Chapter 2. Mechanical, Compositional and Structural Properties of the Decorin Null and
D. Discussion
structural and compositional differences in wildtype, Dcn-/- and Dcn+/- tendons. Chapter 3
addresses Aim 2 and similarly provides the methods, results, and discussion for the
experimental studies performed to characterize the mechanical, structural and
compositional differences in wildtype, Bgn-/- and Bgn+/- tendons. Chapter 4 compares the
properties of decorin and biglycan mutant genotypes. Chapter 5 combines the results in
Aims 1 and 2 in a rigorous multiple regression statistical model to describe relationships
between tendon mechanics, structure and function as described in Aim 3. Finally,
Chapter 6 draws overall conclusions from the previous chapters and discusses potential
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Chapter 2.
Mechanical, Compositional and Structural
Properties of the Decorin Null and Heterozygous Mouse
Patellar Tendon
A. Introduction
Tendon is a dynamic tissue with specialized mechanical properties and complex
composition and structure. Tendon structure can be viewed as a composite of fibers
embedded in an extra-fibrillar ground substance which both individually and interactively
contribute to the overall tendon mechanics. Strength is needed to transmit high forces
from muscle to bone. Viscoelastic tissue characteristics are important to control fine
movements and to conserve energy. Resistance to compressive loading is needed in
tendons that are subjected to compression from other anatomic structures or to resist
compressive forces transverse to the fiber direction due to Poisson’s ratio in tension.
The incidence of tendon injuries in the active and aging population is increasing.31
Both chronic and acute injuries are of concern because they heal slowly and rarely regain
normal function. Surgery, rehabilitation, medication and tendon grafts have all been
employed to improve healing with limited success.3, 9, 32, 44 Problems such as adhesion
formation, reruptures and decreased function are still common, most likely due to the
complexity in recreating the natural tissue architecture and resulting mechanical
behavior.8, 9 Understanding how to best approach these problems, whether through
targeted pharmaceuticals or tissue engineering constructs, demands knowledge of how
properties.
While many studies have been conducted to evaluate the structure-function
relationships in tendon, understanding is still limited. The interactions of small leucine-
rich proteoglycans (SLRPs) with other extracellular matrix (ECM) molecules, such as
collagen fibrils, as well as their association with water and role in fibrillogenesis have
suggested SLRPs may play a part in tendon mechanics. The most common SLRP in
tendon is decorin which accounts for ~80% of the SLRPs in the tensile region of
tendon.35 It is a low molecular weight proteoglycan (PG) and is composed of two regions.
The first region is the single side chain of chondroitin or dermatan sulfate
glycosaminoglycan (GAG) located in the N-terminal region.40 The second region is the
protein core. It consists of a domain of tandem leucine-rich repeats flanked on both sides
by conserved cysteine residues. It has been suggested that this region binds non-
covalently with the collagen fibril39 while the GAG chains interact in the interfibrillar
space.37
Decorin is known to be a regulator during collagen fibrillogenesis and mice
deficient in decorin have previously been shown to have larger and more irregular fibril
diameters than those in wildtype, although the magnitude of these changes are tissue and
age specific.45 These changes in combination with the association of SLRPs with
collagen fibrils and water, have led studies to investigate their role in tendon mechanics.
These studies have shown varying results, again based on specific tendon and age.33, 34, 45
For instance, in 8-10 week old mice, decorin null mice had increased tensile modulus in
the patellar tendon, but not in the tail tendon fascicle or flexor digitorum longus (FDL).33
compressive properties, a study in ligament showed that enzymatic removal of sulfated
GAGs resulted in an increased intrinsic permeability.19 However, it is often unknown if
these mechanical changes were a result of collagen changes or the inherent effect of the
reduction of SLRPs. Knowing that mechanical changes exist is only the first step in
understanding tendon structure-function relationship.
Therefore, the objective of this chapter was to evaluate the structural,
compositional and mechanical changes in decorin null (Dcn-/-) and decorin heterozygote
(Dcn+/-) mouse patellar tendons compared to wildtype (WT). We hypothesized that in the
decorin null mice compared to wildtype, no changes will be seen in tensile elastic, tensile
viscoelastic or compressive elastic properties. In the decorin heterozygotes, tensile
viscoelastic and compressive properties will be decreased. In both null and heterozygote
mice, fibril diameters will be increased with a larger spread. Finally, we hypothesize that
no changes will be seen in total collagen content.
B. Methods
Ninety-nine female, C57Bl/6 mice were used in this IACUC approved study. All
mice were sacrificed at 5 months of age to ensure skeletal maturity before the effects of
aging had begun.7 Three different genotypes were used and included wildtype (WT,
n=34), decorin heterozygotes (Dcn+/-, n=34) and decorin null (Dcn-/-, n=31) mice.10
Whenever possible, mechanical, structural and compositional assays were performed in
the same mouse.
B1. Biomechanical Tests
Patellar tendons were randomly dissected from either the left or right limb for
tensile biomechanical analysis (WT n =14; Dcn+/- n=14; Dcn-/- n=14). Tendons were
removed and carefully dissected under a fine dissection microscope leaving the patella-
tendon-tibia complex intact. The cross sectional area was measured in the tendon
midsubstance using a custom built device consisting of LVDTs, a CCD laser, and
translation stages.12 The tibia was potted in polymethylmethacrylate (PMMA) and a
metal staple placed over the tibial plateau to discourage growth plate failure. The tendon
was coated uniformly with small speckles of Verhoeff stain using a fine bristled brush to
create a textured appearance in the midsubtance for local optical strain analysis. The
potted tibia was gripped and the patella held in a custom fixture which ensured gripping
of the patella bone without pinching the tendon. Specimens were submerged in a 37°C
PBS bath and tensile tested on an Instron 5848 testing machine (Norwood, MA) using a
10N load cell (Fig 2.1). An average gauge length of 3mm was assumed for all calculated
protocol strain levels.
Samples were strained to 1%, preconditioned between 0.5-1.5% strain for 10
cycles at 0.25Hz, and allowed to relax at zero strain for 300 seconds before beginning
viscoelastic testing. A stress relaxation to 4% strain at 5%/s with a 10 minute hold for
relaxation was performed. Immediately following, the tendon was subjected to 10
sinusoidal strain cycles (frequency sweep) at 0.01, 0.1, 1, 5 and 10 Hz with an amplitude
of 0.125% (Fig 2.2). The stress relaxation and frequency sweep were repeated at 6 and
8%.26 Following the frequency sweep at 8% strain, specimens were returned to the
preload displacement and held for 5 minutes before a ramp to failure at 0.1%/s was
applied. Using the applied speckled coating, local tissue strain in the tendon
midsubstance was measured optically with a custom program (MATLAB).
A bilinear fit was applied to the ramp to failure to obtain the elastic properties toe
and linear stiffness, toe and
linear modulus and the
transitional displacement or
strain defined by the
breakpoint in the bilinear fit.
Peak and equilibrium load were determined from the stress relaxation tests at each strain
level and percent relaxation was subsequently calculated from these values. For the
dynamic response to the sinusoidal input, the last 5 load-displacement cycles at each
frequency/strain combination were analyzed by transforming the data into the frequency
domain using a Fast Fourier Transform. The magnitude and phase of the prevailing
frequency for both the displacement and load was determined at each frequency and
strain level using a custom written Matlab program. The same process was applied to the
stress-strain data to determine material properties. Dynamic stiffness,| k*|, was calculated
as the ratio of the magnitude of the load over the displacement, dynamic modulus, |E*|, as
the magnitude of the stress over the strain and phase angle,
φ
, was defined as thePrecondition Hold Hold Incremental step Frequency Sweep S tr a in Time