Osteoinductivity of Commercially Available Demineralized Bone Matrix: Preparations in a Spine Fusion Model

Background: Although autogenous bone is the most widely used graft material for spinal fusion, demineralized bone matrix preparations are available as alternatives or supplements to autograft. They are prepared by acid extraction of most of the mineralized component, with retention of the collagen and noncollagenous proteins, including growth factors. Differences in allograft processing methods among suppliers might yield products with different osteoinductive activities. The purpose of this study was to compare the efficacy of three different commercially available demineralized bone matrix products for inducing spinal fusion in an athymic rat model.

Methods: Sixty male athymic rats underwent spinal fusion and were divided into three groups of eighteen animals each. Group I received Grafton Putty; Group II, DBX Putty; and Group III, AlloMatrix Injectable Putty. A control group of six animals (Group IV) underwent decortication alone. Six animals from each of the three experimental groups were killed at each of three intervals (two, four, and eight weeks), and the six animals from the control group were killed at eight weeks. At each of the time-points, radiographic and histologic analysis and manual testing of the explanted spines were performed.

Results: The spines in Group I demonstrated higher rates of radiographically evident fusion at eight weeks than did the spines in Group III or Group IV (p

Conclusions: This study demonstrated differences in the osteoinductive potentials of commercially available demineralized bone matrices in this animal model.

Clinical Relevance: Comparative clinical testing of demineralized bone matrices is indicated in order to determine which preparations are best suited for promoting successful spinal fusion in humans.

Approximately 200,000 spine fusions are performed each year in the United States, and most are single-level posterolateral lumbar fusions1. Autogenous bone graft is the current gold standard for inducing spinal fusion2. Autogenous bone is readily available from most patients and contains several of the elements that are thought to be critical to promote bone formation, including osteoprogenitor cells, the osteoconductive matrix of the cancellous bone, and osteoinductive signals such as bone morphogenetic proteins3-5. Disadvantages of autogenous bone graft include the morbidity associated with the harvest, the limited supply of the autogenous graft material, and the additional operating room time6-10. Because of the frequent need for bone-grafting in spine surgery, alternatives to autogenous bone are being developed and investigated11-13.

Demineralized bone matrices are a potential alternative or supplement to autogenous bone graft14-16. Demineralized bone matrices are prepared by acid extraction of allograft bone, resulting in loss of most of the mineralized component but retention of collagen and noncollagenous proteins, including growth factors. Demineralized bone matrices do not contain osteoprogenitor cells, but the efficacy of a demineralized bone matrix as a bone-graft substitute or extender may be influenced by a number of factors, including the sterilization process, the carrier, the total amount of bone morphogenetic protein (BMP) present, and the ratios of the different BMPs present17-19. In addition, the osteoconductivity of the demineralized bone matrix-carrier complex may be an important factor since this property promotes migration of osteoprogenitor cells to the bone defect site.

Some demineralized bone matrices are tested chemically or immunologically for BMP content, and certain demineralized bone matrices are tested in vitro to demonstrate their osteoinductive effect on cultured cells. However, many demineralized bone matrices have not been stringently tested in clinically relevant animal models, in part because this has not been required by the United States Food and Drug Administration. To our knowledge, there has never been a prospective, randomized clinical trial to evaluate the efficacies of these agents in a clinical setting.

Despite this lack of data, demineralized bone matrices have been used not only to enhance fusion of the spine20,21 but also to graft nonunions22, osteolytic lesions around total joint implants23, and benign bone cysts24. However, the success rates of different demineralized bone matrices have not been studied in a rigorous fashion, and the efficacy of these agents may vary. The purpose of this study was to compare the efficacy of three different commercially available demineralized bone matrix products in an athymic rat spinal fusion modeP.

Materials and Methods

Demineralized Bone Matrices Tested

Sixty male athymic rats were analyzed in this study. The animals were divided into three experimental groups of eighteen animals each and a control group of six animals. Group I was treated with Grafton Putty (Osteotech, Eatontown, New Jersey); Group II, with DBX Putty (MTF [Musculoskeletal Transplant Foundation], available through Synthes, Paoli, Pennsylvania); Group III, with AlloMatrix Injectable Putty (Wright Medical Technology, Arlington, Tennessee); and Group IV (control group), with decortication alone. Demineralized bone matrices were obtained directly from the manufacturers, and the lot numbers were recorded. For each brand of demineralized bone matrix, at least two different lot numbers were used.

Product Information

The specific details of the preparation and processing of the demineralized bone matrices are proprietary, but each is composed of demineralized human allograft bone combined with a biologically compatible carrier. All of the demineralized bone matrices that we tested can be stored at room temperature and do not have special handling requirements other than maintenance of sterility.

Grafton Putty (Group I) is derived from human banked bone tissue. Donors undergo serologic and microbiologic testing as well as screening on the basis of their medical and social history. The allograft bone is harvested in a sterile manner, washed, sonicated, treated with antibiotics, and demineralized to contain

DBX Putty (Group II) is also derived from human banked bone tissue. Donors undergo screening procedures as dictated by the Musculoskeletal Transplant Foundation (Edison, New Jersey). Allograft tissue is harvested under sterile conditions, washed, and treated with antibiotics. The tissue is then demineralized with hydrochloric acid so that the resulting bone matrix contains

AlloMatrix Injectable Putty (Group III) is derived from human banked bone tissue as well. Donors are screened with serological testing as well as evaluation of their medical and social history. The allograft tissue is processed aseptically by the tissue supplier and undergoes e-beam (electron-beam) sterilization. The allograft bone is demineralized and is combined with calcium sulphate hemihydrate and carboxymethylcellulose. Each lot of AlloMatrix demineralized bone matrix is assayed in vitro tor osteoinductive potential. AlloMatrix Injectable Putty is packaged as a kit containing a powder and a liquid that must be mixed prior to implantation. Once this mixing is performed, the AlloMatrix Putty can be easily molded or pressed into a bone defect.

Arthrodesis in Athymic Nude Rats

Approval was obtained from the Institutional Animal Care and Use Committee before the animal procedures were begun. This spine fusion model has been described previously27. A midline incision was made in the skin, and the transverse processes of L4 and L5 were exposed and decorticated bilaterally with a high-speed burr. After this, 0.3 cm^sup 3^ of graft material was implanted on each side (0.6 cm^sup 3^ total). The appropriate demineralized bone matrix was implanted in the eighteen animals in each of the three experimental groups. The control group underwent decortication alone. Six animals from each of the three experimental groups were killed at each of three intervals (two, four, and eight weeks), and the six animals from the control group were killed at eight weeks.

Radiographic Analysis

Radiographs were made at two, four, and eight weeks after surgery and were examined by t\hree independent observers who were blinded to the treatment group. The amount of bone that had formed between the transverse processes of L4 and L5 was evaluated with use of a scoring system in which 0 indicated minimal or no evidence of new bone formation; 1, immature bone formation, with fusion questionable; and 2, solid-appearing bone, with fusion likely. The radiographic scores of the three observers were summed, with 6 as the maximum score. Spines with a cumulative score of 5 or 6 were considered to have radiographic evidence of fusion.

Manual Palpation

All spines were explanted and assessed for fusion by manual palpation by three observers who were blinded to the type of treatment that the animal had received. Manual palpation has been reported to be the most sensitive and specific method of assessing fusion in this model28,29. All spines were categorized as either fused or not fused. At least two observers had to have considered the spine to be fused for the spine to be deemed fused as demonstrated by manual palpation.

Histologic Techniques

After the animals were killed, all sixty spines were dissected and were fixed in 40% ethanol, dehydrated, and embedded in polymethylmethacrylate. Serial sagittal sections of the transverse processes were cut with a diamond band saw (Exakt, Hamburg, Germany). Sections were mounted on plastic slides, milled, polished, and surface-stained with trichrome or toluidine blue.

Statistical Methods

Scores from the radiographic analysis were assessed with a nonparametric Kruskal-Wallis test comparing the distribution of ranked data in the various groups. Data from the manual palpation assessment were analyzed with use of the Fisher exact test. The kappa statistic was calculated to demonstrate the interobserver reliability of the scoring.


The three experimental groups differed with regard to the fusion rates. These differences were consistently shown radiographically, by manual palpation, and by histologic analysis.

Fig. 1

Radiographs of explanted spines in Group 1 (Grafton Putty) at two, four, and eight weeks. The space between L4 and L5 was initially radiolucent, and bone formation was easily distinguished at the four and eight-week time-points.

Radiographic Analysis

The Grafton Putty demineralized bone matrix (Group I) is extensively demineralized and has a glycerol carrier. New bone formation in the rats treated with the Grafton Putty was the easiest to assess radiographically since the putty material was initially radiolucent. Radiographs of the spines made at two weeks after the surgery showed minimal bone formation between L4 and L5. At the four- week time-point, radiographs clearly showed new bone formation between the transverse processes of L4 and L5 (Fig. 1). Radiographs made at the eight-week time-point indicated that all six of the spines had fused, and even the worst-appearing radiograph of a spine treated with Grafton Putty showed considerable bone formation between the transverse processes.

The DBX Putty (Group II) is less extensively demineralized than is the Grafton Putty and is initially radiopaque. The radiopaque DBX Putty material could be seen on the radiographs made at the two- week time-point, and this made assessment of new bone formation more difficult (Fig. 2). At four weeks, four of the six spines in which DBX had been implanted appeared to be solidly fused. At eight weeks, radiolucent cracks appeared between the bone formed at L4 and L5 in three of the spines, indicating that a pseudarthrosis had occurred. Three of the spines appeared to be fused.

Two weeks after the operative procedure in the spines in Group III (AlloMatrix Injectable Putty), radiopaque material appeared to bridge L4 and L5. However, it was unclear whether this material was residual carrier or new bone. At the four-week time-point, the amount of radiopaque material had increased minimally in comparison with that seen at the two-week time-point, and, by eight weeks, three of the six radiographs showed large radiolucent fissures between L4 and L5, clearly indicating a pseudarthrosis. None of the Group-III spines appeared to be fused on the radiographs made at the eight-week time-point (Fig. 3).

Fig. 2

Radiographs of explanted spines in Group II (DBX Putty) at two, four, and eight weeks. Spines in Group II contained radiopaque material between L4 and L5 at two weeks. However, minimal change was detected between the two and four-week time-points. New bone formation was difficult to distinguish from the background carrier. At eight weeks, three of the six spines in Group II appeared solidly fused, but three appeared to have a pseudarthrosis.

Fig. 3

Radiographs of explanted spines from Group III (AlloMatrix Injectable Putty) at two, four, and eight weeks. Spines in Group III contained radiopaque material between L4 and L5 at the two-week time- point. Bone present at the eight-week time-point appeared to have a more mature appearance, but fusion did not appear to have occurred in any of the spines. The arrowheads point to the transverse process of L5.

There was minimal or no radiographic evidence of bone formation between L4 and L5 in the spines in the control group (Group IV).

There was a high level of agreement with respect to the radiographic scores. The kappa statistic was 0.87. Figure 4 presents the best and worst-appearing radiographs for each study group at eight weeks.

Manual Palpation

At the two-week time-point, no spine in any of the four groups was determined to be fused on manual palpation. However, five of the six spines in Group I had fused by the four-week time-point, and all six had fused by eight weeks. In Group II, two of the six spines were fused at four weeks and three were fused at the eight-week time- point. In Group III, there was no evidence of spine fusion on manual palpation at two, four, or eight weeks after the operative procedure (Table I). There was a significant difference in fusion rates, as noted with manual palpation, between Group I (Grafton) and Group III (AlloMatrix) at both four weeks (five of six compared with zero of six; p = 0.015) and eight weeks (six of six compared with zero of six; p = 0.001). The differences in the fusion rates between Groups I and II and between Groups II and III did not reach significance, probably because of the limited number of animals in the study. No spines in the control group (Group IV) were considered to have fused. The combined kappa statistic was 0.922.

Fig. 4

Best and worst-appearing radiographs of the explanted spines at eight weeks. A: Control spines that underwent decortication alone (Group IV). B: Spines treated with AlloMatrix Injectable Putty (Group III). C: Spines treated with DBX Putty (Group II). D: Spines treated with Grafton Putty (Group I). Grafton Putty is initially radiolucent. AlloMatrix and DBX have radiopaque carriers. The radiopacity between L4 and L5 in the group treated with Grafton Putty was seen to be increased, whereas minimal change could be detected between the two and four-week time-points in the spines treated with AlloMatrix or DBX.

TABLE I Data Derived with Manual Palpation

Histologic Analysis

At two weeks, the spines in Group I (Grafton Putty) demonstrated woven bone on the surfaces of the L4 and L5 transverse processes. In addition, there were strands of collagen, apparently from the carrier, and isolated islands of new bone formation. No endochondral intermediate was detected. By four weeks, these islands of new bone had coalesced, forming networks of woven bone. At eight weeks, no residual demineralized bone matrix was visible and new bone bridged the transverse processes of L4 and L5, resulting in solid fusion in all six spines (Fig. 5).

All six of the spines in Group II (DBX Putty) revealed persistence of the demineralized bone matrix material at the two, four, and eight-week time-points. New bone formation occurred on the surfaces of the transverse processes and in the interstices of the carrier. New bone formation was detected in the two-week specimens, and by eight weeks three of the spines were fused. In the other three spines, there was new bone formation but not complete fusion.

Fig. 5

Sagittal sections of spines at eight weeks (toluidine blue). A: A spine treated with decortication alone (Group IV). B: A spine treated with AlloMatrix Injectable Putty (Group III). C: A spine treated with DBX Putty (Group II). D: A spine treated with Grafton Putty (Group I). Toluidine blue stains the transverse processes (asterisks) and new bone dark blue. Residual carrier in both the AlloMatrix and the DBX Putty-treated spines stains pale blue (arrows). The sagittal section from the animal treated with decortication alone shows no bone between the transverse processes. The section from the spine treated with the AlloMatrix Putty shows some residual carrier and some new bone, which appears to have formed along the surfaces of the decorticated transverse processes and the residual carrier. The section from the DBX Putty-treated spine shows extensive amounts of residual carrier as well as new bone formation in the interstices. No residual carrier is visible in the section from the Grafton Putty-treated group.

In Group III (AlloMatrix Putty), new bone formation was noted originating from the surfaces of the transverse processes. However, the amount of additional bone formation between the two and four- week time-points was minimal. None of the spines had fused by eight weeks, although new bone was present on the dorsal surfaces of the transverse processes. Residual demineralized bone matrix was still detectable in the spines at eight weeks, but less residual carrier was present than had been seen in the two-week spines.

In the control group (decortication only), minimal bone had formed on the surfaces of L4 and L5 at eight weeks (Fig. 5).


We are not aware of any prospective clinical trials comparing demineralized bone matrices, but there i\s some clinical evidence and there are several animal studies indicating that demineralized bone matrices may function as extenders of autogenous bone graft14,30. For example, Johnson et al. combined demineralized bone matrix with autogenous bone graft to effectively treat tibial and femoral nonunions31-33. In their series of thirty problematic femoral nonunions, twenty-four healed within six months after intervention, four required application of a second plate before union occurred, and two patients were lost to follow-up. In a nonrandomized, prospective study of single and multilevel anterior cervical fusions, An et al. compared the results in thirty-eight patients treated with autogenous iliac crest bone graft with those in thirty-nine patents treated with allograft bone and demineralized bone matrix34. At the time of follow-up, at twelve to thirty-one months, the patients treated with the autograft had a lower rate of graft collapse than did those treated with the allograft and demineralized bone matrix (11% compared with 19%) as well as a lower rate of pseudarthrosis (26% compared with 46%), but the differences did not quite reach significance.

Various animal models have been developed to study the osteoinductive potential of demineralized bone matrices and their ability to either substitute for or enhance the biologic activity of autograft bone15,35-38. A general weakness of these studies is that the demineralized bone matrix that was tested was not the same material that is commercially available since the demineralized bone matrix must be made from bone from the same species.

The efficacies of demineralized bone matrices have been assessed in a well-established rabbit posterolateral spine fusion model15,39,40. Morone and Boden demonstrated that decreased autograft volume could be supplemented with demineralized bone matrix gel to yield fusion rates similar to those following use of autograft alone15.

The present study involved an athymic rat posterolateral spine fusion model, a standardized model that enables stringent testing of osteoinductive capacity. An advantage of this model is that the demineralized bone matrix can be evaluated in its commercially available form because the nude rat does not generate an immune response to the human demineralized bone matrix. Results derived from this or any animal model of spine fusion must be interpreted with caution, however. The efficacy of these demineralized bone matrices may be different in other types of models (e.g., a femoral defect model). Also, young healthy animals were used in this study, and the results in such animals may not be applicable to older patients with previous surgical treatment, a history of nicotine use, or poor general health. The final test of the efficacy of demineralized bone matrix is the clinical trial, and success at one level of animal model does not necessarily mean that the demineralized bone matrix will be osteoinductive at the next level. However, failure to induce bone at a lower level of the phylogeny has been suggested to indicate a poor prognosis for the osteoinductive potential of the substance in higher animals and humans1.

While all demineralized bone matrices augment the fusion process by providing an osteoconductive scaffold of variable osteoinductive activity41-44, each manufacturer uses a different system for procuring allografts and for demineralization and sterilization. In addition, demineralized bone matrices are often combined with different carriers such as glycerol, hyaluronic acid, or calcium sulfate. Many demineralized bone matrices have not undergone extensive preclinical or clinical testing, in part because the Food and Drug Administration has not regulated demineralized bone matrices in the same way that medical devices have been regulated. Therefore, it was not surprising that the demineralized bone matrices in this study were found to have different biological activities. In addition to the processing methods, donor quality is another potential source of variability in osteoinductive potential. This could be evaluated by testing different lots from a single graft processor. We did not attempt to compare osteoinductivity among different lots of the same demineralized bone matrix, but, by using at least two lot numbers of each demineralized bone matrix formulation, we hoped to minimize the potential of a systematic error. Several preclinical studies have demonstrated no significant difference among lots from the same manufacturer, even though the donor age in one study ranged from forty-five to sixty-seven years45.

All of the demineralized bone matrices tested in this study are commercially available. Each had excellent handling properties in that they were easily molded and placed onto the decorticated transverse processes of the rats. We did not evaluate the diffusion or solubility of the demineralized bone matrices, and therefore we cannot comment on the duration for which the demineralized bone matrix was present at the desired fusion site after the skin was closed.

This study demonstrated that differences in the osteoinductive potential of commercially available demineralized bone matrices can be detected with the use of this animal model. Whether the differences in fusion rates in athymic rats translate into variable clinical outcomes when the same demineralized bone matrix preparations are used in patients is a matter of speculation. Surgeons should carefully consider the clinical indications for any bone-graft substitute or extender, and comparative clinical testing of demineralized bone matrices is needed to determine which preparations are best suited for promoting successful spinal fusion in humans.

NOTE: The authors thank Fred Dorey, PhD, for his assistance with the statistical analysis in this study.

A commentary is available with the electronic versions of this article, on our web site (www.jbjs.org) and on our quarterly CD-ROM (call our subscription department, at 781-449-9780, to order the CD- ROM).


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Investigation performed at the Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California

Brett Peterson, MD

Peter G. Whang, MD

Roberta Iglesias, MD

Jeff C. Wang, MD

Jay R. Lieberman, MD

Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Center for Health Sciences 76-134, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address for J.R. Lieberman: [email protected]

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the Musculoskeletal Transplant Foundation. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Copyright Journal of Bone and Joint Surgery, Inc. Oct 2004