ARTHRODESIS OF the transverse processes of lumbar vertebrae. Arthrodesis of Lumbar Spine Transverse Processes Using Nacre in Rabbit

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1 JOURNAL OF BONE AND MINERAL RESEARCH Volume 16, Number 12, American Society for Bone and Mineral Research Arthrodesis of Lumbar Spine Transverse Processes Using Nacre in Rabbit MERIEM LAMGHARI, 1 PIERRE ANTONIETTI, 2 SOPHIE BERLAND, 1 ALEX LAURENT, 3 and EVELYNE LOPEZ 1 ABSTRACT This study compares the osteogenic effects of nacre and autogenous bone grafts in a rabbit model of lumbar spine transverse process arthrodesis. A total of 15 rabbits were processed for arthrodesis between the fifth and sixth lumbar vertebrae using nacre powder mixed with autologous blood or autogenous iliac crest bone. Control rabbits were sham operated. Sample vertebrae were removed from the nacre-implanted rabbits at 2, 5, and 11 weeks postsurgery. The autogenous bone graft and sham-operated groups were processed for histological study 11 weeks postsurgery. The results for the three groups were compared at 11 weeks. The nacre-implanted samples taken at 2 weeks showed that the nacre was well tolerated by the host tissue. Endochondral bone formation was seen in the region of the dissolving nacre particles by 5 weeks. The newly formed bone formed a solid fusion between the transverse processes in one-third of the rabbits. There was still new bone formation at 11 weeks at the nacre implant site. Two-thirds of the rabbits had formed a solid fusion. Light microscopy also showed new bone formation 11 weeks after the autologous bone graft. All rabbits had a solid fusion. This initial study indicates that nacre can induce spinal fusion in an acceptable percentage of cases. (J Bone Miner Res 2001;16: ) Key words: biomaterial, nacre, osteogenesis, orthopaedic surgery, axial correction INTRODUCTION ARTHRODESIS OF the transverse processes of lumbar vertebrae is one of the most common forms of spinal fusion used to correct axial deviation. Autogenous iliac crest bone is considered to be the best bone graft material because it gives good clinical results. However, there are limits to the use of autologous bone such as the lack of donor tissue and damage to the donor site. The use of allogenic bone also is associated with complex problems such as the transmission of infectious disease, immunogenicity, and rejection. (1) These restrictions have led numerous authors to look for alternative bone graft materials. (2 4) The mother-of-pearl (nacre) from the shell of the giant oyster Pinctada maxima is a natural biomaterial made up of a mineral phase, aragonite, and a cell-free organic matrix. Our earlier studies on the osteogenic effects of nacre showed that it could stimulate bone regeneration. (5) In vivo studies have shown that it is biocompatible and is recognized by bone cells. (6,7) There also is evidence that it can induce new bone formation. (8 10) In vitro studies have shown that diffusible factors released by the nacre are implicated in the stimulation of bone-forming cells, resulting in the formation of cells with the full osteoblast phenotype and the formation of bone itself. (6,7) We have now used the lumbar arthrodesis model described by Boden et al. to assess the capacity of nacre to stimulate the fusion of transverse processes in the lumbar spine of the rabbit and to compare its effects with those of autologous bone grafts. (11) 1 Laboratoire de Physiologie Générale et Comparée, UMR CNRS 8572, Muséum National d Histoire Naturelle, Paris Cedex 05, France. 2 Département de Chirurgie Orthopédique, Clinique Jouvenet, Square Jouvenet, Paris, France. 3 Service de Neuroradiologie Diagnostique et Interventionnelle, Hôpital Lariboisière, Paris, France. 2232

2 LUMBAR SPINE ARTHRODESIS USING NACRE MATERIALS AND METHODS Animal model This study was done on 15 New Zealand white rabbits (weight, 4 kg) supplied by the Institut National de Recherche Agronomique (INRA; Jouy en Josas, France). Each rabbit underwent bilateral transverse process scraping between the fifth and sixth lumbar vertebrae and each side was implanted with one of two bone graft materials: autogenous iliac crest bone or nacre powder mixed with autologous blood. Control rabbits were sham operated with scraping of the transverse processes but no implantation. Materials Mother-of-pearl (nacre) was obtained from Broome, Northern Australia, and ground by the process of SIERA S.A. (Paris, France) to give a powder (particle size, m). It was sterilized by -irradiation (2.5 rad) and mixed with autologous venous blood to produce a workable paste (graft material volume, 2.5 ml). Autogenous bone graft: Four bone fragments were removed from the iliac crest of each rabbit. The samples were cleaned of soft tissue, fragmented (graft material volume, 2 ml), and implanted between the lumbar transverse processes. Surgery To produce an analogous system, the protocol used was that of Boden et al. (11) Each rabbit was anesthetized by the subcutaneous injection of a mixture of ketamine (35 mg/kg), acepromazine (1 mg/kg), and xylazine (8 mg/kg). Animals were given an initial injection to facilitate shaving the operation site and a second injection immediately before making the initial incision. The surgical approach was median and posterior over the dorsal spines of vertebrae L5 and L6, which were located by palpation and their distance from the iliac crests. The skin was opened by a median incision. This incision was short because the skin is very mobile. The spinal muscles were then retracted and the intraprocess muscles were left in place to prevent loss of graft material. Cautery was unnecessary because spontaneous clotting was very rapid. Then, the graft material was inserted in a space 20 mm thick between the decorticated transverse processes. The muscles were then replaced and the incision was closed. Follow-up The care, housing, and feeding (Center de Recherche en Imagerie Interventionnelle, CRII, Jouy en Josas, France) of the rabbits complied with the regulations governing the conditions for experimental animals. The mobility and condition of all animals were checked daily. All animals were killed by euthanasia in compliance with the requirements of ethical practice. X-rays were taken of the lumbar vertebrae of all rabbits before death to assess the degree of vertebral fusion. The rabbits given the nacre grafts were killed at 2 weeks (n 3), 5 weeks (n 3), and 11 weeks (n 3) postsurgery to determine the time course of the action of the implant. The rabbits given autogenous bone grafts (n 3) and the control sham-operated animals (n 3) were killed at 11 weeks postsurgery. The three groups of rabbits (nacre graft, autogenous graft, and control) were compared at 11 weeks postsurgery. The lumbar vertebrae (L5 L6) were removed, and each fusion was evaluated qualitatively by manual palpation. Then, the samples were prepared for histological analysis. Tissue sampling and histology Lumbar vertebrae (L5 L6) were fixed by immersion in 70% ethanol containing 0.5% basic fuchsin for 30 days, dehydrated in an alcohol series, defatted by immersion in xylene for 30 days, and embedded in methylmethacrylate. The embedded vertebrae were reexamined by X-ray to locate the vertebral fusion zones. Then, serial sections (100 m) were cut in a sagittal plane using a diamond saw. Microradiographs were taken (Massiot-Phillips Pw 1008 X-ray apparatus, 25 kv, 12 ma; Phillips Medical Systems, Paris, France) of the undecalcified contiguous sections using high-resolution contact film (Eastman Kodak Co., Rochester, NY, USA). These microradiographs were examined under transmitted light microscopy to assess qualitatively the mineralization of bone. The 100 m sections were ground down to 15 m between sheets of plate glass and stained with toluidine blue at ph 2.3. The stained sections were examined under transmitted, polarized light microscopy. RESULTS Radiography and manual palpation Radiographs were taken of the spines implanted with nacre at 2, 5, and 11 weeks postimplantation. They revealed bone between the vertebrae starting at 5 weeks postimplantation. Manual palpation at this time indicated a solid fusion in one-third of the implanted rabbits. X-rays of the rabbits with autologous bone grafts (Fig. 1a) and nacre grafts (Fig. 2a) taken 11 weeks postimplantation showed bone between the vertebrae of both groups. The control, sham-operated rabbits showed no bone formation (Fig. 3a). The rate of fusion determined qualitatively by manual palpation of the spines indicated solid fusion in two-thirds of the nacre-implanted rabbits and in all 3 of the boneimplanted rabbits. Light microscopy Comparison of the nacre graft, autogenous bone graft or sham-operated rabbits at 11 weeks postsurgery showed new bone formation within the fusion in the nacre-implanted and bone-implanted rabbits. Similarly, the new bone lamellae were at various stages of maturation (Figs. 1b, 1c, 2b, and 2c). There was no new bone formation between the transverse processes in the sham-operated controls (Figs. 3b and 3c) Histocytology of the graft regions 2233 Autogenous bone graft: The microscope images of the implants with autogenous bone grafts showed both endo-

3 2234 LAMGHARI ET AL. FIG. 1. Autogenous crest iliac bone graft (11 weeks postsurgery). (a) Radiograph of rabbit lumbar spine 11 weeks after transverse process arthrodesis using an autogenous bone graft. Bone fusion can be seen (3) between the fifth and sixth lumbar vertebrae (I, iliac crest). (b) Section through an arthrodesis site 11 weeks after fusion of transverse processes using an autogenous bone graft. There is endochondral bone formation at the implant site ( ). There also is bone formation directly ( ) on the residual fragments of grafted bone ( ). Newly formed bone trabeculae (B; toluidine blue staining; magnification 8). (c) Contact microradiograph of detail shown in panel b ( )., calcified cartilage; B, newly formed bone trabeculae (magnification 16). FIG. 2. Nacre graft (11 weeks postsurgery). (a) Radiograph of rabbit lumbar spine implanted with nacre, taken 11 weeks after surgery. Bone fusion can be seen (3) between the fifth and sixth lumbar vertebrae. (b) Section through an arthrodesis site 11 weeks after nacre graft between the L5 and L6 transverse processes. There is endochondral bone formation in contact with dissolving nacre particles (N). C, cartilage; B, bone trabeculae (toluidine blue staining; magnification 16). (c) Contact microradiograph of a histological section through an arthrodesis site in which a nacre graft was used. The nacre particles (N) lie at the edge of the newly formed bone., Bone trabeculae;, intertrabecular space (magnification 2). chondral and membranous ossification at the implantation site (Fig. 1b). Fragments of residual bone from the autologous graft were still present. Their surfaces were lined with numerous resorption pits. Histological sections showed osteoblastic activity along the periphery of the remaining implant. There also were islands of endochondral bone formation at the implantation site. The new bone forming the fusion was organized as trabeculae around spaces filled with bone marrow (Fig. 1b).

4 LUMBAR SPINE ARTHRODESIS USING NACRE 2235 implant was being dissolved. There was new endochondral bone formation at the implantation site. There was a gradient of cytodifferentiation of cartilage cells starting at the dissolving nacre particles going toward the new bone lamellae (Figs. 4a 4c). There were dense populations of large activated fibroblastic cells with a clear cytoplasm near the nacre particles. Their nuclei were oval, irregular/lobular, and contained large nucleoli. Their plasma membranes were basophilic. These cells also synthesized an extracellular matrix that was stained by toluidine blue, indicating the presence of proteoglycans. The differentiation of these cells into chondroblasts at the implantation site resulted in smaller rounded cells with hyalin cytoplasm (Fig. 4a). These cells had the morphology of chondrocytes. Mature chondrocytes were organized into cartilage-type linear structures and they were present before the formation of new bone (Fig. 4b). The new bone was rich in osteocytes and had osteoid borders lined with plump osteoblasts. It was organized as anastomosing bone lamellae outlining the intertrabecular spaces filled with bone marrow (Fig. 4c). Polarized light microscopy showed both woven and lamellar bone at various stages of maturation. Those rabbits that had a solidly fused mass had more new bone. The histological sections and corresponding microradiographs prepared at 11 weeks postimplantation showed that the new bone had a lamellar structure. The intertrabecular spaces were all filled with bone marrow. There was still endochondral bone formation at this postoperative time (Fig. 4b). The dissolving nacre particles lay around the cartilaginous zones, which were formed before new bone formation (Fig. 4a). Sham operated New bone was formed at the end of the apophyses that had been decorticated. However, the histological sections and contact microradiographs showed no new bone formation within the space between the processes (Figs. 3b and 3c). DISCUSSION FIG. 3. Sham-operated rabbits (control; 11 weeks postsurgery). (a) Radiograph of rabbit lumbar spine 11 weeks after exposure of the transverse processes of the fifth and sixth lumbar vertebrae. There is no new bone formation between the transverse processes (I, iliac crest). (b) Section showing the end of a transverse process 11 weeks after sham operation. The end of the transverse apophysis is closed by spontaneous bone formation (3)., Connective tissue (toluidine blue staining; magnification 32). (c) Contact microradiograph of the end of a transverse apophysis 11 weeks after exposure. The end of the transverse apophysis is closed by new bone formation (3), but there is no new bone formed from the connective tissue ( ; magnification 32). Nacre graft: Histological sections prepared 2 weeks after arthrodesis with nacre showed that the nacre was well tolerated by the host tissue. There was a modest inflammatory reaction at the implantation site. Microradiographs prepared 5 weeks after implantation showed that the nacre This study indicates that nacre powder can be used as a bone graft material for spinal arthrodesis. Implants of nacre from Pinctada maxima shell appear to be able to induce new bone formation between the transverse processes of rabbit vertebrae in very much the same way as autologous bone implants. The newly formed bone is physiologically active; it led to the solid fusion of vertebrae in two-thirds of the rabbits tested. The nacre implanted at sites lacking stem cells or precursor cells led to endochondral bone formation. This suggests that the release of active factors from the nacre as it dissolves leads to the recruitment and differentiation of fibroblasts from the connective tissue between the transverse processes to give rise to bone cells. The histological evidence indicates that the pattern is that of normal endochondral bone formation. Thus, the ectopic formation of cartilage during endochondral bone formation is the first criterion of differentiation indicating that bone induction is taking place. (12) The first phenotypic indication of cartilage cells, the secretion of an extracellular matrix containing proteogly-

5 2236 LAMGHARI ET AL. FIG. 4. The cellular events of endochondral bone formation induced by nacre. (a) Dissolving particles of nacre (N), prechondroblasts (3), and chondroblasts ( ). Magnification 32. (b) Chondroblasts ( ), chondrocyte proliferation ( ), and hypertrophy of these cells ( ) metachromatic extracellular matrix., Endochondral resorption (toluidine blue staining; magnification 32). (c) New bone formation on the cartilage scaffold ( ). New bone trabeculae (B) of the bone bonding the two bridged transverse processes (magnification 32). cans, was indicated clearly by the staining with toluidine blue. (12 16) Such a matrix was found around the differentiating fibroblasts and around the chondroblasts after nacre implantation. Nacre implanted subcutaneously in rats also triggers the formation of new cartilage, but this cartilage remains transient and does not lead to new bone formation. (17) In contrast, nacre implanted between the transverse processes of spinal rabbits induces cartilage formation that then goes on to form bone. The sequences of endochondral bone formation at the nacre and autologous bone implant sites were analogous. Cartilaginous foci were present near the newly forming bone at the autologous bone implant sites up to 11 weeks after implant. Similarly, there were cartilaginous foci at the periphery of newly forming bone 11 weeks after implanting nacre. In the two cases, the new bone consisted of anastomosing trabeculae at various stages of development surrounding intertrabecular areas filled with bone marrow. Autologenous bone grafts and demineralized bone matrix have been used frequently for fusing vertebral transverse processes. Several studies on demineralized bone matrix have shown that it is better than unmodified bone at inducing bone formation. (18 20) Thus, it has been shown that demineralized bone can be used to facilitate the breakdown of implanted biomaterials and to allow the release of inducing proteins such as BMPs from the bone matrix. (20,21) Recently, studies on several animal models have shown that these proteins can be used successfully for the fusion of vertebral transverse processes. (3,4,11,22,23) Boden et al., working on arthrodesis in rabbits, showed that bovine osteoinductive protein, combined with various carriers, led to spinal fusion by 5 weeks postsurgery. (4,11) Implanting human recombinant bone morphogenetic protein 2 (rhbmp-2) resulted in the formation of a bone bridge between transverse processes in 4 weeks in rabbits and in 24 weeks in adult rhesus monkeys. (3,23) In this study, nacre implant led to spinal fusion by 5 weeks. Our earlier in vitro findings suggest that the formation of new bone in association with nacre is stimulated by signal proteins from within the nacre organic matrix. (6,7,10,24 26) These proteins seem to be released in situ during the dissolution of the nacre particles, where they can induce the formation of new endochondral bone between the transverse processes of the lumbar vertebrae in the same way as an autologous bone implant does. The cell-free mineralized matrix of nacre is water soluble, unlike that of bone. This may facilitate the diffusion of bioactive factors in situ, making them accessible for study without the use of any denaturing demineralization step. (24) Our in vitro studies with rat bone marrow stroma cells indicate that a water-soluble extract of nacre organic matrix increases the alkaline phosphatase activity of these cells. (10) Human fibroblasts respond similarly to a water-soluble extract of nacre organic matrix, with a dose-dependent increase in alkaline phosphatase activity. (25) Wolfinbarger obtained similar results with human skin fibroblasts cultured with demineralized bone. (27) The similarity between the osteogenic effects of nacre and boneinducer proteins suggests that the organic matrix of nacre

6 LUMBAR SPINE ARTHRODESIS USING NACRE contains one or more materials that are bone inducers that can lead to the formation of new bone. The proteins in a water-soluble extract of nacre matrix were fractionated and their biological activity was tested. The osteogenic potential was associated with a specific protein fraction, which now is being sequenced. (25) The results of this initial study indicate that the nacre from Pinctada maxima can, like autologous bone, stimulate the formation of a bone bridge between the transverse processes of rabbit lumbar vertebrae. Additional studies will be needed to quantify the improvement in bone stimulation by nacre organic matrix compounds and compare them with other osteoinductive materials. However, the success rate of this prospective investigation appears to be promising. ACKNOWLEDGMENTS This study was supported by a grant from the French Association against Myopathies and funds from the CNRS/ Industry (SIERA S.A., Paris, France). 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J Mater Sci Mater Med 12: Wolfinbarger L Jr, Zheng Y 1993 An in vitro bioassay to assess biological activity in demineralized bone. In Vitro Cell Dev Biol Anim 29A: Address reprint requests to: Prof. Evelyne Lopez Laboratoire de Physiologie Générale et Comparée UMR CNRS 8572 Muséum National d Histoire Naturelle 7 Rue Cuvier Paris Cedex 05, France Received in original form July 26, 2000; in revised form January 29, 2001; accepted April 13, 2001.