THE EFFECT OF MESENCHYMAL STEM CELLS ON ALLOGRAFTS IN A CANINE INTERCALARY BONE
DEFECT
+*Steven A. Lietman, *Jun Fukuroku, *Nozomu Inoue, *Isumi Toda, *Edmund Y.S. Chao
Essential Results: There was no significant change in the dynamic
weight bearing between the stem cell and control groups. The periosteal
callus area was significantly increased (p<0.05) at 6, 8, and 10 weeks
after surgery in the stem cell group (Figure 1). Torsional stiffness and
maximal torque were significantly increased (p<0.05) were significantly
increased in the control group (Figure 2). Fractures were more common
in the allograft in the stem cell group and at the host allograft junction in
the control group (Figure 3).
1000
callus area (mm2 )
800
600
*
*
400
stem cell
control
200
0
-200
2w
4w
6w
8w
1 0w
12w
mean +- SD
weeks after surgery
*
: Statistically difference compared to the control side (paired t
test, P < 0.05)
• Stem
Stem cell
cell >> Control
Control at
at 6,
6, 88 and
and 10
10 weeks.
weeks.
Figure 1: Periosteal Callus Area
RESULTS
RESULTS
Maximum torque
20000
18000
16000
maximum torque(N)
Methods: Sixteen adult hound-type canines ranging from 24 to 35 Kg
(31.4 ± 4.4 (mean ± S.D.)) were used in the study. The allografts were
removed from each animal, wrapped in sterile gauze, and frozen at
-80C° for at least three days between surgeries. Prior to insertion into
the defect, the 4 cm bone segments were debrided of all soft tissues
(periosteum removed and the medullary canal completely curetted of
marrow) and subsequently rinsed in normal saline. Prior to insertion and
fixation in the intramedullary canal an implant consisting of
mesenchymal stem cells in the treatment group and nothing in the
negative control was inserted into the intramedullary aspect of the
allograft. The allografts were fixed as previously described. The
protocol was approved by the Institutional Animal Care and Use
Committee. Load bearing was measured before surgery and at three,
six, and nine weeks postoperatively. During each measurement, a
minimum of six successful runs was obtained for each hindlimb.
Standard radiographs were taken immediately postoperatively and at
two, four, six, eight, ten, and twelve weeks postoperatively to monitor
periosteal callus area. Periosteal callus area was measured directly on
anteroposterior and mediolateral radiographs using an image analyzer
software package (Bioquant System IV; R & M Biometrics, Inc.,
Nashville, TN) and a sonic digitizer (Summa Sketch II Plus;
Summagraphics, Seymour, CT). Animals were euthanized and femora
harvested at postoperative week twelve. The proximal segment of the
host bone -allograft construct was used for mechanical testing (Fig. 2).
The distal segments, as well as the proximal segments after mechanical
testing, were prepared for undecalcified histological analysis. Mineral
apposition rate in the cortical bone area was measured using a double
labeling technique. Labeled osteon density, the density of
fluorochrome-labeled osteons per unit area in a given section, was
measured using sections mounted on microscope slides and illuminated
using an ultraviolet light source. Allograft and host bone porosity was
determined using digitized images of contact microradiographs (Fig. 3).
Mechanical testing and histological data were collected in a blinded
fashion, with the sample code unknown to the evaluator. Paired data
from the treatment and control sides were compared using a paired
Student’s t-test. Time-repeated data, such as gait and callus area
measurements, were analyzed with analysis of variance and Tukey’s
post-hoc test. A difference was considered significant when p values
were less than 0.05.
RESULTS
RESULTS
Periosteal Callus Area
*
Introduction: The treatment of large structural bone defects in
orthopaedics after bone tumor resection or in conjunction with osteolysis
in failed total joint arthroplasty are a clinical challenge with high
complication rates.1,2 Mesenchymal stem cells are pluripotential and
capable of differentiating into cells from a variety of mesenchymal
tissues including: fat, muscle, bone and cartilage. We are interested in
the use of mesenchymal stem cells with bone allografts to heal large
structural bone defects. A process which could upregulate allograft bone
resorption and enhance incorporation into the host bone could reduce the
potential risk of long-term failure, especially in cases in which relatively
large bone defects are bridged. It also would be particularly beneficial if
at the same time the strength of the allograft/host complex were not
significantly compromised. We investigated in this study the effect of
canine mesenchymal stem cells applied to the intramedullary aspect of a
segmental intercalary allograft in a canine femoral defect model.
14000
12000
10000
8000
6000
**
4000
2000
0
stem cell
*
control
mean +- SD
: Statistically significant compared to the control side(student
paired T -test: P < 0.05)
•Stem
•Stem cell group << control
control group
Figure 2: Maximum torque
Radiographs after torsional test
host bone
Location of fracture line
stem cell
control
7
(87.5%)
3
(37.5%)
0
2
(25%)
1
(12.5%)
1
(12.5%)
1
(12.5%)
3
(37.5%)
allograft
on screw hole
allograft
on K-wire hole
junction
host bone
fracture line on screw hole
host bone
screw hole
(no hole)
no line
(junction )
0
0
K-wire hole
allograft
Figure 3: Location of fracture line
Discussion
This study represents an attempt to examine the effects of mesenchymal
stem cells on long bone structural allograft healing. The results indicate
and increase in callus formation and a decrease in fracture at the
allograft host junction in the stem cell treated group. However there was
a decrease in maximum torque and torsional stiffness in the stem cell
treated group which was felt to be due to the weakening of the allograft
bone in the demineralization process in the stem cell treated group.
Further engineering of the pluripotential mesenchymal stem cell could
improve allograft healing and will be investigated in the future.
This study was supported by a grant from Osiris Therapeutics allografts
in the stem cell group underwent.
References
1.Lietman, S. A.; Inoue, N.; Chao, E. Y.; and Frassica, F. J.: Distal
femoral osteoarticular allografts in limb salvage surgery. Ann
Chir Gynaecol, 88(3): 221-5, 1999.
2.Lietman, S. A.; Tomford, W. W.; Gebhardt, M. C.; Springfield, D.
S.; and Mankin, H. J.: Complications of irradiated
allografts in orthopaedic tumor surgery. Clin Orthop, (375):
214-7, 2000.
49th Annual Meeting of the Orthopaedic Research Society
Poster #0852