Cellular and molecular maturation in fetal and adult ovine calcaneal tendons

Abstract

Processes of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid- (2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of mid- and late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models.

Ectopic tissue engineered ligament with silk collagen scaffold for ACL regeneration: A preliminary study

“…During development, tendons are highly plastic tissues in fetus with high cellularity and vascularity, which decrease with tendon maturation [54,55]. Although over-expression of vascular endothelial growth factor (VEGF) has been shown to impede improvements in biomechanical strength after ACL regeneration, blocking VEGF significantly reduced angiogenesis, graft maturation and biomechanical strength [56].…”

Variations during ageing in the three-dimensional anatomical arrangement of fascicles within the equine superficial digital flexor tendon

“…The relatively cellular endotenon/IFM has a more complex protein composition and a higher rate of turnover than the fascicles (Thorpe et al, 2016a;Thorpe et al, 2016b). The endotenon/IFM is present during foetal development at which stage it appears to be thicker than in adult tendons (Russo et al, 2015).…”

Connexins and pannexins in the skeleton: gap junctions, hemichannels and more

“…The same 2 connexins are expressed in equine [28], avian [29] and sheep [30] tendon. The 2 connexins exhibit distinct localization, with Cx43 found in the tip of the cell processes as well as between cell bodies and Cx32 only between cell bodies [27].…”

“…The expression of Cx43 and Cx32 has been studied in fetal and adult tendons obtained from horse [28], rat [27,160] and sheep [30]. These studies show that the 2 connexins are present throughout tendon development, although with a different pattern of expression and localization.…”

“…These studies show that the 2 connexins are present throughout tendon development, although with a different pattern of expression and localization. Their level is high in fetal life and decreases in adult tissue, and these changes accompany the decrease in the proliferation index and cellularity of the tendons, as well as the change from round to elongated cell nuclei [27,28,30,160]. This suggests that Cx43 and Cx32 could be involved in the maturation of the tendon.…”