Elsevier

The Spine Journal

Volume 7, Issue 3, May–June 2007, Pages 318-325
The Spine Journal

Technical Report
rhBMP-6 stimulated osteoprogenitor cells enhance posterolateral spinal fusion in the New Zealand white rabbit

https://doi.org/10.1016/j.spinee.2006.02.005Get rights and content

Abstract

Background context

The nonunion rate after posterolateral spinal fusion can be as high as 35%. This has stimulated interest in the development of techniques for enhancing new bone formation, including the addition of bioactive peptides or the use of cell-based therapies, including genetically modified cells. In previous studies we have demonstrated that exposing autologous, marrow-derived osteoprogenitor cells to a recombinant human bone morphogenetic protein-6 (rhBMP-6) containing extracellular matrix induces osteoblastic differentiation, and that these cells are capable of increasing new bone formation. Growth of autologous cells on a synthetic rhBMP-6 containing matrix yields a population of stimulated osteoprogenitor cells, without the expense of adding large amounts of rhBMP-6 directly, or the risks inherent in the use of genetically altered cells.

Purpose

This study was performed to evaluate the potential of rhBMP-6 stimulated osteoprogenitor cells (stOPC) to enhance the rate and strength of posterolateral spinal fusion.

Study design

Prospective in vivo animal study

Outcome measures

Radiographic evidence of spinal fusion, biomechanical testing of explanted spines, histological analysis of new bone formation

Methods

Single-level posterolateral spinal arthrodeses were performed in 69 New Zealand white rabbits. Autologous marrow stem cells were concentrated and then plated on an rhBMP-6-rich extracellular matrix synthesized by genetically engineered mouse C3H10T1/2 cells. Animals in Groups I (n=18) and II (n=18) received autografts of 30M and 60M rhBMP-6 stOPCs in guanidine extracted demineralized bone matrix (gDBM), respectively, whereas those in Group III (n=13) received iliac crest bone graft (ICBG). Those in Group IV (n=10) received gDBM, and those in Group V (n=10) underwent decortication only. Assessment of fusion was made with serial radiographs, manual palpation of the explanted spines, and biomechanical testing. The fusion masses from two animals each in Groups I, II, and IV were evaluated histologically.

Results

Fifty-three animals were available for analysis at the conclusion of the study. In these animals, the arthrodesis rate was significantly higher after treatment with rhBMP-6 stOPCs (77% for both Groups I and II by palpation) than ICBG, gDBM, or decortication alone (Group III=55%, IV=20% and V=0%, respectively). Similarly, the peak loads to failure of the fusion masses in Groups I and II (212.5±37.8 N and 234.6±45.7 N) were significantly greater than the corresponding values in the other groups (Group III=155.9±36.4N, Group IV=132.7±59.9N, and Group V=92.8±18.4N), though when only the fused specimens in Groups I, II, and III were compared, only Group II was significantly different than Group III (234.6±45.7N and 155.9±36.4N, respectively). The fusion masses in the rhBMP-6 stOPC-treated animals were typified by a thin, fusiform cortical shell, newly formed trabecular bone emanating from the decorticated transverse processes, and residual unremodeled gDBM carrier particles. The fusion masses in the gDBM treated bones were morphologically similar, though they contained less newly formed bone.

Conclusions

The use of rhBMP-6 stOPCs in a carrier of gDBM significantly enhanced the rate and strength of single-level posterolateral spinal arthrodeses in the New Zealand white rabbit, compared with ICBG, gDBM, and decortication alone. Our results confirm that the stimulation of marrow-derived osteoprogenitor cells by growing them on a rhBMP-6 containing extracellular matrix is feasible. Further investigation is warranted to determine the relative contribution of rhBMP-6 stimulation and the number of cells implanted, as well as strategies for optimizing the technique for clinical application.

Introduction

One of the most important factors in achieving spinal fusion is the use of an osteogenic or osteoinductive agent to stimulate the bridging of bone from one vertebra to the next. Currently, autogenous iliac crest bone graft (ICBG) is the material of choice for this application; however, because of the complications and limitations associated with its use [1], [2], several strategies have been explored to enhance its function, or replace it altogether. Early efforts included the use of autograft substitutes, such as allograft bone and calcium phosphate ceramics, while subsequent groups have experimented with cytokines, growth factors, and gene therapy [3], [4], [5], [6], [7]. More recently, several investigators have evaluated strategies involving the use of cell-based therapies [8], [9], [10], [11].

For the past several years, we have been developing a technique for stimulating marrow-derived osteoprogenitor cells by exposing them to a matrix rich in recombinant human bone morphogenetic protein-6 (rhBMP-6) secreted by a transformed murine cell line. Our work is an extension of other studies that have shown that treatment of osteoprogenitor cells with bone morphogenetic proteins (BMPs) in vitro induces differentiation towards an osteoblastic lineage [12], [13], [14]. Our preliminary data, some of which have been published in abstract form (see Babat et al. Trans. 46th Orthopaedic Research Society, 2000;25:363), suggest that this exposure induces osteoblastic differentiation and that the induced cells are capable of increasing new bone formation.

This study was performed to evaluate the osteogenic capacity of two concentrations of BMP-6 stimulated osteoprogenitor cells (rhBMP-6 stOPCs) in posterolateral intertransverse spinal fusions in the New Zealand white rabbit. In particular, we hypothesized that the use of rhBMP-6 stOPCs would improve the rate of fusion and the biomechanical properties of the arthrodesed intertransverse levels in the rabbit spinal fusion model, compared with autogenous iliac crest bone graft.

Section snippets

Materials and methods

After Institutional Animal Care and Use Committee (IACUC) approval, single-level, bilateral intertransverse process spinal fusions were performed on 69, 8-month-old New Zealand white rabbits (3.8±0.5 kg) [15]. The animals were randomly assigned to one of five experimental groups: Groups I and II received osteoprogenitor cells in guanidine extracted demineralized bone matrix (gDBM), Group III received morselized autogenous iliac crest bone graft (ICBG), Group IV received gDBM carrier alone, and

Results

Of the 69 animals originally enrolled in the study, 53 were ultimately available for evaluation; 16 animals were excluded because of technical problems and perioperative morbidity, including anesthetic complications (4), wrong level fusion (3), infection (3), seromas (3), and immediate postoperative hind limb palsy (3).

Palpation. The fusion rate was highest in the two groups of animals that received grafts of rhBMP-6 stOPCs (Groups I and II). In both, the fusion rates were 77%, which was

Discussion

This study was performed to evaluate the osteogenic capacity of autologous osteoprogenitor cells exposed to an extracellular matrix containing rhBMP-6. Single-level (L5–L6) spinal fusions were performed in two groups of rabbits treated with rhBMP-6 stOPCs (30×106 cells and 60×106 cells, respectively), and the fusion rates and biomechanical properties of the fusion masses were compared with those of animals grafted with autogenous iliac crest bone graft, gDBM, and untreated controls. Our results

Acknowledgments

The authors thank Scott A. McAllister, BS, Michael J. Barnum, MD, and L. Brett Babat, MD for their technical assistance, and Jason T. Machan, PhD for consultation regarding the statistical analysis.

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    Funding for this work was provided by the RIH Orthopaedic Foundation, Inc. and University Orthopedics, Inc.

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