Course of Plasmodium infection studied using 2D-COS on human erythrocytes.

BACKGROUND: The threat of malaria is still present in the world. Recognizing the type of parasite is important in determining a treatment plan. The golden routine involves microscopic diagnostics of Giemsa-stained thin blood smears, however, alternative methods are also constantly being sought, in order to gain an additional insight into the course of the disease. Spectroscopic methods, e.g., Raman spectroscopy, are becoming increasingly popular, due to the non-destructive nature of these techniques. METHODS: The study included patients hospitalized for malaria caused by Plasmodium falciparum or Plasmodium vivax, in the Department of Infectious Diseases at the University Hospital in Krakow, Poland, as well as healthy volunteers. The aim of this study was to assess the possibility of using Raman spectroscopy and 2D correlation (2D-COS) spectroscopy in understanding the structural changes in erythrocytes depending on the type of attacking parasite. EPR spectroscopy and two-trace two-dimensional (2T2D) correlation was also used to examine the specificity of paramagnetic centres found in the infected human blood. RESULTS: Two-dimensional (2D) correlation spectroscopy facilitates the identification of the hidden relationship, allowing for the discrimination of Raman spectra obtained during the course of disease in human red blood cells, infected by P. falciparum or P. vivax. Synchronous cross-peaks indicate the processes taking place inside the erythrocyte during the export of the parasite protein towards the cell membrane. In contrast, moieties that generate asynchronous 2D cross-peaks are characteristic of the respective ligand-receptor domains. These changes observed during the course of the infection, have different dynamics for P. falciparum and P. vivax, as indicated by the asynchronous correlation cross-peaks. Two-trace two-dimensional (2T2D) spectroscopy, applied to EPR spectra of blood at the beginning of the infection, showed differences between P. falciparum and P. vivax. CONCLUSIONS: A unique feature of 2D-COS is the ability to discriminate the collected Raman and EPR spectra. The changes observed during the course of a malaria infection have different dynamics for P. falciparum and P. vivax, indicated by the reverse sequence of events. For each type of parasite, a specific recycling process for iron was observed in the infected blood.

Blood-stage antiplasmodial activity and oocyst formation-blockage of metallo copper-cinchonine complex.

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes' midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite.

IN VIVO PERFORMANCE OF THE EXPERIMENTAL CHITOSAN BASED BONE SUBSTITUTE--ADVANCED THERAPY MEDICINAL PRODUCT. A STUDY IN SHEEP.

When evaluating a novel bone substitute material, advanced in vivo testing is an important step in development and safety affirmation. Sheep seems to be a valuable model for human bone turnover and remodeling activity. The experimental material composed with the stem cells is an advanced therapy medicinal product (acc. to EC Regulation 1394/2007). Our research focuses on histological differences in bone formation (guided bone regeneration--GBR) in sheep maxillas after implantation of the new chitosan/tricalcium phosphate/alginate (CH/TCP/Alg) biomaterial in comparison to the commercially available xenogenic bone graft and a/m enhanced with the stem cells isolated from the adipose tissue. Twelve adult female sheep of BCP synthetic line, weighing 60-70 kg were used for the study. The 11 mm diameter defects in maxilla bone were prepared with a trephine bur under general anesthesia and then filled with the bone substitute materials: CH/TCP/Alg, BioOss Collagen, Geistlich AG (BO), CH/TCP/Alg composed with the stem cells (CH/S) or left just with the blood clot (BC). Inbreeding cycle of the animals terminated at 4 months after surgery. Dissected specimens of the maxilla were evaluated histologically and preliminary under microtomography. Histological evaluation showed early new bone formation observed around the experimental biomaterial and commercially available BO. There were no features of purulent inflammation and necrosis, or granulomatous inflammation. Microscopic examination after 4 months following the surgery revealed trabecular bone formation around chitosan based bone graft and xenogenic material with no significant inflammatory response. Different results--no bone recreation were observed for the negative control (BC). In conclusion, the tested materials (CH/TCP/Alg and BO) showed a high degree of biocompatibility and some osteoconductivity in comparison with the control group. Although the handiness, granules size and setting time of CHffCP/Alg may be refined for future clinical tests. The relevant beneficial influence of using the adipose derived stem cells in GBR was not confirmed in this model.

Cytotoxicity Evaluation and Crystallochemical Analysis of a Novel and Commercially Available Bone Substitute Material.

BACKGROUND: Alloplastic biomaterials are an alternative for autologous transplants and xenografts in oral surgery and dental implantology. These non-immunogenic and resorbable materials are becoming the basis for complete and predictable guided bone regeneration in many cases. The chemical composition of a great majority of them is based on calcium phosphate salts. In vivo performance is often variable. OBJECTIVES: The objective was to evaluate the biological and chemical properties of an experimental bone substitute material. MATERIAL AND METHODS: The present research focuses on the cytotoxicity comparison and physiochemical characterization of two biomaterials: a novel chitosan/tricalcium phosphate/alginate composite (CH/TCP/Ag) and a commercially available synthetic bone graft made of HA (60%) and ßTCP (40%) (HA/TCP). The materials were evaluated according to PN-EN ISO 10993 Biological evaluation of medical devices i.e. cytotoxicity on mouse fibroblasts (L929) and, in addition, tests on human osteoblasts (hFOB1.19) and human osteosarcoma (MG-63) were conducted. The crystallochemical analysis was performed using the X-ray powder diffraction method. The Bruker-AXS D8 Advance diffractometer (Karlsruhe, Germany) was used to collect diffractograms. RESULTS: The tested materials showed a close resemblance in chemical composition and a considerable differentiation in cytotoxic response. CONCLUSIONS: The novel composite demonstrated a high degree of cytocompatibility, which is promising in future clinical trials.

Chitosan and composite microsphere-based scaffold for bone tissue engineering: evaluation of tricalcium phosphate content influence on physical and biological properties.

In the hereby presented work the authors describe a technique of high-compression-resistant biodegradable bone scaffold preparation. The methodology is based on the agglomeration of chitosan (CH) and chitosan/ß-tricalcium phosphate (CH/TCP) microspheres and represents a novel approach to 3D matrices design for bone tissue engineering application. The materials were prepared from high deacetylation degree chitosan. The authors describe the method for scaffold fabrication, essential properties of the materials manufactured and the influence of various TCP concentrations on material morphology, mechanical properties (for dry and hydrated materials) and preliminary study on the interaction between CH or CH/TCP scaffolds and within cultured MG-63 osteoblast-like cells. The properties of the obtained materials were significantly affected by the calcium phosphate content, which had a particular influence on the granule microstructure, size distribution and inner biomaterial pore size. The water uptake ability was found to be lower for the materials enriched with the inorganic phase and tended to decrease with the increasing calcium phosphate concentration. The evaluation of mechanical properties has revealed that scaffolds produced with the usage of granule-based technology display a potential to be used as a load-bearing material since the Young's modulus values were limited to the range of 200-500 MPa for dry materials and 15-20 MPa for the hydrated state of the scaffolds. The cell number, identified in three time points (48 h, 7 and 14 days) by Pico Green assay, was lower for the materials enriched with inorganic phase (75 % of control), however cell distribution, when compared to CH only biomaterial, was acknowledged as steadier on the surface of the material containing the highest calcium phosphate concentration.

Bone regeneration potential of the new chitosan-based alloplastic biomaterial.

Over the last few years, alloplastic bone substitute materials are raising much interest as an alternative to autologic transplants and xenogenic materials especially in oral surgery. These non-immunogenic and completely resorbable biomaterials are becoming the basis for complete and predictable guided bone regeneration in many cases. The objective of our research was to evaluate the dynamics of bone formation in rats' skulls after implantation of the new chitosan/tricalcium phosphate/alginate biomaterial in comparison to the commercially available alloplastic bone graft. A total of 45 adult male rats weighing 300-400 g were used for the study. The 85-mm-diameter defects in calvaria bone were prepared with a trephine bur, and then filled with the bone substitute materials: chitosan/tricalcium phosphate/alginate or easy-graft Classic (Degradable Solutions AG) (EA) or left just with the blood clot. Animals were sacrificed at 1 and 3 months for histological, histomorphometrical and micro-tomographic evaluations. Histological evaluation at 1 month showed early new bone formation, observed around the experimental biomaterial (CH/TCP/Alg). There were no features of purulent inflammation and necrosis or granulomatous inflammation. Microscopic examination after 3 months following the surgery revealed trabecular bone formation around chitosan-based bone graft with no significant inflammatory response. Less satisfactory and differing results were observed for the commercially available EA and control blood clot. The tested material (chitosan) showed a high degree of biocompatibility and osteoconductivity in comparison with the control groups. Additionally, it seemed to be a "user-friendly" material for oral surgeons.

Formation and preclinical evaluation of a new alloplastic injectable bone substitute material.

Alloplastic bone substitute materials are raising some more interest as an alternative for autologic transplants and xenogenic materials especially in oral surgery over the last few years. These non-immunogenic and completely resorbable biomaterials are the basis for complete and predictable guided bone regeneration. In the majority of cases, such a material is chosen because of its convenient application by surgeons. The main objective of our project was to design and fabricate an osteoconductive, injectable and readily tolerable by human tissues biomaterial for guided bone regeneration. For this purpose, a self-setting composite consisting of chitosan/tricalcium phosphate microparticles and sodium alginate was made. The material obtained was characterized by microsphere and agglomerate morphology and microstructure. Its features relating to setting time and mechanical properties were precisely investigated. Our material was also evaluated according to PN-EN ISO 10993 Biological evaluation of medical devices, i.e., the in vitro tests for genotoxicity and cytotoxicity were conduced. Then, the following examinations were performed: subchronic systemic toxicity, skin sensitization, irritation and delayed-type hypersensitivity and local effects after implantation. The material tested showed a high degree of cytocompatibility, fulfilled the requirements of International Standards and seemed to be a "user friendly" material for oral surgeons.