Demineralized Bone Matrix and Spinal Arthrodesis Kenneth J.H

The Spine Journal 5 (2005) 217S–223S

Demineralized bone matrix and spinal arthrodesis Kenneth J.H. Lee, MD, Jonathan G. Roper, MD, Jeffrey C. Wang, MD* Department of Orthopaedic Surgery, University of California at Los Angeles, School of Medicine, 1250 16th Street, 7th Floor, Tower #745, Santa Monica, CA 90404, USA

Abstract Spinal fusion is a gold-standard treatment for many disorders of the spine with autogenous bone graft as the gold-standard source for augmenting fusion. However, the morbidity and limitations of autogenous bone grafting has motivated the search for bone graft alternatives. One such alternative is demineralized bone matrix (DBM). The purpose of this paper is to describe and characterize the properties of DBM in addition to reviewing the results of its use in animal and human studies of spinal fusion. A thorough and critical review of the English-language literature was conducted. DBM is both osteoconductive and osteoinductive. Studies have produced variable results with respect to spinal fusion rates. Various studies have demonstrated inferior, equal, or enhanced fusion rates. Some of the differences in these studies include the animal models used, the manner in which DBM was prepared, and the carrier with which DBM was combined. These differences may account for the dissimilar results. DBM is able to function as a graft extender in the human species. Ć 2005 Elsevier Inc. All rights reserved. Keywords: Spinal surgery; Bone grafts; Demineralized bone matrix

Background and introduction attach on to or into it. A wide variety of implants, including screws, rods, plates, wires, and cages are used to enhance the Spinal arthrodesis is one of the more common standard fusion between the unstable motion segments in an attempt surgical approaches for treating the various pathological dis- to stabilize the spine and decrease symptoms. Even in the orders of the spine. It is estimated that approximately presence of internal fixation, however, pseudoarthrosis still 185,000 lumbar spinal fusions occur each year [1]. However, occurs in approximately 10% to 15% of patients undergoing one of the main complications of lumbar spinal arthrodesis posterolateral intertransverse lumbar spinal fusion [4–7]. Al- is its non-union. Although posterolateral lumbar intertransverse process arthrodesis is the most common type of though instrumentation may stabilize the spine initially, it spinal arthrodesis performed, it still has a non-union rate is clear that the ultimate success of any type of spinal fusion is of 10% to 40% in patients with single-level fusions [2,3]. determined by biologic principles that dictate whether the This pseudoarthrosis rate increases when multiple levels of bones will grow together to form a solid mass. The process fusion are attempted. A common clinical approach to de- of bone graft healing can be considered as a race between creasing spinal pseudoarthrosis has been the use of inter- healing of the bone fusion and failure of internal fixation used nal fixation. to immobilize the spine. For every component of the complex spinal anatomy, One of the strategies to help augment spinal fusion rates whether it is the vertebral body, pedicle, facet joint, trans- is the use of bone grafts. A bone graft may be one of verse process, lamina, spinous process, or pars interarticu- several types of implanted material that is used alone or in laris, there is a clinical device commercially available to combination with other substances to help promote the bone healing response. Bone grafts can be characterized as auto- FDA device/drug status: approved for this indication (demineralized graft, allograft (fresh-frozen, freeze-dried, or demineralized), bone matrix [DBM]). osteoconductive carriers, or osteoinductive growth factors. Nothing of value received from a commercial entity related to this Of the myriad of bone grafts available, the preferred graft research. material is autogenous iliac crest bone grafting (ICBG). It is * Corresponding author. UCLA Department of Orthopaedic Surgery, UCLA School of Medicine, 10833 LeConte Ave., 76-124CHS, Box 956902, the most commonly used type of graft associated with pre- Los Angeles, CA 90095-6902. Tel.: (310) 206-8263; fax: (310) 825-1311. dictable bone healing in spinal fusion surgery [8–14]. Autol- E-mail address: [email protected] (J.C. Wang) ogous ICBG is considered the gold standard in bone grafting

1529-9430/05/$ – see front matter Ć 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2005.02.006 218S K.J.H. Lee et al. / The Spine Journal 5 (2005) 217S–223S [15]. It is estimated that approximately 200,000 bone graft harvested, multilevel fusions), graft extenders or enhancers procedures are performed in the United States each year may amplify the volume of bone graft material and aid in [16]. The vast majority of these procedures are performed the stimulation of bone formation. Graft substitutes would for spinal fusions (∼50%), followed by general orthopedic completely replace the autograft and provide equal if not surgery, and cranio-maxillofacial procedures. Autologous improved rates of bone fusion without using any autograft ICBG is popular because it has an excellent success rate, when compared with using autograft alone. there is a low risk of disease transmission, and it is histocom- In an attempt to obviate both the need for autogenous patible [17]. ICBG and its associated donor site morbidity, as well as to Unfortunately, this procedure may also be associated with optimize fusion rates, considerable research and develop- significant morbidity, as complication rates of up to 30% ment has been concentrated on developing either bone graft have been reported [18]. Documented complications include alternatives to ICBG or materials that may enhance the os- excessive bone loss and the associated increased risk of teoinductive activity of a limited supply of autograft, as in the a subsequent fracture, neuropathy, hernia, ureteral injury, case of spine fusions that span multiple vertebral levels. arteriovenous fistula formation, pseudoaneurysm of the One type of bone graft extender is DBM, which is produced pelvic vasculature, pelvic instability, and infection [19,20]. from human allograft tissue. These substances are commer- The most frequently reported complication is chronic donor cially available, commonly used, and require little or no site pain [21]. Aside from its morbidity, limitations of iliac preparation. This article will describe, characterize, and crest autograft include a second operative procedure requir- review the results of the use of DBM in spinal fusion. ing a potential separate incision for harvesting, increased surgical time, limited supply or insufficient amounts of autogenous graft available, especially in cases where patients Demineralized bone matrix have previously undergone iliac crest grafting or where multilevel fusion is required, poor bone quality, and less than Demineralized bone matrix preparations have demon- 100% fusion rates [22,23]. strated a potent effect on the differentiation of osteoprogeni- The ideal bone graft possesses the three properties of tor cells into osteoblasts. Marshall Urist first identified in osteoconductivity, osteogenicity, and osteoinductivity [24]. 1965 an osteoinductive substance while preparing soluble Osteoconductive graft materials provide biocompatible scaf- extracts from demineralized bone [27]. Since that time, folding that supports new bone formation and subsequent the osteoinductive ability of DBM has been well established growth. An osteogenic bone graft contains cellular elements [28–30]. Urist’s pioneering work demonstrated that when at some stage of osteoblastic differentiation (osteoprogenitor DBM was implanted into muscle, ectopic osteogenesis oc- cells) that are able to synthesize new bone at the fusion curred in mice. This demonstration of de novo bone forma- site and thus form new bone directly. Osteoinductive graft tion in ectopic, submuscular sites has become the standard materials facilitate the recruitment and differentiation of stem means of assessing whether different materials contain os- cells into osteoblasts, the bone-forming cells. Autograft con- teoinductive ability. This model is still commonly used today tains all three properties of osteoconductivity, osteogenicity, when testing for the osteoinductive capabilities of certain and osteoinductivity. Allograft is both osteoconductive but DBM products. Urist felt that the proteins contained in these only weakly osteoinductive. Demineralized bone matrix bone extracts contained osteoinductive properties. This work (DBM), a form of allograft, fits into the osteoinductive cate- ultimately led to the identification and cloning of bone mor- gory with a small number of products having some osteocon- phogenic proteins (BMPs). ductive capabilities. The degree of potential osteoinductivity The principal component of DBM responsible for its bone that may be present varies with the method of preparation inductive activity is a group of low-molecular-weight glyco- and sterilization [25,26]. proteins contained within the organic phase, the most im- Alternatives to bone grafts can be characterized as one portant of which are the BMPs. When cortical bone is of three types: graft extenders, graft enhancers, or graft decalcified, these osteoinductive proteins that are buried substitutes. The ideal graft extender would assist in the stim- within the mineralized matrix are exposed, thus enhancing ulation of bone formation and when combined with a de- the bone formation process [27]. Early tissue banks provided creased amount of autologous graft, provide a fusion rate that the first human DBM. As first described by Urist, mild would be comparable to and preferably the same as that of acid extraction of bone removes its mineral component autologous graft alone. In addition, when graft extenders are and yields a mixture of type I collagen and noncollagenous combined with a standard amount of autogenous graft, it proteins, including the BMPs. DBM is mainly comprised of may allow for the arthrodesis of a greater number of levels. collagen (93%), which provides an osteoconductive surface. Graft enhancers, when combined with either a standard or Soluble proteins, such as osteoinductive BMPs and a growth even a decreased amount of autogenous bone, would ideally cocktail of synergistic proteins (transforming growth factor- result in higher fusion rates than those obtained by autoge- beta, insulin-like growth factor, platelet-derived growth nous bone graft alone. Thus, in situations where there is an factor, fibroblast growth factor), only represent approxi- inadequate supply of autogenous graft (eg, limited amount mately 5% of DBM. The remaining 2% of DBM is made K.J.H. Lee et al. / The Spine Journal 5 (2005) 217S–223S 219S up of residual mineralized matrix. In addition to its osteoin- in Grafton (Osteotech). It is highly soluble in water, but ductive ability, DBM also supports new bone formation via studies have shown toxic reactions when high doses were osteoconductive mechanisms [31]. used in rats [35]. Grafton-DBM has been demonstrated to An important point to emphasize is that DBM does not have both osteoinductive and osteoconductive properties directly induce the formation of bone in subcutaneous or [36]. Poloxamer is found in Dynagraft/Orthoblast (Iso Tis). submuscular tissues. In other words, mesenchymal stem It is a polymer gel containing water that becomes more cells do not differentiate into osteoblasts. Rather, DBM rigid with an increase in temperature. Gelatin is denatured, induces chondrogenesis, whereby mesenchymal cells differ- fragmented collagen which is mixed with water to form a gel. entiate into chondroblasts, and not osteoblasts. Although The properties of this carrier are dependent on temperature. cartilage is formed, resorbed, and eventually replaced by Osteofil (RTI) is a porcine-derived gelatin that is stored bone, this DBM sequence of events leading to bone forma- frozen and needs to be heated or hydrated before use. Accell tion differs from that found in “true” or classical enchondral (Iso Tis Orthobiologics) is a gelatin derived from human bone formation. Instead of the chronological differentiation DBM with room temperature storage. Allomatrix (Wright of cartilage cells into hypertrophic, proliferative, and reserve Medical) is a calcium sulfate hemi-hydrate (plaster of Paris) zone layers, followed by osteoclast resorption of calcified mixed with carboxy-methylcellulose. Water is added at the cartilage, and the formation of bone on calcified cartilage, time of surgery. Lecithin, which is derived from soybeans, bone formation by DBM occurs only after cartilage is re- is found in InterGro (Interpore). One study has shown that sorbed and not concomitantly with its resorption [32,33]. Lecithin-DBM improves osteoinductivity [36]. Although hy- A series of studies examined whether the mechanism of aluronic acid is a naturally occurring substance found in bone formation differs depending on the environment into human joints, the DBM putty version is produced in a recom- which DBM is implanted [32–34]. In contrast to the sequence binant fashion in the product form of DBX (MTF/Synthes). of events when DBM is placed into subcutaneous sites, When DBM, with its small amounts of osteoinductive mesenchymal stem cells first differentiate into alkaline proteins, is combined with a carrier, a significant portion of phosphatase-staining osteoblasts when DBM is implanted the complex is the carrier (∼85% carrier and ∼15% DBM). into cranial defects. Bone matrix was then synthesized, The first DBM/carrier products were introduced clinically which was followed by calcification. Thus, is appears that in 1991 and have since become one of the most widely used DBM induces chondrogenesis when placed into subcutane- alternative graft products in spinal fusion surgery [37]. ous and submuscular sites, but induces osteogenesis in cra- Today, there are at least eight manufacturers with greater nial defects. The dominant pathway of DBM osteogenesis than six types of carriers and 25 products on the market. in subcutaneous and submuscular implants is somewhat akin DBM is commercially available in several different forms to enchondral bone formation, whereas in calcarial defects (eg, powder, chips, crushed granules, putty, or gel-filled the mechanism resembles intramembranous bone formation syringes). [34]. This difference is bone formation mechanism highlights Certain unique qualities of DBM make it an attractive bone the importance of the host environment in determining the graft alternative. It is cost-effective and readily available process of DBM osteogenesis. These authors have suggested from human tissue banks. In addition, the demineralization that this distinction in bone formation pathways may result process destroys the antigenic materials in bone, making from the mesenchymal stem cells in the subcutaneous tissues DBM less immunogenic than mineralized bone allograft and calvaria having a predominance of stem cells with differ- [38]. In addition, when autologous autograft or bone marrow ent receptors that selectively bind chondrogenic or osteoge- is combined with DBM, an additional biologic contribution nic proteins respectively. to osteogenesis may be provided from the instant source of DBM exists as a particulate powder once it is extracted osteogenic precursor cells [27,39,40]. This possibility has led from bone. In this form, it is subjected to static charging to much research on its ability to function as an alternative to which makes graft containment and thus graft delivery diffi- ICBG. cult. Graft effectiveness is dependent on graft localization to the fusion site. Human DBM is often combined with other components intended to make DBM easier to handle by Animal studies turning it into a putty or paste. These carriers must be biocompatible with bone (osteocompatible), not reduce the os- The utility of DBM in posterolateral lumbar spinal fusion teoconductivity of DBM, maintain graft containment during has been demonstrated in various animal studies. DBM has irrigation and closing, and maintain graft localization until been widely tested and characterized in the athymic rat the graft site is stabilized. Other desired DBM graft proper- model. The use of the athymic rat model allows for testing ties include a minimally invasive delivery system (eg, be of human products in the exact “off the shelf” formulation extrudable from a syringe) and no special handling issues that would be implanted into humans. Other animal models such as refrigeration or heating. may require the preparation of the DBM formulation using DBM carriers include glycerol, poloxamer, gelatin, cal- species-specific bone material which may allow for some cium sulfate, lecithin, and hyaluronic acid. Glycerol is found additional variations when compared with the actual human 220S K.J.H. Lee et al. / The Spine Journal 5 (2005) 217S–223S product. In addition, meaningful comparisons of one product combined with autograft (70%) [31]. Thus, certain forms to another may not be accurate because the tests involve of DBM may be effective as both a graft extender and a species-specific preparations. graft enhancer. Several rat studies have demonstrated that DBM products One possible explanation for these results is that fiber- can induce a spinal fusion in a dose-dependent manner and containing formulations may be more osteoconductive than may have value as a graft extender [35,41,42]. The products gel-DBM. When the osteoinductive properties of flex-, appear to behave differently with varying fusion rates com- putty-, and gel-DBM were devitalized or removed by guani- paring one product to another. The percentage amount of dine extraction and then compared with one another in terms actual DBM in the substance did not correlate with an in- of bone formation, the devitalized flex- and putty-DBM creased fusion rate. Conclusions from these studies indicate formulations showed significantly more new bone formation that the substances did not perform equally, that some did than devitalized gel-DBM [31]. This finding suggests that not demonstrate any significant bone formation, and that fiber-containing forms of DBM may have enhanced osteo- perhaps the products should be tested more thoroughly conductive ability. before their use in humans. In a rhesus monkey model of posterolateral lumbar fusion, In other animal studies using rabbits [43,44] and dogs [45], an early study demonstrated that it was possible to obtain DBM in combination with autologous bone marrow or spinal fusion by 12 weeks when an osteoinductive growth autograft has produced spinal fusion rates comparable to factor is combined with a DBM carrier [51]. A later study autograft alone. In one such study involving rabbits, examined the effects of two new formulations of Grafton DBM appeared more effective than frozen allograft alone DBM (flex and matrix) [52]. The matrix form of DBM is a in promoting arthrodesis [46]. However, in that same study, new and more porous formulation of flex-DBM. Results DBM did not increase the frequency of fusion when added from this study of rhesus macaques demonstrated that to the standard amount of autograft. In a different rabbit matrix-DBM performed better than the flex-DBM. More model of posterolateral lumbar arthrodesis [47], the gel form importantly, it revealed that osteoinduction was present in of DBM (Grafton) was evaluated against autogenous iliac all monkeys treated with the matrix form, which actually crest graft. Fusion rates between the two were similar. How- improved the fusion success of autograft. One possible ex- ever, when Grafton was combined with a less than the stan- planation for the superior performance of matrix-DBM is dard amount of autograft, fusion rates were comparable to that earlier studies in rabbits had demonstrated this formula- autograft alone. The most effective Grafton to autograft com- tion to be more osteoinductive [31]. Thus, in this nonhuman bination was a 3:1 ratio. From this study, DBM was able to primate model of posterolateral spine fusion, DBM might serve the function of a graft extender. A role for DBM as play a role not only as a graft extender, but also a graft en- a graft extender was further supported by the results of a hancer [52]. dog study [45]. In addition, other studies have suggested In other animal studies, however, DBM has not been that DBM may be effective as graft extenders in the setting shown to be as useful in promoting spinal arthrodesis. In one of limited autograph [47]. Moreover, studies have shown study, DBM alone or in combination with allograft did not that when DBM is combined with autograft bone marrow produce reliable spinal arthrodesis [53] and may have an composites, spinal fusion is more rapid and appears to inhibitory effect on arthrodesis of the spine compared with achieve radiographic and biomechanical stability earlier than autogenous bone alone and with recombinant osteogenic autograft alone [45,48,49]. protein [54]. In addition, other studies have reported signifi- Different formulations of DBM have shown encouraging cantly decreased biomechanical characteristics in the growth results with regard to inducing fusion of the spine. Two new factor–DBM carrier groups [55,56]. Using a unilateral poste- formulations of Grafton-DBM (flex-DBM and putty-DBM) rior lumbar decompression and contralateral spine fusion were studied in a rabbit model of posterolateral spine fusion model in dogs, the effectiveness of five groups to induce [31]. These new types of Grafton are fiber-containing, in bone formation was examined (autograft alone, autograft contrast to gel-DBM, which is particle based. These fiber- with DBM, autograft with type I collagen gel, autograft with containing combinations are easier to handle than gel-DBM. DBM and collagen gel, and autograft with rhBMP-2) [55]. Flex-DBM consists of flexible sheets of Grafton fibers, At 3 months, all groups without DBM demonstrated histo- whereas putty-DBM is more malleable. One study demon- logical and radiographic evidence of fusion, with the strated that all three types of Grafton (gel-, flex-, and putty-) rhBMP2-autograft group showing the largest fusion mass. had equal amounts of osteoinductivity [50]. However, the Furthermore, all groups with DBM (including the autograft flex- and putty-DBM forms demonstrated significantly with DBM group) demonstrated markedly lower rates of higher fusion rates compared with gel-DBM and with auto- fusion and decreased biomechanical strength compared with graft alone (100%, 83%, 58%, and 73%, respectively). Fur- groups that did not contain DBM. thermore, if either flex- or putty-DBM was combined with Other studies have shown that certain formulations of a less than standard amount of autograft, both types had DBM, when used alone, are insufficient to promote spinal significantly superior fusion rates (∼100%) when com- fusion when placed in the more challenging posterolateral pared with autograft alone (33%) and with gel-DBM lumbar spinal fusion model in rabbits. When compared with K.J.H. Lee et al. / The Spine Journal 5 (2005) 217S–223S 221S autologous iliac crest bone graft, allogenic rabbit DBM alone posterolateral fusion were followed for an average of 53 resulted in significantly lower rates of fusion [51,57]. The months. The fusion site was augmented with coralline hy- lower rate of fusion with allogenic DBM alone, as compared droxyapatite with or without DBM. In this study, patients with autograft, was even more pronounced when DBM was who received the Grafton DBM gel had a higher rate of evaluated in the highly challenging nonhuman primate pseudoarthrosis [62]. model of lumbar posterolateral intertransverse process spinal fusion [51]. Drawbacks of DBM Human clinical studies The differing efficacy of DBM on spinal fusion demon- Initial studies on the efficacy of DBM in the human spine strated in these studies is likely a result of the different DBM focused on its application to anterior cervical spine fusion. preparations used. The methods of demineralization used in In a prospective study of 77 patients undergoing anterior some of the studies differed from that originally described cervical spine fusion for cervical disc disease, the fusion by Urist et al. [27]. In addition, some of the animal models rates of freeze-dried allograft augmented with DBM of spinal fusion used in the studies were not ideal (eg, (Grafton) were compared with those of autograft from the unilateral decompression and contralateral intralaminar anterior iliac crest [58]. This study demonstrated that the rates fusion), making extrapolation of these findings to the more of graft collapse and pseudoarthrosis were higher in the common posterolateral intertransverse process model of allograft-DBM group. Although this finding suggested that spinal arthrodesis not as practical and more difficult. the allograft-DBM construct does not offer sufficient osteoin- In addition, the amount of osteogenic activity of a particu- ductive capacity to facilitate reliable arthrodesis, further lar DBM preparation is highly dependent upon the type and analysis demonstrated that these results did not reach statisti- specific preparation of bone used [25,63]. It is obvious that cal significance [58]. donor variability can not be avoided (eg, age, sex, lifestyle A different study examined the fusion rates of a local habits). In addition, its osteoinductive capacity may be af- autograft-DBM construct against that of iliac crest autograft fected by the carrier with which DBM is mixed [28]. Current alone in the setting of lumbar posterolateral spine fusion preparations of DBM which are mixed with a glycerol carrier [59]. One hundred and eight patients were followed for (Grafton) are very acidic. The low pH may have detrimental approximately two years. Fusion rates did not differ between effects on host cells if it is used in large quantities. Other the two groups. The rate of mineralization as assessed by preparations containing hyaluronic acid (DBX) have a more radiographs also was found not to vary between the two neutral pH and thus may be less harmful to host tissues. As groups. This prompted further evaluation of whether DBM a result, the amount of DBM used does not correlate with could serve the function of a graft extender. This proposal efficacy because the different methods of processing and was also suggested in another study [60]. sterilization can affect its osteoinductivity [26,36,64]. A fur- This issue was addressed in a prospective, randomized ther complicating matter is that bone healing in response to study of 120 patients undergoing posterolateral spine fusion DBM is species-specific [65]. at seven different sites [61]. Multilevel fusions of up to three levels were performed. The fusion rates of autogenous iliac crest alone were compared with a combination of three parts DBM (Grafton): one part autogenous iliac crest. Follow-up Summary occurred at the time points of 3, 6, 12, 18, and 24 months. Demineralized bone matrix is an attractive bone graft This study demonstrated that fusion success with Grafton alternative to the gold standard of autologous iliac crest. Nu- DBM gel composite is similar to that of traditional iliac merous studies have documented its osteoinductive ability. crest autograft in posterolateral lumbar spine fusion with The amount of osteoinductive ability may rely on its prepara- respect to mineralization and integrity of the developing tion and the type of carrier with which it is combined. Despite fusion mass. These findings suggest that DBM gel combined its bone-forming capacity, it does not offer any structural with a small amount of autologous bone can provide a suc- or mechanical stability independently of its carrier. Certain cessful fusion with the same frequency as autograft alone. formulations of DBM may function as viable bone graft Specifically, fusion rates were the same as autograft when alternatives in the roles of graft extenders. This role would one third the normal amount of autograft was combined with reduce the amount of harvested autograft required, and po- DBM. That is, DBM may serve as a graft extender in human tentially decrease its associated morbidity. posterolateral spine fusion. This would be beneficial in hosts with compromised osteogenic capacity, such as smokers, as DBM may be used as a supplement to autogenous bone graft [56]. References Not all clinical trials of DBM have been favorable. In a [1] Boden SD. Bone repair and enhancement of clinical trial design: spine retrospective study, 40 patients who underwent instrumented applications. Clin Orthop 1998;355:336–46. 222S K.J.H. Lee et al. / The Spine Journal 5 (2005) 217S–223S

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