www.elsevier.com/locate/addr

Advanced Drug Delivery Reviews 55 (2003) 1613–1629

Collagen sponges for bone regeneration with rhBMP-2

M. Geigera,*, R.H. Lib, W. Friessc

aDrug Product Development, Wyeth BioPharma, One Burtt Road, Andover, MA 01810, USAbWyeth Research, 35 Cambridge Park Drive, Cambridge, MA 02140, USA

cDepartment fuer Pharmazie, Lehrstuhl fuer Pharmazeutische Technologie und Biopharmazie,

Ludwig-Maximilians-Universitaet Muenchen, Butenandtstr. 5–13, 81377 Munich, Germany

Received 17 July 2003; accepted 26 August 2003

Abstract

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

Contents

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.

doi:10.1016/j.addr.2003.08.010

* Corresponding author. Tel.: +1-978-247-1487.

E-mail address: mgeiger@wyeth.com (M. Geiger).

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–16291614

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 Bone represents a complex system of a variety of cell

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.

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 13–30% 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

(20–35%) [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) 1613–1629 1615

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 [7–9].

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 patient’s bone marrow prior to

application.

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 1–15) [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 [13,22–25]. 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).

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

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–16291616

1.3. General requirements for carriers/delivery

systems

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].

Fig. 1. Retention of rhBMP-2 in the rabbit ulna osteotomy model, impla

permission from Elsevier) requires copyright permission since we previou

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 125I-rhBMP-

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 125I-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

delivery.

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

[13].

nted with and without a collagen sponge (reprinted from [13], with

sly published in Trends in Biotechnology [13].

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–1629 1617

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 NeOsteoRbovine 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

regeneration

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

[29–31], 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 [37–40], 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

[53–55].

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) 1613–16291618

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

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

. Cross-linking

(formaldehyde

treatment, physical

methods). Sterilization

(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

implant

(combination

of rhBMP-2

and collagen

sponge)

. Soak time

. Protein

concentration. Buffer

formulation

(pH, composition). RhBMP-2

risk of microbial

contamination;

total protein load,

in vivo retention

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 0.1–0.2 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 binding—the amount

Fig. 2. rhBMP-2 incorporation as a function of collagen sponge mass per cm2 (5 3.1 mg, 3.8 mg, 4.4 mg, 5.1 mg, 5.7 mg, n 6.3 mg)

and 5–50 min waiting time at 0.75 mg/ml rhBMP-2 (with permission from [39]).

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–1629 1619

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

Table 3

Effect of formaldehyde (ch2o) and ethylene oxide (eto) treatment on

free aminogroups, melting temperature Tm, relative collagenase

degradation and rhBMP-2 incorporation of absorbable collagen

sponges

Non-ch2o/

non-eto

ch2o/

non-eto

ch2o/

eto

Free aminogroups (%) 100a 90.5 40

Tm (jC) 49.0 56.5 53.0

Relative degradability (%) 100a < 1 5

rhBMP-2 incorporation (%) 98.0 69.0 58.0

a Set as 100%.

(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

of 125I-rhBMP-2 from non-cross-linked/non-steril-

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–16291620

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 t1/2. 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

(80–140 jC) as well as increasing treatment times

(1–5 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) 1613–1629 1621

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 [55,64–67]. 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

(ACS)

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

fusion.

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) 1613–16291622

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 5–10%

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.

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) 1613–1629 1623

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 5–35% 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

M. Geiger et al. / Advanced Drug Delivery Reviews 55 (2003) 1613–16291624

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

1.77–3.4 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

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) 1613–1629 1625

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 [97–100]. 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

[101].

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 [102–104], 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

development.

Acknowledgements

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.

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