Jelena Rajković

Commercial Carlinae radix herbal drug: Botanical identity, chemical composition and antimicrobial properties

Context: Carlinae radix is an herbal drug, commonly used by the locals in southeastern Serbia for the treatment of respiratory and urogenital diseases and, externally, for various skin conditions. There still seems to be no detailed studies correlating the chemical composition of this drug and its ethnopharmacological uses. Objective: Chemical composition, antimicrobial activity and mode of action of C. radix essential oil, isolated from commercial samples (confirmation of whose true biological identity was also the aim of this work) were analyzed. Antimicrobial potential of decoctions (extracts prepared by boiling plant material in a given solvent), used in ethnomedicine preferentially to the pure essential oil, was also investigated. Materials and methods: The essential oil obtained by hydrodistillation was screened for antimicrobial activity by disc diffusion and broth microdilution methods. Effects of the oil on the growth of Staphylococcus aureus cells were investigated using turbidimetric measurements and visualized using scanning electron microscopy. Analyses of the chemical composition of the oils were done using gas chromatography and gas chromatography/mass spectrometry. Results and discussion: Both the essential oil and the decocts exhibited a very high antimicrobial activity against all tested strains, with S. aureus as the most sensitive one [e.g., for the oil sample the values for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 0.02, 0.04 µL/mL, respectively]. Growth curves of S. aureus demonstrated a significant decrease in turbidity (for the MIC concentration this amounted to ca. 70%) showing a concentration-dependent lysis of the cells, confirmed by scanning electron microscopy. Chemical composition, anatomical and morphological features of the sample pointed to Carlina acanthifolia L. (Asteraceae) instead of Carlina acaulis L. (Asteraceae). Conclusion: The results showed significant antimicrobial effect of the essential oil and the decoctions and support the use of this plant in ethnomedicine for the treatment of various human infections, especially those caused by S. aureus. Adulteration of the drug would not cause significant differences in its biological activity, since chemical composition of the sample showed high similarity with those containing C. acaulis roots.

SEM-EDX Analysis of Bio-Oss® Granules After Incubation In Cell Culture Medium

The aim of this study was to analyze changes in the surface and chemical composition of Bio-Oss® granules after incubation in cell culture medium. Appropriate amount of Bio-Oss® material was incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C for 3 days. After that, granules were dried, coated with gold and analyzed using scanning electron microscope (SEM) equipped with EDX (JEOL, JSM 5300). SEM analysis revealed that the surface of Bio-Oss® particles looks less grainy after incubation in DMEM. The impression is that an incubation in DMEM made erosion of cam bumps or that larger and wider reefs compared to the material before incubation are made by depositing of new material. Semi-quantitative analysis of calcium and phosphorous content in Bio-Oss® before and after incubation in DMEM was determined by EDX spectroscopy and results show that concentration of calcium and phosphorous ions increases after incubation in DMEM.

Enhanced Osteogenesis of Nanosized Cobalt-substituted Hydroxyapatite

Hydroxyapatite (HAp) is an extensively studied material with known biocompatible and osteoconductive properties in bone tissue reconstruction. The improvement of the osteogenetic potential of HAp has been tested through modification of its structure, by replacing Ca2+ ions with Co2+ ions. In our study, we comparatively analyze the osteogenetic potential of the synthesized HAp and Co2+-substituted HAp (HAp/Co) designed on the nano-scale with the aim of specifically stimulating osteogenesis in vivo. We present a quantitative study of the microscopic organization and structure of the newly formed tissue in a bone defect after 12 weeks and 24 weeks. A quantitative analysis of the calcium, magnesium and phosphorus content in the defect and its close environment was used to determine the deposition of minerals after bone reconstruction. The defect reconstructed with HAp/Co nanoparticles (Co2+ content 12 wt%) was filled with a new tissue matrix composed of dense collagen fibers containing centers of mineralization after 24 weeks. The mineral deposition rate was also higher when the defect was reconstructed with HAp/Co than when it was filled with pure HAp. A histological analysis confirmed that the alveolar bone, in which osteoporosis-induced defects were repaired using HAp/Co nanoparticles, was recuperated.

Locally applied cholecalciferol and alfacalcidol act differently on healing of femur defects filled with bone mineral matrix and platelet-rich plasma in ovariectomized rats

Vitamin D and its analogues that are often used in the treatment of osteoporosis have also been shown to affect the healing of bone fractures. To avoid the high-dose systemic treatments, needed to achieve faster healing of fractured bone, the approach of local application of these drugs has become very attractive. The aim of our study was to examine the influence of cholecalciferol and alfacalcidol, locally applied with bovine bone mineral matrix (Bio-Oss), supplemented with platelet-rich plasma on femur defect healing in ovariectomized rats. Experimental osteoporosis was induced by bilateral ovariectomy. To estimate the healing of defects, the formation of the new bone tissue and the resorption of Bio-Oss particles after two and eight weeks of defect healing, we performed biochemical, histological, immunohistochemical, histomorphometric and scanning electron microscopy analyses. Our results showed that locally applied cholecalciferol and alfacalcidol delay early bone healing of defect in ovariectomized rat model. Both of the examined vitamins, particularly alfacalcidol, decreased the resorption of Bio-Oss particles in defects. Only alfacalcidol affected the formation of histologically normal new bone tissue with a good integration into the surrounding osteoporotic bone. Cholecalciferol allowed callus mineralization and led to the formation of new bone with high quantity, with characteristics, similar to those of the control group. Our results suggested that alfacalcidol and cholecalciferol may be promising tools for the local treatment of osteoporotic fractures, but the goal of application should specify the choice of vitamin D form, as well as the composition of scaffolds, with which they are applied.

Bioactive Biomaterials: Potential for Application in Bone Regenerative Medicine

Critical-sized bone defects can be repaired by using bone tissue engineering (BTE) procedures which rely on the combined use of cells, scaffolds and biologically active molecules. Based on their bioreactivity, biomaterials can be bioinert or bioactive. Bioinert biomaterials cause fibrous capsule formation upon implantation which favors the appearance of micromovements in the implant-tissue interface so the prosthesis fails. Bioactive biomaterials elicit a specific biological response thus avoiding fibrous layer formation and are able to interact with the biological environment. Bioactive biomaterials can be natural (bovine bone mineral matrix, hyaluronic acid, collagen, gelatin, fibrin, agarose, alginate, chitosan, silk) or synthetic (ceramics, metals, polymers, hydrogels and composites). Ceramics (bioactive glasses, glass–ceramics, calcium phosphates ceramics and cements) are most frequently used among these biomaterials due to similarity with the bone mineral phase. Another advantage from the use of ceramics is the presence of biologically active hydroxycarbonate apatite layer formed on the surface of these biomaterials, which represents the bonding interface with the tissues. Bioactive biomaterials have wide application as medical devices and in drug delivery systems. Since cells cannot survive without an adequate blood supply, future directions in bioactive biomaterials applications lies in the construction of bioactive and biodegradable 3D scaffolds that have osteogenic and angiogenic features. A possible alternative to improve osteogenic and angiogenic potential of the applied biomaterials is to incorporate bioactive biomolecules (e.g. growth factors) into the scaffold. One of the future perspectives in this area is the construction of smart biomaterials that respond to their environment in predetermined way regarding the protein release, thus allowing release initiated by microenvironmental conditions.