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Collagen sponges for bone regeneration with rhBMP-2

M. Geiger
, R.H. Li
, W. Friess
Drug Product Development, Wyeth BioPharma, One Burtt Road, Andover, MA 01810, USA
Wyeth Research, 35 Cambridge Park Drive, Cambridge, MA 02140, USA
Department fuer Pharmazie, Lehrstuhl fuer Pharmazeutische Technologie und Biopharmazie,
Ludwig-Maximilians-Universitaet Muenchen, Butenandtstr. 513, 81377 Munich, Germany
Received 17 July 2003; accepted 26 August 2003
In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone fractures, alleviate
severe back pain through spinal fusion procedures, or promote healing of non-unions. Many of these procedures involve the use
of bone graft substitutes. An alternative to bone grafts are the bone morphogenetic proteins (BMPs), which have been shown to
induce bone formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to prolong the
residence time of the protein and, in some instances, as support for the invading osteoprogenitor cells. Several factors involved
in the preparation of adequate matrices, specifically collagen sponges, were investigated in order to test the performance in a
new role as an implant providing local delivery of an osteoinductive differentiation factor. Another focus of this review is the
current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an absorbable collagen sponge
(ACS). The efficacy and safety of the combination has been clearly proven in both animal and human trials.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Bone regeneration; Osteoconduction; Osteoinduction; rhBMP-2; Differentiation factors; Tissue engineering; Collagen; Form-
aldehyde cross-linking; Ethylene oxide sterilization; Dehydrothermal treatment
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1614
1.1. Bone regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1614
1.2. Bone morphogenetic proteins (BMPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1615
1.3. General requirements for carriers/delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1616
2. Collagen sponges: general characteristics and impact on performance for use in bone regeneration . . . . . . . . . . . . . 1617
2.1. Interactions of collagen and rhBMP-2 in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618
2.2. Cross-linking of collagen sponges with formaldehyde vapor in a convection chamber and by
dehydrothermal treatment (DHT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620
2.3. Sterilization by gamma- or e-beam (beta-) irradiation and DHT . . . . . . . . . . . . . . . . . . . . . . . . . . 1621
0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +1-978-247-1487.
E-mail address: (M. Geiger).
Advanced Drug Delivery Reviews 55 (2003) 16131629
3. rhBMP-2 and Absorbable Collagen Sponge (ACS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621
3.1. Preparation of the application system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621
3.2. Evaluation in animal models and clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621
3.2.1. Fracture repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622
3.2.2. Healing of critical size defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622
3.2.3. Spinal fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623
3.2.4. Dental and craniofacial reconstruction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1624
4. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1624
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625
1. Introduction
1.1. Bone regeneration
Bone is one of the few tissues in the adult human
body whose ability to regenerate spontaneously has
long been recognized, assuming that the defect does
not exceed a certain limit in size. These critical-sized
defects can either result from congenital deformities,
for example in the skull (cleft palate, facial clefts,
facial asymmetry [1]), trauma or tumor resection, or
degenerative diseases such as osteoarthritis and oste-
omyelitis. Improper osseous healing has potentially
devastating consequences, ranging from disfigure-
ment to loss of function and loss of limb [2]. In
cases of large bone defects where bone is not
expected to regenerate spontaneously, clinicians com-
monly attempt to induce formation of new bone to
bridge the defect using bone graft or bone graft
substitutes. The primary goal is restoration of form
and function [3], ideally by having the defect popu-
lated with material closely resembling the original
bone prior to damage.
One approach to restore form and function is sub-
stituting bony material through the use of permanent
orthopaedic implants made of metals, ceramics, poly-
mers (e.g. polyethylene) or composite materials. Re-
storing function by bone regeneration represents a
fundamentally different approach. By this strategy,
not only can the re-establishment of physical function
be achieved, but also full physiological function may
be realized. Bone regeneration should lead to a cortex
continuous with the surrounding bone, and a marrow
cavity filled with stem cells. Ideally, this construct
would ultimately be indistinguishable from the sur-
rounding host bone by radiography and histology.
Bone represents a complex system of a variety of cell
types embedded in a matrix consisting of collagen and
tightly associated, highly oriented calcium phosphate
crystals. This organization lends bone high resistance
against compression, tension, bending and torsional
forces. The high porosity of bone is an optimal com-
promise between load-bearing capacity and mass.
Bone undergoes constant remodelling by osteoclasts
and deposition of new bone material by osteoblasts
[4,5]. Intervention becomes necessary when this deli-
cate balance is disturbed.
For several decades, the gold standard in bone-
defect management has been autografting, which
involves harvesting healthy bone from one anatomical
site of the patient, most often the iliac crest, and
implanting the material at the defect site. This tech-
nique yields the most predictable results, however
bears considerable risks (donor site pain and morbid-
ity, infection, extra blood loss, and higher cost due to
longer operating times). Additionally, autografting is
ineffective when the defect volume exceeds the vol-
ume of healthy available graft material, a problem
prevalent among the pediatric and geriatric patient
population [2]. In total, unacceptably high failure rates
of 1330% have been reported [3]. The most common
alternative to autograft is human cadaver bone (allo-
graft), which has additional disadvantages, including
potential host reaction, limited supply, excessive re-
sorption, and potential disease transmission. The
reported failure rates exceed those for autografts
(2035%) [3]. Animal bone (xenograft) is rarely used
owing to concerns with immunogenicity and disease
transmission [2].
In the United States alone, approximately 500,000
surgical procedures in this field are performed annu-
ally [6]. Spinal applications now account for almost
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1614
half of the total grafting procedures, followed by
trauma indications, which account for roughly one
quarter [6]. The increasing number of degenerative
disc disease, osteoarthritis, and osteoporosis within an
aging population is expected to contribute to rapid
growth of spinal, joint and trauma segments, respec-
tively, in the future [6]. Therefore there is a growing
need to provide alternatives to traditional bone graft-
ing. In the last decades, the orthopaedic research
community has focused on the four requirements of
bone regeneration: (1) a morphogenetic signal, i.e.
growth and differentiation factors, (2) host cells that
will respond to the signal, i.e. are capable of differ-
entiating into osteoblasts, (3) a biomaterial carrier of
this signal that can deliver the morphogenetic signal to
specific sites and serve as a (degradable) scaffold for
the growth of the responsive host cells, and (4) a
viable, well vascularized host bed [79].
Bone graft substitutes replete with living cells at the
time of delivery would have a major advantage over
acellular substitutes in that the graft is not dependent
on in-vivo cell attachment and invasion, resulting in
potentially faster and more reliable bone formation.
However, cell-based therapies present complex chal-
lenges in the regulatory approval process [7]. One
example for cellular implants is CollagraftR (Neu-
Coll, Campbell, CA, USA), which has been approved
in the US as an alternative to autograft, and is a porous
collagen-calcium phosphate ceramic strip which is
blended with the patients bone marrow prior to
Among the acellular systems are for example
materials derived from natural bone. Demineralized
bone matrix (DBM) consists of the organic part of
human cadaver bone, mainly collagen type I, after
hydrochloric acid extraction of the mineral fraction
[10], and is commercially available in different forms.
Characteristics and applications of DBM have recent-
ly been reviewed [8,11]. Bone graft substitutes man-
ufactured from the mineral phase of human or animal
bone are also available, but considered mainly as
osteoconductive (e.g. providing guidance for the bone
regeneration process at skeletal sites) as opposed to
osteoinductive in nature [12].
Diverse materials, either naturally derived or syn-
thetic, have been tested as bone graft substitutes
(reviewed in Refs. [2,8,13,14]). Whereas a number
of these have been able to successfully bridge
smaller defects by osteoconduction, they generally
do not cause induction of new bone growth
( = osteoinduction). Osteoinductive materials lead to
bone formation even at non-skeletal sites by mim-
icking the processes of hard tissue formation in the
embryonic state [15]. Among the most potent
osteoinductive factors yet discovered are bone mor-
phogenetic proteins (BMPs).
1.2. Bone morphogenetic proteins (BMPs)
In 1965, Urist implanted demineralized bone ma-
trix at intramuscular sites in rodents and rabbits [10].
The sequence of events which followed was reminis-
cent of the bone development process in embryos and
of post-natal endochondral ossification [16,17]. The
term bone morphogenetic protein (BMP) was in-
troduced to describe the substance(s) in the deminer-
alized bone matrix responsible for the phenomenon.
Morphogenesis means generation of form, the
process of tissue and organ construction and assembly
[15]. At least 15 BMPs are currently recognized
(BMPs 115) [18]. The osteoinductive properties of
endogenous BMPs of various origin (e.g. murine,
ovine, bovine, reindeer, primate and human) have
since been evaluated extensively both in vitro and in
vivo (reviewed by Kirker-Head [9]).
Human BMPs are now available more readily and
in substantially larger quantities due to the advent of
recombinant DNA technology. In 1988, Wang et al.
[16] reported the isolation of three polypeptides of
16, 18, and 30 kDa molecular weight from bovine
bone. The encoding human genes were later trans-
fected into Chinese hamster ovary cells and to
Escherichia coli cells [19,20]. Among the recombi-
nant proteins, rhBMP-2 and rhBMP-7 (also termed
(human) osteogenic protein-1 ((h)OP-1) [21]) have
been tested in a number of orthopaedic indications as
well as for application in the dental/maxillofacial
field [13,2225]. Clinical results have recently led to
regulatory approval of both OP-1 (Osigraftk, How-
medica International S. de R.L., Raheen, Limerick,
Ireland) and rhBMP-2 (InFUSEk Bone Graft/LT-
CAGEk Lumbar Tapered Fusion Device, Medtronic
Sofamor Danek, Memphis, TN, USA; InductOsk,
Wyeth Europa, Maidenhead/Berkshire, UK) for some
of these applications by governmental agencies (see
Section 3.2).
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1615
1.3. General requirements for carriers/delivery
It has been shown that rhBMP-2 requires combina-
tion with a biomaterial matrix to attain maximal
efficacy. Such matrices should be characterized by
adequate porosity to allow cell and blood vessel
infiltration, appropriate mechanical stability against
compression and tension, biocompatibility, biodegrad-
ability, amenability to sterilization, adhesiveness to
adjacent bone, affinity for BMPs, and should provide
retention of the protein for a sufficient period of time to
affect the repair (Table 1) [8,9,24,25].
The main role of the delivery system for rhBMP-
2 is to retain the factor at the site for a prolonged
period of time [13]. For example when
2 is soaked into a collagen sponge and implanted in
the rabbit ulna osteotomy model using previously
described methods [26], the local retention is sig-
nificantly prolonged compared to buffer delivery.
Fig. 1 shows that using gamma scintigraphy, 32%
of the initial
I-rhBMP-2 dose remained at the
osteotomy site 7 days after surgery using the
collagen sponge matrix as compared to only 3%
remaining when rhBMP-2 was injected using buffer
It has now become clear that there is probably not
one single desirable pharmacokinetic profile that is
predictive of success. In designing a matrix for
differentiation factor release, it is apparent that the
extremes of release (bolus injections or prolonged
low level release) are not beneficial to bone induc-
tion [13]. A further complicating factor is that
different anatomical sites might require different
kinetics of release for optimal performance. For
example, in either more fluid environments or com-
promised (avascular) sites, BMP clearance might be
faster than the bone-induction response of the host.
In these cases a slow-release system may be re-
quired. It has further to be noted that different animal
species may have varying optimum release profiles
Table 1
Desirable qualities of an ideal rhBMP-2 delivery system (modified
from Refs. [8,9,24,25])
Biocompatibility, low immunogenicity and antigenicity
Biodegradability with biocompatible components, in predictable
manner in concert with bone growth
Adequate porosity for cellular invasion and vascularization
Adequate compressive and tensile strength
Enhancement of cellular attachment (but without inducing soft
tissue growth at the bone/implant interface)
Amenability to sterilization without loss of properties
Affinity to BMPs and host bone
Enhancement of osteogenic activity of BMP with a restrictive
release of BMP at an effective dose during a period coincident
with the accumulation and proliferation of target cells
Adaptability to irregular wound site, malleability
Availability to surgeon on short notice
Fig. 1. Retention of rhBMP-2 in the rabbit ulna osteotomy model, implanted with and without a collagen sponge (reprinted from [13], with
permission from Elsevier) requires copyright permission since we previously published in Trends in Biotechnology [13].
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1616
A wide range of materials has been tested in
combination with BMPs (reviewed in [9,13,27]).
One of the first candidates was demineralized bone
matrix which has intrinsic, limited osteoinductive
properties. Among the osteoconductive carriers have
been poly(a-hydroxy acid) microparticles, foams, or
disks; collagenous materials, e.g. collagen type I
sponges, semi-solid paste, collagen type IV, or type
I collagen/gelatin composites; inorganic ceramic
materials, e.g. calcium phosphate cement, porous
hydroxyapatite (HA), or hydroxyapatite/tricalcium
phosphate (TCP) as blocks and granules; bone or
cartilage derived materials, e.g. inactive collagenous
bone matrix and bovine bone mineral; and compo-
sites, e.g. dentine matrix powder/chondroitin-6-sul-
fate/type I collagen, TCP or coralline HA/type IV
collagen, and poly(a-hydroxy acid)/ carboxymethyl-
cellulose or methylcellulose. BMPs have also been
used in combination with titanium mesh and other
non-degradable metallic orthopaedic implants. Deliv-
ery strategies of rhBMP-2 in commercial products
have concentrated on an absorbable collagen sponge
which is impregnated with protein solution prior to
implantation. RhBMP-7 ( = OP-1; OsigraftR, How-
medica International S. de R.L.) and NeOsteoR
bovine BMP mixture (Sulzer Orthopaedics Biologics,
Wheat Ridge, CO, USA) have also utilized collagen-
based carriers.
2. Collagen sponges: general characteristics and
impact on performance for use in bone
Collagen has received increasing attention over the
last years due to its excellent biocompatibility, degra-
dation into physiological end-products, and suitable
interaction with cells and other macromolecules. The
favorable influence of collagen on cellular infiltration
and wound healing is well known. An additional
benefit is that collagen can be processed on an
aqueous base. A variety of dosage forms have been
in use for years, including aqueous injectable collagen
dispersions, powders and surgical sutures, corneal
shields, tissue and vascular sealants and spongy
implants [28]. Collagen sponges have a long safety
history as hemostatic agents and wound coverings
[2931], and are under investigation as scaffolds in
the emerging field of tissue engineering [31]. A
detailed review on collagen is beyond the scope of
this manuscript, but the reader is referred to other
reviews on this subject [28,32].
The manufacturing of collagen sponge implants
generally starts with the processing of purified colla-
gen material into aqueous solutions or suspensions at
adequate pH (important for swelling and separation of
fiber structures) [28]. Freeze-drying has proven to be
the most advantageous process to manufacture ho-
mogenous porous collagen devices [33,34]. If such
materials are degraded in vivo too quickly, or if the
three-dimensional porous structure cannot be main-
tained sufficiently in the presence of liquid, exoge-
nous cross-linking can be performed, using a variety
of chemical agents [30,31,35,36]. Examples are alde-
hydes [3740], acyl azide [37], carbodiimides
[36,37], hexamethylene-diisocyanate [37], and poly-
epoxy compounds [41]. Alternatively, collagen scaf-
folds can be submitted to physical treatment, i.e.
dehydrothermal treatment (DHT) [42,43], ultra-violet
irradiation [44,45] or g-irradiation [46]. The degree of
cross-linking of collagenous materials is reflected in
several chemical and physicochemical parameters,
e.g. shrinkage temperature [37,47], DSC melting
temperature [38], free amine group content
[37,38,48], enzymatic digestion in vitro [38,49] and
mechanical properties [47,49].
One prerequisite of systems intended for parenter-
al application is sterility [50]. Sterile products can be
obtained with methods using heat (dry heat and
autoclaving) and through cold processes, i.e. micro-
bicidal gases or high-energy irradiation. A decision
tree for sterilization choices has been published by
the EMEA in 2000 [51]. Despite the robustness of
collagen molecules, sterilization with steam or dry
heat of at least 160 jC are not applicable, since the
helices are irreversibly damaged. One alternative to
obtain sterile collagen products is treatment with
ethylene oxide, which is less common for drugs,
but has a place in the sterilization of medical devices
and medicinal products [52]. Gamma-irradiation is
another established sterilization method for collage-
neous products [32] and has extensively been used
for the preparation of human bone and tendon grafts
The combination of rhBMP-2 with a collagen
sponge matrix has proven to be a very promising
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1617
therapeutic in a variety of applications (see Section 3).
Consequently, there are some factors involved in the
preparation process of collagen sponges and the
combination product which require evaluation with
regard to optimal performance in its role as an implant
providing local delivery of an osteoinductive factor
(Table 2). The studies presented here focus on the
effect of cross-linking and sterilization by chemical
means (formaldehyde and ethylene oxide, respective-
ly), plus evaluation of physical treatment (dry heat
and irradiation).
2.1. Interactions of collagen and rhBMP-2 in vitro
and in vivo
For combination products such as rhBMP-2 and a
collagen matrix, it is important to assess not only
efficacy and pharmacokinetics in vivo, but also to
characterize the interactions of protein and matrix in a
variety of in vitro tests. The combination product
should for example be evaluated for loading capacity
and time-course of binding of the protein to the matrix
as well as for subsequent protein release in buffer. Of
specific interest are studies to elucidate the influence
of pH and salts in the protein solution as well as of
potential changes in the matrix due to processing
steps, in our case cross-linking and sterilization of
collagen. Another aspect is the integrity of the protein
after combining it with the matrix, since this is vital
for efficacy, and additional assessments should focus
on the matrix itself.
For clinical application rhBMP-2 is soaked onto a
collagen sponge (see Section 3.1). Consequently, loss
of rhBMP-2 solution due to mechanical manipulation
during implantation as well as potential effects of a
matrix on in vivo retention has to be considered.
Binding studies showed that rhBMP-2 binding to
the sponge was negligible at pH 3 and 4 [39]. At
pH 4.5, significant amounts of rhBMP-2 were bound
which further increased up to 0.10.2 mg rhBMP-2
per mg collagen at pH 5.2 and pH 6.5. The effect may
be explained by the differences in the isoelectric
points of the two proteins (collagen and rhBMP-2).
Depending on the manufacturing process, collagen
exhibits an isoelectric point in the neutral or slightly
acidic pH range [24]. rhBMP-2 has an isoelectric
point of approximately 9 and thus a positive net
charge at the pH of the combination product. This
results in electrostatic attraction forces between
rhBMP-2 and collagen, believed to be a major factor
controlling the protein-matrix interactions. It was
found that the interactions in a phosphate-buffer
environment depended on ionic strength of the medi-
um in in vitro release tests [56]. This finding could be
linked to the fact that the solubility of rhBMP-2 is
known to be dependent on ionic strength, but not salt
specific [57].
In a test soaking a collagen sponge with rhBMP-2,
incorporation of rhBMP-2 into the collagen matrix
reached levels of more than 90% especially at lower
rhBMP-2 concentration (Fig. 2). Incorporation is a
measure for the amount of rhBMP-2 which cannot be
expressed from the soaked matrix. It represents the
combination of rhBMP-2 absorption to collagen plus
the amount of rhBMP-2 dissolved in the liquid which
cannot be removed from the hydrated collagen mate-
rial even by rigorous squeezing. Protein incorporation
could be slightly increased by extending the waiting
time after impregnation. In addition, the amount of
unbound rhBMP-2 was reduced by the use of denser
collagen sponges which provide more binding sites
(Fig. 2).
Cross-linking of collagen, e.g. with formaldehyde,
led to reduced rhBMP-2 incorporation (Table 3) po-
tentially by two mechanisms. One mechanism could
be direct (physical) hindrance of bindingthe amount
Table 2
Factors in the preparation of a rhBMP-2/collagen sponge combi-
nation with potential impact on its performance
Preparation step Factors Potential impact
Manufacturing of
collagen sponge
Sponge mass
treatment, physical
(EtO, irradiation)
Interaction of rhBMP-2
and collagen (direct or
indirect by shift in pH
or ion concentration),
resorption process,
biocompatibility, in
vivo retention of
rhBMP-2, efficacy
Preparation of
the actual
of rhBMP-2
and collagen
Soak time
(pH, composition)
risk of microbial
total protein load,
in vivo retention
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1618
of free amino groups was reduced by 20%. Another
way cross-linking may impact incorporation is by
reducing swelling upon soaking, leading to less sur-
face area and binding sites. In studies referenced here,
it was found that the denaturation temperature of
collagen increased with cross-linking, indicating stron-
ger interactions between the collagen structures [39].
As was shown in vitro, collagenase resistance of the
sponge correlated with the degree of collagen formal-
dehyde exposure (Table 3). Formaldehyde treatment
also increased the tensile strength of the sponges in the
wet state considerably, thus improving the handling
properties [56].
Subsequent sterilization with ethylene oxide caused
a marked decrease in the amount of free amino groups
(approximately 40% of non-sterilized controls), and
the denaturation temperature was lower in sterilized
sponges than in non-sterilized material [38]; however,
binding of rhBMP-2 was only slightly affected (Table
3). Collagenase resistance of the sponge was slightly
reduced by sterilization (Table 3).
Raising the pH of the formulation from 5.0 to 7.0
or increasing the anion concentration led to an
increase in rhBMP-2 incorporation [39]. Both pH
and ion concentrations in the wet implant depend on
the rhBMP-2 formulation and the manufacturing
process of the collagen system, as well as the ratio
of protein formulation to collagen mass. In the
studies referenced here, protein precipitation was a
function of pH and salt concentration, but there
was no effect due to collagen alone [24]. In conclu-
sion of these studies, it was found to be important to
have as little variability in pH, anion concentration,
cross-linking and sponge mass as possible to achieve
consistent or maximum binding and to avoid rhBMP-
2 precipitation.
Studies have demonstrated a positive correlation
between the retention of rhBMP-2 upon implantation
and the osteoinductive activity in a subcutaneous
implantation model in the rat, i.e. systems with a
higher rhBMP-2 retention resulted in significantly
higher bone scores [3,58]. The in vivo release kinetics
I-rhBMP-2 from non-cross-linked/non-steril-
Fig. 2. rhBMP-2 incorporation as a function of collagen sponge mass per cm
(5 3.1 mg, 3.8 mg, 4.4 mg, 5.1 mg, 5.7 mg, n 6.3 mg)
and 550 min waiting time at 0.75 mg/ml rhBMP-2 (with permission from [39]).
Table 3
Effect of formaldehyde (ch2o) and ethylene oxide (eto) treatment on
free aminogroups, melting temperature T
, relative collagenase
degradation and rhBMP-2 incorporation of absorbable collagen
Free aminogroups (%) 100
90.5 40
(jC) 49.0 56.5 53.0
Relative degradability (%) 100
< 1 5
rhBMP-2 incorporation (%) 98.0 69.0 58.0
Set as 100%.
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1619
ized, formaldehyde-cross-linked/non-sterilized and
formaldehyde-cross-linked/ethylene oxide-sterilized
collagen sponges have been investigated to enlighten
the influence of processing conditions. The results
demonstrated small, but significant differences in the
in vivo retention of rhBMP-2 with a mean residence
time between 2.5 and 4 days. In this study, high
initial retention in vivo corresponded to high incor-
poration in vitro [38,58,59]. RhBMP-2 release rate
appeared to be higher for systems which were more
susceptible to collagenase in vitro and degraded
faster. Thus, cross-linking of the matrix (with form-
aldehyde) led to an increase in the in vivo persistence
and prolonged rhBMP-2 mean residence time and
secondary t
. Experiments with different rhBMPs
and modified rhBMP-2 revealed a positive correlation
of protein pI and in vivo retention in collagen
sponges [59]. The pI was found to significantly affect
the initial implant retention of rhBMPs, but not the
subsequent pharmacokinetics. A 100-fold difference
in the implant-retained dose could be observed
depending on the type of rhBMP implanted [59].
Additional studies indicated no significant depen-
dence of the rhBMP-2 pharmacokinetics on the
(limited) variability in the collagenase degradation
rate and the rhBMP-2 incorporation within sponges
cross-linked and sterilized under the same conditions.
In general, the amount of rhBMP-2 incorporated into
collagen sponges had a minimal effect on rhBMP-2
retention in vivo; these results suggest that the release
of rhBMP-2 in vivo is independent of the binding of
rhBMP-2 to the sponge in vitro, and may be diffu-
sion-controlled [60].
2.2. Cross-linking of collagen sponges with form-
aldehyde vapor in a convection chamber and by
dehydrothermal treatment (DHT)
Cross-linking of collagen materials can be achieved
by a variety of processes and chemical reactions (see
above). Instead of pretreatment of collagen or per-
forming chemical cross-linking by treating a collagen
sponge in liquid, two processes present the advan-
tage of preserving the initial collagen sponge struc-
ture after freeze-drying: treatment with formaldehyde
in the gas phase and thermal treatment in presence of
vacuum (dehydrothermal treatment). Cross-linking
by formaldehyde can be performed with vapor cre-
ated from a formalin solution in a diffusion chamber.
Formaldehyde should be homogeneously distributed
throughout the sponge within minutes [24]. Howev-
er, tortuosity of the path through a porous collagen
sponge, initial time necessary to equilibrate the
cross-linking chamber, and loss of formaldehyde
upon diffusion into the collagen matrix by chemical
reaction with the collagen material are potential
factors which could contribute to a concentration
gradient towards the center of the collagen material.
This phenomenon could result in heterogenous cross-
linking over the cross-section of a sponge and an
overall lower cross-linking degree with increasing
sponge thickness or change in pore geometry [24].
Collagen sponges from a lab-scale process using
convection as the driving force have also been
evaluated, where non-cross-linked collagen sponges
were treated in a formaldehyde atmosphere created
by sublimation of paraformaldehyde. 5 min treatment
resulted in material with substantially increased DSC
melting temperature, tensile strength, and resistance
to enzymatic digestion by collagenase. Longer cross-
linking times led to higher melting temperatures, a
plateau being reached at approx. 60 minutes treat-
ment time. Increasing the formaldehyde concentra-
tion 10-fold did not improve the outcome in an
experiment using treatment for 90 min. RhBMP-2
incorporation decreased with cross-linking, which
reflected previous data (see Section 2.1). Analysis
of formaldehyde residues in sponges treated in the
convection system for 60 min revealed that the
critical residual limit of 2000 ppm set by the British
Pharmacopeia for absorbable gelatin sponges could
be met by intensive aeration of the specimens for at
least one day [56].
To avoid the use of chemical cross-linkers, colla-
gen sponges can be physically cross-linked by dehy-
drothermal treatment, which is essentially heating
under vacuum. This procedure leads to the formation
of new amide bonds in protein-based materials
[42,43,61]. With increasing treatment temperatures
(80140 jC) as well as increasing treatment times
(15 days), collagen sponges treated in a lab-scale
system became more resistant to collagenase degra-
dation and to tearing stress. However, processing at
140 jC, especially for extended treatment times (5
days), resulted in a weakening of the material [47].
DHT does not lead to a marked increase in the
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1620
denaturation temperature [42]. As had been seen
with formaldehyde cross-linking, DHT led to a
decrease in rhBMP-2 incorporation [56]. Omura et
al. evaluated a dehydrothermally cross-linked (110
jC for 2 hours) composite sponge of fibrillar and
denatured atelocollagen ( = gelatin). After soaking
with rhBMP-2 solution, the implant resulted in intra-
membraneous ossification in a rat model. Inflamma-
tory and foreign body reactions in the surrounding
connective tissue were minimal [62].
2.3. Sterilization by gamma- or e-beam (beta-)
irradiation and DHT
As mentioned before, sterility of collageneous
matrices cannot be obtained by traditional methods
using heat, since the helices are irreversibly dam-
aged. The use of ethylene oxide for sterilization of
implants is restricted especially in Europe [63].
Therefore, gamma- and electron beam (beta-) irra-
diation were investigated as alternative methods to
sterilize collagen matrices [51]. Both treatments
(irradiation dose 25 kGy, the standard dose recom-
mended by the European Pharmacopeia) led to a
substantial increase in collagen degradation both in
non-cross-linked and DHT-cross-linked sponges, and
to a decrease in denaturation temperature and tensile
strength [56], which confirms results obtained with
other collageneous materials [55,6467]. The cur-
rent understanding of the effect of irradiation is that,
in a dose-dependent manner, peptide bonds are
cleaved and thus collagen molecules are damaged,
at doses which are currently used to sterilize bio-
medical materials. In the presence of (fluid) water,
polypeptide chain scission is accompanied by the
formation of intermolecular cross-links [46,66,68].
DHT can be considered as a sterilization process
with dry heat, using a time/temperature combination
different from the proposed standard conditions
[51]. In order to determine the potential of this
method, biological indicator paper strips loaded with
Bacillus subtilis spores were treated at 110 jC.
Sterility of these indicator strips was achieved
within one day, and likewise in collagen matrices
after the 5-day treatment needed for effective cross-
linking. These findings suggest that collagen materi-
als may be cross-linked and sterilized in one step by
DHT [56].
3. rhBMP-2 and Absorbable Collagen Sponge
3.1. Preparation of the application system
rhBMP-2 (INN: Dibotermin alfa) has been evalu-
ated in a number of clinical trials in combination with
an absorbable collagen sponge (ACS). This combi-
nation has recently gained approval by the U.S. Food
and Drug Administration to be used with a titanium
interbody spine fusion cage for anterior lumbar spinal
fusion (InFUSEk Bone Graft/LT-CAGEk Lumbar
Tapered Fusion Device, Medtronic Sofamor Danek),
and by the EMEA in Europe for treatment of acute
tibia fractures in adults, as an adjunct to standard care
using open fracture reduction and intramedullary nail
fixation (InductOsk, Wyeth Europa).
The active component of the implant consists of a
lyophilized rhBMP-2 preparation which is reconsti-
tuted with the supplied water for injection. The
absorbable collagen sponge is impregnated with pro-
tein solution, and after a waiting time of at least 15
min, the wet implant is removed from the container
and transferred to the prepared implantation site.
RhBMP-2/ACS is either used as an onlay implant
(tibia fracture), or, rolled and placed within an inter-
body fusion cage, implanted for anterior lumbar spine
The ACS was originally developed and approved
for application in hemostasis (Integra Life Sciences-
ILC, Plainsboro, NJ, USA). The manufacturing
involves lyophilization of a dispersion of bovine
Achilles tendon collagen, and cross-linking and ster-
ilization by chemical means [24,39]. The ACS acts as
a carrier for the rhBMP-2 and as a scaffold for new
bone formation [60]. The combination facilitates sur-
gical implantation and rhBMP-2 retention at the
treatment site [69].
3.2. Evaluation in animal models and clinical trials
Due to the high potency of this recombinant
factor, there has been significant success cited in
the literature for small animal species using
rhBMP-2 combined with a number of different mate-
rials. The successes in larger animal models of
fracture healing or segmental defects using a variety
of matrices have been more limited. This is likely due
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1621
to increased challenges of slower healing rates ob-
served for higher order animal species. However,
numerous animal models have demonstrated that
rhBMP-2 delivered in combination with ACS is
efficacious in a variety of applications. Below we
highlight a number of published preclinical and
clinical studies detailing the use of rhBMP-2/ACS
to bridge critical-sized defects, assure and accelerate
fracture repair, repair cranial defects, and promote
healing in spinal applications.
3.2.1. Fracture repair
There are approximately 5.6 million fractures
annually in the USA alone. Approximately 510%
of these exhibit some type of delayed or impaired
healing [70]. Therefore, there is a demonstrated
clinical need for treatments to assure successful
union of the bone gap and accelerate fracture
healing several growth and differentiation factors
are currently targeting this indication. Several pre-
clinical studies have demonstrated that rhBMP-2/
ACS accelerates healing in fracture models. To
demonstrate efficacy, these models compared
rhBMP-2 treated sites with untreated or matrix alone
(control sites), and showed a significant decrease in
healing time. For example, 0.86 mg rhBMP-2 de-
livered in ACS wrapped circumferentially around a
goat tibial fracture, demonstrated superior radio-
graphic healing scores at 6 weeks when compared
to buffer delivered in the sponges [71]. In the same
study, a significant increase in torsional toughness
( p = 0.02), and trends of increased torsional strength
and stiffness ( p = 0.09) was obtained when compared
with fracture controls. In the rabbit ulnar osteotomy
model, rhBMP-2 delivered in ACS demonstrated a
33% acceleration of healing compared to untreated or
buffer/ACS control limbs [26].
These preclinical studies and others paved the
way for evaluation of the rhBMP-2/ACS combina-
tion in pilot human clinical trials for open trauma
fractures. A prospective randomized, controlled, sin-
gle-blind pivotal study was subsequently performed
in 450 patients with open tibial fractures using the
combination of rhBMP-2/ACS plus standard of care
(including intramedullary nailing), or standard of
care (SOC) only. Two different doses were tested
(0.75 and 1.5 mg/ml rhBMP-2). Primary efficacy
endpoint was the proportion of patients who re-
quired a secondary intervention to promote fracture
healing within 12 months of definitive wound
closure (DWC). Secondary endpoints were healing
rate at 6 months, and acceleration of fracture union.
Additional endpoints were the combined clinical and
radiographic endpoint, time to prescription of sec-
ondary intervention, and number and invasiveness
of interventions actually performed. Treatment with
1.5 mg/ml rhBMP-2/ACS produced a significant
reduction in the rate of secondary interventions
prescribed, and in the invasiveness of the second
and subsequent interventions actually performed,
when compared to control (SOC only) patients.
The treatment was associated with a significantly
increased rate of clinical fracture healing at 6
months after DWC, with significant improvement
in fracture healing rates seen as soon as 10 weeks
after DWC, and further confirmed through 12
months after DWC. With respect to safety, data
presented up to date has not revealed any concerns.
For example, there was no difference in the occur-
rence of infections across treatment groups [69,72].
The rhBMP-2/ACS combination (InductOsk,
Wyeth Europa) received approval by the European
regulatory agency (EMEA) for treatment of acute
tibia fractures in adults, as an adjunct to standard
care using open fracture reduction and intramedul-
lary nail fixation, in 2002 [79].
3.2.2. Healing of critical size defects
Animal studies have also demonstrated efficacy in
using rhBMP-2/ACS to heal segmental defects that
are too large to regenerate spontaneously (critical-
sized defects). These defects may be in the long bones
or in the cranium. In one study, 35 Ag rhBMP-2/ACS
was found to be efficacious in healing critical-sized
defects in rabbit radii in 8 weeks compared to either
no treatment or the ACS alone [73]. Several other
studies have shown beneficial effects of combining
the rhBMP-2/ACS with allograft in canine femoral
defect models [74,75] with resulting greater new bone
callus formation compared to controls (bone graft or
ACS alone). Fig. 3 shows the repair of a large
segmental defect in a rhesus monkey radius using
the rhBMP-2/ACS combination. This example shows
that 32 weeks post-implantation, the defect has been
completely bridged by bone and stabilization is no
longer required.
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1622
3.2.3. Spinal fusion
In spinal surgery, approximately 500,000 grafting
procedures are performed annually in the US, of
which half are for spine fusion. The reported non-
union (pseudarthrosis) rate is 535% and clearly this
is another area that would greatly benefit from an
osteoinductive factor therapeutic alternative to tradi-
tional bone grafting [76]. Potential therapeutic areas
for the use of rhBMP-2 in the spine include postero-
lateral spinal fusion or interbody fusion. Lumbar spine
fusion has a relatively high rate of non-union (5
35%) and currently autograft bone is the standard
treatment. Overall the use of rhBMP-2 has shown
promise in a number of animal studies in increasing
fusion success rate and accelerating the time to fusion
compared to autograft [77]. For example in a rabbit
intertransverse process fusion model, treatment with
rhBMP-2/ACS or rhBMP-2/autograft achieved 100%
successful solid fusions whereas only 42% of the
autograft controls were fused [78]. Spinal fusion has
also been achieved in non-human primates with the
rhBMP-2/ACS system (posterolateral intertransverse
process spinal fusion); however, this study suggested
the need to mechanically protect the implanted mate-
rial against soft tissue compression to warrant success
at standard doses [79]. In the application of anterior
interbody fusion cages, the use of these cages as
vehicles for the delivery of bone regeneration factors
(to replace bone graft) has been explored widely [80].
Overall, use of rhBMP-2/ACS instead of bone graft in
the cages resulted in faster radiographic fusions and
greater fusion success rates in both sheep [81] and
monkey [80] models. RhBMP-2/ACS was also shown
superior to autograft in promoting spinal fusion when
placed in an allograft dowel cylinder in a rhesus
monkey model [82].
These preclinical studies, amongst others, led to
the use of rhBMP-2/ACS in human clinical trials for
spine. In a pilot study, Boden et al. [22] determined
that rhBMP-2/ACS-filled interbody fusion cages were
more successful in achieving arthrodesis in patients
compared to autograft with no adverse events. Anoth-
er multi-center study involving 279 patients with
degenerative disk disease compared the use of
rhBMP-2/ACS with autograft in an interbody fusion
procedure using fusion cages. This non-inferiority
Fig. 3. Radiological assessment of a rhesus monkey radius defect regenerated with rhBMP-2/ACS (reprinted from [13], with permission from
Elsevier) requires copyright permission since we previously published in Trends in Biotechnology [13].
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1623
study showed that after 2 years, the lumbar fusion rate
in the rhBMP-2/ACS group remained slightly higher
than that of the control group (94.5% vs. 88.7%); new
bone formation occurred in all investigational patients
[83]. It was concluded that lumbar fusion using
rhBMP-2 and a tapered titanium fusion cage can yield
a solid union and was as effective as the control
treatment, while having the benefit of eliminating
harvesting iliac crest bone graft [60,83]. In 2002, the
FDA approved the use of the rhBMP-2/ACS combi-
nation in a lumbar fusion device to treat degenerative
disk disease at a single involved lumbar spinal level in
skeletally mature patients (InFUSEk Bone Graft/LT-
CAGEk Lumbar Tapered Fusion Device, Medtronic
Sofamor Danek) [60]. Recently, an integrated analysis
of multiple clinical studies was performed using an
analysis of covariance to adjust for preoperative
variables in a total of 679 patients (277 patients
receiving cages combined with rhBMP-2/ACS and
402 patients with autograft from the iliac crest) [84].
The patients treated with rhBMP-2 had statistically
superior outcomes with regard to length of surgery,
blood loss, hospital stay, reoperation rate, median time
to return to work, and fusion rates at 6, 12, and 24
months. Oswestry Disability Index scores and the
Physical Component Scores and Pain Index of the
SF-36 scale also showed statistically superior out-
comes in the rhBMP-2 group [84].
3.2.4. Dental and craniofacial reconstruction
The use of rhBMP-2/ACS has a potentially im-
portant role in the repair of craniofacial defects,
induction of new bone in association with jaw
reconstruction and dental procedures. Patients with
significant loss of aleveolar ridge often require re-
generation of this bone to allow adequate support of
dental implants. The ability of rhBMP-2, in combi-
nation with ACS, to regenerate sufficient bone to
allow placement and osseointegration of titanium
implants has been demonstrated in canines [85,86]
and non-human primates [87] as well as in other
species. In a goat maxillary sinus model, rhBMP-2
impregnated in ACS was used as a substitute for
bone graft and demonstrated substantial bone forma-
tion in the maxillary sinus without adverse events
[88]. Negative control sinuses showed no new bone
growth. In the area of maxillofacial reconstruction,
segmental defects were created in rhesus monkey
mandibles and treated with rhBMP-2/ACS. In all
animals, the alveolar ridges were regenerated com-
pletely indicating that critical-sized hemi-mandibu-
lectomy defects can be regenerated using the rhBMP-
2/ACS combination [89].
These and other successful studies in animals
enabled the testing of rhBMP-2/ACS in humans for
maxillary sinus floor augmentation and alveolar ridge
augmentation. The 16-week open-label feasibility
study for maxillary floor sinus augmentation used
1.773.4 mg rhBMP-2 implanted in ACS [23]. In
this study, CT scans showed significant new bone
growth (mean height 8.51 mm) in all evaluable
patients and allowed the placement of dental implants.
Histologic examination of the core bone biopsies
obtained prior to dental implant placement confirmed
that new bone formed was normal. In a pilot study for
local alveolar ridge preservation and augmentation,
feasibility in using the rhBMP-2/ACS combination to
preserve the alveolar ridge after tooth extraction was
demonstrated [90]. New bone formation was observed
in all alveolar sockets filled with rhBMP-2/ACS;
however, since there was no control group, this study
demonstrated primarily safety of the combination
product and technical feasibility of the procedure.
The 3-year results from this study suggest that
rhBMP-2/ACS (0.43 mg/ml) can be safely used in
tooth extraction sites and in local ridge augmentation
procedures, and that endosseous dental implants
placed in bony areas treated with rhBMP-2/ACS are
stable and can be functionally restored without com-
plication [91].
4. Outlook
Future work on novel delivery systems for rhBMP-
2 for bone regeneration is focused on injectable
formats which would allow percutaneous applica-
tion without requiring an open procedure. These
injectable formats include versions of hyaluronic
acid gels [92], calcium phosphate pastes [93],
collagen-based delivery systems [94], temperature-
sensitive poly(N-isopropylacrylamide) polymers
[95], and poly (ethyleneglycol) (PEG)-based hydro-
gels [96].
Since bone is often formed via a transitional car-
tilage intermediate, the same factors that regenerate
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1624
bone might also be beneficial in cartilage repair. For
example, researchers have demonstrated that rhBMP-
2 has cartilage repair capabilities in the rabbit knee
cartilage full-thickness defect model [97100]. Fig.
4 shows an example of the hyaline cartilage regen-
erated by rhBMP-2/ACS in a rabbit model compared
to buffer/ACS which shows fibrous tissue repair.
Regeneration of cartilage is an exciting clinical
target since unlike bone, cartilage does not possess
self-repair properties. rhBMPs have also undergone
first evaluation in tendon repair in rats and rabbits
A different approach to novel bone graft substitutes
using the BMP concept is gene therapy, which can be
realized either by systems containing genetically
modified cells, or by integrating encoding DNA into
an osteoconductive scaffold [102104], e.g. collagen
sponges [105]. This strategy is based on the hypoth-
esis that gene transfer may lead to a prolonged
delivery of the signal triggering the formation of
new bone. A number of questions have yet to be
clarified, especially on safety and reliability of the
gene therapy concept [104]. Researchers have also
combined BMPs with other molecules for synergistic
effects, e.g. phosphate-diesterase inhibitors [106] and
transforming growth factor-beta1 [107]. The combi-
nation with angiogenic factors might be one approach
to further improve outcome by rapidly inducing
vascularization of newly formed tissue. As recently
reported, BMP-2 and bFGF can synergistically medi-
ate mesenchymal stem cell differentiation toward
bone formation and promote proliferation in vitro
and in two in vivo models [108]. Thus, several
perspectives for combinations of collagen with differ-
entiation or growth factors are outlined for future
The authors would like to thank M.L. Bell, B.
Perez, R. Riedel and J. Wozney for helpful dis-
cussions in the preparation of the manuscript.
[1] P.J. Boyne, Application of bone morphogenetic proteins in
the treatment of clinical oral and maxillofacial osseous de-
fects, J. Bone Jt. Surg., Am. Vol. 83-A (Suppl. 1 (Pt 2)) (2001)
[2] R.A. Kenley, Y. Kalvin, J. Abrams, R. Eyal, T. Turek, L.J.
Hollinger, J.O. Hollinger, Biotechnology and bone graft
substitutes, Pharm. Res. 10 (10) (1993) 13931401.
[3] S.R. Winn, H. Uludag, J.O. Hollinger, Sustained release em-
phasizing recombinant human bone morphogenetic protein 2,
Adv. Drug Deliv. Rev. 31 (1998) 303318.
[4] H. Lippert, Lehrbuch Anatomie, 4. Auflage, Urban und
Schwarzenberg, Munchen, 1996, p. 27.
[5] G. Thews, E. Mutschler, P. Vaupel, Anatomie Physiologie
Pathophysiologie des Menschen, 5. Auflage, Wissenschaft-
liche Verlagsgesellschaft, Stuttgart, 1999, p. 44.
[6] Datamonitor, US Bone Substitutes, Datamonitor, New York,
NY, USA, 1999.
[7] S.P. Bruder, B.S. Fox, Tissue engineering of bone-cell based
strategies, Clin. Orthop. 367-S (1999) S68S83.
[8] K.J.L. Burg, S. Porter, J.F. Kellam, Biomaterial develop-
ments for bone tissue engineering, Biomaterials 21 (2000)
[9] C.A. Kirker-Head, Potential applications and delivery strat-
Fig. 4. 3 3 mm Full thickness cartilage defect. A= ACS Control, B= ACS+ 5 Ag rhBMP-2 ( = 0.25 mg/ml concentration).
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1625
egies for bone morphogenetic proteins, Adv. Drug Deliv.
Rev. 43 (2000) 6592.
[10] M.R. Urist, Bone formation by autoinduction, Science 150
(1965) 893.
[11] M. Zhang, R.M. Powers, L. Wolfinbarger, Effect(s) of
the demineralization process on the osteoinductivity of
demineralized bone matrix, J. Periodontol. 68 (11) 1997,
pp. 10851092.
[12] S. Joschek, B. Nies, R. Krotz, A. Goepferich, Chemical and
physicochemical characterization of porous hydroxyapatite
ceramics made of natural bone, Biomaterials 21 (2000)
[13] R.H. Li, J.M. Wozney, Delivering on the promise of bone
morphogenetic proteins, Trends Biotechnol. 19 (7) (2001)
[14] J. Glowacki, Tissue response to bone-derived materials, in:
M.B. Habal, A.H. Reddi (Eds.), Bone Grafts: From Basic
Science to Clinical Application, W.B. Saunders, Philadel-
phia, PA, 1992, pp. 8492.
[15] M.R. Urist, Bone morphogenetic protein in biology and med-
icine, in: T.S. Lindholm (Ed.), Bone Morphogenetic Proteins:
Biology, Biochemistry and Reconstructive Surgery (Tissue
Engineering), Academic Press, San Diego, CA, USA,
1996, pp. 727.
[16] E.A. Wang, V. Rosen, P. Cordes, R.M. Hewick, M.J. Kitz,
D.P. Luxenberg, P.S. Sibley, J.M. Wozney, Purification and
characterization of other distinct bone-inducing factors, Proc.
Natl. Acad. Sci. U. S. A. 85 (1988) 94849488.
[17] J.M. Wozney, V. Rosen, A.J. Celeste, L.M. Mitsock, M.J.
Whitters, R.W. Kriz, R.M. Hewick, E.A. Wang, Novel reg-
ulators of bone formation: molecular clones and activities,
Science 242 (4885) (1988) 15281534.
[18] J.M. Wozney, V. Rosen, Bone morphogenetic protein and
bone morphogenetic protein gene family in bone formation
and repair, Clin. Orthop. 346 (1998) 2637.
[19] K. Bessho, Y. Konishi, S. Kaihara, K. Fujimura, Y. Okubo, T.
Iizuka, Bone induction by Eschericia coli-derived recombi-
nant human bone morphogenetic protein-2 compared with
Chinese hamster ovary cell-derived recombinant human bone
morphogenetic protein-2, Br. J. Oral Maxillofac. Surg. 38 (6)
(2000) 645649.
[20] N.R. Kubler, J.F. Reuther, G. Faller, T. Kirchner, R. Ruppert,
W. Sebald, Inductive properties of recombinant human BMP-
2 produced in a bacterial expression system, Int. J. Oral
Maxillofac. Surg. 27 (4) (1998) 305309.
[21] T.K. Sampath, J.C. Maliakal, P.V. Hauschka, W.K. Jones, H.
Sasak, R.F. Tucker, K.H. White, J.E. Coughlin, M.M. Tuck-
er, R.H. Pang, et al., Recombinant human osteogenic protein-
1 (hOP-1) induces new bone formation in vivo with a spe-
cific activity comparable with natural bovine osteogenic pro-
tein and stimulates osteoblast proliferation and differentiation
in vitro, J. Biol. Chem. 267 (28) (1992) 2035220362.
[22] S.D. Boden, T.A. Zdeblick, H.S. Sandhu, S.E. Heim, The use
of rhBMP-2 in interbody fusion cages: definitive evidence of
osteoinduction in humans: a preliminary report, Spine 25 (3)
(2000) 376381.
[23] P.J. Boyne, R.E. Marx, M. Nevins, G. Triplett, E. Lazaro,
L.C. Lilly, M. Alder, P. Nummikoski, A feasibility study
evaluating rhBMP-2/absorbable collagen sponge for maxil-
lary sinus floor augmentation, Int. J. Periodontics Restor.
Dent. 17 (1) (1997) 1125.
[24] W. Friess, Drug Delivery Systems Based on Collagen,
Berichte aus der Pharmazie, Shaker Verlag, Aachen,
2000, pp. 94137.
[25] J.H. Brekke, J.M. Toth, Principles of tissue engineering ap-
plied to programmable osteogenesis, J. Biomed. Mater. Res.
(Applied Biomaterials) 43 (1998) 380398.
[26] M.L. Bouxsein, T.J. Ture, C.A. Blake, D. DAugusta, X. Li,
M. Stevens, H.J. Seeherman, J.M. Wozney, Recombinant
human bone morphogenetic protein-2 accelerates healing in
a rabbit ulnar osteotomy model, J. Bone Jt. Surg. 83-A (8)
(2001) 12191230.
[27] J.E. Babensee, L.V. McIntire, A.G. Mikos, Growth factor
delivery for tissue engineering, Pharm. Res. 17 (5) (2000)
[28] W. Friess, Collagen-biomaterial for drug delivery, Eur. J.
Pharm. Biopharm. 45 (1998) 113136.
[29] M. Chvapil, Collagen sponge: theory and practice of medical
applications, J. Biomed. Mater. Res. 11 (1977) 721741.
[30] M. Geiger, W. Friess, Collagen sponge implants: applica-
tions, characteristics and evaluation: Part I, Pharm. Tech.
Europe 14 (2002 February) 4856.
[31] M. Geiger, W. Friess, Collagen sponge implants: applica-
tions, characteristics and evaluation: Part II, Pharm. Tech.
Europe 14 (2002 April) 5866.
[32] S.D. Gorham, Collagen, in: D. Byrom (Ed.), Biomaterials.
Novel Materials from Biological Sources, Stockton Press,
New York, 1991, pp. 55122.
[33] N. Dagalakis, J. Flink, P. Stasikelis, J.F. Burke, I.V. Yannas,
Design of an artificial skin: Part III. Control of pore structure,
J. Biomed. Mater. Res. 14 (1980) 511528.
[34] P. Tewes-Schwarzer, Manufacturing principles of freeze
dried collagen sponges: characteristics and applications, in:
L. Rey, J.C. May (Eds.), Freeze Drying/Lyophilization Phar-
maceutical and Biological Products, Drugs and the Pharma-
ceutical Sciences, vol. 96, Marcel Dekker, New York, 1999.
[35] E. Khor, Methods for the treatment of collagenous tissues for
bioprostheses, Biomaterials 18 (1997) 95105.
[36] K. Tomihata, K. Burczak, K. Shiraki, Y. Ikada, Cross-linking
and biodegradation of native and denatured collagen, in: S.
Shalaby, Y. Idada, R. Langer, J. Williams (Eds.), Polymers of
Biological and Biomedical Significance, ACS Symposium
Series, vol. 540, ACS, Washington, DC, 1994, pp. 275286.
[37] P.B. van Wachem, M.J.A. van Luyn, L.H.H. Olde Damink,
P.J. Dijkstra, J. Feijen, P. Nieuwenhuis, Biocompatibility and
tissue regenerating capacity of cross-linked dermal sheep
collagen, J. Biomed. Mater. Res. 28 (1994) 353363.
[38] W. Friess, H. Uludag, S. Foskett, R. Biron, Bone regeneration
with rhBMP-2 using absorbable collagen sponges: Influence
of processing on ACS characteristics and formulation, Pharm.
Dev. Technol. 4 (3) (1999) 387396.
[39] W. Friess, H. Uludag, S. Foskett, R. Biron, C. Sargeant, Char-
acterization of absorbable collagen sponges as rhBMP-2
carriers, Int. J. Pharm. 187 (1999) 9199.
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1626
[40] R.J. Ruderman, C.W.R. Wade, W.D. Shepard, F. Leonard,
Prolonged resorption of collagen sponges: vapor-phase treat-
ment with formaldehyde, J. Biomed. Mater. Res. 7 (1973)
[41] R. Zeeman, P.J. Dijkstr, P.B. van Wachem, M.J.A. van Luyn,
M. Hendriks, P.T. Cahalan, J. Feijen, Cross-linking and mod-
ification of dermal sheep collagen using 1,4-butanediol-digly-
cidyl-ether, J. Biomed. Mater. Res. 46 (3) (1999) 424433.
[42] S.D. Gorham, N.D. Light, A.M. Diamond, M.J. Willins, A.J.
Bailey, T.J. Wess, N.J. Leslie, Effect of chemical modifica-
tions on the susceptibility of collagen to proteolysis: II. De-
hydrothermal cross-linking, Int. J. Biol. Macromol. 14
(1992) 129138.
[43] K.S. Weadock, E.J. Miller, E.L. Keuffel, M.G. Dunn, Effect
of physical cross-linking methods on collagen fiber durabil-
ity in proteolytic solutions, J. Biomed. Mater. Res. 32 (1996)
[44] K.S. Weadock, R.M. Olson, F.H. Silver, Evaluation of colla-
gen cross-linking techniques, Biomater. Med. Dev. Artif. Or-
gans 11 (4) (198384) 293318.
[45] K.S. Weadock, E.J. Miller, L.D. Bellincampi, J.P. Zawadsky,
M.G. Dunn, Physical cross-linking of collagen fibers: com-
parison of ultraviolet irradiation and dehydrothermal treat-
ment, J. Biomed. Mater. Res. 29 (1995) 13731379.
[46] D.T. Cheung, N. Perelman, D. Tong, M.E. Nimni, The effect
of y-irradiation on collagen molecules, isolated a-chains, and
cross-linked native fibers, J. Biomed. Mater. Res. 24 (1990)
[47] M. Geiger, W. Friess, Cross-linking of porous collagen carrier
systems by dehydrothermal treatment, Proc. 3rd World Meet-
ing APV/APGE, Berlin, 3/6 April, 2000, Arbeitsgemein-
schaft fur Pharmazeutische Verfahrenstechnik e.V., Mainz,
2000, pp. 239240.
[48] W. Friess, G. Lee, Basic thermoanalytical studies of insolu-
ble collagen matrices, Biomaterials 17 (1996) 22892294.
[49] L.H.H. Olde Damink, P.J. Dijkstra, M.J.A. van Luyn, P.B.
van Wachem, P. Nieuwenhuis, J. Feijen, In vitro degradation
of dermal sheep collagen cross-linked using a water-soluble
carbodiimide, Biomaterials 17 (1996) 679684.
[50] European Pharmacopeia, 4th ed., Council of Europe, Stras-
bourg, Cedex, France, 2002.
[51] The European Agency for the Evaluation of Medicinal Prod-
ucts (EMEA), Decision Trees for the Selection of Sterilisa-
tion Methods, CPMP/QWP/054/98 corr, 2000.
[52] R. Peacock, Ethylene oxide sterilization: the way ahead,
Med. Device Technol., (1999 July/August) 2425.
[53] A.J. Hamer, I. Stockley, R.A. Elson, Changes in allograft
bone irradiated at different temperatures, J. Bone Jt. Surg.,
Br. 81-B (1999) 342344.
[54] S. Hepple, A. Hamer, I. Stockley, A.P. Weetman, Early im-
mune response to frozen, gamma-irradiated bone allograft,
J. Bone Jt. Surg., Br. 81-B (Suppl. II) (1999) 151.
[55] A. Salehpour, D.L. Butler, F.S. Proch, H.E. Schwartz, S.M.
Feder, C.M. Doxey, A. Ratcliffe, Dose dependent response of
gamma irradiation on mechanical properties and related bio-
chemical composition of goat bone patellar tendon bone al-
lografts, J. Orthop. Res. 13 (6) (1995) 898906.
[56] M. Geiger, Porous Collagen/Ceramic Composite Carriers for
Bone Regeneration Using Recombinant Human Bone Mor-
phogenetic Protein-2 (rhBMP-2), Dissertation Universitaet
Erlangen-Nuernberg, Germany, 2001.
[57] S.E. Abbatiello, T.J. Porter, Anion-mediated precipitation of
recombinant human bone morphogenetc protein (rhBMP-2)
is dependent upon the heparin-binding N-terminal region,
Protein Sci. 6 (Suppl. 2) (1997) 99.
[58] H. Uludag, T. Gao, T.J. Porter, W. Friess, J.M. Wozney,
Delivery systems for BMPs: factors contributing to protein
retention at an application site, J. Bone Jt. Surg., Am. Vol.
83-A (Pt 2) (2001) 128135(Suppl).
[59] H. Uludag, W. Friess, D. Williams, T. Porter, G. Timony, D.
DAugusta, C. Blake, R. Palmer, B. Biron, J.M. Wozney,
rhBMP-collagen sponges as osteoinductive devices: effects
of in vitro sponge characteristics and protein pI on in vivo
rhBMP pharmacokinetics, Ann. N.Y. Acad. Sci. 875 (1999)
369378(Bioartificial Organs II).
[60] FDA information on InFUSEk, CDRH, PMA No. P000058
(2002), available from:
P000058.html (08 May 2003).
[61] J. Bello, H. Riese-Bello, Liaisons transversales de la gelatine
sous linfluence de la chaleur, Sci. Ind. Photogr. 19 (10)
(1958) 361364.
[62] S. Omura, N. Miziki, R. Kawabe, S. Ota, S. Kobayashi, K.
Fujita, A carrier for clinical use of recombinant human
BMP-2: dehydrothermally cross-linked composite of fibril-
lar and denatured atelocollagen sponge, Int. J. Oral Maxil-
lofac. Surg. 27 (1998) 129134.
[63] EMEA, Note for Guidance on Limitations to the Use of
Ethylene Oxide in the Manufacture of Medicinal Products,
CPMP/QWP/159/01, 2001.
[64] T.J. Rasmussen, S.M. Feder, D.L. Butler, F.R. Noyes, The
effects of 4 Mrad of gamma-irradiation on the initial mechan-
ical properties of bone-patellar tendon-bone grafts, Arthro-
scopy 10 (2) (1994) 188197.
[65] L.H.H. Olde Damink, P.J. Dijkstra, M.J.A. van Luyn, P.B.
van Wachem, P. Nieuwenhuis, J. Feijen, Changes in the me-
chanical properties of dermal sheep collagen during in vitro
degradation, J. Biomed. Mater. Res. 29 (1995) 139147.
[66] A.J. Bailey, D.N. Rhodes, Irradiation-induced cross-linking
of collagen, Radiat. Res. 22 (1964) 606621.
[67] E.M. Noah, J. Chen, X. Jiao, I. Heschel, N. Pallua, Impact of
sterilization on the porous design and cell behavior in colla-
gen sponges prepared for tissue engineering, Biomaterials 23
(14) (2002) 28552861.
[68] R.A. Grant, R.W. Cox, C.M. Kent, The effects of gamma
irradiation on the structure and reactivity of native and cross-
linked collagen fibres, J. Anat. 115 (1973) 2943.
[69] The European Agency for Evaluation of Medicinal Products
(EMEA), European Public Assessment Report (EPAR) on
InductOsk, CPMP/3188/02 (2002), available from: http://
inductos.htm (08 May 2003).
[70] C.M. RP-651147, Orthopedic and musculoskeletal markets:
biotechnology and tissue engineering, Med. Data Int., (2000)
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1627
[71] R.D. Welch, A.L. Jones, R.W. Bucholz, C.M. Reinert, J.S.
Tjia, W.A. Pierce, J.M. Wozney, X.J. Li, Effect of recombi-
nant human bone morphogenetic protein-2 on fracture heal-
ing in a goat tibial fracture model, J. Bone Miner. Res. 13 (9)
(1998) 14831490.
[72] BESTT (The BMP-2 Evaluation in Surgery for Tibial Trau-
ma Study Group), Recombinant human bone morphogenetic
protein-2 for open tibial fractures: a prospective, controlled,
randomized study in 450 patients, J. Bone Jt. Surg. 84A
(2002) 21232134.
[73] J. Hollinger, J.M. Schmitt, D.C. Buck, R. Shannon, S.P. Joh,
H.D. Zegzula, J. Wozney, Recombinant human bone mor-
phogenetic protein-2 and collagen for bone regeneration,
J. Biomed. Materi. Res. 43 (4) (1998) 356364.
[74] A. Zabka, G.E. Pluhar, R.B. Edwards III, P.A. Manley, K.
Hayashi, J.P. Heiner, V.L. Kalscheur, H.J. Seeherman, His-
tomorphometric description of allograft bone remodeling and
union in a canine segmental femoral defect model: a compar-
ison of rhBMP-2, cancellous bone graft, and absorbable col-
lagen sponge, J. Orthop. Res. 19 (2) (2001) 318327.
[75] G. Pluhar, P.A. Manley, J.P.R.V. Heiner Jr., H.J. Seeherman,
M.D. Markel, The effect of recombinant human bone mor-
phogenetic protein-2 on femoral reconstruction with an in-
tercalary allograft in a dog model, J. Orthop. Res. 19 (2)
(1999) 308317.
[76] S.C. Ludwig, J.M. Kowalski, S.D. Boden, Osteoinductive
bone graft substitutes, Eur. Spine J. 9 (Suppl. 1) (2000)
[77] S. Yoon, S. Boden, Osteoinductive molecules in orthopae-
dics: basic science and preclinical studies, Clin. Orthop. Re-
lat. Res. 395 (2002) 3343.
[78] J.H. Schimandle, S.D. Boden, W.C. Hutton, Experimental
spinal fusion with recombinant human bone morphogenetic
protein-2, Spine 20 (1995) 13261337.
[79] G.J. Martin Jr., S.D. Boden, M.A. Marone, M.A. Marone,
P.A. Moskovitz, Posterolateral intertransverse process spinal
fusion arthrodesis with rhBMP-2 in a non-human primate-
important lessons learned regarding dose, carrier, and safety,
J. Spinal Disord. 12 (1999) 179186.
[80] S.D. Boden, G.J. Martin Jr., W.C. Horton, T.L. Truss, H.S.
Sandhu, Laparoscopic anterior spinal arthrodesis with
rhBMP-2 in a titanium interbody threaded cage, J. Spinal
Disord. 11 (1998) 95101.
[81] H. Sandhu, J.M. Toth, A.D. Diwan, H.B. Seim III, L.E. Ka-
nim, J.M. Kabo, A.S. Turner, Histologic evaluation of the
efficacy of rhBMP-2 compared with autograft bone in sheep
spinal anterior interbody fusion, Spine 27 (2002) 567575.
[82] B.P. Hecht, J.S. Fischgrund, H.N. Herkowitz, L. Penman,
J.M. Toth, A. Shirkhoda, The use of recombinant human bone
morphogenetic protein 2 (rhBMP-2) to promote spinal fusion
in a nonhuman primate anterior interbody fusion model,
Spine 24 (1999) 629636.
[83] J.K. Burkus, M.F. Gornet, C.A. Dickman, T.A. Zdeblick,
Anterior lumbar interbody spine fusion using rhBMP-2 with
tapered interbody cages, J. Spinal Disord. Tech. 15 (2002)
[84] J.K. Burkus, S.E. Heim, M.F. Gornet, T.A. Zdeblick, Is IN-
FUSE bone graft superior to autograft bone? An integrated
analysis of clinical trials using the LT

CAGE lumbar tapered

fusion device, J. Spinal Disord. 16 (2) (2003) 113122.
[85] T.J. Sigurdsson, E. Fu, D.N. Tatakis, M.D. Rohrer, U.M.
Wikesjo, Bone morphogenetic protein-2 for peri-implant
bone regeneration and osseointegration, Clin. Oral Implants
Res. 8 (5) (1997) 367374.
[86] T.J. Sigurdsson, L. Nygaard, D.N. Tatakis, E. Fu, T.J. Turek,
L. Jin, J.M. Wozney, U.M. Wikesjo, Periodontal repair in
dogs: evaluation of rhBMP-2 carriers, Int. J. Periodontics
Restor. Dent. 16 (1996) 525537.
[87] O. Hanisch, D.N. Tatakis, M.D. Rohrer, P.S. Wohrle, J.M.
Wikesjo, U.M. Wikesjo, Bone formation and osseointegration
stimulated by rhBMP-2 following subantral augmentation
procedures in nonhuman primates, Int. J. Oral Maxillofac.
Implants 12 (1997) 785792.
[88] M. Nevins, C. Kirker-Head, M. Nevins, J. Wozney, R. Palm-
er, D. Graham, Bone formation in the goat maxillary sinus
induced by absorbable collagen sponge implants impreg-
nated with recombinant human bone morphogenetic pro-
tein-2, Int. J. Periodontics Restor. Dent. 16 (1) (1996) 819.
[89] P. Boyne, Animal studies of application of rhBMP-2 in max-
illofacial reconstruction, Bone 19 (Suppl. 1) (1996) 83S92S.
[90] T.H. Howell, J. Fiorellini, A. Jones, M. Alder, P. Nummikos-
ki, M. Lazaro, L. Lilly, D. Cochran, A feasibility study eval-
uating rhBMP-2/absorbable collagen sponge device for local
alveolar ridge preservation or augmentation, Int. J. Periodon-
tics Restor. Dent. 17 (1997) 124139.
[91] D.L. Cochran, A.A. Jones, L.C. Lilly, J.P. Fiorellini, H. Ho-
well, Evaluation of recombinant human bone morphogenetic
protein-2 in oral applications including the use of endosseous
implants: 3-year results of a pilot study in humans, J. Perio-
dontol. 71 (8) (2000) 12411257.
[92] H.D. Kim, D.A. DAugusta, R. Li, M. Bouxsein, C. Blake,
K. Ammirati, C. Luppen, H. Seeherman, J. Li, M. Stevens, J.
Golden, J. Wozney, Biodistribution and efficacy of rhBMP-2
in hyaluronan-based carriers, 26th International Symposium
of the Controlled Release Society, Controlled Release Soci-
ety, Minneapolis, MN, 1999, pp. 100101.
[93] H. Seeherman, R. Li, C. Blake, M.L. Bouxsein, J. Cooper, D.
Gavin, J. Li, J. Wozney, A single injection of rhBMP-2/cal-
ciumphosphate paste given one week after surgery accelerates
osteotomy healing by 50% in a non-human primate fibula
osteotomy model, Transactions of the 48th Annual Meeting
of the Orthopaedic Research Society, Dallas, TX, 27, 2002,
[94] R.H. Li, H. Seeherman, M. Bouxsein, D. DAugusta, C.
Blake, K. Ammirati, C. Luppen, M. Stevens, J. Li, H.
Kim, J. Wozney, Acceleration of fracture healing with
rhBMP-2 delivered in an injectable gelfoam delivery system,
Society for Biomaterials, Sixth World Biomaterials Congress
Transactions, Society for Biomaterials USA, Minneapolis,
MN, 2000, p. 560.
[95] H. Uludag, B. Norrie, N. Kousinioris, T. Gao, Engineering
temperature-sensitive poly (N-isopropylacrylamide) poly-
mers as carriers of therapeutic proteins, Biotechnol. Bioeng.
3 (6) (2001) 510521.
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1628
[96] M.P. Lutolf, F.E. Weber, H.G. Schmoekel, J.C. Schense, T.
Kohler, R. Mueller, J.A. Hubbell, Nat. Biotechnol. 21 (2003)
[97] R.S. Sellers, D. Peluso, E.A. Morris, The effect of recombi-
nant human bone morphogenetic protein-2 (rhBMP-2) on the
healing of full-thickness defects of articular cartilage, J. Bone
Jt. Surg., Am. 79 (1997) 14521463.
[98] R.S. Sellers, R. Zhang, S.S. Glasson, H.D. Kim, D. Peluso,
D.A. DAugusta, K. Beckwith, E.A. Morris, Repair of artic-
ular cartilage defects one year after treatment with recombi-
nant human bone morphogenetic protein-2 (rhBMP-2),
J. Bone Jt. Surg., Am. 82 (2) (2000) 151160.
[99] S. Glasson, et al., Efficacy of different matrices in rabbit full-
thickness cartilage defects, 46th Annual Meeting of the Or-
thopaedic Research Society, Orlando, FL. 2000.
[100] M. Grgic, M. Jelic, V. Basic, N. Basic, M. Pecina, S. Vuki-
cevic, Regeneration of articular cartilage defects in rabbits by
osteogenic protein-1 (bone morphogenetic protein-7), Acta
Med. Croat. 51 (1) (1997) 2327.
[101] P. Aspenberg, C. Forslund, Tendon healing stimulated with
BMPs, Meeting Abstract: First European Conference on Bone
Morphogenetic Proteins, Oct. 711, Zagreb, Croatia. 1998.
[102] S.D. Boden, Bioactive Factors for bone tissue engineering,
Clin. Orthop. 367-S (1999) S84S94.
[103] J. Bonadio, Tissue engineering via local gene delivery: up-
date and future prospects for enhancing the technology, Adv.
Drug Deliv. Rev. 44 (2000) 185194.
[104] S.R. Winn, Y. Hu, C. Sfeir, J.O. Hollinger, Gene therapy
approaches formodulating bone regeneration, Adv. Drug De-
liv. Rev. 44 (2000) 121138.
[105] J.Y. Lee, D. Musgrave, D. Pelinkovic, K. Fukushima, J.
Cummins, A. Usas, P. Robbins, F.H. Fu, J. Huard, Effect
of bone morphogenetic protein-2-expressing muscle-derived
cells on healing of critical-sized bone defects in mice,
J. Bone Jt. Surg. 83-A (7) (2001) 10321039.
[106] H. Horiuchi, N. Saito, T. Kinoshita, S. Wakabayashi, N.
Yotsumoto, K. Takaoka, Effect of phosphodiesterase inhib-
itor-4, rolipram, on new bone formations by recombinant
human bone morphogenetic protein-2, Bone 30 (4) (2002)
[107] O. Matsaba, L.N. Ramoshebi, J. Crooks, U. Ripamonti,
Transforming growth factor-beta1 supports the rapid mor-
phogenesis of heterotopic endochondral bone initiated by
human osteogenic protein-1 via the synergistic upregulation
of molecular markers, Growth Factors 19 (2) (2001) 7386.
[108] S. Akita, M. Fukui, T. Fujii, K. Akino, Interaction Between
Mesenchymal Stem Cells and Osteogenic Cytokines in Vitro
and in Vivo, ETRS (European Tissue Repair Society) Focus
Meeting Nice, Sep. 1214, 2002, ETRS Bulletin vol. 9
(2),Oxford, UK.
M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 16131629 1629