition of tail tendon fascicles, nor did it significantly affect the tensile material properties obtained at either slow (5%/s) or fast (50%/s) strain rates. Further, no difference was found between genotypes in the strain rate sensitivity of any tensile material property. Consistent with the compositional analyses, QRT-PCR data did not reveal any differences of twofold or greater in the gene expression levels of collagens I, III, V, nor in the proteoglycans decorin, fibromodulin, lumican, biglycan, versican, or aggrecan. Gdf5 expression was upregulated twofold in GDF-7 deficient tail tendons, and Bmp7 expression was downregulated twofold. No notable differences in expression levels for Bmp1-6 or Gdf6 were detected. GDF-5 protein levels were 50% higher in GDF-7 deficient tail tendon compared to wild type tail tendon. The results of this study support the intriguing possibility that compensation by Gdf-5 may be at least in part responsible for the absence of a strong phenotype in GDF-7 deficient mice.

Achilles tendon characterization in GDF-7 deficient mice.

Growth/differentiation factors (GDFs) play a significant role in numerous skeletal tissues and processes. Previous work using the brachypod mouse has suggested that GDF-5 affects Achilles tendon composition, ultrastructure, and material behavior, as well as tendon repair. The aim of the present study was to examine the role of a related GDF family member, GDF-7 (BMP-12), in intact tendon by studying the Achilles tendon of genetically engineered knockout mice. Achilles tendons from 16-week-old GDF-7 -/- mice contained 14% less GAG/DNA than did wild type littermates (p = 0.0481), although collagen content was comparable to controls. Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) results show that GDF-5 was upregulated two-threefold in response to the absence of GDF-7 protein. GDF-6 was also upregulated in knockouts, but to a lesser extent (twofold, p = 0.0013). On an ultrastructural level, GDF-7 deficient Achilles tendons exhibited a shift towards smaller diameter fibrils which resulted in a small but significant reduction in mean fibril diameter (-8%, p = 0.05). GDF-7 deficiency did not noticeably affect the expression of fibrillar collagens (I, III, V) or tendon proteoglycans (decorin, fibromodulin, lumican, biglycan, versican, aggrecan). Differences in tendon composition and ultrastructure were not biologically significant enough to have a noticeable effect on the structural or material behavior of the tendons. These results demonstrate that GDF-7 deficiency has a subtle effect on the composition and ultrastructure of murine Achilles tendon. The small magnitude of the observed differences may be due to overcompensation by related GDF family members.