Human Platelet Rich Plasma Study


The animals in both human platelet rich plasma and simvastatin study groups did not show obvious signs of systemic illness throughout the study period. Wound infection, dehiscence, or other adverse effects were not encountered in any of the animals in the present study. However, six animals developed diarrhea, five animals developed scabs, and eight animals of different treatment groups were died. They were all excluded and replaced by other animals.
4.1 Human Platelet Rich plasma Study
4.1.1 Amount of sagittal suture expansion
All the animals tolerated the expansion appliance, with no appliance failure. Table (4.1) shows the mean and standard deviation for amount of expansion of the sagittal suture after one week of expansion. One way ANOVA showed that there was no significant difference in the amount of expansion among the hPRP- treated groups and control group (Table 4.2). The mean value of expansion was 3.05 mm (?? 0.55mm).
Table (4.1): Mean and standard deviation for amount of sagittal suture expansion in control, single- and double hPRP treated groups
Amount of sagittal suture expansion (mm)
Group No. Mean St.D.
Control 20 3.05a 0.42
Single hPRP 20 2.91a 0.65
Double hPRP 20 3.18 a 0.58
Total 60 3.05 0.55
Means with the same letters are not significantly different (Duncan test)

Table (4.2): One way ANOVA of amount of sagittal suture expansion in control, single and double hPRP treated groups
P-value F Mean Square df Sum of Square
0.806* 0.220 0.89
2 0.177
between groups

0.403 12
4.835
within groups
14 5.012 Total

*significant difference existed at P ' 0.05

4.1.2 Relapse ratio of the expanded sagittal suture
The mean and standard deviation for relapse ratio of the expanded sagittal suture in the control group and both hPRP groups is presented in table (4.3). One way ANOVA showed that the relapse ratio was significantly lower in both hPRP groups than the control group. Although, the relapse ratio was less in the double hPRP group than single hPRP group, but the difference was not significant (Table 4.4).
Table (4.3): Mean and standard deviation for relapse ratio of the expanded sagittal suture in the control,Single and double hPRP treated groups
Relapse ratio (%)
Groups No. Mean St.D.
Control 5
23.66a 7.27
Single hPRP
5 10.12b 2.77
Double hPRP 5 6.88b 2.99
Means with the same letters are not significantly different (Duncan test)

Table (4.4): One way ANOVA for relapse ratio of the expanded sagittal suture in the control,Single and double hPRP treated groups
P-value F Mean Square df Sum of Square
0.000* 17.071 396.165 2 792.329 between groups

23.207 12 278.488 within groups
14 1070.817 Total

*significant difference existed at P ' 0.05

4.1.3 Radiological Density of the Sagittal Suture
4.1.3.1 Density of the Sagittal Suture After Two Weeks of Retention
Table (4.5) shows the mean and standard deviation for the density of the expanded sagittal suture in the control, single and double hPRP groups after two weeks of retention. One way ANOVA showed that the density was significantly higher in the double hPRP group than the single hPRP and control groups. Although the density was greater in the single hPRP group than control group, the difference was not statistically significant (Table 4.6).

Table( 4.5): Mean and standard deviation for radiographic density of the sagittal suture (in pixels) after two weeks of retention in the control, single and double hPRP treated groups
Radiographic density (Pixel)
Group No. Mean St.D.
Control 5 38.16 b 3.76
Single hPRP 5 41.45 b 2.00
Double hPRP 5 46.47 a 1.31
Means with the same letters are not significantly different (Duncan test)

Table (4.6): One way ANOVA for radiographic density of the sagittal suture (in pixels) after two weeks of retention in the control, single and double hPRP treated groups
P-value F Mean Square df Sum of Squar
.001* 13.250 87.563 2 175.127 between groups

6.609 12 79.302 within groups
14 254.429 Total

*significant difference existed at P ' 0.05
4.1.3.2 Density of the Sagittal Suture After Four Weeks of Retention
Table (4.7) shows the mean and standard deviation for the density of the expanded sagittal suture after four weeks of retention. One way ANOVA showed that the density was significantly higher in the double hPRP group than the single hPRP and control groups. Although the density was greater in the single hPRP group than control group, the difference was not statistically significant (Table 4.8).
Table (4.7): Mean and standard deviation for radiographic density of the sagittal suture (in pixels) after four weeks of retention in the control, single and double hPRP treated groups
Radiographic density (Pixel)
Groups No. Mean St.D.
Control 5 42.55 b 3.25
Single PRP 5 44.68 b 1.11
Double PRP 5 49.19 a 0.92

P-value F Mean
Square df Sum of Square
.001* 13.607 57.422 2 114.843 between groups

4.220 12 50.640 within groups
14 165.484 Total

Table (4.8): One way ANOVA for radiographic density of the sagittal suture (in pixels) after four weeks of retention in the control, single and double hPRP treated groups
*significant difference existed at P ' 0.05
4.1.3.3 Density of the sagittal suture after six weeks of retention
Table (4.9) shows the mean and standard deviation for the density of the expanded sagittal suture after six weeks of retention. One way ANOVA showed that the density was significantly higher in the double hPRP group than the single hPRP and control groups. In addition, the density was significantly greater in the single hPRP group than the control group (Table 4.10).
Table (4.9): Mean and standard deviation for radiographic density of the sagittal suture(in pixels) after six weeks of retention in the control, single and double hPRP treated groups
Radiographic density (Pixel)
Groups No. Mean St.D.
Control 5 44.5 c 2.85
Single hPRP 5 49.22 b 2.52
Double hPRP 5 53.63 a 2.53
Means with the same letters are not significantly different (Duncan test)

Table (4.10): One way ANOVA for radiographic density of the sagittal suture(in pixels) after six weeks of retention in the control, single and double hPRP treated groups
P-value F Mean Square df Sum of Square
.001*0 14.917 103.894 2 207.787 between groups

6.965 12 83.576 within groups
14 291.364 Total

*significant difference existed at P ' 0.05

Figure (4.1) shows the radiographic density of the expanded sagittal suture in the control , single- and double hPRP treated groups after two, four and six weeks of retention.

Figure (4.1): Radiographic density of the sagittal suture (in pixels) after two, four and six weeks of retention in the control, single- and double hPRP treated groups

4.1.4 Histological and Histomorphometric Study
4.1.4.1 Histological Study
The bone specimens included regenerated tissue in the expanded suture line and the two sutural bone edges.
4.1.4.1.1 Histological Features of the Normal Sagittal Suture
The sagittal suture of adult rabbits consists of the bones from either side of the suture edges and fibrous tissue occupying the suture. The bones were very close to each other and the fibrous tissue consisted of mesenchymal cells and thick bundles of collagen fibers. Few thin-walled blood vessels were seen in the fibrous tissue and they appeared as empty or congested vessels (Fig.4.2).

4.1.4.1.2 Histological Features of the Expanded Sagittal Suture after Two Weeks of Retention
The microscopical examination of the expanded suture from control group revealed that there was a large quantity of fibrous tissue joining the two edges of expanded gap, this tissue consisted of irregularly arranged collagen fibers with fibroblasts and newly-formed vascular channels in various stages of maturation. At both edges of the suture, there was large number of osteocytes and few number of active osteoblasts with absence of osteoclasts cells (Fig.4.3).
The microscopic examination of the expanded sutural gap from experimental group that was treated with single dose of hPRP revealed the presence of large quantity of fibrous tissue joining the two edges of expanded suture consisting of a mixture of fibroblasts, fibrocytes, blood channels and dense collagen fibers that were vertically oriented to the expanded suture. Both edges of the sutural gap exhibited osteoblastic activity represented by proliferation of osteoblasts. There were some new bone growths from both edges of sutural gap (Fig.4.4).
The histological study of the expanded sutural gap from experimental group that was treated with double dose of hPRP showed that the mature fibrous tissue consisting of various numbers of fibrocytes with huge number of large congested blood channels filling the expanded gap. In addition, there were some bone islands made of osteoid and present among the fibrous tissue. The osteoblasts, present on the surface of these newly- formed bony spicules, eventually became enveloped. There were some longitudinal new bone growths extended from the edges of both sutural margins (Fig.4.5).





4.1.4.1.3 Histological Features of the Expanded Sagittal Suture after Four Weeks of Retention
The histological examination of sagittal suture specimens from control group showed that there was a large quantity of fibrous tissue joining the two edges of suture line. This tissue consisted of large number of fibrocytes and few number of blood channels. This tissue intermingled with new bone growths which extended from both sides going up and down the suture line. Some clusters of osteoid material surrounded with osteoblast-like cells were seen near the two edges of the suture in addition to the presence of active osteoblasts along the sutural edge (Fig.4.6).
The histological examination of sagittal suture specimens from experimental group that was treated with single hPRP showed that new bone growths projected from both sides of suture line were joining together by thick mature fibrous tissue consisting of large quantity of collagen fibers and few numbers of fibrocytes. A thin mass of osseous tissue was seen extending horizontally from both ends of suture. A number of cavities was seen within the suture. These cavities were larger than those in original adjacent bone (Fig.4.7).
The histological examination of sagittal suture specimens from experimental group that was treated with double dose of hPRP showed that new bone growths were seen projecting from both edges of suture intermingled with dense mature fibrous tissue with presence of osteoid masses through suture tissue. Vascular channels were seen in the fibrous tissue and there was moderate osteoblastic activity at the borders of bones on either side of the suture (Fig.4.8).



4.1.4.1.4 Histological Features of the Expanded Sagittal Suture after Six Weeks of Retention
The histological examination of sagittal suture specimens from control group showed that the suture was of reduced size and the two ends of the suture line were joined by thick fibrous tissue consisting of fibroblasts, bundles of collagen fibers and newly formed vascular channels. Osteoblastic activity was seen at borders of the bones from either side of the suture. Osteoclasts were not seen at the edges of suture lines (Fig.4.9).
The histological examination of sagittal suture specimens from experimental group that was treated with single dose of hPRP showed vascularized dense mature fibrous tissue joining the two edges of the suture line. The finger- like projections from both sides of the suture were close to each other (Fig.4.10).
The histological examination of sagittal suture specimens from experimental group that was treated with double hPRP showed a reduced suture and the presence of well vascularised islands of osseous tissue joining the two sides of suture. Minimal fibrous tissue was seen and it was undergoing ossification (Fig 4.11).


4.1.4.2 Histomorphometric Study
4.1.4.2.1 Histomorphometric Parameters of the Expanded Sagittal Suture After Two Weeks of Retention
The mean and standard deviation for the numbers of osteoblasts and blood vessels in the control, single hPRP, and double hPRP groups after two weeks of retention are shown in table (4.11).
One way ANOVA showed that the number of osteoblasts in the hPRP groups was higher than that of control group, but the difference was not significant. Although, the number of osteoblasts was more in the double hPRP group than single hPRP group, but the difference was not significant. The number of blood vessels were significantly higher in the double hPRP group than the single hPRP and control groups, with no significant difference between the two latter groups(Table 4.12).
The mean and standard deviation for the osteoid area(O.Ar) and total area(T.Ar) of the expanded sagittal suture in the control, single hPRP, and double hPRP groups after two weeks of retention are shown in table (4.13).
The T.Ar was less in the double hPRP group than the single hPRP and control groups. One way ANOVA showed that the difference among the three groups was significant(Table 4.14). O.Ar were noted only in the double hPRP group, but not in the single hPRP and control groups. The percentage of O.Ar/T.Ar in the double hPRP group was 16.8%.
Table (4.11):Mean (?? standard deviation) of the numbers of osteoblasts and blood vessels in the control, single-and double hPRP treated groups after two weeks of retention
Parameters
(Numbers) Control
Mean?? St.D. Single hPRP
Mean?? St.D. Double hPRP
Mean?? St.D.
Osteoblasts 44.80 a ?? 6.72 63.20 a ?? 22.48 67.80 a ?? 18.50
Blood vessels 8.00 b ?? 1.58 9.00 b ?? 0.71 12.00 a ?? 1.58
Means with the same letters horizontally are not significantly different(Duncan test)

P-value F Mean Square df Sum of Square
*0.125 2.489 740.600 2 1481.200 between groups Osteoblast

297.533 12 3570.400 within groups

14 5051.600 Total
*0.001 11.818 21.667 2 43.333 between groups Blood vessel

1.833 12 .00022 within groups
14 .33365 Total
Table (4.12): One way ANOVA for the numbers of osteoblasts and blood vessels in
the control, single-and double hPRP treated groups after two weeks of retention
significant difference existed at P ' 0.05
Table (4.13): Mean (?? standard deviation) of osteoid and total areas of the expanded sagittal suture in the control, single- and double hPRP treated groups after two weeks of retention
Parameters
Area (mm2) Control
Mean?? St.D. Single hPRP
Mean?? St.D. Double hPRP
Mean?? St.D.
Osteoid area Nil Nil 0.0588 ?? 0.0236
Total area 0.6180 a ?? 0.0411 0.5322 b ?? 0.0537
0.3682 c ?? 0.0508
Percentage of Osteoid area/Total area Nil Nil 16.8 ?? 8.3
Means with the same letters horizontally are not significantly different (Duncan test)
Table (4.14): One way ANOVA for osteoid and total areas of the expanded sagittal
suture in the control, single- and double hPRP treated groups after two weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.012 2 0.006 30.939 0.000*
within groups
0.002 12 0.000
Total 0.014 14
Total area between groups 0.161 2 0.81 33.774 0.000*
within groups
0.029 12 0.002
Total 0.190 14
Percentage of osteoid area/Total area between groups 940.800 2 470.400 20.247 0.000*
within groups
278.800 12 23.233
Total 1219.600 14

*significant difference existed at P ' 0.05

4.1.4.2.2 Histomorphometric Parameters of the Expanded Sagittal Suture After Four Weeks of Retention
The mean and standard deviation for the numbers of osteoblasts and blood vessels are shown in table (4.15).
The numbers of osteoblasts at four weeks increased in both hPRP groups as compared to those at 2 weeks. One way ANOVA showed that the number of osteoblasts in the two hPRP groups at four weeks was significantly higher than the control group. Although, the number of osteoblasts was more in the double hPRP group than single hPRP group, but the difference was not significant. The number of blood vessels decreased at four weeks, as compared to two weeks, in the single hPRP and control groups. At four weeks, the number of blood vessels was significantly higher in the double hPRP group than single hPRP and control groups(Table 4.16).
The mean and standard deviation for the osteoid and total areas of the expanded sagittal suture are shown in table (4.17).
The total suture area decreased in the three groups at four weeks, as compared to two weeks. At four weeks, one way ANOVA showed that the T.Ar was significantly less in the double hPRP group than the control group. The difference between the the single hPRP and control group as well as between single-and double hPRPgroups was not significant. The O.Ar start to appear at four weeks in both control and single hPRP groups. The O.Ar. is significantly more in hPRP groups than the control group,but with no significant difference between the two hPRP groups. The percentage of O.Ar/T.Ar was more in the double hPRP than the single hPRP and control groups. The difference among the three groups was significant(Table 4.18) .

Table (4.15): Mean(?? standard deviation) of the numbers of osteoblasts and blood vessels in the control, single-and double hPRP treated groups after four weeks of retention
Parameters
(Numbers) Control
Mean ?? St.D. Single hPRP
Mean ?? St.D. Double hPRP
Mean ?? St.D.
Osteoblasts 46.4b ?? 6.39 72a ?? 8.60 82.6a ?? 13.89
Blood vessels 5.0 b ?? 1.58 6b ?? 2.55 12.0a ?? 2.92
Means with the same letters horizontally are not significantly different (Duncan test)

Table (4.16): One way ANOVA for the numbers of osteoblasts and blood vessels in the control, single-and double hPRP treated groups after four weeks of retention
P-value F Mean Square df Sum of Square
*0.000 16.890 1731.800 2 3463.600 between groups Osteoblast

102.533 12 1230.400 within groups

14 4694.000 Total
*0.001 12.286 71.667 2 143.333 between groups Bloodvessels

5.8333 12 70.000 within groups
14 213.333 Total

*significant difference existed at P ' 0.05

Table (4.17): Mean(?? standard deviation) of the steoid and total areas of the expanded sagittal suture in the control, single- and double hPRP treated groups after four weeks of retention

Parameters
Area(mm2) Control
Mean ?? St.D. Single hPRP
Mean ?? St.D. Double hPRP
Mean ?? St.D.
Osteoid area 0.0263 b ??0.0129 0.0765 a
??0.0424 0.1119 a
?? 0.0149
Total area 0.2773 a
?? 0.0467 0.2621 ab
?? 0.0391
0.2139 b
?? 0.0240
Percentage of Osteoid area/Total area 9.4c
?? 4.5 28.2 b
?? 14.6 52.8 a
?? 7.2
Means with the same letters horizontally are not significantly different (Duncan test)

Table (4.18): One way ANOVA for the steoid and total areas of the expanded sagittal suture in the control, single- and double hPRP treated groups after four weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.019 2 09.00 12.689 0.001*
within groups
0.009 12 0.001
Total 0.027 14
Total area between groups 0.011
2 0.005 3.824 0.052*
within groups
0.017 12 0.001
Total 0.028 14
Percentage of osteoid area/Total area between groups 4736.933 2 2368.467 24.654 0.000*
within groups
1152.800 12 96.067
Total 5889.733 14

*significant difference existed at P ' 0.05

4.1.4.2.3 Histomorphometric Parameters of the Expanded Sagittal Suture After Six Weeks of Retention
The mean and standard deviation for the numbers of osteoblasts and blood vessels in the control, single hPRP and double hPRP groups after six weeks of retention are shown in table (4.19).
The numbers of osteoblasts at six weeks reduced in both single and double hPRP groups, as compared to those at four weeks. At six weeks, the number of osteoblasts in the single- and double hPRP groups was less than that in the control group, but one way ANOVA showed that the difference was not significant. The number of blood vessels decreased at six weeks, as compared to that in four weeks. One way ANOVA showed that the number of blood vessels at six weeks is still significantly higher in the double hPRP group than the single hPRP and control groups, with no significant difference between the two latter groups(Table 4.20).
The mean and standard deviation for the osteoid and total areas of the expanded sagittal suture in the control, single hPRP and double hPRP groups after six weeks of retention are shown in table (4.21).
The total suture area at six weeks was reduced in the three groups as compared to that at four weeks. One way ANOVA showed that the T.Ar was significantly less in the double hPRP group than the control group, with no siginificant difference between the control and single hPRP groups as well as between the single- and double hPRP. At six weeks, One way ANOVA showed that the osteoid area is significantly higher in the single- and double hPRP groups than the control group, with no significant difference between the two hPRP groups. The percentage of O.Ar/T.Ar in the both hPRP groups was significantly higher than that of the control group. In addition, the percentage of O.Ar/T.Ar was significantly higher in the double- than single hPRP groups(Table 4.22).
Table (4.19): Mean (?? standard deviation) of the numbers of osteoblasts and blood vessels in the control, single- and double hPRP treated groups after six weeks of retention
Parameters
(Numbers) Control
Mean ?? St.D. Single hPRP
Mean ?? St.D. Double hPRP
Mean ?? St.D.
Osteoblasts 48.8 a ?? 13.63 40.60 a ??10.06 35.60 a ??8.29
Blood vessels 3.0 b ?? 0.71 4.20 b ?? 0.84 10.00 a ??1.58
Means with the same letters horizontally are not significantly different (Duncan test)

Table (4.20): One way ANOVA for the numbers of osteoblasts and blood vessels in the control, single- and double hPRP treated groups after six weeks of retention
P-value F Mean Square df Sum of Square
*0.196 1.872 222.067 2 444.133 between groups Osteoblast

118.600 12 1423.200 within groups

14 1867.333 Total
*0.000 56.811 70.067 2 140.133 between groups Blood vessels

1.233 12 14.800 within groups
14 154.933 Total
*significant difference existed at P ' 0.05
Table (4.21):Mean (??standard deviation) of the osteoid and total areas of the expanded sagittal suture in the control, single- and double hPRP treated groups after six weeks of retention
Parameters
Area(mm2) Control
Mean ?? St.D. Single hPRP
Mean ?? St.D. Double hPRP
Mean ?? St.D.
Osteoid area 0.0583b ?? 0.0204 0.0857 a ??0.0083 0.0907a ??0.0132
Total area 0.2453a ?? 0.0058 0.2230ab ?? 0.0328 0.1831b ??0.0404
Percentage of Osteoid area/Total area 23.8 c ?? 8.3 39.2 b ?? 6.5 51.0a ?? 9.8
Means with the same letters horizontally are not significantly different (Duncan test)
Table (4.22): One way ANOVA for the osteoid and total areas of the expanded sagittal suture in the control, single- and double hPRP treated groups after six weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.003 2 0.002 6.913 0.010*
within groups
0.003 12 0.000
Total 0.006 14
Total area between groups 0.010 2 0.005 5.412 0.021*
within groups
0.011 12 0.001
Total 0.021 14
Percentage of osteoid area/Total area between groups 1860.400 2 930.200 13.263 0.001*
within groups
841.600 12 70.133
Total 2702.000 14

*significant difference existed at P ' 0.05
Figure ( 4.12) shows the number of osteoblasts and figure (4.13)shows the number of blood vessels in the expanded sagittal suture of the control , single- and double hPRP groups after two, four and six weeks of retention.
Figures (4.14) shows the osteoid area and and figure (4.15) shows the total area of the expanded sagittal suture in the control , single- and double hPRP groups after two, four and six weeks of retention.

Figure( 4.12) : Number of osteoblasts in the expanded sagittal suture of the control, single- and double hPRP treated groups after two, four and six week of retention
Figure (4.13): Number of blood vessels in the expanded sagittal suture of the control, single- and double hPRP treated groups after two, four and six week of retention


Figure (4.14): Osteoid areas(mm2) in the expanded sagittal suture of control, single- and double hPRP treated groups after two, four and six week of retention

Figure (4.15): Total areas (mm2) in the expanded sagittal suture of the control, single- and double hPRP treated groups after two, four and six week of retention
4.1.5 Serum Markers of Bone Formation of hPRP Study
The mean and standard deviation for the serum markers of a non-expansion and non treated group of animals are shown in table (4.23).This group was taken as representative of normal values of those markers in normal rabbits.
Non-expansion group No. Calcium
(nm) Phosphorous
(nm) ALP
(nm) Bone ALP(U/L) Osteocalcin
(ng/L)
5 2.67
??0.51 1.87
??0.06 127.80
??14.72 12.67
??2.65 113.73
?? 25.09
Table( 4.23): Mean (?? standard deviation) of serum calcium, phosphorous, total alkaline phosphtase, bone-specific alkaline phosphtase and osteocalcin in non expansion rabbits
4.1.5.1 Serum Markers of Bone Formation After Two Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphtase, bone-specific alkaline phosphtase and osteocalcin after two weeks of retention are shown in table (4.24).
One way ANOVA showed that the serum calcium and phosphorous levels were not statistically significant between the control , single and double hPRP groups. The calcium level was lower in the hPRP groups than control group. The phosphorous level was lower in the single hPRP group than double hPRP group.
The total alkaline phosphatase level was higher in the hPRP treated groups than control group without any significant difference among the three groups.The bone-specific alkaline phosphatase and osteocalcin levels were significantly higher in the double hPRP group than the single hPRP and control groups without any significant difference between the two latter groups(Table 4.25).
Table (4.24): Mean (??standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control and hPRP treated groups after two weeks of retention
Group No Calcium
(nm) Phosphorous(nm) TALP
(nm) Bone ALP
U/L Osteocalcin
ng/L
Control 5 3.17 a
??0.40 1.53 a
??0.07 130.20 a
?? 12.36 14.36 b
??1.76 130.88 b
?? 12.31
Single hPRP 5 2.66 a
??0.29 1.29 a
??0.14 143.80 a
??13.31 15.10 b
??2.05 144.85 b
??16.74
Double hPRP 5 2.93 a
??0.48 1.54 a
??0.56 147.51 a
??10.96 21.91 a
?? 2.92 184.34 a
??25.09
Means which share the same letters vertically are not significantly different (Duncan test)
Table (4.25): One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control and hPRP treated groups after two weeks of retention
Sum of Square df Mean Square F P-value
Calcium between groups .650 2 .325 2.063
.170'

within groups 1.892 12 .158
Total 2.542 14
Phosphoros between groups .197 2 .099 .877
.441'

within groups 1.349 12 .112
Total 1.546 14
TALP
between groups 830.376 2 415.188 2.768
.103'

within groups 1800.057 12 150.005
Total 2630.433 14
Bone ALP between groups 173.447 2 86.724 16.417
.000'

within groups 63.391 12 5.283
Total 236.838 14
Osteocalcin
between groups 7687.562 2 3843.781 10.862
.002'

within groups 4246.600 12 353.883
Total 11934.162 14
'significant difference existed at P ' 0.05
4.1.5.2 Serum Markers of Bone Formation After Four Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin after four weeks of retention are shown in table (4.26).
One way ANOVA showed that there was no significant difference in serum calcium and phosphorous levels between the control group and hPRP treated groups. Their values were lower in the single and double hPRP treated groups than the control group.
The serum total alkaline phosphatase level showed no significant difference between the control group and hPRP treated groups. The single hPRP and double hPRP groups demonstrated higher value than the control group.
The serum bone specific alkaline phosphatase level was significantly higher in the double hPRP group than the control group. Although, the single hPRP group showed a higher value of bone alkaline phosphatase than the control group, the difference was not significant.
The serum osteocalcin level was higher in both hPRP groups than control group with no significant difference(Table 4.27).The double hPRP group demonstrated a higher value than the single hPRP group.
Table (4.26): Mean (??standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control group and hPRP treated groups after four weeks of retention
Group No. Calcium (nm) Phosphoros (nm) ALP
(nm) Bone ALP
U/L Osteocalcin (ng/L)
Control 5 2.83 a
??0.30 1.42 a
??0.11 132.60 a
??8.91 13.84 b
?? 2.09 116.42 a
??21.33
Single hPRP 5 2.73 a
??0.35 1.23 a
??0.15 140.37 a
?? 11.41 15.70 ab
??1.70 123.51 a
?? 31.43
Double hPRP 5 2.68 a
??0.41 1.38 a
??0.23 138.04 a ??17.29 18.14 a
??2.05 135.48 a
?? 25.06
Means which share the same letters vertically are not significantly different (Duncan test)
Sum of Square df Mean Square F P-value
Calcium between groups .058 2 .029 0.230
.798'

within groups 1.519 12 .127
Total 1.578 14
Phosphorous between groups .094 2 .047 1.581
.246'

within groups .358 12 .030
Total .453 14
TALP
between groups 157.939 2 78.969 0.466
.638'

within groups 2034.129 12 169.511
Total 2192.068 14
Bone ALP
between groups 46.638 2 23.319 6.061
.015'

within groups 46.169 12 3.847
Total 92.806 14
Osteocalcin
between groups 928.019 2 464.010 0.672
.529'

within groups 8285.186 12 690.432
Total 9213.206 14
Table (4.27): One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control group and hPRP treated groups after four weeks of retention
'significant difference existed at P ' 0.05

4.1.5.3 Serum Markers of Bone Formation after Six Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin after six weeks of retention are shown in table (4.28).
One way ANOVA showed that there was no significant difference in the serum calcium and phosphorous levels between the control group and hPRP treated groups.
Similarly, no significant difference in total alkaline phosphatase, bone-specific alkaline phosphatase, and osteocalcin levels between the control and hPRP treated groups(Table 29). However, their values are higher in the double hPRP group than the single hPRP group.

Table (4.28) : Mean (??standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control group and hPRP treated groups after six weeks of retention
Group No. Calcium
(nm) Phosphorous(nm) ALP
(nm) Bone ALP
U/L Osteocalcin
ng/L
control 5 2.56 a
??0.47 1.20 a
??0.24 128.20 a
??17.88 9.87 a
??1.16 79.46 a
??15.18
Single hPRP 5 2.61 a
??0.44 1.30 a
??0.17 131.80 a
?? 9.28 9.91 a
??1.30 92.94 a
??17.55
Double hPRP 5 2.70 a
??0.40 1.26 a
??0.28 144.60 a
??19.01 11.04 a
?? 2.65 101.01 a
??15.36
Means which share the same letters vertically are not significantly different (Duncan test)

Table (4.29): One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control group and hPRP treated groups after six weeks of retention
Sum of Square df Mean Square F P-value
Calcium between groups .048 2 .024 .123
.885'

within groups 2.314 12 .193
Total 2.362 14
Phosphorous between groups .022 2 .011 .203
.819'

within groups .659 12 .055
Total .682 14
TALP
between groups 930.200 3 310.067 1.261
.32'

within groups 3935.600 16 245.975
Total 4865.800 19
Bone ALP
between groups 4.408 2 2.204 .656
.536'

within groups 40.299 12 3.358
Total 44.707 14
Osteocalcin
between groups 1185.646 2 592.823 2.297
.143'

within groups 3097.447 12 258.121
Total 4283.092 14
'significant difference existed at P ' 0.05

The means serum bone-specific alkaline phosphatase of the control and hPRP treated groups after two, four and six weeks of retention are shown in figure (4.16). The level of serum osteocalcin of the control and hPRP treated groups after two, four and six weeks of retention is shown in figure (4.17).


Figure (4.16): Serum bone-specific alkaline phosphatase in the control and hPRP treated groups after two, four and six weeks of retention


Figure (4.17): Serum osteocalcin in the control and hPRP treated groups after two, four and six weeks of retention

4.2 Simvastatin Study
4.2.1 Amount of Sagittal Suture Expansion
The mean and standard deviation for the amount of sagittal suture expansion in the simvastatin study are presented in table (4.30).One way ANOVA showed that there was no significant difference in the amount of suture expansion among the control , methylcellulose gel , 0.5mg simvastatin , and 1.0 mg simvastatin groups(Table 4.31). The mean of sagittal suture expansion of the four groups was 3.15mm (??0.62 mm).

Table (4.30): Mean and standard deviation for amount of sagittal suture expansion (mm) in simvastatin study
Amount of sagittal suture expansion(mm)
Group No. Mean St.D.
Control 20 3.05a 0.42
Methylcellulose gel 20 3.33 a 0.39
0.5mg simvastatin 20 3.00 a 0.79
0.1mg simvastatin 20 3.20 a 0.74
Total 80 3.15 0.62
Means which share the same letters are not significantly different (Duncan test)
Table (4.31): One way ANOVA of amount of sagittal suture expansion (mm) in simvastatin study
P-value F Mean Square df Sum of Square
0.859* 0.251 0.111
3 0.333
between groups

0.441 16
7.061
within groups
19 7.394 Total

*significant difference existed at P ' 0.05
4.2.2 Relapse Ratio of the Expanded Sagittal Suture
The Mean and standard deviation of relapse ratios of the expanded sagittal suture in the control, methylcellulose carrier gel, simvastatin gel 0.5 mg and 1.0 mg groups are presented in table (4.32).
One way ANOVA showed that the relapse ratios of the simvastatin gel treated groups (both 0.5mg and 1.0 mg) were significantly less than the control and methylcellulose carrier gel groups(Table 4.33). In the 1.0 mg simvastatin gel group, the relapse ratio was less than the 0.5mg simvastatin gel group, but the difference was not significant.

Table (4.32): Mean and standard deviation of relapse ratios of the expanded sagittal suture in the control, methylcellulose carrier gel, 0.5 mg and 1.0 mg simvastatin gel treated groups
Relapse Ratio (%)
Groups No. Mean St.D.
Control 5 23.66a 7.27
Methylcellulose gel 5 22.11a 3.98
Simvastatin gel (0.5mg) 5 13.40b 4.04
Simvastatin gel (1.0mg) 5 8.53 b 4.43
Means which share the same letters are not significantly different (Duncan test)
Table (4.33): One way ANOVA of relapse ratios of the expanded sagittal suture in the control, methylcellulose carrier gel, 0.5 mg and 1.0 mg simvastatin gel treated groups

P-value F Mean Square df Sum of Square
*0.001 9.874 258.582 3 775.747 between groups

26.188 16 419.005 within groups
19 1194.752 Total

*significant difference existed at P ' 0.05

4.2.3 Radiographic Density of the Expanded Sagittal Suture

4.2.3.1 Density of the Sagittal Suture After Two Weeks of Retention
Table (4.34) shows the mean and standard deviation of density of expanded sagittal suture in the control, methylcellulose carrier gel, 0.5 mg and 1.0 mg simvastatin gel groups after two weeks of retention. One way ANOVA showed that the density of the sagittal suture was significantly higher in the 0.5 mg and 1.0 mg simvastatin gel than the control groups. No significant difference was noted between 0.5 mg and 1.0 mg simvastatin gel groups (Table 4.35).
Table(4.34): Mean and standard deviation of radiographic density of sagittal suture in the control, methylcellulose, 0.5mg and 1.0mg simvastatin gel treated groups after two weeks of retention
Radiographic density (pixel)
Group No. Mean St.D.
Control 5 38.16 b 3.76
Methylcellulose gel 5 36.50 b 5.18
Simvastatin gel (0.5mg) 5 43.95 a 2.63
Simvastatin gel (1.0mg) 5 46.25 a 1.87
Means which share the same letters are not significantly different (Duncan test)

Table(4.35): One way ANOVA of radiographic density of sagittal suture in the control, methylcellulose, and 0.5mg and 1.0mg simvastatin gel treated groups after two weeks of retention
P-value F Mean Square df Sum of Square
.001' 8.365 107.322 3 321.967 between groups

12.830 16 205.283 within groups
19 527.251 Total

*significant difference existed at P ' 0.05

4.2.3.2 Density of the sagittal suture after four weeks of retention
Table (4.36) shows the mean and standard deviation of the density of expanded sagittal suture in the control, methylcellulose carrier gel, 0.5 mg and 1.0 mg simvastatin gel groups after four weeks of retention. One way ANOVA showed that the density of the sagittal suture was significantly higher in 1.0 mg simvastatin gel group than the control and methylcellulose carrier gel groups. No significant difference was noted between 0.5 mg and 1.0 mg simvastatin gel groups (Table 4.37).

Table (4.36): Mean and standard deviation of radiographic density of sagittal suture in the control, methylcellulose, 0.5 mg and 1.0 mg simvastatin gel treated groups after four weeks of retention
Radiographic density (pixel)
Group No. Mean St.D.
Control 5 42.55 b 3.25
Methylcellulose gel 5 42.18 b 4.29
Simvastatin gel (0.5mg) 5 47.12 ab 3.32
Simvastatin gel (1.0mg) 5 50.04 a 3.47
Means which share the same letters are not significantly different (Duncan test)
Table (4.37): One way ANOVA of radiographic density of sagittal suture in the control, methylcellulose, 0.5 mg and 1.0 mg simvastatin gel treated groups after four weeks of retention
P-value F Mean Square df Sum of Square
009'.0 5.505 71.627 3 214.881 between groups

13.010 16 208.164 within groups
19 423.045 Total

*significant difference existed at P ' 0.05

4.2.3.3 Density of the sagittal suture after six weeks of retention
Table (4.38) shows the mean and standard deviation of density of expanded sagittal suture in the control, methylcellulose carrier gel, 0.5 mg and 1.0 mg simvastatin gel groups after six weeks of retention. One way ANOVA showed that the density of the sagittal suture was significantly higher in the 0.5 mg and 1.0 mg simvastatin gel than the control and methylcellulose carrier gel groups. Although, the density in the 1.0 mg simvastatin gel group is higher than the 0.5 mg simvastatin gel group , this difference was not significant(Table 4.39).

Table (4.38): Mean and standard deviation of radiographic density of the sagittal suture in the control, methylcellulose, 0.5 mg and 1.0 mg simvastatin gel treated groups after six weeks of retention
Radiographic density (Pixel)
Group No. Mean St.D.
Control 5 44.51 b 2.85
Methylcellulose gel 5 45.55 b 2.62
Simvastatin gel (0.5mg) 5 51.17 a 2.40
Simvastatin gel (1.0mg) 5 54.46 a 2.94
Means which share the same letters are not significantly different (Duncan test)
Table (4.39): One way ANOVA of radiographic density of the sagittal suture in the control, methylcellulose, 0.5 mg and 1.0 mg simvastatin gel treated groups after six weeks of retention
P-value F Mean Square df Sum of Square
.000' 15.092 110.867 3 332.601 between groups

7.346 16 117.535 within groups
19 450.136 Total

*significant difference existed at P ' 0.05

Figure (4.18) shows the density of expanded sagittal suture in the control , methylcellulose carrier gel , 0.5 mg and 1.0 mg simvastatin gel groups after two, four and six weeks of retention.

Figure (4.18):Radiographic density of sagittal suture in the control, methylcellulose , 0.5 mg and 1.0 mg simvastatin gel groups after two, four and six weeks of retention

4.2.4 Histological and Histomorphometric Study
4.2.4.1 Histological Study
4.2.4.1.1 Histological Features of Sagittal Suture after Two Weeks of Retention

The histological appearance of expanded sagittal suture from control group that was treated with methylcellulose gel alone showed the presence of haphazardly arranged collagen fibers between sutural margins. The fibroblasts cells, and newly-formed vascular channels were seen throughout the expanded gap. There were various numbers of osteocytes and osteoblasts present along the sutural edges (Fig. 4.19).
Histological examination of sutural specimens from experimental group which was treated with 0.5mg of simvastatin, revealed the presence of thick fibrous tissue consisting of few fibroblasts, newly- formed blood channels and dense bundles of collagen fibers joining the two edges of the expanded suture. Among the fibrillar matrix collagen in the middle area of the expanded suture, there were islands of osteoid-like tissue which were surrounded with osteoblasts. There were some longitudinal new bone trabeculae extended from both sutural edges as finger-like projections. (Fig.4.20).
The histological study of sagittal suture specimens from experimental group treated with 1.0 mg of simvastatin showed the presence of fair quantities of mature fibrous tissue joining the tips of bones from either side of the suture which was rich in fibrillar matrix collagen, blood channels and fibrocytes with few number of fibroblasts.In addition, there were some bone islands made of osteoid present among the fibrous tissue. The finger like projections of newly formed bone emerged from both edges of expanded suture were clearly seen and gradually decreasing the expanded suture gap (Fig. 4.21).






4.2.4.1.2 Histological Features of Sagittal Suture after Four Weeks of Retention
The histological appearance of sagittal suture from control group that was treated with methylcellulose gel showed the presence of dense fibrous tissue consisting of fibrocytes, blood channels and collagen fibers intermingled with bone growths from both sides of suture line. There were some osteoid masses near the old bone suture edges with active osteoblasts seen along the sutural margin (Fig.4.22).
The histological study of sutural specimens from experimental group that was treated with 0.5mg simvastatin revealed the presence of dense fibrous tissue joining both edges of suture line together with large number of bony papillary projections across the suture line, reducing the overall expanded suture space with the presence of small and large sized mass of new bone through the sutural tissue (Fig.4.23 ).
Microscopical examination of sagittal suture specimens from group treated with 1.0 mg simvastatin gel showed a small amount of a dense mature fibrous tissue mainly made up of collagen fibers plus few number of fibrocytes joining both sides of sutural margin with the presence of various numbers of cavities. The primary bony growths became more mature by gradual increase in mineralization of the osteoid. The growths were large and elongated from the two edges of native bone margins. These longitudinal new growths of bones seemed as if they were trying to bridge the expanded gap and decreasing the space between old bone margins (Fig.4.24).

4.2.4.1.3 Histological Features of Sagittal Suture after Six Weeks of Retention
The histological examination of sagittal suture specimens from experimental group that was treated with methylcellulose gel showed
fibrous tissue joining the two edges of suture and it was more abundant but looser than that of the control group and blood channels were more numerous. The finger-like bony projections were close to each other and separated by well vascularized fibrous tissue. Islands of new bones were seen (Fig.4.25).
The histological examination of sagittal suture specimens from experimental group that was treated with 0.5 mg simvastatin showed that the two edges of the suture were joined by bone growths projecting from either sides of suture plus fibro-osseous tissue and the suture was more compact than the control group. Some cavities are present within fibrous tissue. Osteoblasts were seen particularly in close vicinity of the upper and lower edges of the suture (Fig. 4.26).
The histological examination of sagittal suture specimens from experimental group that was treated with 1.0 mg simvastatin gel demonstrated that the suture line was cemented by thick mature fibrous tissue. The majority of this fibrous tissue showed ossification and there was intermingling of the osseous projections from either sides of suture. The ossified fibrous tissue was greater in quantity than that seen in the 0.5 mg simvastatin treated group. A large quantity of osseous tissue was seen connecting between the two edges of the suture. The suture space is no more evident and was completely occupied by osseous tissue resulting from the union of bone growths from both suture edges (Fig. 4.27).




4.2.4.2 Histomorphometric Study
4.2.4.2.1 Histomorphometric Parameters of the Expanded Sagittal Suture after Two Weeks of Retention
The mean and standard deviation for the numbers of osteoblasts and blood vessels are shown in table (4.40).
At two weeks, one way ANOVA showed that the number of osteoblasts is significantly higher in the 0.5mg and 1.0 mg simvastatin treated groups than control and methylcellulose carrier gel groups. The 1.0 mg simvastatin group showed a significantly higher number of osteoblasts than in the 0.5 mg simvastatin group.
The number of blood vessels is significantly higher in the 0.5 mg and 1.0 mg simvastatin groups than control and methylcellulose carrier gel groups.The 1.0mg simvastatin group showed a significantly higher number of blood vessels than the 0.5mg simvastatin group(Table 4.41).
The mean and standard deviation of osteoid and total areas of the expanded sagittal suture are shown in table (4.42).
One way ANOVA showed that the total suture area was significantly less in the 0.5 mg and 1.0 mg simvastatin treated groups than control and methylcellulose carrier gel group.The 1.0 mg simvastatin group showed a significantly less total suture area than 0.5 mg simvastatin group. The osteoid area was more in the 1.0 mg than 0.5 mg simvastatin group, but the difference was not significant. No osteoid area was noted in the control group and methylcellulose carrier gel groups. The percentage of O.Ar/T.Ar was significantly higher in the 1.0 mg simvastatin group than 0.5 mg simvastatin group (Table 4.43).

Table (4.40): Mean (?? standard deviation) of the number of osteoblasts and blood vessels in the control, methylcellulose and simvastatin groups after two weeks of retention
Parameters
(Numbers) Control Methylcellulose gel
Simvastatin gel (0.5mg) Simvastatin gel (1mg)
Osteoblasts 44.80 c ??6.72 38.80 c ??12.05 69.60 b ??19.57 89.20 a ??15.72
Blood vessels 8.00 c ??1.58 8.40 c ?? 1.14 11.60 b ?? 1.29 15.40 a ?? 1.95
Means with the same letters horizontally are not significantly different (Duncan test)
Table (4.41): One way ANOVA of the number of osteoblasts and blood vessels in the control, methylcellulose and simvastatin groups after two weeks of retention
P-value F Mean Square df Sum of Square
*0.000 13.196 2706.400 3 8119.200 between groups Osteoblast

205.100 16 8281.600 within groups

19 11400.800 Total
*0.000 26.509 58.983 3 176.950 between groups Blood vessels

2.225 16 35.600 within groups
19 212.550 Total
*significant difference existed at P ' 0.05

Table (4.42): Mean (?? standard deviation) of osteoid and total areas of the expanded sagittal suture in control, methylcellulose and simvastatin groups after two weeks of retention
Parameters
Area (mm2) Control Methylcellulose gel
Simvastatin
gel (0.5mg) Simvastatin gel (1.0 mg)
Osteoid area Nil Nil 0.0394a ??0.0094 0.0466 a
??0.0155
Total area 0.6180 a
?? 0.04111
0.5819 a
?? 0.1033
0.4778b ??0.0320
0.3741c
??0.0939
Percentage of osteoid area/Total area Nil Nil 8.4b
??2.3 12.2 a
??2.5
Means with the same letters horizontally are not significantly different (Duncan test)

Table (4.43): One way ANOVA of osteoid and total areas of the expanded sagittal suture in control, methylcellulose and simvastatin groups after two weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.009 3 0.003 38.039 0.000*
within groups
0.001 16 0.000
Total 0.011 19
Total area between groups 0.181 3 0.060 10.884 0.000*
within groups
0.089 16 0.006
Total 0.270 19
Percentage of osteoid area/Total area between groups 566.550 3 188.850 62.950 0.000*
within groups
48.000 16 3.000
Total 614.550 19
*significant difference existed at P ' 0.05

4.2.4.2.2 Histomorphometric Parameters of the Expanded Sagittal Suture After Four Weeks of Retention
The mean and standard deviation of the numbers of osteoblasts and blood vessels in the sagittal suture at four weeks of retention are shown in table (4.44).
One way ANOVA showed that the number of osteoblasts at four weeks was significantly higher in the 0.5 mg and 1.0 mg simvastatin groups than the methylcellulose carrier gel and control groups.No significant difference was noted between 0.5 mg and 1.0 mg simvastatin groups. No significant difference was noted in the number of blood vessels among the four groups (Table 4.45).
The mean and standard deviation of total suture area and osteoid area in the sagittal suture at four weeks of retention are shown in table (4.46).
The total suture area decreased in the four groups at four weeks, as compared to two weeks. At four weeks, one way ANOVA showed that the total suture area was significantly less in the 1.0 mg simvastatin group than both control groups. Meanwhile,the 0.5 mg simvastatin group showed a significantly less T.Ar than methylcellulose carrier gel group with no significant difference with control and 1.0 mg simvastatin groups. The osteoid area start to appear at four weeks in both control and mehylcellulose carrier gel group. The 0.5 mg and 1.0 mg simvastatin groups showed a significantly larger O.Ar. than the control and methylcellulose carrier gel group, with no significant difference between the 0.5 mg and 1.0 mg simvastatin groups. The percentage of O.Ar/T.Ar was significantly more in the o.5 mg and 1.0 mg simvastatin groups than the control and methylcellulose carrier gel groups, with no significant difference between the 0.5 mg and 1.0 mg simvastatin groups(Table 4.47).
Table (4.44): Mean(?? standard deviation) of numbers of osteoblasts and blood vessels in control, methylcellulose, and simvastatin groups after four weeks of retention
Parameters
(Numbers) Control Methylcellulose gel
Simvastatin gel(0.5mg) Simvastatin gel(1mg)
Osteoblasts 46.4 b ?? 6.39 44.4 b ??7.50 74.6 a ??17.27 81.40 a ??15.39
Blood vessels 5.0 a ?? 1.58 4.0 a?? 1.41 4.6 a?? 0.89 5.00 a ?? 0.71
Means with the same letters horizontally are not statistically significant(Duncan test)

Table (4.45): One way ANOVA of numbers of osteoblasts and blood vessels in control, methylcellulose, and simvastatin groups after four weeks of retention
P-value F Mean Square df Sum of Square
*0.000 11.472 1813.133 3 5439.400 between groups Osteoblast

158.050 16 2528.800 within groups

19 7968.200 Total
*0.527 0.770 1.117 3 3.350 between groups Blood vessels

1.450 16 23.200 within groups
19 26.550 Total

*significant difference existed at P ' 0.05

Table (4.46): Mean (?? standard deviation) of osteoid and total areas of the expanded sagittal suture in control, methylcellulose and simvastatin groups after four weeks of retention
Parameters
Area(mm2) Control Methylcellulose gel
Simvastatin gel (0.5mg) Simvastatin gel(1mg)
Osteoid area 0.0263 b
??0.0129 0.0129 b
??0.0056 0.0747 a
?? 0.0546 0.0747 a
?? 0.0184
Total area 0.2773 ab
??0.0467 0.2890 a
??0.0572
0.2131 bc
?? 0.0594 0.1987 c
?? 0.0409
Percentage of osteoid area/ Total area 9.4 b
??4.5 8.6 b
??2.9 38.4 a
??18.7 47.2 a
??8.3
Means with the same letters horizontally are not significantly different (Duncan test)
Table (4.47): One way ANOVA of osteoid and total areas of the expanded sagittal suture in control, methylcellulose and simvastatin groups after four weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.018 3 0.006 20.154 0.000*
within groups
0.005 16 0.000
Total 0.023 19
Total area between groups 0.031 3 0.010 3.834 0.030*
within groups
0.043 16 0.003
Total 0.073 19
Percentage of osteoid area/ Total area between groups 5907.400 3 1969.133 17.500 0.000*
within groups
1800.400 16 112.525
Total 7707.800 19

*significant difference existed at P ' 0.05
4.2.4.2.3 Histomorphometric Parameters of the Expanded Sagittal Suture after Six Weeks of Retention
The mean and standard deviation of the numbers of osteoblasts and blood vessels in the sagittal suture after six weeks of retention are shown in table (4.48).
The number of osteoblasts at six weeks was reduced in the simvastatin groups and increased in both control groups as compared to those at four weeks. At six weeks, one way ANOVA showed that there was no significant difference in the number of osteoblasts among the four groups. In the four groups, the number of blood vessels decreased at six weeks, as compared to that in four weeks,with no significant difference was noted between 0.5 mg and 1.0 mg simvastatin groups. The number of blood vessels at six weeks was significantly higher in 1.0 mg simvastatin groups than the methylcellulose carrier gel and control groups with no significant difference being noted between 0.5 mg simvastatin and both control groups(Table 4.49).
The mean and standard deviation of the osteoid and total areas of the expanded sagittal suture after six weeks of retention are shown in table (4.50).
The T.Ar at six weeks was reduced in the four groups as compared to that at four weeks. One way ANOVA showed that the T.Ar was significantly less in the 1.0 mg simvastatin groups than the control and methylcellulose carrier gel groups. The T.Ar was less in the 0.5 mg simvastatin group than the control group, but the difference was not significant. Although, the T.Ar was less in the 1.0 mg simvastatin gel group than 0.5 mg simvastatin group, but the difference was not significant.
At six weeks, the osteoid area was significantly higher in the 0.5 mg simvastatin groups than the two control groups. The osteoid area was significantly higher in the 1.0 mg simvastatin group than passive control group with no significant difference with the 0.5 mg simvastatin and methylcellulose gel groups. The percentage of O.Ar/T.Ar in the 1.0 mg simvastatin group was significantly higher than that of both control groups. No significant difference was noted in the O.Ar/T.Ar between 0.5 mg and 1.0 mg simvastatin groups(Table 4.51).

Table (4.48): Mean (??standard deviation) of numbers of osteoblasts and blood vessels in the control, methylcellulose and simvastatin groups after six weeks retention
Parameters
(Numbers) Control Methylcellulose gel
Simvastatin gel (0.5mg) Simvastatin gel(1mg)
Osteoblast 48.8 a ?? 21.1
47.2 a ??11.82 39.4 a ??10.45 34 a ?? 7.38
Blood vessels 3.0 b ?? 0.71 2.8 b?? 0.84 4.0 ab ??1.0 4.6 a ?? 1.57
Means with the same letters horizontally are not statistically significant(Duncan test)
P-value F Mean Square df Sum of Square
*0.161 1.956 239.250 3 717.750 between groups Osteoblast

122.300 16 1956.800 within groups

19 2674.550 Total
*0.052 3.200 3.600 3 10.800 between groups Blood vessels

1.125 16 18.000 within groups
19 28.800 Total
Table (4.49): One way ANOVA of numbers of osteoblasts and blood vessels in the control, methylcellulose and simvastatin groups after six weeks retention

*significant difference existed at P ' 0.05

Parameters
Area(mm2) Control
Methylcellulose gel
Simvastatin gel (0.5mg)
Simvastatin gel(1mg)

Osteoid area 0.0583 c ??0.0204 0.0745cb
?? 0.0173 0.0975a
?? 0.0104 0.0888 ba
??0.0080
Total area 0.2453a ??0.0058 0.2476 a
?? 0.0487
0.2088ab
?? 0.0231 0.1827 b
?? 0.0449
Percentage of Osteoid area/ Total area 23.8 c ??8.3 31.2 bc ?? 9.2 47.0 ab ?? 6.1 53.0 a ?? 21.7
Table (4.50): Mean (??standard deviation)of osteoid and total areas of the expanded sagittal suture in the control, methylcellulose and simvastatin groups after six weeks of retention
Means with the same letters horizontally are not significantly different (Duncan test)

Table (4.51): One way ANOVA of osteoid and total areas of the expanded sagittal suture in the control, methylcellulose and simvastatin groups after six weeks of retention
Sum of Square df Mean Square F P-value
Osteoid area between groups 0.004 3 0.001 6.605 0.004'
within groups
0.004 16 0.000
Total 0.008 19
Total area between groups 0.015 3 0.005 3.913 0.029'
within groups
0.020 16 0.001
Total 0.034 19
Percentage of osteoid area/ Total area between groups 2758.150 3 919.383 5.539 0.008'
within groups
2655.600 16 165.975
Total 5413.750 19

*significant difference existed at P ' 0.05

Figure (4.28) shows the number of osteoblasts and figure ( 4.29) shows the number of blood vessels in the expanded sagittal suture of the control , methylcellulose and simvastatin groups after two, four and six weeks of retention. Figure (4.30) shows osteoid area and figure (4.31) shows the total area in the expanded sagittal suture of the control, methylcellulose and simvastatin groups after two, four and six weeks of retention.


Figure ( 4.28) : Number of osteoblasts in the expanded sagittal suture of the control, methylcellulose and simvastatin groups after two, four and six week of retention


Figure (4.29) :Number of blood vessels in the expanded sagittal suture of control, methylcellulose and simvastatin groups after two, four and six week of retention

Figure (4.30) :Osteoid area in the expanded sagittal suture of control, methylcellulose, and simvastatin groups after two, four and six week of retention

Figure (4.31):Total area in the expanded sagittal suture of control, methylcellulose and simvastatin groups after two, four and six week of retention

4.2.5 Serum Markers of Bone Formation in Simvastatin Study
4.2.5.1 Serum Markers after Two Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin after two weeks of retention are shown in table (4.52).
The serum calcium level was lower in the 0.5 mg simvastatin and 1.0 mg simvastatin groups than the control and methylcellulose gel groups. One way ANOVA showed that there was no significant difference in the serum calcium level among the four groups.
The serum phosphorous level was lower in the methylcellulose group and 1.0 mg simvastatin group than the control and 0.5 mg simvastatin groups. There was no significant difference in the serum phosphorous level among the four groups.
The serum total alkaline phosphatase level was higher in the 0.5 mg and 1.0 mg simvastatin treated groups than the control and methylcellulose groups with no significant difference being noted among the four groups.
The serum bone-specific alkaline phosphatase level was significantly higher in the 1.0 mg simvastatin group than the control and methylcellulose groups. Although, the bone alkaline phosphatase was higher in the 1.0 mg simvastatin group than 0.5 mg simvastatin group, the difference between them was not significant. Also, BALP was higher in the 0.5 mg simvastatin group than both control groups but, the difference between them was not significant
Morever, one way ANOVA showed that the serum osteocalcin level was significantly higher in the 0.5 mg and 1.0 mg simvastatin treated groups than the control and methylcellulose groups .Although, the osteocalcin level in the 1.0 mg simvastatin group was higher than the 0.5 mg simvastatin group, but, the difference between them was not significant(Table 4.53).
Table (4.52): Mean(??standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after two weeks of retention
Group No Calcium
(nm) Phosphorous
(nm) TALP
(nm) Bone ALP (U/L) Osteocalcin
(ng/L)
Control 5 3.17 a
?? 0.40 1.53 a
??0.07
130.20 a
?? 12.36 14.36 a
?? 1.76 130.88 a
?? 12.31
Methyl-
cellulose gel 5 3.60 a
?? 0.27 1.33 a
??0.07
137.00 a
?? 17.36 14.22 a
?? 2.38 135.08 a
?? 21.06
Simvastatin
gel (0.5mg) 5 3.04 a
?? 0.50 1.55 a
??0.30
147.00 a
?? 12.43 17.16 ab
?? 2.15 166.87 b
?? 23.87
Simvastatin
gel (1.0 mg) 5 3.03 a
?? 0.32 1.32 a
?? 0.22 142.00 a
?? 17.93 19.36 b
??2.76 191.06 b
??27.04
Means which share the same letters vertically are not significantly different (Duncan test)
Table (4.53): One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after two weeks of retention
Sum of Square df Mean Square F P-value
Calcium between groups .131 3 .044 1.040
.402'

within groups .671 16 .042
Total .801 19
Phosphorous between groups .609 3 .203 1.763
.195'

within groups 1.843 16 .115
Total 2.453 19
TALP
between groups 772.150 3 257.383 1.107
.375'

within groups 3720.800 16 232.550
Total 4492.950 19
Bone ALP
between groups 91.150 3 30.383 5.797
.007'

within groups 83.859 16 5.241
Total 175.009 19
Osteocalcin
between groups 12080.111 3 4026.704 8.495
.001'

within groups 7583.938 16 473.996
Total 19664.048 19
'significant difference existed at P ' 0.05

4.2.5.2 Serum Markers after Four Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin after four weeks of retention are shown in table (4.54).
The serum calcium level was lower in the 1.0 mg simvastatin group and methylcellulose than the control and 0.5 mg simvastatin groups, with no significant difference being noted among the four groups by one way ANOVA test.
The serum total alkaline phosphatase was higher in the 0.5 mg and 1.0 mg simvastatin groups than the control and methylcellulose groups , with no significant difference being noted among the four groups.
The serum level of bone-specific alkaline phosphatase was higher in the 1.0mg simvastatin group than the other groups. The difference was significant between the 1.0 mg simvastatin in comparison with control and methylcellulose groups, but not with the 0.5 mg simvastatin group. Although, BALP was higher in the 0.5 mg simvastatin group than the control and methylcellulose groups, but no significant difference being noted among the three groups.
The serum osteocalcin level was higher in the simvastatin groups than the control and methylcellulose groups, but no significant difference being noted among the four groups(Table 4.55).

Table (4.54): Mean (?? standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after four weeks of retention
Group No. Calcium
(nm) Phosphorous
(nm) Total ALP
(nm) Bone-ALP
(U/L) Osteocalcin
(ng/L)
Control 5 2.83 a
?? 0.30 1.42 a
?? 0.11 132.60 a
?? 8.91 13.84 a
?? 2.09 116.42 a
?? 21.33
Methyl -
cellulose gel 5 2.71 a
?? 0.33 1.30 a
?? 0.15 129.40 a
?? 19.83 14.04 a
?? 1.26 119.42 a
?? 15.53
Simvastatingel (0.5mg) 5 2.92 a
?? 0.30 1.50 a
?? 0.36 142.20 a
?? 17.80 16.14 ab
?? 2.56 122.82 a
?? 23.97
Simvastatin gel (1.0mg) 5 2.55 a ??0.27 1.34 a
?? 0.05 145.20 a
?? 6.83 17.76 b
?? 2.11 136.40 a
?? 14.37
Means which share the same letters vertically are not significantly different (Duncan test)
Table(4.55): One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after four weeks of retention
Sum of Square df Mean Square F P-value
Calcium between groups .952 3 .317 2.183
.130'

within groups 2.326 16 .145
Total 3.278 19
Phosphorous between groups .235 3 .078 2.154
.133'

within groups .582 16 .036
Total .817 19
TALP
between groups 854.550 3 284.850 1.363
.290'

within groups 3344.000 16 209.000
Total 4198.550 19
Bone ALP
between groups 52.218 3 17.406 4.108
.024'

within groups 67.790 16 4.237
Total 120.008 19
Osteocalcin
between groups 1166.808 3 388.936 1.053
.396'

within groups 5909.599 16 369.350
Total 7076.407 19
'significant difference existed at P ' 0.05

4.2.5.3 Serum Markers after Six Weeks of Retention
The mean and standard deviation of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin after six weeks of retention are shown in table (4.56).
The serum calcium level was higher in the 0.5 mg and 1.0 mg simvastatin groups than the control and methylcellulose groups, with no significant difference being noted among the four groups by one way ANOVA test.
The serum phosphorous level showed no significant difference among the four groups and the values in the three treated groups were nearly close to the control group.
The serum total alkaline phosphatase level was higher in the simvastatin groups than the two control groups, but with no significant difference being noted among the four groups.
The serum bone-specific alkaline phosphatase and osteoclacin levels were higher in the simvastatin groups than the two control groups, but with no significant difference being noted among the four groups(Table 4.57).
Group No Calcium
(nm) Phosphorous
(nm) Total-ALP
(nm) Bone ALP
(U/L) Osteocalcin
(ng/L)
Control 5 2.56 a
?? 0.47 1.20 a
?? 0.24 128.20 a
??17.88 9.87 a
?? 1.16 79.46 a
?? 15.18
Methyl '
Cellulose gel 5 2.59 a
?? 0.31 1.32 a
?? 0.15 130.80 a
??18.14 10.67 a
?? 1.38 82.24 a
?? 14.80
Simvastatin gel (0.5mg) 5 2.97 a
?? 0.37 1.33 a
?? 0.23 135.67 a
??11.28 10.19 a
?? 2.15 85.28 a
?? 13.83
Simvastatingel (1.0mg) 5 2.87 a
?? 0.07 1.22 a
?? 0.19 146.98 a
?? 12.07 10.82 a
??1.85 97.07 a
?? 25.22
Table (4.56):Mean(??standard deviation) of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after six weeks of retention
Means which share the same letters vertically are not significantly different (Duncan test)

Table (4.57):One way ANOVA of serum calcium, phosphorous, total alkaline phosphatase, bone-specific alkaline phosphatase and osteocalcin in the control, methylcellulose and simvastatin groups after six weeks of retention
Sum of Square df Mean Square F P-value
Calcium between groups .609 3 .203 1.763
0.195'

within groups 1.843 16 .115
Total 2.453 19
Phosphorous between groups 0.388 3 .129 1.413
0.276'

within groups 1.466 16 .092
Total 1.855 19
TALP
between groups 1036.261 3 345.420 1.499
0.253'

within groups 3687.412 16 230.463
Total 4723.673 19
Bone ALP
between groups 2.855 3 .952 0.337
0.799'

within groups 45.137 16 2.821
Total 47.992 19
Osteocalcin
between groups 900.360 3 300.120 0.940
0.444'

within groups 5107.260 16 319.204
Total 6007.619 19
'significant difference existed at P ' 0.05

Figure (4.32) shows the serum level of bone-specific alkaline phosphatase and figure (4.33) shows the serum level of osteocalcin in the control, methylcellulose and simvastatin groups after two, four and six weeks of retention .

Source: Essay UK - http://www.essay.uk.com/free-essays/science/human-platelet-plasma-study.php


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