الفهرس 
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Abstract
The current investigation goaled to assess the efficacy of nanohydroxyapatite (nHA), chitosan/hydroxyapatite nanocomposites (nCh/HA) and silver/hydroxyapatite nanoparticle (nAg/HA) in reconstruction of bone in osteoporotic rat model induced by ovariectomy.
The characterisation of the manufactured nanomaterials (nHA, nCh/HA and nAg/HA) by TEM and SEM approved their nanoscale with rod-shape.
The PDI values extracted from zeta potential and size distribution measurements illustrated the present nanomaterials as highly polydisperse. Zeta potential measurements provided an indication of the predominance of nHA in the nCh/HA nanocomposite and denoted silver substitutions for calcium into the hydroxyapatite lattice.
FT-IR curves formed at the appropriate wavelength specifies that the formed nHA does not possess any impurities and proves the incorporation of chitosan or silver within nCh/HA or nAg/HA composites.
The XRD peaks matched well the data for standard hydroxyapatite phase. All in all, the general characterisation of the tested nanomaterials elucidates the absence of impurities
and establishes the incorporation of chitosan or silver within
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Summary and Conclusion
nCh/HA or nAg/HA composites with the predominance of nHA.
The biological experimentation in the present research was conducted on forty-eight adult female rats which were assigned into 6 groups (8 rats/group) as follows:
group (1): Sham-operated control group in which the rats were subjected to surgical operation without removing the ovaries.
group (2): Osteoporotic (OVX) group in which the rats were subjected to surgical operation to remove the ovaries.
group (3): nHA group in which the OVX rats were treated intravenously with nanohydroxyapatite (8 mg/kg B.wt.) once monthly for three months.
group (4): nCh/HA group in which the OVX rats were treated intravenously with chitosan/hydroxyapatite nanocomposite (8 mg/kg B.wt.) once monthly for three months.
group (5): nAg/HA group in which the OVX rats were treated intravenously with silver/hydroxyapatite nanoparticle (8 mg /kg B.wt.), once monthly for
three months.
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Summary and Conclusion
group (6): Fosamax® group in which the OVX rats treated orally with Fosamax® (1 mg /kg B.wt.) once weekly for three months.
At the end of the experimental period, all animals were fasted overnight (12 hours) and the blood specimens were withdrawn from the retro-orbital venous plexus under diethyl ether anaesthesia. The blood specimens were centrifuged to separate the sera, which were divided into aliquots of 500 μL, and frozen at -80°C pending for the biochemical analysis. After sacrificing the rats by cervical dislocation, the long bones of the hindlimb, including the femur were harvested promptly from each rat and the muscle tissue was scraped with sterile ophthalmic scissors and forceps. Some femur bones were immersed in 0.9% saline solution for measuring bone mineral density (BMD) and bone mineral content (BMC), some were fixed in formalin saline (10%) for the histopathological investigation, and others were preserved in liquid nitrogen then stored at -80°C for molecular genetics evaluations.
Sera SOST, BALP, BSP and Osx levels were estimated by ELISA. Gene expression levels of RANKL, CtsK, BMP-2 and SMAD1 were quantified by RT-PCR. The BMD and BMC levels in femur bones were measured by DEXA. Histopathological examination of femur bone tissue sections
was carried out by using optical microscope.
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Summary and Conclusion
The outcomes of this study conferred a solid foundation for the success of the investigated nanoformulations in the alleviation of osteoporosis in the experimental model. The documents of bone loss in the osteoporotic rats in the current experiment are specified via a high serum SOST, BALP and BSP concentrations and low serum Osx level. Also, the over expression of RANKL and CtsK mRNA and down-regulation of BMP-2 and SMAD1 gene expression level detected the imbalance between bone resorption and bone formation processes. Additionally, the histopathological examination of femur bone tissue sections of the osteoporotic rats implied the deterioration in bone microstructure; including the appearance of eroded bone trabeculae, active osteoclasts, and multiple areas of resorption cavitation and vacuolation within faintly stained bone matrix indicating conspicuous bone resorption.
Treatment of osteoporotic groups with nHA, nCh/HA or nAg/HA succeeded to protect against bone damage in the experimental model of osteoporosis via reducing serum SOST level and decreasing bone turnover rate which is evidenced by the decline in serum BALP and BSP levels. Moreover, these nanostructures showed their potency in suppressing osteoclastogenesis which is confirmed via the down-regulation of RANKL and CtsK gene expression levels. Furthermore, the
enhancement of serum Osx level, up-regulation of bone BMP-2
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Summary and Conclusion
and SMAD1 genes expression level as well as strengthening of BMD and BMC may represent the potential mechanisms of these nanoplatforms in counteracting osteoporosis. This could provide clues for the therapeutic effect of these nanomaterials on osteoporosis from the perspective of bone formation. The histopathological observations appreciate the biochemical, molecular and densitometry findings as they revealed a noticeable refinement of bone microarchitecture in the treated groups.
In conclusion, this study delivers a new direction and an experimental basis for the attenuation of osteoporosis as the present findings ascertained the effectiveness of nHA, nCh/HA and nAg/HA as promising nanobiomaterials for resisting bone resorption as well as rejuvenating bone regeneration. This is particularly evident for the nHA-treated group followed by nCh/HA then nAg/HA-treated groups. The superior impact of nHA could be ascribed to its nano-size and shape, which more closely mimics that of HA crystals in the natural bone.
As leading results, administration of nAg/HA revealed both; protection against accelerated bone turnover and
promotion of bone formation in osteoporotic rats.