- Email: [email protected]
Cytotoxicity and MMP-9 activation induced in human mononuclear cells by UHMWPE oxidation
Biomaterials 23 (2002) 3645–3650
Cytotoxicity and MMP-9 activation induced in human mononuclear cells by UHMWPE oxidation F. Reno" a, P. Braccob, L. Costab, M. Cannasa,* a
Human Anatomy Laboratory, Department of Medical Sciences, Independent University of Eastern Piedmont ‘‘A.Avogadro’’, Via Solaroli 17, 28100 Novara, Italy b Chemistry Department I.F.M., Via Zuretti 29, University of Turin, 10125 Turin, Italy Received 18 June 2001; accepted 12 March 2002
Abstract Ultra high molecular weight polyethylene (UHMWPE) used in orthopedic prosthesis can be oxidized during sterilization processes such as g-ray irradiation. Oxidation alters UHMWPE structure and mechanical properties and it has been suggested that this alteration is the first step in the loosening process. Direct effects of UHMWPE oxidation on the cellular and tissue response to the implant have not been previously investigated. We used heat-oxidized UHMWPE, whose oxidative state is comparable to the one induced in g-ray irradiated polymer, in order to observe the peripheral blood mononuclear cells (PBMCs) behavior after 24 and 48 h exposure to the oxidized and non-oxidized polymers. Flow cytometric analysis showed a cytotoxic effect of oxidized UHMWPE after 48 h compared with the control samples. Moreover, gelatin zymography of PBMCs conditioned media showed a strong increase in MMP-9 (gelatinase B) release and activation in oxidized UHMWPE samples. This first evidence of a direct effect of the UHMWPE oxidative status on the cellular behavior suggests that oxidation alters not only the UHMWPE physical–mechanical properties, but it can also be responsible for altered tissue response. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oxidation; UHMWPE; PBMC; Cytotoxicity; MMP-9
1. Introduction Ultra high molecular weight polyethylene (UHMWPE) is widely used in orthopedic prosthetic implants [1]. UHMWPE sterilization using high-energy radiation in air atmosphere induces some unwanted side effects such as a strong oxidation [2] that decreases the molecular mass [3] and consequently decreases the mechanical properties and increases wear and prosthesis failure [4]. No reports about cell and tissue interaction with oxidized UHMWPE are available. The interaction of cells and tissue with biomaterials following the implantation has been widely studied and it has been observed that a key step in the healing process is the specific leukocytes inflammatory response [5]. Usually, the chemotactic recruitment of leukocytes around an implant promotes tissue repair since such biomedical devices are inert, but mechanisms by which peripheral blood mononuclear cells (PBMCs) adhere to *Corresponding author. Tel./fax: +39-321-660-632. E-mail address: [email protected] (M. Cannas).
polymeric artificial surfaces through the protein layer, are not fully understood and these interactions could play an important role in up or down regulating the inflammatory response. The recruitment of leukocytes into a site of tissue damage depends on their activation by cytokines and mobility through the endothelial barrier and the extracellular matrix with contemporary expression of adhesion molecules and matrix-degrading enzymes such as matrix metalloproteinase (MMPs) [6]. The aim of this work was to quantify the effects of oxidation in orthopedic UHMWPE on PBMCs viability and MMPs release in order to clarify the tissue response to oxidized polymeric biomaterial.
2. Experimental 2.1. Materials A compression-molded plate of prosthetic UHMWPE (Gur 1020, Poly Hi Solidur, Germany) was used. The
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 9 7 - 2
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F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
material, in compliance with ASTM F 648-98 was without additives. The plate was initially sawn to a size suitable for microtomy (5 3 7 cm3). A PolyCuts Microtome (Reichert-Jung) was used. Microtomy was performed at a cutting speed of 20 mm/s in air at room temperature. Slices of about 200 mm height with a surface of about 2 cm2 were recovered. Half of the slices (n ¼ 38) were oxidized in air in oven at 1201C for 80 h. The behavior of heat-oxidized polyethylene is very similar to the g-ray oxidized ones [7]; moreover, oxidation levels obtained using thermal treatment are easily measurable and reproducible.
2.2.3. Flow cytometry analysis All experiments were performed using a FACScan cytometer (Becton-Dickinson) equipped with a 15 mW air-cooled argon ion laser operating at 488 nm. PI was added to cell suspension at a final concentration of 1 mg/ ml for 30 min at 371C. PI red fluorescence was measured through a 620 nm BP filter and displayed on a fourdecade log scale. A minimum of 10,000 events was collected per sample at a low sample flow rate setting (12 ml/min). As the PI is an exclusion dye, the proportion of fluorescent cells means the number of dead cell [8]. Data analysis was performed with WinMDI software version 2.7.
2.2. Techniques 2.2.1. UHMWPE surface analysis The original and oxidized samples were analyzed with infrared spectroscopy in attenuate total reflection (ATR). An FTIR microscope (Perkin-Elmer System 2000, Autoimage) equipped with an ATR objective (Germanium, incidence angle of the IR beam 451, 100 100 mm2 nominal surface area) was used. ATR spectra were collected using 64 scans. The ATR spectra were corrected for wavelength dependence of the beam penetration by a computer program (Atrcorr program, Grams 32, Galactic) assuming 1.5 to be the refractive index of UHMWPE. 2.2.2. Peripheral blood mononuclear cells isolation and treatment (1) EDTA-collected human peripheral venous blood (10 ml) from 7 healthy donors (4 men and 3 women, average age7SE=2672) was layered onto a Ficoll– Hypaque density gradient to separate mononuclear cells from erythrocytes and granulocytes. Mononuclear cells were obtained by blood centrifugation on a Percoll gradient. PBMCs were centrifuged twice in sterile phosphate buffer (PBS), counted in optical microscopy using trypan blue exclusion test, and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco) containing penicillin (100 mg/ml), streptomycin (100 mg/ml) and l-glutamine (2 mm) (Sigma). PBMCs were seeded at a concentration of 1 105 cell/cm2 in a 6-well plate, UHMWPE disks (PE) or UHMWPE oxidized disks (PEOx). After 24 and 48 h of exposure, serum-free DMEM (1 ml) was added to the samples and cells were ‘‘washed’’ from the samples surface and collected. Few cells were observed on the surface after washing (5–10 cells per field when samples were observed at 16 magnification in the optical microscope). Collected cells were centrifuged at 1200 rpm for 5 min. Supernatants were collected for gelatin zymography and cell were resuspended in 2 ml PBS at pH 7.4, stained with propidium iodide (PI) and their viability analyzed using flow cytometry.
2.2.4. Gelatin zymography Medium samples were collected after 1, 4, 24 and 48 h and used for measurements of MMPs activity by zymography [9]. Latent and active gelatinase A (MMP-2) and B (MMP-9) activity was detected by zimogram analysis using SDS–polyacrylamide gels copolymerized with 0.2% gelatin. These enzymes become dissociated from tissue inhibitor metalloproteinases (TIMPs) by the presence of SDS during electrophoresis. Removal of SDS following electrophoresis allows the proenzymes to renature in an active or partially active conformation. This permits their detection and the detection of lower molecular mass activated forms. In brief, conditioned medium was mixed with sample buffer and electrophoresed directly without boiling or reduction. Following electrophoresis, SDS was extracted from the polyacrylamide gel with Triton X-100, and the gel was incubated in 0.05 m Tris, pH 7.5, containing 5 mm CaCl2, and 5 mm ZnCl2 at 371C overnight. Gels were stained with Coomassie blue and destained. Both proenzyme and active gelatinase were detected as clear bands against the blue background of the stained gelatin. Positive control for gelatinase A and B (Chemicon International) was used to identify the two enzymes and their activated forms. Unconditioned medium samples were used as negative control. A densitometric analysis of the bands seen on gels was performed using the NIH Image 1.62 software and results were expressed as arbitrary units of optical density (O.D.). The relative amount of MMPs with respect to the control samples was calculated using the following formula: total sample O.D. (lower band+upper band)/total control O.D. (lower band+ upper band) 100. The percentage of MMPs activation was calculated using the following formula: O.D. lower band/O.D. upper band+O.D. lower band 100. The statistical analysis of data was performed using the SPSS software for Windows. Student’s t-test was used for data coupled with a significance of po0:05:
F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
3. Results 3.1. Surface characterization The ATR spectra of PE and PEOx are reported in Fig. 1. The ester groups (1740 cm1) were present on the surface of the PE samples. This small level of oxidation depends on the microtoming process [10]. The ATR spectrum of PEOx (Fig. 1) showed the presence of OH groups (3450–3350 cm1) due to alcohol and acid compounds, ester groups (1740 cm1), ketones (1718 cm1) and acid (1710 cm1) [11].
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24 h. In the control samples the total MMP-9 activity and the percentage of the active form (60–70% total activity) increased at 48 h (Fig. 3, Ct). PBMCs seeded onto UHMWPE disks for 24 and 48 h released MMP-9 enzyme in the same amount and activity observed for the control samples (Fig. 3, PE). Also oxidized UHMWPE did not induce alteration in MMP-9 secretion after 24 h. After 48 h PBMCs exposed to the oxidized polymer released a MMP-9 amount ranging from 120% to 160% of both control and UHMWPE samples and with a higher percentage of activation (range 85–90%) (Fig. 3, PEOx).
3.2. Cytotoxicity 4. Discussion PBMCs cultured in the presence of 10% FCS showed a low percentage of dead cells after 24 (3.470.4%) and 48 h (4.571%) culture on the controls (Fig. 2, first row). Surprisingly, UHMWPE was able to increase dead cells percentage at 24 (5.770.5%) and 48 h (7.571.2%) (Fig. 2, second row). The observed increase in cytotoxicity was statistically significant (po0:05). After 24 h oxidized UHMWPE induced a percentage of dead cells (5.670.4%) not different from UHMWPE, while after 48 h its induced cytotoxicity increased reaching a value of 1372.5% (Fig. 2, third row). 3.3. MMP-9 release and activation No MMP-9 activity was measurable in the PBMCs medium 1 and 4 h after seeding while MMP-2 activity was always present and derived from the serum added (data not shown). In fact MMP-2 activity levels did not change during the entire experimental period (Fig. 3). MMP-9 released in the medium from PBMCs seeded onto polystyrene was present both in the active (range 40–50% of the total activity) and inactive form after
Oxidized species such as ester and acids originating from the decomposition of primary peroxides, which can be formed by the mechano-oxidation during the shaping of PE samples [10], were present on the samples surface. Decomposition of secondary hydroperoxides produced by g-ray sterilization process in air produces large amounts of alcohol, ketones and acids [7]. UHMWPE oxidation has been indicated as responsible for increased in vivo biodegradation of prosthesis [12] but no data are available about the oxidative phenomenon effect/s on the cellular environment surrounding the prosthesis. In order to mimic the ‘‘oxidized scenario’’ UHMWPE disks treated with a thermal oxidative process [13], have been used to test PBMCs cellular behavior. The oxidative effect was that the apolar surface of PE has been changed into a polar surface in PEOx. Moreover, oxidized UHMWPE induced a strong PBMCs toxicity after 48 h of exposure. This toxicity could be due to the presence of free radicals and peroxides that are able to induce necrosis and apoptosis in PBMCs [14]. In fact cells stained by PI can
.5
1718
.4
3400
.2
1710
1740
.3
1740
PEOx .1
PE 0 4000
3500
3000
2500
2000
1500
1000
Absorbance / Wavenumber (cm-1) Fig. 1. ATR spectrum of UHMWPE (PE) and UHMWPE thermally oxidized (PEOx).
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3648 F. Reno" et al. / Biomaterials 23 (2002) 3645–3650 Fig. 2. Dot plot of forward scatter versus PI fluorescence (Red fluorescence) for PBMCs seeded onto polystyrene plates (Ct), UHMWPE (PE) and UHMWPE oxidized disks (PEOx) for 24 and 48 h, respectively. Dead cells (PI positive) have been counted in the upper left quadrant.
F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
3649
demonstrated that human fibroblasts express different amounts of MMP-2 (gelatinase A) when seeded on titanium with a different groove surface topography [20]. It is interesting to note that marked cytotoxicity and MMP-9 release both occurred only after 48 and 24 h during which no dramatic phenomenon occurred, suggesting the presence of a complex molecular multistep mechanism in which both integrins and cytokines could be involved. Moreover, to better understand the role of the oxidative phenomenon in the PBMCs response it could be of interest to investigate if the presence of anti-oxidant agents in the culture medium will be able to prevent or reverse both cytotoxicity and MMP-9 release. In conclusion this report had focused on the effects of UHMWPE oxidation on PBMCs cellular behavior showing that both cytotoxicity and MMP-9 release and activation were increased through a mechanism not yet clarified but that could be triggered directly by the oxidative phenomenon.
Fig. 3. Representative gelatin zymography obtained from 24 (A) and 48 (B) h PBMCs conditioned medium in the presence of polystyrene (CT), UHMWPE (PE) and UHMWPE oxidized disks (PEOx). MMP2 (gelatinase A) and both inactive and active MMP-9 (gelatinase B). Both inactive (pro-MMP2 and 9) and active (MMP-2 and 9) forms are indicated along with molecular weight markers for inactive forms.
be both necrotic cells and late apoptotic cells [8]. Also, non-oxidized UHMWPE can induce a light PBMCs toxicity compared to the cells grown onto polystyrene, but this phenomenon can be addressed to the presence of a small quantity of oxidized species on the polymer surface. Another effect of oxidized UHMWPE was the induction of MMP-9 release by PBMCs. MMP-9 (gelatinase B) is a member of matrix metalloproteinase family particularly involved in the lymphocytes migration [15] and it is expressed and produced by natural killer cells [16], B cells [17] and neutrophils [18]. The production and activation of matrix-degrading proteinases such as MMP-9 by PBMCs is likely to be an important factor in facilitating trafficking through the endothelial barrier and the extracellular matrix during inflammation. The increased PBMCs MMP-9 release could alter the periprosthetic environment starting dangerous inflammation-like phenomena. MMP-9 production in human lymphocytes can be induced by cytokines such as TNF-a and IL-1b [15] and integrin activation [15]. Even if peroxides can induce MMP-9 expression in cardiac fibroblasts [19] no information are available about a similar mechanism in PBMCs. It is possible that the surface of oxidized samples presented a different microstructure responsible for the different cellular response observed in PBMCs, in fact it has been
Acknowledgements The authors would like to thank Dr. P. Grazianetti and Mr. V. Pretato from the Human Anatomy Laboratory for their precious technical collaboration.
References [1] Li S, Burnstein AH. Ultra high molecular weight polyethylene. The material and its use in total joint implants. J Bone Jt Surg Am 1994;76A:1080–8. [2] Costa L, Luda MP, Trossarelli L, Brach del Prever EM, Crova M, Gallinaro P. Oxidation in orthopedic UHMWPE sterilized by gamma-radiation and ethylene oxide. Biomaterials 1998;19: 659–68. [3] Premnath V, Harris WH, Jasty M, Merrill EW. Gamma sterilization of UHMWPE articular implants: an analysis of the oxidation problem. Biomaterials 1996;17:1741–53. [4] Jasty M, Bragdon C, Lee K, Hanson A, Harris WH. Wear of polyethylene in total joint arthroplasty: experimental studies. In: Morrey BF, editor. Biological, material and mechanical consideration of joint replacement. New York: Raven Press, 1993. p. 103–18. [5] Tang L, Eaton JW. Inflammatory response to biomaterials. Am J Clin Pathol 1995;103:466–71. [6] Xia M, Leppert D, Hauser SL, Sreedharan SP, Nelson PJ, Krensky AM, Goetzl EJ. Stimulus specificity of matrix metalloproteinase dependence of human T-cell migration through a model basement membrane. J Immunol 1996;156:160–7. [7] Lacoste J, Carlsoon DJ. Gamma-, foto-, and thermally-initiated oxidation of linear low density polyethylene: a quantitative comparison of oxidation products. J Polym Sci 1992;30:493–500. [8] Vitale M, Zamai L, Mazzotti G, Cataldi A, Falcieri E. Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes. Histochemistry 1993;100:223–9. [9] Arumugam S, Jang YC, Chen-Jensen C, Gibran NS, Isik FF. Temporal activity of plasminogen activators and matrix
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F. Reno" et al. / Biomaterials 23 (2002) 3645–3650 metalloproteinases during cutaneous wound repair. Surgery 1999;125:587–93. Jacobson K, Costa L, Bracco P, Augustsson N, Stenberg B. Effects of microtoming on oxidation of ultra high molecular weight polyethylene (UHMWPE). Polym Degrad Stab 2001;73: 141–50. Jacobson K, Costa L, Luda MP, Trossarelli L. Ultra high molecular weight polyethylene: I. Mechano-oxidative degradation. Polym Degradation Stability 1997;55:329–38. Brach del Prever EM, Crova M, Costa L, Dellera A, Camino G, Gallinaro P. Unacceptable biodegradation of polyethylene in vivo. Biomaterials 1996;17:873–8. Costa L, Luda MP, Trossarelli L. Ultra high molecular weight polyethylene: II. Thermal- and photo-oxidation degradation. Polym Degradation Stability 1997;58:41–54. Fenech M, Crott J, Turner J, Brown S. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleous assay: description of the method and results for hydrogen peroxide. Mutagenesis 1999;14:605–12.
[15] Sterler-Stevenson M, Mansoor A, Lim M, Fukushima P, Marti G, Ptaszynski K, Wang J, Sterler-Stevenson WG. Cytokines and chemochine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J Immunol 1997;158:2327–33. [16] Albertsson P, Kim MH, Jonges LE, Kitson RP, Kuppen PJ, Johansson BR, Nannmark U, Goldfarb RH. Matrix metalloproteinase of human NK cell. In Vivo 2000;14:269–76. [17] Trocme C, Gaudin P, Berthier S, Barro C, Zaoui P, Morel F. Human B lymphocytes synthetise the 92-kDa gelatinase, matrix metalloproteinase-9. J Biol Chem 1998;273:20677–84. [18] Wise J, Sopata I, Smerdel A, Maslinski S. Ligation of selectin L and integrin CD11b/CD18 (Mac-1) induces release of gelatinase B (MMP-9) from human neutrophils. Inflamm Res 1998;47:325–7. [19] Siwik DA, Pagano PJ, Colucci WS. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblast. Am J Physiol Cell Physiol 2001;280:53–60. [20] Chou L, Firth JD, Uitto VJ, Brunette DM. Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblast. J Biomed Mater Res 1998;39:437–45.
Cytotoxicity and MMP-9 activation induced in human mononuclear cells by UHMWPE oxidation F. Reno" a, P. Braccob, L. Costab, M. Cannasa,* a
Human Anatomy Laboratory, Department of Medical Sciences, Independent University of Eastern Piedmont ‘‘A.Avogadro’’, Via Solaroli 17, 28100 Novara, Italy b Chemistry Department I.F.M., Via Zuretti 29, University of Turin, 10125 Turin, Italy Received 18 June 2001; accepted 12 March 2002
Abstract Ultra high molecular weight polyethylene (UHMWPE) used in orthopedic prosthesis can be oxidized during sterilization processes such as g-ray irradiation. Oxidation alters UHMWPE structure and mechanical properties and it has been suggested that this alteration is the first step in the loosening process. Direct effects of UHMWPE oxidation on the cellular and tissue response to the implant have not been previously investigated. We used heat-oxidized UHMWPE, whose oxidative state is comparable to the one induced in g-ray irradiated polymer, in order to observe the peripheral blood mononuclear cells (PBMCs) behavior after 24 and 48 h exposure to the oxidized and non-oxidized polymers. Flow cytometric analysis showed a cytotoxic effect of oxidized UHMWPE after 48 h compared with the control samples. Moreover, gelatin zymography of PBMCs conditioned media showed a strong increase in MMP-9 (gelatinase B) release and activation in oxidized UHMWPE samples. This first evidence of a direct effect of the UHMWPE oxidative status on the cellular behavior suggests that oxidation alters not only the UHMWPE physical–mechanical properties, but it can also be responsible for altered tissue response. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oxidation; UHMWPE; PBMC; Cytotoxicity; MMP-9
1. Introduction Ultra high molecular weight polyethylene (UHMWPE) is widely used in orthopedic prosthetic implants [1]. UHMWPE sterilization using high-energy radiation in air atmosphere induces some unwanted side effects such as a strong oxidation [2] that decreases the molecular mass [3] and consequently decreases the mechanical properties and increases wear and prosthesis failure [4]. No reports about cell and tissue interaction with oxidized UHMWPE are available. The interaction of cells and tissue with biomaterials following the implantation has been widely studied and it has been observed that a key step in the healing process is the specific leukocytes inflammatory response [5]. Usually, the chemotactic recruitment of leukocytes around an implant promotes tissue repair since such biomedical devices are inert, but mechanisms by which peripheral blood mononuclear cells (PBMCs) adhere to *Corresponding author. Tel./fax: +39-321-660-632. E-mail address: [email protected] (M. Cannas).
polymeric artificial surfaces through the protein layer, are not fully understood and these interactions could play an important role in up or down regulating the inflammatory response. The recruitment of leukocytes into a site of tissue damage depends on their activation by cytokines and mobility through the endothelial barrier and the extracellular matrix with contemporary expression of adhesion molecules and matrix-degrading enzymes such as matrix metalloproteinase (MMPs) [6]. The aim of this work was to quantify the effects of oxidation in orthopedic UHMWPE on PBMCs viability and MMPs release in order to clarify the tissue response to oxidized polymeric biomaterial.
2. Experimental 2.1. Materials A compression-molded plate of prosthetic UHMWPE (Gur 1020, Poly Hi Solidur, Germany) was used. The
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 9 7 - 2
3646
F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
material, in compliance with ASTM F 648-98 was without additives. The plate was initially sawn to a size suitable for microtomy (5 3 7 cm3). A PolyCuts Microtome (Reichert-Jung) was used. Microtomy was performed at a cutting speed of 20 mm/s in air at room temperature. Slices of about 200 mm height with a surface of about 2 cm2 were recovered. Half of the slices (n ¼ 38) were oxidized in air in oven at 1201C for 80 h. The behavior of heat-oxidized polyethylene is very similar to the g-ray oxidized ones [7]; moreover, oxidation levels obtained using thermal treatment are easily measurable and reproducible.
2.2.3. Flow cytometry analysis All experiments were performed using a FACScan cytometer (Becton-Dickinson) equipped with a 15 mW air-cooled argon ion laser operating at 488 nm. PI was added to cell suspension at a final concentration of 1 mg/ ml for 30 min at 371C. PI red fluorescence was measured through a 620 nm BP filter and displayed on a fourdecade log scale. A minimum of 10,000 events was collected per sample at a low sample flow rate setting (12 ml/min). As the PI is an exclusion dye, the proportion of fluorescent cells means the number of dead cell [8]. Data analysis was performed with WinMDI software version 2.7.
2.2. Techniques 2.2.1. UHMWPE surface analysis The original and oxidized samples were analyzed with infrared spectroscopy in attenuate total reflection (ATR). An FTIR microscope (Perkin-Elmer System 2000, Autoimage) equipped with an ATR objective (Germanium, incidence angle of the IR beam 451, 100 100 mm2 nominal surface area) was used. ATR spectra were collected using 64 scans. The ATR spectra were corrected for wavelength dependence of the beam penetration by a computer program (Atrcorr program, Grams 32, Galactic) assuming 1.5 to be the refractive index of UHMWPE. 2.2.2. Peripheral blood mononuclear cells isolation and treatment (1) EDTA-collected human peripheral venous blood (10 ml) from 7 healthy donors (4 men and 3 women, average age7SE=2672) was layered onto a Ficoll– Hypaque density gradient to separate mononuclear cells from erythrocytes and granulocytes. Mononuclear cells were obtained by blood centrifugation on a Percoll gradient. PBMCs were centrifuged twice in sterile phosphate buffer (PBS), counted in optical microscopy using trypan blue exclusion test, and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco) containing penicillin (100 mg/ml), streptomycin (100 mg/ml) and l-glutamine (2 mm) (Sigma). PBMCs were seeded at a concentration of 1 105 cell/cm2 in a 6-well plate, UHMWPE disks (PE) or UHMWPE oxidized disks (PEOx). After 24 and 48 h of exposure, serum-free DMEM (1 ml) was added to the samples and cells were ‘‘washed’’ from the samples surface and collected. Few cells were observed on the surface after washing (5–10 cells per field when samples were observed at 16 magnification in the optical microscope). Collected cells were centrifuged at 1200 rpm for 5 min. Supernatants were collected for gelatin zymography and cell were resuspended in 2 ml PBS at pH 7.4, stained with propidium iodide (PI) and their viability analyzed using flow cytometry.
2.2.4. Gelatin zymography Medium samples were collected after 1, 4, 24 and 48 h and used for measurements of MMPs activity by zymography [9]. Latent and active gelatinase A (MMP-2) and B (MMP-9) activity was detected by zimogram analysis using SDS–polyacrylamide gels copolymerized with 0.2% gelatin. These enzymes become dissociated from tissue inhibitor metalloproteinases (TIMPs) by the presence of SDS during electrophoresis. Removal of SDS following electrophoresis allows the proenzymes to renature in an active or partially active conformation. This permits their detection and the detection of lower molecular mass activated forms. In brief, conditioned medium was mixed with sample buffer and electrophoresed directly without boiling or reduction. Following electrophoresis, SDS was extracted from the polyacrylamide gel with Triton X-100, and the gel was incubated in 0.05 m Tris, pH 7.5, containing 5 mm CaCl2, and 5 mm ZnCl2 at 371C overnight. Gels were stained with Coomassie blue and destained. Both proenzyme and active gelatinase were detected as clear bands against the blue background of the stained gelatin. Positive control for gelatinase A and B (Chemicon International) was used to identify the two enzymes and their activated forms. Unconditioned medium samples were used as negative control. A densitometric analysis of the bands seen on gels was performed using the NIH Image 1.62 software and results were expressed as arbitrary units of optical density (O.D.). The relative amount of MMPs with respect to the control samples was calculated using the following formula: total sample O.D. (lower band+upper band)/total control O.D. (lower band+ upper band) 100. The percentage of MMPs activation was calculated using the following formula: O.D. lower band/O.D. upper band+O.D. lower band 100. The statistical analysis of data was performed using the SPSS software for Windows. Student’s t-test was used for data coupled with a significance of po0:05:
F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
3. Results 3.1. Surface characterization The ATR spectra of PE and PEOx are reported in Fig. 1. The ester groups (1740 cm1) were present on the surface of the PE samples. This small level of oxidation depends on the microtoming process [10]. The ATR spectrum of PEOx (Fig. 1) showed the presence of OH groups (3450–3350 cm1) due to alcohol and acid compounds, ester groups (1740 cm1), ketones (1718 cm1) and acid (1710 cm1) [11].
3647
24 h. In the control samples the total MMP-9 activity and the percentage of the active form (60–70% total activity) increased at 48 h (Fig. 3, Ct). PBMCs seeded onto UHMWPE disks for 24 and 48 h released MMP-9 enzyme in the same amount and activity observed for the control samples (Fig. 3, PE). Also oxidized UHMWPE did not induce alteration in MMP-9 secretion after 24 h. After 48 h PBMCs exposed to the oxidized polymer released a MMP-9 amount ranging from 120% to 160% of both control and UHMWPE samples and with a higher percentage of activation (range 85–90%) (Fig. 3, PEOx).
3.2. Cytotoxicity 4. Discussion PBMCs cultured in the presence of 10% FCS showed a low percentage of dead cells after 24 (3.470.4%) and 48 h (4.571%) culture on the controls (Fig. 2, first row). Surprisingly, UHMWPE was able to increase dead cells percentage at 24 (5.770.5%) and 48 h (7.571.2%) (Fig. 2, second row). The observed increase in cytotoxicity was statistically significant (po0:05). After 24 h oxidized UHMWPE induced a percentage of dead cells (5.670.4%) not different from UHMWPE, while after 48 h its induced cytotoxicity increased reaching a value of 1372.5% (Fig. 2, third row). 3.3. MMP-9 release and activation No MMP-9 activity was measurable in the PBMCs medium 1 and 4 h after seeding while MMP-2 activity was always present and derived from the serum added (data not shown). In fact MMP-2 activity levels did not change during the entire experimental period (Fig. 3). MMP-9 released in the medium from PBMCs seeded onto polystyrene was present both in the active (range 40–50% of the total activity) and inactive form after
Oxidized species such as ester and acids originating from the decomposition of primary peroxides, which can be formed by the mechano-oxidation during the shaping of PE samples [10], were present on the samples surface. Decomposition of secondary hydroperoxides produced by g-ray sterilization process in air produces large amounts of alcohol, ketones and acids [7]. UHMWPE oxidation has been indicated as responsible for increased in vivo biodegradation of prosthesis [12] but no data are available about the oxidative phenomenon effect/s on the cellular environment surrounding the prosthesis. In order to mimic the ‘‘oxidized scenario’’ UHMWPE disks treated with a thermal oxidative process [13], have been used to test PBMCs cellular behavior. The oxidative effect was that the apolar surface of PE has been changed into a polar surface in PEOx. Moreover, oxidized UHMWPE induced a strong PBMCs toxicity after 48 h of exposure. This toxicity could be due to the presence of free radicals and peroxides that are able to induce necrosis and apoptosis in PBMCs [14]. In fact cells stained by PI can
.5
1718
.4
3400
.2
1710
1740
.3
1740
PEOx .1
PE 0 4000
3500
3000
2500
2000
1500
1000
Absorbance / Wavenumber (cm-1) Fig. 1. ATR spectrum of UHMWPE (PE) and UHMWPE thermally oxidized (PEOx).
500
3648 F. Reno" et al. / Biomaterials 23 (2002) 3645–3650 Fig. 2. Dot plot of forward scatter versus PI fluorescence (Red fluorescence) for PBMCs seeded onto polystyrene plates (Ct), UHMWPE (PE) and UHMWPE oxidized disks (PEOx) for 24 and 48 h, respectively. Dead cells (PI positive) have been counted in the upper left quadrant.
F. Reno" et al. / Biomaterials 23 (2002) 3645–3650
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demonstrated that human fibroblasts express different amounts of MMP-2 (gelatinase A) when seeded on titanium with a different groove surface topography [20]. It is interesting to note that marked cytotoxicity and MMP-9 release both occurred only after 48 and 24 h during which no dramatic phenomenon occurred, suggesting the presence of a complex molecular multistep mechanism in which both integrins and cytokines could be involved. Moreover, to better understand the role of the oxidative phenomenon in the PBMCs response it could be of interest to investigate if the presence of anti-oxidant agents in the culture medium will be able to prevent or reverse both cytotoxicity and MMP-9 release. In conclusion this report had focused on the effects of UHMWPE oxidation on PBMCs cellular behavior showing that both cytotoxicity and MMP-9 release and activation were increased through a mechanism not yet clarified but that could be triggered directly by the oxidative phenomenon.
Fig. 3. Representative gelatin zymography obtained from 24 (A) and 48 (B) h PBMCs conditioned medium in the presence of polystyrene (CT), UHMWPE (PE) and UHMWPE oxidized disks (PEOx). MMP2 (gelatinase A) and both inactive and active MMP-9 (gelatinase B). Both inactive (pro-MMP2 and 9) and active (MMP-2 and 9) forms are indicated along with molecular weight markers for inactive forms.
be both necrotic cells and late apoptotic cells [8]. Also, non-oxidized UHMWPE can induce a light PBMCs toxicity compared to the cells grown onto polystyrene, but this phenomenon can be addressed to the presence of a small quantity of oxidized species on the polymer surface. Another effect of oxidized UHMWPE was the induction of MMP-9 release by PBMCs. MMP-9 (gelatinase B) is a member of matrix metalloproteinase family particularly involved in the lymphocytes migration [15] and it is expressed and produced by natural killer cells [16], B cells [17] and neutrophils [18]. The production and activation of matrix-degrading proteinases such as MMP-9 by PBMCs is likely to be an important factor in facilitating trafficking through the endothelial barrier and the extracellular matrix during inflammation. The increased PBMCs MMP-9 release could alter the periprosthetic environment starting dangerous inflammation-like phenomena. MMP-9 production in human lymphocytes can be induced by cytokines such as TNF-a and IL-1b [15] and integrin activation [15]. Even if peroxides can induce MMP-9 expression in cardiac fibroblasts [19] no information are available about a similar mechanism in PBMCs. It is possible that the surface of oxidized samples presented a different microstructure responsible for the different cellular response observed in PBMCs, in fact it has been
Acknowledgements The authors would like to thank Dr. P. Grazianetti and Mr. V. Pretato from the Human Anatomy Laboratory for their precious technical collaboration.
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