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Adsorption of serum and urine proteins to polyurethanes
S178
Poster Session P3: Tuesday I7 Septentber
Candida tropicalis (CT) and Candida krusei (CK) are two yeasts which pose a threat to the successful integration of medical devices into the human host. Endotracheal tubes and voice box prostheses are composed of PVC and silicone respectively. The adherence potential of CT and CK to these two biomaterials was investigated to assess the risk of fungal colonisation of medical devices. Cultures of CT and CK were grown in air or 5%COa for 16 hours at 37°C and then treated with PBS or saliva for 30 minutes. Discs of PVC and silicone were then incubated in Candida suspensions at 37E for time periods up to 24 hours. At selected time points, discs were removed and viable counts of adherent organisms were determined; image analysis was used to determine surface coverage of discs. Surface roughness of the two materials was measured using atomic force microscopy. Candida adherence to and surface coverage of PVC and silicone after 24 hours incubation is shown in the table below (meantid) for two of the environments studied. A significant difference was observed in the surface roughness of PVC and silicone, 40.24f0.25nm and 20.08fl.24nm respectively. Material
Organism
Environment
Surface coverage (%l 1.56M.09 1.28M.02 13.23K). 19 1.95ti.05 1.02iO.03 4.68M.06 1.25+~0.04 8.98fo.50 2.51iO.19 1.56M.14 3.69M.11 7.69M.50 21.51ztO.58 1.94iO.12 3.96zt0.27 23.601t0.89
Adherence
%
WC
CT CK
Silicone
CT CK
Air, PBS S%CC$, saliva Air. PBS 5%C&, saliva Air, PBS 5%C&, saliva Air, PBS 5%CD7, saliva
Candida growth in 5%COa and saliva-treated exhibited maximal adherence to PVC and silicone, indicating the importance of oropharyngeal conditions on yeast colonisation of oral biomaterials.
Protein adsorption onto biomaterial surfaces is believed to be the earliest event occurring after implantation and will effect the subsequent biocompatibility of the device. For example, alpha-lmicroglobulin has been implicated in the immune response.1 As a resuit of the wfde range of devices which are implanted at various sites in the body, biomaterfals will be exposed to differing biological fluids. At present the protein adsorption studies have concentrated on serum protein only. There is therefore a need to investigate the uptake of proteins from other body fluids which may also come into contact with medical devices. Serum, cerebrospinal fluid (CSF), urine and used continuous ambulatory peritoneal dialysis fluid (CAPD) were adsorbed onto polystyrene employed as a model biomaterial surface. The treated surfaces were sequentially immersed in water and increasing concentrations of isopropanol (IsoPOH)/water mixes to selectively desorb the proteins based on their binding strength. Desorption profiles were obtained by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The presence of alpha-l microglobulin in the desorption washes was confirmed by immunoblotting. SDS-PAGE profiles showed that there were similarities in the protein composition of the biobgioal fluids, with alpha-l -microglobulin present However, desorption of the adsorbed alpha-lin each fluid. microglobulin indicated that the binding characteristics were different in each fluid. Serum- and urine-derived alpha-I-microglobulin was desorbed from polystyrene with 30% IsoPOH. while CSF-derived alpha-I-microglobulin required 50% IsoPOH and CAPD-derived alpha-I-microglobulin was removed only with 70% IaoPOH. The composition and behaviour of a conditioning film on a biomaterial surface, and subsequent events relating to biocompatibility, appear to be influenced by the composition of the suspending biological fluid. Thisworkwassupponedby 1.
EU Brim-Euram
pmgfamme
B.Akerstrom.FoliaHistochemCyiobiol.1992.
ElRE2-CT92-0233
30:183-186
On insertion, intrauterine devices (IUDs) become coated with a mucus biofilm which offers protection to vaginal bacteria which may be introduced into the uterus (Malhi et a/.. 1989). We have previously reported the development of an IUD capable of prolonged release of chlorhexidine acetate (CHDA) in vitro and capable of reducing the number of bacteria in surrounding mucus (Reynolds et a/., 1991). This study assessed the toxicity of such devices in vitro and in viva. Ln vitro tox cl& Hollow, nylon fibres were loaded with CHDA (2 - 2Opg in 2&l 70% ethanol) and placed on to a cultured monolayer of RENT4 rat endometrial cells. 4 days later cell viability was assessed by dye exclusion and determination of mitochondrial dehydrogenase, lysosomal and golgi body activity. Studies were performed in triplicate on 3 separate occassions. lo wvo b&i& Identical fibres (containing 1.0 or 2.8 mg CHDA in 2Opl 70% ethanol, n=6) were inserted into the guinea pig uterus following laparotomy. After 5 days both uterine horns were removed for extensive histological examination The degree of cell death and mitosis in the horn containing the fibre was compared with that of the untreated, contralateral horn. CHDA reduced viability of endometrial cells in vitro (IDss: 14-2Opg; dependent upon method of assessment). In viva the fibre containing 2.8 mg caused significant cell death within the stroma (pcO.05, MannWhitney) but had no significant effects on the epithelium or glandular epithelium; there were no effects on mitosis. There was no significant toxicity when the fibres contained 1.Omg. These results, taken together with those previously published (Reynolds et al., 1991) suggest that, despite the unfavourable results for assessments of toxicity in viva. incorporation of CHDA into the structure of IUDs may reduce the incidence of device-related infections without causing significant damage to the adjacent uterine tissue. Malhi, J.S. et al., (1989) J. Pharm. Pharmac. 41 (suppl.) 11OP Reynolds, J.P. et al., (1991) J. Pharm. Pharmacol. &j (suppl) 90
Polyurethanes src a class of biomaterisl widely used in the production of vascular and urological devices. Although polyurethancs possess good mechanical properties their biocompatibility is compromised by the hydrophobic nature of the material. Hydrothane (PolyMedica Inc. USA) is a novel polyurethane with good mechanical and hydrophilic properties. However, no information is available concerning the biological characterization of this polyuredtane. The biocompatibility of an implanted device will be h~fluenced by the adsorption of proteins from the surrounding bcdy fluid. Therefore, in this study we have investigated the adsorption of mine and serum proteins onto Hydrothane, comparing the results with a second, commercially available polyurethane, Chronoflex (PolyMedica Inc, USA). Funher, we have assessed me anti-bactetiaJ adherence potential of the polyttrethanes. Discs of Hydrothane and Chronoflex were incubated with serum or urine. Bound proteins were desodxd with sodium dodecyl sulphate. polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and sepamted by SDS-PAGE. The presence of adsorbed human serum albumitt (HSA), fibrinogen (Fg). alpha-lmicroglobuIin (a-l-m), complement (C3) and fitaonectin (Fn) was cottfirmed by immunoblotting. The adhesion of Pseudomo~s aeruginosu J34to serum- and urine-treated discs was assessed by an adenosine triphosphate (ATP) bioluminescence assay.1 The adsorption pattern of serum and urine proteins to Hydrothane and Chronoflex were similar. The presence of HSA. Fg, a-l-m, C3 and Fn was appatent on serum-treated discs while HSA, a-l-m and trxe amounts of Fn were adsorbed from urine. Adhesion of Ps.aeruginosa B4 to serum-treated Hydrothanc and Chronoflex was significantly increased when compared to the control disc (Mann-Whitney rank sum; PsZO.05). Bacterial adherence to minetreated Hydrothane was also enhanced by the adsorbed proteins. For both polymers, bacteria adhered to the serum-tmated disc in significantly greater numbers than w the urine-ueated smface. The findings indicate that the biocompatible properties of a polymer are significantly affect& by the smmunding body fluid. The nam of the resulting conditioning film may influence factors such as me propensity for bacterial colonisation, which will have a beating on susceptibility w infection and the subsequent success of the device. llus workwassupportedby EU
B&e-Eumm -me
BREZ-Cl92-0233
I. M.A.Was.sall c, al J Pham~ F’hammcol. 1995. In press
Poster Session P3: Tuesday I7 Septentber
Candida tropicalis (CT) and Candida krusei (CK) are two yeasts which pose a threat to the successful integration of medical devices into the human host. Endotracheal tubes and voice box prostheses are composed of PVC and silicone respectively. The adherence potential of CT and CK to these two biomaterials was investigated to assess the risk of fungal colonisation of medical devices. Cultures of CT and CK were grown in air or 5%COa for 16 hours at 37°C and then treated with PBS or saliva for 30 minutes. Discs of PVC and silicone were then incubated in Candida suspensions at 37E for time periods up to 24 hours. At selected time points, discs were removed and viable counts of adherent organisms were determined; image analysis was used to determine surface coverage of discs. Surface roughness of the two materials was measured using atomic force microscopy. Candida adherence to and surface coverage of PVC and silicone after 24 hours incubation is shown in the table below (meantid) for two of the environments studied. A significant difference was observed in the surface roughness of PVC and silicone, 40.24f0.25nm and 20.08fl.24nm respectively. Material
Organism
Environment
Surface coverage (%l 1.56M.09 1.28M.02 13.23K). 19 1.95ti.05 1.02iO.03 4.68M.06 1.25+~0.04 8.98fo.50 2.51iO.19 1.56M.14 3.69M.11 7.69M.50 21.51ztO.58 1.94iO.12 3.96zt0.27 23.601t0.89
Adherence
%
WC
CT CK
Silicone
CT CK
Air, PBS S%CC$, saliva Air. PBS 5%C&, saliva Air, PBS 5%C&, saliva Air, PBS 5%CD7, saliva
Candida growth in 5%COa and saliva-treated exhibited maximal adherence to PVC and silicone, indicating the importance of oropharyngeal conditions on yeast colonisation of oral biomaterials.
Protein adsorption onto biomaterial surfaces is believed to be the earliest event occurring after implantation and will effect the subsequent biocompatibility of the device. For example, alpha-lmicroglobulin has been implicated in the immune response.1 As a resuit of the wfde range of devices which are implanted at various sites in the body, biomaterfals will be exposed to differing biological fluids. At present the protein adsorption studies have concentrated on serum protein only. There is therefore a need to investigate the uptake of proteins from other body fluids which may also come into contact with medical devices. Serum, cerebrospinal fluid (CSF), urine and used continuous ambulatory peritoneal dialysis fluid (CAPD) were adsorbed onto polystyrene employed as a model biomaterial surface. The treated surfaces were sequentially immersed in water and increasing concentrations of isopropanol (IsoPOH)/water mixes to selectively desorb the proteins based on their binding strength. Desorption profiles were obtained by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The presence of alpha-l microglobulin in the desorption washes was confirmed by immunoblotting. SDS-PAGE profiles showed that there were similarities in the protein composition of the biobgioal fluids, with alpha-l -microglobulin present However, desorption of the adsorbed alpha-lin each fluid. microglobulin indicated that the binding characteristics were different in each fluid. Serum- and urine-derived alpha-I-microglobulin was desorbed from polystyrene with 30% IsoPOH. while CSF-derived alpha-I-microglobulin required 50% IsoPOH and CAPD-derived alpha-I-microglobulin was removed only with 70% IaoPOH. The composition and behaviour of a conditioning film on a biomaterial surface, and subsequent events relating to biocompatibility, appear to be influenced by the composition of the suspending biological fluid. Thisworkwassupponedby 1.
EU Brim-Euram
pmgfamme
B.Akerstrom.FoliaHistochemCyiobiol.1992.
ElRE2-CT92-0233
30:183-186
On insertion, intrauterine devices (IUDs) become coated with a mucus biofilm which offers protection to vaginal bacteria which may be introduced into the uterus (Malhi et a/.. 1989). We have previously reported the development of an IUD capable of prolonged release of chlorhexidine acetate (CHDA) in vitro and capable of reducing the number of bacteria in surrounding mucus (Reynolds et a/., 1991). This study assessed the toxicity of such devices in vitro and in viva. Ln vitro tox cl& Hollow, nylon fibres were loaded with CHDA (2 - 2Opg in 2&l 70% ethanol) and placed on to a cultured monolayer of RENT4 rat endometrial cells. 4 days later cell viability was assessed by dye exclusion and determination of mitochondrial dehydrogenase, lysosomal and golgi body activity. Studies were performed in triplicate on 3 separate occassions. lo wvo b&i& Identical fibres (containing 1.0 or 2.8 mg CHDA in 2Opl 70% ethanol, n=6) were inserted into the guinea pig uterus following laparotomy. After 5 days both uterine horns were removed for extensive histological examination The degree of cell death and mitosis in the horn containing the fibre was compared with that of the untreated, contralateral horn. CHDA reduced viability of endometrial cells in vitro (IDss: 14-2Opg; dependent upon method of assessment). In viva the fibre containing 2.8 mg caused significant cell death within the stroma (pcO.05, MannWhitney) but had no significant effects on the epithelium or glandular epithelium; there were no effects on mitosis. There was no significant toxicity when the fibres contained 1.Omg. These results, taken together with those previously published (Reynolds et al., 1991) suggest that, despite the unfavourable results for assessments of toxicity in viva. incorporation of CHDA into the structure of IUDs may reduce the incidence of device-related infections without causing significant damage to the adjacent uterine tissue. Malhi, J.S. et al., (1989) J. Pharm. Pharmac. 41 (suppl.) 11OP Reynolds, J.P. et al., (1991) J. Pharm. Pharmacol. &j (suppl) 90
Polyurethanes src a class of biomaterisl widely used in the production of vascular and urological devices. Although polyurethancs possess good mechanical properties their biocompatibility is compromised by the hydrophobic nature of the material. Hydrothane (PolyMedica Inc. USA) is a novel polyurethane with good mechanical and hydrophilic properties. However, no information is available concerning the biological characterization of this polyuredtane. The biocompatibility of an implanted device will be h~fluenced by the adsorption of proteins from the surrounding bcdy fluid. Therefore, in this study we have investigated the adsorption of mine and serum proteins onto Hydrothane, comparing the results with a second, commercially available polyurethane, Chronoflex (PolyMedica Inc, USA). Funher, we have assessed me anti-bactetiaJ adherence potential of the polyttrethanes. Discs of Hydrothane and Chronoflex were incubated with serum or urine. Bound proteins were desodxd with sodium dodecyl sulphate. polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and sepamted by SDS-PAGE. The presence of adsorbed human serum albumitt (HSA), fibrinogen (Fg). alpha-lmicroglobuIin (a-l-m), complement (C3) and fitaonectin (Fn) was cottfirmed by immunoblotting. The adhesion of Pseudomo~s aeruginosu J34to serum- and urine-treated discs was assessed by an adenosine triphosphate (ATP) bioluminescence assay.1 The adsorption pattern of serum and urine proteins to Hydrothane and Chronoflex were similar. The presence of HSA. Fg, a-l-m, C3 and Fn was appatent on serum-treated discs while HSA, a-l-m and trxe amounts of Fn were adsorbed from urine. Adhesion of Ps.aeruginosa B4 to serum-treated Hydrothanc and Chronoflex was significantly increased when compared to the control disc (Mann-Whitney rank sum; PsZO.05). Bacterial adherence to minetreated Hydrothane was also enhanced by the adsorbed proteins. For both polymers, bacteria adhered to the serum-tmated disc in significantly greater numbers than w the urine-ueated smface. The findings indicate that the biocompatible properties of a polymer are significantly affect& by the smmunding body fluid. The nam of the resulting conditioning film may influence factors such as me propensity for bacterial colonisation, which will have a beating on susceptibility w infection and the subsequent success of the device. llus workwassupportedby EU
B&e-Eumm -me
BREZ-Cl92-0233
I. M.A.Was.sall c, al J Pham~ F’hammcol. 1995. In press