The survival and proliferation of fibroblasts on orthodontic miniscrews with different surface treatment: an in vitro study.

It is of fundamental importance for prosthodontic and orthodontic applications that there is a short osseointegration time of dental implants without inflammation of the surrounding tissue. In addition to the chemical properties of the implant material, the surface morphology is an equally critical parameter. The objective of this work was to study the effect of two simple surface treatments on the survival and proliferation of fibroblasts. Three groups of orthodontic miniscrews (Mondeal) were used. One group was given an airflow (EMS, Schweiz) treatment, the second was sand-blasted in the area of the threading and a third group served as a control. After preparation sterilised screws were cultured in vitro with fibroblasts (L-929). The metabolic cell activity on the implant surface was determined after 24, 48 and 120 hours using the alamarBlue assay and a count of DAPI labelled fibroblasts was performed with a fluorescence microscope. After 24 hours, but not at 48 hours and 120 hours, the metabolic activity of the fibroblasts was slightly decreased for the airflow screw group. Generally, no significant difference was found regarding metabolic activity and proliferation of fibroblasts within the different groups.

Wound management after the application of bone grafting substitutes in the orofacial region.

Surgical dressing after the application of bone grafting material depends on the type and size of the defect. A complete and tension-free wound closure has proved to be successful. In this context the infection problem needs special attention. Bone graft substitutes with an adequate surface structure, porosity and chemical properties, in combination with sufficient blood circulation, hold osteoconductive potential. They serve as a guide rail for the osteoblast-induced formation of new bone tissue, which at best may lead to complete replacement of the grafting material.

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

The biodegradation of hydroxyapatite bone graft substitutes in vivo.

Hydroxyapatite (HA) ceramics are widely used for bone reconstruction. They are osteoconductive and serve as structural scaffolds for the deposition of new bone. Generally, scaffold materials should be degradable as they affect the mechanical properties of the reconstructed bone negatively. Degradation by osteoclasts during the bone remodelling process is desirable but often does not take place. In the current study we analysed by light microscopy the degradation of two granular HA implants in critically sized defects in the mandibula of Goettingen mini-pigs five weeks after implantation. Bio-Oss consists of sintered bovine bone and NanoBone is a synthetic HA produced in a sol-gel process in the presence of SiO2. We found that both biomaterials were degraded by osteoclasts with ruffled borders and acid phosphatase activity. The osteoclasts created resorption lacunae and resorptive trails and contained mineral particles. Frequently, resorption surfaces were in direct contact with bone formative surfaces on one granule. Granules, especially of NanoBone, were also covered by osteoclasts if located in vascularised connective tissue distant from bone tissue. However, this usually occurred without the creation of resorption lacunae. The former defect margins consisted of newly formed bone often without remnants of bone substitutes. Our results show that the degradation of both biomaterials corresponds to the natural bone degradation processes and suggest the possibility of complete resorption during bone remodelling.

Bone functions and the requirements for bone grafts and substitutes in the orofacial region.

Bone is the largest calcium storage, has distinctive plasticity and adaptability and is part of the supporting tissue. An adequate composition is thus necessary. The bone matrix consists of organic and anorganic structures. Osteoblasts, osteoclasts and osteocytes are responsible for bone formation, resorption and metabolism. The periosteum, endosteum and bone tissue are a functional unit and provide protection, nutrition and growth. Bone is subject to continuous remodelling.

Critical considerations on the diagnostic appraisal, adaptation and remodelling of bone graft substitutes.

The diagnostic assessment of skeletal defects has a long-standing tradition. As a result of the development of new bone grafting materials, the demands on diagnostic assessment have also increased. The mode and quality of diagnostic appraisal are crucial to further clinical use and outcome prediction. Alongside traditional clinical and biological techniques, molecular biological methods have gained a broad scope of application and will be used even more frequently in the future.

Evidence for perforin-like activity associated with earthworm leukocytes.

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms.

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüssler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem. 272, 20 884-20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immune system destroy non-self components.

Cell adhesion and the immune system: a case study using earthworms.

In the earthworm's immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non-self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effectors show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK-like effectors. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization.

Isolation and characterization of earthworm hemolysins and agglutinins.

Hemolytic activity in coelomic fluid of Eisenia fetida (ECF) is due to three proteins H1, H2, H3 with molecular weights of 46, 43 and 40 kD, respectively. These proteins were isolated by preparative PAGE. H1 and H2 were shown to be stable in SDS and alpha-2-ME whereas H3 splits into two fragments with molecular weights of 18 and 21 kD after SDS treatment. IEF indicates that each protein consists of different isoforms with pIs between 5.1 and 6.2 H3 was demonstrated to be a bifunctional protein that can lyse and agglutinate erythrocytes. At 56 degrees C hemolytic activity of all three proteins was inactivated, but the agglutination activity of H3 was stable. Intracoelomic injection of erythrocytes reduced the number of hemolysins from three to two. Monospecific antisera were raised against the isolated hemolysins H1,2 and 3. The use of these antibodies and of carbohydrates as inhibitors of the biological activity of the molecules demonstrates the close structural relationship of agglutinins and hemolysins in the CF of E. fetida.

Interaction of earthworm hemolysin with lipid membranes requires sphingolipids.

Lytic activity in the coelomic fluid of earthworm (Eisenia fetida fetida) has been ascribed to eiseniapore, a hemolytic protein of 38 kDa. Since receptors for eiseniapore on target cell membranes are not known, we used lipid vesicles of various composition to determine whether specific lipids may serve as receptors. Lytic activity of eiseniapore was probed by the relief of fluorescence dequenching from the fluorophore 8-aminonaphthalene-1,3, 6-trisulfonic acid originally incorporated into the vesicle lumen as a complex with p-xylene-bis-pyridinium bromide. Hemolysin binds to and disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcholine or galactosyl as well as the ceramide backbone, e.g. sphingomyelin, are present. Cholesterol enhances eiseniapore lytic activity toward sphingomyelin-containing vesicles probably due to interaction with sphingomyelin. Leakage of vesicles was most efficient when the lipid composition resembled that of the outer leaflet of human erythrocytes. Presumably, an oligomeric protein pore formed by six monomers is responsible for leakage of sphingomyelin-containing vesicles. The secondary structure of eiseniapore did not change upon binding to lipid membranes. The lytic activity of eiseniapore was completely abolished after its denaturation or after preincubation with polyclonal antibodies. Our results suggest that the presence of specific sphingolipids is sufficient to mediate lytic activity of eiseniapore. This action contributes to our understanding of earthworm immune responses.

Crossreactivity of hemolytic and hemagglutinating proteins in the coelomic fluid of lumbricidae (Annelida).

Oligospecific antisera against the hemolytic and hemagglutinating compounds of the coelomic fluid (CF) of the earthworm species Eisenia fetida, Lumbricus terrestris and Aporrectodea caliginosa were prepared. The proteins were bound to the membranes of erythrocytes and injected into rabbits. By the use of these antisera the following results were obtained: 1) The antisera RaL T and RaAC reached a titer of 1:64,000, the detection limit of RaEF was 1:512,000. RaEF was demonstrated to be oligospecific only against three hemolytic proteins by Western blotting. 2) RaEF is able to inhibit the biological activity not only of hemolysins (E. fetida, A. caliginosa) but also of hemagglutinins (L. terrestris, L. rubellus, D. rubidus). 3) Complex carbohydrates, but not simple sugar compounds, are able to inhibit hemolytic and agglutinating activities in the CF of the investigated species. Fet. and alpha 1ac. GP were demonstrated to be strong inhibitors both of the hemolytic and of the hemagglutinating activity, whereas N-acetylglucosamine was only able to inhibit the hemagglutinating activity. 4) The investigated compounds were shown to crossreact in ELISA experiments. 5) By the use of Western blotting, the crossreacting molecules in the CF of the investigated lumbricid species were identified.

Cytotoxic activity in the coelomic fluid of the annelid Eisenia foetida Sav.

The annelid Eisenia foetida not only causes hemolysis of red blood cells of several vertebrate species, but also has a toxic effect on a variety of cell types, such as chicken fibroblasts, guinea-pig polymorphonuclear leukocytes and insect hemocytes. However, it has no influence on the vitality of the coelomocytes of Lumbricus terrestris and other lumbricides, nor on the hemocytes of the snail Helix pomatia, the mussels Anodonta cygnea and Unio tumidus, free cells of the turbellarian Euplanaria sp. or whole Rhabditis oxycerca (nematode) and the protozoons Paramaecium caudatum and an amoeba of the Proteus-type. By electrofocussing the hemolytic activity of pooled coelomic fluid was separated into 7 hemolytic bands. Three of them are cytotoxic. The cytotoxic effect is a result of the destruction of the cell membrane, as shown by measuring the release of lactate dehydrogenase (LDH). The bacteriostatic and bacteriocidal action of the coelomic fluid of E. foetida against a broad spectrum of gram positive and gram negative bacteria was tested. An antibacterial activity could be observed only against Proteus vulgaris and Bacillus megaterium. It was noted that the so-called Eisenia foetida-factor acts on an antigenic structure at the cell surface when anti-sheep-E-antibody was used under competitive conditions. The binding between the Eisenia foetida-factor and this membrane structure is relatively strong as it cannot be removed by subsequent treatment with anti-sheep-E-antibody or 2 M KCl.

Comparative analysis of hemolytic and hemagglutinating activities in the coelomic fluid of Eisenia foetida and Lumbricus terrestris (Annelida, Lumbricidae).

The CF of Lumbricus terrestris and Eisenia foetida was analyzed with regard to the protein patterns by isoelectric focusing. The CF of E. foetida results in a variety of protein bands; after separation: seven of these with isoelectric points at pH 5.1, 5.2, 5.4, 5.6, 5.8, 6.0 and 6.2 cause the hemolysis of red blood cells. The protein pattern of normal CF of L. terrestris is relatively poor. Most of the protein bands are located at the top of the separation (pH less than 5.1) near the anode. However, the number of protein bands is drastically enhanced within 24 hours after an intracoelomic injection of foreign material. Some of the induced proteins show a similar pattern at comparable pH ranges to the CF of E. foetida. The induced proteins with isoelectric points at pH 4.7, 5.0, 5.1, 5.2, 5.4, 5.8, 6.0 and 6.2 effect the hemolysis of vertebrate erythrocytes. After stimulation with rabbit erythrocytes an enhanced bactericidal and bacteriostatic activity against Proteus vulgaris can be proven and seems to be related to the induced hemolytic proteins. The antibacterial activity can be completely adsorbed by incubation of the CF with rabbit erythrocytes, which indicates its binding ability to marker molecules on the erythrocytes surface.