Morphology and Gene Expression Changes in Rat Achilles Tendon Aging
Takintope Akinbiyi, M.Sc., Lili Xu, MD, Zuping Zhou, Ph.D, Damien Laudier, B.S., H.T.L., Peter Taub, MD, Hui Bin Sun, Ph.D.
The Mount Sinai School of Medicine, New York, NY, USA.
BACKGROUND:
Tendons are the connective tissue comprised mostly of type I collagen that transmit force from skeletal muscles to bone, and are therefore crucial in generating motion. With age, an increase in tendon damage due to numerous changes including hyaline degeneration, hypoxic injury, fatty deposition, as well as fibrocartilagenous metaplasia are seen. This damage accompanies a decrease in the proliferative capacity of stem cells that occurs with age. Both of these important changes could serve to reduce the quality of tendon healing after surgical repair.
Cited2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is a transcriptional co-regulator that is known to inhibit expression of matrix metalloproteinases (MMPs 1 & 13) and is essential for cell proliferation. As such, adequate levels of Cited2 are required to maintain tendon homeostasis and cellularity. Characterization of these changes will provide insight into the development of clinical therapies to optimize tendon healing, especially in aged populations.
METHODS:
Young and old (3-6 and 24-28 months old, respectively) male Sprague-Dawley rats were sacrificed using CO2 inhalation, and the Achilles tendons were harvested. Tendons were either fixed in 10% formalin for morphological and immunohistochemical (IHC) analysis, or the tendon derived stem cells (TDSCs) were isolated and cultured to passage 3, then harvested for RNA extraction and quantitative PCR analysis. Three pairs of IHC images (200x magnification) were taken from equivalent areas in the mid-substance of each Achilles tendon.
RESULTS:
Morphological changes. Older Achilles tendon had significantly fewer cells (68.0±17.78) per field-of-view (0.15mm2) than young tendons had (90.3±1.53), p<0.05 (Fig.1A). The average number of cells in linear clusters was significantly greater in older tendon (2.53±2.18) than in young tendon (2.06±1.21) suggesting focalized hypercellularity, possibly to compensate for reduced functionality, p<0.05 (Fig.1B). The relative average nuclear surface area (a surrogate for nuclear size) was significantly greater in older tendon (418.2±193.8) than in young tendon (330.95±134.3) indicating enlarging nuclei with age, a possible compensatory adaptation, p<0.05 (Fig.1C). 
Cited2 expression. Cited2 gene expression in old TDSCs was decreased versus young TDSCs (Fig. 2A) and protein expression was likewise reduced in IHC staining of old tendon (Fig. 2B&C). 
CONCLUSIONS:
With age, definite morphological changes were noted in tendon specimens. In addition to an overall decrease in tenocyte number, older individual tenocytes were shown to be larger and more juxtaposed, which possibly serves to overcome functional deficiencies in individual cells that occur with age. Young tendons also appeared more homogenous along their axis than their older counterparts. Importantly, changes in Cited2 gene expression, and the resultant protein expression, were noted to decrease with age suggesting a possible target for therapeutic intervention in the future.
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