Richard Chiu

Ultrahigh molecular weight polyethylene wear debris inhibits osteoprogenitor proliferation and differentiation in vitro

Polyethylene wear debris induces progressive osteolysis by increasing bone degradation and suppressing bone formation. Polyethylene particles inhibit the function of mature osteoblasts, but whether polyethylene particles also interfere with the proliferation and differentiation of osteoprogenitor cells is unknown. In this study, we investigated the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on the osteogenic activity of primary murine bone marrow osteoprogenitors and MC3T3-E1 preosteoblastic cells in vitro. Submicron-sized UHMWPE particles generated from wear simulator tests were isolated from serum-containing solution by density gradient centrifugation. The particles were coated onto the surface of culture wells at concentrations of 0.038, 0.075, 0.150, 0.300, and 0.600% v/v in a layer of type I collagen matrix. Primary murine bone marrow cells and MC3T3-E1 preosteoblasts were seeded onto the particle-collagen matrix and induced to differentiate in osteogenic medium for 20 days. Exposure of both cell populations to UHMWPE particles resulted in a dose-dependent decrease in mineralization, proliferation, alkaline phosphatase activity, and osteocalcin production when compared with control cells cultured on collagen matrix without particles. Complete suppression of osteogenesis was observed at particle concentrations > or =0.150% v/v. This study demonstrated that UHMWPE particles inhibit the osteogenic activity of osteoprogenitor cells, which may result in reduced periprosthetic bone regeneration and repair.

Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5.

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3‐E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3‐E1 cells treated with PMMA particles over a 72‐h period showed a significant reduction in cell viability and proliferation as indicated by a dose‐ and time‐dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose‐dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V‐stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3‐E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose‐dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5‐induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation.

Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3‐E1 osteoprogenitor cells

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3‐E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3‐E1 cultures challenged with PMMA particles showed a gradual dose‐dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038–0.150% v/v) and complete reduction of these outcome parameters at high particle doses (≥0.300% v/v). MC3T3‐E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3‐E1 cells grown in conditioned medium from particle‐treated MC3T3‐E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3‐E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation.

OP-1 (BMP-7) stimulates osteoprogenitor cell differentiation in the presence of polymethylmethacrylate particles

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation, proliferation, and mineralization of osteoprogenitor cells in vitro. In this study, we investigated the effects of OP-1 (BMP-7) on the osteogenesis of MC3T3-E1 osteoprogenitor cells exposed to PMMA particles in vitro. MC3T3-E1 cells challenged with PMMA particles on the 1st day of differentiation in osteogenic culture showed a significant dose-dependent decrease in mineralization and alkaline phosphatase expression over a 20-day culture period. Exposure of these cells to OP-1 (200 ng/mL) during days 1-4, 1-20, and 4-20 in the presence of PMMA particles resulted in significant increases in mineralization and alkaline phosphatase expression at all particle doses. Addition of OP-1 to MC3T3-E1 cultures challenged with PMMA particles on the 4th day of differentiation in osteogenic media also resulted in significant increases in mineralization and alkaline phosphatase expression. This study has shown that OP-1 stimulates osteogenesis in MC3T3-E1 osteoprogenitor cells that have been inhibited by PMMA particles. Local administration of OP-1 to the site of osteolysis may be a potential adjunctive therapy to reverse the bone destruction due to wear particles.

Polymethylmethacrylate particle exposure causes changes in p38 MAPK and TGF-β signaling in differentiating MC3T3-E1 cells

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris

Total joint replacements are one of the most commonly performed orthopaedic procedures worldwide, with over 700,000 surgeries performed annually in the US to treat arthritic conditions of the hip and knee. One of the major complications of total joint replacement is implant wear and osteolysis, a process that involves continuous shedding of micronand submicron-sized particles from implant components. Implant particles elicit cascades of inflammatory, osteolytic, and granulomatous reactions from macrophages, osteoclasts, and fibroblasts, causing the prosthesis to become unstable. Since the mid 1990s, in vitro studies have shown that wear debris particles inhibit the osteogenic function of osteoblasts and osteoprogenitor cells of human and rodent species. Osteolysis and implant loosening involve not only increased bone resorption by osteoclasts and inflammatory cells, but also reduced bone formation by osteoblasts and their progenitors. This disruption of proliferation, differentiation, function, and survival of osteoblasts prevents the implant from properly integrating with surrounding bone. The inhibitory effects of implant wear debris on osteoblasts and osteoprogenitors have been demonstrated using particles of metallic (titanium, cobalt chrome), polymeric (polyethylene, PMMA), and ceramic (alumina, zirconia) implants. Human and rodent primary osteoblasts and osteoblast cell lines, such as MG-63 cells, treated with titanium and polyethylene particles in culture, uniformly show reduced type I collagen synthesis with evidence of particle phagocytosis and morphological changes consistent with cell injury and cytoskeletal disorganization on microscopy. Selected studies also show that particles impair osteoblast viability, proliferation, adhesion, extracellular matrix production, and osteogenic protein expression (e.g., alkaline phosphatase). Implant particles uniformly stimulate expression of NF-κB and IL-6, IL-8, PGE2, RANKL, M-CSF, and MCP-1, pro-inflammatory factors known to recruit monocyte-macrophages or induce osteoclast differentiation and activity. These studies also indicate that the effect of particles on osteoblasts depends on particle size and composition and the maturational state of the cell. Metal implants such as cobalt chromium and titanium alloys pose an additional risk of metal ion toxicity. Wear debris particles also inhibit the osteogenic activity of osteoprogenitors and marrow stromal cells (MSCs). Human bone marrow-derived MSCs exposed to titanium particles exhibit reduced proliferation, type I collagen expression, viability, and matrix mineralization with evidence of particle phagocytosis and structural and biochemical changes indicative of

INHIBITION OF MARROW STROMAL CELL OSTEOGENESIS BY POLYMETHYLMETHACRYLATE WEAR PARTICLES AND SOLUBLE FACTORS RELEASED FROM POLYMETHYLMETHACRYLATE PARTICLE-ACTIVATED MACROPHAGES.: 482

Purpose Implant loosening of total joint arthroplasty may involve reduced bone formation resulting from the effects of prosthetic wear debris on osteoblast precursors. The inhibition of osteoblastic differentiation by wear debris may occur via a direct effect of wear particles on osteoblast progenitors or via an indirect effect of inhibitory cytokines released from particle-activated inflammatory cells. This study determined whether the direct exposure of marrow stromal cells to polymethylmethacrylate (PMMA) cement particles inhibits the ability of these cells to differentiate into osteoblasts and whether macrophages exposed to PMMA particles produce soluble factors that can indirectly inhibit osteogenesis. Methods Osteogenesis was induced by growing murine marrow stromal cells (MSCs) in osteogenic medium containing 10−7 M dexamethasone, 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate for 15 days. Throughout their 15-day culture period in osteogenic medium, MSCs were either directly challenged with PMMA particles (0.30% v/v) or incubated in conditioned medium from Raw264.7 macrophage cultures that had been pretreated with PMMA particles (0.30% v/v) for 24 hours. To determine whether the effects of PMMA particles on MSC osteogenesis were reversible, particles were removed from MSC cultures after 1, 3, and 5 days of particle treatment, and cell growth in osteogenic medium was continued in the absence of particles for 15 more days. MSC cultures were assessed for the levels of cell proliferation, alkaline phosphatase expression, and bone mineralization at the end of the 15-day osteogenic period. Results MSCs treated with PMMA particles throughout the 15-day culture period showed a 90 to 95% reduction in the levels of proliferation, alkaline phosphatase expression, and mineralization. MSCs treated with PMMA for only 5 days also showed a 90 to 95% reduction in these outcome parameters, whereas MSCs treated for ≤ 3 days showed a significantly diminished inhibitory response. MSCs incubated in conditioned medium from particle-treated macrophages showed a significant 53% reduction in mineralized nodules. Conclusions This study has demonstrated that the direct exposure of MSCs to PMMA particles causes complete, irreversible suppression of osteogenesis. Macrophages exposed to PMMA particles also release soluble factors that can inhibit mineralization. The inhibition of bone growth during implant loosening is therefore due to the effects of wear particles and macrophage-released cytokines on osteoblast progenitors.

521 POLYMETHYLMETHACRYLATE PARTICLES INHIBIT OSTEOBLASTIC DIFFERENTIATION OF BONE MARROW OSTEOPROGENITOR CELLS IN VITRO.

Purpose Particulate debris generated from orthopedic implants induces osteolysis at the bone-implant interface, causing the prosthesis to become unstable. Bone regeneration in the prosthetic bed depends on the activity of osteoblasts and their differentiation from osteoprogenitors in the bone marrow. This study investigated the effects of wear particles of polymethylmethacrylate (PMMA), a prosthetic material, on the ability of bone marrow osteoprogenitor cells to differentiate into functional osteoblasts in vitro. Methods Bone marrow cells isolated from the femurs and tibias of C57 mice were grown in osteogenic medium containing 10-7 M dexamethasone, 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate. Cell cultures were challenged with PMMA particles at concentrations of 0.038%, 0.075%, 0.150%, 0.300%, and 0.600% v/v on the initial day (day 0) of growth in osteogenic medium. Three additional cultures were challenged with particles on the 5th, 10th, and 15th day of growth in osteogenic medium to assess the particle effects at later stages of differentiation. All cultures were grown for 15 days past the time of particle addition, after which the cells were measured for the levels of bone mineralization, alkaline phosphatase expression, and cell proliferation. In a time course experiment, measurements were taken at 5-day intervals for the cultures challenged with 0.300% v/v PMMA on the initial day of growth in osteogenic medium. Results Addition of PMMA particles to bone marrow cells on the first day (day 0) of growth in osteogenic medium resulted in a dose-dependent decrease in bone mineralization, alkaline phosphatase expression, and cell proliferation, with complete suppression of osteoblastic phenotype observed at particle concentrations ≥ 0.300% v/v. Cells challenged with 0.300% v/v PMMA on the first day of differentiation showed no rise in bone mineralization, alkaline phosphatase expression, and cell proliferation throughout the entire culture period. Cells treated with particles on the 5th, 10th, and 15th day of growth in osteogenic medium showed insignificant reductions in bone mineralization and alkaline phosphatase expression. Conclusions This study has demonstrated that PMMA particles suppress osteoprogenitor differentiation and function. The strongest inhibitory effects are observed when the cells are challenged within the first 5 days of osteoblastic differentiation. Beyond this time frame, the osteoprogenitors become less sensitive to particle treatment as they develop from relatively undifferentiated progenitors into mature osteoblasts.