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Department of Orthopaedics, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands
b
IsoTis NV, Prof. Bronkhorstlaan 10D, PO Box 98, 3729 AB Bilthoven, Netherlands
Abstract
In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and
highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs
potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many
other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the
cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a
level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the
increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further
investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations
on design and assessment of outcome parameters.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Bone tissue engineering; Animal model; Spinal surgery; Cell viability
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fracture repair,
osteoinduction,
medullar bone formation, and
bone formation in autografts.
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Fig. 2. Micrographs of hybrid constructs implanted 9 weeks intramuscularly in goats. (a) Low-magnication micrograph of a
7 7 7 mm hybrid construct. New bone is clearly present as pink
bright red on the biphasic calcium phosphate (BCP) ceramic scaffold
surface. Note the typical distribution of bone that is present only in the
interior of the scaffold (bar=1.3 mm). (b) High-magnication micrograph showing newly formed bone lined by osteoblasts (arrows), on
the surface of the BCP scaffold (bar=100 mm). (c) High magnication
with uorescent microscopy showing the sequential uorochrome
labels in newly formed bone: C=calcein green at 4 weeks; X=xylenol
orange at 6 weeks; T=tetracyclin at 8 weeks. Mineralization was
initiated around 4 weeks and was directed centripetally (bar=200 mm).
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centrally [94,95]. Largely unknown, but crucial for cellbased TE, is the mechanism of bone formation in
autologous bone grafts, especially with regard to
osteogenicity as a function of residing osteoprogenitor
cells.
Bone tissue engineering is primarily a biologic process
in which various other aspects such as biomechanics,
play an important role. Therefore, to study the
feasibility of this technique in spinal fusion, biologic
similarity and non-union rates analogous to the human
situation are considered to be of utmost importance
[2,96,97]. Furthermore, understanding of the many
factors that inuence spinal fusion is essential to
optimize and identify the effect of TE. In Table 1a and
b we summarize some potential posterior and anterior
spine fusion models.
4.1. Factors that influence experimental spinal fusion
There are numerous factors that inuence the success
rate of experimental spinal fusion. These can be divided
into animal- and surgery-related factors. Animal-related
factors are the variations between different species, as a
result of evolutionary complexity and the varying pace
and degree of skeletal maturity. For example, in rats the
fusion rate is much higher than in mature non-rodent
mammals, including humans, where spontaneous fusion
after decortication of the facet joints and lamina is
exceptional [98]. Surgery-related factors include the
location and number of fused segments [91,99,100],
and the use and rigidity of instrumentation [101103].
For instance, the lumbosacral junction is exceptionally
prone to non-union, potentially as a result of excessive
motion [99]. Vascularization is critical for the osteogenic
process and is greatly inuenced by the regime of
decortication. In the case of PLF, the pars interarticularis preferentially is not decorticated [92], contrary to
the facet joints and transverse processes, where decortication facilitates vascular ingrowth into the fusion
mass [93]. Medication is another important issue, for
example
non-steroidal
anti-inammatory
drugs
(NSAIDs), that are normally used for post-operative
pain relief, have shown to negatively affect osteogenesis
[104106]. Finally, post-operative handling also appeared to inuence the outcome, as was shown by the
negative effect of regularly lifting rabbits that underwent
non-instrumented PLF from their cages in comparison
to a non-disturbed group [107].
4.2. Posterolateral fusion (Table 1a)
Spine surgeons are aware of the difculties of
achieving successful fusion; therefore, they meticulously
decorticate the facet joints, transverse processes and
laminae, and commonly use rigid instrumentation to
keep the segments xed during the critical period in
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Table 1
Selected animal models for (a) posterolateral and (b) anterior spinal fusions
Species (surg. model)
Segment
Follow up time
(weeks)
Conditions
L5-L6
210
Sham7decort, AGa
110
5
AG
AG, DBMb, ceramics, 7BMPc
L3-L4
12
Dog-Mongrel (decort.
non-instr.)
T1-8
AG, DBM
T6-13
T13-L7
3, 6, 12
6, 12, 26
AG, PLAd7BMP
AG, collagen7BMP, no graft
L4-5
12
AG, PLA7BMPs
4, 8, 12
Dog-beagle (decort.
non-instr.)
10 Consecutive T Levels
L3-5
6, 12
12
Sheep (decort7instr.)
16
8, 16
16
4, 8, 12, 16
20
24
12, 20, 36, 72
L4-5
12
24
DBM7BMP
AG, ceramics7BMP
C2-3 5-6
124
6, 12, 26
12
(Decort+instr.)
Sheep
Non-human primate
a
C2-3 5-6
2x separate cervical 2x
separate lumbal
12, 24
6, 12, 24
Stand-alone ceramics
Stand-alone AG, ceramics
Sham, AG, alloe
AG, allo, ceramics7plate
Cyl. cage with AG or BMP
Stand-alone AG, Ceramic
Stand-alone AG, ceramics
L4-L5
L3-4
24, 48, 96
12, 24
16
L2-3 4-5
L4-5
8, 32
8, 16, 32
24
L7-S1
L6-S1
12, 24
12, 24
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6. Discussion
In the present paper, we reviewed the current knowledge and achievements on cell-based bone tissue
engineering with regard to spinal fusion. Because cell
survival in large graft applications is questionable, and
therefore a simple cell-derived bone formation is
unlikely, emphasis was put on the potential mechanisms
by which the technique should function. Although the
exact mechanism is not elucidated, there are indications
that TE bone formation resembles the appositional
osteogenesis described to occur in ectopic autologous
bone grafts [76,79] and in disrupted bone marrow
cavities [6365]. In both processes, viable cells were
shown to be crucial. This might be a serious limitation
for the TE technique, because in many studies substantial loss of viability has been reported in the weeks
after transplantation of autologous bone grafts that,
with respect to cell survival, can be considered as cell
constructs [74,81,142]. Furthermore, in studies where
autografts were implanted orthotopically for several
months, the added value of viable cells in the grafts was
considered insignicant. In these studies, viable autografts were compared to autografts that were devitalized
by freezing in liquid nitrogen, before transplantation
[74,143145]. On the other hand, the functioning of
hybrid constructs might be possible with relatively few
surviving cells, as an increasing number of reports of
successful critical-size defect studies in large animals
have appeared [29,4043]. These ndings are promising
and make spinal fusion studies a logical next step in
research on feasibility of the technique.
Although AIF with the application of cages has many
advantages, researchers should realize that the ability to
discriminate between the long-term effects of TE and
simple osteoconduction may diminish. Therefore, the
more challenging posterolateral model seems initially
more appropriate. Besides construct osteogenicity,
successful spinal fusion will also rely on functionality
of the newly formed bone and integration in the
surrounding bone. This is a concern, because hybrid
constructs have the tendency to form individual ossicles
[34,6669] with mainly central bone formation and only
occasional bridging of individually implanted granules
(Fig. 2) [26]. Therefore, not only the percentage of bone,
but also these aspects of functional behavior should be
studied.
As soon as the feasibility of the technique has been
established, the question will arise whether safety and
efcacy studies in non-human primates are necessary, as
was carried out extensively for BMPs [132,133].
According to our opinion, the need for primate models
is defendable for BMP studies, where the risks of
(uncontrolled) bone formation may be higher as
compared to cell-based TE. Especially when nonstimulated cells are applied, this risk seems reasonably
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[15]
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Acknowledgements
The authors acknowledge The Netherlands Technology Foundation (STW; grant UGN. 4966) for nancial
support.
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