Abstract
Background: Although autogenous bone is the most widely used graft
material for spinal fusion, demineralized bone matrix preparations are
available as alternatives or supplements to autograft. They are prepared by
acid extraction of most of the mineralized component, with retention of the
collagen and noncollagenous proteins, including growth factors. Differences in
allograft processing methods among suppliers might yield products with
different osteoinductive activities. The purpose of this study was to compare
the efficacy of three different commercially available demineralized bone
matrix products for inducing spinal fusion in an athymic rat model.
Methods: Sixty male athymic rats underwent spinal fusion and were
divided into three groups of eighteen animals each. Group I received Grafton
Putty; Group II, DBX Putty; and Group III, AlloMatrix Injectable Putty. A
control group of six animals (Group IV) underwent decortication alone. Six
animals from each of the three experimental groups were killed at each of
three intervals (two, four, and eight weeks), and the six animals from the
control group were killed at eight weeks. At each of the time-points,
radiographic and histologic analysis and manual testing of the explanted
spines were performed.
Results: The spines in Group I demonstrated higher rates of
radiographically evident fusion at eight weeks than did the spines in Group
III or Group IV (p < 0.05). Manual testing of the spines at four weeks
revealed variable fusion rates (five of six in Group I, two of six in Group
II, and none of six in Group III). At eight weeks, all six spines in Group I,
three of the six in Group II, and no spine in Group III or IV had fused.
Histologic analysis of the spines in Groups I, II, and III demonstrated
varying amounts of residual demineralized bone matrix and new bone formation.
Group-I spines demonstrated the most new bone formation.
Conclusions: This study demonstrated differences in the
osteoinductive potentials of commercially available demineralized bone
matrices in this animal model.
Clinical Relevance: Comparative clinical testing of demineralized
bone matrices is indicated in order to determine which preparations are best
suited for promoting successful spinal fusion in humans.
Approximately 200,000 spine fusions are performed each year in the United
States, and most are single-level posterolateral lumbar
fusions1. Autogenous
bone graft is the current gold standard for inducing spinal
fusion2. Autogenous
bone is readily available from most patients and contains several of the
elements that are thought to be critical to promote bone formation, including
osteoprogenitor cells, the osteoconductive matrix of the cancellous bone, and
osteoinductive signals such as bone morphogenetic
proteins3-5.
Disadvantages of autogenous bone graft include the morbidity associated with
the harvest, the limited supply of the autogenous graft material, and the
additional operating room
time6-10.
Because of the frequent need for bone-grafting in spine surgery, alternatives
to autogenous bone are being developed and
investigated11-13.
Demineralized bone matrices are a potential alternative or supplement to
autogenous bone
graft14-16.
Demineralized bone matrices are prepared by acid extraction of allograft bone,
resulting in loss of most of the mineralized component but retention of
collagen and noncollagenous proteins, including growth factors. Demineralized
bone matrices do not contain osteoprogenitor cells, but the efficacy of a
demineralized bone matrix as a bone-graft substitute or extender may be
influenced by a number of factors, including the sterilization process, the
carrier, the total amount of bone morphogenetic protein (BMP) present, and the
ratios of the different BMPs
present17-19.
In addition, the osteoconductivity of the demineralized bone matrix-carrier
complex may be an important factor since this property promotes migration of
osteoprogenitor cells to the bone defect site.
Some demineralized bone matrices are tested chemically or immunologically
for BMP content, and certain demineralized bone matrices are tested in vitro
to demonstrate their osteoinductive effect on cultured cells. However, many
demineralized bone matrices have not been stringently tested in clinically
relevant animal models, in part because this has not been required by the
United States Food and Drug Administration. To our knowledge, there has never
been a prospective, randomized clinical trial to evaluate the efficacies of
these agents in a clinical setting.
Despite this lack of data, demineralized bone matrices have been used not
only to enhance fusion of the
spine20,21
but also to graft
nonunions22,
osteolytic lesions around total joint
implants23, and
benign bone
cysts24. However,
the success rates of different demineralized bone matrices have not been
studied in a rigorous fashion, and the efficacy of these agents may vary. The
purpose of this study was to compare the efficacy of three different
commercially available demineralized bone matrix products in an athymic rat
spinal fusion
model25.
Demineralized Bone Matrices Tested
Sixty male athymic rats were analyzed in this study. The animals were
divided into three experimental groups of eighteen animals each and a control
group of six animals. Group I was treated with Grafton Putty (Osteotech,
Eaton-town, New Jersey); Group II, with DBX Putty (MTF [Musculoskeletal
Transplant Foundation], available through Synthes, Paoli, Pennsylvania); Group
III, with AlloMatrix Injectable Putty (Wright Medical Technology, Arlington,
Tennessee); and Group IV (control group), with decortication alone.
Demineralized bone matrices were obtained directly from the manufacturers, and
the lot numbers were recorded. For each brand of demineralized bone matrix, at
least two different lot numbers were used.
Product Information
The specific details of the preparation and processing of the demineralized
bone matrices are proprietary, but each is composed of demineralized human
allograft bone combined with a biologically compatible carrier. All of the
demineralized bone matrices that we tested can be stored at room temperature
and do not have special handling requirements other than maintenance of
sterility.
Grafton Putty (Group I) is derived from human banked bone tissue. Donors
undergo serologic and microbiologic testing as well as screening on the basis
of their medical and social history. The allograft bone is harvested in a
sterile manner, washed, sonicated, treated with antibiotics, and demineralized
to contain <0.5% calcium phosphate. It is then combined with glycerol,
which results in a putty-like consistency. Grafton Putty also undergoes a
validated proprietary production process that has been shown to inactivate
human immunodeficiency virus-1, hepatitis-B virus, hepatitis-C virus,
cytomegalovirus, and poliomyelitis
virus26. Grafton
Putty is packaged in a ready-to-use form; it can be molded or packed directly
into a bone defect.
DBX Putty (Group II) is also derived from human banked bone tissue. Donors
undergo screening procedures as dictated by the Musculoskeletal Transplant
Foundation (Edison, New Jersey). Allograft tissue is harvested under sterile
conditions, washed, and treated with antibiotics. The tissue is then
demineralized with hydrochloric acid so that the resulting bone matrix
contains <8% calcium. The demineralized bone is combined with 4% sodium
hyaluronate carrier prior to packaging. DBX is packaged in a syringe; it can
be easily measured and is injected directly into a bone defect site.
AlloMatrix Injectable Putty (Group III) is derived from human banked bone
tissue as well. Donors are screened with serological testing as well as
evaluation of their medical and social history. The allograft tissue is
processed aseptically by the tissue supplier and undergoes e-beam
(electron-beam) sterilization. The allograft bone is demineralized and is
combined with calcium sulphate hemihydrate and carboxymethyl-cellulose. Each
lot of AlloMatrix demineralized bone matrix is assayed in vitro for
osteoinductive potential. AlloMatrix Injectable Putty is packaged as a kit
containing a powder and a liquid that must be mixed prior to implantation.
Once this mixing is performed, the AlloMatrix Putty can be easily molded or
pressed into a bone defect.
Arthrodesis in Athymic Nude Rats
Approval was obtained from the Institutional Animal Care and Use Committee
before the animal procedures were begun. This spine fusion model has been
described
previously27. A
midline incision was made in the skin, and the transverse processes of L4 and
L5 were exposed and decorticated bilaterally with a high-speed burr. After
this, 0.3 cm3 of graft material was implanted on each side (0.6
cm3 total). The appropriate demineralized bone matrix was implanted
in the eighteen animals in each of the three experimental groups. The control
group underwent decortication alone. Six animals from each of the three
experimental groups were killed at each of three intervals (two, four, and
eight weeks), and the six animals from the control group were killed at eight
weeks.
Radiographic Analysis
Radiographs were made at two, four, and eight weeks after surgery and were
examined by three independent observers who were blinded to the treatment
group. The amount of bone that had formed between the transverse processes of
L4 and L5 was evaluated with use of a scoring system in which 0 indicated
minimal or no evidence of new bone formation; 1, immature bone formation, with
fusion questionable; and 2, solid-appearing bone, with fusion likely. The
radiographic scores of the three observers were summed, with 6 as the maximum
score. Spines with a cumulative score of 5 or 6 were considered to have
radiographic evidence of fusion.
Manual Palpation
All spines were explanted and assessed for fusion by manual palpation by
three observers who were blinded to the type of treatment that the animal had
received. Manual palpation has been reported to be the most sensitive and
specific method of assessing fusion in this
model28,29.
All spines were categorized as either fused or not fused. At least two
observers had to have considered the spine to be fused for the spine to be
deemed fused as demonstrated by manual palpation.
Histologic Techniques
After the animals were killed, all sixty spines were dissected and were
fixed in 40% ethanol, dehydrated, and embedded in polymethylmethacrylate.
Serial sagittal sections of the transverse processes were cut with a diamond
band saw (Exakt, Hamburg, Germany). Sections were mounted on plastic slides,
milled, polished, and surface-stained with trichrome or toluidine blue.
Statistical Methods
Scores from the radiographic analysis were assessed with a nonparametric
Kruskal-Wallis test comparing the distribution of ranked data in the various
groups. Data from the manual palpation assessment were analyzed with use of
the Fisher exact test. The kappa statistic was calculated to demonstrate the
interobserver reliability of the scoring.
The three experimental groups differed with regard to the fusion rates.
These differences were consistently shown radiographically, by manual
palpation, and by histologic analysis.
Radiographic Analysis
The Grafton Putty demineralized bone matrix (Group I) is extensively
demineralized and has a glycerol carrier. New bone formation in the rats
treated with the Grafton Putty was the easiest to assess radiographically
since the putty material was initially radiolucent. Radiographs of the spines
made at two weeks after the surgery showed minimal bone formation between L4
and L5. At the four-week time-point, radiographs clearly showed new bone
formation between the transverse processes of L4 and L5
(Fig. 1). Radiographs made at
the eight-week time-point indicated that all six of the spines had fused, and
even the worst-appearing radiograph of a spine treated with Grafton Putty
showed considerable bone formation between the transverse processes.
The DBX Putty (Group II) is less extensively demineralized than is the
Grafton Putty and is initially radiopaque. The radiopaque DBX Putty material
could be seen on the radiographs made at the two-week time-point, and this
made assessment of new bone formation more difficult
(Fig. 2). At four weeks, four
of the six spines in which DBX had been implanted appeared to be solidly
fused. At eight weeks, radiolucent cracks appeared between the bone formed at
L4 and L5 in three of the spines, indicating that a pseudarthrosis had
occurred. Three of the spines appeared to be fused.
Two weeks after the operative procedure in the spines in Group III
(AlloMatrix Injectable Putty), radiopaque material appeared to bridge L4 and
L5. However, it was unclear whether this material was residual carrier or new
bone. At the four-week time-point, the amount of radiopaque material had
increased minimally in comparison with that seen at the two-week time-point,
and, by eight weeks, three of the six radiographs showed large radiolucent
fissures between L4 and L5, clearly indicating a pseudarthrosis. None of the
Group-III spines appeared to be fused on the radiographs made at the
eight-week time-point (Fig.
3).
There was minimal or no radiographic evidence of bone formation between L4
and L5 in the spines in the control group (Group IV).
There was a high level of agreement with respect to the radiographic
scores. The kappa statistic was 0.87.
Figure 4 presents the best and
worst-appearing radiographs for each study group at eight weeks.
Manual Palpation
At the two-week time-point, no spine in any of the four groups was
determined to be fused on manual palpation. However, five of the six spines in
Group I had fused by the four-week time-point, and all six had fused by eight
weeks. In Group II, two of the six spines were fused at four weeks and three
were fused at the eight-week time-point. In Group III, there was no evidence
of spine fusion on manual palpation at two, four, or eight weeks after the
operative procedure (Table I).
There was a significant difference in fusion rates, as noted with manual
palpation, between Group I (Grafton) and Group III (AlloMatrix) at both four
weeks (five of six compared with zero of six; p = 0.015) and eight weeks (six
of six compared with zero of six; p = 0.001). The differences in the fusion
rates between Groups I and II and between Groups II and III did not reach
significance, probably because of the limited number of animals in the study.
No spines in the control group (Group IV) were considered to have fused. The
combined kappa statistic was 0.922.
Histologic Analysis
At two weeks, the spines in Group I (Grafton Putty) demonstrated woven bone
on the surfaces of the L4 and L5 transverse processes. In addition, there were
strands of collagen, apparently from the carrier, and isolated islands of new
bone formation. No endochondral intermediate was detected. By four weeks,
these islands of new bone had coalesced, forming networks of woven bone. At
eight weeks, no residual demineralized bone matrix was visible and new bone
bridged the transverse processes of L4 and L5, resulting in solid fusion in
all six spines (Fig. 5).
All six of the spines in Group II (DBX Putty) revealed persistence of the
demineralized bone matrix material at the two, four, and eight-week
time-points. New bone formation occurred on the surfaces of the transverse
processes and in the interstices of the carrier. New bone formation was
detected in the two-week specimens, and by eight weeks three of the spines
were fused. In the other three spines, there was new bone formation but not
complete fusion.
In Group III (AlloMatrix Putty), new bone formation was noted originating
from the surfaces of the transverse processes. However, the amount of
additional bone formation between the two and four-week time-points was
minimal. None of the spines had fused by eight weeks, although new bone was
present on the dorsal surfaces of the transverse processes. Residual
demineralized bone matrix was still detectable in the spines at eight weeks,
but less residual carrier was present than had been seen in the two-week
spines.
In the control group (decortication only), minimal bone had formed on the
surfaces of L4 and L5 at eight weeks (Fig.
5).
We are not aware of any prospective clinical trials comparing demineralized
bone matrices, but there is some clinical evidence and there are several
animal studies indicating that demineralized bone matrices may function as
extenders of autogenous bone
graft14,30.
For example, Johnson et al. combined demineralized bone matrix with autogenous
bone graft to effectively treat tibial and femoral
nonunions31-33.
In their series of thirty problematic femoral nonunions, twenty-four healed
within six months after intervention, four required application of a second
plate before union occurred, and two patients were lost to follow-up. In a
nonrandomized, prospective study of single and multilevel anterior cervical
fusions, An et al. compared the results in thirty-eight patients treated with
autogenous iliac crest bone graft with those in thirty-nine patents treated
with allograft bone and demineralized bone
matrix34. At the
time of follow-up, at twelve to thirty-one months, the patients treated with
the autograft had a lower rate of graft collapse than did those treated with
the allograft and demineralized bone matrix (11% compared with 19%) as well as
a lower rate of pseudarthrosis (26% compared with 46%), but the differences
did not quite reach significance.
Various animal models have been developed to study the osteoinductive
potential of demineralized bone matrices and their ability to either
substitute for or enhance the biologic activity of autograft
bone15,35-38.
A general weakness of these studies is that the demineralized bone matrix that
was tested was not the same material that is commercially available since the
demineralized bone matrix must be made from bone from the same species.
The efficacies of demineralized bone matrices have been assessed in a
well-established rabbit posterolateral spine fusion
model15,39,40.
Morone and Boden demonstrated that decreased autograft volume could be
supplemented with demineralized bone matrix gel to yield fusion rates similar
to those following use of autograft
alone15.
The present study involved an athymic rat posterolateral spine fusion
model, a standardized model that enables stringent testing of osteoinductive
capacity. An advantage of this model is that the demineralized bone matrix can
be evaluated in its commercially available form because the nude rat does not
generate an immune response to the human demineralized bone matrix. Results
derived from this or any animal model of spine fusion must be interpreted with
caution, however. The efficacy of these demineralized bone matrices may be
different in other types of models (e.g., a femoral defect model). Also, young
healthy animals were used in this study, and the results in such animals may
not be applicable to older patients with previous surgical treatment, a
history of nicotine use, or poor general health. The final test of the
efficacy of demineralized bone matrix is the clinical trial, and success at
one level of animal model does not necessarily mean that the demineralized
bone matrix will be osteoinductive at the next level. However, failure to
induce bone at a lower level of the phylogeny has been suggested to indicate a
poor prognosis for the osteoinductive potential of the substance in higher
animals and
humans1.
While all demineralized bone matrices augment the fusion process by
providing an osteoconductive scaffold of variable osteoinductive
activity41-44,
each manufacturer uses a different system for procuring allografts and for
demineralization and sterilization. In addition, demineralized bone matrices
are often combined with different carriers such as glycerol, hyaluronic acid,
or calcium sulfate. Many demineralized bone matrices have not undergone
extensive preclinical or clinical testing, in part because the Food and Drug
Administration has not regulated demineralized bone matrices in the same way
that medical devices have been regulated. Therefore, it was not surprising
that the demineralized bone matrices in this study were found to have
different biological activities. In addition to the processing methods, donor
quality is another potential source of variability in osteoinductive
potential. This could be evaluated by testing different lots from a single
graft processor. We did not attempt to compare osteoinductivity among
different lots of the same demineralized bone matrix, but, by using at least
two lot numbers of each demineralized bone matrix formulation, we hoped to
minimize the potential of a systematic error. Several preclinical studies have
demonstrated no significant difference among lots from the same manufacturer,
even though the donor age in one study ranged from forty-five to sixty-seven
years45.
All of the demineralized bone matrices tested in this study are
commercially available. Each had excellent handling properties in that they
were easily molded and placed onto the decorticated transverse processes of
the rats. We did not evaluate the diffusion or solubility of the demineralized
bone matrices, and therefore we cannot comment on the duration for which the
demineralized bone matrix was present at the desired fusion site after the
skin was closed.
This study demonstrated that differences in the osteoinductive potential of
commercially available demineralized bone matrices can be detected with the
use of this animal model. Whether the differences in fusion rates in athymic
rats translate into variable clinical outcomes when the same demineralized
bone matrix preparations are used in patients is a matter of speculation.
Surgeons should carefully consider the clinical indications for any bone-graft
substitute or extender, and comparative clinical testing of demineralized bone
matrices is needed to determine which preparations are best suited for
promoting successful spinal fusion in humans.
Note: The authors thank Fred Dorey, PhD, for his assistance with
the statistical analysis in this study.
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