DIRECT AND INDIRECT INDUCTION OF APOPTOSIS IN HUMAN MESENCHYMAL STEM CELLS IN RESPONSE TO TITANIUM PARTICLES *Wang, ML; *Tuli R; **Manner PA; ***Sharkey, PF; *Hall DJ; +*Tuan RS +* Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, Bethesda, MD INTRODUCTION The bone marrow stroma contains mesenchymal stem cells (MSCs), multipotential cells capable of differentiating along various mesenchy- mal lineages, including osteogenesis; and, in a procedure such as total hip arthroplasty (THA), MSCs are likely to be exposed to high concen- trations of implant wear particles along the femoral stem of the prosthe- sis. We have previously reported that the osteogenic differentiation of human mesenchymal stem cells (hMSCs) is suppressed upon exposure to titanium particles accompanied by reduced bone sialoprotein (BSP) gene expression, diminished production of collagen type I and BSP, de- creased cellular viability and proliferation, and inhibition of extracellular matrix mineralization. In this study, we further investigate hMSC cyto- toxicity upon exposure to submicron particles of commercially pure tita- nium (cpTi) and zirconium oxide (ZrO2). MATERIAL AND METHODS Particle Preparation and Characterization : cpTi and ZrO2 particles (Sigma-Aldrich) were sterilized and resuspended in specific particle concentrations. Limulus assay of cpTi and ZrO2 particulate suspensions excluded endotoxin levels exceeding 0.06 EU/ml. Laser scattering par- ticle analysis (Horiba LA-910; EMD) revealed mean particle sizes of 0.939±0.380 and 0.876±0.540 μm for cpTi and ZrO2 respectively. MSCs Isolation and Culture : hMSCs were isolated from bone marrow aspirates of patients undergoing THA or total knee arthroplasty (TKA) for primary osteoarthritis and culture expanded in vitro. This study was approved by the NIH Office of Human Subjects Research and the IRB of George Washington University School of Medicine. Viability Assay : Cellular viability was determined based on exclusion of Trypan Blue staining. In Situ Cell Death Characterization : To determine if particles caused cell death by means of apoptosis or necrosis, cleavage of ge- nomic DNA during apoptosis was assessed based on the TUNEL method. Cells were cultured with 500 particles/cell or conditioned me- dium (CM) for 72 h prior to determining apoptotic rate. Particle-free and serum-free medium were used as negative and positive controls re- spectively. Western Analysis : Rabbit IgG polyclonal antibodies (Santa Cruz) reactive to human full length p53, human p73α, and human β- actin were used. Data Analysis : All values are expressed as the mean of three separate experiments ± the standard error of the mean (SEM). Sta- tistical significance between control values and treatment values was de- termined by a pairwise Student’s t-test. RESULTS AND DISCUSSION The purpose of this study is to test the hypothesis that exposure to wear particles compromises the viability of hMSCs through the induction of apoptosis, and that this effect may be mediated by soluble factors re- leased by hMSCs in response to particulate stress. We demonstrate here that direct exposure to submicron cpTi and ZrO2 submicron particles ad- versely affects hMSC viability through the induction of apoptosis, elicit- ing increased expression of the tumor suppressor proteins p53 and p73 in a manner dependent on material composition, particle dosage, and expo- sure time. In addition, conditioned medium containing soluble factors released by hMSCs in response to cpTi particles, but not ZrO2 particles, is capable of compromising cellular viability, inducing apoptosis in the absence of particle exposure. Increased expression of p53 can be in- duced by a variety of cellular stresses, and the ability of particle expo- sure and serum deprivation to induce increased p53 and p73 expression and subsequent apoptosis, suggests that these two distinct cellular insults may activate the p53 pathway through similar mechanisms. Addition- ally, our findings suggest that the p53-mediated apoptosis observed here may also be associated with the activation of death receptors by cyto- kines within the conditioned medium produced in response to particulate stress. At present, this possible mechanism of particle cytotoxicity re- mains unexamined and warrants further investigation. This study repre- sents the first report to demonstrate that exposure of hMSCs to wear par- ticles can compromise cellular viability through the direct and indirect induction of apoptosis, suggesting that long-term exposure of marrow- derived hMSCs to wear debris may reduce the population of viable os- teoblastic progenitors, potentially contributing to poor periprosthetic bone quality and implant loosening. A B 0 25 50 75 100 0 1000 2000 3000 4000 5000 Particles/cell Viability (%) cpTi ZrO2 0 25 50 75 100 0 24 48 72 Time (hours) Viability (%) ZrO2 cpTi CT Figure 1. Trypan Blue exclusion assay. (A) Culture of hMSCs exposed to increasing concentrations of both ZrO2 and cpTi particles exhibited decreased cellular viability in a dose-dependent manner. (B) Exposure of hMSCs to both particles (500 part/cell) decreased cellular viability in a time-dependent manner compared to non-particle loaded control cells (CT) (p<0.01). The level of cytotoxicity in cpTi -loaded cells was higher than that in cells cultured with ZrO2 (p<0.01). Figure 2. Western analysis of p53 and p73 expression. (A) hMSCs cul- tured with increasing concentrations of cpTi particles for 72 hours exhibited increased expression of the tumor suppressor proteins p53 and p73 under particulate loads of 500 and 5000 particles/cell compared to control, while a concentration of 50 particles/cell did not induce an in- crease in p53 or p73. (B, C) hMSCs cultured with ZrO2 and cpTi parti- cles exhibited increased (B) p53 and (C) p73 expression at 24 hours. Cells cultured with cpTi or serum-free (SF) medium exhibited increased p53 and p73 levels compared to ZrO2-loaded cells. A B 0 10 20 30 40 50 CT ZrO2 cpTi SF Apoptosis (%) * * ** 0 10 20 30 40 50 CT ZrO2 50% cpTi cpTi Apoptosis (%) ** Figure 3. TUNEL assay. (A) hMSCs cultured with ZrO2, cpTi, or se- rum-free (SF) conditions exhibited increased levels of apoptosis com- pared to non-particle loaded cells (CT). Cells loaded with cpTi exhib- ited elevated levels of apoptosis compared to ZrO2-loaded cells. (B) hMSCs cultured in conditioned medium (CM) collected from cpTi - loaded cell cultures (cpTi) exhibited increased apoptosis compared to cells cultured in CM from non-particle loaded control cells (CT). Dilu- tion (1:1) of cpTi -CM with fresh medium (50% cpTi) abolished this apoptotic inductive effect. Cells cultured in CM from ZrO2-loaded cells (ZrO2) did not exhibit elevated levels of apoptosis compared to control. (*, p<0.05; **, p<0.01) **George Washington University, Washington, D.C. ***Thomas Jefferson University, Philadelphia, PA 49th Annual Meeting of the Orthopaedic Research Society Paper #0284 49th Annual Meeting of the Orthopaedic Research Society 49th Annual Meeting of the Orthopaedic Research Society 49th Annual Meeting of the Orthopaedic Research Society 49th Annual Meeting of the Orthopaedic Research Society