- Contrasting the response to glucocorticoids in shaft fractures versus metaphyseal
bone regeneration.- Metaphyseal and shaft bone healing responds differently to
glucocorticoids.- Stable metaphyseal healing tended to be stimulated by glucocorticoids.- Stability did not influence the response to glucocorticoids
in shaft fractures.Strength: this study is the first to compare metaphyseal bone
regeneration and shaft fracture healing in mechanical terms.Limitation: mechanical testing of so different fractures required
different techniques and timing.
Introduction
Inflammation is thought to play a crucial role in the initiation
of fracture healing. Inhibition of inflammation with non-steroidal
anti-inflammatory drugs (NSAIDs) has a strong detrimental effect
on the healing of diaphyseal fractures, but a weak effect on metaphyseal
healing.[1] This
suggests that inflammation plays different roles in fractures in the
metaphysis and the shaft. This might explain the perplexing discrepancy
between the seemingly harmless clinical use of NSAIDs in patients
with fractures, and the clearly detrimental effect of such treatment
in animal models, as humanfractures usually occur in metaphyses, while
almost all animal models deal with shaft fractures.Fractures in cancellous metaphyseal bone tend to heal by direct
bone formation within the injured marrow compartment,[2,3] suggesting that mesenchymal cells
residing locally in the marrow might be sufficient for the bone regenerative
process. In contrast, shaft fractures, at least in part, depend
on the ability to recruit competent cells from surrounding tissues
or circulation.[4] It
is possible that inflammation is crucial for this recruitment. If
so, dampening of the inflammation would be detrimental to healing
in a shaft fracture, but not to metaphyseal healing. We have previously
tested this hypothesis using an NSAID to reduce inflammation.[1] However, that study
could not separate the roles of stability and location, i.e., the
findings could be explained by a difference in stem cell availability,
a difference in mechanical environment or a difference in the amount
of new tissue that needed to be formed. We therefore now studied
the role of inflammation by use of dexamethasone for inhibition,
taking mechanical stability into account.We hypothesised first that a stable metaphyseal bone healing
model would be less impeded by a dampened inflammation than would
an unstable, conventional diaphyseal fracture model. We then hypothesised
that a difference would also appear if the diaphyseal model was mechanically
stabilised.
Patients and Methods
Animals and surgery
In total, 120 male C57Bl/6 mice, then at ten weeks old, were
used (Scanbur, Sollentuna, Sweden). All surgery was undertaken in
a sterile fashion by a surgeon blinded to drug treatment. Isoflurane
gas (Forene, Abbot Scandinavia, Solna, Sweden) was used in order
to sedate. The animals received 0.1 mg/kg buprenorphine (Temgesic,
Schering-Plough, Brussels, Belgium) every 12 hours for 36 to 72
hours (depending on the fracture model), starting just before surgery.
Antibiotics, 0.2 mg/kg oxytetracycline (Engemycin, Intervet, Boxmeer,
Holland) was also given by subcutaneous injection before surgery.The animals had unrestricted access to food and water and were
housed four per cage, in a 12:12 hour light cycle. All experiments
were in accordance with ethical approvals 85-12 and 54-14 from the
Regional Animal Ethics Board.
Stable metaphyseal injury model
We used a screw in the metaphyseal region as a metaphyseal injury
model. The pull-out force of a screw one week after implantation, with
or without various drug treatments, correlates with the amount of
newly formed bone both around the screw and in empty drill holes.[5] In total, 20 mice
received a custom-made screw implant (Rydahl Precision Components, Karlstad,
Sweden) into the fronto-medial aspect of the proximal right tibia.
First, a 0.6 mm diameter drill hole was made. A screw made of titanium
with a length of 1 mm and thread M 0.7, was then screwed in place
(Fig. 1). The mice also received a drill hole 0.6 mm in diameter at
the corresponding site on the contralateral tibia, here without
a screw. These techniques have been previously described in greater
detail,[1] and
can be considered mechanically stable. The relevance of this model
for fracture healing is discussed below.MicroCT scan of a screw newly
inserted in the tibial metaphysis.The animals were randomised to receive either dexamethasone (Vorenvet,
Boehringer Ingelheim, Germany) or control injections. Dexamethasone
was given subcutaneously at a dose of 2 mg/kg three times per week,
starting on the day of surgery. Control animals received an equal
volume of saline. One week after surgery, the animals were killed
with carbon dioxide, and both tibias were harvested for evaluation.
The first 20 mice in the metaphyseal model showed a trend (p = 0.06)
towards increased pull-out resistance in the dexamethasone group.
Therefore, the experiment was repeated twice, with 24 and 30 mice,
bringing the total number of mice used for this model up to 74.
As there was no significant difference in outcome between the groups
(p = 0.4), and no difference in the effect of dexamethasone between
the groups (p = 0.2), they were pooled.
Unstable shaft fracture model
A total of 26 mice had their right knee opened, the patella dislocated
medially, and an intramedullary pin, with a diameter of 0.4 mm,
inserted into the femur. The femur was then cut transversely in
the shaft, using a custom-made pair of tongs. The patella was sutured
back in place and the skin closed. The process has been described
in greater detail elsewhere.[1]The mice were randomised to receive dexamethasone or control
injections, as described above, for the metaphyseal fracture group.
The animals were killed after 17 days, the femur harvested, the
intramedullary pin pulled out, and the femur frozen at -20°C for
later analysis.
Stable shaft fracture model
A total of 20 mice had an external fixator (MouseExFix simple
XL, RISystem, Davos, Switzerland) inserted. After opening the skin over the left femur and applying the
external fixator using four threaded pins, the shaft of the femur
was transected using a 0.22 mm diameter Gigli wire, leaving a 0.22
mm osteotomy. The muscle and the skin were then sutured, with the
screws sticking out through the suture and the plate outside the
skin.The animals were randomised to dexamethasone or control injections,
and given doses as described above. After 17 days the animals were
killed and the femurs harvested. The fixator was removed and the
femurs were frozen at -20°C for analysis.
Mechanical testing
All tests were performed on the same computerised materials testing
machine (100R; DDL Inc., Eden Prairie, Minnesota). All experiments
used force at failure as the primary outcome variable.Immediately after harvest, the tibias containing a metaphyseal
screw were placed in a custom-made mount on the materials testing
machine. The screw was attached to the force transducer by the use
of a clamp and was pulled out by the machine at a cross-head speed
of 0.01 mm/s.Both stable and unstable shaft femurs were thawed and tested
with three-point bending in the sagittal plane. The support bars
were 6 mm apart, and the loading bar was applied on the frontal
area of the bone with a cross-head speed of 0.05 mm/s. Before the
mechanical testing, the samples were analysed by microCT (µCT) as described
below.
µCT morphometry
A µCT machine (type1174; Bruker, Boston, Massachusetts) was used,
with a 0.25 mm Al filter, 50 kV, in 180° scans. Beam hardening and
ring artifacts were corrected for. Reconstruction and analysis were undertaken
with NRecon and CTAn (Bruker). For all experiments, standards with
0.25 g and 0.75 g of calcium hydroxyapatite per cm3 were
used to assess mineral density.The metaphyseal drill hole of the left tibia was analysed for
new bone formation using a pixel size of 8 µm, a rotation step of
0.5°, and a frame averaging of three. The volume of interest (VOI)
was defined as a 0.5 mm diameter cylinder covering where the drill
had been passed through, starting at the endosteum and extending
1 mm into the marrow compartment (Fig. 2). This volume was analysed
for bone volume (BV) and tissue mineral density (TMD).MicroCT scans with the volumes of
interest (VOI) extracted. In order to ensure that the images in
this figure are representative, the figure shows the specimen with
the median value for bone volume (BV), or bone area, from each treatment
group. Areas with decreased opacity in the unstable shaft VOIs indicate
areas that were excluded from calculations of BV and tissue mineral density.The two diaphyseal fracture models were analysed with a pixel
size of 12 um, a frame averaging of two, and a rotation step of
0.4° for the unstable model, and 0.3° for the stable model. For
the unstable model, the VOI was defined as a segment delineated
by two planes 2 mm apart, perpendicular to the longitudinal axis
of the femur and centred on the osteotomy. Old bone and the volume corresponding
to the original marrow space were excluded manually (Fig. 2). The
remaining VOI was analysed for BV and TMD. For the stable diaphysis,
the VOI comprised the newly formed callus in the fracture gap, excluding
old bone, also done manually (Fig. 2). The VOI was analysed for
TMD and mean bone area, i.e. BV per mm gap (since different animals
had slightly different lengths of the gap, total BV could not be
used).
Statistical analysis
Data were tested with the Shapiro–Wilk test for normality, and
parametric or non-parametric tests were used accordingly. Data not
normally distributed are noted as such in the results section. Hodges–Lehmann
was used to estimate the 95% confidence interval (CI) for the non-parametric
data, and t-tests were used to estimate the CIs
for the parametric data. A p-value of < 0.05 was considered significant.
Results
Excluded samples
The exclusions in the experiment are presented in Table I.The exclusions and cause thereofExFix, stable external fixation; IM-nail; unstable
nailingThe pull-out force of the metaphyseal screws was increased by
18% by dexamethasone, after pooling results from the three metaphyseal
experiments (95% CI 0.12 to 36; p = 0.049; Fig. 3). The 95% CI suggests
that inhibition owing to dexamethasone is highly unlikely. In contrast,
the force at failure of the shaft was decreased by 50% in the unstable
model (95% CI 19 to 81; p = 0.003). In the stable model, data were
not normally distributed. Median decrease was 51% (95% CI 15 to
74; p = 0.02).Graph demonstrating that the dexamethasone
had different effects on force at failure in the metaphysis and
the shaft. In the metaphysis an 18% increase (p = 0.049), and in
the stable and unstable shafts, a decrease by 50% and 51% (p = 0.02
and p = 0.003). There was no discernible difference between the
effect on the stable and the unstable shaft fractures.The metaphyseal drill hole BV and TMD were not normally distributed.
The median BV was decreased by 11% by dexamethasone (Figs 2 and
4, p = 0.006, 95% CI 3.6 to 19). The median TMD was decreased by
16% (95% CI 5.4 to 24; p = 0.005). In a few specimens, small localised
areas with low density were seen within the newly formed bone. In
the model of the unstable shaft fracture, dexamethasone decreased
the BV of the callus by 33% (Figs 2 and 4, 95% CI 16 to 49; p < 0.001).
The TMD of the unstable shaft fracture was not normally distributed.
The median TMD was decreased by 8% (95% CI 2.3 to 12; p = 0.004).
In the stable diaphyseal model there was no detectable effect on mean
bone area (Figs 2 and 4, 95% CI -12 to 29; p = 0.36) or TMD (95%
CI -6 to 25; p = 0.20).Graphs demonstrating the microCT
data for the amount of newly formed bone in the three models. a)
In the metaphysis there was an 11% decrease in bone volume (BV)
of the drill hole; b) the mean bone area of the callus of the stable
shaft fracture was not significantly affected, whereas c) the BV
of the central 2 mm callus of the unstable shaft was reduced by 33%
with dexamethasone injection*p < 0.05.
Discussion
Stable metaphyseal bone healing was not impaired by dexamethasone,
while there was a detrimental effect in both stable and unstable
healing of a shaft fracture.The result can be partly explained by three differences between
metaphyseal and shaft injuries. First, metaphyseal marrow is rich
in stem cells compared with the shaft marrow, the former also being
more committed towards an osteogenic fate.[6] If a shaft fracture is dependent on
stem cell recruitment from distant sources,[4] and inflammation is required for this
process, then dampening of inflammation would be harmful. In contrast,
the marrow compartment in the metaphysis has a stem cell supply,
which might already be sufficient.Second, metaphyseal fractures heal with less new tissue formation,
and with a much smaller callus. This means that fewer progenitor
cells are needed for new tissue formation. Hence, a drug that decreases
progenitor cell recruitment could be expected to have smaller effects in
a metaphyseal fracture.Third, shaft fractures heal via an endochondral phase which is
not usually found in metaphyseal fractures, neither in drill holes
in mice, nor in proximal tibial osteotomies in rabbits, or distal
radial fractures in patients.[2,3] Blocking glucocorticoid
receptor signaling in chondrocytes increases cartilage formation
in endochondral bone formation after fracture, suggesting that inflammation
is needed for cartilage formation after fracture.[7]Dexamethasone tended to improve the healing of metaphyseal injury,
as measured by the pull-out force. However, long-term glucocorticoid
treatment is known to have a negative effect on bone formation[8] and is a leading cause
of secondary osteoporosis,[9] possibly
because of induction of osteoblast and osteocyte apoptosis.[10] Yet, glucocorticoids,
together with other anti-inflammatory drugs, have also demonstrated
positive effects on bone metabolism in patients with upregulated
inflammation such as in rheumatoid arthritis.[11] In line with our
findings, a recent in vivo study showed that dexamethasone
treatment improved the quality
of metaphyseal bone while impairing the same in shaft bone, in both
ovariectomised (OVX) and non-OVX mice.[12] The positive effect in the metaphysis
was dependent on the presence of lymphocytes, as the effect was
absent in mice with severe combined immunodeficiency (SCID). An
NSAID, carprofen, did not have the same positive effect in the metaphysis as glucocorticoids
did.[12] This
corresponds well with previous findings from our lab that the NSAID
indomethacin had no positive effect in our metaphyseal screw model.[1]The main limitation of this study is that different models had
to be used in order to enable mechanical evaluation of shaft and
metaphyseal fractures. Comparing a screw pull-out model to a three-point
bending model is not ideal, but both give a measure of the most
important variable for a patient with a fracture, namely mechanical strength.The relevance for screw fixation as a measure of bone healing
requires some discussion. The pull-out force immediately after insertion
is minimal, and then increases over time as new bony threads are
formed.[1] This
bone formation is a response to trauma and occurs also in the absence
of a screw in the drill hole. The mechanical testing estimates the
strength of this newly formed bone, and the pull-out force correlates
with the amount of this newly formed bone.[5] As the amount and quality of the new
bone in metaphyseal fracture healing are most important in clinical
cases, we consider this screw model relevant for metaphyseal fracture
healing. It is a practical and simple model that allows for mechanical
evaluation. In contrast, a screw model in shaft bone would not yield
relevant information as fixation would largely be dependent on the
pre-existing cortex, and not on newly formed bone.The predetermined primary outcome variable of this study was
force at failure. The results of the morphometric analysis were
slightly different. There was a small decrease in bone density in
the metaphyseal drill hole, which contrasts with the increased pull-out
force. A possible explanation could be related to the observation
that small areas devoid of bone occurred within the VOI in some
dexamethasone samples. Another explanation could be that the implant
influenced the bone formation response, compared with the drill
hole without a screw. Furthermore, there was no morphometric to
correlate to the dramatic decrease in force at failure with dexamethasone
in the stable model. It appears that force at failure is a more
sensitive outcome variable, summarising the effects of subtle morphological
changes owing to treatment.In this experiment, the interaction between stability and dexamethasone
on metaphyseal healing was not studied. It is known that relative
stability has an effect on metaphyseal fractures. A very stable
situation has been demonstrated to lead to slower healing compared
with a less stable situation.[13] However,
since we could not see an effect of stability in the shaft, and
since instability is not usually a property of metaphyseal fractures,
we did not investigate this avenue.Because shaft and metaphyseal fractures do not heal at the same
pace, we had to use different healing periods before performing
the measurements. All time points were chosen to optimise the ability
to reveal the effect of impaired fracture healing, based on pilot
experiments. For the screw model, the time point followed the dramatic
early rise in pull-out force that occurs during the first week.[1] For the shaft models,
the time point corresponded to the earliest time when there was
bony bridging and a distinct failure point at the mechanical testing curves.In conclusion, inhibition of inflammation by use of dexamethasone
had strikingly different effects in metaphyseal versus shaft
fractures. Stable and unstable shaft fractures responded similarly.
These results underline the difference between metaphyseal and shaft
healing, and suggest a different role for inflammation in healing
at the two sites.
Table I
The exclusions and cause thereof
Model
Dexamethasone
Control
Comment
Metaphyseal
1
1
Computer error
Shaft (ExFix)
3
2
Infection or faulty surgical procedure
Shaft (IM-nail)
3
1
IM-nail protruded distally and relative stability was lost
Authors: Jinwen Tu; Holger Henneicke; Yaqing Zhang; Shihani Stoner; Tegan L Cheng; Aaron Schindeler; Di Chen; Jan Tuckermann; Mark S Cooper; Markus J Seibel; Hong Zhou Journal: Bone Date: 2014-09-03 Impact factor: 4.398