ANTECEDENTES DE LA INVESTIGACIÓN

Fluorescence profiles were obtained by FCM after immunostaining the M, RT, PL and gingival cells for the connective tissue proteins collagen type I, fibronectin, tenascin and decorin. Antigen levels expressed by the PL cells, representing the cell population derived from normal healthy PL tissue, was used as a baseline for comparison with the GTR-associated M and RT cells. A solid vertical line was drawn arbitrarily on the histograms presented in this chapter to indicate the mean of each of the PL profiles.

Nearly 50% of cells within the cultures examined were found to be positive for collagen I. The representative histograms (Figure 5.2) show that the expression of collagen I was narrow and uniformly distributed. This observation suggested that all the cells in each of the different cultures had very similar collagen levels, although there were differences in collagen expression between the different cell cultures. Thus, it was noted that both the M and RT profiles were shifted to higher fluorescence intensity units (to the right) of the vertical line, showing that the M and RT cells had higher levels than the PL cells. The results of a number of experiments are summarised in Table 5.2 and show that the differences in collagen I expression between all the cell types were statistically significant, using the

Kruskal-Wallis test (p < 0.05). In order to determine which two cell types were different from each other, the Mann-Whitney U test was carried out. The results of this statistical analysis revealed that, although the GTR- associated M and RT cells (APIs of 75 and 73, respectively) had higher collagen levels than the normal PL cells (AFI of 66), these differences were not significant (p > 0.05). The M and RT cells also had elevated levels of this protein compared with the gingival cells (AFI of 40), but only the M cells were significantly higher (p < 0.01).

Table 5.2. Expression of soft connective tissue proteins by membrane-associated (M), regenerated tissue (RT), periodontal ligament (PL) and gingival (G) cells.

Protein M cells n=6 RT cells n=4 AFI PL cells n=5 G cells n=9 p value* Collagen 1 75 73 66 40 0.047 (56/118) (31/129) (42/92) (25/53) Fibronectin 290 272 188 97 0.014 (231/562) (84/552) (148/362) (50/121) Tenascin 62 63 53 34 0.154 (37/120) (30/102) (43/67) (25/54) Decorin 41 61 50 29 0.081 (35/65) (40/116) (45/65) (23/48)

The values are arbitrary. Data are presented as the median and the numbers in brackets are the distribution of the data presented as 25/75 percentile.

*Data were analysed using the Kruskal-Wallis test and p < 0.05 is considered to be statistically significant.

AFI = Average fluorescence intensity

o M cells o L RT cells M— o <D E ° i 1 : j PL cells ■^1 , ^4 ; oo Gingival cells Fluorescence Intensity

Figure 5.2. Representative histograms of collagen I expression by M (bold blue line), RT (bold red line), PL (bold green line) and gingival cells (bold orange line). The corresponding negative control profiles are presented as dashed lines. The vertical black line shows the mean of the PL histogram, which is used to indicate the collagen I level expressed by the normal PL cells. Note high expression of collagen I in the M and RT cells, and low expression in the gingival cells relative to the PL cells. Fluorescence intensity units are arbitrary.

As with collagen I, fibronectin expression in the M and RT cells was higher than in the PL cells, as shown in the representative histograms in Figure 5.3. Unlike collagen I, however, fibronectin distribution among the individual cells within each culture was relatively wide and, moreover, was not bell-shaped but skewed to levels of fluorescence which were greater than the mean. This non-uniform distribution observed with the M, RT and PL cultures is indicative of the presence of a heterogeneous cell population in the culture, i.e., the presence of sub-populations of cells expressing different levels of antigen. Detailed analysis of these fluorescence profiles was therefore carried out to examine whether the M, RT and PL cultures comprised distinct sub-populations of cells, as described in section 5.3.4 below.

The results of fibronectin expression are summarised in Table 5.2 and show that the differences in the expression of this antigen among the cell types were statistically significant, using the Kruskal-Wallis test (p = 0.014). However, the GTR-associated M and RT cells expressed higher levels of fibronectin (APIs of 290 and 272, respectively) than the cells derived from normal PL (AFI of 188), although these differences were not statistically significant (p > 0.05) as determined by the Mann-Whitney U test. The M and RT cells also expressed fibronectin at levels 2.9- and 2.8-fold higher than the gingival cells, although only the difference between the M and gingival cells was statistically significant (p < 0.001).

M cells s CnI - OO RT cells S- «4— o <D E 3 z PL cells a - Glnglval cells

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Figure 5.3. Representative histograms of fibronectin expression by M, RT, PL and gingival cultures. Histograms representing fibronectin-positive cells and the negative control cells are shown in bold and dashed coloured lines, respectively. The majority of the M and RT profiles are shifted to the right of the vertical black line, indicating that these cells express fibronectin at higher levels than the PL cells.

As with the expression of collagen type I and fibronectin, the representative profiles for tenascin expression by the M and RT cells demonstrated higher fluorescence levels than the PL cells, as shown in Figure 5.4. The histograms also showed that the GTR cells had a relatively wider distribution of tenascin compared to the PL cells. The observation that the M and RT profiles were skewed to the higher fluorescence levels suggested the presence of sub-populations of cells in these cultures.

The results summarised in Table 5.2 also confirmed that the GTR- associated cells expressed tenascin at higher levels (APIs of 62 and 63 in the M and RT cells, respectively) than in the normal PL cells (AFI of 53). Gingival cells had an AFI of 34. Although both of the GTR-associated cells had approximately 1.2- and 2-fold higher tenascin levels than the PL and gingival cells, respectively, statistical analysis revealed that there were no significant differences between the cell types (p > 0.05). However, while 53% of the RT cells were positive for tenascin, only 32% of the normal PL cells were tenascin-positive. The proportions of tenascin-positive cells in the M and gingival cultures were also relatively low (34 and 43%, respectively).

The decorin profiles of all cell types (data not shown) appeared to be narrow and uniform, similar to collagen I profiles. The results in Table 5.2 show that the RT cells (AFI of 61) had the highest decorin expression among the cell types examined, although unlike the expression of the other connective tissue proteins examined, decorin was expressed at lower levels in the M cells (AFI of 41) than in the PL cells (AFI of 50). The gingival cultures again had the lowest AFI level, which was 29. Despite apparent differences in decorin AFI levels between the cell types, these were not statistical significant (p > 0.05).

Negative controls for the above experiments were the M, RT, PL and gingival cells treated with non-specific mouse lgGi as primary antibody. Such cells had very low AFI levels, (10, 14, 8 and 6, respectively) and the profiles of these cells were very similarly narrow and uniform, as shown by the dashed lines in Figures 5.2-4.

M cells RT cells S-

8

Fluorescence Intensity

Figure 5.4. Representative histograms of tenascin expression by M, RT, PL and gingival cultures. Histograms representing tenascin-positive cells and the negative control cells are shown in bold and dashed coloured lines, respectively. The vertical solid black line indicates the mean fluorescence intensity of the PL histogram. Note the higher levels and wider distributions of tenascin in the M and RT cells compared with the PL cells.

5.3.3 Expression of bone-associated proteins

In order to further characterise the GTR-associated cells, the expression of proteins known to be involved in the formation of hard connective tissue, such as bone and cementum, were investigated by FCM. The FCM profiles of osteonectin expression were found to be relatively narrow and uniformly distributed in all cultures (data not shown). However, the RT cells were found to have a higher level of osteonectin (AFI of 67) than the M and PL cells (AFIs of 45 and 56, respectively), as shown in Table 5.3. Gingival cells had an AFI of 38, although none of these differences were statistically significant (p > 0.05).

Differential expression of osteocalcin was evident in the representative histogram profiles, which showed that osteocalcin in the M, RT and PL cultures had a non-uniform distribution (Figure 5.5). These skewed profiles suggested the possibility that heterogeneous sub­ populations of cells were present among the cells in these cultures, which were analysed further below (section 5.3.4). The mean fluorescence intensity of the PL cells, indicated by the vertical line in Figure 5.5, shows that the RT, but not the M cells, express higher osteocalcin levels than the PL cells. The differences in osteocalcin expression among the cell types are presented in Table 5.3 and were statistically significant (p < 0.05). However, the Mann-Whitney U test revealed that the 2.4- and 1.2-fold higher expression of osteocalcin in the RT cells was not statistically greater than in the M and normal PL cells, respectively (p > 0.05). The RT cells expressed this protein at a significantly higher level than the gingival cells (p < 0.05).

The BSP profiles of all the cell types were observed to be narrow and uniform (data not shown), although the RT cells (AFI of 56) had slightly higher levels than the M and PL cells (AFIs of 51 and 52, respectively) (Table 5.3). BSP in gingival cells was very low (AFI of 29). These differences were, however, not statistically significant (p > 0.05).

The representative osteopontin profiles of the M and RT cells exhibited markedly non-uniform distributions relative to the PL profile, as shown in Figure 5.6. Moreover, the profiles of both the GTR-associated cells appeared to contain more than one distinct peak, clearly indicating the

presence of heterogeneous sub-populations of cells. These were analysed below (section 5.3.4). In contrast, osteopontin distribution in the normal PL cells was uniform. Table 5.3 shows, however, that as with all other bone- associated proteins, the RT cultures expressed elevated levels of osteopontin (AFI of 58) compared with the other types of cells. Both the M and PL cultures expressed this protein at the same level (AFI of 46), while the gingival cells again expressed the lowest level (AFI of 30) among the cell types examined. However, statistical analysis showed that the differences were not statistically significant (p > 0.05).

Negative controls for the above experiments were the M, RT, PL and gingival cells which were incubated only with the FITC-conjugated secondary antibody. These cells had very low AFI levels (approximately between 5 - 1 0 ) and their fluorescence profiles were observed to be very narrow and uniform, as presented as dotted lines in Figures 5.5 and 5.6.

Table 5.3. Expression of bone-associated proteins by membrane-associated (M), regenerated tissue (RT), periodontal ligament (PL) and gingival (G) cells.

Protein M cells n=6 AFI RT cells n=4 PL cells n=5 G cells n=9 p value* Osteonectin 45 67 56 38 0.113 (33/72) (47/131) (51/67) (31/53) Osteocalcin 41 100 81 40 0.031 (27/67) (79/173) (73/98) (31/56) BSP 51 56 52 29 0.084 (28/103) (34/104) (47/63) (17/45) Osteopontin 46 58 46 30 0.143 (33/63) (35/109) (43/62) (21/48)

Data are presented as the median and the numbers in brackets represent the distribution as the 25/75 percentile.

*Data were analysed using the Kruskal-Wallis test and p < 0.05 is considered to be statistically significant.

AFI = average fluorescence intensity BSP = bone sialoprotein

M cells RT cells <D E 3

z

Fluorescence intensity

Figure 5.5. Representative profiles of osteocalcin expression (solid lines) by M, RT and PL cells. The dotted lines represent the fluorescence profiles of negative control cells of each culture where the primary antibody was omitted. The vertical black line indicates the mean fluorescence intensity of osteocalcin in the PL cells. Note the relatively lower expression in the M cells and higher in the RT cells compared with the PL cells.

o : lO o <D E 3

z

Fluorescence intensity

Figure 5.6. Representative profiles of osteopontin expression (solid lines) by M, RT and PL cells. The dotted lines represent the profiles of negative control cells. The vertical line indicates the mean fluorescence intensity of osteopontin in the PL cells. Note the higher expression in the RT cells than in the PL cells which are similar to the M cells.

5.3.4 Sub-populations of GTR-associated cells and normal PL cells The fluorescence profiles of all the cultures were further analysed to examine possible heterogeneity among the cells. As noted above, the presence of a non-uniform, skewed distribution of fluorescence or the presence of more than one peak in the profiles is considered to indicate that sub-populations of cells are present in the cultures. Such profiles were identified and different levels of antigen expression were selected by gating. The cells within each arbitrary gate were then analysed for their size and granularity as well as their specific AFI level.

Among the soft-connective tissue proteins, the profiles of collagen I in all cell types were uniform, indicating that no subsets of collagen-expressing cells were present in the cultures. Similar results were obtained for decorin. The tenascin profiles of the M and RT cells demonstrated some skew but an apparent second peak was not evident. In contrast, the fibronectin profiles of the M, RT and normal PL cells were very broad, markedly skewed to higher fluorescence levels and appeared to have more than one peak of fibronectin expression. This suggested that a substantial proportion of the cells in the cultures have a higher level of fibronectin than other cells in the same culture. The profiles of fibronectin were therefore arbitrarily gated into sub-populations designated as fibronectin'®'" and fibronectin^'®^ cells, as shown in Figure 5.7. The fibronectin'®'" cells were defined as having AFI values approximately between 4 and 560 and the fibronectin'^'®^ cells between 560 and 4500 AFI units.

(f) ~Q> Ü Q> E ? fibronectin'®'^ fibronectin"'*'* O O

Fluorescence intensity

Figure 5.7. Fibronectin'®'" and fibronectin'"^" cells in a culture of M cells. The vertical dotted lines represent the borders of the selected gates which are identified manually.

T h e s e regions, com prising the fibronectin'®'" and fibronectin"'®" cells in the M, R T and PL cultures w ere then ‘b a ck -a n a lys e d ’, as shown in T a b le 5.4, to d eterm ine th e A FI levels of each of the cells as well as their size and granularity. T h e fibronectin'®'" cells com prised 6 9 % and 4 4 % of the total M and R T cells, respectively. T h e s e fibronectin'®'" G T R -a s s o c ia te d cells w e re found to h ave m arkedly higher flu o rescen ce intensity valu es (A F Is of 1 5 9 and 170, respectively) com pared with the fibronectin'®'" PL cells (A FI of 4 3 ), which com prised 6 0 % of total M cells. Similarly, the fibronectin"'®" M and R T sub-populations had much higher levels (1 0 5 6 and 9 8 9 , respectively) than the fibronectin"'®" PL cells (A FI of 2 8 9 ). B ack-analysis also re ve a le d that the fibronectin"'®" cells w ere larger and m ore g ran u lar than th e fibronectin'®'^'' counterparts in all the M, R T and PL cultures, although th ere w ere no ap p aren t d ifferences in eith er the size or granularity b etw een the fibronectin'®''''-expressing M, R T and PL cells or the fibronectin"'®"-expressing M, R T and PL cells. Figure 5 .8 show th e dot plots of th e M cells from F ig u re 5 .8 w hich w ere d esig n ated as fibronectin'®'" and fibronectin"'®". S im ilar distributions w e re also observed for the R T and PL sub-populations (not shown).

Table 5.4. Comparison of sub-populations in fibronectin-positive membrane- associated (M). regenerated tissue (RT) and periodontal ligament (PL) cells.

Fibronectin'®'''

Cell type AFI Size Granularity % of total cells M 159 ± 7 8 454 ± 76 390 ± 95 6 9 ± 11 RT 170 ± 8 9 4 4 8 ± 126 332 ± 143 44 ± 2 PL 43 ± 1 9 487 ± 79 371 ± 75 6 0 ± 15 Fibronectin'’'®'’ M 1056 ± 5 1 0 562 ± 62 525 ± 49 31 ± 1 1 RT 989 ± 235 5 3 5 ± 179 427 ± 82 56 ± 3 PL 2 8 9 ± 181 609 ± 40 494 ± 55 4 0 ± 15

Data are presented as the mean ± standard deviation. The average fluorescence intensity (AFI), size and granularity units are arbitrary.

i 5 § (D =5 <=> C 03 O s i Fibronectin'®'^ M c ells Flbronectin^'^h M c e lls 200 400 000 S iz e 800 lo o o

Figure 5.8. Dot plots of the fibronectin'®^ and fibronectin^'^'’ M cells.

Among the bone-associated proteins, the fluorescence profiles of osteonectin and BSP were found to be uniform with no apparent second peak. In contrast, osteocalcin profiles of the M, RT and PL cells indicated heterogeneity of these cells in these cultures (see Figure 5.5). For back- analysis, the osteocalcin histograms were arbitrarily gated, as described above for fibronectin, and the cells within the gates designated as osteocalcin'®'^ and osteocalcin^'®^. The osteocalcin'*^ sub-population in the RT cultures had a lower antigen level (AFI of 13) than that in the PL cultures (AFI of 35). In contrast, the osteocalcin^'®'’ sub-population in the RT cells was found to express this bone-associated protein at higher levels (AFI of 64) than that in the PL cells (AFI of 51). Osteocalcin expression in the M sub-populations, however, was somewhat lower than those of both the RT and PL cells.

The osteopontin histograms of the PL cells appeared to be wide with a single peak with no apparent second peak (see Figure 5.6). In marked contrast, the histograms of osteopontin-expressing cells in the M and RT cultures were observed to have two peaks which were subsequently gated, as shown in Figure 5.9, and designated as the osteopontin'®'"' (AFI values between 3 - 22) and osteopontin'"®'’ (AFI values between 22 - 300) sub­ populations. Further analysis showed that both the ‘low* and ‘high’ RT sub­ populations (AFIs of 15 and 50) expressed osteopontin very similar to the ‘low" and ‘high’ M sub-populations (AFIs of 15 and 58, respectively). The osteopontin'®'"' cells were smaller and less granular than their osteopontin'"®'’ counterparts in all cultures, similar to sub-populations expressing fibronectin at low and high levels (see Figure 5.7).

osteopontin***^ (/) e - (D Ü O R- CD E R-

z

Fluorescence intensity

Figure 5.9. Osteopontin'®^ and osteopontin'”^*’ cells in a representative profile of the RT cells. The vertical dotted lines represent the borders of the selected gates.