The effect of ultra-high molecular weight polyethylene wear debris on MG63 osteosarcoma cells in vitro

BACKGROUND: Focal osteolysis due to ultra-high molecular weight polyethylene wear debris involves effects on both bone resorption and bone formation. METHODS: The response of MG63 osteoblast-like osteosarcoma cells to ultra-high molecular weight polyethylene wear debris isolated by enzymatic digestion of granulomatous tissue obtained from the sites of failed total hip arthroplasties was examined. Scanning electron microscopy, particle-size analysis, and Fourier transform infrared spectroscopy were used to characterize the number, morphology, size distribution, and chemical composition of the particles. Cell response was assessed by adding particles at varying dilutions to confluent cultures and measuring changes in cell proliferation (number of cells and [3H]-thymidine incorporation), osteoblast function (alkaline-phosphatase-specific activity and osteocalcin production), matrix production (collagen production and proteoglycan sulfation), and local cytokine production (prostaglandin-E2 production). RESULTS: The mean size of the particles was 0.60 micrometer, and 95 percent of the particles had a size of less than 1.5 micrometers. The number of particles per gram of tissue ranged from 1.39 to 3.38x10(9). Three of the four batches of particles were endotoxin-free. Exposure of the cells to particles of wear debris significantly increased the number of cells (p<0.05) and the [3H]-thymidine incorporation (p<0.05) in a dose-dependent manner. In contrast, the addition of particles decreased alkaline-phosphatase-specific activity and osteocalcin production. Collagen production and proteoglycan sulfation were also decreased, while prostaglandin-E2 synthesis was increased by the addition of particles. CONCLUSIONS: Ultra-high molecular weight polyethylene particles isolated from human tissue stimulated osteoblast proliferation and prostaglandin-E2 production and inhibited cell differentiation and matrix production. These results indicate that particles of wear debris inhibit cell functions associated with bone formation and that osteoblasts may produce factors in response to wear debris that influence neighboring cells, such as osteoclasts and macrophages. CLINICAL RELEVANCE: Particles of wear debris, especially ultra-high molecular weight polyethylene, have been implicated in the loosening of implants and the development of osteolysis. The present study shows that particles of ultra-high molecular weight polyethylene isolated from human tissue inhibit osteoblast functions associated with bone formation. In addition, particles of wear debris induced osteoblasts to secrete factors capable of influencing neighboring cells, such as osteoclasts and macrophages. These results suggest that osteoblasts may play a role in the cascade of events leading to granuloma formation, osteolysis, and failure of orthopaedic implants.     

Effect of submicron polyethylene particles on an osseointegrated implant

In a rabbit model of a weight bearing, articulating prosthetic joint we repeatedly injected submicron particles of Ultra-High-Molecular-Weight-Polyethylene (UHMWPE) produced in a hip simulator. The contralateral knee with the same prosthesis was injected with carrier (NaCl) without UHMWPE. Histomorphometrical studies on undecalcified cut and ground sections at 26 and 42 weeks involved quantifications of the entire bone to metal contact and the bone area around each implant. We found no statistically significant differences between test and control groups, and the UHMWPE debris did not induce any significant osteolysis, indicating that an osseointegrated implant with a sealed interface may not be affected by UHMWPE debris or progress to aseptic loosening.     

Effect of submicron polyethylene particles on an osseointegrated implant: an experimental study with a rabbit patello-femoral prosthesis

In a rabbit model of a weight bearing, articulating prosthetic joint we repeatedly injected submicron particles of Ultra-High-Molecular-Weight-Polyethylene (UHMWPE) produced in a hip simulator. The contralateral knee with the same prosthesis was injected with carrier (NaCl) without UHMWPE. Histomorphometrical studies on undecalcified cut and ground sections at 26 and 42 weeks involved quantifications of the entire bone to metal contact and the bone area around each implant. We found no statistically significant differences between test and control groups, and the UHMWPE debris did not induce any significant osteolysis, indicating that an osseointegrated implant with a sealed interface may not be affected by UHMWPE debris or progress to aseptic loosening.     

磨损颗粒引发假体周围骨溶解的机制及药物治疗进展

目的对磨损颗粒引发假体周围骨溶解的机制及药物治疗进展进行了综述介绍。方法对国外近期相关文献进行综述。结果及结论人工关节置换是治疗晚期骨关节炎及老年股骨颈骨折的有效方法,能达到解除关节疼痛、重建活动功能及提高生活质量的目的。人工关节无菌性松动是影响人工关节使用寿命和远期疗效的最重要的并发症。磨损颗粒诱发假体周围组织细胞产生一系列生物学反应是导致假体周围骨溶解及假体无菌性松动的重要因素。磨损颗粒刺激假体周围的巨噬细胞、成骨细胞、成纤维细胞等产生多种细胞因子,形成破骨细胞性骨吸收,同时影响成骨细胞的分化及功能,抑制骨形成。药物在预防和治疗磨损颗粒引起的人工关节松动方面能起到积极的作用。     

Ultrahigh molecular weight polyethylene particles have direct effects on proliferation,differentiation, and local factor production of MG63 osteoblast-like cells

Small particles of ultrahigh molecular weight polyethylene stimulate formation of foreign-body granulomas and bone resorption. Bone formation may also be affected by wear debris. To determine if wear debris directly affects osteoblasts, we characterized a commercial preparation of ultrahigh molecular weight polyethylene (GUR4150) particles and examined their effect on MG63 osteoblast-like cells. In aliquots of the culture medium containing ultrahigh molecular weight polyethylene, 79% of the particles were less than 1 μm in diameter, indicating that the cells were exposed to particles of less than 1 μm. MG63 cell response to the particles was measured by assaying cell number. [³H]thymidine incorporation, alkaline phosphatase specific activity, osteocalcin production, [³⁵S]sulfate incorporation, and production of prostaglandin E₂ and transforming growth factor-β. Cell number and [³H]thymidine incorporation were increased in a dose-dependent manner. Alkaline phosphatase specific activity, a marker of cell differentiation for the cultures, was significantly decreased, but osteocalcin production was not affected. [³⁵S]sulfate incorporation, a measure of extracellular matrix production, was reduced. Prostaglandin E₂ release was increased, but transforming growth factor-β production was decreased in a dose-dependent manner. This shows that ultrahigh molecular weight polyethylene particles affect MG63 proliferation, differentiation, extracellular matrix synthesis, and local factor production. These effects were direct and dose dependent. The findings suggest that ultrahigh molecular weight polyethylene wear debris particles with an average size of approximately 1 μm may inhibit bone formation by inhibiting cell differentiation and reducing transforming growth factor-β production and matrix synthesis. In addition, increases in prostaglandin E₂ production may not only affect osteoblasts by an autocrine pathway but may also stimulate the proliferation and activation of cells in the monocytic lineage. These changes favor decreased bone formation and increased bone resorption as occur in osteolysis.     

Polyethylene and methyl methacrylate particle-stimulated inflammatory tissue and macrophages up-regulate bone resorption in a murine neonatal calvaria in vitro organ system.

There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. This study examined the effect of inflammatory membranes stimulated with methyl methacrylate and polyethylene on bone resorption, using the murine air pouch model. The capacity of RAW 264.7 mouse macrophages exposed to polymer particles to produce factors affecting bone metabolism was also studied. Neonatal calvaria bones were co-cultured with either pouch membranes or conditioned media from activated macrophages. Bone resorption was measured by the release of calcium from cultured bones, and the activity of tartrate-resistant acid phosphatase in both bone sections and culture medium was also assayed. Results showed that inflammatory pouch membrane activated by methyl methacrylate and polyethylene enhanced osteoclastic bone resorption. Conditioned media from particles stimulated mouse macrophages also stimulated bone resorption, although this effect was weaker than resorption induced by inflammatory pouch membranes. The addition of the particles directly into the medium of cultured calvaria bones had little effect on bone resorption. Our observations indicate that both inflammatory tissue and macrophages provoked by particles can stimulate bone resorption in cultured mouse neonatal calvaria bones. This simple in vitro bone resorption system allows us to investigate the fundamental cellular and molecular mechanism of wear debris induced bone resorption and to screen potential therapeutic approaches for aseptic loosening.     

Polyethylene particles from a hip simulator cause (45)Ca release from cultured bone

Periprosthetic osteolysis is a dominant factor in the success or failure of total hip prostheses. Polyethylene wear debris has been implicated in the process of bone resorption and subsequent implant loosening. The present study is the first to examine the effect of ultra high molecular weight polyethylene (UHMWPE) wear debris produced by a hip simulator on calvarial bone resorption in vitro. (45)Ca release was measured in cultured mouse calvarial bone samples. Although short-term exposure to UHMWPE particles (2 h) decreased (45)Ca release, longer-term exposure for 1-2 days increased release in a dose-dependent manner. After one-day exposure to 7.5 x 10(6) particles per mL, 18% more (45)Ca was released from cultured calvarial bone than from control samples. It was concluded that UHMWPE wear particles either directly or indirectly stimulated osteoclasts to activate bone resorption. Polyethylene wear debris contributes to the osteolytic process at the bone-implant interface.     

微小假体磨损颗粒诱导置入物旁骨溶解的扫描电镜观察

为比较不同人工关节微小磨损颗粒对假体－骨界面处骨整合及骨整合及骨溶解的影响,探讨假体松动的机制。方法本实验采用扫描电镜技术对钛事金、钴－铬－钼与聚乙烯（ｕｌｔｒａｈｉｇｈｍｏｌｅｃｕｌａｒｗｅｉｇｈｔｐｏｌｙｅｔｈｙｌｅｎｅ,ＵＨＭＷＰＥ）三种微小颗粒诱导的假体周围骨组织改变进行超微结构观察。结果研究发现,直径２．５μｍ的Ｔｉ－６Ａｌ－４Ｖ颗粒诱导置入物旁骨吸收或骨溶解的程度明显低于相同直径的Ｃｏ     

The interaction of the macrophage and the osteoblast in the pathophysiology of aseptic loosening of joint replacements

Macrophage phagocytosis of cement particles with production of inflammatory mediators is a component of the underlying mechanism of aseptic loosening of joint prostheses. Prostaglandin E₂ (PGE₂), a bone resorbing mediator, has been implicated in the loosening process. Investigations have shown that macrophage phagocytosis of cement particles leads to production of bone-resorbing mediators other than PGE₂. In this study, conditioned medium from macrophages exposed to crushed simplex cement particles stimulated osteoblasts to release radiolabeled arachidonic acid and metabolites. Incubation of osteoblasts in conditioned medium from macrophages exposed to cement particles small enough to be phagocytized increased PGE₂ release 80-fold over unexposed osteoblasts (P<0.001). Incubation of osteoblasts in conditioned medium from macrophages exposed to particles too large to be phagocytized, or to bone cement filtrate, did not stimulate PGE₂ release. We propose that the role of the macrophage in aseptic loosening is primarily to recognize the mechanical failure of the cement mantle by phagocytosis of cement particles and subsequent production of small amounts of specific mediators. These mediators stimulate surrounding osteoblasts to secrete PGE₂, which then amplifies the inflammatory response and ultimately results in bone resorption and aseptic loosening.     

Nitric oxide and prostaglandin E2 production in response to ultra-high molecular weight polyethylene particles depends on osteoblast maturation state

BACKGROUND: Recent studies have shown that osteoblast-like cells respond directly to ultra-high molecular weight polyethylene particles in culture, suggesting that they may be involved in aseptic loosening of endoprostheses. We tested the hypothesis that the state of cell maturation plays a role in the response of osteogenic cells to ultra-high molecular weight polyethylene particles. METHODS: MG63 cells (immature osteoblast-like cells), OCT-1 cells (mature secretory osteoblast-like cells), and MLO-Y4 cells (osteocyte-like cells) were treated for twenty-four hours with commercial ultra-high molecular weight polyethylene particles with an average diameter of 1 mm. The effect of particle treatment on cell proliferation was assessed by measuring the number of cells, whereas the effects on differentiation and local factor production were assessed by measuring the production of osteocalcin, prostaglandin E2, and nitric oxide. The effect of particles on apoptosis was also evaluated. RESULTS: The addition of ultra-high molecular weight polyethylene particles increased the number of MG63 cells, did not affect the number of OCT-1 cells, and led to a decrease in the number of MLO-Y4 cells. The observed changes in cell number were not due to programmed cell death, as no more than 3% of the cells in cultures treated with the highest concentration of particles were undergoing apoptosis. Osteocalcin production was not affected by the addition of particles. Prostaglandin E2 production was increased in all three types of cultures, but the effect was greatest in OCT-1 cell cultures, as was the absolute amount of prostaglandin E2 produced. Nitric oxide production was unaffected in MG63 cell cultures, but it was stimulated in OCT-1 and MLO-Y4 cell cultures. CONCLUSIONS: The results of the present study support the hypothesis that osteoblast cell maturation state plays an important role in the response to ultra-high molecular weight polyethylene particles and that the terminally differentiated osteocyte may be involved in the bone response to wear debris in vivo.     

Osteoclastic differentiation by mononuclear phagocytes containing biomaterial particles

Aseptic loosening of implant components is a common and important complication of both cemented and uncemented prosthetic joint replacements. Wear particles derived from organic polymer and metal implant biomaterials are commonly found within macrophages and macrophage polykaryons in the fibrous membrane between loose implant components and the host bone undergoing resorption. In order to determine whether biomaterial particle-containing, foreign-body macrophages may contribute to periprosthetic bone resorption, we cultured murine monocytes that had phagocytosed particles of biomaterials commonly employed in bone implant surgery [polymethylmethacrylate (PMMA), ultra-high molecular weight polyethylene (PE), titanium and chromium-cobalt] on bone slices and glass coverslips with UMR 106 osteoblast-like stromal cells in the presence of 1,25-dihydroxy-vitamin D₃. Under these conditions, all biomaterial particle-containing, foreign-body macrophages differentiated into osteoclastic cells, i.e. tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells capable of extensive lacunar bone resorption. This study shows that particle phagocytosis by macrophages does not abrogate the ability of these cells to undergo osteoclast differentiation. These findings emphasise the importance of the foreign-body macrophage response to biomaterial wear particles in the pathogenesis of aseptic loosening.     

Implant wear induces inflammation, but not osteoclastic bone resorption, in RANK(-/-) mice.

Signaling of RANK (receptor activator of nuclear factor kappa B) through its ligand RANKL appears critical in osteolysis associated with aseptic loosening (AL). The purpose of this study was to investigate the role of RANK in a murine osteolysis model developed in RANK knockout (RANK(-/-)) mice. Ultra high molecular weight polyethylene (UHMWPE) debris was introduced into established air pouches on RANK(-/-) mice, followed by implantation of calvaria bone from syngeneic littermates. Wild type C57BL/6 (RANK(+/+)) mice injected with either UHMWPE or saline alone were included in this study. Pouch tissues were collected 14 days after UHMWPE inoculation for molecular and histology analysis. Results showed that UHMWPE stimulation induced strong pouch tissue inflammation in RANK(-/-) mice, as manifested by inflammatory cellular infiltration, pouch tissue proliferation, and increased gene expression of IL-1beta, TNFalpha, and RANKL. However, the UHMWPE-induced inflammation in RANK(-/-) mice was not associated with the osteoclastic bone resorption observed in RANK(+/+) mice. In RANK(+/+) mice subjected to UHMWPE stimulation, a large number of TRAP(+) cells were found on the implanted bone surface, where active osteoclastic bone resorption was observed. No TRAP(+) cells were found in UHMWPE-containing pouch tissues of RANK(-/-) mice. Consistent with the lack of osteoclastic activity shown by TRAP staining, no significant UHMWPE particle-induced bone resorption was found in RANK(-/-) mice. A well preserved bone collagen content (Van Gieson staining) and normal plateau surface contour [microcomputed tomography (microCT)] of implanted bone was observed in RANK(-/-) mice subjected to UHMWPE stimulation. In conclusion, this study provides the evidence that UHMWPE particles induce strong inflammatory responses, but not associated with osteoclastic bone resorption in RANK(-/-) mice. This indicates that RANK signaling is essential for UHMWPE particle-induced osteoclastic bone resorption, but does not participate in UHMWPE particle-induced inflammatory response.     

New insight into the mechanism of hip prosthesis loosening: effect of titanium debris size on osteoblast function.

The incidence of rheumatoid arthritis and osteoarthritis is on the rise due to our expanding elderly population. Total joint arthroplasty is the most successful, prevalent treatment modality for these and other degenerative hip conditions. Despite the wide array of prosthetic devices commercially available, hip prostheses share a common problem with a gradual and then accelerating loss of bone tissue and bone-implant interface integrity, followed by implant instability and loosening. Implant failure is largely the result of inevitable wear of the device and generation of wear debris. To provide information for the development of improved prosthetic wear characteristics, we examined the effects of size-separated titanium particles on bone forming cell populations. We demonstrate unequivocally that particle size is a critical factor in the function, proliferation, and viability of bone-forming osteoblasts in vitro. In addition, we have elucidated the time-dependent distribution of the phagocytosed particles within the osteoblast, indicating an accumulation of particles in the perinuclear area of the affected cells. The report finds that particle size is a critical factor in changes in the bone formation-related functions of osteoblasts exposed to simulate wear debris, and that 1.5-4 microm titanium particles have the greatest effect on osteoblast proliferation and viability in vitro. The size of titanium particles generated through wear of a prosthetic device may be an important consideration in the development of superior implant technology.     

Human osteoblast-like cells phagocytose metal particles and express the macrophage marker CD68 in vitro

Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages.     

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.     

The application potential of sintered beta-dicalcium pyrophosphate in total joint arthroplasty

An in vitro bone cell culture model was used to evaluate the potential application of sintered beta-dicalcium pyrophosphate (SDCP) in arthroplasty surgery. Primary osteoclasts and osteoblasts were cocultured with different sizes of SDCP particles. The changes in cell counts and the synthesis and secretion of alkaline phosphatase, acid phosphatase, and prostaglandin E(2) in response to the SDCP particles were monitored. When bone cells were cultured with SDCP particles smaller than 53 microm, both the osteoblast and osteoclast cell counts decreased significantly. When the SDCP particles were larger than 177 microm, although the osteoblast population increased significantly, the osteoclast population decreased significantly. Simultaneously, the titer of prostaglandin E(2) in the medium and the cytoplasmic prostaglandin E(2) increased significantly. We concluded that SDCP is a potentially useful bioceramic for the prevention of osteoclast-mediated periprosthetic osteolysis.     

Inflammatory Response to Implant Particulates in a Macrophage/Osteoblast Coculture Model

The purpose of this study was to further define the cellular response to titanium and polymethylmethacrylate (PMMA) particles in aseptic loosening, and to determine if the use of pamidronate may be effective in inhibiting bone resorption associated with this response. Macrophages and osteoblasts were cocultured to simulate the environment around an aseptically loose prosthesis. Macrophages were plated on the bottom of six well plates and osteoblasts were plated on culture dish inserts, and placed into the wells with the macrophages. Incubation of macrophages with PMMA in this system led to release of prostaglandin E (PGE₂), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-6 (IL-6). Incubation with titanium led to release of tumor necrosis factor (TNF) and IL-6. Exposure of calvaria to media from cells exposed to either PMMA or titanium led to release of calcium 45. Incubation of calvaria with pamidronate was able to inhibit release of calcium 45 associated with exposure to the macrophage/osteoblast/particle conditioned medium. Bone resorption at the interface between implant and bone is a consistent feature leading to loosening of orthopedic implants. By inhibiting bone resorption associated with the inflammatory response to implant particulates, pamidronate or other bisphosphonates may have clinical utility in the treatment or prevention or aseptic loosening. Received: 22 December 1995 / Accepted: 3 May 1996     

Third-body wear of highly cross-linked polyethylene in a hip simulator

The wear performance of a radiation cross-linked melted ultrahigh-molecular-weight polyethylene (UHMWPE) articulating against 28-mm cobalt chrome femoral heads in the presence of third-body particulate debris was investigated in a hip simulator and compared with the wear of conventional UHMWPE. Particles of aluminum oxide or bone cement containing barium sulfate were added to the serum. In the presence of aluminum oxide particles, the incremental wear rates of conventional UHMWPE averaged as high as 149 +/- 116 mg/million cycles compared with 37 +/- 38 mg/million cycles for the highly cross-linked components. The difference in the average weight loss was statistically significant at P <.01. With bone cement particles, the conventional UHMWPE components had an average incremental wear rate of 19 +/- 5mg/million cycles, and the wear rate of the highly cross-linked UHMWPE components was 0.5 +/- 0.7 mg/million cycles.