| | The effect of spinal instrumentation particulate wear debris: an in vivo rabbit model and applied clinical study of retrieved instrumentation cases☆☆☆★Received 2 March 2002; accepted 28 May 2002. Abstract Study design: The current study was undertaken to determine if the presence of spinal instrumentation wear particulate debris deleteriously influences early osseointegration of posterolateral bone graft or disrupts an established posterolateral fusion mass. Objectives: Using an in vivo animal model, the first phase (basic science) of this study was to evaluate the effect(s) of titanium wear particulate on a posterolateral spinal arthrodesis based on serological, histological and immunocytochemical analyses. The second phase (clinical) was to perform the same analysis of soft tissue surrounding spinal instrumentation in 12 symptomatic clinical patients. Summary of background data: The effect of unintended wear particulate resulting from micromotion between the interconnection mechanisms in spinal instrumentation remains a clinical concern. Methods: Thirty-four New Zealand White rabbits were randomized into two groups based on postoperative time periods of 2 months (Group 1, n=14) and 4 months (Group II, n=20). Group I underwent a posterolateral arthrodesis (PLF) at L5–L6 using tricortical iliac autograft or tricortical iliac autograft plus titanium particulate. Group 2 all received iliac autograft at the initial surgery and were reoperated on after 8 weeks and treated with PLF exposure alone or titanium particulate. Postoperative analysis included serological quantification of systemic cytokines. Postmortem microradiographic, immunocytochemical and histopathological assessment of the intertransverse fusion mass quantified the extent of osteolysis, local proinflammatory cytokines, osteoclasts and inflammatory infiltrates. Clinical aspect of study: Over the last 2 years, 12 patients more than 0.4 years after spinal instrumentation presented with painful paraspinal inflammation. At surgical exploration, the cultures were negative for infection and the surrounding soft tissue was examined for cytokine reactions. There was loosening of implants and osteolysis in the location of the wear debris in 8 of 12 patients. Results: Basic science phase: serological analysis of systemic cytokines indicated no significant differences in cytokine levels (p>.05) between the titanium or autograft treatments. Immunocytochemistry indicated increased levels of local cytokines: TNF-α at the titanium-treated PLF sites at both time periods (p<.05). Osteoclast cell counts and regions of osteolytic resorption lacunae were higher in the titanium-treated versus autograft-alone groups (p<.05), and the extent of cellular apoptosis was markedly higher in the titanium-treated sites at both time intervals. Electron microscopy indicated definitive evidence of phagocytized titanium particles and foci of local, chronic inflammatory changes in the titanium-treated sites. Clinical aspect: Eleven of 12 clinical cases demonstrated elevated TNF-α levels and an increased osteoclastic response in the vicinity of wear debris caused by dry frictional wear particles of titanium or stainless steel. Osteolysis most commonly involved loose transverse connectors. Resection of the wear debris and surrounding fibroinflammatory glycocalyx resulted in resolution of clinical symptoms in all 12 cases. Conclusions: Titanium particulate debris introduced at the level of a spinal arthrodesis elicits a cytokine-mediated particulate-induced response favoring proinflammatory infiltrates, increased expression of intracellular TNF-α, increased osteoclastic activity and cellular apoptosis. This is the first basic scientific study and the first clinical study demonstrating associations of spinal instrumentation particulates wear debris and increased cytokines and increased osteoclastic activity. Osteolysis is the number one cause of failure of orthopedic implants in the appendicular skeleton. Spinal surgeons need to increase their awareness of this destructive process.
Introduction  The effect of unintended wear particulate resulting from micromotion between the interconnection mechanisms in spinal instrumentation remains a clinical concern. Recently, there have been a number of retrospective clinical studies describing the histologic response of wear particulate generated from spinal implants and the clinical consequence of this material on posterolateral arthrodesis procedures 3, 6, 9, 22, 25, 26, 33, 34, 36. Dubousset et al. [9] were among the first to describe a late “infection” complication in patients managed with posterior stainless steel Cotrel-Dubousset (C-D) instrumentation (18 patients, average 34 months after surgery). Histopathology revealed an acute and chronic inflammation with granuloma formation at the instrumentation transverse connector site, necessitating hardware removal. The etiology of this complication was considered a sterile inflammatory reaction secondary to fretting corrosion of the instrumentation. Cook et al. [6] retrospectively compared the reoperation rate in 190 consecutive patients after primary posterior instrumentation for idiopathic scoliosis. Evaluating three different stainless steel instrumentation systems, the authors introduced the concept of “late operative site pain” of unknown etiology as the most frequent indication (8%, 14 patients) for reoperation. The most frequently noted surgical observation at the time of implant removal was corrosion at the level of the interconnection mechanisms (9 of 14 patients), which was associated with tissue discoloration and intracellular metallic debris. Moreover, Wang et al. [34] described the incidence of metal debris associated with the use of titanium implants in nine patients. It was observed that tissue concentrations of titanium were highest in patients with a pseudarthrosis, whereas patients with a solid fusion had negligible levels of titanium. According to Wang et al., these particles activated a macrophage cellular response in the spinal tissues similar to that seen in joint prostheses. A number of other retrospective clinical studies have documented an inflammatory, foreign-body reaction in soft-tissue structures adjacent to spinal implants 3, 10, 22, 36. However, the specific etiology (eg, cytokine expression and concentration) of this pathology remains in question. Interestingly, despite these aforementioned studies, there have been no controlled basic science studies or applied clinical studies undertaken to determine if wear particulate from titanium or stainless steel spinal instrumentation directly influences the maturation process/success of a posterolateral spinal arthrodesis, or deleteriously affects an established fusion mass by means of cellular mediators capable of bone resorption. With these issues presented, the current study was undertaken to investigate the biological response of local and systemic tissues to titanium wear particulate introduced into a posterolateral spinal arthrodesis. Using an in vivo rabbit model and based on perioperative serological and postmortem radiographical, histological and immunocytochemical analyses, the fundamental objectives of this study were threefold: Basic science study objectives
1.Determine if titanium particulate debris introduced at the level of a spinal arthrodesis elicits a cytokine-mediated particulate-induced response, leading to increased concentrations of systemic and local cellular mediators capable of bone resorption.
2.Quantify and compare the levels of systemic/local cytokines and osteoclastic activity between titanium versus autograft alone posterolateral arthrodesis sites.
3.Compare the extent of local proinflammatory infiltrates, macrophage activity and cellular apoptosis in titanium-treated versus autograft posterolateral arthrodeses.
Clinical correlation
1.Using concurrent clinical information, there were 12 patients presenting to our spinal services with inflammation secondary to spinal instrumentation, causing pain, in the absence of infection. The surrounding soft tissue adjacent to the instrumentation at the time of operative removal was analyzed for the same cytochemical analysis: cytokine assays, osteoclast counts and macrophage characterization.
Materials and methods: basic science phase Animal model and surgical preparation All surgical and experimental procedures commenced after protocol approval by the appropriate institutional animal care and use committees. A total of 34 skeletally mature Harlan Sprague Dawley New Zealand White rabbits (4.1 to 4.5 kilograms) were included in this study and randomized into two groups based on postoperative survival periods of 8 weeks (Group 1, n=14) and 16 weeks (Group 2, n=20). After normal health status determination, each animal was sedated with subcutaneous injection of ketamine (35 mg/kg), xylazine (5 mg/kg) and acepromazine (.75 mg/kg) anesthetic medications, followed by endotracheal intubation and general anesthesia using 0.5% to 1.0% isoflurane. With the animal positioned prone, the posterior lumbar region was shaved, aseptically prepared and draped in sterile fashion. Prophylactic antibiotics (trimethoprimsulfazine .2 mg/kg, oral) and analgesics (butorphenol .125 mg/kg, intramuscular (IM) were administered pre- and postoperatively. Surgical technique and treatment groups A localization radiograph, obtained before surgical intervention, ensured exposure of the appropriate L5–L6 vertebral levels. After a single midline skin incision, two separate fascial incisions permitted blunt dissection and exposure of the L5–L6 facet joints and transverse processes. After exposure, the posterior cortices of the L5–L6 transverse processes were decorticated using a rongeur until punctate bleeding was observed, followed by thorough irrigation to remove residual osseous debris. Animals in Group 1 (n=14) underwent a posterolateral arthrodesis at L5–L6 using the following techniques: 1) tricortical iliac autograft (2 g; n=7) or 2) tricortical iliac autograft (2 g plus 200 mg titanium particulate; n=7). All animals in Group 2 received iliac autograft at the initial procedure and were reoperated on after 8 weeks and treated with one of the following: 1) posterolateral exposure of the fusion site (sham, n=10) or 2) exposure with the addition of 200 mg titanium particulate (100 mg per side; n=10). The treatment composites were placed into the right and left posterolateral gutters in direct apposition with each of the transverse processes at L5–L6, spanning the intertransverse space. For the autograft group, 2 g of autologous tricortical iliac bone was equally distributed to the bilaterally decorticated arthrodesis site (1 g per side) at the L5–L6 level. For the titanium treatments, 200 mg of titanium was thoroughly mixed with autograft (Group 1) or deposited directly on the posterolateral fusion mass (Group 2). After the surgical procedure, all incisions were closed in an interrupted fashion using 2.0 vicryl. Observations of ambulatory activities and wound healing were monitored daily, and all animals received analgesics and prophylactic antibiotics for the first 10 days after surgery. Material specifications: titanium powder The implanted commercially pure titanium powder (1 to 5 μm diameter) was obtained from Alfa Aesar (Johnson Matthey Company, Ward Hill, MA). The particle size (95% <5 μm diameter), concentration and methods of sterilization were based on previous investigations [28]. Before implantation, the titanium particles were packaged in 200-mg samples and steam sterilized at 270 Celsius for 30 minutes. Sterilized samples were sent for Kinetic QCL (quantitative colorimetric lAL) testing and shown to be free of endotoxin (<0.05 EU/ml) according to Food and Drug Administration–established guidelines for limulus assay analysis (Standard Operating Procedure 162.6; BioWhittaker, Inc., Walkersville, MD). A total of 200 mg titanium (approximately 6×108 particles) was uniformly distributed per fusion site (unilateral intertransverse area=6 cm2, 100 mg titanium). The concentration was based on previous basic and clinical investigations 28, 34. Serological cytokine analyses To quantify the levels of systemic cytokines, blood serum samples were collected preoperatively and postoperatively at weekly intervals for all animals. The levels of serum cytokines—interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 were quantified using ELISA antibody pairs (IFN-γ and IL-6, R&D Systems, Minneapolis, MN; IL-8 and TNF-α, BD Pharmingen, Inc., San Diego, CA). These sandwich ELISAs were conducted in triplicate following the protocol provided for IFN-γ, TNF-α, IL-6, and IL-8 matched pair antibodies by R&D Systems and BD Pharmingen 1, 16. The detection limits within serum for each of the tested cytokines were 10 pg/ml for IFN-γ, 25 pg/ml for TNF-α and 3 pg/ml for IL-6 and IL-8. Radiological analysis Status of the posterolateral spinal fusion sites was evaluated on the 8-week postsacrifice anteroposterior radiographs for Group 1 and 8- and 16-week radiographs for Group 2 using the four-point grading scale documented by Lenke et al. [19]. The fusion status was compared between the autograft and titanium treatment groups and evaluated by three reviewers blinded to the treatment. The percentage of successful fusions (A or B) or nonunions (C or D) were determined for each treatment group. Histological techniques The bilateral posterolateral fusion sites, including the transverse processes, were transected at the vertebral body border and randomly assigned to one of two treatments. One side (right or left) was designated for methylmethacrylate embedding and the remaining site for paraffin processing. The fibrous/granulation tissue/muscle layer overlying the posterolateral fusion mass was processed using three techniques: immunocytochemistry, electron microscopy and stained for cellular apoptosis. Using plain and polarized light microscopy, histopathological assessment for all tissues included assessment of trabecular architecture, osteoclastic activity, cytokine levels, as well as any signs of macrophage/foreign body giant cell/granulomas inflammatory reactions or cellular apoptosis. Histology: methylmethacrylate-embedded tissue The posterolateral specimens designated for methylmethacrylate were dehydrated in 80% ethanol, stained using the Villanueva Osteochrome Bone Stain (Polysciences, Inc., Warrington, PA), processed using undecalcified technique and embedded in polymethylmethacrylate. Using the EXAKT Microgrinding Device (EXAKT Technologies, Oklahoma City, OK), the embedded specimens were cut into 300- to 500-μm-thick sections, ground and polished to 75 μm in thickness. Microradiographs were then obtained using a Faxitron X-ray Unit (Faxitron X-Ray Corporation, Buffalo Grove, IL) and Konica Graphic Arts Film (Konica Imaging, U.S.A., Inc., Glen Cove, NY). The slide-mounted specimens were placed 12 inches from the beam and exposed for 2 minutes, using a technique of 25 kiloVoltspeak and 3 milliamperes while in direct contact with single-emulsion high-resolution graphics arts film. The high-resolution microradiographs permitted confirmation of the plain film radiographic findings and determination of titanium particle distribution within the trabecular structure. Histology: paraffin-embedded tissue analysis The remaining posterolateral hemisections were fixed in 10% formalin solution, paraffin processed and slide mounted. Using thin-sectioning microtomy, the paraffin-embedded posterolateral fusion specimens were sectioned (3 to 5 μm in thickness) and stained using the following techniques: 1) hematoxylin and eosin (decalcified tissue); 2) acid phosphatase enzymatic stain; using the Diagnostics Acid Phosphatase Kit (Sigma Chemical, Inc., St. Louis, MO), the tartrate-resistant acid phosphatase method permitted osteoclast identification (nondecalcified). Histology: overlying fibrous/muscle tissue analysis The tissue band overlying the posterolateral fusion mass was processed using immunocytochemistry (local cytokine concentration), electron microscopy (titanium phagocytosis) and cellular apoptosis (programmed cell death). Immunocytochemistry Using standard immunocytochemistry techniques, the tissue layer overlying the posterolateral fusion mass was processed to quantify the levels of local cytokines. The specimens were trimmed and excess bone fragments were removed to produce a 2-cm2 piece of tissue. Tissues were then placed in cassettes and covered with embedding compound, placed in cooled isopentane, sectioned with a cryostat, fixed in anhydrous acetone for 20 seconds and stored at −70 Celsius. The endogenous peroxides within the samples were blocked with peroxide (H202), using a two-step method previously documented [1]. Using primary and secondary antibodies combined with the avidin-biotin complex (ABC)-horseradish peroxidase technique, immunohistochemical localization of macrophage intracellular and membrane-bound cytokines included the following: IL-1, IL-2, IL-6, IL-8, TNF-α and prostaglandin E2 (PGE2). Electron microscopy Random sections of the fibrous/granulation tissue layer/muscle overlying the fusion mass were processed for transmission electron microscopy to assess titanium particle distribution within cellular structures. The specimens were fixed in 4% glutaraldehyde on 0.1 M cacodylate buffer (pH 7.2 for 1 to 4 hours), post-fixed in osmium teroxide 1% in 0.1 M cacodylate buffer for 1 hour, embedded in capsules and heat cured. The resin samples were sectioned at 1 μm thickness and analyzed using transmission electron microscopy. Cell apoptosis Sections of tissue overlying the fusion mass were processed to qualitatively assess the levels of apoptotic cells, comparing titanium-treated versus autograft-alone–treated samples. The ApopTag Kit plus peroxidase (Intergen Company, Purchase, NY), distinguishes cellular apoptosis (programmed cell death) from necrosis (accidental cell death) by specifically detecting DNA cleavage by means of peroxidase binding and chromatin condensation associated with apoptosis. Histomorphometric evaluation Quantification of local cytokines and osteoclasts was performed using the BioQuant Image Analysis System (R&M Biometrics, Nashville, TN). One section per specimen was analyzed using 30 consecutive fields per sample at ×40 magnification. At this magnification and field number, the entire trabecular area within the posterolateral site (osteoclasts) and tissue overlying the site (cytokines) was analyzed. Data and statistical analysis Histomorphometric data represent the mean number of osteoclasts and cytokine-expressing macrophages within the trabecular structures and overlying tissues, respectively. All data are shown as mean±1 standard deviation and were statistically compared using a Student's t test. Statistical comparisons at p<.05 were considered significant. Materials and methods: parallel clinical study Twelve patients presented to our clinical spinal services fitting the criteria of periprosthetic osteolysis or what Cook et al. [6] described as “Late Operative Site Pain (LOSP) following primary posterior spinal instrumentation”. Seven patients had titanium implants, and five patients had stainless steel implants (Fig. 1). All 12 cases were symptomatic of painful pseudarthrosis. All were culture negative at the time of surgical exploration a mean of 4.03 years after the initial posterior instrumentation procedure (range, 0.4 to 11 years postoperatively). After removal of the instrumentation, the surrounding granulomatous material, called a glycocalyx, was resected and sent for histological analysis, osteoclast count, and so forth (Fig. 2). Eight of 12 cases had grossly visible osteolysis and required bone grafting to repair pseudarthrosis in the site of obvious black staining of soft tissues resulting from loosening, fretting and abrasion of loose or broken rods, screws or crosslinks. Results: basic science phase Surgical procedures Intraoperative anesthetic complications (n=2) and postoperative iliac crest morbidity (n=2) accounted for the loss of four animals in Group 1. Otherwise, there was no incidence of neurologic or infectious complications. In Group 2, one animal was lost because of infection (n=1, autograft alone) and three to postoperative morbidity (n=3) within 2 weeks after the initial surgical procedure. The remaining animals were characterized as having a normal recovery throughout the 16-week postoperative period and tolerated the second operative procedure without complication. At the time of scheduled necropsy, there were no gross signs of infection in any animals. Serological cytokine analyses The levels of systemic cytokines were quantified preoperatively (baseline) and postoperatively at weekly intervals for animals in both study groups. The levels of serum cytokines in all cases and time points for IFN-γ, TNF-α, IL-6 and IL-8 were below sample detection limits of 10 pg/ml, 25 pg/ml, 3 pg/ml and 3 pg/ml, respectively. No differences in circulating cytokine concentrations could be detected as a result of the surgical procedure or introduction of titanium particulate at the posterolateral arthrodesis site (p>.05). Radiography Based on plain film radiography, no differences were observed between the autograft-alone and autograft-plus-titanium treated sites in Group 1. Both treatments indicated successful arthrodeses in 60% of the cases by the 8-week endpoint. Group 2 indicated similar fusion success (55% to 57%) between the two treatments. Importantly, at this 8-week time period for Group 2, the titanium was not implanted, so all animals represent autograft treatments alone. After implantation of the titanium and sham procedures, both treatments indicated improvements in fusion success by the 16-week period. The autograft-alone treatment increased in fusion success from 57% to 85%, whereas the titanium-treated fusions marginally improved from 55% to 66% (Table 1). Decalcified and undecalcified bone histomorphology Group 1: 8-week time interval Based on plain and polarized light microscopy of the autograft-alone procedures, there was significant evidence of normal bone remodeling, with trabecular bone extending between the transverse processes in cases of successful arthrodeses. The nonunions indicated the presence of corticocancellous bone fragments within the intertransverse space, with a connective tissue interface primarily consisting of collagen bundles. The autograft-plus-titanium treated specimens exhibited titanium particles embedded within the marrow cavities with disruption of marrow cellularity, evidence of granulation tissue, histiocytes and thinning trabeculae. There was no evidence of polymorphonuclear cells, normally associated with toxic reactions; however, the histiocytic response was characteristic of a local, chronic inflammatory reaction (Fig. 3). Group 2: 16-week time interval By 16 weeks, the iliac crest autograft treatments, in many cases, demonstrated complete intertransverse fusion bridges, containing more lamellar than woven trabeculae. Moreover, there was no evidence of a histiocytic reaction within the intended arthrodesis sites. In contrast, the addition of titanium particles to the 8-week postoperative arthrodeses resulted in titanium penetration of the fibrous tissue layer and posterolateral fusion mass. The inflammatory reaction was concentrated along the trabecular borders with significant evidence of mononuclear cell (macrophage) infiltration and particulate phagocytosis. Similar to the 8-week groups, the inflammatory reaction was considered chronic, without evidence of polymorphonuclear cells. However, the level of osteoclastic activity and incidence of resorption lacunae were inordinately high along the endosteal surfaces of the intertransverse trabeculae. Acid phosphatase: osteoclastic activity Identification of osteoclastogenesis was performed using acid phosphatase enzymatic stain and quantitative histomorphometry. Both the 8- and 16-week autograft plus titanium treatments indicated significantly more osteoclasts than corresponding autograft-alone treatments (p<.05). At 8 weeks, the mean osteoclast cell count for autograft plus titanium was 38.2±5.15 versus autograft alone 23.1±8.41 (p<.05). By 16 weeks, the number of osteoclasts increased for the titanium groups (43.8±15.53) but remained essentially unchanged for the autograft (sham procedure) 25.8±10.26 (p<.05; Fig. 4). The tartrate-resistant acid phosphatase method permitted osteoclast identification, which appeared as large red-stained cells along the endosteal surface. For the titanium-treated specimens, the cells were arranged in clusters and in many cases demonstrated a “ruffled border” or scalloping effect, indicating osteoclastic activity (Fig. 5). Cytokine analyses: local tissues Using immunocytochemistry techniques, a 2-cm2 tissue layer overlying the posterolateral fusion mass was processed to quantify the levels of local cytokines. The membrane-bound or intracellular cytokines (antigens) localized on macrophages produced a yellow to brown chromagen label in response to the primary and secondary antibodies, when combined with the avidan-biotin complex (ABC)-horseradish peroxidase technique. These regions of active cytokines were quantified using light microscopy. There was no detectable evidence of local IL-1, IL-2, IL-6, IL-8 and PGE2 cytokine activity within the region overlying the posterolateral fusion site for autograft controls or titanium-treated specimens, despite regions heavily laden with titanium particulate. In contrast to these findings, local concentrations of TNF-α were significantly higher in the titanium-treated sites in the 8 and 16-week treatment groups (p<.05; Fig. 6). At 8 weeks, the TNF-α cytokine count for autograft plus titanium was 18.91±7.45 cells versus autograft alone 3.26±2.58 (p<.05). The mean number of TNF-α cytokines increased slightly for the autograft plus titanium groups by 16 weeks (21.42±9.96) but remained essentially unchanged for the autograft (sham procedure) 2.66±2.16 (p<.05; Fig. 7). Electron microscopy In corroboration with the light microscopic histologic findings, transmission electron microscopic evaluation of the titanium-treated posterolateral sites indicated significant evidence of phagocytized titanium particles by macrophages within the posterolateral fibrous tissue layer (Fig. 8). Despite the dense accumulation and distribution of particulate matter, there was only minor evidence of particulate within the endothelial cells. Apoptosis One of the interesting findings in this investigation was the extent of cellular apoptosis (programmed cell death) in the titanium-treated animals versus autograft alone in both the 8 and 16-week treatments (Fig. 9). There was considerable apoptosis of fibrocytes and endothelial cell linings of fascia tissue overlying the fusion mass. Moreover, there was minor evidence of apoptosis of the infiltrating macrophages, localized between tissue layers. The autograft control treatments at both time intervals indicated little to no evidence of apoptosis or inflammatory infiltrates. Results: clinical correlation phase The clinical symptoms and pain in all 12 cases resolved soon after resection of the loose instrumentation. In total joint arthroplasty this has been attributed to the reduction in particle load, which in turn alleviates the inflammatory response. All cases had been covered by perioperative antibiotics for only 48 hours, but these were not clinical infections. The granulomatous response did not extend the length of the incisions but instead coated the metal implants as an adherent biofilm or glycocalyx (Fig. 10). Furthermore, the intraoperative cultures for aerobic, anaerobic, fungal and tuberculous organisms were all negative. The histologic analysis did not show polymorphonuclear cells, which would have indicated a pyogenic infection; instead there was a macrophage response. Eleven of the 12 cases demonstrated elevated cytokines; TNF-α ranged from a low of 67 to a high of 525 cells. Notice that this is an order of magnitude higher than the levels of TNF-α noted in the rabbit model. We attribute this to a greater length of time the patients were followed postoperatively and exposed to the inflammatory wear debris. The single patient in our clinical series without an elevated cytokine count was a patient who did not have osteolysis observed at the time of surgical reexploration. In fact, the main reason for the surgical exploration was “transitional disease” or spinal stenosis just cephalad to the spinal instrumentation. Independent histologists who were blinded as to the origin of the tissue observed apoptosis and elevated osteoclasts. Furthermore, similar to the rabbit model, the location of the osteoclastic response was in the area of the particulate debris on the slide (Fig. 11). Discussion To our knowledge, the current project serves as the first to investigate the effect of titanium and stainless steel wear particulate in clinical cases and with an applied animal model. Wear debris in spinal surgery is the product of spinal implant interconnection loosening and has deleterious effects on the development and maintenance of a posterolateral arthrodesis. The issue of wear particulate secondary to orthopedic implants is not a new phenomenon. Review of the joint prosthesis literature highlights a plethora of articles describing local tissue reaction to metallic wear debris 7, 8, 17, 35 as well as the in vitro human macrophage 18, 20 and fibroblast [37] response to retrieved titanium alloy particles. Other studies have indicated that corrosion is continually changing the shape, size and chemical composition of implanted alloys, and that this may alter the biochemical tissue environment surrounding an implant, favoring bone resorption 29, 30. The concentration of titanium powder implanted at the posterolateral site (100 mg/6 cm2) in the current study represents a mid-range load level based on previous studies 14, 24, 28. Moreover, particles of phagocytosable size (5 μm or less) used in the current study were found to be the most reactive based on previous investigations [2]. Further clinical and basic investigations are necessary to quantify the load levels and concentrations of wear particulate secondary to titanium and stainless spinal instrumentation systems. Based on the plain film radiography, the introduction of titanium into a “new” arthrodesis does not seem to influence the success of fusion outcome. Interestingly, the same material implanted on a developing or successful arthrodesis 8 weeks postoperatively appears to hinder further development of the fusion mass but apparently does not disrupt the existing posterolateral arthrodesis (Table 1ß). Increasing the postoperative time interval may offer differing results in terms of posterolateral osteolysis, particularly in light of the increasing osteoclastic activity. In all cases, a localized, chronic inflammatory reaction characterized the histopathologic condition for all treatments containing titanium particulate. In contrast, the autograft controls indicated a normal healing process, without significant histopathologic changes or evidence of inflammatory infiltrates. Importantly, there was no evidence of an acute inflammatory reaction with polymorphonuclear cell response observed in any specimens. Light microscopic and corroborative electron photomicrographs of the titanium-treated sites indicated definitive evidence of particulate phagocytosis by the infiltrating monocytes (macrophages). The comparative levels of systemic and local cytokines offered interesting results in the current project. Based on ELISA analysis, all serum cytokines assayed—IFN-γ, TNF-α, IL-6 and IL-8—indicated concentrations below detectable limits. In similarity, there was no detectable evidence of local IL-1, IL-2, IL-6, IL-8 and PGE2 cytokine activity within regions overlying the posterolateral fusion site for autograft controls or titanium-treated specimens, despite the high concentration of titanium particulate. However, there was clear evidence of increasing concentrations of local TNF-α in both titanium groups (8 and 16 weeks), which were significantly higher than corresponding autograft treatments. The lack of systemic TNF-α despite its presence in local tissues is consistent with an “innate” immune response 1, 31. Specifically, the local release of TNF-α serves to stimulate recruitment of neutrophils and monocytes to the site of infection (eg, particulate activation) using two mechanisms: 1) stimulation of vascular cells to express new surface receptors and 2) stimulation of endothelial cells and macrophages to secrete chemokines. At high concentrations (ie, 0.0001 mM detectable by ELISA), TNF-α catastrophically affects the brain, liver, bone marrow, heart and blood vessels. Therefore, TNF-α is highly regulated to produce a local response and is undetectable in peripheral serum unless other evidence of systemic effects is present. In addition to elevated concentrations of TNF-α, the titanium-treated sites indicated a significant increase in number of osteoclasts within the posterolateral arthrodesis region when compared with autogenous controls. The method used for osteoclast quantification reliably detects osteoclasts and provides an indirect measure of osteoclastic activity [11]. The elevated number of osteoclasts may be secondary to increased concentrations of local TNF-α. Based on previous studies, TNF-α has been shown to be the first cytokine produced in response to particulate phagocytosis by macrophages 2, 4, is known to activate osteoclasts to resorb bone 12, 27 and is critically involved in wear debris osteolysis in vivo [21]. In the current project, phagocytosis of the titanium particles by infiltrating macrophages most likely accounts for the release of local TNF-α, leading to increased osteoclastogenesis within the posterolateral fusion mass. Apoptosis is a form of programmed cell death, which serves to eliminate compromised or superfluous cells by means of phagocytosis by macrophages as well as normal adjacent cells and is distinct from cellular necrosis (accidental cell death) 12, 32. The mechanistic basis underlying the apoptotic cells observed in the titanium-treated sites in the current study may be secondary to macrophage exhaustion, the release of cytokines (TNF-α) and production of reactive oxygen intermediates within local tissues. The presence of foreign bodies have shown to trigger oxygen radical release, which exhausts macrophages and undermines the monocytes' ability to initiate a second burst [23]. The combined histomorphologic and cytokine results of the current study corroborate the concept of a self-perpetuating immunoincompetent fibroinflammatory zone described by Gristina [13]. The fundamental concept expanded by Gristina is that wear particulate generated from orthopedic implants initiates a macrophage reaction, leading to cytokine and oxygen free radical release. The continued macrophage activation, secondary to particulate “overload,” eventually leads to immune exhaustion, chronic inflammation and increased susceptibility to infection. In summary, titanium and stainless steel wear particulate localized at the posterolateral arthrodesis site elicits a macrophage-mediated inflammatory response leading to increased levels of local proinflammatory cytokine TNF-α production, subsequent osteoclastogenesis and cellular apoptosis. In the current study, we were unable to document an increased incidence of osteolysis secondary to activated osteoclasts in rabbits 15, 28. However, in 8 of 12 patients there was gross visible osteolysis in the vicinity of particulate wear debris. The adjacent soft tissue demonstrated the same spectrum of cytokine elevation profile as in the rabbit model. In the rabbit study, increasing the postoperative survival period may offer differing results in terms of osteolysis. With the introduction of modular artificial disc replacements and new materials for orthopedic spinal implants, the effects of implant-fretting corrosion on local spinal and systemic tissues will remain a clinical concern. Based on the current project, the presence of titanium particulate debris, secondary to motion between spinal implants, may serve as the impetus for late-onset inflammatory/infectious complications and long-term osteolysis of an established posterolateral fusion mass in the clinical setting.5 Acknowledgements The authors would like to thank the University of Maryland Biotechnology Institute, Animal Core Facility and Research Staff for their assistance with the animal husbandry procedures. References 1.
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☆ FDA device/drug status: not applicable. ☆☆ Nothing of value received from a commercial entity related to this research. ★ Research funding for this project was provided by Orthopaedic Associates Research Foundation, Inc., Towson, Maryland. PII: S1529-9430(02)00443-6 © 2003 Elsevier Science Inc. All rights reserved. | |
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