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International Poster Journal of Dentistry and Oral Medicine
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Int Poster J Dent Oral Med 5 (2003), No. 3     15. Sep. 2003

Int Poster J Dent Oral Med 2003, Vol 5 No 03, Poster 193

In vitro-colonization of Sulfat-Reducing Bacteria (SRB) on resorbable membranes for periodontal regeneration

Quantitative SEM-evaluation

Language: English

Authors:
ZÄ Katharina Grimm, Univ.-Prof. Dr. med. dent. habil. Wolf-Dieter Grimm
Abteilung für Parodontologie, Fakultät für Zahn-, Mund- und Kieferheilkunde, Universität Witten/Herdecke;
Prof. Dr. emerit. Johannes v.d. Hoeven
Fakultät für Zahn-, Mund- und Kieferheilkunde, Orale Biologie, Universität Witten/Herdecke;
PhD Petra Langendijk
Department re Biochimie medicale, Universite de Geneve, Faculte de Medicine, Centre Medical Universitaire

Date/Event/Venue:
06.-09.03.02
80th General Session, International Association for Dental Research, San Diego, USA, 2002
San Diego, USA,

Introduction

A role in the pathogenesis of periodontal disease is played by P. gingivalis and Sulfat-reducing bacteria (SRB). The term sulfate-reducing bacteria (SRB) describes strictly anaerobic microbes that accomplish the dissimilatory reduction of sulfate to hydrogen sulfide. The 16S rDNA sequence showed a high similarity of 99.7% with the 16S rDNA of the proposed species 'Desulfovibrio fairfieldensis'.

Objectives

The aim of this study was to examine in an in vitro assay the colonization of 2 types of resorbable membranes for guided tissue regeneration by strains of SRB and P .gingivalis.

Table 1: Quantitative SEM analyzes of bacterial load on resorbable membranes

In vitro assay

Strains of SRB were isolated from periodontal pockets using enrichment techniques. The strains were preliminary characterized by phylogenetic analysis of 16 S rDNA sequences. Pieces of membrane were submerged in batchcultures of Desulfovibrio spp. and P. gingivalis FDC381 in reduced growth medium specific for SRB or PY medium, and incubated for 1-8 days lagtime. The membranes used were polylactic acid (GUIDOR) and glycolide and lactide copolymer (RESOLUT). The dry weight of membranes was determined after 2, 5 hours and 3, 6, 12, and 24 weeks.

Material and Methods

Measurement of bacterial using the software system Scion Image for Windows

After incubation, membranes were prepared for SEM analysis. Bacterial density was measured quantitatively at 500x magnification for selecting region of interests (ROI). Five 20x25µm fields were randomly selected for each specimen using the software system Scion Image for Windows. In "Threshold Mode" all pixels are equal or greater than a single threshold level. In "Density Slice Mode" all pixels between a lower and upper threshold are highlighted in red.

Fig. 4:
Quantitative SEM analyzes of resorbable membranes using SCION Images for Windows-Analyze particles
Fig. 5:
Quantitative SEM analyzes of resorbable membranes using SCION Images for Windows-Density slice mode
Fig. 6:
Quantitative SEM analyzes of resorbable membranes using SCION Images for Windows-Threshold Mode
Fig. 7:
Quantitative SEM analyzes of resorbable membranes using SCION Images for Windows-Threshold Mode-Density Plot Profile

Fig. 8a-c:
Biofilm formation on resorbable membranes (SEM analzes): Mixed Culture (SRB)
Fig. 8d-f:
Biofilm formation on resorbable membranes (SEM analzes): GW0706
Fig. 8g-i:
Biofilm formation on resorbable membranes (SEM analzes): M2509
Fig. 8j-l:
Biofilm formation on resorbable membranes (SEM analzes): P. gingivalis

Ultramorphological analysis of the surfaces was carried out using a Philips scanning electron microscope (Philips XL 30 FEG) at 20 kV. As detector the secondary electron detector was used. Prior to SEM investigation the membranes were washed with water, fixed in glutaraldehyde, dehydrated in ethanol, critical-point dried sputtered with gold-paladium using a Bal-Tec Scd 050 sputter to achieve a better resolution.

Fig. 1, 2:
SEM analyzes of resorbable membranes

  Fig. 3:
SEM analyzes of resorbable membranes

Biodegradation of resorbable membranes

After incubation the hydrolysis of Gore Resolut membranes was much faster than that of Guidor membranes. After 3 weeks the Gore membranes had largely dissolved, while Guidor membranes were still intact. After 6 weeks of incubation Gore Resolut membranes were degraded for the larger part, Guidor membranes remained their mass twice as long, only after 12 weeks their mass had degraded to this extend.

Fig. 9a,b:
Biodegradation of resorbable mebranes

Table 2: Biodegradation of resorbable mebranes
  t=0 2.5h, 0.01w 3 weeks 6 weeks 12 weeks 24 weeks
    rel. decrease rel. decrease rel. decrease rel. decrease rel. decrease
    Resolut Giudor Resolut Giudor Resolut Giudor Resolut Giudor Resolut Giudor
medium 100 10 3 nd 2 68 29 100 69    
GW0706 100 10 8 46 4 70 12 98 68 100 60
M2509 100 12 4 60 6 77 18 95 79 100 82
P.g. 91 100 13 1 57 5 68 30 100 71 100 62
P.i. 655 100 17 0 56 5 92 7 99 53 98 52
mix SRB+ 100 12 2 44 2 45 30 93 71 98 63

Results

SEM analysis revealed differences in the accumulation of SRB strains and P. gingivalis on the two investigated membranes using quantitative evaluation of bacterial density. The bacterial cells adhering to the membranes surface were depending on the degree of membranes mass loss over the time interval. SEM analysis: SEM analysis revealed differences in the accumulation of SRB strains and P. gingivalis on the 2 types of investigated membranes. The deep SRB invasion at the 3d week and frequent presence of internal bacteria at the 6th week on the PLA with copolymer membranes underlined the difference obtained quantitative evaluation of bacterial density. The difference between the bacterial layers was statistically significant (Kruskal-Wallis-Test, p=0.05).

Conclusions

  • The present investigation examined the relationship between the degrees of PL membrane degradation and the amount of SRB colonization observed after up to 24 weeks undisturbed growth.
  • The bacterial cells adhering to the membranes surface was depended on the degree of membranes mass loss over the time interval.
  • Interestingly, the results suggest an active role of SRB the degradation of the resorbable PLA membranes.

 

This Poster was submitted by Univ.-Prof. Dr. med. dent. habil. Wolf-Dieter Grimm.

Correspondence address:
ZÄ Katharina Grimm
Universität Witten/Herdecke
Fakultät für Zahn-, Mund- und Kieferheilkunde
Abteilung für Parodontologie
A.-Herrhausen-Str.50
58448 Witten
Germany