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7.2.4 Gait analysis for functional recovery
Rats with ACLR and ipsilateral flexor tendon donor site injury with weekly
intra-articular injections (from 2
nd
to 5
th
week post operation) significantly idled the
injured limb during walking at 6 week and 12 week post operation (Figure 7.14). As
compared to pre-injury levels, target print ratio (TPR) (p=0.006) (Figure 7.14B),
swing duration ratio (SWR) (p<0.001) (Figure 7.14C) and Limb Idleness index (LII)
(p<0.001) (Figure 7.14D) were significantly altered, but the temporal changes in
anchor print ratio (APR) (p=0.702) (Figure 7.14A) were not significant. There were
no significant differences among experimental groups in all gait parameters (p=0.576,
0.726, 0.896, 0.597 for APR, TPR, SWR and LII respectively). At 6 weeks post
operation, 30% of rats in each group got LII larger than 1.3. At 12 weeks post injury,
percentages of LII>1.3 in saline, 0.3 mg/ml and 3 mg/ml GHK-Cu groups were 30%,
30% and 50% respectively but the difference was not statically significant
(Likelihood Ratio test, p=0.477).
7.3 Chapter summary
We found that biological modulation of early healing stages in ACLR by
intra-operative vitamin C irrigation could promote graft healing as shown by
improvement in restoration of A-P knee laxity at 6 week post operation. However,
the positive effects were only observed in low dose (3 mg/ml) vitamin C irrigation
solution. Moreover, biological modulation by post-operative intra-articular injection
of GHK-Cu also showed positive effects on tissue remodeling of the graft and
improved restoration of A-P knee laxity at 6 week post operation, but the treatment
effect became in significant at 12 week post operation after cessation of GHK-Cu
supplementation at 5 week post operation.
The results of section 7.1 and section 7.2 have been submitted as conference
abstracts and peer-reviewed journals. (Appendix IV)
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Chapter 8
Discussion
8.1 Validity of animal model and outcome measure
8.1.1 Determination of initial graft tension for ACLR in a rat model
In this part of study, it is shown that higher initial graft tensioning is more favorable
for graft healing in ACLR using free tendon graft. An initial graft tension of 6 N has
been tried but the fixation failed at periosteum suture, thus 4 N initial graft tensioning
was used in the subsequent investigations of biological modulation in ACLR. Our
findings echoed previous animal studies using BPTB grafts in the ways that higher
initial tensioning was beneficial [186] and that the effect of initial tensioning was
diminished at later healing stages as compared to time zero [187]. However, our
finding is contrary to a study in dog ACLR model [188]. As bilateral operation with
different graft tensions on each limb was performed in this study [188], a
compensatory redistribution of loading may confound the results. Moreover, as
compared to the studies in rat (4 N), rabbit (17.5 N) and goat (35 N), the graft tension
used in the dog model (39 N) might be too high even accounting for the size of the
animal, which may hamper normal tissue remodeling [189] and cause excessive
tibial femoral compressive force [190]. The present study showed that severe graft
degeneration at the intra-articular mid-substance may contribute to the lower knee
stiffness at 2 weeks post-operation. As graft degradation is presumably mediated by
infiltrating healing cells, we speculate that the tension-induced changes in collagen
fibrillar ultrastructure [191] and the tissue reorganization associated with Poisson
contraction [192] may retard the infiltration of healing cells and better preserve graft
integrity. At 6 weeks post-operation, significant graft remodeling (ligamentization)
was initiated and the effect of initial graft tensioning became insignificant. It
suggests that graft tensioning may only affect early stages of graft healing. In
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139
contrast to previous animal studies which used bone
–
patellar tendon
–
bone (BPTB)
grafts, this study investigates the effect of graft tension on interface healing in ACLR
using free tendon graft. Initial graft tensioning appeared to affect the graft-to-bone
interactions, and adverse peri-graft bone changes such as tunnel enlargement may
negatively affect the strength of graft-to-bone attachment in ACLR [193]. Though
the small size of rat may cause some technical difficulties on the precision of the
surgical procedures, rat model of ACLR was still used by other researchers [194]. In
our study, the variations of failure parameters and laxity measures were not
particularly large as compared to ACLR studies in larger animals [187, 189]. Our
choice of graft tension (2 or 4 N) corresponded to 5
–
10 % of ultimate strength of rat
ACL (~40 N), which is similar to the range used in clinical practice (80
–
150 N) [195]
that corresponded to 4
–
7 % of intact human ACL (2,160 N) [196]. In our study, the
measurement of A-P knee laxity entailed larger random variations as compared to the
knee stiffness data, probably due to the variations in defining neutral position in knee
laxity test. Further improvement in standardization of the neutral position will be
favorable, thus the knee laxity test was modified and applied in the study of the
effect of GHK-Cu. Although 6 weeks in adult rat was roughly equivalent to 4 years
in human [197], knee laxity was not completely restored at 6 weeks post operation.
The time frame in this study may be similar to the follow-up period in previous
clinical studies of graft pre-tensioning [198-200], but a longer follow-up may be
necessary for studies of ligamentization and chondral lesions.
8.1.2 Histological evaluation of the temporal changes in graft healing in the
rat model of ACLR
As compared to previous studies (animal studies included in systematic review in
Chapter 3) on ACLR, the present study adapted a histological sectioning method that
was more standardized. Previous studies mainly employed transverse or longitudinal
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140
sections of the bone tunnels, which involved visual identification of tunnel exits
during trimming of samples before embedding. It may be subject to larger variations
and more sectioning works would be needed. With sagittal section of the whole knee
segment, histological examination of defined region of interests (femoral tunnel,
tibial tunnel and intra-articular mid-substance) could be identified with the help of
other anatomical landmarks. It is possible to examine changes in other intra-articular
tissues such as meniscus, articular cartilage, PCL and ACL remnant; so that the effect
of biological modulation was better evaluated. Moreover, the variations of tunnel
placement could also be visualized in the sagittal sections. In the present studies with
rat ACLR model, it appeared that the position of tibial tunnel were consistent but the
extra-articular exit of the femoral tunnel subjected to larger variation, as drill guide is
not available for small animal surgery. It might be a major source of intra-group
variations.
Histological examination showed that graft degeneration started at the first week post
operation, it supported that biological modulation to reduce graft degeneration should
be applied as early as possible; thus intra-operative supplementation of bioactive
agents would be a proper strategy. Although autogenic tendon grafts were used, cell
viability in the graft was low and the graft remained hypocellular in the first week
post operation. Cell recruitment [68] is regarded to initiate the healing process but
cell repopulation into the dense collagenous matrix in the tendon graft also sacrifice
the structural integrity. The healing cells recruited into graft-tunnel interface may be
originated from bone marrow while the cell infiltration in the intra-articular
mid-substance was probably derived from synovial origin [201]. As cell recruitment
started at day 7 post operation, biological modulation that affect cell activities or cell
recruitment should be delivered or remain active at this time point. Thus
intra-operative delivery of biologics intended to modulate inherent cell activities
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should consider controlled drug delivery that last for more than 1 weeks post
operation. In the present study, vitamin C was supplemented intra-operatively which
may only act only the initiating events of graft healing in the first week; GHK-Cu
was used to boost up cell activities for matrix remodeling, thus repeated
intra-articular injections were given after 1 week post operation in order to keep
biological stimulation on the healing cells recruited to the tendon graft.
At the tunnel graft interface, the extent of graft incorporation and graft degeneration
were probably interacting in a reciprocal manner: if the graft remained intact, only
minimal graft incorporation would be possible by exposing the collagen fibres from
the graft surface for anchorage into bone (Figure 8.1). Extensive graft incorporation
inevitably involved loosening/restructuring of collagen fibres on the graft surface.
Because the collagen fibre runs in parallel in the tendon graft, successful graft
incorporation between graft surface and tunnel wall would lead to the need of
incorporation of the next deeper layers of collagen fibres inside the graft. Therefore,
tunnel closure caused by mineralization of the bone graft junction would create “new”
tunnel graft interface that needs further graft incorporation. It may probably explain
why we observed poor graft incorporation at day 84 as compared to day 42 post
operation. Quality of bone healing was also essential for graft incorporation as tunnel
enlargement would prevent graft incorporation. A bony shell was observed at the
tunnel graft interface at longer time points (day 42, 84), in which tunnel closure was
achieved by mineralization at graft-tunnel interface. Ligamentization was observed
as regain of structural integrity of graft at longer time point (day 84). In summary,
histological examination showed that the remodeling process was still ongoing up to
day 84 post operation in the rat model. In order to attain good healing outcomes such
as stable graft anchorage and mechanically strong graft mid-substance, there is room
for further improvement by biological augmentation.
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8.1.3 Establishment of gait analysis to monitor functional deficit related to
ACL deficiency
The results of this section of the PhD Study showed that Limb idleness index (LII) is
useful to reveal OA-related pain in ACLT destabilized knees. The coefficient of
variation for LII is 13.3%, indicating acceptable reliability of the measurement. LII is
Figure 8.1
Diagrammatic presentation of graft-tunnel interaction with
(A) transverse and (B) longitudinal section, showing the
collagen fibre incorporation into bone at the graft surface but
not for the collagen fibres inside the graft. Insertion of collagen
fibres into bone requires disruption of collagen fibres on the
graft surface (C), while mineralization of the graft-to-bone
insertion may lead to tunnel closure (D), but another round of
incorporation of deep collagen fibres inside the graft was
necessary to maintain stable anchorage.
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143
in essence a measure of symmetry in walking gait. LII> 1 means that the target limb
was idled; in contrast, LII < 1 reflects less activity in the contralateral limb. At
pre-injury time point (n=18), the mean LII was 0.97 with a range of 0.75-1.20. In the
reversal test with buprenorphine injection, all rats with LII
1.20 were responsive to
analgesic treatment, indicating limb idleness on the target sides was associated with
pain. On the contrary, one rat from Sham group (Rat59) with LII
0.75 indicated
significant limb idling on the contralateral side. Rat59 also responded to
buprenorphine in a reversed way as compared to other painful cases in target limbs
(Figure 6.12). Examination on histological samples from Rat59 showed that the
OARSI score in contralateral side (4.5) was higher than the target side (1). It
indicates that Rat59 may experience pain associated with osteoarthritic changes on
the contralateral side. These observations suggest that LII is sensitive to detect the
relative mechanical allodynia of target and contralateral sides. Thus it is necessary to
consider both target and contralateral sides when we examine the relationship of LII
and OA changes. The significant correlation between LII and the difference of
OARSI scores (target to contralateral sides) supports that LII is capable of detecting
the relative severity of OA changes in both knees in rat model with ACL
insufficiencies.
This is the first study measuring OA pain in a ACLT animal model with a long
follow-up (6 months), while all previous animal studies of OA pain were carried out
in a relatively shorter duration (maximum follow-up ranging from 2 weeks [202] to
10 weeks [203]). In those studies with shorter follow-up time, pain due to acute joint
inflammation rather than chronic arthritic changes was measured [204]. On the other
hand, as compared to previous studies on rat ACLT model of post-traumatic knee
OA [205, 206], our data showed a slower development of OA (symptomatic at 6
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144
months post operation), which might be due to the use of female rats (221.5±11.9 g
at pre-injury and 286.4±23.0g at 6 months post-operation), as heavier and more
active male rats (around 300 g at 8 weeks old) are used in previous studies [206]. Yet
we also detected similar cartilage degeneration [205, 206]. With such a long
follow-up time, some spontaneous OA [207] were observed in the un-traumatized
knees. The observed outcomes in ACLT group were probably contributed by both
post-traumatic and primary OA. The significant intra-group variations in our data
may affect the detection of between-subject group differences. However, these
variations helped to explore the relationship of the painful responses as detected by
gait adaptation and the osteoarthritic changes as revealed by histology. The
correlation of LII to OARSI scores suggested that the gait adaptation to pain was
associated with the OA-specific joint degeneration, with an outlier which is also
explainable by the co-existence of OA and tendon calcification (Rat48). As abnormal
LII was responsive to buprenorphine treatment, the use of LII in ACLT rat model
may be helpful for evaluation of the effect of biological modulation in ACLR, since
knee pain is also an important outcome measure in clinical evaluation of recovery in
ACLR.
Gait analysis has been used to measure this movement-evoked pain in OA using
Catwalk system [208]. All the built-in gait parameters did not show significant
association with OA pain except for the percentage of ipsilateral paw intensity at
standing, which is similar to the calculation of anchor print ratio and target print ratio
in this study. We calculated mean integrated paw print intensity to estimate the extent
of limb loading during ambulation, which might be more representative than
maximum or mean print intensities at any time point during the stance phase (built-in
gait parameters). As limb idleness can also result from decreasing time of loading
(increasing paw elevation time) in addition to reducing loading during stance phase,
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145
LII presents a new parameter that integrates both mechanisms to avoid mechanical
allodynia. The three component ratios of LII are chosen because they represent three
different aspects to idle a target limb during walking, thus LII would be able to detect
“limb idleness” when only one of these ratios was increased. For example,
Rat 53
would be regar
ded as “no change in limb idleness” if we accessed anchor print ratio
and swing duration ratio; but with a higher target print ratio, we concluded that Rat53
also experienced limb idleness on target limb at 6 month post injury as revealed by
LII (Figure 6.11), which also responded to buprenorphine treatment (Figure 6.12).
Alternatively, false-positive
cases of “limb idleness” detected by single ratio could be
excluded when other ratios were changed in different directions. For example, Rat55
got a high target print ratio but a low swing duration ratio, thus it would be difficult
to judge whether Rat55 was idling its target limb if we only based on single ratio. By
combining these component ratios to yield LII, Rat55 was still within the normal
range and it did not respond to buprenorphine treatment (Figure 6.12). This may
explain why individual component ratios cannot detect the group difference at 6
month post injury in contrast to LII, because the rats in one group may not use the
same strategy to achieve limb idleness. Nevertheless, individual ratios should still be
presented along with LII in the gait pattern analysis to measure OA-related pain
development in the ACLT model. If there is a switch of limb idling strategies or
compensatory changes, evaluations on individual ratios and LII will provide more
information.
With the development and validation of animal gait analysis to monitor functional
deficit as reflected by gait asymmetry during walking, this method was used to
evaluate functional recovery after ACLR in a rat model as post-traumatic OA is
probably associated with ACL injury [145].
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146
8.2 Possible mechanisms of treatment effects
8.2.1 Effect of vitamin C irrigation saline on ACLR
Our results show that intra-operative supplementation of low dose vitamin C at
3mg/ml effectively improves graft integrity and A-P knee laxity, but the ultimate load
of the femur-graft-tibia complex was not improved. It is possible that vitamin C
modulated cell recruitment at early time points, safeguarded graft integrity and
reduced loss in graft tension. However, the actual mechanisms of the effectiveness of
vitamin C irrigation still need further exploration. Vitamin C irrigation significantly
reduced serum CRP levels at day 1 post operation at all tested doses, indicating that
the extent of post-operative inflammation was suppressed. However, it may not
sufficiently account for the observed effects of vitamin C irrigation on ACLR, as the
beneficial effect of vitamin C irrigation on ACLR was only observed in low dose (3
mg/ml). Our previous report [160] also showed that higher doses of vitamin C did
not exert positive effects on healing outcomes on tendon adhesion despite oxidative
stress was antagonized. We speculated that abolishment of reactive oxygen species
(ROS) does not guarantee a better healing outcome, because ROS is still necessary
for normal function of healing responses. This speculation is supported by the
observation that overexpression of antioxidant enzymes (catalase) and application of
high dose antioxidants (such as N-acetylcysteine) to wound may impair wound
healing [209] and angiogenesis [210, 211]. There is no significant difference in gait
patterns among different experimental groups at 6 week post ACL reconstruction,
indicating that the observed differences in knee laxity did not affect limb functions in
walking. However, increased percentage of highly asymmetric walking gait (with
LII>1.3) may suggest some side effects associated with higher dose vitamin C
irrigation. Application of vitamin C or antioxidant treatment to promote
post-operative functional recovery may need further investigation on safety dose
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