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  • 1.
    Mladenovic, Zivko
    Umeå University, Faculty of Medicine, Department of Odontology.
    Biological interface of bone graft substitute materials: experimental studies on interactions between biomaterials and bone cells2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bone graft substitute materials are becoming more common as alternative therapy strategies when bone defects in patients need to be treated. The interaction between bone cells and biomaterials occur at the surface of the materials. A great deal is known about the importance of surface topography and physic-chemical properties of biomaterials. It is also known that cells require proteins in order to interact with biomaterials. Less is known about how material properties and proteins interact forming the biological interface that cells will be exposed to, and that might determine if new bone is formed or not in the patient. The overall aim of the present thesis was to systematically investigate bone graft substitute material surface reactions and the interface in order to better understand how biomaterials may promote bone formation. Bio-Oss (BO) is a commonly used bone graft substitute material in reconstruction of periodontal and dentoalveolar bone defects. BO is mainly considered to be “osteoconductive”, but we could show that it does interact with a biological fluid (α-MEM cell culture medium) through dissolution/precipitation reactions. A significant reduction of calcium and phosphate levels in the medium was obtained even with low concentrations of BO. A release of silicon from the material was also demonstrated. An osteogenic response was seen in close contact to the BO particles when cultured with different types of pre-osteoblastic cells (Paper I). X-Ray Photoelectron Spectroscopy (XPS) with fast-frozen sample technique was used to further characterize the surface of BO, Frios Algipore (AP) and 45S5 Bioglass (BG). These three bone graft substitute materials are used as “model systems”, because they have all demonstrated newly formed bone on the surface after implantation in patients. From the XPS analysis it can be concluded that AP and BG acquired a positively charged surface while BO gained a negatively charged surface. Only AP and BG adsorb organic components (amino acids) from the medium (Paper II). Next we investigated initial surface reactions and the formation of a biological interface in the presence of proteins (serum) for the three biomaterials. The major findings were that in the presence of proteins BO underwent a surface charge reversal, all three biomaterials adsorbed proteins on the surface and all three biomaterials altered the chemical composition of the cell culture medium (Paper III-IV). Silicon (Si), which was released from BO as well as from BG, is interesting in relation to bone health. Positive effects of BG Si dissolution products on osteoblasts have been reported earlier. In the present study inhibitory interactions of Si on the RANK/RANKL/OPG signaling pathway as well as with gap junction intercellular communication in vitro are reported. These new findings implicate that Si could potentially be beneficial for patients with imbalance in bone remodeling (osteoporosis) and treatments of bone defects (Paper V). In conclusion, biomaterials of different origins interact with a solution resembling the extracellular tissue fluid. The dissolution-precipitation reactions are influenced by the material concentration used and should be taken into consideration when designing experiments and when biomaterials are used clinically. The presence of proteins will influence surface reactions, the formation of the biological interface and have implications on cellular responses. Possible dissolution products from the biomaterials should be investigated.  Si, a dissolution product, is shown to have an inhibitory effect on osteoclastogenesis and bone resorption in vitro. Potential clinical value of Si in treatment of patients with bone defects should be further investigated.

  • 2.
    Mladenovic, Zivko
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Willman, Britta
    Umeå University, Faculty of Medicine, Department of Odontology.
    Shahabi, Kaveh
    Umeå University, Faculty of Medicine, Department of Odontology.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology.
    Silicon inhibits signaling pathways and cell-cell communication important for osteoclast formation and bone resorption in vitroManuscript (preprint) (Other academic)
    Abstract [en]

    Silicon containing materials are used in bone regeneration, and some of the materials, e.g. Bioactive glass 45S5 (BG), release silicon (Si) ions to the surrounding tissue after implantation. The role of Si in bone biology is debated; nevertheless findings suggest that Si is beneficial for bone formation. A majority of the experimental studies on Si and bone have focused on osteoblasts. The effects of Si on osteoclast formation and function have not been directly addressed. In the present study, we show that ionic dissolution extract from BG inhibit osteoclast bone resorption in an organ culture system as well as osteoclast formation in a mouse bone marrow system and in the RAW264.7 cell line. Si containing cell culture medium was prepared to address the issue whether or not the inhibitory effects with BG dissolution extract were Si ion dependent. The results suggest that the inhibitory effects of Si act directly on osteoclast precursors, by interactions with the RANK/RANKL/OPG signaling pathway as well as with gap junction intercellular communication. However, regulation via osteoblasts cannot be excluded. The inhibitory effect of Si on osteoclasts could be useful for future therapies or treating bone loss in patients, provided that molecular mechanisms are established.

  • 3.
    Mladenovic, Zivko
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sahlin-Platt, Annika
    Umeå University, Faculty of Medicine, Department of Odontology, Ortodontics.
    Andersson, Britta
    Department of Medicine Solna, Karolinska Institutet, S-171 76 Stockholm,Sweden.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology.
    In vitro study of the biological interface of Bio-Oss: implications of the experimental setup2013In: Clinical Oral Implants Research, ISSN 0905-7161, E-ISSN 1600-0501, Vol. 24, no 3, p. 329-335Article in journal (Refereed)
    Abstract [en]

    Objectives To systematically investigate the biological interface of Bio-Oss by analysing dissolution–precipitation behaviour and osteogenic responses using in vitro experimental systems.

    Material and methods Different concentrations (1–100 mg/ml) of Bio-Oss were incubated in cell culture medium for 24 h before elemental concentrations for calcium, phosphorus and silicon in the medium were analysed with inductive coupled plasma-optical emission spectroscopy. Radioactive calcium-45 isotope labelling technique was used to study possible precipitation of calcium on the Bio-Oss particle. Biological interface of Bio-Oss was studied in osteogenic experiments using mineralization medium and three different sources of cells (primary mouse bone marrow stromal cells, primary rat calvarial cells and MC3T3-E1 mouse pre-osteoblast cell line). Cells were fixed and stained with Toulidine blue, von Kossa or Alizarin Red staining for confirmation of extracellular matrix mineralization.

    Results Elemental analysis of the cell culture medium demonstrated a significant decrease of calcium and phosphorus and a dose-dependent release of silicon to the medium after incubation with Bio-Oss. A significant decrease of calcium and phosphorus in the medium occurred even at low concentrations of Bio-Oss. Uptake of calcium on the Bio-Oss particle was confirmed with radioactive calcium-45 isotope labelling technique. In osteogenic experiments with Bio-Oss (<1 mg/ml), matrix mineralization around the Bio-Oss particles were demonstrated in all three cell types with von Kossa and Alizarin Red staining.

    Conclusion Dissolution–precipitation reactions occur at the surface of Bio-Oss, and osteogenic responses are seen at the biological interface. The concentration of Bio-Oss is a key factor for the experimental in vitro results, and may also have implications for the clinic.

  • 4.
    Mladenovic, Zivko
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sahlin-Platt, Annika
    Umeå University, Faculty of Medicine, Department of Odontology.
    Andersson,, Martin
    Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden.
    Shchukarev, Andrey
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology.
    Investigation of surface reactions and solid-solution interfaces of three bonegraft substitute materials incubated in cell culture mediumArticle in journal (Other academic)
  • 5.
    Mladenovic, Zivko
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sahlin-Platt, Annika
    Umeå University, Faculty of Medicine, Department of Odontology.
    Bengtsson, Åsa
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology.
    Shchukarev, Andrey
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Surface characterization of bone graft substitute materials conditioned in cell culture medium2010In: Surface and Interface Analysis, ISSN 0142-2421, E-ISSN 1096-9918, Vol. 42, no 6-7, p. 452-456Article in journal (Refereed)
    Abstract [en]

    Biomaterials are widely used in clinical practice as bone graft substitutes for treating patients with bone defects. A molecular level understanding of the chemical processes at the interface between the biomaterial and the biological environment is crucial to succeed in tissue regeneration and to predict the treatment outcome. In this study, we used three different bone graft substitute materials (BioGlass 45S5—synthetic, Bio-Oss—bovine derived and Algipore—derived from algae) which were incubated in an α-minimum essential medium (α-MEM) during 1, 3 and 7 days. Initial surface composition of the biomaterials and the chemistry of their solid–solution interface were monitored by XPS with a fast-frozen samples technique. The XPS analysis showed that the equilibrium at the solid-solution interface is reached within 24 h. The Na/Cl atomic ratio at equilibrium indicates a negatively charged surface for Bio-Oss. In contrast, the other two materials gained a positive surface charge, which resulted in pronounced adsorption of amino acids at the interface from the medium. The surface chemical reconstruction and charge generation mechanism responsible for this effect are discussed with regard to bulk composition of the materials and possible proliferation and differentiation cell patterns that could be expected at the interface. Copyright © 2010 John Wiley & Sons, Ltd.

  • 6.
    Sahlin-Platt, Annika
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Cell Biology.
    Örtengren, Ulf
    Mladenovic, Zivko
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Cell Biology.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Cell Biology.
    Effects of Dyract AP and released ionic products on periodontal ligament cells and bone marrow cultures2008In: Dental Materials, ISSN 0109-5641, E-ISSN 1879-0097, Vol. 24, no 12, p. 1623-1630Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: The aim of this work was to investigate the release of inorganic ionic products from specimens of the polyacid-modified composite resin Dyract AP (DAP) and furthermore, to analyze the biological effect of DAP and the medium extract in human periodontal ligament (PDL) cells and mouse bone marrow cell (BMC) cultures.

    METHODS: Ion release from DAP specimens immersed in cell culture medium was analyzed with inductively coupled plasma optical emission spectroscopy (ICP-OES). Cells were cultured with either DAP specimens or with DAP media extract and effects on cell proliferation, osteoblastic gene expression and mineralization capacity were analyzed with direct-contact tests, neutral red (NR) uptake, quantitative real-time PCR and a bone nodule formation assay.

    RESULTS: ICP-OES analysis of DAP extract demonstrated a significant increase in fluoride, strontium and silica. PDL cells demonstrated normal growth pattern in the direct-contact tests with the material. DAP extracts produced a dose-dependent stimulation of cell proliferation and concomitant inhibition of osteoblast specific markers and nodule formation.

    SIGNIFICANCE: The compomer may have possible bioactive properties due to ions leaching out from the filler component.

  • 7.
    Shchukarev, Andrey
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mladenovic, Zivko
    Umeå University, Faculty of Medicine, Department of Odontology, Ortodontics.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology, Ortodontics.
    Surface characterization of bone graft substitute materials conditioned in cell culture medium. 2. Protein adsorption2012In: Surface and Interface Analysis, ISSN 0142-2421, E-ISSN 1096-9918, Vol. 44, no 8, p. 919-923Article in journal (Refereed)
    Abstract [en]

    Three bone graft substitute materials (Bioglass 45S5, Bio-Oss (R) and Algipore (R)) were conditioned in a-minimum essential medium (alpha-MEM), with the addition of 10% fetal bovine serum (FBS), for 1 and 7?days. The chemistry of their solid-solution interface was monitored by X-ray photoelectron spectroscopy, using fast-frozen sample technique, and compared to that reported for original alpha-MEM. FBS added to the biological medium causes significant changes in the interface after only 1day of conditioning. Interfacial chemical composition and N 1s spectra show immediate adsorption of proteins at the surface of all three biomaterials, independent of their surface charge and chemical composition. However, the atomic ratio C/N and the C 1s spectra indicate a different orientation of adsorbed serum proteins, which is dependent on the particle's surface charge. Moreover, the adsorption of serum proteins at the surface of Bio-Oss causes a charge reversal at the interface, as evidenced by the change in the atomic ratio of Na/Cl. In addition to the particle's surface charge, the formation of the protein interfacial layer at the surface of the biomaterial seems to be the second major phenomenon important for subsequent cell recognition and the initiation of biomineralization. Copyright (c) 2012 John Wiley & Sons, Ltd.

  • 8.
    Shchukarev, Andrey
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ransjö, Maria
    Umeå University, Faculty of Medicine, Department of Odontology.
    Mladenović, Živko
    Umeå University, Faculty of Medicine, Department of Odontology.
    To build or not to build: the interface of bone graft substitute materials in biological media from the view point of the cells2011In: Biomaterials science and engineering / [ed] Rosario Pignatello, Published by InTech , 2011, p. 287-308Chapter in book (Refereed)
    Abstract [en]

    These contribution books collect reviews and original articles from eminent experts working in the interdisciplinary arena of biomaterial development and use. From their direct and recent experience, the readers can achieve a wide vision on the new and ongoing potentials of different synthetic and engineered biomaterials. Contributions were not selected based on a direct market or clinical interest, than on results coming from very fundamental studies which have been mainly gathered for this book. This fact will also allow to gain a more general view of what and how the various biomaterials can do and work for, along with the methodologies necessary to design, develop and characterize them, without the restrictions necessarily imposed by industrial or profit concerns. The book collects 22 chapters related to recent researches on new materials, particularly dealing with their potential and different applications in biomedicine and clinics: from tissue engineering to polymeric scaffolds, from bone mimetic products to prostheses, up to strategies to manage their interaction with living cells.

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