Structural Differences Between Distinct Tendon Types Arise During Fetal Development

Abstract

Positional tendons and energy-storing tendons from the forelimbs of quadrupeds are an excellent model to assess mechanical/mechanopathological differences between two functionally distinct classes of ten- dons. The common digital extensor (CDE, positional) and superficial digital flexor (SDF, energy-storing) are ana- tomically proximate, but experience significantly different mechanical loading. It is reasonable to suspect that the well-documented structuro-mechanical differences between these two tendons arises via mechanoregulation under functional loading in gait; however, their differences may have more complex roots. In this study we have ex- plored the collagen structure of CDE and SDF tendons during fetal development, prior to any differential mechan- ical loading that might arise from gait. We have shown that structural differentiation between these functionally distinct tendons arises well before birth, and thus may be biologically rather than mechanically driven.

Full text

Structural Differences Between Distinct Tendon Types Arise During Fetal Development
S Sparavalo1, CAM Bray1, TM Brock-Fisher1, NM Easton1, CA Guinard1, SM Wells1,2, JM Lee1,3, & SP Veres1,4
1School of Biomedical Engineering, 2Department of Physics & Atmospheric Science, & 3Department of Applied
Oral Sciences, Dalhousie University, & 4Division of Engineering, Saint Mary’s University, Halifax, Canada.
Introduction: Positional tendons and energy-storing tendons from the forelimbs of quadrupeds are an excellent
model to assess mechanical/mechanopathological differences between two functionally distinct classes of ten-
dons. The common digital extensor (CDE, positional) and superficial digital flexor (SDF, energy-storing) are ana-
tomically proximate, but experience significantly different mechanical loading. It is reasonable to suspect that the
well-documented structuro-mechanical differences between these two tendons arises via mechanoregulation under
functional loading in gait; however, their differences may have more complex roots. In this study we have ex-
plored the collagen structure of CDE and SDF tendons during fetal development, prior to any differential mechan-
ical loading that might arise from gait. We have shown that structural differentiation between these functionally
distinct tendons arises well before birth, and thus may be biologically rather than mechanically driven.
Methods: Bovine fetuses were collected from an abattoir. Crown-to-rump length was measured and used to de-
termine gestational age. Paired CDE and SDF tendons were removed from one forelimb of each fetus (8 fetuses,
114 to 229 days old). A subsample of each tendon was analyzed using differential scanning calorimetry (DSC),
run against an empty pan at 5°C/min using a Q200 DSC (TA Instruments). The remaining portion of each tendon
was analyzed using hydrothermal isometric tension testing (HIT) in a custom-built multisample tester. Samples
were isometrically constrained and heated in water from room temperature to 90°C while force-time-temperature
data were recorded. Under DSC, endotherm heat-flow vs. temperature data were analyzed using Universal Analy-
sis software (4.5A, TA Instruments). Under HIT, force-temperature data were analyzed using Microsoft Excel.
Fetal data were compared with re-analyzed data from adult bovine CDE and SDF tendons (n = 7), tested identical-
ly for a separate study. Statistical analyses were performed using JMP software (version 11.0, SAS Institute).
Results & Discussion: Even in fetal life, adjacent flexor and extensor tendons are markedly different. DSC endo-
therm onset and peak temperatures were lower for SDF tendons compared to matching CDE tendons: that is, the
energy-storing tendons had collagen which was less thermally stable (Fig. 1A). This may indicate greater collagen
turnover. In direct contrast, HIT (which assesses mechanically functional crosslinking) showed that fetal SDF
tendons had higher denaturation temperatures (Fig. 1B). Therefore, the collagen in the energy-storing tendons was
more resistant to water solvation, perhaps due to greater crosslink-induced molecular packing. There was a posi-
tive linear correlation between denaturation temperature and fetal age for the energy-storing SDF tendons
(p=0.003, R2=0.79), but not for positional CDE tendons. Under HIT, the fetal SDF tendons also sustained load
under much higher temperatures than could the CDE tendons (Fig. 1C). Both of these results indicate that func-
tional crosslinking forms more completely in SDF tendons during fetal development. Thus, even before gait be-
gins, energy-storing and positional tendons diverge structurally—as seen in adults (Fig. 1A-C).
Figure 1. DSC (A) and HIT (B & C) analyses of bovine fetal CDE and SDF tendons. Data for adult tendons are also shown.
Conclusions: Our results clearly demonstrate that different collagen structures form within bovine positional
(CDE) and energy-storing (SDF) tendons during fetal development, in the absence of differences in functional
mechanical loading. The structural and mechanical differences that exist between functionally distinct adult ten-
dons may be more biologically than mechanically driven. Understanding the developmental path of tendons may
have important consequences for tailored therapeutics and for tissue engineering of tendons.
CDE SDF CDE SDF
Fetal Adult
CDE SDF CDE SDF
Fetal Adult
CDE SDF CDE SDF
Fetal Adult
62
63
64
65
66
67
68
HIT Denaturation Temp. (°C)
61
62
63
64
65
DSC Onset Temperature (°C)
65
70
75
80
85
90
95
HIT Temp. of Max Force (°C)
*
**
**
p ≤ 0.05
*p ≤ 0.005
** p ≤ 0.0005
***
*
***
*** *** ***
A B C
*
all survived to 90 °C
two survived to 90 °C