9. results 9.1 experimental animals · pdf file9.1 experimental animals ... the cold stimulus...

9. RESULTS

9.1 Experimental animals Two animals died during the first experimental treatment session due to

suspected respiratory distress associated with excess pressure across the chest

from the restraint system used. After the method of restraining the rat on the

holding board was altered slightly to avoid any pressure on the thoracic area, all

of the remaining animals survived the procedures, gained weight during the

observation period and showed no obvious long-term adverse effects. The two

dead animals were replaced so that the final sample size remained at 30.

9.2 Anaesthesia The Hypnorm®/Hypnovel® anaesthetic combination used during the application of

the cold stimulus was found to provide rapid and predictable anaesthesia, with

the animals showing signs of regaining consciousness within 30 minutes to one

hour after administration. All rats were noted to have resumed normal behaviour

when observed the day after the experimental intervention. The Nembutal®

anaesthetic agent used for animal sacrifice required a much longer period for

onset, and the effects were not as predictable for each individual animal. In some

cases, additional doses were required to produce sufficiently deep anaesthesia

as indicated by absence of the corneal and plantar reflexes. It was not

uncommon for a delay of 10 to 15 minutes to occur between initial administration

of the drug and onset of adequate anaesthesia.

9.3 Specimen preparation and sectioning Although care was taken during initial trimming of the maxillae at the time of

animal sacrifice, embedding of the tissue specimens in the paraffin wax, and

trimming of the wax block prior to sectioning on the microtome, it was evident that

the plane of section was not always in the true coronal plane. It was noted when

viewing some of the sections that the furcation region was only visible on the

control or experimental side and not the other side. In a small number of

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sections, only one or other of the experimental or control teeth were included.

This generally was seen only in sections taken from the extreme mesial or distal

of the furcation areas. Occasionally, the furcation region was not visible on either

the experimental or control side in sections from the beginning or end of the

series for a particular animal. In such cases where the furcation was not visible,

no epithelial cell or nerve fibre counts were possible, and the relevant field in the

scoring spreadsheet was marked as missing data.

In addition to the plane of section, tissue damage was sometimes observed

within the furcation region. This generally consisted of a slight separation of the

soft tissue of the periodontal ligament from the hard tissue of the root surface.

When this occurred, it was usually a minor problem and did not affect the

analysis of the relevant section. In the rare cases where the furcation area was

damaged to an extent where accurate counting was judged to be impossible, the

relevant spreadsheet entry was again marked as missing data.

9.4 H&E stained sections 9.4.1 General overview

Examination of the H&E sections revealed that ankylosis within the periodontal

ligament of the experimental teeth was not present in all five animals at any of

the time periods studied. At least one animal from each time group exhibited

ankylosis in one or more sections, but in four time groups ankylosis was noted in

only the one animal out of the five in the group (Table 1). Ankylosis was not

observed in any of the control teeth in any of the animals from any of the

observation groups.

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Table 1 Distribution of ankylosis in experimental teeth (H&E stained sections)

Time Period Animals with ankylosis of

experimental teeth

Number of sections

affected

7 days Animal 1 11

10 days Animal 4 2

14 days Animal 2

Animal 3

Animal 4

13

3

10

18 days Animal 4 13

21 days Animal 2

Animal 3

Animal 5

6

9

1

28 days Animal 1 8

When present in sections taken from animals in the shorter time groups (ie 7 and

10 days), ankylosis within the furcation of the experimental teeth tended to

consist of fine bony trabeculae extending from the alveolar bone to the root

surface. It was noted that the ankylotic areas became denser and less

trabeculated in the later time groups, sometimes occupying almost the entire

furcation region.

Root resorption within the furcation area was found in both control and

experimental teeth, but was more prevalent on the experimental side. When

resorption was observed in control teeth, it was usually quite localised with

generally no more than 10-50µm of root surface affected. In experimental teeth,

the resorption was often widespread throughout the furcation, affecting up to

180µm of the root surface. Resorption in experimental teeth was commonly seen

immediately adjacent to areas of ankylosis, although the root surface at the site

of the actual ankylotic union was usually seen to be intact and free of signs of

resorption. In a similar manner to the distribution of ankylosis over the various

time periods, resorption in experimental teeth appeared to be more extensive

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and affecting a larger area of root surface within the furcation in the later time

periods.

9.4.2 7 days observation group

One of the five animals from this group exhibited ankylosis in 11 out of 15

experimental tooth sections viewed. The ankylosis consisted of fine, finger-like

projections extending through the periodontal ligament from the alveolar bone to

the root surface, with ankylotic union seen at various times in the buccal, middle

and palatal areas of the furcation. The greatest amount of root surface affected

by ankylosis in any one furcation was 100 µm, which comprised approximately

one third of the furcation. Connective tissue cells and blood vessels were seen

interspersed amongst the ankylotic projections (Figure 5). The cementum surface

was generally seen to be intact although several small, discreet areas of active

root resorption were observed in one section (Figure 6). Resorption within the

furcation on the control side was noted in three out of the five animals, but this

was seen in only a few sections and the largest single area measured involved

only 30µm of the root surface. In all of the sections from animals in this time

group, the pulp tissue of the experimental teeth appeared to be necrotic, with

decreased cellularity and tissue disorganisation.

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Figure 6 Ankylosis at 7 days Figure 5 Ankylosis at 7 days Note localised resorption adjacent to ankylotic

area. H&E stain, 20X magnification H&E stain, 10X magnification


Ankylosis was seen in one animal from this group, but in only two out of the 15

sections. The ankylotic areas were similar in appearance to those seen in the 7

days group, with connective tissue cells and blood vessels amongst the ankylotic

material (Figure 7). The largest area of root surface affected by ankylotic union in

this time group was 50µm. Root resorption was seen in both sections adjacent to

the areas of ankylosis (Figure 8). Resorption was also seen in teeth which were

not affected by ankylosis, including two control teeth. All of these areas were

quite small, with the largest surface affected in any one furcation being 60µm.

The pulp tissue of the ankylosed tooth in this observation group appeared to be

more cellular compared to the 7 days specimen, although still disorganised

compared to the corresponding control tooth.

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Figure 7 Ankylosis at 10 days H&E stain, 10X magnification

Figure 8 Ankylosis at 10 days

Higher power view of previous section, showing resorption adjacent to ankylotic

area. H&E stain, 20X magnification


Three out of the five animals from this group developed ankylosis, which was

observed in 13 sections in one animal, 10 in another and 3 in the third specimen.

The ankylosis in the animals of this group was similar in appearance to the 7

days group, with blood vessels and connective tissue cells often observed within

the ankylotic areas. The ankylosis was, however, generally more extensive,

occasionally affecting the entire furcation (Figure 9). Resorption was also more

common, and was usually seen affecting the root surface adjacent to the

ankylotic area, although it was sometimes seen within the zone of ankylosis. The

pulp tissue of the experimental teeth in this group was more cellular than that of

the 7 days group, and some sections showed changes within the pulp chamber

which appeared to be a form of reactive hard tissue formation although there did

not appear to be any tubule structures present as would be expected in the case

of secondary dentine (Figure 10). Occasionally cells were seen to be trapped

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within this material within the pulp chamber, leading to an appearance similar to

cellular cementum.


Note greater extent of ankylosis and resorption. H&E stain, 20X magnification

Figure 10 Resorption at 14 days

Note changes within pulp chamber. H&E stain, 20X magnification


One animal from this group developed ankylosis, which was observed in 13 out

of the 15 sections viewed. The ankylotic areas seen in this time group differed

from that found in the earlier groups, with a dense, solid area of bone occupying

the entire furcation in most of the sections (Figure 11). In contrast to the

specimens from the earlier time groups, there did not appear to be any

connective tissue cells or blood vessels incorporated within the ankylotic

material. Sections taken from the distal end of the furcation region still exhibited

solid bony ankylosis, but the area of contact with the root surface here was

smaller and restricted to the buccal and middle areas of the furcation. Resorption

of the root surface adjacent to the ankylotic area was a common feature, often

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widespread throughout the furcation (Figure 12), and in some cases it was seen

affecting the buccal and palatal roots. Repaired resorption was noted in one of

the sections in which the whole furcation was affected by ankylosis.


Note dense ankylotic material occupying entire furcation. H&E stain, 10X

magnification

Figure 12 Resorption at 18 days. This section was taken from the distal extreme

of an ankylotic furcation H&E stain, 40X magnification


Three animals from this group exhibited ankylosis, with nine, six and one section

respectively showing evidence of ankylotic union. The type of ankylosis seen in

these animals consisted of either solid bony material or fine trabeculae extending

from the alveolar bone to the root surface. Where the solid form of ankylosis was

seen, the contact area with the root surface was quite small and did not occupy

the whole furcation, unlike the case for the 18 days group. Widespread root

resorption was seen throughout the furcation in two of the animals whilst the third

animal, in which ankylosis was observed in only one section, did not exhibit any

resorption within the ankylotic furcation. The pulpal changes first noted in the

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experimental teeth in the animals from the 14 days observation group were more

pronounced in the 21 days group (Figure 13).

Figure 13 Ankylosis and resorption at 21 days

Note widespread resorption adjacent to ankylotic area, and pulp changes. H&E stain, 10X magnification


One animal from this group developed ankylosis, which was seen in eight of the

15 sections. The appearance of the ankylotic area was similar to that seen in the

21 days group, with solid bony material spanning the periodontal ligament but

with small areas of contact with the root surface. Root resorption was a common

feature in this animal. It was often widespread throughout the furcation and was

also seen to affect the area of ankylotic union. The pulpal changes in the

experimental teeth of this group were similar to those seen in the 21 days group.

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There was some tissue damage noted in the sections from this time period, but a

reasonable view of the furcation structures was still possible (Figure 14).

Figure 14 Ankylosis and resorption at 28 days

H&E stain, 40X magnification

9.5 Immunostaining 9.5.1 Cytokeratin AE1/AE3

At the dilution of 1:500, consistent staining results were obtained with the

cytokeratin AE1/AE3 antibody as shown by strong staining of the epithelium of

the palatal mucosa and the epithelial cells in the control sections (Figure 15).

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Background staining was also minimal, facilitating identification and counting of

the epithelial cell rests of Malassez. All staining with this antibody was carried out

using the multi-species detection kit.

Figure 15 Cytokeratin AE1/AE3 stain.

Note strong positive staining of gingival epithelium and epithelial cell clusters, 10X magnification

9.5.2 PGP 9.5

Although it was effective in staining nerve fibres, the PGP 9.5 immunostain did

not produce the same consistent results as the cytokeratin AE1/AE3 antibody. At

a dilution of 1:20,000, PGP 9.5 stained nerve fibres well, as evidenced by strong

staining of the large nerve bundles within the mucosa of the hard palate (Figure

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16). However, background staining of the periodontal ligament tissues was noted

to some degree in all sections stained, although this occurred to a much lesser

degree in the sections stained in the immunocytochemistry laboratory of the

IMVS. The intensity of the overall staining result was also somewhat variable,

with some sections appearing very pale throughout. In the sections stained in the

IMVS laboratory, it was also noted that the nerve fibres stained darker than those

treated using the multi-species detection kit.

Figure 16 PGP 9.5 stain.

Note strong staining of major palatine nerve bundles, 10X magnification

9.6 Epithelial cells Epithelial cells or clusters of cells were generally observed close to the root

surface in both the control and experimental teeth, and often appeared to be

almost in contact with the cementum (Figure 17). In a small number of sections,

the epithelial cells appeared to have been incorporated into the cementum, with

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positively stained cells visible below the root surface. In control teeth, several

clusters comprising four or five cells each were often seen spaced at intervals

along the cementum surface of the furcation. In most cases, this pattern

extended along the entire length of the furcation (Figure 18), although sometimes

the cell clusters were restricted to one part of the furcation only (Figure 19).

Occasionally, the epithelial cell clusters appeared elongated, where they

extended for a distance along the root surface without encroaching any further

than usual into the periodontal ligament space (Figure 20).

Figure 17 Epithelial cell clusters on control side at 7 days

Cytokeratin AE1/AE3 stain, 40X magnification

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Figure 18 Epithelial clusters along length of furcation on control side at 10 days Cytokeratin AE1/AE3 stain, 10X magnification

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Figure 19 Epithelial clusters on control side at 14 days Note restriction of clusters to middle part of furcation. Cytokeratin AE1/AE3 stain, 10X

magnification

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Figure 20 Elongated epithelial cell cluster on control side at 21 days


In both experimental and control teeth, but particularly on the experimental side,

small cell-sized particles which exhibited a staining reaction with the cytokeratin

antibody were seen. These particles were observed throughout the tissue

sections, including the periodontal ligament, pulp and alveolar bone. On close

inspection, they appeared to have the general structure of small cells, with darkly

stained granular material within the cytoplasm (Figure 21). It was thought that

these structures may be phagocytic cells, although it was not clear if they actually

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represented cells or whether they were artefactual. In some cases, their

presence complicated the identification of the epithelial cells during counting.

Figure 21 Cytokeratin-positive particles

Experimental tooth from 7 days observation group showing unidentified positively stained particles and ankylosis. Cytokeratin AE1/AE3 stain, 40X magnification

Preliminary examination of the sections indicated that the number of epithelial

cells within the periodontal ligament of the furcation region was higher in the

control teeth compared to the experimental teeth, regardless of whether

ankylosis was present or absent. In the control teeth, the number of epithelial

cells and clusters visible in each furcation was quite variable, with counts ranging

from one cell/cluster up to more than 20. In contrast, the furcations of

experimental teeth were, in many instances, completely devoid of any visible

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epithelial cell rests even when the tooth was free of ankylosis (Figure 22). Where

epithelial cell rests were observed in the furcations of experimental teeth, they

often consisted of a small number of cells or even single cells, whereas larger

groups of strongly stained cells were usually seen on the control side (Figure 23,

Figure 24). When similar, larger groups of epithelial cells were present on the

experimental side, usually in the absence of ankylosis, they tended to exhibit

weaker staining and thus have a faded appearance (Figure 25). Statistical

analysis of the cell counts will be presented later.

Figure 22 Absence of epithelial cells in experimental tooth at 7 days

Note lack of epithelial cells in furcation. Cytokeratin AE1/AE3 stain, 10X magnification

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Figure 23 Epithelial clusters on control side at 18 days Note several darkly stained epithelial cell clusters. Cytokeratin AE1/AE3 stain, 10X magnification

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Figure 24 Epithelial clusters on experimental side at 18 days Note the presence of a single epithelial cell cluster. This photomicrograph is taken from the same

section as the previous figure. Cytokeratin AE1/AE3 stain, 10X magnification

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Figure 25 Faded epithelial clusters on experimental side at 10 days


In experimental teeth which had developed ankylosis, there were generally no

epithelial cells or clusters detected immediately adjacent to the ankylotic area

and in many instances there were no epithelial cells visible at all within the entire

furcation (Figure 26). In one case, a single epithelial cell cluster was seen in

close proximity to an area of root resorption which bordered the ankylotic area

(Figure 27). In several ankylosed teeth, epithelial cells and clusters were often

seen along the root surfaces away from the furcation region, and sometimes

within the furcation itself but distant to the ankylotic material (Figure 28).

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Figure 26 Ankylosis and epithelial clusters at 14 days Note lack of epithelial cell clusters in the furcation of this experimental tooth. Cytokeratin

AE1/AE3 stain, 10X magnification

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Figure 27 Ankylosis and epithelial clusters at 10 days Note ankylotic area with single epithelial cluster nearby in this experimental tooth. Note resorption

bay adjacent to ankylotic area. Cytokeratin AE1/AE3 stain, 40X magnification

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Figure 28 Epithelial clusters distant to ankylotic area on experimental side at 14 days


9.7 Nerve fibres A general overview of the sections stained with PGP 9.5 revealed that the density

of the visible nerve structures was much greater in the connective tissue of the

gingiva and palatal mucosa, when compared to the periodontal ligament tissue.

Thick nerve fibres were visible deeper in the connective tissue of the gingiva and

palatal mucosa, with numerous fine nerve fibres and endings visible closer to the

epithelium (Figure 29). The nerve fibres identified within the periodontal ligament

consisted mainly of fine fibres, although thicker structures were occasionally

seen. These larger fibres were generally found close to the alveolar bone

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surface. With regard to the number and density of the fibres within the ligament, it

was noted that large areas of the ligament within the furcation region often

appeared to be devoid of nerve fibres (Figure 30). This pattern was somewhat

variable however, with some sections exhibiting large numbers of fibres (Figure

31). The distribution of the nerve fibres did not appear to differ greatly between

the control and experimental sides (Figure 32, Figure 33), although initial

examination of the sections suggested that on the experimental side there were

fewer nerve fibres in the area of the ligament closest to the tooth root.

Figure 29 Densely innervated gingival tissue.

PGP 9.5 stain, 40X magnification

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Figure 30 Sparse innervation in furcation on control side at 18 days PGP 9.5 stain, 40X magnification

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Figure 31 Nerve fibres on experimental side at 10 days Note large number of fibres. PGP 9.5 stain, 40X magnification

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Figure 32 Nerve fibre distribution on control side at 14 days PGP 9.5 stain, 40X magnification

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Figure 33 Nerve fibre distribution on experimental side at 14 days

Note similar appearance and distribution of nerve fibres compared with control tooth in previous figure. PGP 9.5 stain, 40X magnification

Due to the small size of the nerves, 40X magnification was required for adequate

identification and examination of the structures. Single fibres were sometimes

seen, but it was more common to see groups of fibres clustered close together

(Figure 34). The majority of the nerve fibres seen within the periodontal ligament

of the furcation were located close to blood vessels, and in many cases they

appeared to be within the walls of the vessels themselves (Figure 35). Branching

of the nerve fibres was sometimes observed (Figure 36). With respect to their

relationship to the alveolar bone and root surface, the nerve fibres were most

often seen closer to the bone surface or in the middle zone of the ligament.

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Relatively few nerve fibres were observed close to the cementum of the root

surface, especially within the periodontal ligament of the experimental teeth.

Occasionally, nerve fibres were noted in close proximity to areas of ankylosis

(Figure 37), and they were also sometimes seen near resorption bays (Figure

38), although this was less common.

Figure 34 Nerve fibres and blood vessels on control side at 10 days This grouping of nerve fibres around a blood vessel near the bone surface was a common

finding. PGP 9.5 stain, 40X magnification

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Figure 35 Nerve fibres and blood vessels on experimental side at 10 days Note nerve fibres in close proximity to blood vessels and within blood vessel walls. PGP 9.5 stain,

40X magnification

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Figure 36 Branching nerve fibres on experimental side at 21 days Note the characteristic location of nerve fibres adjacent to blood vessels as well as the presence

of a branching fibre. PGP 9.5 stain, 40X magnification

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Figure 37 Nerve fibres and ankylosis at 21 days

Note that several small nerve fibres are visible within the ankylotic area. PGP 9.5 stain, 40X magnification

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Figure 38 Nerve fibres and resorption on experimental side at 21 days

Several nerve fibres are visible close to the resorbed root surface, with one situated less than 50µm from the root. PGP 9.5 stain, 60X magnification

9.8 Statistical analysis 9.8.1 Epithelial cells

The histogram plot of the epithelial cell count data showed that it was not

normally distributed (Appendix 12.6.1). The initial univariate Poisson GEE

regression analyses found that all but one of the variables assessed were of

sufficient influence with regard to epithelial cell count to be included in the final

multivariate analysis. The criteria used for this determination was a p-value of

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less than 0.20 in the univariate analysis. The multivariate analysis allowed for

interpretation of the influence of each variable after adjusting for all other

variables included in the model. The variables included for analysis were: side

(experimental versus control), location (buccal, middle, and palatal), observation

time period, and the presence or absence of ankylosis. A side by time interaction

was included in the initial univariate analyses to control for the effect of side (ie

experimental versus control tooth), but the relationship was not significant

(p=0.21) and so this interaction was not included in the final analysis.

The results of the multivariate analysis (Table 2, 3) indicated that there was a

significant difference in epithelial cell count between the control and experimental

teeth (p<0.0001), with a larger number of epithelial cells on the control side. With

regard to location, there were significant differences between buccal, middle and

palatal, with the epithelial cell count being higher in the middle section, followed

by the palatal then the buccal region. An analysis of the side by location

interaction also identified several significant relationships. The absence or

presence of ankylosis also had a significant effect on epithelial cell count

(p=0.0149), with the count being higher when ankylosis was not present. There

was no significant relationship identified between epithelial cell count and time

(p=0.0804).

Table 2 Significance of model variables for multivariate analysis of epithelial cell count, showing significance of each variable (significant p values indicated in bold type)

Variable Estimated cell count

p value

Side expt ctrl

0.721.69

<0.0001

Location buc mid pal

0.931.261.13

0.0020

Time 7 d 1.34

0.0804

99

10 d 14 d 18 d 21 d 28 d

1.731.140.770.930.94

Ankylosis non-ankylosed ankylosed

1.280.95

0.0149

Side by location ctrl x buc ctrl x mid ctrl x pal expt x buc expt x mid expt x pal

1.551.901.660.560.840.78

0.0348

Table 3 Breakdown of individual results of multivariate analysis for epithelial cell count, showing the significance of pairwise comparisons between variables (significant p values indicated in bold type)

Effect Contrast p value

Side ctrl vs expt <0.0001

Location buc vs mid buc vs pal mid vs pal

<0.0001 0.0096 0.0396

Ankylosis non-ankylosed vs ankylosed 0.0035

Side by location

ctrl x buc vs ctrl x mid ctrl x buc vs ctrl x pal ctrl x buc vs expt x buc ctrl x buc vs expt x mid ctrl x buc vs expt x pal ctrl x mid vs ctrl x pal ctrl x mid vs expt x buc ctrl x mid vs expt x mid ctrl x mid vs expt x pal ctrl x pal vs expt x buc ctrl x pal vs expt x mid ctrl x pal vs expt x pal expt x buc vs expt x mid expt x buc vs expt x pal expt x mid vs expt x pal

0.0059 0.3448

<0.0001 <0.0001 <0.0001

0.0011 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

0.0022 0.3480

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Ankylosis was dichotomised into a binary yes/no variable for the purposes of

assessing its relationship with epithelial cell count. The results of the log binomial

GEE regression model which was used for this analysis indicated a significant

relationship (p=0.0027, Table 4). By taking the exponential of the estimate for

epithelial cell count (exp(-1.4045) = 0.245) the model indicated that for every one

unit increase in epithelial cell count, the probability of ankylosis in the relevant

section decreased by 75.5 per cent.

Table 4 Results of regression analysis investigating the relationship between epithelial cell count and ankylosis

Log binomial GEE regression analysis

Parameter EstimateStandard

Error95% Confidence

Limits p value

Epithelial cells

-1.4045 0.4679 -2.3216 -0.4873 0.0027

9.8.2 Nerve fibres

As was the case with the epithelial cell count data, the nerve fibre data were not

normally distributed (Appendix 12.6.2). The initial univariate analyses indicated

that the side by time interaction, which was introduced to account for any

influence of side on the relationship between time and nerve fibre count, was not

significant at the p=0.20 level (p=0.32). However, there was a weak relationship

overall between time and nerve fibre count (p=0.19), so time was included in the

final multivariate analysis but not the side by time interaction. The variables

included in the final model were: side, location (buccal, middle, palatal), PDL area

(relationship of nerves to bone and tooth root – ie bone, middle or root areas),

side by PDL area, side by location, and time.

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The results of the multivariate model (Table 5) indicated that there was a

significant difference in nerve fibre count between sides, with more fibres

recorded on the control side compared to the experimental side. With regard to

bucco-lingual location within the periodontal ligament, the overall interaction was

not significant (p=0.052). A significant difference was noted between nerve

counts in the region of the periodontal ligament closest to the tooth root

compared to both the middle (p<0.0001) and bone areas (p<0.0001). The latter

two PDL areas did not differ significantly from each other. When side and PDL

area were considered together, there were significant differences between

control and experimental teeth with regard to nerve fibre count in the middle

(p=0.048) and particularly the root thirds (p<0.0001) of the periodontal ligament,

with higher counts on the control side. No significant relationship was found for

time, or side by location.

Table 5 Significance of model variables for multivariate analysis of nerve fibre count, showing significance of each variable (significant p values indicated in bold type)

Variable Estimated cell count

p value

Side ctrl expt

1.621.39

0.0185


1.551.391.58

0.0527

PDL area bone mid root

2.072.170.75

<0.0001

Side by PDL area ctrl x bone ctrl x mid ctrl x root expt x bone expt x mid expt x root

2.102.050.982.052.300.58

0.0014

Side by location 0.0566

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ctrl x buc ctrl x mid ctrl x pal expt x buc expt x mid expt x pal

1.601.471.791.501.311.38

Time 7 d 10 d 14 d 18 d 21 d 28 d

1.481.561.721.561.381.36

0.2081

Table 6 Breakdown of individual results of multivariate analysis for nerve fibre count, showing the significance of pairwise comparisons between variables (significant p values indicated in bold type)


Side ctrl vs expt 0.0094 PDL area bone vs mid

bone vs root mid vs root

0.3794 <0.0001 <0.0001

Side by PDL area

ctrl x bone vs ctrl x mid ctrl x bone vs ctrl x root ctrl x bone vs expt x bone ctrl x bone vs expt x mid ctrl x bone vs expt x root ctrl x mid vs ctrl x root ctrl x mid vs expt x bone ctrl x mid vs expt x mid ctrl x mid vs expt x root ctrl x root vs expt x bone ctrl x root vs expt x mid ctrl x root vs expt x root expt x bone vs expt x mid expt x bone vs expt x root expt x mid vs expt x root

0.7572 <0.0001

0.7693 0.0862

<0.0001 <0.0001

0.9729 0.0483

<0.0001 <0.0001 <0.0001 <0.0001

0.1363 <0.0001 <0.0001

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9.8.3 Resorption

To assess the relationship between it and several other variables, resorption was

dichotomised as a yes/no variable. The variables included in the multivariate

analysis were: side, location (buccal, middle, palatal) and epithelial cell count.

Overall time, side by time and side by location interactions were also investigated

but were not significant and hence were omitted from the final analysis. There

was a significant relationship between side and resorption (p=0.0005), with more

resorption recorded on the experimental side. With regard to location within the

periodontal ligament, there was a significant difference in resorption between the

middle and palatal areas (p=0.0004), but not between middle and buccal or

buccal and palatal (Table 7, Table 8).

Table 7 Significance of model variables for multivariate analysis of resorption, showing significance of each variable (significant p values indicated in bold type)

Variable Estimated prevalence (%) p value

Side ctrl expt

2.2510.77

0.0005


5.386.593.37

0.0111

Table 8 Breakdown of individual results of multivariate analysis for resorption, showing the significance of pairwise comparisons between variables (significant p values indicated in bold type)


Side ctrl vs expt <0.0001 Location buc vs mid

buc vs pal mid vs pal

0.2126 0.0745 0.0004

There was also a significant relationship between resorption and epithelial cell

count (p<0.0001). This relationship was similar to that found between ankylosis

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and epithelial cell count, and indicated that for every one unit increase in

epithelial cell count, the probability of resorption in the relevant section

decreased by 55% (Table 9). This was calculated by taking the exponential of the

estimate of the epithelial cell count (ie exp(-0.7897) = 0.45).

Table 9 Results of regression analysis investigating the relationship between resorption and epithelial cell count

Log binomial GEE regression analysis

Parameter EstimateStandard

Error95% Confidence

Limits p value

Epithelial cells

-0.7897 0.1364 -1.0571 -0.5223 <0.0001

9.8.4 Relationship between nerve fibres and epithelial cells

The initial regression model employed to assess this relationship adjusted for

side (control versus experimental) and location within the periodontal ligament

(buccal, middle, palatal). This analysis indicated that there was no relationship

between epithelial cell count and nerve fibre count (p=0.44). A second model was

then set up to examine epithelial cell and nerve fibre counts directly without

adjusting for other variables. This analysis also found no significant relationship

between the two counts (p=0.36).

9.8.5 Method error

The results of the weighted kappa statistics were K=0.6105 (95% confidence

limits 0.5078 – 0.7132) for epithelial cells and K=0.7351 (95% confidence limits

0.6896 – 0.7806) for nerve fibres. Kappa statistic values in the range 0.61 – 0.80

indicate good agreement between the two sets of data being tested 96.

105

9. results 9.1 experimental animals · pdf file9.1 experimental animals ... the cold stimulus...

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