JBJS | Titanium Particles Stimulate Bone Resorption by Inducing Differentiation of Murine Osteoclasts

The Journal of Bone & Joint Surgery, Volume 83, Issue 4

Articles | April 01, 2001

Titanium Particles Stimulate Bone Resorption by Inducing Differentiation of Murine Osteoclasts

Yanming Bi, PhD; R. Renee VanDeMotter, MS; Ashraf A. Ragab, MD; Victor M. Goldberg, MD; James M. Anderson, MD, PhD; Edward M. Greenfield, PhD

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Investigation performed at the Departments of Orthopaedics and Pathology, Case Western Reserve University, Cleveland, Ohio
Yanming Bi, PhD R. Renee Van De Motter, MS Ashraf A. Ragab, MD Victor M. Goldberg, MD James M. Anderson, MD, PhD Edward M. Greenfield, PhD Departments of Orthopaedics (Y.B., R.R.V.D.M., A.A.R., V.M.G., and E.M.G.) and Pathology (J.M.A.), Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-5000. E-mail address for E.M. Greenfield: emg3@po.cwru.edu
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were National Institutes of Health Grants AR43769, HL33849, and AR07505.

The Journal of Bone & Joint Surgery. 2001; 83:501-501

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Abstract

Background:

Loosening of orthopaedic implants is mediated by cytokines that elicit bone resorption and are produced in response to phagocytosis of implant-derived wear particles. This accelerated bone resorption could be due to increased osteoclastic activity, survival, or differentiation. Although a number of in vitro studies have shown that wear particles increase osteoclastic activity, the increase was less than twofold in all cases. The objective of the current study was to test the hypothesis that wear particles stimulate bone resorption by inducing osteoclast differentiation.

Methods:

Conditioned media were prepared from murine marrow cells or human peripheral blood monocytes incubated in the presence or absence of titanium particles. The effects of conditioned media on osteoclast differentiation were examined with use of a recently developed assay in which osteoclast precursors are co-cultured with mesenchymal support cells.

Results:

The present study showed that titanium particles induced both murine marrow cells and human peripheral blood monocytes to produce factors that stimulated osteoclast differentiation. The mean increase in osteoclast differentiation was 29.3 ± 9.4-fold. The stimulation of osteoclast differentiation led to a parallel increase in bone resorption. The amount of stimulation was regulated in a dose-dependent manner by the concentration of both titanium particles and conditioned media. The stimulation of osteoclast differentiation required interactions between the cells and the particles themselves and, therefore, was not due to metal ions, soluble contaminants released from the particles, or submicrometer particles. In contrast, conditioned media from control cells incubated in the absence of titanium particles had no detectable effect on any of the examined parameters.

Conclusions:

The present study showed that titanium particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation. In contrast, the titanium particles had only small effects on osteoclast activity or survival.

Clinical Relevance:

The present study provides strong support for the hypothesis that osteoclast differentiation is an important factor in the development of aseptic loosening. The development of therapeutic interventions to reduce osteoclast differentiation may be a useful approach for improving the performance of orthopaedic implants.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

A septic loosening of orthopaedic implants is a serious problem that frequently leads to pain, loss of function, and revision surgery. Most cases of aseptic loosening arise from the local resorption of bone surrounding the implant, a process known as osteolysis¹. An understanding of the mechanisms involved in this process is crucial to the development of new methods to prevent implant loosening.

Several types of studies have suggested that wear particles induce osteolysis. For example, histological examination of tissue surrounding revised implants has revealed the formation of a synovial-like membrane containing macrophages and fibroblasts¹. This synovial-like membrane has been found to contain large numbers of implant-derived particles of a phagocytosable size^2,3. Wear particles have been shown to stimulate the production of a variety of bone resorptive cytokines in vitro, including TNF (tumor necrosis factor), IL-1 (interleukin-1), and IL-6 (interleukin-6)^1,4-10. Similarly, cytokines, which elicit bone resorption, have been identified in the membrane that forms around loose implants in vivo¹. More conclusively, higher levels of TNF, IL-1, and IL-6 have been found in regional lymph nodes than in contralateral lymph nodes¹¹. However, the production of bone resorptive cytokines does not demonstrate that bone resorption is increased by this mechanism, since wear particles also induce the production of cytokines that inhibit bone resorption, including IL-4 (interleukin-4), IL-10 (interleukin-10), and IFN-g (interferon-g)^12-14. Thus, it is not possible to predict a priori whether the mixture of factors produced in response to wear particles will have a net stimulatory or inhibitory effect on bone resorption. In fact, in a previous study, we showed that conditioned media from unstimulated marrow cells had a net inhibitory effect on osteoclastic activity¹⁵. In the current study, therefore, we focused on the functional effects of conditioned media from marrow cells that had been incubated with orthopaedic wear particles rather than on the measurement of specific cytokines.

The induction of osteolysis by wear particles could reflect increased bone resorptive activity of extant osteoclasts, increased differentiation of new osteoclasts from their precursors, and/or increased survival of mature osteoclasts. We hypothesized that osteolysis induced by wear particles was primarily due to stimulation of osteoclast differentiation, a multistep process that includes precursor proliferation, commitment to the osteoclastic lineage, expression of osteoclastic marker genes such as tartrate-resistant acid phosphatase (TRAP), and fusion into multinucleated cells. To test this hypothesis, it was necessary to examine a model in which osteoclast differentiation was stimulated by the bone resorptive cytokines induced by wear particles. We recently developed an osteoclast differentiation assay that is stimulated by all cytokines that have been tested—namely, TNF, IL-1, IL-6, and IL-11 (interleukin-11)^16,17. In this assay, osteoclast precursors are co-cultured with conditionally immortalized murine calvaria-4 (CIMC-4) mesenchymal support cells isolated from transgenic mice that express a form of the SV40 (simian virus 40) large T antigen that is both temperature-sensitive and inducible by IFN-g. In the current study, the stimulation of osteoclast differentiation by conditioned media from murine marrow cells and human peripheral blood monocytes exposed to titanium particles was examined with use of the osteoclast precursor-CIMC-4 cell co-culture assay system.

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Fig. 1:: Conditioned media from murine marrow cells incubated with titanium particles increased the number of both tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) and TRAP-positive mononuclear cells. The number of TRAP-positive multinucleated cells (A) and TRAP-positive mononuclear cells (B) was assessed every three days in cultures containing 25% conditioned media from marrow cells incubated with (solid circles) or without (open circles) titanium particles. N = 6 for all groups. A single asterisk (p < 0.02) or a double asterisk (p < 0.0001) indicates a significant difference between cultures containing conditioned media from marrow cells incubated with and without particles at the same time-point.

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Fig. 2:: The increased number of TRAP-positive multinucleated cells (MNC) and TRAP-positive mononuclear cells induced by conditioned media from murine marrow cells incubated with titanium particles was due to increased osteoclast differentiation rather than to increased osteoclast survival. The number of TRAP-positive multinucleated cells (A) and TRAP-positive mononuclear cells (B) was assessed daily during days 6 through 9 in cultures containing 25% conditioned media from marrow cells incubated with (solid circles) or without (open circles) titanium particles. N = 6 for all groups. An asterisk indicates a significant difference (p < 0.0002) between cultures containing conditioned media from marrow cells incubated with and without particles at the same time-point.

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Fig. 3:: The increased number of TRAP-positive multinucleated cells (MNC) was dependent on the dose of the conditioned media from marrow cells incubated with titanium particles. The number of TRAP-positive multinucleated cells was determined in cultures containing the indicated concentrations of conditioned media from marrow cells incubated with (solid circles) or without (open circles) titanium particles. All groups received enough mock conditioned medium so that the total concentration of conditioned medium was 50%. N = 5 or 6 for all groups. An asterisk indicates a significant difference (p < 0.0005) between cultures containing the same percentage of conditioned media from marrow cells incubated with and without particles.

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Fig. 4:: Titanium particles increased the number of TRAP-positive multinucleated cells (MNC) in a dose-dependent manner. The number of TRAP-positive multinucleated cells was determined in cultures containing 25% conditioned media from marrow cells incubated with the indicated concentrations of titanium particles (cpTi). N = 5 or 6 for all groups. A single asterisk (p < 0.001) or a double asterisk (p < 0.0001) indicates a significant difference between cultures containing conditioned media from marrow cells incubated with and without particles.

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Fig. 5:: Conditioned media from marrow cells incubated with titanium particles significantly increased the formation of resorption lacunae. The number of TRAP-positive multinucleated cells (MNC) on ivory slices (A) and the percentage of the total area of ivory slices that were covered by resorption lacunae (B) were assessed after twelve days in cultures containing 25% conditioned media from marrow cells incubated with or without titanium particles (cpTi) or with mock conditioned media. N = 5 or 6 for all groups. A single asterisk indicates a significant difference (p < 0.0001) between cultures containing conditioned media from marrow cells incubated with and without particles.

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Fig. 6:: The increase in the number of TRAP-positive multinucleated cells (MNC) required direct contact between the marrow cells and the titanium particles (cpTi). The number of TRAP-positive multinucleated cells was assessed in cultures containing 25% conditioned media from marrow cells incubated with (second bar) or without (fourth bar) titanium particles or with particle filtrate (fifth bar). The number of TRAP-positive multinucleated cells also was assessed in cultures containing 25% conditioned media from titanium particles incubated without marrow cells (third bar). Particle filtrates were prepared by incubating titanium particles for twenty-four hours at the same concentration used in the marrow cell cultures and were processed as described in the methods for conditioned media. In this experiment, titanium particles stimulated a forty-onefold increase in the formation of TRAP-positive multinucleated cells (the second compared with the fourth bar); the asterisk indicates a significant difference (p < 0.0001). N = 4, 5, or 6 for all groups.

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Fig. 7:: Conditioned media from human peripheral blood mononuclear cells or adherent monocytes incubated with titanium particles (cpTi) increased the number of TRAP-positive multinucleated cells (MNC). The number of TRAP-positive multinucleated cells was determined after seven days in cultures containing conditioned media from either human mononuclear cells (second and third bars) or adherent monocytes (fourth and fifth bars) incubated without (second and fourth bars) or with (third and fifth bars) titanium particles. N = 6 for all groups. An asterisk indicates a significant difference (p < 0.0001) between cultures containing 25% conditioned media from the same preparation of cells incubated with and without particles.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

All animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under the supervision of our Institutional Animal Care and Use Committee¹⁸. Human peripheral blood samples were obtained from normal adult volunteers in accordance with the guidelines established by the Institutional Review Board of University Hospitals of Cleveland. 1a,25-dihydroxyvitamin D3 was obtained from Biomol (Plymouth Meeting, Pennsylvania); dexamethasone, from Sigma (St. Louis, Missouri); and ascorbic acid, from GIBCO BRL (Gaithersburg, Maryland). The culture medium used throughout was phenol red-free minimum essential medium (GIBCO BRL) containing nonessential amino acids (Mediatech, Herndon, Virginia), 2 mM L-glutamine (Mediatech), 10% fetal bovine serum (HyClone Laboratories, Logan, Utah), penicillin (100 U/mL) (Mediatech), and streptomycin (100 g/mL) (Mediatech). All of these reagents were tested for endotoxin with use of the high-sensitivity version of the Chromogenic Limulus Amoebocyte Lysate Test (QCL-1000; BioWhittaker, Walkersville, Maryland), and they were from lots that contained the lowest concentration of endotoxin available.

Particle Preparation

Commercially pure titanium particles (number 00681, lot G11G04; Johnson Matthey, Ward Hill, Massachusetts) were used. Approximately 75% of these particles were shown by analysis with a Coulter Counter (Coulter Electronics, Hialeah, Florida) and scanning electron microscopy to be smaller than 6.5 m in diameter¹⁹. The titanium particles were sterilized by incubation in 70% ethanol for one hour, washed three times with phosphate-buffered saline solution, and stored at a concentration of 1.26 ¥ 10⁷ particles per mL of phosphate-buffered saline solution containing penicillin (100 U/mL) and streptomycin (100 g/mL). For experiments, this particle stock solution was diluted with media to provide the indicated numbers of titanium particles. The diluted particle suspensions were preincubated with 5% carbon dioxide at 37°C in Petri dishes during preparation of marrow cells. Negative controls were prepared with an equivalent dilution of phosphate-buffered saline solution with antibiotics but without particles.

Conditioned Media Preparation

Marrow cells were flushed from the femora and tibiae of six to twelve-week-old C57BL/6 mice with use of phosphate-buffered saline solution with antibiotics. Red blood cells were lysed in 0.83% ammonium chloride and 10-nM Tris-HCI (pH 7.4). Cells were washed twice with phosphate-buffered saline solution and resuspended in the culture medium described above. Nucleated marrow cells (2.5 ¥ 10⁵ cells/cm²) were added to the Petri dishes with preincubated titanium particles in a total volume of medium of 0.25 mL/cm². Petri dishes were used instead of tissue-culture dishes in order to reduce nonspecific activation of marrow cells by adherence. After twenty-four hours of incubation in 5% carbon dioxide at 37°C, the conditioned media were harvested, centrifuged at 2300 times gravity for twenty-five minutes at 4°C, passed through a 0.2-m filter (Gelman Sciences, Ann Arbor, Michigan), aliquoted, and stored at —80°C until used. Immediately before osteoclast differentiation assays, conditioned media were thawed and centrifuged at 10,000 times gravity for fifteen minutes at 4°C.

Human peripheral blood mononuclear cells were isolated from the whole blood of normal adult volunteers as previously described²⁰. These preparations, which contained 75% to 80% monocytes and 20% to 25% lymphocytes, were further purified by adherence as previously described²⁰. We confirmed that more than 95% of the adherent cells were monocytes as assessed by histochemical staining for nonspecific esterase. Titanium particles diluted in culture medium as described above were added to individual cultures of either peripheral blood mononuclear cells or adherent monocytes; control cultures received an equivalent dilution of phosphate-buffered saline solution with antibiotics but without particles. After twenty-four hours of incubation in 5% carbon dioxide at 37°C, the conditioned media were harvested and processed as described above.

Osteoclast Differentiation Assay

The effects of the conditioned media were determined with use of our previously described osteoclast differentiation assay^16,17, in which osteoclast precursors are co-cultured with CIMC-4 mesenchymal support cells. CIMC-4 cells were grown under permissive conditions (at 33°C with IFN [100 g/mL]) to allow expression of SV40 large T antigen. Prior to the experiments, CIMC-4 cells were cultured under nonpermissive conditions (at 37°C without IFN-g) for one week to allow degradation of SV40 large T antigen. Afterward, CIMC-4 cells were plated at a density of 10⁴ cells/cm² in 1-cm² wells. After incubation of the CIMC-4 cells for one day in 5% carbon dioxide at 37°C, nucleated spleen cells as a source of osteoclast precursors were added at a density of 5 ¥ 10⁵ cells/cm² with conditioned media, 100 nM dexamethasone, ascorbic acid (50 g/mL)¹⁶, and either 0.2 or 0.3 nM 1a,25-dihydroxyvitamin D3. This low concentration of 1a,25-dihydroxyvitamin D3 was added to the osteoclast differentiation assays in order to facilitate stimulation by the conditioned media¹⁷. The media were changed every three days. On the indicated days, the cells were stained histochemically for TRAP, and the number of TRAP-positive multinucleated cells containing three or more nuclei was determined as previously described¹⁶. In selected experiments, TRAP-positive mononuclear cells also were determined¹⁶.

To further confirm the osteoclast phenotype, the formation of resorption lacunae was measured in selected osteoclast differentiation assays. For this purpose, cultures were performed in 0.3-cm² wells containing 3 ¥ 10⁴ CIMC-4 cells/cm², 5 ¥ 10⁵ nucleated spleen cells/cm², and round slices of elephant ivory (the kind gift of Dr. T. Bettinger and Dr. A. Lewandowski, Cleveland Metroparks Zoo, Cleveland, Ohio). The number of TRAP-positive multinucleated cells was counted with use of light microscopy¹⁶, and the extent of resorption on the ivory slices was determined by computer-assisted histomorphometry as previously described¹⁶.

All data are presented as the mean and the standard error of the mean and are representative of multiple experiments. Statistical analyses were performed with analysis of variance with Bonferroni-Dunn (control) post hoc tests when multiple groups were compared with a single control group (Figs. 4, 5, and 6) and the Fisher protected least-significant-difference post hoc tests when multiple groups were compared (Figs. 1, 2, 3, and 7).

Results

Introduction | Materials and Methods | Results | Discussion | References

In order to determine whether wear particles induce marrow cells to produce factors that stimulate osteoclast differentiation, we studied the effect of conditioned media from marrow cells incubated with or without titanium particles on the number of TRAP-positive multinucleated cells that formed in our osteoclast differentiation assay¹⁷. Figure 1, A shows that the number of TRAP-positive multinucleated cells increased on days 9 and 12 in cultures containing conditioned media from marrow cells incubated with titanium particles. In this experiment, the maximal stimulation was a sevenfold increase in the number of such cells compared with that in cultures containing conditioned media from marrow cells incubated without particles. To determine whether the increase in TRAP-positive multinucleated cells was associated with an increase in the abundance of osteoclast precursors, we also quantified the number of TRAP-positive mononuclear cells. Figure 1, B shows that the conditioned media from marrow cells incubated with titanium particles also increased the number of TRAP-positive mononuclear cells. As expected, the appearance of the osteoclast precursors preceded the TRAP-positive multinucleated cells by a few days.

Increased osteoclast differentiation is the most likely mechanism responsible for the increase in TRAP-positive multinucleated cells depicted in Figure 1. However, it is possible that TRAP-positive multinucleated cells might form and die rapidly between days 6 and 9 of culture. Thus, extensive osteoclast differentiation might occur irrespective of whether conditioned medium is added, and the conditioned media from marrow cells incubated with titanium particles might increase the number of TRAP-positive multinucleated cells by increasing their survival. To test this possibility, we determined the number of TRAP-positive multinucleated cells at shorter intervals to preclude the possibility of overlooking rapid osteoclast differentiation and rapid cell death. Daily intervals were chosen since twenty-four hours is required for the fusion of TRAP-positive mononuclear cells into multinucleated cells²¹ and an additional eighteen to twenty-four hours is required for the apoptosis of mature osteoclasts^22,23. Figure 2, A shows that conditioned media from marrow cells incubated without particles had no detectable effect even when the number of TRAP-positive multinucleated cells was determined at daily intervals. In contrast, the number of TRAP-positive multinucleated cells progressively increased each day in the cultures containing conditioned media from marrow cells incubated with titanium particles (Fig. 2, A). Thus, this approach confirmed that the increased number of TRAP-positive multinucleated cells induced by conditioned media from marrow cells cultured with titanium particles was due to the stimulation of osteoclast differentiation rather than to an increase in osteoclast survival. Since the formation of TRAP-positive mononuclear cells also requires at least twenty-four hours²⁴, this experiment also allowed us to assess the roles of increased differentiation and survival on the increase in the number of TRAP-positive mononuclear cells found in cultures containing conditioned media from marrow cells incubated with titanium particles. Figure 2, B shows that this process was also due to the stimulation of differentiation rather than to an increase in survival.

Figure 2, A shows a maximal seventy-eightfold increase in the number of TRAP-positive multinucleated cells when conditioned media from marrow cells incubated with titanium particles were compared with conditioned media from marrow cells incubated without particles. Since the maximal stimulation was observed on day 9 in the experiments represented in both Figures 1, A and 2, A, staining for TRAP was performed on day 9 in all of the other experiments unless otherwise indicated.

The effect of the conditioned media from marrow cells cultured with titanium particles was dose-dependent (Fig. 3). In contrast, none of the tested concentrations of conditioned media from marrow cells incubated without particles had a detectable effect on the number of TRAP-positive multinucleated cells. In this experiment, the maximal stimulation was a twelvefold increase in the number of such cells when conditioned media from marrow cells incubated with particles were compared with conditioned media from marrow cells incubated without particles. Since the maximal stimulation was observed in cultures with 25% conditioned media, this concentration was used in all of the other experiments.

Titanium particles increased the number of TRAP-positive multinucleated cells in a dose-dependent manner (Fig. 4). All conditioned media from marrow cells cultured with titanium particle concentrations greater than 1 × 10⁴ particles/cm² significantly (p < 0.001) increased the number of osteoclasts compared with that in conditioned media from marrow cells incubated without particles. The maximal stimulation (an eighteenfold increase in the number of TRAP-positive multinucleated cells) was observed in association with concentrations of 4.2 ×¥ 10⁴ and 8.5 × 10⁴ particles/cm². All other experiments, therefore, were performed with a concentration of 8.5 × 10⁴ titanium particles/cm², which was equal to 3.4 × 10⁵ titanium particles per mL of tissue-culture medium.

To further confirm the osteoclast phenotype of the TRAP-positive multinucleated cells, we also examined the formation of resorption lacunae on slices of elephant ivory. TRAP-positive multinucleated cells were found to be closely associated with resorption lacunae. Moreover, quantitative histomorphometry demonstrated that conditioned media from marrow cells incubated with titanium particles stimulated a significant increase in the formation of resorption lacunae that paralleled the increase in the number of TRAP-positive multinucleated cells (Fig. 5). Thus, conditioned media from marrow cells incubated with titanium particles stimulated a fifteenfold increase in the number of TRAP-positive multinucleated cells and a twenty-fourfold increase in the formation of resorption lacunae compared with conditioned media from marrow cells incubated without particles.

Taken together, the results described above show that the increase in the number of TRAP-positive multinucleated cells induced by conditioned media from marrow cells incubated with titanium particles in our in vitro assay was due to increased osteoclast differentiation. In order to determine whether direct interactions between the marrow cells and the titanium particles were required to induce the formation of TRAP-positive multinucleated cells, we first examined the effect of conditioned media from cultures containing particles but not marrow cells. A comparison of the second and third bars in Figure 6 shows that the presence of marrow cells was required for titanium particles to induce the formation of TRAP-positive multinucleated cells. Wear particles are subject to corrosion and dissolution²⁵. Thus, the observed effects of the titanium particles potentially could be due to released metal ions. In fact, filtrates of titanium particles have been reported to stimulate cytokine production⁷. Therefore, we determined whether filtrates of titanium particles could mimic the ability of the particles to induce the formation of TRAP-positive multinucleated cells. A comparison of the fourth and fifth bars in Figure 6 shows that the filtrates did not have this capacity. Together with our other findings, this experiment demonstrated that the increase in the number of TRAP-positive multinucleated cells was not due to released metal ions, soluble contaminants, or submicrometer particles. Instead, the increase in the number of TRAP-positive multinucleated cells required direct contact between the marrow cells and the titanium particles.

In order to determine whether human cells can also be stimulated by titanium particles to secrete factors that stimulate osteoclast differentiation, we tested conditioned media from human peripheral blood mononuclear cells cultured in the presence and absence of titanium particles. The first three bars in Figure 7 show that the human cells responded like the murine marrow cells did in the experiments represented in Figures 1 through 6. Conditioned media from human mononuclear cells cultured with titanium particles stimulated a thirty-fourfold increase in the formation of TRAP-positive multinucleated cells compared with conditioned media from cultures without particles (Fig. 7). Moreover, conditioned media from human mononuclear cells cultured without particles had no detectable effect on the formation of TRAP-positive multinucleated cells. Use of these human-cell cultures also allowed us to determine which cell type was responsible for secreting the factors that stimulated osteoclast differentiation. For this purpose, we compared mononuclear cells, consisting of a mixture of monocytes and lymphocytes, with a cell preparation obtained by adherence to tissue-culture plastic. Histochemical staining for nonspecific esterase confirmed that >95% of the adherent cells were monocytes. A comparison of the third and fifth bars in Figure 7 shows that the conditioned media from the adherent monocytes stimulated the formation of as many TRAP-positive multinucleated cells as did the conditioned media from the mixed cell population. These results indicate that monocytes are the primary cell type responsible for production of the conditioned medium that stimulates osteoclast differentiation.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

Our results demonstrated that titanium particles induce murine marrow cells and human peripheral blood monocytes to produce conditioned media that stimulate osteoclast differentiation. The mean stimulation in the representative experiments shown in Figures 1 through 7 was a 29.3 ± 9.4-fold increase in the number of TRAP-positive multinucleated cells. The stimulation of osteoclast differentiation by titanium particles required interactions between the marrow cells and the particles themselves and, therefore, was not mimicked by particle filtrates that may have contained metal ions, soluble contaminants released from the particles, or submicrometer particles. The amount of stimulation was regulated in a dose-dependent manner over a wide range of concentrations of both titanium particles and conditioned media. In contrast, conditioned media from cells incubated without particles had no significant effect on any of the examined parameters. Our in vitro results are consistent with the hypothesis that wear particles from orthopaedic implants induce osteolysis by stimulating osteoclast differentiation. Also consistent with this hypothesis are the reports of increased numbers of osteoclasts and their precursors in animal studies of wear particles and studies of tissues surrounding retrieved implants^1,26-29. In what we believe to be the most comprehensive study of retrieved implants to date, Kadoya et al.²⁷ found a twentyfold increase in the number of osteoclasts and a twenty-fivefold increase in the number of CD11b-positive cells on the bone surfaces surrounding revised implants. Since the CD11b-positive cells exhibited many osteoclastic characteristics, including multinucleation, expression of TRAP, and formation of a ruffled border at the bone resorption site²⁷, these cells were most likely monocyte-macrophage lineage cells that were differentiating along the osteoclastic pathway. Our hypothesis is also supported by the finding that synovial fluid from patients with loose implants contained elevated levels of TRAP, presumably because of the secretion of this enzyme by the increased number of osteoclasts³⁰.

The increase in osteoclast differentiation induced by titanium particles led to a parallel increase in bone resorption. Thus, the resorptive activity per osteoclast was not substantially increased—that is, the results were consistent with those in other studies that have shown that wear particles have only a small effect on osteoclastic activity (less than a twofold increase in all cases)^5,30-34.

In the present study, we examined commercially available particles. Although it was impossible to determine whether the particles perfectly mimicked the physicochemical properties of particles that are produced in vivo¹, they were chosen because they were well characterized¹⁹ and mimicked the ability of authentic wear particles^9,10 to induce the production of bone resorptive cytokines^4-8,10 and in vivo osteolysis²⁸.

It was recently reported that mesenchymal cells capable of supporting osteoclast differentiation can be found in the pseudocapsule from patients with a loose implant³⁵. These pseudocapsule mesenchymal cells, as well as the CIMC-4 cells used in the present study, likely induce osteoclast differentiation in response to factors produced by marrow cells stimulated with wear debris by up-regulating the production of RANK (receptor activator of NF-kappa B) ligand. This recently described membrane-bound member of the TNF superfamily is required for osteoclast differentiation and is produced in response to a growing list of bone resorptive agents, including 1a,25-dihydroxyvitamin D3, parathyroid hormone, IL-1, IL-6, and IL-11¹². Its role in osteoclast differentiation induced by wear particles is currently under investigation in our laboratory^36,37.

We know of only one pair of previous in vitro studies in which the effect of orthopaedic wear particles on osteoclast differentiation was examined^38,39. In those studies, orthopaedic wear particles stimulated a two to fourfold increase in osteoclast differentiation. This small degree of stimulation compared with that observed in our study was likely due to technical differences between the studies. For example, osteoclast differentiation may have been almost maximally stimulated in the previous studies even in the cultures without particles since all cultures included 10 nM 1a,25-dihydroxyvitamin D3^38,39, a concentration that elicits maximal stimulation in most osteoclast differentiation systems^17,40. In fact, we have been unable to demonstrate titanium-induced stimulation of osteoclast differentiation in cultures containing 10 nM 1a,25-dihydroxyvitamin D3 (data not shown). Moreover, our osteoclast differentiation assay was specifically chosen because it responds to a variety of bone resorptive cytokines that are likely produced in response to wear particles (as already described). Thus, the small degree of stimulation found in the previous studies^38,39 may have been due to the use of an assay that was not responsive to some of these cytokines that presumably are produced by marrow cells activated by particles. This possibility is especially likely since the effect of the cytokines on osteoclast differentiation is highly synergistic¹⁷.

Net bone loss during osteolysis is due to the rate of bone resorption exceeding the rate of bone formation²⁸. Thus, it is also important to consider the effect of orthopaedic wear debris on bone formation. In fact, a number of cell-culture, animal, and human studies have demonstrated that wear particles inhibit osteoblast differentiation or activity^27,29,41-43. However, in all cases, the inhibition was modest (less than fourfold) and substantially less than the twenty to thirtyfold effect of wear particles on osteoclast differentiation shown in our study as well as in the study of tissue surrounding loose implants in patients²⁷. Taken together, these results indicate that the stimulation of osteoclast differentiation is quantitatively more important in the development of aseptic loosening than is the inhibition of bone formation.

In summary, both the in vivo studies described in the previous paragraphs and our in vitro results provide strong support for the hypothesis that osteoclast differentiation is an important factor in the development of aseptic loosening. This understanding of the cellular mechanisms involved in aseptic loosening suggests that the development of therapeutic interventions to reduce osteoclast differentiation is a promising approach for improving the performance of orthopaedic implants.

Note: The authors are grateful to E. Colton for preparation of the human peripheral blood mononuclear cells; to S. Kaar for assistance with the histomorphometry; and to C. Carlin, J. Ninomiya, and L. Zhang for many helpful discussions.

References

Introduction | Materials and Methods | Results | Discussion | References

Friedman RJ; Black J; Galante JO; Jacobs JJ; and Skinner HB: Current concepts in orthopaedic biomaterials and implant fixation. Inst Course Lect,1994.43: 233-55, 43233 1994

Kobayashi A; Freeman MA; Bonfield W; Kadoya Y; Yamac T; Al-Saffar N; Scott G; and Revell PA: Number of polyethylene particles and osteolysis in total joint replacements. A quantitative study using a tissue-digestion method. J Bone Joint Surg Br,1997.79: 844-8, 79844 1997 [PubMed]

Margevicius KJ; Bauer TW; McMahon JT; Brown SA; and Merritt K: Isolation and characterization of debris in membranes around total joint prostheses. J Bone Joint Surg Am,1994.76: 1664-75, 761664 1994 [PubMed]

Blaine TA; Rosier AN; Puzas JE; Looney RJ; Reynolds PR; Reynolds SD; and O’Keefe RJ: Increased levels of tumor necrosis factor-alpha and interleukin-6 protein and messenger RNA in human peripheral blood monocytes due to titanium particles. J Bone Joint Surg Am.,1996.78: 1181-92, 781181 1996 [PubMed]

Glant TT, and Jacobs JJ: Response of three murine macrophage populations to particulate debris: bone resorption in organ cultures. J Orthop Res,1994.720-31, 720 1994

Gonzales JB; Purdon MA; and Horowitz SM: In vitro studies on the role of titanium in aseptic loosening. Clin Orthop.,1996.330: 244-50, 330244 1996 [PubMed]

Harada Y; Brown S; Merritt K; Wang JT; Doppalapudi VA; Willis AA; Jasty M; Harris WH; and Goldring SR: Effects of metal particles and their corrosion products on human monocyte/macrophages in vitro. Trans Orthop Res Soc,1995.20: 776, 20776 1995

Kane KR; Mochel DM; DeHeer DH; Beebe JD; Marks TR; Hamstra DJ; and Swanson AB: Influence of titanium particle size on the in vitro activation of macrophages. Contemp Orthop,1994.28: 249-61, 28249 1994

Maloney WJ; James RE; and Smith RL: Human macrophage response to retrieved titanium alloy particles in vitro. Clin Orthop,1996.322: 268-78, 322268 1996 [PubMed]

Shanbhag AS; Jacobs JJ; Black J; Galante JO; and Glant TT: Human monocyte response to particulate biomaterials generated in vivo and in vitro. . J Orthop Res,1995.13: 792-801, 13792 1995 [PubMed]

Hicks DG; Judkins AR; Sickel JZ; Rosier RN; Puzas JE; and O’Keefe RJ: Granular histiocytosis of pelvic lymph nodes following total hip arthroplasty. The presence of wear debris, cytokine production, and immunologically activated macrophages. J Bone Joint Surg Am,1996.78: 482-96, 78482 1996 [PubMed]

Greenfield EM; Bi Y; and Miyauchi A: Regulation of osteoclast activity. Life Sci,1999.65: 1087-102, 651087 1999 [PubMed]

Pollice PF, Hsu J, Hicks DG, Bukata S, Rosier RN, Reynolds PR, Puzas JE, O’Keefe RJ. : Interleukin-10 inhibits cytokine synthesis in monocytes stimulated by titanium particles: evidence of an anti-inflammatory regulatory pathway. J Orthop Res,1998.16: 697-704, 16697 1998 [PubMed]

Weyand CM; Geisler A; Brack A; Bolander ME; and Goronzy JJ: Oligoclonal T-cell proliferation and interferon-gamma production in periprosthetic inflammation. Lab Invest,1998.78: 677-85, 78677 1998 [PubMed]

Ninomiya JT; Bi Y; Banks MA; Lavish SA; Goldberg VM; and Greenfield EM: Bone marrow cells produce soluble factors that inhibit osteoclast activity. J Orthop Res,1999.17: 51-8, 1751 1999 [PubMed]

Ragab AA; Lavish SA; Banks MA; Goldberg VM; and Greenfield EM: Osteoclast differentiation requires ascorbic acid. J Bone Miner Res,1998.13: 970-7, 13970 1998 [PubMed]

Van De Motter RR; Ragab A; and Greenfield EM: Synergistic stimulation of osteoclast differentiation by bone resorptive hormones and cytokines in the presence of conditionally immortalized murine calvaria (CIMC) cells [abstract]. Bone,1998.23: 330, 23330 1998

Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council:Guide for the care and use of laboratory animals. Publication 86-23, revised 1985. Washington, DC: United States Department of Health and Human Services, Public Health Service, National Institutes of Health; 1985.

Ragab AA; Van De Motter R; Lavish SA; Goldberg VM; Ninomiya JT; Carlin CR; and Greenfield EM: Measurement and removal of adherent endotoxin from titanium particles and implant surfaces. J Orthop Res,1999.17: 803-9, 17803 1999 [PubMed]

McNally AK, and Anderson JM: Interleukin-4 induces foreign body giant cells from human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells. Am J Pathol,1995.147: 1487-99, 1471487 1995 [PubMed]

Wesolowski G; Duong LT; Lakkakorpi PT; Nagy RM; Tezuka K; Tanaka H; Rodan GA; and Rodan SB: Isolation and characterization of highly enriched, prefusion mouse osteoclastic cells. Exp Cell Res,1995.219: 679-86, 219679 1995 [PubMed]

Jimi E; Shuto T; and Koga T: Macrophage colony-stimulating factor and interleukin-1 alpha maintain the survival of osteoclast-like cells. Endocrinology,1995.136: 808-11, 136808 1995 [PubMed]

Udagawa N; Takahashi N; Jimi E; Matsuzaki K; Tsurukai T; Itoh K; Nakagawa N; Yasuda H; Goto M; Tsuda E; Higashio K; Gillespie MT; Martin TJ; and Suda T: Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-kappa B ligand. Bone,1999.25: 517-23, 25517 1999 [PubMed]

Tsurukai T; Takahashi N; Jimi E; Nakamura I; Udagawa N; Nogimori K; Tamura M; and Suda T: Isolation and characterization of osteoclast precursors that differentiate into osteoclasts on calvarial cells within a short period of time. J Cell Physiol,1998.177: 26-35, 17726 1998 [PubMed]

Jacobs JJ; Gilbert JL; and Urban RM: Corrosion of metal orthopaedic implants. J Bone Joint Surg Am,1998.80: 268-82, 80268 1998 [PubMed]

Sabokbar A; Fujikawa Y; Neale S; Murray DW; and Athanasou NA: Human arthroplasty derived macrophages differentiate into osteoclastic bone resorbing cells. Ann Rheum Dis.,1997.56: 414-20, 56414 1997 [PubMed]

Kadoya Y; Revell PA; al-Saffar N; Kobayashi A; Scott G; and Freeman MA: Bone formation and bone resorption in failed total joint arthroplasties: histomorphometric analysis with histochemical and immunohistochemical technique. J Orthop Res,1996.14: 473-82, 14473 1996 [PubMed]

Kaar SG; Ragab AA; Kaye SJ; Kilic BA; Jinno T; Goldberg VM; Bi Y; Stewart MC; Carter JR; and Greenfield EM: Rapid repair of titanium particle-induced osteolysis is dramatically reduced in aged mice. J Orthop Res,2001.19: 171-8, 19171 2001 [PubMed]

Goodman S; Aspenberg P; Song Y; Knoblich G; Huie P; Regula D; and Lidgren L: Tissue ingrowth and differentiation in the bone-harvest chamber in the presence of cobalt-chromium-alloy and high-density-polyethylene particles. J Bone Joint Surg Am,1995.77: 1025-35, 771025 1995 [PubMed]

Kim KJ; Hijikata H; Itoh T; and Kumegawa M: Joint fluid from patients with failed total hip arthroplasty stimulates pit formation by mouse osteoclasts on dentin slices. J Biomed Mater Res,1998.43: 234-40, 43234 1998 [PubMed]

Algan SM; Purdon M; and Horowitz SM: Role of tumor necrosis factor alpha in particulate-induced bone resorption. J Orthop Res,1996.14: 30-5, 1430 1996 [PubMed]

Herman JH; Sowder WG; Anderson D; Appel AM; and Hopson CN: Polymethylmethacrylate-induced release of bone resorbing factors. J Bone Joint Surg Am,1989.71: 1530-41, 711530 1989 [PubMed]

Kim KJ; Itoh T; Tanahashi M; and Kumegawa M: Activation of osteoclast-mediated bone resorption by the supernatant from a rabbit synovial cell line in response to polyethylene particles. J Biomed Mater Res,1996.32: 3-9, 323 1996 [PubMed]

Murray DW, and Rushton N: Macrophages stimulate bone resorption when they phagocytose particles. J Bone Joint Surg Br,1990.72: 988-92, 72988 1990 [PubMed]

Sakai H; Jingushi S; Yasuda K; Harada H; Shuto T; Kukita A; Kukita T; and Iwamoto Y: Pseudocapsule from revision THA hip with osteolysis includes stromal cell like fibroblasts which induce osteoclast differentiation. Trans Orthop Res Soc,1999.24: 914, 24914 1999

Nalepka JL; Lowe RW; and Greenfield EM: The role of ODF/OPGL in stimulation of osteoclast differentiation by TNF-alpha. Trans Orthop Res Soc,2001.26: 959, 26959 2001

Van De Motter RR; Lowe RW; Bi Y; Goldberg VM; and Greenfield EM: Bone resorptive cytokines produced in response to wear particles induce osteoclast differentiation by stimulating ODF/OPGL expression by mesenchymal support cells. Trans Orthop Res Soc,2000.25: 75, 2575 2000

Sabokbar A; Fujikawa Y; Brett J; Murray DW; and Athanasou NA: Increased osteoclastic differentiation by PMMA particle-associated macrophages. Inhibitory effect by interleukin 4 and leukemia inhibitory factor. Acta Orthop Scand,1996.67: 593-8, 67593 1996 [PubMed]

Sabokbar A; Fujikawa Y; Murray DW; and Athanasou NA: Radio-opaque agents in bone cement increase bone resorption. J Bone Joint Surg Br,1997.79: 129-34, 79129 1997 [PubMed]

Udagawa N, Takahashi N, Akatsu T, Sasaki T, Yamaguchi A, Kodama H, Martin TJ, Suda T. : The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology,1989.125: 1805-13, 1251805 1989 [PubMed]

Allen MJ; Myer BJ; Millett PJ; and Rushton N: The effects of particulate cobalt, chromium and cobalt-chromium alloy on human osteoblast-like cells in vitro. J Bone Joint Surg Br,1997.79: 475-82, 79475 1997 [PubMed]

Dean DD; Schwartz Z; Liu Y; Blanchard CR; Agrawal CM; Mabrey JD; Sylvia VL; Lohman CH; and Boyan BD: The effect of ultra-high molecular weight polyethylene wear debris on MG63 osteosarcoma cells in vitro. J Bone Joint Surg Am,1999.81: 452-61, 81452 1999 [PubMed]

Yao J; Cs-Szabo G; Jacobs JJ; Kuettner KE; and Glant TT: Suppression of osteoblast function by titanium particles. J Bone Joint Surg Am,1997.79: 107-12, 79107 1997 [PubMed]

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