The aim of this study was to determine the role of the perlecan (Hspg2) heparan sulphate (HS) side chains on cell and matrix homeostasis in tail and Achilles tendons in 3 and 12 week old Hspg2 exon 3 null HS deficient (Hspg2Δ3-/Δ3-) and C57 BL/6 Wild Type (WT) mice. Perlecan has important cell regulatory and matrix organizational properties through HS mediated interactions with a range of growth factors and morphogens and with structural extracellular matrix glycoproteins which define tissue function and allow the resident cells to regulate tissue homeostasis. It was expected that ablation of the HS chains on perlecan would severely disrupt normal tendon organization and functional properties and it was envisaged that this study would better define the role of HS in normal tendon function and in tendon repair processes. Tail and Achilles tendons from each genotype were biomechanically tested (ultimate tensile stress (UTS), tensile modulus (TM)) and glycosaminoglycan (GAG) and collagen (hydroxyproline) compositional analyses were undertaken. Tenocytes were isolated from tail tendons from each mouse genotype and grown in monolayer culture. These cultures were undertaken in the presence of FGF-2 to assess the cell signaling properties of each genotype. Total RNA was isolated from 3-12 week old tail and Achilles tendons and qRT-PCR was undertaken to assess the expression of the following genes Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1. Type VI collagen and perlecan were immunolocalised in tail tendon and collagen fibrils were imaged using transmission electron microscopy (TEM). FGF-2 stimulated tenocyte monolayers displayed elevated Adamts4, Mmp2, 3, 13 mRNA levels compared to WT mice. Non-stimulated tendon Col1A1, Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1 mRNA levels showed no major differences between the two genotypes other than a decline with ageing while LTBP2 expression increased. Eln expression also declined to a greater extent in the perlecan exon 3 null mice (P < 0:05). Type VI collagen and perlecan were immunolocalised in tail tendon and collagen fibrils imaged using transmission electron microscopy (TEM). This indicated a more compact form of collagen localization in the perlecan exon 3 null mice. Collagen fibrils were also smaller by TEM, which may facilitate a more condensed fibril packing accounting for the superior UTS displayed by the perlecan exon 3 null mice. The amplified catabolic phenotype of Hspg2Δ3-/Δ3- mice may account for the age-dependent decline in GAG observed in tail tendon over 3 to 12 weeks. After Achilles tenotomy Hspg2Δ3-/Δ3- and WT mice had similar rates of recovery of UTS and TM over 12 weeks post operatively indicating that a deficiency of HS was not detrimental to tendon repair.
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- Extracellular matrix
- Fibroblast growth factor
- Heparan sulphate