الفهرس 
Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Purpose of the study:
The principal goal of this study was to evaluate the osteoinductive efficacy of the selected nanomaterials (Au-NPs, Au/HA-NPs, C/HA-NPs, HA-NPs and C-NPs) on the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into osteoblasts. Moreover, this study was extended to elucidate the therapeutic role of osteoblasts’ infusion, generated from culturing BM-MSCs in either osteogenic medium alone or supplied with the selected nanomaterials, in alleviating the bone remodeling events in primary osteoporosis induced in the adult female rats. This was achieved through utilizing biochemical, molecular genetics, DEXA and histological techniques.
Material and Methods
The current study included 2 parts; firstly, the in vitro study including: (1) characterization of the selected nanomaterials by TEM and Zetasizer to determine the shape, size and zeta potentials, respectively. (2) Identification of the cultured BM-MSCs by morphology using inverted microscope, flow cytometry analysis of a cluster of surface markers (CD 34, CD45, CD73, CD90 and CD105) and evaluating multi-lineage differentiation capacity (adipogenesis, chondrogenesis and osteogenesis). (3) MTT assay to determine the cytotoxic effect of the selected nanomaterials with six different concentrations (2 - 25) µg/ml) on the BM-MSCs over 24h, 48h and 72h incubation periods. (4) Osteogenic differentiation of BM-MSCs by their culturing in either osteogenic medium (OS), containing 5.0 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 0.1 μM dexamethasone, alone or combined with the selected nanostructures for 14 days. (5) Gene expression analysis of osteogenesis-related genes (Runx-2 and BMP-2) and alizarin red S assay to confirm the identity of the generated osteoblasts.
Secondly, the in vivo study which was conducted on sixty-four adult female Wistar rats which were divided into eight groups; each group included eight rats as follows:
1) The first group: served as a sham group (negative control), 3 months later, this group was intravenously injected with a single dose of PBS.
2) The second group: represented the untreated ovariectomized group in which rats were subjected to ovariectomy and left for 3 months for primary osteoporosis induction. After 3 months, this group was intravenously injected with a single dose of PBS.
3) The third group: included the gold group in which ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of osteogenic medium combined with Au-NPs) and left for 3 months.
4) The fourth group: included gold/hydroxyapatite group in which the ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of the osteogenic medium combined with Au/HA-NPs) and left for 3 months.
5) The fifth group: referred to the chitosan/hydroxyapatite group in which ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of the osteogenic medium combined with C/HA-NPs) and left for 3 months.
6) The sixth group: represented hydroxyapatite group in which ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of osteogenic medium combined with HA-NPs) and left for 3 months.
7) The seventh group: included chitosan group in which ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of osteogenic medium combined with C-NPs) and left for 3 months.
8) The eighth group: included osteogenic group in which ovariectomized rats were intravenously injected with a single dose of cultured osteoblasts (3 × 106 cells/rat) suspended in PBS (cultured osteoblasts derived from BM-MSCs through the action of osteogenic medium alone) and left for 3 months.
At the end of the experimental period (6 months), SRY gene detection in the bone tissue was applied for tracking the transplanted osteoblasts to the femur bone of diseased rats. In addition, serum bone formation marker (osterix), bone turnover marker (BALP), bone resorption markers (SOST and BSP) levels were analyzed. Also, the gene expression analysis of the regulator of the bone remodeling cycle (RANKL and OPG) and osteoclastic enzyme (cathepsin K) were assessed in the bone tissue of the rat femur bones in different groups using qRT-PCR. Moreover, bone mineral density and bone mineral content were evaluated using DEXA. Furthermore, histological investigation of the bone tissue sections was carried out. Eventually, the statistical analysis of the obtained data was performed.
Results:
The obtained results could be summarized as follows:
1. In vitro results:
- TEM imaging and zetasizer analysis denoted that HA-NPs have a rod shape with size of 80–125.4 nm in length and 35–45.7 nm in width and zeta potential of −3.18 mV, Au-NPs and C-NPs have a spherical shape with a size of [(30.2–40.7), (35.8–64.3) nm], respectively and zeta potentials of −29.3 and 34.8 mV, respectively. While, TEM analysis of Au/HA and C/HA-NPs nanostructures revealed that both Au-NPs and C-NPs were successfully adsorbed on HA-NPs surface, forming nanocomposites with zeta potentials of −21.3 and −31.5 mV, respectively
- Morphological and flow cytometry characterization confirmed the identity BM-MSCs, besides their multi-potency capacity.
- MTT study revealed that all selected nanomaterials have insignificant toxicity on BM-MSCs over 24h, 48h and 72h incubation periods.
- Runx-2 displayed significant up-regulated levels in differentiated BM-MSCs cultured in OS supplemented with nanomaterials (Au-NPs and Au/HA-NPs) versus those cultured in OS alone. Similarly, BMP-2 showed significant elevated levels in differentiated BM-MSCs cultured in OS supplemented with nanomaterials (Au-NPs, Au/HA-NPs or C/HA-NPs) versus those cultured in OS alone as documented by gene expression analysis using qRT-PCR.
- Finally, alizarin red S assay demonstrated the successful osteogenic differentiation of BM-MSCs cultured in either OS alone or OS combined with the nanomaterials (Au-NPs, Au/HA-NPs, C/HA-NPs, HA-NPs, or C-NPs) for 21 days, as confirmed by the remarkable red staining of calcium nodules deposited in the extracellular matrix.
2. In vivo findings:
- The transplanted osteoblasts successfully migrated to the femur bones of the ovariectomized rats in all osteoblast-infused groups (except OS and C-NPs groups), as confirmed by SRY gene detection in the bone tissues.
- Biochemical results revealed a significant decline in the serum levels of osterix and elevation of BALP, SOST and BSP in the untreated ovariectomized group as compared with the sham group. Also, the results of gene expression analysis showed significant up-regulation in the levels of cathepsin K, RANKL and significant down-regulation in OPG gene expression level, resulting in a significant elevation in the overall ratio of RANKL/OPG in the untreated ovariectomized group versus the sham group. Moreover, DEXA analysis of the femur bones of the untreated ovariectomized group showed a significant depletion in BMD and BMC relative to the sham group. Histological investigation of the bone tissue sections of rats in the untreated ovariectomized group exhibited severe bone resorption.
- Osteoblasts intravenous infusion, resulted from BM-MSCs cultured in either OS alone or OS combined with (HA-NPs, Au-NPs, Au/HA-NPs or C/HA-NPs) in the ovariectomized groups produced a significant elevation in osterix and depletion in BALP, SOST and BSP serum levels as compared to the untreated ovariectomized group. Also, molecular genetic analysis of the bone tissue of the ovariectomized rats infused with osteoblasts, derived from culturing BM-MSCs in either OS alone or OS combined with (HA-NPs, Au-NPs, Au/HA-NPs or C/HA-NPs) displayed a significant down-regulation in the gene expression levels of cathepsin K and RANKL when compared with the untreated ovariectomized group. However, OPG gene expression level showed insignificant change in the bone tissues of all osteoblast-infused groups. This resulted in an overall significant decrease in RANKL/OPG ratio. Moreover, all osteoblast-infused groups displayed a remarkable augmentation in BMD and BMC as compared to the untreated ovariectomized rats.
- Furthermore, all osteoblast-infused groups displayed an improvement in the bone histoarchitecture as compared to the untreated ovariectomized group (except OS and C-NPs groups).