Search results for: mice bone

Authors: Archana Sharma, Vijayashree Nayak, Ashok Kumar

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Three-dimensional matrices were fabricated from blend of natural-natural polymers like carrageenan-gelatin and synthetic -natural polymers such as PEG- gelatin (PEG of different molecular weights (2,000 and 6,000) using two different crosslinkers; glutaraldehyde and EDC-NHS by cryogelation technique. Blends represented a feasible approach to design 3-D scaffolds with controllable mechanical, physical and biochemical properties without compromising biocompatibility and biodegradability. These matrices possessed interconnected porous structure, good mechanical strength, biodegradable nature, constant swelling kinetics, ability to withstand high temperature and visco-elastic behavior. Hemocompatibility of cryogel matrices was determined by coagulation assays and hemolytic activity assay which demonstrated that these cryogels have negligible effects on coagulation time and have excellent blood compatibility. In vitro biocompatibility (cell-matrix interaction) inferred good cell adhesion, proliferation, and secretion of ECM on matrices. These matrices provide a microenvironment for the growth, proliferation, differentiation and secretion of ECM of different cell types such as IMR-32, C2C12, Cos-7, rat bone marrow derived MSCs and human bone marrow MSCs. Hoechst 33342 and PI staining also confirmed that the cells were uniformly distributed, adhered and proliferated properly on the cryogel matrix. An ideal scaffold used for tissue engineering application should allow the cells to adhere, proliferate and maintain their functionality. Neurotransmitter analysis has been done which indicated that IMR-32 cells adhered, proliferated and secreted neurotransmitters when they interacted with these matrices which showed restoration of their functionality. The cell-matrix interaction up to molecular level was also evaluated so to check genotoxicity and protein expression profile which indicated that these cryogel matrices are non-genotoxic and maintained biofunctionality of cells growing on these matrices. All these cryogels, when implanted subcutaneously in balb/c mice, showed no adverse systemic or local toxicity effects at implantation site. There was no significant increase in inflammatory cell count has otherwise been observed after scaffold implantation. These cryogels are supermacroporous and this porous structure allows cell infiltration and proliferation of host cells. This showed the integration and presence of infiltrated cells into the cryogel implants. Histological analysis confirmed that the implanted cryogels do not have any adverse effect in spite of host immune system recognition at the site of implantation, on its surrounding tissues and other vital host organs. In vivo biocompatibility study after in vitro biocompatibility analysis has also concluded that these synthesized cryogels act as important biological substitutes, more adaptable and appropriate for transplantation. Thus, these cryogels showed their potential for soft tissue engineering applications.

Keywords: cryogelation, hemocompatibility, in vitro biocompatibility, in vivo biocompatibility, soft tissue engineering applications

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Authors: Pinar Erturk, Sevde Altuntas, Fatih Buyukserin

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In order to obtain an effective integration between an implant and a bone, implant surfaces should have similar properties to bone tissue surfaces. Especially mimicry of the chemical, mechanical and topographic properties of the implant to the bone is crucial for fast and effective osseointegration. Titanium-based biomaterials are more preferred in clinical use, and there are studies of coating these implants with oxide layers that have chemical/nanotopographic properties stimulating cell interactions for enhanced osseointegration. There are low success rates of current implantations, especially in craniofacial implant applications, which are large and vital zones, and the oxide layer coating increases bone-implant integration providing long-lasting implants without requiring revision surgery. Our aim in this study is to examine bone-cell behavior on titanium implants with an aluminum oxide layer (AAO) on effective osseointegration potential in the deformation of large zones with difficult spontaneous healing. In our study, aluminum layer coated titanium surfaces were anodized in sulfuric, phosphoric, and oxalic acid, which are the most common used AAO anodization electrolytes. After morphologic, chemical, and mechanical tests on AAO coated Ti substrates, viability, adhesion, and mineralization of adult bone cells on these substrates were analyzed. Besides with atomic layer deposition (ALD) as a sensitive and conformal technique, these surfaces were coated with pure alumina (5 nm); thus, cell studies were performed on ALD-coated nanoporous oxide layers with suppressed ionic content too. Lastly, in order to investigate the effect of the topography on the cell behavior, flat non-porous alumina layers on silicon wafers formed by ALD were compared with the porous ones. Cell viability ratio was similar between anodized surfaces, but pure alumina coated titanium and anodized surfaces showed a higher viability ratio compared to bare titanium and bare anodized ones. Alumina coated titanium surfaces, which anodized in phosphoric acid, showed significantly different mineralization ratios after 21 days over other bare titanium and titanium surfaces which anodized in other electrolytes. Bare titanium was the second surface that had the highest mineralization ratio. Otherwise, titanium, which is anodized in oxalic acid electrolyte, demonstrated the lowest mineralization. No significant difference was shown between bare titanium and anodized surfaces except AAO titanium surface anodized in phosphoric acid. Currently, osteogenic activities of these cells on the genetic level are investigated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis results of RUNX-2, VEGF, OPG, and osteopontin genes. Also, as a result of the activities of the genes mentioned before, Western Blot will be used for protein detection. Acknowledgment: The project is supported by The Scientific and Technological Research Council of Turkey.

Keywords: alumina, craniofacial implant, MG-63 cell line, osseointegration, oxalic acid, phosphoric acid, sulphuric acid, titanium

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Authors: Shuofeng Yuan, Bojian Zheng

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Assembly of the heterotrimeric polymerase complex of influenza virus from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the N-terminal of PB1 (PB1N) and the C terminal of PA (PAC) may be a desired target for antiviral development. In this study, we first compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization (FP) assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors, which blocked PA and PB1 interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of PAC-PB1N interaction and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which would cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.

Keywords: influenza, antiviral, viral polymerase, compounds

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Authors: Bui Phuong Linh, Yuki Sakakibara, Ryuto Tanaka, Elizabeth H. Pigney, Taishi Hashiguchi

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NASH is an increasingly prevalent chronic liver disease that can progress to hepatocellular carcinoma and now is attracting interest worldwide. The STAM™ model is a clinically-correlated murine NASH model which shows the same pathological progression as NASH patients and has been widely used for pharmacological and basic research. The multiple parallel hits hypothesis suggests abnormalities in adipocytokines, intestinal microflora, and endotoxins are intertwined and could contribute to the development of NASH. In fact, NASH patients often exhibit gut dysbiosis and dysfunction in adipose tissue and metabolism. However, the analysis of the STAM™ model has only focused on the liver. To clarify whether the STAM™ model can also mimic multiple pathways of NASH progression, we analyzed the organ crosstalk interactions between the liver and the gut and the phenotype of adipose tissue in the STAM™ model. NASH was induced in male mice by a single subcutaneous injection of 200 µg streptozotocin 2 days after birth and feeding with high-fat diet after 4 weeks of age. The mice were sacrificed at NASH stage. Colon samples were snap-frozen in liquid nitrogen and stored at -80˚C for tight junction-related protein analysis. Adipose tissue was prepared into paraffin blocks for HE staining. Blood adiponectin was analyzed to confirm changes in the adipocytokine profile. Tight junction-related proteins in the intestine showed that expression of ZO-1 decreased with the progression of the disease. Increased expression of endotoxin in the blood and decreased expression of Adiponectin were also observed. HE staining revealed hypertrophy of adipocytes. Decreased expression of ZO-1 in the intestine of STAM™ mice suggests the occurrence of leaky gut, and abnormalities in adipocytokine secretion were also observed. Together with the liver, phenotypes in these organs are highly similar to human NASH patients and might be involved in the pathogenesis of NASH.

Keywords: Non-alcoholic steatohepatitis, hepatocellular carcinoma, fibrosis, organ crosstalk, leaky gut

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Authors: Gulsharat A. Baigonakova, Ekaterina S. Marchenko, Venera R. Luchsheva

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The preferred materials for bone grafting are titanium-nickel alloys. They have a porous, permeable structure similar to that of bone tissue, can withstand long-term physiological stress in the body, and retain the scaffolding function for bone tissue ingrowth. Despite the excellent functional properties of these alloys, there is a possibility of post-operative infectious complications that prevent the newly formed bone tissue from filling the spaces created in a timely manner and prolong the rehabilitation period of patients. In order to minimise such consequences, it is necessary to use biocompatible materials capable of simultaneously fulfilling the function of a long-term functioning implant and an osteoreplacement carrier saturated with drugs. Methods to modify the surface by saturation with bioactive substances, in particular macrocyclic compounds, for the controlled release of drugs, biologically active substances, and cells are becoming increasingly important. This work is dedicated to the functionalisation of the surface of porous titanium nickelide by the deposition of macrocyclic compounds in order to provide titanium nickelide with antibacterial activity and accelerated osteogenesis. The paper evaluates the effect of macrocyclic compound deposition methods on the continuity, structure, and cytocompatibility of the surface properties of porous titanium nickelide. Macrocyclic compounds were deposited on the porous surface of titanium nickelide under the influence of various physical effects. Structural research methods have allowed the evaluation of the surface morphology of titanium nickelide and the nature of the distribution of these compounds. The method of surface functionalisation of titanium nickelide influences the size of the deposited bioactive molecules and the nature of their distribution. The surface functionalisation method developed has enabled titanium nickelide to be deposited uniformly on the inner and outer surfaces of the pores, which will subsequently enable the material to be uniformly saturated with various drugs, including antibiotics and inhibitors. The surface-modified porous titanium nickelide showed high biocompatibility and low cytotoxicity in in vitro studies. The research was carried out with financial support from the Russian Science Foundation under Grant No. 22-72-10037.

Keywords: biocompatibility, NiTi, surface, porous structure

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Authors: Ezaldin A. M. Mohammed, Youssef K. H. Abdalhafid, Masoud. M. Zatout

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The study aims to clarify the toxic effect of plant hormones, which are widely used in agriculture. One of these is the plant hormones (indole acetic acid); has been ٳata hormone to rats at 100 ppm salt solution of 0.2 per day after day for a period of forty days before conception until the fourteenth day or sixteenth or childbirth. Treatment brought about a marked shortage in the rate of increase in the weight of mice., And a percentage of the weight of the liver there was a distinct increase in the relative weight of the liver. As well as the increase in pathological changes and increase the size of the nuclei and Kupffer cell, as noted widespread and dense clusters of inflammatory cells accompanied by about the erosion of liver tissue and blood ٳrchah. Biochemical analyzes showed a marked decrease of the liver in antioxidant enzymes and an increase in the rate of free radicals. It was also noted an increase in cases of abortion. The owner of so many birth defects. It was also noted the lack of body weight in fetuses and increase the absorption rate of embryos in fetuses of mothers treatment compared to the control group. Showed microscopic examinations of the liver of mice born in the transaction and the decay in the presence of hepatic cells and edema, blood vessels and increase the rate of cell death.

Keywords: indol acetic acid, liver, pathological changes, albino rats

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Authors: Hamidreza Khodaei, Ali Daryabeigi Zand

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Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid. Despite the fact that 28 different isomers of CLA have already been identified, but the main isomer found in natural diets more than ninety percent CLA on intake of food constitutes demonstrates. CLA is known to be a substance that readily available by rumen microorganisms in some ruminants such as cattle and sheep would likely be made. The main objective of this research was to evaluate the impacts of CLA on lipid metabolism and enhanced fat around the muscle durability by reducing the process of oxidation. In order to implement this research, 80 female mice of the Balb/C, with 55 days of age were employed in the experiment. Treatments include various levels of CLA. Over the course of this study blood samples was also taken from the tail vein of the studied mice. Some other relevant parameters such as serum concentrations of triglycerides, total cholesterol, LDL, HDL and liver enzymes were also determined. The oxidative stability of fats TBARS technique was investigated at different intervals. The findings of the research were analyzed by statistical software of SAS 98. The results, CLA had no significant effect on liver enzymes (P > 0.05). However, it showed a statistically significant impact on triglycerides and total cholesterol. Ratio of LDL to HDL declined remarkably. Histological studies demonstrated reduced accumulation of fat in the tissues surrounding muscles.

Keywords: conjugated linoleic acid, fat metabolism, fat retention, oxidation process

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Authors: Mehieddine Bouatrous

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Recently bioactive ceramic materials have been applied in the biomedical field as bulk, granular, or coating materials for more than half a century. More recently, bone tissue engineering scaffolds made of highly porous bioactive ceramic, glass-ceramic, and composite materials have also been created. As a result, recent bioactive ceramic structures have a high bioactivity rate, an open pores network, and good mechanical characteristics simulating cortical bone. Cyclowollastonite frameworks are also suggested for use as a graft material. As a porogenous agent, various amounts of the polymethyl methacrylate (PMMA) powders were used in this study successfully to synthesize a highly interrelated, nanostructured porous cyclowollastonite with a large specific surface area where the morphology and porosity were investigated. Porous cyclowollastonite bioactive ceramics were synthesized with a cost-effective and eco-friendly wet chemical method. The synthesized biomaterial is bioactive according to in vitro tests and can be used for bone tissue engineering scaffolds where cyclowollastonite sintered dense discs were submerged in simulated body fluid (S.B.F.) for various periods of time (1-4 weeks), resulting in the formation of a dense and consistent layer of hydroxyapatite on the surface of the ceramics, indicating its good in vitro bioactivity. Therefore, the cyclowollastonite framework exhibits good in vitro bioactivity due to its highly interconnecting porous structure and open macropores. The results demonstrate that even after soaking for several days, the surface of cyclowollastonite ceramic can generate a dense and consistent layer of hydroxyapatite. The results showed that cyclowollastonite framework exhibits good in vitro bioactivity due to highly interconnecting porous structure and open macropores.

Keywords: porous, bioactive, biomaterials, S.B.F, cyclowollastonite, biodegradability

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Authors: Matej Patzelt, Jan Dudak, Frantisek Krejci, Jan Zemlicka, Vladimir Musil, Jitka Riedlova, Viktor Sykora, Jana Mrzilkova, Petr Zach

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Introduction: Micro-CT is well used for examination of bone structures and teeth. On the other hand visualization of the soft tissues is still limited. The goal of our study was to create a new fixation method for soft tissue imaging in micro-CT. Methodology: We used organs of 18 mice - heart, lungs, kidneys, liver and brain, which we fixated in ethanol after meticulous preparation. We fixated organs in different concentrations of ethanol and for different period of time. We used three types of ethanol concentration - 97%, 50% and ascending ethanol concentration (25%, 50%, 75%, 97% each for 12 hours). Fixated organs were scanned after 72 hours, 168 hours and 336 hours period of fixation. We scanned all specimens in micro-CT MARS (Medipix All Resolution System). Results: Ethanol method provided contrast enhancement in all studied organs in all used types of fixation. Fixation in 97% ethanol provided very fast fixation and the contrast among the tissues was visible already after 72 hours of fixation. Fixation for the period of 168 and 336 hours gave better details, especially in lung tissue, where alveoli were visualized. On the other hand, this type of fixation caused organs to petrify. Fixation in 50% ethanol provided best results in 336 hours fixation, details were visualized better than in 97% ethanol and samples were not as hard as in fixation in 97% ethanol. Best results were obtained in fixation in ascending ethanol concentration. All organs were visualized in great details, best-visualized organ was heart, where trabeculae and valves were visible. In this type of fixation, organs stayed soft for whole time. Conclusion: New ethanol method is a great option for soft tissue fixation as well as the method for enhancing contrast among tissues in organs. The best results were obtained with fixation of the organs in ascending ethanol concentration, the best visualized organ was the heart.

Keywords: x-ray imaging, small animals, ethanol, ex-vivo

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Authors: Seyedmahdi Mousavihashemi

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Natural Akermanite (NAM) has been successfully prepared by a modified sol-gel method. Optimization in calcination temperature and mechanical ball milling resulted in a pure and nano-sized powder which characterized by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared Spectroscopy (FT–IR). We hypothesized that nano-sized Akermanite (AM) would mimic more efficiently the nanocrystal structure and function of natural bone apatite, owing to the higher surface area, compare to conventional micron-size Akermanite (AM). Accordingly, we used the unique advantage of nanotechnology to improve novel nano akermanite particles as a potential candidate for bone tissue regeneration whether as a per implant filling powder or in combination with other biomaterials as a composite scaffold. Pure Akermanite (PAM) powders were successfully obtained via a simple sol-gel method followed by calcination at 1250 °C. Mechanical grinding in a ceramic ball mill for 7 hours resulted in akermanite (AM) nanoparticles in the range of about 30- 45 nm.

Keywords: biomedical engineering, nano composite, SEM, TEM

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Authors: G. Voicu, C. D. Ghitulica, A. Cucuruz, C. Busuioc

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In the field of tissue engineering, the compositional and microstructural features of the employed materials play an important role, with implications on the mechanical and biological behaviour of the medical devices. In this context, the development of calcium phosphate-natural biopolymer composites represents a choice of many scientific groups. Thus, tricalcium phosphate powders were synthesized by a wet method, namely co-precipitation, starting from high purity reagents. Moreover, the substitution of calcium with magnesium have been approached, in the 5-10 wt.% range. Afterwards, the phosphate powders were integrated into two types of composites with chitosan, different from morphological point of view. First, 3D porous scaffolds were obtained by a freeze-drying procedure. Second, uniform compact films were achieved by film casting. The influence of chitosan molecular weight (low, medium and high), as well as phosphate powder to polymer ratio (1:1 and 1:2) on the morphological properties, were analysed in detail. In conclusion, the reported biocomposites, prepared by a straightforward route are suitable for bone substitution or repairing applications.

Keywords: bone reconstruction, chitosan, composite scaffolds, tricalcium phosphate

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Authors: Cecília M. Antão, Paulo Jorge Nogueira

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Breast cancer (BC) is the first cause of cancer mortality among Portuguese women. This retrospective observational study aimed at identifying comorbidities associated with BC female patients admitted to Portuguese public hospitals (2010-2018), investigating the effect of comorbidities on BC mortality rate, and building a predictive model using logistic regression. Results showed that the BC mortality in Portugal decreased in this period and reached 4.37% in 2018. Adjusted odds ratio indicated that secondary malignant neoplasms of liver, of bone and bone marrow, congestive heart failure, and diabetes were associated with an increased chance of dying from breast cancer. Although the Lisbon district (the most populated area) accounted for the largest percentage of BC patients, the logistic regression model showed that, besides patient’s age, being resident in Bragança, Castelo Branco, or Porto districts was directly associated with an increase of the mortality rate.

Keywords: breast cancer, comorbidities, logistic regression, adjusted odds ratio

Procedia PDF Downloads 87

Authors: Shuafeng Yuan, Bojian Zheng

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The RNA-dependent RNA polymerase of influenza a virus comprises conserved and independently folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PAN) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PAN endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitor assay with the DNA gel-based endonuclease inhibitor assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted ANA-0 bound the endonuclease cavity of PAN by interacting with the metal-binding and catalytic residues. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.

Keywords: anti-influenza, novel compound, inhibition of endonuclease, PA

Procedia PDF Downloads 245

Authors: Samantha Shallop, Hakinya Karra, Tytus Bernas, Gladys Shaw, Gretchen Neigh, Jeffrey Dupree, Mathula Thangarajh

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Abnormal neuronal homeostasis is considered a structural correlate of cognitive deficits in Duchenne Muscular Dystrophy. Neurons are highly polarized cells with multiple dendrites but a single axon. Trafficking of cellular organelles are highly regulated, with the cargo in the somatodendritic region of the neuron not permitted to enter the axonal compartment. We investigated the molecular mechanisms that regular organelle trafficking in neurons using a multimodal approach, including high-resolution structural illumination, proteomics, immunohistochemistry, and computational modeling. We investigated the expression of ankyrin-G, the master regulator controlling neuronal polarity. The expression of ankyrin G and the morphology of the axon initial segment was profoundly abnormal in the CA1 hippocampal neurons in the mdx52 animal model of DMD. Ankyrin-G colocalized with kinesin KIF5a, the anterograde protein transporter, with higher levels in older mdx52 mice than younger mdx52 mice. These results suggest that the functional trafficking from the somatodendritic compartment is abnormal. Our data suggests that dystrophin deficiency compromised neuronal homeostasis via ankyrin-G-based mechanisms.

Keywords: neurons, axonal transport, duchenne muscular dystrophy, organelle transport

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Authors: Dike H. Ogbuagu, Etsede J. Oritsematosan

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This work investigated possible inductions of CaC₂, often misused by fruit vendors to stimulate artificial ripening, on mammalian sperm morphology and viability. Thirty isogenic strains of male albino mice, Mus musculus (age≈ 8weeks; weight= 32.5±2.0g) were acclimatized (ambient temperature 28.0±1.0°C) for 2 weeks and fed standard growers mash and water ad libutum. They were later exposed to graded toxicant concentrations (w/w) of 2.5000, 1.2500, 0.6250, and 0.3125% in 4 cages. A control cage was also established. After 5 weeks, 3 animals from each cage were sacrificed by cervical dislocation and the cauda epididymis excised. Sperm morphology and viability were determined by microscopic procedures. The ANOVA, means plots, Student’s t-test and variation plots were used to analyze data. The common abnormalities observed included Double Head, Pin Head, Knobbed Head, No Tail and With Hook. The higher toxicant concentrations induced significantly lower body weights [F(829.899) ˃ Fcrit(4.19)] and more abnormalities [F(26.52) ˃ Fcrit(4.00)] at P˂0.05. Sperm cells in the control setup were significantly more viable than those in the 0.625% (t=0.005) and 2.500% toxicant doses (t=0.018) at the 95% confidence limit. CaC₂ appeared to induced morphological abnormalities and reduced viability in sperm cells of M. musculus.

Keywords: artificial ripening, calcium carbide, fruit vendors, sperm morphology, sperm viability

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Authors: Alsulami H., Alghamdi N., Alasker A., Almohen N., Shome D.

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Most conventional chemotherapeutic agents which are used for the treatment of cancers not only eradicate cancer cells but also affect normal hematopoietic Stem cells (HSCs) that leads to severe pancytopenia during treatment. Therefore, a need exists for novel approaches to treat cancer without or with minimum effect on normal HSCs. Parthenolide (PTL), a herbal product occurring naturally in the plant Feverfew, is a potential new chemotherapeutic agent for the treatment of many cancers such as acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). In this study we investigated the effect of different PTL concentrations on the viability of normal HSCs and also on the ability of these cells to form colonies after they have been treated with PTL in vitro. Methods: In this study, 24 samples of bone marrow and cord blood were collected with consent, and mononuclear cells were separated using density gradient separation. These cells were then exposed to various concentrations of PTL for 24 hours. Cell viability after culture was determined using 7ADD in a flow cytometry test. Additionally, the impact of PTL on hematopoietic stem cells (HSCs) was evaluated using a colony forming unit assay (CFU). Furthermore, the levels of NFҝB expression were assessed by using a PE-labelled anti-pNFκBP65 antibody. Results: this study showed that there was no statistically significant difference in the percentage of cell death between untreated and PTL treated cells with 5 μM PTL (p = 0.7), 10 μM PTL (p = 0.4) and 25 μM (p = 0.09) respectively. However, at higher doses, PTL caused significant increase in the percentage of cell death. These results were significant when compared to untreated control (p < 0.001). The response of cord blood cells (n=4) on the other hand was slightly different from that for bone marrow cells in that the percentage of cell death was significant at 100 μM PTL. Therefore, cord blood cells seemed more resistant than bone marrow cells. Discussion &Conclusion: At concentrations ≤25 μM PTL has a minimum or no effect on HSCs in vitro. Cord blood HSCs are more resistant to PTL compared to bone marrow HSCs. This could be due to the higher percentage of T-lymphocytes, which are resistant to PTL, in CB samples (85% in CB vs. 56% in BM. Additionally, CB samples contained a higher proportion of CD34+ cells, with 14.5% of brightly CD34+ cells compared to only 1% in normal BM. These bright CD34+ cells in CB were mostly negative for early-stage stem cell maturation antigens, making them young and resilient to oxidative stress and high concentrations of PTL.

Keywords: stem cell, parthenolide, NFKB, CLL

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Authors: Trad Khodja Rafik, Mahtout Amine, Ghoul Rachid, Benbouali Amine, Boulahlib Amine, Hariza Abdelmalik

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Cranioplasty with custom implant realized using 3D printing technology. Cranioplasty is a surgical act that aims restoring cranial bone losses in order to protect the brain from external aggressions and to improve the patient aesthetic appearance. This objective can be achieved with taking advantage of the current technological development in computer science and biomechanics. The objective of this paper it to present an approach for the realization of high precision biocompatible cranial implants using new 3D printing technologies at the lowest cost. The proposed method is to reproduce the missing part of the skull by referring to its healthy contralateral part. Once the model is validated by the neurosurgeons, a mold is 3D printed for the production of a biocompatible implant in Poly-Methyl-Methacrylate (PMMA) acrylic cement. Using this procedure four patients underwent this procedure with excellent aesthetic results.

Keywords: cranioplasty, cranial bone loss, 3D printing technology, custom-made implants, PMMA

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Authors: Predrag Kavaric, Vladimir L. Jubic, Maxim Cadenovic

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The patient was transferred from his dentist to our tertiary medical institution. In anamnesis, we got information that his dental intervention was two years ago when he got dental implants but because of the coronavirus pandemic event, he didn’t finish the whole procedure. After two years, he decided that he will continue his work at his dentist, then his dentist noticed that there is no earlier inserted implant in the upper jaw on the right side. They do Panoramic X-ray and find that the implant is all in the maxillary sinus cavity. The flour of the maxilla was intact without any fistula on the place where the implant was inserted in the maxilla bone, After that initial diagnostic they sent the patient to maxillofacial surgery and otorhinolaryngology. We asked for a CT scan of paranasal sinuses, which confirmed the foreign body in the right maxillary sinus. The plan was that in general anesthesia we do FESS and try to find a foreign body in the maxillary sinus or in case of failure to do Caldwel Luc on that side. After preoperative preparation in GA, we do FESS. In inspection, we find small polyps and chronically changed mucosa of osteomeatal complex and right maxillary sinus. After removing polyps we did uncinectomy and medial maxillectomy. With Heuweiser Antrum grasping forceps after several attempts we managed to extract a foreign body from the bottom of the right maxillary sinus. On the first postoperative day we did detamponade, and then we discharge the patient from hospital. The Covid pandemic has contributed to the postponement of a large number of planned operations, which has resulted in various complications in the treatment of a number of patients. In this case, it happened that the implant was most likely rejected by the bone but in the direction of the maxillary sinus, which is not a common cause. On the other hand, the success was that less traumatic intervention was able to remove the foreign body from the maxillary sinus in which it was located. Since the sinus floor is free of bone defects, it can be continued relatively quickly with dental procedures.

Keywords: x-ray, surgery, maxillar sinus, complication, fees

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Authors: Engin Berber, Nurettin Canakoglu, Ibrahim Sozdutmaz, Merve Caliskan, Shaikh Terkis Islam Pavel, Hazel Yetiskin, Aykut Ozdarendeli

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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The CCHFV genome consists of three molecules of negative-sense single-stranded RNA, each encapsulated separately. The virion particle contains viral RNA polymerase (L segment), surface glycoproteins Gn and Gc (Msegment), and a nucleocapsid protein NP (S segment). CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Eastern Europe. Clinical CCHF was first recognized in Turkey in 2002. The numbers of CCHF cases have gradually increased in Turkey making the virus a public health concern. Between 2002 and 2014, more than 8000 the CCHF cases have been reported in Turkey and mortality rate is around 5%. So, Turkey is one of the countries where the epidemy has become spread to the wider geography and the biggest outbreaks of CCHF have occurred in the world. We have recently developed an inactivated cell-culture based vaccine against CCHF. We have showed that the Balb/c mice immunized with the CCHF vaccine induced the high level of neutralizing antibodies. In this study, we aimed to determine the immunodominant regions of nucleoprotein (NP) CCHFV Kelkit06 strain which stimulate T cells. For this purpose, pools of overlapping NP were used for an IFN- γ ELISPOT assay. Balb/c mice were divided into two groups for the experiment. Two groups (n = 10 each) were immunized via the intraperitoneal route with 5, or 10μg of the cell culture-based vaccine. The control group (n = 6) was mock immunized with PBS. Booster injections with the same formulation were given on days 21 and 42 after the first immunization. The higher reactivity against the CCHFV NP pools 31-40 and 80-90 was determined in the two dose groups. In order to analyze the vaccine-induced T cell responses in Balb/c mice immunized with varying doses of the vaccine, we have been also currently working on CD4+, CD8+ and CD3 + T cells by flow cytometry.

Keywords: Crimean-Congo hemorrhagic fever virus, immunodominant regions of NP, T cell response, vaccine

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Authors: Alireza Hassanshahi, Xanthe Strudwick, Zlatko Kopecki, Allison J Cowin

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Studies have shown that Flightless (Flii) is elevated in human wounds, including burns, and reducing the level of Flii is a promising approach for improving wound repair and reducing scar formation. The most effective approach has been to neutralise Flii activity using localized, intradermal application of function blocking monoclonal antibodies. However, large surface area burns are difficult to treat by intradermal injection of therapeutics, so the aim of this study was to investigate if a systemic injection of a monoclonal antibody against Flii could improve healing in mice following burn injury. Flii neutralizing antibodies (FnAbs) were labelled with Alxa-Fluor-680 for biodistribution studies and the healing effects of systemically administered FnAbs to mice with burn injuries. A partial thickness, 7% (70mm2) total body surface area scald burn injury was created on the dorsal surface of mice (n=10/group), and 100µL of Alexa-Flour-680-labeled FnAbs were injected into the intraperitoneal cavity (IP) at time of injury. The burns were imaged on days 0, 1, 2, 3, 4, and 7 using IVIS Lumina S5 Imaging System, and healing was assessed macroscopically, histologically, and using immunohistochemistry. Fluorescent radiance efficiency measurements showed that IP injected Alexa-Fluor-680-FnAbs localized at the site of burn injury from day 1, remaining there for the whole 7-day study. The burns treated with FnAbs showed a reduction in macroscopic wound area and an increased rate of epithelialization compared to controls. Immunohistochemistry for NIMP-R14 showed a reduction in the inflammatory infiltrate, while CD31/VEGF staining showed improved angiogenesis post-systemic FnAb treatment. These results suggest that systemically administered FnAbs are active within the burn site and can improve healing outcomes. The clinical application of systemically injected Flii monoclonal antibodies could therefore be a potential approach for promoting the healing of large surface area burns immediately after injury.

Keywords: biodistribution, burn, flightless, systemic, fnAbs

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Authors: Ane Escobar, Paula Angelomé, Mihaela Delcea, Marek Grzelczak, Sergio Enrique Moya

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The antibacterial capacity of bone-anchoring implants can be improved by the use of antibiotics that can be delivered to the media after the surgery. Mesoporous films have shown great potential in drug delivery for orthopedic applications, since pore size and thickness can be tuned to produce different surface area and free volume inside the material. This work shows the synthesis of mesoporous titania films (MTF) by sol-gel chemistry and evaporation-induced self-assembly (EISA) on top of glass substrates. Pores with a diameter of 12nm were observed by Transmission Electron Microscopy (TEM). A film thickness of 100 nm was measured by Scanning Electron Microscopy (SEM). Gentamicin was used to study the antibiotic delivery from the film by means of High-performance liquid chromatography (HPLC). The Staphilococcus aureus strand was used to evaluate the effectiveness of the penicillin loaded films toward inhibiting bacterial colonization. MC3T3-E1 pre-osteoblast cell proliferation experiments proved that MTFs have a good biocompatibility and are a suitable surface for MC3T3-E1 cell proliferation. Moreover, images taken by Confocal Fluorescence Microscopy using labeled vinculin, showed good adhesion of the MC3T3-E1 cells to the MTFs, as well as complex actin filaments arrangement. In order to improve cell proliferation Bone Morphogenetic Protein-2 (BMP-2) was adsorbed on top of the mesoporous film. The deposition of the protein was proved by measurements in the contact angle, showing an increment in the hydrophobicity while the protein concentration is higher. By measuring the dehydrogenase activity in MC3T3-E1 cells cultured in dually functionalized mesoporous titatina films with gentamicin and BMP-2 is possible to find an improvement in cell proliferation. For this purpose, the absorption of a yellow-color formazan dye, product of a water-soluble salt (WST-8) reduction by the dehydrogenases, is measured. In summary, this study proves that by means of the surface modification of MTFs with proteins and loading of gentamicin is possible to achieve an antibacterial effect and a cell growth improvement.

Keywords: antibacterial, biocompatibility, bone morphogenetic protein-2, cell proliferation, gentamicin, implants, mesoporous titania films, osteoblasts

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Authors: Aoxue Yang, Weili Tian, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Glucocorticoids (GCs) are used to treat GBM-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and BTSCs. Identifying markers specifically expressed by brain tumor stem cells (BTSCs) may enable specific therapies that spare their regular tissue-resident counterparts. By ribosome profiling analysis, we have identified that glycerol-3-phosphate dehydrogenase 1 (GPD1) is expressed by dormant BTSCs but not by NSCs. Through different stress-induced experiments in vitro, we found that only dexamethasone (DEXA) can significantly increase the expression of GPD1 in NSCs. Adversely, mifepristone (MIFE) which is classified as glucocorticoid receptors antagonists, could decrease GPD1 protein level and weaken the proliferation and stemness in BTSCs. Furthermore, DEXA can induce GPD1 expression in tumor-bearing mice brains and shorten animal survival, whereas MIFE has a distinct adverse effect that prolonged mice lifespan. Knocking out GR in NSC can block the upregulation of GPD1 inducing by DEXA, and we find the specific sequences on GPD1 promotor combined with GR, thus improving the efficiency of GPD1 transcription from CHIP-Seq. Moreover, GR and GPD1 are highly co-stained on GBM sections obtained from patients and mice. All these findings confirmed that GR could regulate GPD1 and loss of GPD1 Impairs Multiple Pathways Important for BTSCs Maintenance GPD1 is also a critical enzyme regulating glycolysis and lipid synthesis. We observed that DEXA and MIFE could change the metabolic profiles of BTSCs by regulating GPD1 to shift the transition of cell dormancy. Our transcriptome and lipidomics analysis demonstrated that cell cycle signaling and phosphoglycerides synthesis pathways contributed a lot to the inhibition of GPD1 caused by MIFE. In conclusion, our findings raise concern that treatment of GBM with GCs may compromise the efficacy of chemotherapy and contribute to BTSC dormancy. Inhibition of GR can dramatically reduce GPD1 and extend the survival duration of GBM-bearing mice. The molecular link between GPD1 and GR may give us an attractive therapeutic target for glioblastoma.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides

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Authors: Mansi Patel, Priti Mehta

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Background: Ionizing radiations have detrimental effects on humans, and the growing technological encroachment has increased human exposure to it enormously. So, the safety issues have emphasized researchers to develop radioprotector from natural resources having minimal toxicity. A substance having anti-inflammatory, antioxidant, and immunomodulatory activity can be a potential candidate for radioprotection. One such plant with immense potential i.e. Bamboo was selected for the present study. Purpose: The study aims to evaluate the potential of Indian bamboo leaves for protection against the clastogenic effect of gamma radiation. Methods: The protective effect of bamboo leaf extract against gamma radiation-induced genetic damage in human peripheral blood lymphocytes (HPBLs) was evaluated in vitro using Cytokinesis blocked micronuclei assay (CBMN). The blood samples were pretreated with varying concentration of extract 30 min before the radiation exposure (4Gy & 6Gy). The reduction in the frequency of micronuclei was observed for the irradiated and control groups. The effect of various concentration of bamboo leaf extract (400,600,800 mg/kg) on the development of radiation induced sickness and altered mortality in mice exposed to 8 Gy of whole-body gamma radiation was studied. The developed symptoms were clinically scored by multiple endpoints for 30 days. Results: Treatment of HPBLs with varying concentration of extract before exposure to a different dose of γ- radiation resulted in significant (P < 0.0001) decline of radiation induced micronuclei. It showed dose dependent and concentration driven activity. The maximum protection ~ 70% was achieved at nine µg/ml concentration. Extract treated whole body irradiated mice showed 50%, 83.3% and 100% survival for 400, 600, and 800mg/kg with 1.05, 0.43 and 0 clinical score respectively when compared to Irradiated mice having 6.03 clinical score and 0% survival. Conclusion: Our findings indicate bamboo leaf extract reduced the radiation induced cytogenetic damage. It has also increased the survival ratio and reduced the radiation induced sickness and mortality when exposed to a lethal dose of gamma radiation.

Keywords: bamboo leaf extract, Cytokinesis blocked micronuclei (CBMN) assay, ionizing radiation, radio protector

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Authors: H. Maachou, A. Chagnes, G. Cote

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The present work reports the properties of chitosan/hydroxyapatite (Cs/HA: 100/00, 70/30 and 30/70) composite microspheres obtained by emulsification processing route. The morphology of chitosane microspheres was observed by a scanning electron microscope (SEM) which shows an aggregate of spherical microspheres with a particle size, determined by optical microscope, ranged from 4 to 10 µm. Thereafter, a biomimetic approach was used to study the in vitro biomineralization of these composites. It concerns the composites immersion in simulated body fluid (SBF) for different times. The deposited calcium phosphate was studied using X-ray diffraction analysis (XRD), FTIR spectroscopy and ICP analysis of phosphorus. In fact, the mineral formed on Cs/HA microspheres was a mixture of carbonated HA and β-TCP as showed by FTIR peaks at 1419,5 and 871,8 cm-1 and XRD peak at 29,5°. This formation was induced by the presence of HA in chitosan microspheres. These results are confirmed by SEM micrographs which chow the Ca-P crystals growth in form of cauliflowers. So, these materials are of great interest for bone regeneration applications due to their ability to nucleate calcium phosphates in presence of simulated body fluid (SBF).

Keywords: hydroxyapatite, chitosan, microsphere, composite, bone regeneration

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Authors: Jaspal Rana

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Oxidative stress has been identified as an underlying cause of ischemia-reperfusion (IR) related cognitive dysfunction and brain damage. Therefore, antioxidant based therapies to treat IR injury are being investigated. Allium cepa L. (onion) is used as culinary medicine and is documented to have marked antioxidant effects. Hence, the present study was designed to evaluate the effect of A. cepa outer scale extract (ACE) against IR induced cognition and biochemical deficit in mice. ACE was prepared by maceration with 70% methanol and fractionated into ethylacetate and aqueous fractions. Bilateral common carotid artery occlusion for 10 min followed by 24 h reperfusion was used to induce cerebral IR injury. Following IR injury, ACE (100 and 200 mg/kg) was administered orally to animals for 7 days once daily. Behavioral outcomes (memory and sensorimotor functions) were evaluated using Morris water maze and neurological severity score. Cerebral infarct size, brain thiobarbituric acid reactive species, reduced glutathione, and superoxide dismutase activity was also determined. Treatment with ACE significantly ameliorated IR mediated deterioration of memory and sensorimotor functions and rise in brain oxidative stress in animals. The results of the present investigation revealed that ACE improved functional outcomes after cerebral IR injury which may be attributed to its antioxidant properties.

Keywords: ischemia-reperfusion, neuroprotective, stroke, antioxidant

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Authors: Mariane G. Tadros, Shahira M. Ezzat, Maha M. Salama, Mohamed A. Farag

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In this study, the in vitro anticholinesterase activity of the volatile oils of both O. basilicum and O. africanum was investigated and both samples showed significant activity. As a result, the major constituents of the two oils were isolated using several column chromatography. Linalool, 1,8-cineol and eugenol were isolated from the volatile oil of O. basilicum and camphor was isolated from the volatile oil of O. africanum. The anticholinesterase activity of the isolated compounds were also evaluated where 1,8-cineol showed the highest inhibitory activity followed by camphor. To confirm these activities, learning and memory enhancing effects were tested in mice. Memory impairment was induced by scopolamine, a cholinergic muscarinic receptor antagonist. Anti-amnesic effects of both volatile oils and their terpenoids were investigated by the passive avoidance task in mice. We also examined their effects on brain acetylcholinesterase activity. Results showed that scopolamine-induced cognitive dysfunction was significantly attenuated by administration of the volatile oils and their terpenoids, eugenol and camphor, in the passive avoidance task and inhibited brain acetylcholinesterase activity. These results suggest that O. basilicum and O. africanum volatile oils can be good candidates for further studies on Alzheimer’s disease via their acetylcholinesterase inhibitory actions.

Keywords: Ocimum baselicum, Ocimum africanum, GC/MS analysis, anticholinesterase

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Authors: Ahmad Nazrun Shuid, Isa Naina Mohamed, Nurul Izzah Ibrahim, Norazlina Mohamed

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Studies have shown that oral tocotrienol, a potent vitamin E, promoted fracture healing of osteoporotic bone. In this study, tocotrienol was combined with a polymer carrier (PLGA), and injected to the fracture site. The slow and constant release of tocotrienol particles would promote fracture healing of post-menopausal osteoporosis rat model. Fracture healing was assessed using micro-CT. Twenty-four Sprague-Dawley rats were ovariectomised or sham-operated and the left tibiae were fractured and fixed with plate and screws. The fractures were created at the upper third of the left tibiae. The rats were divided into 3 groups: sham-operated (SO), ovariectomised-control (OVxC) and PLGA-incorporated tocotrienol treatment (OVx + TT) groups. After 4 weeks, the OVx + TT group showed significantly better callus fracture healing than the OVxC group. In conclusion, tocotrienol-incorporated PLGA was able to promote fracture healing of osteoporotic bone.

Keywords: osteoporosis, micro-CT, tocotrienol, PLGA, fracture

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Authors: Georgeta Voicu, Cristina Daniela Ghitulica, Andreia Cucuruz, Cristina Busuioc

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In the field of tissue engineering, the compositional and microstructural features of the employed materials play an important role, with implications on the mechanical and biological behaviour of the medical devices. In this context, the development of apatite - natural biopolymer composites represents a choice of many scientific groups. Thus, hydroxyapatite powders were synthesized by a wet method, namely co-precipitation, starting from high purity reagents (CaO, MgO, and H3PO4). Moreover, the substitution of calcium with magnesium have been approached, in the 5 - 10 wt.% range. Afterward, the phosphate powders were integrated in two types of composites with chitosan, different from morphological point of view. First, 3D porous scaffolds were obtained by a freeze-drying procedure. Second, uniform, compact films were achieved by film casting. The influence of chitosan molecular weight (low, medium and high), as well as apatite powder to polymer ratio (1:1 and 1:2) on the morphological properties, were analysed in detail. In conclusion, the reported biocomposites, prepared by a straightforward route are suitable for bone substitution or repairing applications.

Keywords: bone reconstruction, chitosan, composite scaffolds, hydroxyapatite

Procedia PDF Downloads 322

Authors: Isabela S. Gonçalves, Vitor G. P. Lima, Tiago M. B. Campos, Marcos Jacobovitz, Luana M. R. Vasconcellos, Ivone R. Oliveira

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Periodontitis is an infectious disease of multifactorial origin, which originates from a periodontogenic bacterial biofilm that colonizes the surfaces of the teeth, resulting in an inflammatory reaction to microbial aggression. In the absence of adequate treatment, it can lead to the gradual destruction of the periodontal ligaments, cementum and alveolar bone. In guided tissue/bone regeneration therapy (GTR/GBR), a barrier membrane is placed between the fibrous tissues and the bone defect to prevent unwanted incursions of fibrous tissues into the bone defect, thus allowing the regeneration of quality bone. Currently, there are a significant number of biodegradable barrier membranes available on the market. However, a very common problem is that the membranes are not bioactive/osteogenic, that is, they are incapable of inducing a favorable osteogenic response and integration with the host tissue, resulting in many cases in displacement/expulsion of the membrane, requiring a new surgical procedure and replacement of the implant. Aiming to improve the bioactive and osteogenic properties of the membrane, this work evaluated the production of membranes that integrate the biocompatibility of the hydrophilic synthetic polymer (polyvinyl alcohol - PVA) with the osteogenic effects of chlorinated bioactive glasses (BG58S-Cl), using the electrospinning equipment (AeroSpinner L1.0 from Areka) which allows the execution of spinning by high voltage and/or blowing in solution and with a high production rate, enabling development on an industrial scale. In the formulation of bioactive glasses, the replacement of nitrates by chlorinated molecules has shown to be a promising alternative, since the chloride ion is naturally present in the body and, with its presence in the bioactive glass, the biocompatibility of the material increases. Thus, in this work, chlorinated bioactive glasses were synthesized by the sol-gel route using the compounds tetraethyl orthosilicate (TEOS), calcium chloride dihydrate and monobasic ammonium phosphate with pH adjustments with 37% HCl (1.5 or 2.5) and different calcination temperatures (500, 600 and 700 °C) were evaluated. The BG-58S-Cl powders obtained were characterized by pH, conductivity and zeta potential x time curves and by SEM/FEG, FTIR-ATR and Raman tests. The material produced under the selected conditions was evaluated in relation to the milling procedure, obtaining particles suitable for incorporation into PVA polymer solutions to be electrospun (D50 = 22 µm). Membranes were produced and evaluated regarding the influence of the crosslinking agent content as well as the crosslinking treatment temperature (3, 5 and 10 wt% citric acid) and (130 or 175 oC) and were characterized by SEM/FEG, FTIR, TG and DSC. From the optimization of the crosslinking conditions, membranes were prepared by adding BG58S-Cl powder (5 and 10 wt%) to the PVA solutions and were characterized by SEM-FEG, DSC, bioactivity in SBF and behavior in cell culture (cell viability, total protein content, alkaline phosphatase, mineralization nodules). The micrographs showed homogeneity of the distribution of BG58S-Cl particles throughout the sample, favoring cell differentiation.

Keywords: barrier membranes, chlorinated bioactive glasses, polyvinyl alcohol, tissue regeneration.

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Authors: D. Ficai, A. Ficai, D. Gudovan, I. A. Gudovan, I. Ardelean, R. Trusca, E. Andronescu, V. Mitran, A. Cimpean

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In the last years, scientists struggled hardly to mimic bone structures to develop implants and biostructures which present higher biocompatibility and reduced rejection rate. One way to obtain this goal is to use similar materials as that of bone, namely collagen/hydroxyapatite composite materials. However, it is very important to tailor both compositions but also the microstructure of the bone that would ensure both the optimal osteointegartion and the mechanical properties required by the application. In this study, new collagen/hydroxyapatites composite materials doped with Cu, Li, Mn, Zn were successfully prepared. The synthesis method is described below: weight the Ca(OH)₂ mass, i.e., 7,3067g, and ZnCl₂ (0.134g), CuSO₄ (0.159g), LiCO₃ (0.133g), MnCl₂.4H₂O (0.1971g), and suspend in 100ml distilled water under magnetic stirring. The solution thus obtained is added a solution of NaH₂PO₄*H2O (8.247g dissolved in 50ml distilled water) under slow dropping of 1 ml/min followed by adjusting the pH to 9.5 with HCl and finally filter and wash until neutral pH. The as-obtained slurry was dried in the oven at 80°C and then calcined at 600°C in order to ensure a proper purification of the final product of organic phases, also inducing a proper sterilization of the mixture before insertion into the collagen matrix. The collagen/hydroxyapatite composite materials are tailored from morphological point of view to optimize their biocompatibility and bio-integration against mechanical properties whereas the addition of the dopants is aimed to improve the biological activity of the samples. The addition of transitional metals can improve the biocompatibility and especially the osteoblasts adhesion (Mn²⁺) or to induce slightly better osteoblast differentiation of the osteoblast, Zn²⁺ being a cofactor for many enzymes including those responsible for cell differentiation. If the amount is too high, the final material can become toxic and lose all of its biocompatibility. In order to achieve a good biocompatibility and not reach the cytotoxic effect, the amount of transitional metals added has to be maintained at low levels (0.5% molar). The amount of transitional metals entering into the elemental cell of HA will be verified using inductively-coupled plasma mass spectrometric system. This highly sensitive technique is necessary, because, at such low levels of transitional metals, the difference between biocompatible and cytotoxic is a very thin line, thus requiring proper and thorough investigation using a precise technique. In order to determine the structure and morphology of the obtained composite materials, IR spectroscopy, X-Ray diffraction (XRD), scanning electron microscopy (SEM), and Energy Dispersive X-Ray Spectrometry (EDS) were used. Acknowledgment: The present work was possible due to the EU-funding grant POSCCE-A2O2.2.1-2013-1, Project No. 638/12.03.2014, code SMIS-CSNR 48652. The financial contribution received from the national project “Biomimetic porous structures obtained by 3D printing developed for bone tissue engineering (BIOGRAFTPRINT), No. 127PED/2017 is also highly acknowledged.

Keywords: collagen, composite materials, hydroxyapatite, bone tissue engineering

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