Jeffrey M. Toth

Effective doses of recombinant human bone morphogenetic protein-2 in experimental spinal fusion.

Study Design Nineteen dogs underwent L4‐L5 intertransverse process fusions with either 58 μg, 115 μg, 230 μg, 460 μg, or 920 μg of recombinant human bone morphogenetic protein‐2 carried by a polylactic acid polymer. A previous study (12 dogs) compared 2300 μg of recombinant human bone morphogenetic protein‐2, autogenous iliac bone, and carrier alone in this model. All fusions subsequently were compared. Objectives To characterize the dose‐response relationship of recombinant human bone morphogenetic protein‐2 in a spinal fusion model. Summary of Background Data Recombinant osteoinductive morphogens, such as recombinant human bone morphogenetic protein‐2, are effective in vertebrate diaphyseal defect and spinal fusion models. It is hypothesized that the quality of spinal fusion produced with recombinant human bone morphogenetic protein‐2, above a threshold dose, does not change with increasing amounts of inductive protein. Methods After decortication of the posterior elements, the designated implants were placed along the intertransverse process space bilaterally. The fusion sites were evaluated after 3 months by computed tomography imaging, high‐resolution radiography, manual testing, mechanical testing, and histologic analysis. Results As in the study using 2300 μg of recombinant human bone morphogenetic protein‐2, implantation of 58–920 μg of recombinant human bone morphogenetic protein‐2 successfully resulted in intertransverse process fusion in the dog by 3 months. This had not occurred in animals containing autograft or carrier alone. The cross‐sectional area of the fusion mass and mechanical stiffness of the L4‐L5 intersegment were not dose‐dependent. Histologic findings varied but were not related to rhBMP‐2 dose. Inflammatory reaction to the composite implant was proportional inversely to the volume of the fusion mass. Conclusions No mechanical, radiographic, or histologic differences in the quality of intertransverse process fusion resulted from a 40‐fold variation in dose of recombinant human bone morphogenetic protein‐2.

The use of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote spinal fusion in a nonhuman primate anterior interbody fusion model.

Study Design. A study on the efficacy of recombinant human bone morphogenetic protein 2 (rhBMP‐2) in a nonhuman primate anterior interbody fusion model. Objectives. To investigate the efficacy of rhBMP‐2 with an absorbable collagen sponge carrier to promote spinal fusion in a nonhuman primate anterior interbody fusion model. Summary of Background Data. RhBMP‐2 is an osteoinductive growth factor capable of inducing new bone formation in vivo. Although dosage studies using rhBMP‐2 have been performed on species of lower phylogenetic level, they cannot be extrapolated to the primate. Dosage studies on nonhuman primates are essential before proceeding with human primate application. Methods. Six female adult Macaca mulatta (rhesus macaque) monkeys underwent an anterior L7‐S1 interbody lumbar fusion. All six sites were assigned randomly to one of two fusion methods: 1) autogenous bone graft within a single freeze‐dried smooth cortical dowel allograft cylinder (control) or 2) rhBMP‐2‐soaked absorbable collagen sponges within a single freeze‐dried smooth cortical dowel allograft cylinder also soaked in rhBMP‐2. The animals underwent a baseline computed tomography scan followed by 3‐ and 6‐month postoperation scans. Anteroposterior and lateral radiographs of the lumbosacral spine were performed monthly. After the monkeys were killed, the lumbar spine fusion sites were evaluated. Histologic evaluation of all fusion sites was performed. Results. The three monkeys receiving rhBMP‐2‐ soaked collagen sponges with a freeze‐dried allograft demonstrated radiographic signs of fusion as early as 8 weeks. The control animals were slower to reveal new bone formation. The computed tomography scans revealed extensive fusion of the L7‐S1 lumbar vertebrae in the group with rhBMP‐2. A pseudarthrosis was present in two of the control animals. Conclusions. This study was able to document the efficacy of rhBMP‐2 with an absorbable collagen sponge carrier and a cortical dowel allograft to promote anterior interbody fusion in a nonhuman primate model at a dose of 0.4 mg per implant site (1.5 mg/mL concentration). The rate of new bone formation and fusion with the use of rhBMP‐2 and cortical dowel allograft appears to be far superior to that of autogenous cancellous iliac crest graft with cortical dowel allograft.

Evaluation of rhBMP-2 with an OPLA carrier in a canine posterolateral (transverse process) spinal fusion model.

Study Design Posterolateral L4-L5 transverse process fusions were done on 14 adult beagles. Six were implanted with recombinant human bone morphogenetic protein-2 carried by open-cell polylactic acid polymer delivery vehicle. Six received autogenous iliac bone graft. Two received carrier alone. Eleven were killed 3 months after implantation. One in each group was maintained for 8 months. Objectives. To compare recombinant human bone morphogenetic protein-2 and open-cell polylactic acid polymer with autogenous iliac bone for inducing transverse process fusion in the canine by 3 months and to determine whether transverse process decortication and implantation of carrier alone causes spontaneous transverse process fusion in the canine. Summary of Background Data Recombinant human bone morphogenetic proteins have healed segmental long bone defects in several models. They have induced interlaminar and facet fusions in canines. Interlaminar and facet fusions have occurred after sham decortications in canines. Recombinant human bone morphogenetic protein-2 has not been evaluated for transverse process fusion in canines. Transverse process fusion is a preferred clinical method for achieving posterior lumbar fusion. Methods Fusion sites were evaluated by serial computed tomography scans. After the dogs were killed, explanted spines were subjected to manual testing, mechanical testing, high resolution radiography, and histologic analysis. Results One hundred percent of recombinant human bone morphogenetic protein-2-implanted sites had solid transverse process fusion by 3 months according to all measures. No autografted sites were fused at this interval. Osseous bridging of posterolateral gutters occurred in the recombinant human bone morphogenetic protein-2-implanted sites after 2 months, the earliest radiographic measure. None of the carrier-only sites showed bone formation. Conclusions Recombinant bone morphogenetic protein-2 carried by open-cell polylactic acid polymer is superior to autogenous iliac bone for producing radiographically and mechanically solid transverse process fusions in canines by 3 months. Spontaneous transverse process fusion does not occur in canines after decortication and open-cell polylactic acid polymer implantation.

Prospective Comparison of Autograft vs. Allograft for Adult Posterolateral Lumbar Spine Fusion: Differences Among Freeze-Dried, Frozen, and Mixed Grafts

The use of allografts continues today for the purpose of spinal fusion. The literature is conflicting on the effectiveness of allografts in spinal fusion as compared with autografts. Numerous variables affect fusion, including age, sex, smoking status, type of surgery, and anterior versus posterior surgery. Therefore, we have undertaken a prospective study in which comparison was made between allograft and autograft in the same individual. The purpose of this prospective study was to compare autografts, frozen allografts, freeze-dried allografts, and a mixture of allograft and autograft in the same patient undergoing an instrumented posterolateral lumbar spine fusion. Twenty patients (nine men and 11 women with ages ranging from 29-72 years and a mean age of 43.5 years) underwent posterolateral fusions of the lumbar spine with pedicle screw instrumentation. An autogenous posterior iliac crest bone graft was placed on one side in each patient and an allograft on the other side. Assessing bone fusion quality from grades 1 to 4, the autograft side had a grade 1 solid fusion in 16 of 20 cases (80%). On the other hand, freeze-dried grafting resulted in grade 4 resorption in all seven cases. Frozen allografts resorbed in three of five cases, and partial fusions were achieved in the remaining cases. When a mixture of autograft and freeze-dried allograft was used, grade 1 solid fusion was achieved in four of eight cases and partial fusions were achieved in the others. Bone densitometry results also showed that autograft sites gave significantly greater bone density, followed by mixture, frozen allografts, and freeze-dried allografts in this order.(ABSTRACT TRUNCATED AT 250 WORDS)

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part I.

The study presented here investigated hydroxyapatite biomaterials implanted in soft-tissue sites in adult sheep to determine whether these materials are osteoinductive and whether the rate of osteoinduction can be increased by manipulating the composition and porosity of the implants. For the study, 16.8-mm x 5-mm discs were prepared from mixtures of hydroxyapatite and beta-tricalcium phosphate. Five mixtures of hydroxyapatite-ceramic and hydroxyapatite-cement paste forms were studied: 100 percent hydroxyapatite-ceramic (Interpore), 60 percent hydroxyapatite-ceramic, 100 percent hydroxyapatite-cement paste, 60 percent hydroxyapatite-cement paste, and 20 percent hydroxyapatite-cement paste. Biomaterials were implanted in subcutaneous and intramuscular soft-tissue pockets in 10 adult sheep. Cranial bone grafts of equal dimension were implanted as controls. One year after implantation, the volume of all biomaterials and bone grafts was determined from a computed tomographic scan, and porosity and bone formation were determined using backscatter electron microscopy. Cranial bone and the 20 percent hydroxyapatite-cement paste implants demonstrated significant volume reduction in all sites after 1 year (p < 0.001). No significant difference in volume of the remaining four biomaterials was found. There was no significant change in pore size in the ceramic implants (range, 200 to 300 micro) and in the cement-paste implants containing 60 percent hydroxyapatite or more (range, 3 to 5 nm). Pore size in the cement-paste implants containing 20 percent hydroxyapatite increased significantly with resorption of the tricalcium-phosphate component, reaching a maximum of 200 to 300 micro in the periphery, where the greatest tricalcium-phosphate resorption had occurred. Both ceramic biomaterials demonstrated lamellar bone deposition within well-formed haversian systems through the entire depth of the implants, ranging from a mean of 6.6 percent to 11.7 percent. There was minimal bone formation in the cement-paste implants containing 60 percent hydroxyapatite or more. In contrast, cement-paste implants containing 20 percent hydroxyapatite demonstrated up to 10 percent bone replacement, which was greatest in the periphery of the implants where the greatest tricalcium-phosphate resorption had occurred. This study confirms the occurrence of true osteoinduction within hydroxyapatite-derived biomaterials, when examined using backscatter techniques. In this study, the rate of osteoinduction was greatest when a porous architecture was maintained, which was best achieved in ceramic rather than cement-paste forms of hydroxyapatite. Porosity and resultant bone formation in cement-paste implants can be improved by combining hydroxyapatite with a rapidly resorbing component, such as tricalcium phosphate.

Principles of Tissue Engineering Applied to Programmable Osteogenesis

This article presents a strategy for design, engineering, and fabrication of a bioresorbable, manufactured bone graft substitute (BGS) device. The approach is based on established precepts of osteogenesis, molecular biology of hyaluronic acid and osteoinductive proteins, and theoretical preformance criteria for such a device collated from the literature of 1991 to 1996. Application of this design and engineering strategy results in a composite device consisting of a D,D-L,L-polylactic acid macrostructure optimized to the architecture of cancellous bone, a microstructure composed of a filamentous velour of hyaluronan and a recombinant human bone morphogenetic protein 2 (rhBMP-2). The performance of this construct was tested in vivo in the dog, intertransverse process, spinal fusion model and in a critical sized defect of the rabbit radius. Data from these studies are used to illustrate principle points of the design and engineering concept.

Experimental spinal fusion with recombinant human bone morphogenetic protein-2 without decortication of osseous elements

Study Design. L4-L5 intertransverse process fusions were produced with 58 μg, 230 μg, or 920 μg of recombinant human bone morphogenetic protein-2 in 20 dogs. Eleven had traditional decortication of posterior elements before insertion of the implant. Nine were left undecorticated. All animals were evaluated 3 months after surgery. Objectives. To determine whether decortication is a prerequisite for successful fusion in the presence of osteoinductive proteins such as bone morphogenetic protein-2. Summary of Background Data. Recombinant osteoinductive proteins can induce de novo bone in ectopic soft-tissue sites in the absence of bone marrow elements. Traditional methods for achieving spinal fusion rely on exposure of bone marrow through decortication to facilitate osteogenesis. It is hypothesized that the presence of an implanted osteoinductive protein obviates the need for exposure and release of host inductive factors. Methods. Recombinant human bone morphogenetic protein-2-induced intertransverse process fusions were performed with and without decortication. Fusion sites were evaluated by computed tomography imaging, high-resolution radiography, manual testing, mechanical testing, and histologic analysis. Results. One hundred percent of decorticated spines and 89% of undecorticated spines were clinically fused by 3 months. Ninety-one percent of decorticated spines and 78% of undecorticated specimens exhibited bilateral transverse process osseous bridging. The only spines that failed to achieve solid bilateral arthrodesis were in the lowest dose group. With the higher two doses, there was histologic evidence of osseous continuity between the fusion mass and undecorticated transverse processes. Conclusions. There were no statistical differences in clinical and radiographic fusion rates between decorticated and undecorticated sites. With higher doses of recombinant human bone morphogenetic protein-2, there was little histologic distinction between fusions in decorticated versus undecorticated spines.

Histologic evaluation of the efficacy of rhBMP-2 compared with autograft bone in sheep spinal anterior interbody fusion.

Study Design. The sheep anterior lumbar spinal fusion model was used to study the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2)–collagen composite in comparison with autograft to enhance spinal interbody fusion. Comparisons were drawn from temporal radiographic and end-point biomechanical and histologic data. Objective. To analyze histologically the ability of rhBMP-2 to achieve complete arthrodesis between vertebral bodies. Summary of Background Data. Studies using rhBMP for enhancement of anterior interbody fusion have used numerous endpoints. However, systematic histologic evaluation of the fusion has not been conducted. Methods. Twelve sheep underwent single-level anterior lumbar interbody fusion performed with a cylindrical fenestrated titanium interbody fusion device (INTER FIX, Medtronic Sofamor Danek, Inc., Memphis, TN). The device was filled either with rhBMP-2–collagen (n = 6) or autogenous iliac crest bone graft (n = 6). Radiologic evaluation was carried out at 2-month intervals, and all sheep were killed 6 months after surgery. Nondestructive biomechanical testing for stiffness to flexion, extension, and lateral bending moments, un-decalcified histology, and qualitative and quantitative histologic evaluation were performed. Results. Radiographs revealed a bony bridge anterior to the cage in five of six rhBMP-2-treated animals, whereas it was present only in one of five in the autogenous bone graft group. Segments treated with rhBMP-2 were 20% stiffer in flexion than autograft-treated segments at 6 months. Six of six in the rhBMP-2 group and two of six in the autograft group showed complete fusion. There was a significantly higher rate of bony continuity observed at the fenestrations of the rhBMP-2 group. Three times more number of cage fenestrations in the rhBMP-2 group demonstrated “all-bone” when compared with the autograft group (P < 0.001). Further, the scar tissue in and around the autograft-treated cages was 16-fold more (P < 0.01) than that seen for rhBMP-2-treated cages. Conclusions. The study demonstrates that rhBMP-2 can lead to earlier radiologic fusion and a more consistent increased stiffness of the segments when compared with autograft in sheep anterior lumbar interbody fusion. Furthermore, a three times higher histologic fusion rate is attainable with significantly reduced fibrous tissue around the implant when rhBMP-2 is used.

Evaluation of Porous Biphasic Calcium Phosphate Ceramics for Anterior Cervical Interbody Fusion in a Caprine Model

Study Design. This study compared the efficacy of characterized 50/50 hydroxyapatite/β-tricalcium phosphate ceramics of 30%, 50%, and 70% porosity and autograft to promote interbody spinal fusion at C2-C3 and C5-C6 in 24 goats: 12 at 3 months and 12 at 6 months. Objectives. Radiographs, histology, dual energy x-ray absorptiometry analysis, and biomechanical testing were used to evaluate the ability of the 30%, 50%, and 70% porous 50/50 hydroxyapatite/β-tricalcium phosphate ceramics and autograft to promote cervical interbody fusion. Summary of Background Data. The conundrum in the use of calcium phosphates for interbody fusion is what porosity is most effective to promote ingrowth yet strong enough to resist compressive stresses found in the spine? It is known that the ability for bone ingrowth increases and the compressive strength decreases as porosity of the ceramic is increase. Dense ceramics remain intact but may be surrounded by fibrous tissue. Porous ceramics have good ingrowth but may fracture. Methods. Radiographs were evaluated for fusion and fracture or collapse of the ceramics or autograft. Dual energy x-ray absorptiometry was used to evaluate the fusion mass. Treated motion segments underwent biomechanical testing to quantify the flexibility of the segment. Undecalcified and decalcified histologic analysis were performed to evaluate the presence or absence of a bony union. Results. Thirty percent, 50% and 70% porous ceramics had better radiographic fusion scores than the autograft at 3 and 6 months. Incidence of ceramic fracture did not increase with porosity and was equivalent to the collapse of autograft, although ceramics maintained disc height when fracture occurred. No statistically significant differences were found between autograft and the porous ceramics with biomechanical testing and peri-implant bone mineral density values as measured by dual energy x-ray absorptiometry. At 3 months, histologic analysis showed a union rate of 0% for autograft and 30% porous ceramic, 67% for 50% porous ceramic, and 83% for 70% porous ceramic. At 6 months, the union rate was 67% for the 30%, 50%, and 70% porous ceramics and 50% for autograft. Conclusions. Thirty percent, 50%, and 70% porous ceramics performed equal to or better than autogenous bone after 3 and 6 months. There may be promise for the use of 50/50 hydroxyapatite/β-tricalcium phosphate in spine surgery as the need to harvest autograft from the iliac crest is obviated, and complications and cost associated with the harvest are avoided.

Short-term Osteoclastic Activity Induced by Locally High Concentrations of Recombinant Human Bone Morphogenetic Protein–2 in a Cancellous Bone Environment

Study Design. An experimental study investigating osteoclastic activity induced by rhBMP-2 in sheep. Objective. To examine the effects of increasing local rhBMP-2 concentration on osteoclastic response and peri-implant bone resorption. Summary of Background Data. Level I clinical studies have established the safe and effective volume and concentration of rhBMP-2 delivered on an absorbable collagen sponge. However, peri-implant bone resorption appearing as decreased mineral density has been observed radiographically in rare instances after implantation of rhBMP-2 on an absorbable collagen sponge (rhBMP-2/ACS). Methods. Bilateral corticocancellous defects were created in the distal femora of 30 adult sheep. Combinations of rhBMP-2/ACS implant volume (V) (1V = normal fill or 2V = overfilled) and rhBMP-2 solution concentration (×) (1× = normal concentration or 3.5× = hyperconcentrated) resulted in local rhBMP-2 concentrations of 0×, 1×, 2×, 3.5×, and 7× the estimated effective concentration for this model. Faxitron radiography, quantitative CT, histology, and quantitative histomorphometry were conducted in a blinded fashion to analyze the effect of the treatments. Results. At 1 week, the normal fill-normal concentration implants (1×) produced the least transient osteoclastic activity resulting in limited peri-implant resorption. Overfilled-hyperconcentrated implants (2×, 3.5×) demonstrated moderate resorption zones. Overfilled-hyperconcentrated implants (7×) demonstrated extensive osteoclastic activity and marked resorption. Results at 4 and 8 weeks revealed dense osteoid and bone in the voids with progressive bony healing. Control defects showed no osteoclastic activity with little to no bony healing. Conclusion. Increasing the local rhBMP-2 concentration by overfilling the defect with rhBMP-2/ACS or hyperconcen-trating the rhBMP-2 solution on the absorbable collagen sponge led to a concentration-dependent osteoclastic resorption of peri-implant bone. The osteoclastic effect was transient, and progressive healing took place over the 8-week survival period.