Collagen sponges for bone regeneration with rhBMP-2

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.

Introduction

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 osteomyelitis. Improper osseous healing has potentially devastating consequences, ranging from disfigurement to loss of function and loss of limb [2]. In cases of large bone defects where bone is not expected to regenerate spontaneously, clinicians commonly 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 populated with material closely resembling the original bone prior to damage.

One approach to restore form and function is substituting bony material through the use of permanent orthopaedic implants made of metals, ceramics, polymers (e.g. polyethylene) or composite materials. Restoring 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 surrounding 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 compromise 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 delicate 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 technique yields the most predictable results, however bears considerable risks (donor site pain and morbidity, infection, extra blood loss, and higher cost due to longer operating times). Additionally, autografting is ineffective when the defect volume exceeds the volume 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 (allograft), which has additional disadvantages, including potential host reaction, limited supply, excessive resorption, 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 annually [6]. Spinal applications now account for almost 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, respectively, in the future [6]. Therefore there is a growing need to provide alternatives to traditional bone grafting. 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 differentiating 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], [8], [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 challenges in the regulatory approval process [7]. One example for “cellular implants” is Collagraft® (NeuColl, 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 recently been reviewed [8], [11]. Bone graft substitutes manufactured 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 synthetic, have been tested as bone graft substitutes (reviewed in [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 mimicking the processes of hard tissue formation in the embryonic state [15]. Among the most potent osteoinductive factors yet discovered are bone morphogenetic proteins (BMPs).

In 1965, Urist implanted demineralized bone matrix at intramuscular sites in rodents and rabbits [10]. The sequence of events which followed was reminiscent of the bone development process in embryos and of post-natal endochondral ossification [16], [17]. The term “bone morphogenetic protein” (BMP) was introduced to describe the substance(s) in the demineralized 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 transfected into Chinese hamster ovary cells and to Escherichia coli cells [19], [20]. Among the recombinant 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], [23], [24], [25]. Clinical results have recently led to regulatory approval of both OP-1 (Osigraft™, Howmedica International S. de R.L., Raheen, Limerick, Ireland) and rhBMP-2 (InFUSE™ Bone Graft/LT-CAGE™ Lumbar Tapered Fusion Device, Medtronic Sofamor Danek, Memphis, TN, USA; InductOs™, Wyeth Europa, Maidenhead/Berkshire, UK) for some of these applications by governmental agencies (see Section 3.2).

It has been shown that rhBMP-2 requires combination 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, biodegradability, 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 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 significantly 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 induction [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 compromised (avascular) sites, BMP clearance might be faster than the bone-induction response of the host. In these cases a slow-release system may be required. It has further to be noted that different animal species may have varying optimum release profiles [13].

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(α-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 composites, e.g. dentine matrix powder/chondroitin-6-sulfate/type I collagen, TCP or coralline HA/type IV collagen, and poly(α-hydroxy acid)/ carboxymethyl-cellulose or methylcellulose. BMPs have also been used in combination with titanium mesh and other non-degradable metallic orthopaedic implants. Delivery 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; Osigraft®, Howmedica International S. de R.L.) and NeOsteo® bovine BMP mixture (Sulzer Orthopaedics Biologics, Wheat Ridge, CO, USA) have also utilized collagen-based carriers.