1. INTRODUCTION
High energy trauma to a synovial joint causes an array of
mechanical, cellular, and biochemical responses that can ultimately
lead to posttraumatic osteoarthritis (PTOA). Mechanical damage to the
cartilage, such as surface fissures [1] and microstructural damage [2],
have been observed at impact sites immediately following trauma.
However, the degree to which this damage renders the tissue more
susceptible to wear and contributes to the progression of PTOA is
unknown. Additionally, no treatments are currently available to
strengthen cartilage after joint trauma and protect the tissue from
subsequent wear. Our previous work has investigated collagen
crosslinking of cartilage using genipin as a potential therapeutic
treatment [3]. We showed that genipin crosslinking improves the wear
resistance of healthy, intact cartilage in vitro and significantly protects
the tissue from collagenase digestion at doses that are non-toxic to
chondrocytes [3]. The purposes of this study were to investigate the
role of mechanical damage in cartilage wear and friction after an
impact, and to determine whether genipin crosslinking is an effective
treatment to improve the wear of impacted cartilage.
METHODS
Cartilage/bone specimens from thawed bovine stifles were
impacted via a drop tower instrumented with a load cell and
accelerometer, using a 3.2 cm spherical impact head from a height of
25 cm with a total mass of 499 g. Alcian blue staining was performed
to investigate glycosaminoglycan (GAG) levels post-impact.
Immunohistochemistry was performed to assess denatured collagen
(COL2-3/4M) and collagenase cleavage (COL2-3/4Cshort). Crosslinked
and control specimens were incubated post-impact for 15 min in 0, 2,
or 10 mM genipin (Challenge Bioproducts, Taiwan) solutions in PBS
at 37 ºC, then were transferred to genipin-free PBS at 37 ºC to bring
the total time to 24 h. Stress-relaxation tests were performed via
indentation initially, then again after impact, and finally after
crosslinking with a Hysitron TI950 TriboIndenter (Minneapolis, MN)
equipped with a 3-D OmniProbe® transducer and a 750 µm diameter
flat punch probe. The indentation protocol, which had previously been
shown to produce repeatable stiffness measurements of cartilage that
were sensitive to changes due to crosslinking [4], consisted of a 20-sec
loading period, a 50-sec hold at a peak total displacement of 70 µm,
and a 1-sec unloading period; three separate locations on each of four
specimens were indented. The unloading stiffness was calculated for
each indentation, and a standard linear solid model was fit to the
holding phase of the stress-relaxation tests (n = 12) [5].
Impacted/crosslinked specimens and control specimens underwent
reciprocating pin-on-disk friction tests for 30 minutes using a
Universal Micro-Tribometer (Bruker, Campbell, CA); the initial
coefficient of friction was reported (n = 4). Separate specimens
underwent identical wear tests for 48 hours in a square pattern (n = 6)
against stainless steel discs using an Orthopod (AMTI, Watertown,
MA) as previously described [6]. To visualize damage from the wear
test, surfaces were stained with india ink. To quantify the amount of
collagen released to the hydrating fluid during the wear test of each
specimen, a hydroxyproline assay was performed. Differences were
determined using a one-way ANOVA with Tukey’s post-hoc test and
significance set at p<0.05.
RESULTS
The impact energy from the drop tower was 0.89 ± 0.30 J and
resulted in shallow fissures at the articular surface (Fig. 1A). Alcian
blue staining of impacted cartilage revealed that a large amount of
GAG’s were retained by the cartilage post-impact (Fig. 1A).
Immunohistochemistry indicated that collagen became denatured
under the impact (Fig. 1B), which was not due to collagenase cleavage
(data not shown). The cartilage unloading stiffness decreased by
40.2% after impact (p<0.001) and was not significantly different after
crosslinking in either genipin concentration. Similarly, equilibrium
modulus from the standard linear solid model decreased 42.4% upon
impact, and did not significantly change with crosslinking. The initial
modulus from the standard linear solid model also decreased after
impact, was not significantly changed with 2 mM crosslinking, but
increased with the 10 mM treatment (data not shown). The initial
coefficient of friction rose by 88% after impact and was not
significantly changed by crosslinking. India ink staining suggested that
SB
3
C2015
Summer Biomechanics, Bioengineering and BIotransport Conference
June 17-20, 2015,Snowbird Resort, Utah, USA
THE EFFECT OF IMPACT AND GENIPIN CROSSLINKING ON THE FRICTION AND
WEAR OF ARTICULAR CARTILAGE
C.M. Bonitsky (1), M. Selep (1), M.E. McGann (1), T.C. Ovaert (1), S.B. Trippel (2), D.R. Wagner (1)
(1) Aerospace and Mechanical Engineering
University of Notre Dame
Notre Dame, Indiana, USA
(2) Department of Orthopaedic Surgery
Indiana University
Indianapolis, Indiana, USA

2. impacted specimens with 0 mM genipin treatment sustained the most
wear while the fissures due to impact did not noticeably worsen in the
specimens that had been crosslinked in 2 and 10 mM genipin (Fig.
2A). The wear quantification confirmed these results, indicating that
the impacted specimens released significantly more collagen to the
hydrating fluid during the wear test than those that had not been
impacted, and that the 2 and 10 mM genipin crosslinking treatments
reduced the wear of the impacted specimens to levels that were
comparable to that of the non-impacted controls (Fig. 2B).
DISCUSSION
This study investigated a novel method to improve articular
cartilage wear resistance after a traumatic injury. We used genipin, a
natural plant extract, to crosslink the collagen network in a clinically
relevant 15 minute time span. The impact protocol caused fissures and
microstructural damage at the articular surface and decreased the
cartilage stiffness via indentation testing. Although we have previously
shown that our crosslinking protocol in 2 and 10 mM genipin increases
the unloading stiffness of intact cartilage by 16 and 64%, respectively
[3], no significant increase in unloading stiffness was observed when
the impacted specimens were crosslinked with these same treatments.
The results suggest that the impact damage dominates the indentation
stiffness measurement, perhaps due to the rupture of collagen fibers in
the superficial zone. Immunohistochemistry staining demonstrated
collagen network denaturation and microarchitectural damage in the
superficial zone, while Alcian blue straining revealed that a large
amount of GAGs were retained by the cartilage post-impact.
Microarchitectural damage coupled with GAG retention may cause the
swelling and increased water content that has been reported in
impacted cartilage [7] due to the decreased ability of the damaged
collagen network to restrict proteoglycan expansion through the
absorption of water. The microarchitectural damage and subsequent
swelling may also be observed in the decreased moduli of the cartilage
post-impact and may lead to decreased fluid pressurization and
increased cartilage friction [8], as was observed in the current study.
The wear resistance of impacted articular cartilage that had not been
crosslinked was less than that of undamaged cartilage in the in vitro
wear test, suggesting that mechanical damage that is immediately
induced by impact loading may directly contribute to the progression
of PTOA. The wear resistance was improved by the crosslinking
treatments, and was about equal with the 2 and 10 mM genipin
protocols. As previous studies have demonstrated that the 2 mM
genipin crosslinking treatment is non-toxic to chondrocytes [3], it may
be appropriate to adapt this protocol for clinical use to prevent
accelerated wear after a patient experiences injurious joint trauma.
ACKNOWLEDGEMENTS
Supported by the US Army Medical Research & Materiel
Command W81XWH-07-0662. We thank Malcolm Cabello for his
help in fabricating test specimens.
REFERENCES
[1] Repo, R, et al., J. Bone Joint Surg. Am., 59(8):1068-1076, 1977.
[2] Wilson, W et al., J. Orth. Res., 24(2):220-228, 2006.
[3] McGann, M et al., in review.
[4] McGann, M et al., J. Mech. Beh. Biomed. Mat., 34:264-272, 2014.
[5] Cheng, L et al., J. Polymer Physics, 38(1):10-22, 2000
[6] McGann, M et al., Proc Inst Mech Eng Part H J Eng Med,
226(8):612-622, 2012.
[7] Torzilli, P et al., J. Biomech. Eng., 121(5):433-441, 1999.
[8] Ateshian, G, J. of Biomech., 42:1163-1176, 2009.
FIGURE 2: A) INDIA INK STAINING BEFORE AND AFTER
WEAR TESTING. B) EFFECT OF IMPACT AND GENIPIN
CROSSLINKING ON HYDROXYPROLINE (HYP) RELEASED
TO THE HYDRATING FLUID DURING WEAR TESTING
(*:p<0.05, **:p<0.01).
FIGURE 1: A) ALCIAN BLUE STAINED ARTICULAR
CARTILAGE AFTER IMPACTING THEN INCUBATING IN
PBS AT 37°C FOR 24 HRS. B) IMMUNOHISTOCHEMISTRY
OF SURFACE DAMAGE DUE TO A SINGLE IMPACT
SHOWING DENATURED COLLAGEN (COL2-3/4M). BAR
REPRESENTS 200 μm.