الفهرس 
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Abstract
Tissue engineering offers a potential avenue to overcome the limitation related to auto-and allograft tissues and to enhance the body repair system by combining many strands (Matrix / Scaffold), cells and biologically active molecules and converting them together into functional tissues. The scaffold plays a key role in the tissue engineering field for cells to proliferate and maintain their differentiated function, and its architecture defines the ultimate shape of the new bone. Bioactive glasses are the most interesting bioceramic materials for bone defects and soft tissue treatments during the last decades. That is because of their unique ability to convert to hydroxyapatite (HA) in vivo and their strongly bonding to bone. Additionally, they have the ability to increase the proliferation and differentiation of osteoblasts.
The first bioactive glass which was discovered by Hench and cowork-ers in the late 1960’s and early 1970’s then developed thereafter silicate-based glass, such as borate- and phosphate-based compositions has opened a new research field by using glasses as implants and bone tissue engineer-ing applications. The composition of such glass was based on 45.0 SiO2 - 24.5 CaO - 24.5 Na2O - 6.0 P2O5 glass system in wt%. This glass proved to form chemical bonds with host tissue through formation of new hydroxyapatite layer.
Some shortages were discovered from synthesis of bioactive glasses by conventional melting method. As a result, there have been different methods developed to prepare nano-bioactive glass (NBG) (1-100 nm particle size), such as, sol-gel technique using of bioactive glass nanoparticles in fabrication of composite scaffolds. Based on these advantages, the development and discovering of new bioactive glass fillers for the scaffold materials are still a critical issue to optimize the properties of such scaffolds. Also, Synthesis of new nanocomposite scaffolds seeded with Mesenchymal stem cells for tissue engineering application is still under investigation and development.
The preset work was mainly designed to evaluate biocompatibility of noval composite scaffolds based on collagen and chitosan polymer blend used as a polymer matrix for nanobioactive glass doped with different ratios of CeO2 ranged from 0 to 10 mol%. Composite scaffolds collagen/chitosan reinforced by 30% weight nano-bioglass particles and encoded as Cl/CH, CL/CH/C0, CL/CH/C5 and CL/CH/C10. The characterization of the glasses and the nanocomposite scaffolds using different analytical techniques, such as DTA, TEM, SEM-EDX and FTIR. Also, the non-cellular in vitro bioactivity test was performed in simulated body fluids for periods 1, 3, 10, 20 and 30 d by measuring PH, weight loss (%), water uptake and the calcium and phosphate ion concentrations by SEM/EDX analysis. After that, all four characterized composite scaffolds were transferred to the third part of study with osteoblast cells which isolated in the second part of the present study from rabbit bone marrow mesenchymal stem cells. In this part, Rabbit bone marrow MSCs were isolated from the femurs and tibia of sex rabbits with an (average age: 6-8 weeks and average weight: 0.75-1 kg).
The animals were scarified without complication to animals in accord-ance with animal care policies, followed by initiation of the tissue culture process with isolation of mesenchymal stem cells from rabbit’s bone marrow then characterized by flow cytometric analysis. The aim of this was to choose the best isolated MSCs were cultured and expanded for increasing the possibility of isolating clonally expanded cells with higher success rates and utilizing their high potential for osteogenic differentiation. This section was subjected to isolate, culture and expansion with morphological characterization study, finally osteogenic differentiation by bioactive factors which responsible for osteogenic differentiation of osteoblast cells (Dexamethasone, β-glycerophospate ascorbic acid2-phosphate, and b-FGF). The selected factor was shown to be extremely important for proliferation and osteogensis process of BMMSCS followed by characterization of osteogenic potential through three weeks of differentiation by histological staining; like, ALP activity, Alizarin Red and von Kossa stains were done to confirm the osteogenic differentiation of mesenchymal stem cells.
Isolation of MSC-like cells was successful in all samples, and they were successfully expressed typical stem cells markers positive expression of CD90, CD146, CD73 and CD105 but negative expression for the marker CD34 by flowcytometery analysis as well as osteogenic differentiation potential, cells showed positive expression of ALP starting from day 7 of subculture as evidence of presence of preosteoblast and osteoblast cells in subculture and ALP activity has been regarded as an early marker for the osteogenic differentiation of MSCs or osteoblasts. The results of ALP were revealed a high number of ALP positive cells at day 7 followed by a progressive decline from day 14 to 21. The decline in ALP staining intensity and ALP activity corresponded to appearance of mineralization which was evident by day 14 with calcium deposition histochemical staining and increased progressively with time till day 21.Hence, calcium deposition is a marker for late stage osteogenic differentiation. In this study, Alizarin Red and von Kossa staining methods were used to confirm the presence of hydroxyapatite (HAp), which is the mineral part of the bone that consists predominantly of calcium and phosphate. Silver nitrate used in von Kossa staining is known to react specifically with phosphate deposits only, which is not enough to confirm the existence of HAp. Therefore, an additional Alizarin Red staining method was used here to detect calcium deposits since each of these staining techniques detects a different deposit and since each may have different nonspecific binding, it was possible that the two methods produce slightly different outcomes.
After that, the cells which successfully isolated and expanded in culture without degenerative change in their morphology were transferred to the third part of the study for assessment of biocompatibility of four composite scaffolds which were fabricated and characterized in the first part of this study. The biocompatibility of the four composite scaffolds CL/CH, CL/CH/C0, CL/CH/C5 and CL/CH/C10 was evaluated in vitro by observing the behavior of the first passage mineralization-induced MSCs cultured in close contact with the scaffolds around and in the vicinity of scaffolds surfaces (direct contact) and (indirect contact) using extracts of composite scaffolds. Different nanocomposite scaffolds with osteoblast cells were directly examined as explant without fixation while they were still in their culture flasks. Cell behaviors were monitored daily and MTT assay for direct contact of cells/scaffolds twice in week, trypan blue and hemocytometer cell counting for indirect contact of cells/scaffolds extracts at day 7. The results showed that at 24 h after direct contact with four composite scaffolds and indirect contact (extracts) using cell counting , all composite scaffolds and extracts showed proliferation of cells > 50% and increased in density at day 7 and cell proliferation was increased as the percentage of cerium increased in the glass incorporated into the scaffold at days 3 and 7..
Also, MTT assay revealed that none of the the tested composite scaffolds affected the viability of the cells (inhibition value <25 %), and few death of cells was observed.the inhibition was ordered from high to low as; CL/CH> CL/CH/C0 > CL/CH/C5 > CL/CH/C10. Accordingly, osteoblasts proliferation was significantly higher by incorporation of bioactive glass nanoparticles with polymer than plain polymer, which indicated that the bioactive glass had stimulating effects to promote cell proliferation rate. Hence, the biocompatibility of the scaffold was increased as the percentage of CeO2 in bioactive glass nanoparticles increased.These two combined analyses give an overview for direct correlation between toxicity, cell death, reduced cell proliferation, altered morphology. Also, the bioactivity of expanded and differentiated osteoblast cells was studied by histochemical staining with Alizarin Red and von Kossa to confirm the osteogenic differentiation process besides the anticancer activity of the scaffolds was studied against cancer osteosarcoma cell lines using Sulforhodamine B (SRB) assay.
In the present study, we observed different behavior between two types of cells osteoblast cells (normal cells) and osteosarcoma cells (cancer cells).The effect of high concentration level of CeO2 on osteoblast cells protected cells from oxidative stress and enhance proliferation and osteogenic induction property more than low concentration level. This means that an excellent bioactivity of scaffolds was showed with the highest CeO2 content (CL/CH/C10) then (CL/CH/C5) relative to other composite, whereas in osteosarcoma cellsMG-63, we evaluated the cytotoxic effect of the novel synthesized Ce-depoed bioactive glass nanocomposte scaffolds using Sulforhodamine B (SRB) method and the result was showed that after 24hr of treatment with two concentrations of CeO2, significant viability decrease was observed compared with control cells. Among the composites, having CL/CH/C5 showed the highest cytotoxic effect and reduced survival rate of osteosarcoma cells to 75.68%. The subsequent increase of CeO2 concentration was also effective but its effect was less to CeO2 concentration level of 5 and more than other two composites without CeO2 particles and control cells.
Conclusion
Nanobioactive glass supported the attachment and proliferation of BM-MSCs, and provided an appropriate environment for the cell proliferation. Incorporation of CeO2 in the glass composition which used as a bioactive filler of Collagen/Chitosan scaffolds increased cell viability and proliferation. The composite scaffold with the highest content of CeO2 content (CL/CH/C10) showed the least toxic effect to BM-MSCs directed to osteogenic lineage. Ce ions were enhanced proliferation, and osteogenic differentiation of the cells and collagen formation.
Collectively, the present study showed that the highest concentration of cerium oxide paricles BG-C10 scaffolds on the normal cells exhibiting good biocompatibility and stimulating both the proliferation of rBM-MSCs and the osteogenic differentiation of osteoblast cells while, the lowest concentration BG-C5 was the trend toxicity on osteosaroma cells.
Thus, these findings showed that the prepared hybrid Ce-doped glass/Collagen/Chitosan bioactive scaffolds were biocompatible and hold great potential for basic research, bone tissue engineering applications and cancer treatment diseases.