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initialtension (39 N) was detrimental tohealing, while the
studies in rabbit [8] showed improvements in healing
outcomes with a higher initial graft tensioning. On the
other hand, a study in goat did not report difference in
long-term healing outcomes [1]. Since all these animal
studiesusedBPTBgrafts, thereis noinformationaboutthe
effects of graft tension on the healing process in ACLR
using free tendon graft, in which thegraft–tunnel interface
healing presents a major challenge for further improve-
ment. Our purpose is to evaluate the effect of initial graft
tensioningonthehealingoutcome ina ratmodelof ACLR
using flexor tendon graft. It is hypothesized that a higher
initial tensioning favours restoration of knee laxity and
improves biological healing.
Materials and methods
Theanimalexperiments inthis studywereapprovedbythe
Animal ExperimentationEthicsCommitteeinThe Chinese
University of Hong Kong (Ref No. 10/077/MIS). Experi-
mentaldesignof the present studywasillustratedinFig.1.
The procedures of ACLR were adapted from our previous
studies in rabbit [15] with the introduction of controlled
initial graft tensioning. Fifty-five male Sprague–Dawley
rats (12 weeks old, 400–450 g) were used. Unilateral
operation was performed on the right knee. The flexor
digitorum longus tendon (25 mm in length and 1 mm in
diameter) was harvested from right heel. The knee joint
capsule was opened, and native ACL was excised. Tibial
and femoral tunnels of 7 mm in length and 1.2 mm
diameter (by 1.1 mm drill bit) were created from the
footprints of thenative ACL tothe medialside of tibiaand
the lateral femoral condyle, respectively. The tendon graft
was inserted through both bone tunnels and fixed to the
tibial periosteum [2, 4] first. Through a pulley system, a
freely suspended weight was used to provide a constant
tensioning force of 2 or 4 N tothe graft during its fixation
totheneighbouringfemoral periosteum (Fig.2a). Animals
were given 0.05 ml Temgesic once after operation and
were allowed free cage movements. At 2 and 6 weeks
post-operation, knee samples were harvested for static
knee laxity test and graft pull-out test (n = 6) and histo-
logical examination with H&E staining (n = 4). For vali-
dation of laxity test at time 0, a group of 15 rats were
operated on both limbs, which were randomly assigned to
‘‘sham’’ group (with medial parapatella arthrotomy),
‘‘intact’’ group (with non-operated knees), ‘‘deficient’’
group (with ACL transected without repair), 2 N pre-ten-
sioning and 4 N pre-tensioning group (6 knees in each
group). Intact and sham group served as positive controls,
while deficient group served as negative controls for the
laxity test.
Static antero-posterior (A-P) knee laxity test
The laxity test protocol is developed with reference to the
Lachman test and KT-2000 knee arthrometer. Fresh knee
samples weretrimmedtoexpose 1.5 cm of tibia andfemur
shafts for fixation in adhesive polymer (1:1:2 of UREOL
5202-1A, UREOL 5202-1B, Filler DT-082. Ciba Specialty
Chemicals, Cambridge, UK). The samples were then
mounted to custom-made jigs by a mechanical testing
machine(H25KM,TinusOlsen,PA,USA),witha50 Nload
cell (H25KM, Tinus Olsen) (load measurement accuracy:
?0.5 %ofmax. load).Thejigs werepositionedtokeepthe
knee inneutralA-P position (forcezero) withnaturalvarus
of rat kneeat approximately 10. Witha pilotstudyondif-
ferent applied shear loads (2 or 3 N) and different knee
flexion angles (55, 70, 85, 100) (data not shown), we
found that a shear load of 2 N was safe to prevent graft
failureduringlaxitytest, whilepositioningatakneeflexion
angle of 70 can reveal the largest difference in A-P
Table 1 Histological scoringsystem
Score
Graft degeneration
a
Nodegenerative changes
0
Minimaldegenerationinside graftand/orattunnelinterface 1
Significant degeneration inside graft and/or at tunnel
interface
2
Extensive degenerationinside graftand/orat tunnel
interface
3
Interfacehealing and adverse peri-graft bonechanges
b
Direct graft incorporation
c
without adverse peri-graft bone
changes
0
Direct graft incorporation with adverse peri-graft bone
changes
1
Indirectgraftincorporation
d
withoutadverseperi-graft bone
changes
2
Indirect graft incorporation with adverse peri-graft bone
changes
3
Nograft incorporation
4
a
Graftdegenerationwasscoredinmicrographsunderbrightfieldand
polarizedillumination.‘‘Nodegeneration’’wasscoredtosamples that
did not reveal anytraits of matrixdegeneration. ‘‘Minimal degener-
ation’’ referred to a lack of cell infiltration into graft with bright
collagen birefringence. Cell infiltration intograft with a loss of col-
lagenbirefringencewasscoredas‘‘significantdegeneration’’(\50%
ofregionof interest) or ‘‘extensive degeneration’’ ([50% of region
ofinterest)
b
Adverse peri-graft bone changes were defined as the presence of
bone intrusion into the tendon graft, peri-graft bone erosion or the
presence of cysts in peri-graft interface
c
Directgraftincorporationwas definedasdirectconnectionbetween
boneandtendonwithoutafibrousinterface(includingSharpey’sfibre
andfibrocartilage zone)
d
Indirect graft incorporation was defined as connection between
bone andtendon graft with a loose fibrous interface
Knee Surg Sports Traumatol Arthrosc
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Appendix IIIA
