الفهرس 
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Abstract
7. Summary and conclusion
This study was done to evaluate the healing process of calvarial bony defects using milled teeth bone graft material (tooth ash) versus beta tricalcium phosphate bone graft material (β-TCP).
Materials and Methods
The study was conducted on 45 adult male albino rats, weighing 150-200 g. Animals are divided into three main groups; the control group and the two experimental groups. Under general anesthesia, in both control and experimental groups an approximately 1.5 cm incision was done down to expose the cranial bone. Then two noncritical size bone defects (3 mm diameter) were created one in each parietal side of the rat calvarial bone. The calvarial defects were created primarily by dental trephine with a dental drill against the superficial aspect of the calvarium using low speed (1000 rpm) surgical drill and under copious saline irrigation.
Animals were randomly divided into three groups (n=15)
group I (control group): Consisted of 15 rats, bilateral calvarial bony defects (30 bony defects) were prepared in the parietal side of the calvarial bone and were left to heal spontaneously. Five rats were sacrificed after 7, 15 and 30 days subsequently. This group was considered as a reference for the normal bone healing where no bone graft or bone substitute was placed.
group II: Consisted of 15 rats, bilateral calvarial bony defects (30 bony defects) were prepared in the parietal side of the calvarial bone and completely filled with beta tricalcium phosphate bone graft material (β-TCP). Five rats were sacrificed after 7, 15, and 30 days postoperatively.
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group III: Consisted of 15 rats, bilateral calvarial bony defects (30 bony defects) were prepared in the parietal side of the calvarial bone and completely filled with Milled Teeth (tooth ash). Five rats were sacrificed after 7, 15, and days postoperatively.
Histological examination:
Specimens from one of the operation sites (right side) of both control and experimental groups were taken and fixed in 10% neutral buffered formalin, then decalcified in 10% EDTA solution and processed according to a standardized protocol to obtain paraffin sections that stained and analyzed with:
1. Haematoxylin and Eosin (Hx&E).
2. Masson’s trichrome stain.
3. Histomorphometric analysis
Transmission electron microscopic (TEM) examination:
The specimens from the other operation sites (left side) of both control and experimental groups were fixed in 3% phosphate buffered glutaraldehyde for 4 hours, washed in the buffer for 24 hours at 4°c then they were decalcified in 10% ethylene diamine tetra acetic acid ( EDTA) PH 7-7.3
Results:
1. Histological Results:
The histological sections stained with Haematoxylin &Eosin and Masson’s trichrome stains revealed that;
In the control group (group I); healing of the calvarial bony defects proceeded in a normal sequence of bone healing starting at day 7 and
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increased up to day 30. At day 7 the bone defect was found to be filled with granulation tissue which was replaced by bony trabeculae radiating from the periphery towards the center of the bone defect. These bony trabeculation appeared at day 15, and increased at day 30 postsurgically.
In experimental group II; healing of calvarial bony defects started at day 7 faster than its counterparts of the control group as revealed by the increased number and thickness of bony trabeculae from day 15 to day 30 also reduced size of bone marrow spaces at day 15 and the prominent appearance of their coalescence at day 30 postsurgically.
In experimental group III; the newly formed bone trabeculae appeared at day 7 and, increased in number & thickness from day 15 to day 30 postsurgically. Increased maturity of bone trabeculae at day 30 and narrowing of calvarial bony defect. The activity and maturity of the bone defect was ahead in experimental group III as compared to the other groups.
2. Transmission electron microscopic (TEM) results :
In the control group (group I): active secretory fibroblasts and osteoblasts were clearly seen at day 7. At day 15, a lot of blood capillaries containing blood cells and lined with endothelium were encountered. Different types of bone cells; osteoprogenitor cells, bone lining cells and osteoblasts were arranged on the surface of new bone around the blood capillaries. At day 30, more new bone formation around blood vessel was seen. Increase in the number of bone cells; osteoprogenitor cells, bone lining cells, osteoblasts and osteocytes entrapped in lacunae on the new bone surface was observed.
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In experimental group II: At day 7; multinucleated osteoclast cell with prominent vacuolated cytoplasm and ruffled border in Howship’s lacunae were frequently seen. Different stages of osteoblastic differentiation were clearly seen in osteoid tissue. At day 15, increase the number of active osteoblasts arranged around β-TCP material with large prominent nuclei and abundant secretory vesicles were seen. Young rounded osteocytes entrapped in lacunae and having cytoplasmic processes inside canaliculi were seen. At day 30, more new bone formation and bone cells were seen around β-TCP material which was still prominently found. Release of secretory granules by active osteoblast produced the electron dense area of the newly formed bone.
In experimental group III: At day 7, new bone formation around tooth ash was seen. Dentin and dentinal tubules were prominently found. Different types of cells appeared more active: multiple lymphocytes, fibroblast, osteoprogenitor cells, osteoblasts and blood vessels were observed. Multinucleated osteoclasts with prominent vacuolated cytoplasm and ruffled border in Howship’s lacunae were clearly seen. At day 15, an increase in new bone formation and more active bone cells were seen. Multinucleated odontoclasts with prominent vacuolated cytoplasm were located on the partially resorbed tooth ash surface. At day 30, disappearing of the tooth ash and infiltration of different types of cells; osteoprogenitor cells, bone lining cells, and osteoblasts and the presence of blood vessels containing blood cells into tooth ash spaces was outstanding feature. Mature cuboidal osteoblasts were arranged on the surface of disappearing tooth ash. Spindle shaped osteocytes entrapped in lacunae within mineralized new bone were clearly observed.
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3. Histomorphometric and Statistical Analysis
The Histomorphometric and Statistical Analysis revealed the following results:
1. The newly formed bone in the two study groups was higher than that of the control group all over the study period, moreover there was a significant increase in the amount of newly formed bone in the tooth ash group than the β-TCP group at 7, 15, 30 days postsurgically
.