A study on the structure and morphologic development of calcaneal tendon and triceps surae muscle in human fetuses during the fetal period and the evaluation of clinical importance of calcaneal tendon / Estudio sobre la estructura y desarrollo morfológico del tendón calcáneo y el músculo tríceps sural en fetos humanos durante el período fetal y evaluación de la importancia clínica del tendón calcáneo

It was aimed that the morphometric development of calcaneal tendon and the structures building it up in human fetuses during the fetal period be anatomically studied and that its clinical importance be evaluated. The study comprised a total of 102 fetus legs (51 human fetuses: 26 male and 25 female) whose ages varied between 15-40 gestational week, without external pathology or anomaly. The fetuses were divided in groups according to gestational weeks, trimesters and months. In the wake of the general external measurements of fetuses, leg dissection was performed. Afterwards, the morphometric parameters of gastrocnemius muscle, soleus muscle and calcaneal tendon were measured. The averages and the standard deviations of the measured parameters were determined according to gestational weeks, trimesters and months. There was a significant correlation between the measured parameters and the gestational age (p<0.001). There was no difference between sexes in terms of parameters (p>0.005). All the obtained results were discussed by making a comparison between them and the previous studies made. We are of the opinion that the data obtained in our study will be of use to the involved clinicians in the evaluation of the development of calcaneal tendon and the structures constituting it during the fetal period and in clinical studies and applications as well.

El objetivo de esta investigación consiste en el estudio del desarrollo morfométrico del tendón calcáneo y las estructuras que se desarrollan con él durante el período fetal humano y evaluar su importancia clínica. El estudio comprendió un total de 102 piernas de fetos (51 fetos humanos: 26 masculinos y 25 femeninos) cuyas edades variaron entre 15 a 40 semanas de edad gestacional, sin presencia de patología externa o anomalía. Los fetos fueron divididos en grupos de acuerdo con las semanas de gestación, trimestres y meses. Luego de realizar mediciones generales externas en los fetos, se procedió a la disección de las piernas. Se midieron parámetros morfométricos correspondientes a los músculos gastrocnemio, sóleo y tendón calcáneo. Los promedios y las desviaciones estándar de los parámetros medidos se determinaron de acuerdo a la edad gestacional de los fetos, en semanas trimestres y meses. Hubo una correlación significativa entre los parámetros medidos y la edad gestacional (p<0,001). No hubo diferencias entre los sexos en términos de parámetros (p>0,005). Todos los resultados obtenidos se discutieron haciendo una comparación entre ellos y los estudios previos realizados. Los datos obtenidos en nuestro estudio serán de utilidad para los médicos en la evaluación del desarrollo del tendón calcáneo y las estructuras que lo constituyen, durante el período fetal, para la utilización de este conocimiento en estudios clínicos y diversas aplicaciones.

Cellular and molecular maturation in fetal and adult ovine calcaneal tendons.

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-ß~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.

Development of the human Achilles tendon enthesis organ.

The attachment of the Achilles tendon is part of an 'enthesis organ' that reduces stress concentration at the hard-soft tissue interface. The organ also includes opposing sesamoid and periosteal fibrocartilages, a bursa and Kager's fat pad. In addition, the deep crural and plantar fasciae contribute to Achilles stress dissipation and could also be regarded as components. Here we describe the sequence in which these various tissues differentiate. Serial sections of feet from spontaneously aborted foetuses (crown rump lengths 22-322 mm) were examined. All slides formed part of an existing collection of histologically sectioned embryological material, obtained under Spanish law and housed in the Universidad Complutense, Madrid. From the earliest stages, it was evident that the Achilles tendon and plantar fascia had a mutual attachment to the calcaneal perichondrium. The first components of the enthesis organ to appear (in the 45-mm foetus) were the retrocalcaneal bursa and the crural fascia. The former developed by cavitation within the mesenchyme that later gave rise to Kager's fat pad. The tip of the putative fat pad protruded into the developing bursa in the 110-mm foetus and fully differentiated adipocytes were apparent in the 17-mm foetus. All three fibrocartilages were first recognisable in the 332-mm foetus--at which time adipogenesis had commenced in the heel fat pad. The sequence in which the various elements became apparent suggests that bursal formation and the appearance of the crural fascia may be necessary to facilitate the foot movements that subsequently lead to fibrocartilage differentiation. The later commencement of adipogenesis in the heel than in Kager's pad probably reflects the non-weight environment in utero. The direct continuity between plantar fascia and Achilles tendon that is characteristic of the adult reflects the initial attachment of both structures to the calcaneal perichondrium rather than to the skeletal anlagen itself.

Mechanical factors influence the expression of endostatin--an inhibitor of angiogenesis--in tendons.

Avascular zones of tendons are predisposed for degenerative changes and spontaneous rupture. Therefore, we analyzed the expression of the endogenous angiogenesis inhibiting factor endostatin in human fetal and adult tendons by immunohistochemical and biochemical methods. Moreover, to elucidate factors involved in the regulation of vascularity, we exposed primary cultures of rat tendon cells to intermittent hydrostatic pressure (0.2 MPa, 0.1 Hz for 24 h), and measured the endostatin content by ELISA and the effect of the conditioned medium to the proliferation of human umbilical vein endothelial cells (HUVEC). In fetal tendons high endostatin levels could be quantified by ELISA whereas low levels were found in adult tissue. In fetal tendons endostatin could also be immunostained in endothelial cells but mainly in fibroblasts. In adult Achilles tendons endostatin immunostaining was restricted to endothelial cells. In the tibialis posterior tendon--as an example for "wrap around"--endostatin immunostaining remained positive in the fibrocartilage adjacent to the medial malleolus. Fibrochondrocytes of the type II collagen positive fibrocartilage were intensively stained with the endostatin antibody. Factor VIII immunostaining showed that this region was largely avascular. Monolayer cultures of tendon cells released measurable amounts of endostatin into their culture supernatants. Application of intermittent hydrostatic pressure increased endostatin expression significantly. The conditioned media of tendon fibroblasts cultivated under intermittent hydrostatic pressure inhibited the proliferation of HUVEC in a dose dependent way. The spatial expression of endostatin in adult gliding tendons suggests that mechanical factors are involved in the regulation of this anti-angiogenic factor. In accordance, tendon cells exposed to intermittent hydrostatic pressure inhibit endothelial cell proliferation via humoral factors and produce endostatin. These findings support the view that the development and maintenance of avascular zones in tendons might be caused by a mechanically induced upregulation of anti-angiogenic factors.

Angiogenesis in fetal tendon development: spatial and temporal expression of the angiogenic peptide vascular endothelial cell growth factor.

In traction tendons, whose line of action corresponds to that of the muscle, few blood vessels are uniformly distributed within the tendon tissue. In gliding tendons, which change their direction of pull, an avascular zone is normally found in the region where the tendon wraps around the pulley. This avascular fibrocartilaginous gliding zone is predisposed for degenerative changes and spontaneous rupture. Since factors regulating angiogenesis in tendons are largely unknown, we analyzed the expression of the vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR) in human fetal and adult tendon tissue by immunohistochemical, biochemical, and molecular biology methods. In order to elucidate whether mechanical stress might influence VEGF expression in tendon tissue we loaded primary cultures of rat tenocytes with intermittent hydrostatic pressure in a special cell culture chamber (amplitude: 0.2 Mpa, frequency: 0.1 Hz, time period: 5 h/day) and measured VEGF expression using ELISA. In fetal tendons high VEGF levels could be quantified by ELISA, whereas negligible ones were found in adult tissue. VEGF could be immunostained in tenocytes and endothelial cells. In the tibialis posterior tendon - as an example for a gliding tendon - VEGF immunostaining decreased in the gliding zone adjacent to the bony hypomochlion between week 20 and week 24 after gestation. This region remained largely avascular during the fetal period. In the peritendineum and in regions proximally and distally of the gliding zone immunostaining for VEGF was positive and factor VIII-positive microvessels could be detected. In these vessels, the VEGFR-1 (flt-1) and the VGEFR-2 could also be visualized. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the results regarding VEGF expression and showed further that the splice variants VEGF(121) and VEGF(165) are expressed exclusively during angiogenesis in fetal tendons. Monolayer cultures of tendon cells released measurable amounts of VEGF. Application of intermittent hydrostatic pressure decreased VEGF expression significantly. Thus, the angiogenic peptide VEGF is present in human fetal tendons, which are exposed to traction, but not in the avascular zone of gliding tendons, which are predominantly exposed to compressive and shearing forces. These findings support the view that the development of avascular zones in tendons might be caused by a mechanically induced downreglation of VEGF expression.

Anatomy of the Achilles tendon and plantar fascia in relation to the calcaneus in various age groups.

Ten adult cadaver feet, three neonatal feet, and the feet of two fetuses were dissected to investigate whether an anatomical continuity exists between the fibers of the Achilles tendon and the plantar fascia. Histologic sections of the feet were done in three age groups: neonate, persons in their mid-20s, and the elderly. As the foot ages, there appears to be continued diminution of the number of fibers connecting the Achilles tendon and plantar fascia. The neonate has a thick continuation of fibers, while the middle-aged foot has only superficial periosteal fibers that continue from tendon to fascia. The elderly feet show simply an insertion of fibers of both structures into the calcaneus with periosteum in between.

Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon.

We describe by routine histology and by immunohistochemistry three phenotypically and developmentally distinct fibrocartilages associated with the Achilles tendon of the rat. All the fibrocartilages develop after birth and show significant age-related changes in the composition of their extracellular matrix. Attachment-zone fibrocartilage occurs at the insertion of the tendon on the calcaneus. It derives from the cartilage rudiment of the calcaneus and from the region where the tendon merges with the perichondrium. The extracellular matrix contain type II collagen and chondroitin sulphate. Compressive tendon fibrocartilage occurs in the deep part of the tendon where it presses against the calcaneus, and is derived by metaplasia of tendon cells. The cells label strongly for the intermediate filament vimentin, and the extracellular matrix contains chondroitin and keratan sulphates, but type II collagen only in very old animals (> 2 years). Calcaneal fibrocartilage covered the posterior surface of the calcaneus where it was in contact with the Achilles tendon. It labelled intensely for type II collagen and contained chondroitin and keratan sulphates. The cells were rich in vimentin. This fibrocartilage was derived from the calcaneal perichondrium.