The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes.

Tendons and ligaments are connective tissues that guide motion, share loads, and transmit forces in a manner that is unique to each as well as the anatomical site and biomechanical stresses to which they are subjected. Collagens are the major molecular components of both tendons and ligaments. The hierarchical structure of tendon and its functional properties are determined by the collagens present, as well as their supramolecular organization. There are 28 different types of collagen that assemble into a variety of supramolecular structures. The assembly of specific supramolecular structures is dependent on the interaction with other matrix molecules as well as the cellular elements. Multiple suprastructural assemblies are integrated to form the functional tendon/ligament. This chapter begins with a discussion of collagen molecules. This is followed by a definition of the supramolecular structures assembled by different collagen types. The general principles involved in the assembly of collagen-containing suprastructures are presented focusing on the regulation of tendon collagen fibrillogenesis. Finally, site-specific differences are discussed. While generalizations can be made, differences exist between different tendons as well as between tendons and ligaments. Compositional differences will impact structure that in turn will determine functional differences. Elucidation of the unique physiology and pathophysiology of different tendons and ligaments will require an appreciation of the role compositional differences have on collagen suprastructural assembly, tissue organization, and function.

No abstract

Specific niches may affect how cells from different regions contribute to tendon biology, particularly in regard to the healing of certain tendinopathies. The objectives of this study are to determine whether distinct subpopulations of stem/progenitor cells are found within the tendon proper and the epi- and paratenon, the peritenon, as well as to characterize these stem/progenitor cell populations. In this study, we hypothesized that tendon stem/progenitor cells exist in each region, that these populations possess distinct features, and that these populations while multipotent could have differing potentials. To test this hypothesis, stem/progenitor cells were isolated and characterized from the peritenon and tendon proper of mouse Achilles tendons. Colony-forming unit and multipotency assays, as well as flow cytometry, and real-time quantitative polymerase chain reaction analyses of stem cell markers were performed. Significantly, more stem/progenitor cell colonies were observed from cells derived from the tendon proper relative to the peritenon. Analysis of surface markers for stem/progenitor cells from both regions indicated that they were Sca1(+) (stem cell marker), Cd90(+) and Cd44(+) (fibroblast markers), Cd18(-) (leukocyte marker), Cd34(-) (hematopoietic and vascular marker), and Cd133(-) (perivascular marker). Tendon proper stem/progenitor cells had increased expression levels for tenomodulin (Tnmd) and scleraxis (Scx), indicative of enrichment of stem/progenitor cells of a tendon origin. In contrast, cells of the peritenon demonstrated relative increases in the vascular (endomucin) and pericyte (Cd133) markers relative to cells from the tendon proper. Stem/progenitor cells from both regions were multipotent (adipogenic, chondrogenic, osteogenic, and tenogenic). These findings demonstrated that different progenitor populations exist within discrete niches of the Achilles tendon-tendon proper versus peritenon. Overall, these data support the hypothesis that the progenitor pools from both regions have distinct properties and contain enriched progenitor subpopulations of different origins. Moreover, in considering their roles in tendon healing more broadly, they are potential cell sources that may differentially contribute to intrinsic and extrinsic tendon repair mechanisms. That is, intrinsic repair may require a progenitor class with predominant tendon marker expression, while extrinsic repair may involve a progenitor class recruited from perivascular cells of the peritenon.

Recent studies have demonstrated that the small leucine-rich proteoglycans (SLRPs) biglycan and decorin impact tendon development, aging and healing in mature mice. However, despite the increased risk of tendon injury in the elderly, the role of SLRPs in tendon repair has not been investigated in aged animals. Therefore, our objective was to elucidate the influences of bigylcan and decorin on tendon healing in aged mice to relate our findings to previous work in mature mice. Since the processes of aging and healing are known to interact, our hypothesis was that aging mediates the role of biglycan and decorin on tendon healing. Patellar tendons from wild-type, biglycan-null and decorin-null mice were injured at 270 days using an established model. At 3 and 6 weeks post-surgery, structural, mechanical and biochemical analyses were performed and compared to uninjured controls. Early stage healing was inferior in biglycan-null and decorin-null mice as compared to wild type. However, tendons of all genotypes failed to exhibit improved mechanical properties between 3 and 6 weeks post-injury. In contrast, in a previous investigation of tendon healing in mature (i.e., 120 day-old) mice, only biglycan-null mice were deficient in early stage healing while decorin-null− mice were deficient in late-stage healing. These results confirm that the impact of SLRPs on tendon healing is mediated by age and could inform future age-specific therapies for enhancing tendon healing.

Defining the constituent regulatory molecules in tendon is critical to understanding the process of tendon repair and instructive to the development of novel treatment modalities. The purpose of this study is to define the structural, expressional, and mechanical changes in the tendon injury response, and elucidate the roles of two class I small leucine-rich proteoglycans (SLRPs). We utilized biglycan-null, decorin-null and wild type mice with an established patellar tendon injury model. Mechanical testing demonstrated functional changes associated with injury and the incomplete recapitulation of mechanical properties after six weeks. In addition, SLRP deficiency influenced the mechanical properties with a marked lack of improvement between three and six weeks in decorin-null tendons. Morphological analyses of the injury response and role of SLRPs demonstrated alterations in cell density and shape as well as collagen alignment and fibril structure resulting from injury. SLRP gene expression was studied using RT-qPCR with alterations in expression associated with the injured tendons. Our results show that in the absence of biglycan initial healing may be impaired while in the absence of decorin later healing is clearly diminished. This suggests that biglycan and decorin may have sequential roles in the tendon response to injury.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ2=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.