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Comparative Efficacy of Commonly Available Human Bone Graft Substitutes as Tested for Posterolateral Fusion in an Athymic Rat Model

ABSTRACT Background: Insufficient data exist on bone graft substitute materials efficacy; two thirds lack any clinical data.1,2 This prospective animal study identified efficacy differences among commercially available materials of several classes. Methods: Historically validated muscle pouch osteoinduction study (OIS) and posterolateral fusion (PLF) were performed in an athymic rat model. Grafting material products implanted were demineralized bone matrix (DBM)-based allografts (Accell EVO3, DBX Mix, DBX Strip, Grafton Crunch, Grafton Flex, Grafton Matrix, Grafton Putty, Magnifuse, and Progenix Plus), allografts (OsteoSponge, MinerOss), cellular allograft (Osteocel Plus), ceramics (Mozaik Strip), or activated ceramics (Actifuse ABX Putty, Vitoss BA). After 4 weeks, OIS specimens were evaluated ex vivo by histologic osteoinductivity. After 8 weeks, PLF ex vivo specimens were evaluated for fusion by manual palpation (FMP), radiography (FXR), and histology (FHISTO). Results: OIS: No materials exhibited a rejection reaction on histology. All DBM-based materials exhibited osteoinductive potential as new bone formation at > 88% of implanted sites. One plain allograft (OsteoSponge) formed bone at 25% of sites. No bone formed for one ceramic (Mozaik Strip), three activated ceramics (Actifuse ABX Putty), or one cellular allograft, regardless of human bone marrow aspirate (hBMA) when added. PLF: Among the 10 DBMs, 6 had FMP of 100% (Accell EVO3, DBX Mix, DBX Strip, Grafton Flex, Grafton Putty, Magnifuse), 2 had FMP of 94% (Grafton Crunch, Grafton Matrix), and 2 conditions had FMP of 0% (Progenix Plus, Progenix Plus + athymic rat iliac crest bone graft [arICBG]). Ceramics (Mozaik Strip), activated ceramics (Actifuse ABX Putty, Vitoss BA), plain allograft (OsteoSponge, MinerOss (PLF study), and cellular allograft (Osteocel Plus) demonstrated 0% FMP. ArICBG demonstrated 13% FMP. Conclusions: Eight DBM-based materials (Accell EVO3, DBX Mix, DBX Strip, Grafton Crunch, Grafton Flex, Grafton Matrix, Grafton Putty, Magnifuse) demonstrated excellent (> 90% FMP) efficacy in promoting fusion via bone healing. Two DBM conditions (Progenix Plus, Progenix Plus + arICBG) showed no manual palpation fusion (FMP). Systematically, over the 2 studies (OIS and PLF), cellular (Osteocel Plus), plain allografts (OsteoSponge, MinerOss; PLF study), ceramic (Mozaik Strip), and activated ceramics (Actifuse ABX Putty, Vitoss BA) demonstrated poor FMP efficacy (< 10%). Clinical Relevance: When selecting DBMs, clinicians must be cognizant of variability in DBM efficacy by product and lot. While theoretically osteoinductive, cellular allograft and activated ceramics yielded poor in vivo efficacy. Whole allograft and ceramics may provide osteoconductive scaffolding for mixed-material grafting; however, surgeons should be cautious in using them alone. Direct clinical data are needed to establish efficacy for any bone graft substitute.

Iliac crest bone graft (ICBG) remains the gold standard graft. Autograft provides an osteoconductive scaffold, osteoinductive cellular chemoattractants, and osteogenic progenitor cells. However, autograft has drawbacks: potential harvest-related complications, limited volume, and poor bone quality in certain patients limits utility. 4,11 The incidence of long-term pain following ICBG harvesting is from 2% to 25%, increasing morbidity for back pain patients, who already represent 40% of opioid prescriptions. [12][13][14][15] The American Academy of Orthopaedic Surgeons (AAOS) identified 63 bone graft substitutes, although many are used despite a lack of clinical evidence on their efficacy. 2,16 Research continues to find the ideal substitute that would be ''biocompatible, bioresorbable, osteoconductive, osteoinductive.'' 6 Current options, discussed below, include whole and cellular allograft, 2,17 demineralized bone matrix (DBM), hydroxyapatite (HA) and calcium ceramics/bioactive glasses, bone morphogenetic proteins (BMPs), and combinations of these.

Ceramic and Synthetic Materials
Used for decades, HA or tricalcium phosphates (TCP) grafts are available in many forms. They are cleared by the US Food and Drug Administration (FDA) for marketing on ''substantial equivalence'' to existing materials, without clinical efficacy data.
Silica, phosphate, and borate based bioactive glasses are in-development. 10,[18][19][20] These induce a hydroxycarbonate apatite surface layer for bone binding, and possibly create osteoinduction by solubilization of silica and calcium ions. [21][22][23] However, clinical use is limited by brittleness causing low fracture strength and difficulty in synthesizing porous scaffolds.

Whole Allografts
Whole allograft may be cortical or cancellous; incorporation is not as rapid or as reliable as autograft. 24 Disease transmission is rare, with no viral transmissions since 2002. 10 Xenograft is similar but carries a higher risk of rejection. 18 Demineralized Bone Matrix-based Products DBM is created via acid extraction of the mineral components of allograft, leaving osteoconductive matrix with osteoinductive native BMPs. Their concentration varies among individual donor-based lots of the same product. 7,25 Materials are heterogeneous, with different particle sizes and shapes, levels of demineralization and structure, and nonallograft additives for handling.

Study Objectives
Despite extensive clinical utilization of grafting adjuncts, there is little comparative efficacy data. Limited evidence suggests different DBMs facilitate different spinal fusion rates. 11,[50][51][52] Each material class also poses its own risks: including chronic pain from autograft harvest, rejection or disease transmission from allograft, and wound or neurologic complications with BMP. 3,10,40,42 Better understanding of efficacy and osteoinductivity will help clinicians balance risk/benefit in grafting material selection.
The specific objectives of this study were to: 1. Screen for osteoinductivity and immunologic reaction of human and synthetic materials in

MATERIALS AND METHODS
The Institutional Animal Care Use Committee approved all procedures. Table 1 describes implant materials.

Design
Osteoinductivity of 16 materials was evaluated via rat intermuscular bone formation, listed in Tables 1  and 2.

Subjects
Sixty-eight athymic nude rNu À rNu À male rats (Harlan Laboratories, Indiana), 7-to 8-week-old, and 200 to 350 grams were used. Athymic nude rats minimize immune responses to the human derived materials.

Procedure
Prophylaxis included buprenorphine for pain and ampicillin for infection (same as surgical description below). After general anesthesia and skin preparation, a small incision was made over the hind-limb. Then 0.2 cm 3 of material was placed between muscular bundles, close to the femur but not touching bone, and wounds sutured closed. [53][54][55] Evaluation of Osteoinductivity At 4 weeks, rats were killed. Material-tissue explants were decalcified and paraffin embedded. Sections were hematoxylin and eosin (H&E) stained. Osteoinductivity was a ''1'' indicating the presence of bone forming cells, cartilage, or new bone occupying a minimum of at least 5% of the implant area for . 50% of implanted sites; or a ''0'' indicating the absence of bone forming cells, chondrogenic activity, or new bone.

Posterolateral Fusion
Design Bilateral 2-level posterolateral fusion (PLF) were performed at L3-L4-L5. Eight rats were implanted per each of 18 materials/conditions (Table 1). Positive controls employed were athymic rat ICBG (arICBG) as a gold standard ICBG equivalent and 2 doses of rhBMP-2/ACS (0.1 mg/mL, 0.006 mg/mL). Surgery alone (sham) was a negative control. Materials were prepared according to product instructions and applied in accordance with a previously generated randomization schedule. Rats were observed daily and killed at 8 weeks after surgery. Fusion assessments included manual palpation, radiography, and histopathology.
A 4-to 6-cm dorsal midline skin incision was made from L1 to L2 to the iliac crests. Twocentimeter paramedian incisions were made. Paraspinal musculature was retracted to expose the transverse processes (TPs) of L3 to L5 bilaterally, and then irrigated with 1.5 mL of normal saline. L3 to L5 TPs were decorticated bilaterally via motorized burr until bleeding bone was observed. 0.6 cm 3 of material was implanted each side over L3 to L5 TP bleeding bone as shown in Appendix 1 (1.2 cm 3 implant per animal). For sham surgery, TPs were decorticated with no material implanted.
Two-layer closure was performed using monofilament polyglyconate (4-0 Maxon, Tyco Healthcare). Allograft: Particles of a DBM-based material (Magnifuse) and a cellular allograft (Osteocel) were aseptically scaled down using a rongeur to approx-imately 1 mm 3 particles (accommodate to smaller size of fusion site in the rat).
Athymic rat ICBG: Iliac crest was aseptically harvested from donor athymic rats, rongeur morselized to 2 to 3 mm 3 , and used within 4 hours of harvest.

Statistical Analysis
PLF: For manual palpation fusion (F MP ) and radiographic fusion (F XR ), each was calculated as percent of fused spinal levels over total levels implanted per material. Ninety-five percent exact confidence intervals (CIs) were calculated. Histologic fusion (F HISTO ) was calculated as percent of fused right and left sides over total sides implanted per material. Intrarelationships among dependent variables (F MP , F HISTO , F XR ) were examined by Spearman correlation coefficients by material with significance if P , 0.05.
OIS: These data are the sites showing osteoinductivity divided by the total implanted sites. The coefficient of variation (CV%) was calculated when . 1 unique lot was tested per material. For frequency data, the Fisher's exact test was used.

RESULTS
All animals tolerated the OIS (n ¼ 68) and PLF procedures (n ¼ 184) and gained weight, with no adverse events.

PLF Study
For F MP , F XR , and F HISTO outcomes, see Figure  1 and data presented in Appendix 2. Figure 2 presents radiographs of radiodense materials that appear on x-ray but with 0% F MP . Figures 3 to 10 present high-resolution ex vivo radiographs paired with decalcified histologic sections. The sections were H&E stained and had original magnifications of 34 to 37. Examples were subjectively selected as the best two in class and the single worst in class. When a single rat is described, it is labeled as ''#,sample number.'', with ''R'' or ''L'' for right or left side if relevant.

Histopathology
Histological evaluation revealed no adverse findings, evidence of tissue rejection, or poor biocompatibility in any of the study groups. Details are presented in Figures 3 to 10.
Osteoinductivity results (OIS) and manual testing fusion (PLF) results were significantly positively correlated across materials tested in spite of differing lots (r ¼ 0.72, P , 0.002, 16 common test materials between OIS and PLF). Two exceptions

DISCUSSION
Successful union after a multilevel instrumented spinal fusion procedure is a clinical challenge that usually necessitates use of graft extenders. Novel biomaterials, bone grafting substitutes, and adjuvants are rapidly designed and launched without proven preclinical efficacy (recent clinical review and updated guidelines). 2 In order to address this, we present radiographic and histological images for each individual material tested. Individual growth factors and DBMs have been studied with similar methods. 7,11,17,25,48,[50][51][52][59][60][61][62][63][64] However, none compared multiple DBMs, whole and cellular allografts, and ceramics and activated ceramics. Our data provide the most comprehensive comparative efficacy evaluation of fusion adjuncts in an athymic rat model to date. Unlike prior single-level athymic rat studies, a 2-level fusion procedure was used, 17,50,65,66 allowing efficacy assessment in multilevel fusions similar to commonly performed clinical spine surgery.

Ceramics and Activated Ceramics
These ceramics resulted in insufficient in vivo ossification for manual palpation fusion (F MP ), with 73% average residual graft and fibrous tissue on histology. No ceramics have shown in vivo osteoinductive effects; some literature recommends using these only as graft extenders, not solo grafts. 2,23,66 The lack of a ceramic þ arICBG mix graft in this study may negatively bias results, as this combination may be attempted clinically. However, as the ceramic þ hBMA group failed to fuse, this suggests hBMA does not substitute for autologous bone graft with ceramics.

Whole and Cellular
Osteocel cell-based allograft material failed to fuse. The ability of fat and marrow derived stem cells to survive in athymic nude mice has been shown. 60,73 Also, other human-derived tissues function in this animal model 74,75 ; however, this rodent model may not be a reliable and valid test of this class of grafting materials. DBM containing viable cells (Trinity Evolution Orthofix) failed to induce rigid fusions in athymic rats in another study as well. 76 Results herein suggest a poor osteogenic effect for Figure 2. Selected in vivo radiographs of rats at 0-, 4-, and 8-week time points with radiodense materials having F MP of 0%. Despite the lack of fusion as determined by manual palpation (F MP ) and histology (F HISTO ), these ceramic and activated ceramic materials have high radiodensity that mimics a fusion mass on radiography. Multifocal changes to the radiodensity pattern from 0 to 8 weeks suggest remodeling activity. Careful radiographic review is necessary to note the transverse radiolucent fissures and lack of continuous bone bridges. Figure 3. Bilateral robust mineralized fusion masses span L3 to L5 (0.1 mg/mL rhBMP-2/ACS). Radiography shows uniform density with defined margins demonstrating formed bone with cortex (0.006 mg/mL rhBMP-2/ACS). Number 692: histology shows a thin tract of wispy basophilic material (scored as residual material with mineralization) infiltrated with fibrous connective tissue and a few multinucleated giant cells. Radiography shows a fusion region with a thin rim of lamellar bone that forms a complete border continuous with TPs L3 to L4 to L5. Number 660: on histology the fusion is robust with lamellar bone continuous with L3 to L4 to L5 TPs. Trabeculae near TPs are mildly robust, and some portions of the mass are devoid of trabeculae. Particulate dense mineral exists in the L3 to L4 to L5 TP interspaces on radiography (arICBG). Histology shows 69% new bone and marrow formation, suggesting bioactivity and early remodeling.      this material, potentially questioning clinical efficacy to justify the added cost.
OsteoSponge histology was unique: the implant compressed in total volume significantly with implantation. This biased new bone formation evaluation; analysis stated 60%, however, this is misleading as volume of new bone is small due to the graft compression. The small volume of bone was unable to support fusion across TPs. This demonstrates the clinical importance of structural support to material selection.

Demineralized Bone Matrix-based
Prior studies of allograft-DBMs in single-level fusion procedure in athymic rats have demonstrated fusion rates between 20% and 80%. 7,50 When comparing only to materials tested within our study and at similar doses, 2 versions (Grafton Putty and Grafton Flex) have demonstrated fusion rates of 65% to 100% and 1 (DBX) a rate of 50%. 11,51 One study demonstrated a fusion rate of 39% for 1 putty (Grafton Putty), but the dose used was 0.4 cm 3 /level instead of the 0.6 cm 3 /level used here and in other referenced studies. 63 Accell Evo3 demonstrated greater fusion on manual palpitation than via histologic analysis in this study, possibly indicating an ongoing process of ossification revealed in the histologic sections. Similarly high fusion results for 2 DBMs (Accell Evo3 and Grafton Putty) were recently reported by Brecevich et al. 77 Here, these allograft-DBMs resulted in F MP . 88%.
Results demonstrate these graft materials perform in a 2-level fusion despite the increase in strain. Three allograft-DBMs failed, suggesting low material efficacy, although lot variability exists.

Controls arICBG
In a study by Lee et al, 50 rat tail autograft failed to promote PLF in an athymic rat model. Despite selection of arICBG over rat tail autograft, minimal F MP was observed, suggesting an opportunity to refine arICBG application, particularly as histology demonstrated early bone formation that had not yet progressed to bridging bone.

rhBMP-2
It was previously demonstrated that rhBMP-2 concentrations of 0.032 to 0.160 mg/mL resulted in 100% manually fused single-level L4-5 PLF in mature female Lewis rats; at 0.006 mg/mL, fusion rate was 33% to 50%. 17 The current study found a concentration of 0.006 mg/mL achieved 100% manually fused (F MP ) in this 2-level model.

Current Experience and Limitations
The common feature in materials with high fusion rates appears to be the availability of concentrated osteoinductive factors. DBMs induced fusion positively correlated with the concentration of BMPs in each DBM sample using a similar model. 7,17,50,63 The ability of a material to successfully fuse in the PLF was positively correlated with osteoinductivity via OIS (r ¼ 0.72). It may be inferred that successful bone healing is primarily assisted by the osteoinductive component of a material assuming a favorable biocompatible scaffold is in place.
We selected male rats to minimize variance secondary to hormonal fluctuations. However, this limits comparison with historic experiments using female rats. We also note that we used an 8-week time point, whereas some referenced studies used 6 weeks.

Future Research
Some materials were downsized to fit the animal; however, the ''ideal'' particle size for rats is unknown. Fusion rates show dependency on particle size (unpublished pilot studies 78 ). Histology showed residual acellular graft, likely oversized, in some nonfusing materials. Autograft success in larger rabbits supports this concept. 65 The volume of bone graft material, area and depth of TP decortication, extent of surgical dissection and time point to sacrifice have not been standardized. 17,50,63,65,79,80 Overdissection dilutes bioactive molecule effect; rhBMP-2 in a muscle pouch only creates bone when adhered to its collagen sponge (or other carrier/scaffold) and not freely diluted away. 81 Other experimental options include interbody fusion.

CONCLUSION
In summary, this is a comprehensive attempt testing a multitude of commercially available bone graft substitutes across several classes in a side-byside comparison, in an animal model where fusion is a challenge to achieve and relies on effective grafting products. We demonstrated BMPs and DBMs fused at a higher rate compared with the alternative graft options: cellular allografts (Osteocel) and synthetics (Vitoss, Mosaic, Actifuse). These results were generally consistent across the OIS study (objective 1) and the PLF study (objective 2), as evidenced by statistical measurement of consistency (objective 3). Two materials were exceptions, 1 DBM-based product (Progenix Plus) and 1 plain allograft (OsteoSponge). These animal model data provide an indication of clinical efficacy.
Currently, surgeons are challenged to provide the best and most efficacious grafting options for our patients at reasonable costs. Graft adjuncts are often combined in clinical use, and may be mixed with autograft (local bone) to optimize bone healing and fusion. Future testing of combinations such as these may reveal synergies not apparent on the single material experiment presented here. However, the results of this study help to address the paucity of clinical data 82 and preclinical data to support decisions on selection of individual grafting materials to optimize bone healing in spinal fusion.