Bone Regenerative Potentiality of Nanostructured Biphasic Hydroxyapatite / Tricalcium Phosphate Prepared from Eggshell as a Bone Graft in Vivo ISSN : 1812 – 1217

Aims: to evaluate the biological response to nano sized biphasic hydroxyapatite and tricalcium phosphate synthesized from eggshell. Materials and methods: Egg shell burned in furnace at 1000 C° for 1 hour. The part of resultant powder was treated chemically with phosphoric acid to form HA, other part of heated egg shell treated with nitric oxide and di-ammonium hydrogen phosphate to form TCP then the two products mixed mechanically at ratio 80%/20% (HA/TCP). The mixture turned to nanoparticles via mechanical attrition and sieving membrane and sent for TEM, the resultant material characterization checked by FTIR spectroscopy. Two defects of 2 mm prepared in the femoral bone of 20 rabbits one filled with prepared material (group A) and the other left empty as negative control (group B). Results: greater bone formation was founded in group A where statistically significant. Conclusions: biphasic hydroxyapatite and tricalcium phosphate nano particles synthesized from eggshell is effective in bone regeneration and could be used as bone substitute derived from source of no economic value.


INTRODUCTION
Nano material defined as materials that have at least one external dimension of 1-100 nm (1) . Major trauma and disease like osteoporosis may lead to comminuted fracture which its treatment is a challenging task.
Comminuted fracture defect is beyond the ability of bone regeneration capacity to promote healing of bone. Bone graft is used to bridge the defect to act as scaffold to provide support and promote bone healing (2) .
Autogenous bone graft is the old standard graft because it provides osteoinduction, osteoconduction and osteoprogenitors cells, but it still has some certain effects like limited graft volume, donor site morbidity and increasing time of operation (3,4) Allogenic bone graft and xenograft are still involved in the clinical use, but they are associated with many problems like possibility of cross infection and chance of immune rejection of recipient (5) . Recently, various types of synthetic bone substitutes have been introduced for clinical applications (6) .
Calcium phosphate is mostly used as synthetic bone graft (7) . Within group of calcium phosphate, hydroxyapatite (HA) and tricalcium phosphate (TCP) are mostly used as synthetic bone substitute (8) . Hydroxyapatites possess excellent biocompatibility, and they exhibit low biodegradation rate brittle nature (9) . TCP provides good osteoconductivity and possesses high biodegradation rate (Walsh et al 2008).
Therefore, biphasic calcium phosphate (BCP) has been used to take the advantages of HA and TCP providing appropriate dissolution and good bioactivity, cell attachment, proliferation, and differentiation for regeneration of bone (1) .
Nanotechnology nowadays had a great role in orthopedics because it offers a greater rivaling to nanostructure of natural bone (10) .
Nanomaterials (which are material with dimensions less than 100nm) can decrease implant infection, increase bone growth, and inhibit inflammation without addition of pharmaceutical materials (11) .

Formation of nano particles:
The resultant material changed to nano form The healing evaluated at different time interval at 3 days, 7 days, 14 days, and 28 days.

Specimen collection:
At the end of each time interval, the rabbits were sacrificed and the femoral bone was

Statistical analysis:
Computer package (SPSS version 26) was used to conduct the histomorphometrical analysis.
Data were presented as means ± SE (standard error) of mean and were analyzed using independent t test at significant level set on < 0.05.

IR spectra of nano prepared material
The product then was analyzed by FTIR spectroscopy at room temperature at region (500-4000 cm -1 ) and the product examined by transmission electron microscope. The spectrum of the synthetic nanostructured biphasic HA/TCP showed in (Figure 1).

Transmission electron microscope
The transmission electron microscope examination showed that the particles size of

Three days after surgery:
All three defects were easily detected. They appeared like round radiolucencies, the control group showed no bone formation, while the group of prepared material shows higher radioopacities as shown in (Figure 4).

Seven days after surgery:
The control group was again easily detected with slight increase in radio-opacity. The other group showed better signs of healing and greater radio-opacity. As shown in (Figures 5).       The defect was filled with compact bone with no spongy bone and the osteocyte represented the most cells as shown in ( Figure   15).

Figure (15):
Histological section of 4X magnification of prepared nano-structured material at 28 days after surgery. Arrows show the compact bone closing the defect area.

Inflammatory response:
At three days after surgery the control group showed sever inflammatory response, while the synthetic nano material showed only mild inflammatory response At one week after surgery the control group showed moderate inflammatory response, while prepared nano material showed no inflammatory response At two weeks and four weeks' period, no inflammatory response saw at the two groups.

Statistical analysis:
In all statistical analysis, small letters reflect where change in the capital letter mean there is statistically significant deference at p ≤0.05.

Statistical analysis of radiological results
The nano prepared material shows greater radioopacity as compared with control and there was statistically significant difference till the 28 days, as shown in (Table 1).

Nanostructured HA/TCP Prepared from Eggshell
Al -Rafidain Dent J Vol. 20, No2, 2020 Small letters refer to comparison within group, their changes reflect statistically significant difference.Capital letters refer to comparison between groups their change reflect statistically significant difference.

1.Statistical analysis of newly bone
Statistical analysis showed that there was an increased significant difference within each group with period. The group A which treated with nanostructured material showed greater bone formation than control group throughout the periods of study with significant difference statistically, as shown in (Table 2).

Table (2):
Show the statistical analysis of newly bone area formed, mean ± stander error.
Small letters refer to comparison within group, their changes reflect statistically significant difference. Capital letters refer to comparison between groups their change reflect statistically significant difference.

2.Statistical analysis of number of osteoblasts found:
The statistical analysis showed that there was significant difference within each group with period. At three days, one week and two weeks'  (Table 3).  Small letters refer to comparison within group, their changes reflect statistically significant difference. Capital letters refer to comparison between groups their change reflect statistically significant difference.

3.Statistical analysis of numbers of osteoclast founded
The statistical analysis showed that there was  (12,13) Preparation of bone graft with excellent biocompatibility, osteogenic capacity without immune rejection is a big challenge for researchers (14) . The prepared nano material  (19) .

CONCLUSIONS
The prepared nano structured biphasic hydroxyapatite and tricalcium phosphate synthesized from eggshell showed a good bone regeneration capacity and excellent biocompatibility with very little inflammatory response and it could be used as bone substitute material prepared from eggshell of no economic value.