Margit Wiedmann-Al-Ahmad

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials.

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.

Biofilm formation and composition on different implant materials in vivo

Biofilm formation was evaluated on the following titanium and zirconia implants in vivo: machined titanium (Ti-m), modified titanium (TiUnite), modified zirconia (ZiUnite), machined alumina-toughened zirconia (ATZ-m), sandblasted alumina-toughened zirconia (ATZ-s), and machined zirconia (TZP-A-m). Bovine enamel slabs were used as controls. Surface morphologies were examined by atomic force (AFM) and scanning electron microscopy (SEM). The surface wettability was also determined. Twelve healthy volunteers wore a splint system with the tested materials. After 3 and 5 days the materials were examined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). The levels of Streptococcus spp., Veillonella spp., Fusobacteriaum nucleatum, and Actinomyces naeslundii were quantitatively determined. The biofilm thickness was found to be between 19.78 and 36.73 μm after 3 days and between 26.11 and 32.43 μm after 5 days. With the exception of Ti-m the biofilm thickness after 3 days was correlated with surface roughness. In addition to Streptococcus spp. as the main component of the biofilm (11.23-25.30%), F. nucleatum, A. naeslundii, and Veillonella spp. were also detected. No significant differences in biofilm composition on the implant surfaces could be observed. In total, the influence of roughness and material on biofilm formation was compensated by biofilm maturation.

Bone regeneration in sinus lifts: comparing tissue-engineered bone and iliac bone.

Lifting of the sinus floor is a standard procedure for bony augmentation that enables dental implantation. Although cultivated skin and mucosal grafts are often used in plastic and maxillofacial surgery, tissue-engineered bone has not achieved the same success. We present the clinical results of dental implants placed after the insertion of periosteum-derived, tissue-engineered bone grafts in sinus lifts. Periosteal cells were isolated from biopsy specimens of periosteum, resuspended and cultured. The cell suspension was soaked in polymer fleeces. The cell-polymer constructs were transplanted by sinus lift 8 weeks after harvesting. The patients (n=35) had either one or both sides operated on. Seventeen had a one-stage sinus lift with simultaneous implantation (54 implants). In 18 patients the implants were inserted 3 months after augmentation (64 implants). Selected cases were biopsied. A control group (41 patients: one stage=48 implants, two stage=135 implants) had augmentation with autologous bone only. They were followed up clinically and radiologically for at least 24 months. Both implants and augmentation were significantly more successful in the control group. Failure of augmentation of the tissue-engineered bone was more common after large areas had been augmented. Eleven implants were lost in the study group and only one in the control group. Lifting the sinus floor with autologous bone is more reliable than with tissue-engineered transplants. Although lamellar bone can be found in periosteum-derived, tissue-engineered transplants, the range of indications must be limited.

In vivo study of the initial bacterial adhesion on different implant materials.

OBJECTIVE Biofilm formation on implant materials plays a major role in the aetiology of periimplantitis. The aim of this study was to examine in vivo the initial bacterial adhesion on six different implant materials. METHODS The implant materials Ti-m, TiUnite®, ZiUnite®, ATZ-m, ATZ-s, TZP-A-m were tested using bovine enamel slabs as controls. All materials, fixed on splint systems, were examined after 30 min and 120 min of oral exposure. DAPI staining was used for quantitative analysis of the initially adherent microorganisms. Initial adherent microorganisms were visualised by fluorescence In situ-hybridisation (FISH) and quantified by confocal laser scanning microscopy (CLSM). The targets of the oligonucleotide probes were Eubacteria, Veillonella spp., Fusobacterium nucleatum, Actinomyces naeslundii and Streptococcus spp. RESULTS DAPI analysis showed that increasing the time of oral exposure resulted in an increasing amount of initial adherent bacteria. The highest level of colonisation was on ZiUnite®, with the lowest occurring on the bovine enamel, followed by Ti-m. This early colonisation correlated significantly with the surface roughnesses of the materials. FISH and CLSM showed no significant differences relating to total bacterial composition. However, Streptococcus spp. was shown to be the main colonisers on each of the investigated materials. CONCLUSION it could be shown that within an oral exposure time of 30 min and 120 min, despite the salivary acquired pellicle initial biofilm formation is mainly influenced directly or indirect by the material surface topography. Highly polished surfaces should minimise the risk of biofilm formation, plaque accumulation and possibly periimplantitis.

Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro

OBJECTIVE Sodium benzoate (SB), potassium sorbate (PS) and sodium nitrite (SN) are commonly used food preservatives. In this in vitro study, the effects of these substances on biofilm formation of Streptococcus mutans were analysed. METHODS In addition to the microtiter plate test (MPT), a biofilm reactor containing bovine enamel slabs (BES) was used to study the influence of food preservatives on biofilm formation in 5 independent periods of 4 days each. These included one period with chlorhexidine digluconate (CHX) as a positive control as well as a period with growth medium alone as a negative control. The vitality of the biofilm on BES was detected using live/dead staining and confocal laser scanning microscopy. Additionally, the number of colony forming units (CFU) was determined. RESULTS In MPT 0.12% SN significantly reduced the biofilm formation. PS at a concentration of 0.4% tended to inhibit biofilm formation, whereas the inhibition for 0.8% PS was significant. Less inhibition was caused by 0.8% SB. In the biofilm reactor 0.06% of SN, 0.1% of SB and 0.1% PS significantly reduced the covering grade as well as the CFU of the biofilm. Biofilm vitality was reduced significantly by CHX to a level of 32.5% compared to the control. Only SB reduced the vitality to a level of 19.1%. SN and PS showed no influence on biofilm vitality. CONCLUSION This study indicates the potential of food preservatives as inhibitory agents in S. mutans biofilm formation, which should be kept in mind when studying the effects of conserved food on dental plaque biofilm in situ.

Influence of iron restriction on Chlamydia pneumoniae and C. trachomatis.

Iron is an essential metabolite for pathogenic bacteria, and the specificity exhibited by bacteria for host-iron chelates may be correlated with host and tissue tropism. The effect of iron restriction on Chlamydia pneumoniae and C. trachomatis was studied by use of the iron-chelating compound deferoxamine. Growth of C. pneumoniae was inhibited much more than that of C. trachomatis and the effect of iron restriction largely depended on the cell line used for propagation. This might reflect differences in tissue tropism of the two chlamydial species. As iron levels are usually higher in men than in women, this might also be connected with the higher prevalence rate of C. pneumoniae antibodies in males, observed in all populations studied so far.

Endodontic and Salivary Isolates of Enterococcus faecalis Integrate into Biofilm from Human Salivary Bacteria Cultivated In Vitro

INTRODUCTION The aim of this study was to examine whether Enterococcus faecalis isolates from endodontic patients (from saliva and from a root canal) are able to prevail against salivary bacteria when grown in coculture in a biofilm reactor. METHODS Saliva that was tested to be free of E. faecalis was used as the inoculum. The fate of E. faecalis was examined by using culture techniques and fluorescence in situ hybridization (FISH). RESULTS The root canal isolate accounted for 37.4% of the biofilm and 31.9% of the planktonic phase when examined by the culture technique, whereas the proportions examined by FISH showed 15.3% in the biofilm and 11.7% in the planktonic phase. The saliva isolate (as examined by the culture technique) accounted for 32.4% in the biofilm and 27.1% in the planktonic phase, respectively, compared with 14.1% in the biofilm and 9.5% in the planktonic phase when examined by FISH analysis. CONCLUSIONS These results led to the suggestion that E. faecalis could persist in the biofilm of the human oral cavity. Because of the ubiquitous presence of E. faecalis, root canal infections may arise from different sources.

Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials

Rapid prototyping (RP)-produced scaffolds are gaining increasing importance in scaffold-guided tissue engineering. Microbial adhesion on the surface of replacement materials has a strong influence on healing and long-term outcome. Consequently, it is important to examine the adherence of microorganisms on RP-produced scaffolds. This research focussed on manufacturing of scaffolds by 3D-bioplotting and examination of their microbial adhesion characteristics. Tricalciumphosphate (TCP), calcium/sodium alginate, and poly(lactide-co-glycolic acid) (PLGA) constructs were produced and used to study the adhesion of dental pathogens. Six oral bacterial strains, one Candida strain and human saliva were used for the adhesion studies. The number of colony forming units (CFU) were determined and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were performed. Microorganisms adhered to all scaffolds. All strains, except for Streptococcus oralis, adhered best to PLGA scaffolds. Streptococcus oralis adhered to each of the biomaterials equally. Streptococcus mutans and Enterococcus faecalis adhered best to PLGA scaffolds, followed by alginate and TCP. Prevotella nigrescens, Porphyromonas gingivalis, Streptococcus sanguis, and Candida albicans showed the highest adherence to PLGA, followed by TCP and alginate. In contrast, the microorganisms of saliva adhered significantly better to TCP, followed by PLGA and alginate. SEM observations correlated with the results of the CFU determinations. CLSM detected bacteria within deeper sheets of alginate. In conclusion, because of the high adherence rate of oral pathogens to the scaffolds, the application of these biomaterials for bone replacement in oral surgery could result in biomaterial-related infections. Strategies to decrease microbial adherence and to prevent infections due to oral pathogens are discussed.

Airborne microbes in different dental environments in comparison to a public area.

OBJECTIVE The aim of this study was to investigate quantitatively and qualitatively the airborne microbial load in a multi-chair dental clinic, a normal dental practice and a non-dental public area over a time period of four days and at different time points to estimate the risk of infections during dental surgery. METHODS A multi-chair and a single chair treatment room each were examined in comparison to a non-medical public area over a period of four days. The colony forming units m(-3) (CFUs) were determined and isolated bacteria were characterised by morphological and biochemical analysis, gas chromatography and by 16S rRNA-gene sequencing. In the analyses enterococci were selectively searched for. RESULTS The CFUs in the multi-chair treatment room were between 20 and 1050 CFU m(-3). During treatment the maxima reached were below 800 CFU m(-3). The values in the dental practice were between 200 and 600 CFU m(-3) and remain slightly but not significantly below the levels of the clinic (p > 0.05). In the common area, the CFUs were between 200 and 800 CFU m(-3). The proportion of micrococci was 56.8% in the clinic, 56.07% in the practice and 69.67% in the public area Coagulase-negative staphylococci constituted 35% at the dental clinic, 25% at the bank and 38% at the dental practice. No significant differences amongst the units were detected in the microbial composition of their dental aerosols (p > 0.05). CONCLUSION Although, the bacterial counts in dental room were not significantly higher than the bacterial counts in a public area, the risk from dental clinic might be higher than a public area due to the type of micro-organisms, host susceptibility and the exposure time.