Adhesion and Colonisation Intensity of Staphylococcus
epidermidis and Pseudomonas aeruginosa on a Composite
Material Surface of Hydroxyapatites and Titanium Dioxide

Ingus Skadins; Aigars Reinis; Juta Kroica; Agnese Pura; Liga Berzina-Cimdina; Dmitrijs Jakovlevs; Dagnija Rostoka; Natalija Berza; Janis Vetra

Adhesion and Colonisation Intensity of Staphylococcus epidermidis and Pseudomonas aeruginosa on a Composite Material Surface of Hydroxyapatites and Titanium Dioxide

Ingus Skadins , Aigars Reinis , Juta Kroica , Agnese Pura , Liga Berzina-Cimdina , Dmitrijs Jakovlevs , Dagnija Rostoka , Natalija Berza , Janis Vetra

Ingus Skadins ^1*, Aigars Reinis ¹, Juta Kroica ¹, Agnese Pura ², Liga Berzina-Cimdina ², Dmitrijs Jakovlevs ², Dagnija Rostoka ¹, Natalija Berza ¹, Janis Vetra ³

Department of Biology and Microbiology, Riga Stradin? University, Latvia
Biomaterial Development and Innovation Centre, Riga Technical University, Latvia
Institute of Anatomy and Anthropology, Riga Stradin? University, Latvia

Corresponding Author: Ingus Skadins Riga, Dzirciema street 16, LV-1007, Latvia Email: Ingus.Skadins@rsu.lv

Related article at Pubmed, Scholar Google

Visit for more related articles at International Journal of Collaborative Research on Internal Medicine & Public Health

Abstract

Introduction: The use of biomaterial implants in medicine is becoming ever more popular, and on many occasions the use of biomaterial implants becomes a lifesaving procedure. A primary obstacle to a wider use of biomaterial implants is their risk of infection and unsuccessful tissue integration with the surface of biomaterials. For the creation of new substances various combinations of materials are used nowadays, thus forming composite materials. Composite materials are materials which consist of two or more components or phases, which according to their characteristics vary considerably, but they are mutually dissolvent or little solvent, and they have a distinct border surface.1 Most often titanium (Ti) and titanium fused implants are covered, as they are bioinert materials.2-4 HAp/TiO2 composite ceramic layer shows better linkage strength with the material if compared with pure HAp. By increasing TiO2 amount in the composite, its strengths also enhances, as well as TiO2 addition can decrease attachment of bacteria to the biomaterial, thus decreasing the possible infection risk of the implant; however infection risk around TiO2 containing implant is still a problem in medicine.5

Objective: The objective of the study was to determine Staphylococcus epidermidis (S.epidermidis) and Pseudomonas aeruginosa (Ps.aeruginosa) adhesion and colonisation intensity on hydroxyapatite (HAp) and titanium dioxide (TiO2) commercial composite material surfaces originally synthesised by Biomaterial Development and Innovation Centre of Riga Technical University. Materials used: Composite material samples used: No 1 100% HAp – burned 1000oC, No 2 50% HAp and 50% TiO2 – burned at 1000oC, No 3 80% HAp and 20% TiO2 – burned at 1000oC, No 4 100% TiO2 – burned at 1000oC, No 5 20% HAp and 80% TiO2 – burned at 1000oC, No 6 100% TiO2 – burned at 1200oC, No 7 20% HAp and 80% TiO2 – burned at 1200oC, No 8 80% HAp and 20% TiO2 – burned at 1200oC, No 9 50% HAp and 50% TiO2 – burned at 1200oC, No 10 100% HAp – burned at 1200oC

Method: Ps.aeruginosa ATCC 27853, S.epidermidis ATCC 12228 reference cultures were used in the study. Bacterial suspensions were prepared from bacterial pure cultures of 1 ml TSB (Trypto-Casein-Soy Broth) volume with a concentration of 10, 102 and 103 CFU/ml (colony forming units). Samples were cultivated at 37oC temperature for 2 h in order to determine adhesion intensity, and a sample with a concentration of 102 CFU/ml for 24 h – to determine colonisation intensity. For adhesion evaluation and determination of colonisation amount, sonication and culture method was used. For determination of colonisation intensity sonicationculture method was used as well as scanning electron microscope.

Result: In general, adhesion intensity on HAp and TiO2 composite material surface is not big. S.epidermidis adhesion starts at 10 CFU/ml/2h/37oC exposition only for biomaterial types 2, 3, 4 (from 0.0027-0.003 CFU/mm2), at 102 CFU/ml/2h/37oC – better adhesion is observed on surfaces of biomaterials 3 and 4, and worse adhesion on surfaces of biomaterials 6, 7, 8. At 103 CFU/ml/2h/37oC exposition the greatest adhesion is observed on surfaces of biomaterials 3 and 10 (0.093 and 0.094 CFU/mm2 respectively). Ps.aeruginosa adhesion intensity was lower than S.epidermidis and at 10 CFU/ml/2h/37oC exposition happened only on biomaterial surfaces 1 and 4 (0.028 and 0.001 CFU/mm2 respectively). At 102 CFU/ml/2h/37oC exposition also demonstrated a low adhesion level, and at 103 CFU/ml/2h/37oC exposition the lowest adhesion intensity was demonstrated on biomaterials 3, 6, 9, 10, and the greatest one on biomaterial 1.

Conclusion: Used bacteria have low adhesion abilities on HAp and TiO2 containing biomaterials. Pseudomonas aeruginosa show even lower adhesion ability on biomaterials with HAp and TiO2 mixture. Optimal HAp/TiO2 composite ceramics composition, which ensures the lowest contamination risk of microorganisms is with a 50% and 80% TiO2 content after a thermal processing at 1200 ?C.

Keywords

S.epidermidis, Ps. aeruginosa, Titanium Dioxide, Composite Material, Hydroxyapatite, Adhesion, Colonisation.

INTRODUCTION

For the creation of new substances various combinations of materials are used nowadays, thus forming composite materials. Composite materials are materials which consist of two or more components or phases, which according to their characteristics vary considerably, but they are mutually dissolvent or little solvent, and they have a distinct border surface. The use of composites in medicine is a relatively new discovery and many of these materials are still being studied.1 However, already now their use and manufacturing in implants and medical appliances, e.g. catheters, is much wider, if compared with simple homogenous materials. It is important to note that bacterial adhesion and colonisation intensity must be determined in vitro and in vivo as each component and biomaterial must be biologically compatible6 and there’s a question of how they will interact with the organism of a living being (including humans)7 as well as how great is the risk of implant infection.8-11 Within the last 10 years metallic implants have been widely used and they have been covered with a bioactive HAp/TiO2 layer in order to enhance fixation between the living bone and implant, and the formation of new bone cells, as well as in order to prevent metal corrosion. Most often titanium (Ti) and titanium fused implants are covered, as they are bioinert materials.2-4 HAp/TiO2 composite ceramic layer shows better linkage strength with the material if compared with pure HAp. By increasing TiO2 amount in the composite, its strengths also enhances,12 as well as TiO2 addition can decrease attachment of bacteria to the biomaterial,13 thus decreasing the possible infection risk of the implant; however infection risk around TiO2 containing implant is still a problem in medicine.5

Objective

The objective of the study was to determine Staphylococcus epidermidis (S.epidermidis) and Pseudomonas aeruginosa (Ps.aeruginosa) adhesion and colonisation intensity on hydroxyapatite (HAp) and titanium dioxide (TiO2) commercial composite material surfaces originally synthesised by Biomaterial Development and Innovation Centre of Riga Technical University.

Material and Method

Materials used:

Composite material samples used:

No 1 100% HAp – burned 1000oC

No 2 50% HAp and 50% TiO2 – burned at 1000oC

No 3 80% HAp and 20% TiO2 – burned at 1000oC

No 4 100% TiO2 – burned at 1000oC

No 5 20% HAp and 80% TiO2 – burned at 1000oC

No 6 100% TiO2 – burned at 1200oC

No 7 20% HAp and 80% TiO2 – burned at 1200oC

No 8 80% HAp and 20% TiO2 – burned at 1200oC

No 9 50% HAp and 50% TiO2 – burned at 1200oC

No 10 100% HAp – burned at 1200oC

Ps.aeruginosa ATCC 27853, S.epidermidis ATCC 12228 reference cultures were used in the study. Bacterial suspensions were prepared from bacterial pure cultures of 1 ml TSB (Trypto- Casein-Soy Broth) volume with a concentration of 10, 102 and 103 CFU/ml (colony forming units). Samples were cultivated at 37oC temperature for 2 h in order to determine adhesion intensity, and a sample with a concentration of 102 CFU/ml for 24 h – to determine colonisation intensity. For adhesion evaluation and determination of colonisation amount, sonication and culture method was used.14-15 For determination of colonisation intensity sonication-culture method was used14-15 as well as scanning electron microscope.

Sonication-culture method:14-15 After incubation unattached microorganisms were rinsed off. In order to detach bacteria that had not attached to the surface of biomaterials, discs were processed for 1 minute in ultrasonic bath (at 45 kHz frequency) and for 1 minute at a maximum velocity in a Vortex centrifuge. In order to determine the total amount of microorganisms, cultures were prepared from each sample on a TSA (Trypto-Casein-Soy agar) culture and they were cultured for 24 hours in a temperature of 37oC.

Scanning electron microscope: TESCAN manufactured SEM Mira LMU was used. Prior to analysis, biomaterial samples were dried and fixated in ethyl alcohol ether (1:1) mixture, and then a thin layer of gold was dusted onto the samples for better electron conduction in order to make qualitative SEM micrographs.

Acquired data was processed, using Microsoft Office Excel.

Results

In general, adhesion intensity on HAp and TiO2 composite material surface is not so high. S.epidermidis adhesion starts at 10 CFU/ml/2h/37oC exposition only for biomaterial types 2, 3, 4 (from 0.0027-0.003 CFU/mm2), at 102 CFU/ml/2h/37oC – better adhesion is observed on surfaces of biomaterials 3 and 4, and worse adhesion on surfaces of biomaterials 6, 7, 8. At 103 CFU/ml/2h/37oC exposition the greatest adhesion is observed on surfaces of biomaterials 3 and 10 (0.093 and 0.094 CFU/mm2 respectively).

Ps.aeruginosa adhesion intensity was lower than S.epidermidis and at 10 CFU/ml/2h/37oC exposition happened only on biomaterial surfaces 1 and 4 (0.028 and 0.001 CFU/mm2 respectively). At 102 CFU/ml/2h/37oC exposition also demonstrated a low adhesion level, and at 103 CFU/ml/2h/37oC exposition the lowest adhesion intensity was demonstrated on biomaterials 3, 6, 9, 10, and the greatest one on biomaterial 1.

Adhesion intensity results are shown in diagrams 1a, 1b, 2a, 2b and table 1.

Bacterial colonisation on composite materials used in the study is various. Very low colonisation intensity was observed in HAp material burned at 1000oC, and a very great colonisation intensity on HAp material burned at 1200oC. Various colonisation intensities were observed on materials with a various HAp and TiO2 composition, and preparation technology (Table 2, Diagram 3a and 3b).

Analysing SEM Figures it was determined that on the surface of biomaterial 1 S.epidermidis was practically not observed, whereas on the rest of composite materials S.epidermidis did not form its characteristic biofilm, instead colonising material surface in a dispersed manner, in types of colonies (Figure 1)

Ps.aeruginosa colonised composite materials similarly as staphylococci, colonising, without forming a biofilm, rather forming a dispersed bacterial film (Figure 2).

Discussion

From the experimental data you can see that a clean TiO2 has very low bacterial adhesion compared to other samples, which coincides with A. Pavlovas study carried out in 2011. This can be explained by the fact that TiO2 has a hydrophilic surface, whereas both bacterial surfaces are hydrophobic. Since microorganisms with hydrophobic characteristics attach better to hydrophobic surfaces, composite materials with a greater amount of TiO2 contain less adhesion. It was observed that S.epidermidis bacteria has greater tendency to attach to synthesised HAp. Sample surfaces burned at 1200⁰C demonstrate lower levels of adhesion, because bacteria find it easier to attach to porous materials, therefore adhesion intensity of microorganisms on denser samples is much lower, because microstructure of materials burned at 1200°C is less porous.

In diagrams 2a and 2b you can see that composite materials HAp with 50%TiO2 and HAp with 80% TiO2 burned at 1200 °C have the lowest microorganism adhesion. Tendency of Ps.aeruginosa bacteria to attach to composite material surfaces is lower than that of S.epidermidis.

Experimental data in diagram 3 shows that tendency of Ps.aeruginosa bacteria to colonise ceramic surfaces of the samples is slightly less than that of S.epidermidis. Lower microorganism colonisation can be observed on sample surfaces burned at 12000C because of decrease of pores.16 Therefore in case of S.epidermidis composite ceramics with HAp composition of 80% TiO2 chances of colonisation are lower at 12000C, whereas in case of Ps.aeruginosa it is characteristic of all composites at both burning temperatures.

Conclusion

Used bacteria have low adhesion abilities on HAp and TiO2 containing biomaterials. Pseudomonas aeruginosa show even lower adhesion ability on biomaterials with HAp and TiO2 mixture.

Optimal HAp/TiO2 composite ceramics composition, which ensures the lowest contamination risk of microorganisms is with a 50% and 80% TiO2 content after a thermal processing at 1200 0C.

For future studies and applications - biomaterials that demonstrated low bacterial adhesion will be implanted in laboratory animals to determine the minimal infective dose of biomaterial, as well as research expression of bacterial-biomaterial interactions on inflammatory mediators (IL- 10, TNF-α, β-defensin-2 ) in tissues.

Acknowledgment

This work has been partly supported by the European Social Fund within the project “Multidisciplinary Research in Biomaterials Technology of New Scientist Group”, No.2009/0199/1DP/1.1.1.2.0/09/APIA/VIAA/090, (PVS ID1380).processing at 1200 0C.

Conflict of Interest: None declared.

References

Hin TS. Engineering Materials for Biomedical Applications. World Scientific Publishing Company: USA, 2004, p.350.
Heimann RB, Schurmann N, Muller RT. In vitro and in vivo performance of Ti6Al4V implants with plasma-sprayed osteoconductive hydroxylapatite- bioinert titania bond coat „duplex” systems: an experimental study in sheep. J. Mater. Sci.: Mater. Med. 2004, 15, 1045-1052.
Harle J, Kim HW, Mordan N, Knowles JC, Salih V. Initial responses of human osteoblasts to sol–gel modifed titanium with hydroxyapatite and titania composition. Acta Biomaterialia. 2006, 2, 547–556.
Xiao XF, Liub RF, Zheng YZ. Characterization of hydroxyapatite/titania composite coatings codeposited by a hydrothermal–electrochemical method on titanium. Surf. Coat. Technol., 2006, 200, 4406–4413.
Zhao L, Chu PK, Zhang Y, Wu Z. Preparation and characteristics of micro-arc oxidation film on novel Ti-3Zr-2Sn-3Mo-25Nb biomedical alloy. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2009, 470-480.
Park J, Lakes RS. Biomaterials. An Introduction. Springer: New York, 2007, p.561.
Слуцкий Л, Ветра Я. Биологические вопросы биоматериаловедения.- Рига; Латвийская Медицинская академия, 2001. pp 25-43.
O`Gara JP, Humphryes H. Staphylococcus epidermidis biofilms: importance and implications. J.Med.Microbiol., Jul. 2001, vol.50, 7:582-587.
Trampuz A, Zimmerli W. Prosthetic joint infections: update in diagnosis and treatment. Swiss Med. Wkly. 2005, 135:243–251.
Handbook of Bacterial Adhesion, Edited by Yuehuei HA, Friedman RJ. – Towota; Humana Press, 2000. – P. 11-20.
Hetrick EM, Schoenfisch MH. Reducing implant-related infections: active release strategies. Chem. Soc. Rev., 2006, 35:780-789
Kim HW, Kim HE, Salih V, Knowles JC. Hydroxyapatite and Titania Sol–Gel Composite Coatings on Titanium for Hard Tissue Implants; Mechanical and In Vitro Biological Performance. J. Biomed. Mater. Res., Part B, 2005, 72B (1-8), 1-8.
Pavlova A, Reinis A, Berzina-Cimdina L, Kroica J, Burlakova A, Rubenis K. Staphylococcus epidermidis and Pseudomonas aeruginosa adhesion intensity on a TiO2 ceramic in an in vitro study. Advanced Materials Research, 2011, 222, 301-304.
Trampuz A, Piper KE, Jacobson MJ, et al. Sonication of removed hip and knee prostheses for diagnosis of infection. N Engl J Med. 2007;357:654-663.
Sampedro MF, Huddleston PM, Piper KE, et al. A biofilm approach to detect bacteria on removed spinal implants. Spine 2010 May 20;35(12):1218-24.
Yuehuei HA, Friedman RJ. Concise Review of Mechanisms of Bacterial Adhesion to Biomaterial Surfaces. Applied Biomaterials, 1998, 43, 338-348.