**3. Results**

*Photodynamic Therapy - From Basic Science to Clinical Research*

the remaining solution.

**2.8 Microbiological analysis**

rial cells from the implant surfaces.

inoculated to Brucella agar plates.

**2.9 Scanning electron microscopy analysis**

colonies.

The implants belonging to the positive control group (PC) were put in 0.2% chlorhexidine gluconate solution (Curasept ADS® Curaden International AG, Kriens, Switzerland) for a duration of 60 seconds. After their removal from the chlorhexidine solution, the implants were only rinsed with sterile saline to remove

After treatment procedures each implant was placed in a tube containing 500 μl of phosphate buffered saline (PBS). The tubes were vortexed for 60 seconds (Vortex, Genius 3, IKA, Germany). This was done to remove the remaining bacte-

From each tube, 100 μl were taken and using a 96-well microtiter plates a ten-fold dilution was performed and 30 μl of suspension from each well was then

The plates were placed in anaerobic conditions and after 72 hours and the colony forming units per milliliter (CFU/ml) were counted (**Figure 4**). MALDI Biotyper (Bruker Daltonics, Germany) was used to macroscopically differentiate distinctive

Scanning electron microscopy (SEM) was performed on one randomly selected implant from each of the treatment groups and one sterile non-treated implant. The implants for SEM analysis were stored for 2 hours in 2% paraformaldehyde and, later on dehydrated in increasing concentrations of ethanol (60%, 75% and 95%), for 30 minutes in each and dried overnight. The surfaces of the prepared implants

**156**

**Figure 4.**

*Visible colonies of bacteria on Brucella agar plate.*

To determine the difference among the groups and between the two types of implants, multivariate analysis of variance test was applied.

The comparison between the two types of implants: titanium and zirconia, regardless of the study groups, showed that there was a significantly lower number of bacteria on zirconia implants for all three types of bacteria separately, as well as for the total number of bacteria (**Table 4**).

For the comparison among the study groups regardless of the type of implant, Tukey test was applied. Regarding the total number of bacteria, the least bacteria were found in PDT1 and PDT2. These two groups were followed by PDT3 and PC without significant difference among them. The negative control group (NC) as expected, had the largest number of bacteria compared to the other groups. The same results were obtained for the number of each bacteria separately.

The total number of bacteria for every group and both implant types are shown in **Figure 5** in schematic form. The difference between zirconia implants and titanium implants was not the same for all groups. The smallest difference between both types of implants in the number of bacteria was for the control group. The impact was almost the same for PDT1, PDT2, PC and TB, while the largest difference between titanium and zirconia implants were in the PDT3 group. The results for each of the bacteria separately are shown in **Figures 6**–**8**.

#### **3.1 Titanium implants**

There were statistically significant differences among the groups for each of the bacteria separately and also for the total number of bacteria (p = 0.0022). These results are presented in **Table 5** in logarithmic form. Regarding the total number of bacteria, the largest reduction was observed in the PDT1 (98.3%) and PDT2 (97.8%) groups. These two groups had statistically significant difference when compared to NC (p < 0.05). In the PDT3 group there was a 68.7% bacterial reduction, without statistically significant difference when compared to NC (**Table 5**).

When each bacteria was compared separately, the PDT1 and PDT2 groups also showed the largest bacterial reduction. PDT1 group, was significantly more effective in the eliminating *A. actinomycetemcomitans* and *P. gingivalis* (p < 0.05).


**Table 4.**

**159**

**Figure 7.**

**Figure 5.**

**Figure 6.**

*Evaluation of the Antimicrobial Efficacy of Different Types of Photodynamic Therapy…*

*The total number of bacteria in logarithmic form, for both types of implants and for each study groups.*

*The number of A. actinymycetemcomitans in logarithmic form for both types of implants and the study groups.*

*The number of P. gingivalis in logarithmic form for both types of implants and the study groups.*

*DOI: http://dx.doi.org/10.5772/intechopen.94268*

*Number of bacteria by implant type and treatment groups in logarithmic form.*

**158**

*Evaluation of the Antimicrobial Efficacy of Different Types of Photodynamic Therapy… DOI: http://dx.doi.org/10.5772/intechopen.94268*

**Figure 5.** *The total number of bacteria in logarithmic form, for both types of implants and for each study groups.*

**Figure 7.** *The number of P. gingivalis in logarithmic form for both types of implants and the study groups.*

*Photodynamic Therapy - From Basic Science to Clinical Research*

**158**

Factor

**Implants**

Zirconia Titanium

**Group**

PDT1 PDT2 PDT3

TB PC NC Factor

**Implants**

Zirconia Titanium

**Group**

PDT1 PDT2 PDT3

TB PC NC *\*p-value for MANOVA test.*

**Table 4.**

*Number of bacteria by implant type and treatment groups in logarithmic form.*

24 24 24 24 24 24

6.4

(1.8) *abc - result of post-hoc comparison (Tukey test). Having the same letter means that there is no statistically significant difference.*

3.2

(2.7)

ab

4.5

(2.9)

a

3.1

(3.2)

ab

2.0

(2.3)

b

2.6

(2.5)

b

**<0.0001**

24 24 24 24 24 24

7.1

(1.5)

3.6

(2.7)

5.0

(2.7)

a

ab

3.6

(3.2)

ab

2.3

(2.4)

b

2.8

(2.6)

b

**<0.0001**

72 72

5.3

(2.7)

2.0

(2.1)

**<0.0001**

72 72

5.8

(2.5)

2.3

(2.3)

N

mean

st.d.

p\*

N

mean

st.d.

p\*

**<0.0001**

24 24 24 24 24 24

6.2

(1.3) *Prevotella intermedia*

2.9

(2.7)

ab

4.3

(2.7)

a

3.1

(2.9)

ab

1.8

(2.0)

b

1.9

(2.2)

b

**<0.0001**

24 24 24 24 24 24

6.2

(1.6)

**Total**

2.8

(2.6)

4.0

(2.7)

a

ab

2.9

(2.8)

ab

1.6

(2.1)

b

2.0

(2.5)

b

**<0.0001**

72 72

4.9

(2.5)

1.9

(2.1)

**<0.0001**

72 72

4.9

(2.6)

1.6

(2.1)

N

mean

st.d.

**Aggregatibacter actynomycetemcomitans**

p\*

N

mean

st.d.

p\*

**<0.0001**

**Porphyromonas gingivalis**

**Figure 8.** *The number of P. intermedia in logarithmic form for both types of implants and the study groups.*

As for *P.intermedia* the PDT1 group showed no significant difference compared to NC group. On the other hand the PDT2 group was significantly more effective in the elimination of each of the bacteria separately when compared to the NC group (p < 0.05).

The least effective among the groups, when compared to the NC group, was the TB group (62.4%). Compared to NC there was no significant difference neither for the total number nor for each bacteria separately.
