Chitosan/hydroxyapatite hybrid scaffold for bone tissue engineering.

BACKGROUND: To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of such techniques remain the availability of the autologous material and the second surgical site, inducing pain and morbidity. OBJECTIVE: Our aim was to investigate the biocompatibility in vitro of three dimensions hybrid biodegradable scaffolds combining osteoconductive properties of hydroxyapatite and anti-inflammatory properties of chitosan. METHODS: Hybrid scaffolds were characterized by microscopic observations, equilibrium swelling ratio and overtime weight loss measurements. In vitro studies were performed using primary human bone cells cultured for 7, 14 and 21 days. Cell viability, proliferation, morphology and differentiation through alkaline phosphatase (ALP) activity measurement were assessed. RESULTS: Characterization of our scaffolds demonstrated porous, hydrophilic and biodegradable characteristics. In vitro studies showed that these scaffolds have induced slight decrease in cell death and proliferation comparing to the culture plastic substrate control condition, as well as increased short term osteoinductive properties. CONCLUSIONS: In this study, we have provided evidence that our hybrid hydroxyapatite/chitosan scaffolds could be suitable for bone filling.

Interfacial reactions of glasses for biomedical application by scanning transmission electron microscopy and microanalysis.

Short-term physico-chemical reactions at the interface between bioactive glass particles and biological fluids are studied for three glasses with different bioactive properties; these glasses are in the SiO(2)-Na(2)O-CaO-P(2)O(5)-K(2)O-Al(2)O(3)-MgO system. Our aim is to show the difference between the mechanisms of their surface reactions. The relation between the composition and the bioactive properties of these glasses is also discussed. The elemental analysis is performed at the submicrometer scale by scanning transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy. After different immersion times (ranging from 0 to 96 h) of bioactive glass particles in a simulated biological solution, results show the formation of different surface layers at the glass periphery in the case of two bioactive glasses (A9 and BVA). For the third glass (BVH) we do not observe any surface layer formation or any modification of the glass composition. For the two other glasses (A9 and BVA), we observe the presence of different layers: an already observed (Si, O, Al) rich layer at the periphery, a previously demonstrated thin (Si, O) layer formed on top of the (Si, O, Al) layer and a (Ca, P) layer. We determine the different steps of the mechanisms of the surface reactions, which appear to be similar in these glasses, and compare the physico-chemical reactions and kinetics using the different immersion times. The A9 glass permits the observation of all important steps of the surface reactions which lead to bioactivity. This study shows the important relationship between composition and bioactivity which can determine the medical applicability of the glass.

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings.

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.

Sensitivity of bacterial biofilms and planktonic cells to a new antimicrobial agent, Oxsil 320N.

The effective concentrations of disinfectants were determined for planktonic bacteria using the norms EN 1040 and NF T 72-150. This concentration corresponds to biocide efficacy after 5 min of contact, followed by neutralization. However, micro-organisms often colonize a substratum and form microcolonies or biofilms where they are enclosed in exopolymer matrices. Biofilms are commonly resistant to a broad range of antimicrobial agents, and resistance mechanisms involve exopolymer matrices, changes in gene expression and metabolic alterations. Due to these different resistance mechanisms, it is difficult to select and titrate antimicrobial agents to be effective against biofilms. In this context, SODIFRA developed a new disinfectant, Oxsil 320N (French patent 94 15 193). Oxsil 320N is an association of three active principles: hydrogen peroxide, acetic acid/peracetic acid and silver. This biocide was tested on planktonic bacteria and on 24-h biofilms formed on AISI 304 stainless steel surfaces. The effective concentration of Oxsil 320N was also determined on biofilms using SODIFRA recommendations (without neutralization of the biocide). Data showed that the antimicrobial efficacy measured on planktonic bacteria is not a reliable indicator of performance when biofilm is present. When biofilms were exposed to Oxsil 320N, the concentration needed to achieve a 10(5)-fold decrease in concentration was 10 times higher than that for bacterial suspensions (0.313% Oxsil 320N). An effective concentration of Oxsil 320N of 3.13% was required.

Preparation and characterization of an electrodeposited calcium phosphate coating associated with a calcium alginate matrix.

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

The rate of fibrinolysis is increased by free retraction of human gingival fibroblast populated fibrin lattices.

We previously demonstrated that human gingival fibroblasts (HGF), but not their dermal counterparts, when seeded in retracting fibrin lattices induced intense fibrinolysis that was observed at the earliest stages of contraction and led to complete matrix degradation by day 7 of culture. Our aim was to examine the influence of mechanical forces in such fibrinolytic processes. HGF were seeded in retracting (R) e.g. free floating or non retracting (NR) e.g. anchored fibrin lattices (FL). Cultures were analysed from day 1-12 by phase contrast microscopy and scanning electron microscopy (s.e.m.). Levels of fibrin degradation products (FDP) and tissue plasminogen activator (tPA) accumulating in culture media were quantified by ELISA. Urokinase (uPA) and gelatinase A (MMP2) were identified by zymographic techniques. At the s.e.m. level, vacuolization around some HGF was noticed at the earliest stages of culture for RFL and complete degradation of lattices occurred at day 7. Formation of lysed matrix cavity was far less intense in NRFL even after 12 days of culture. FDP amounts at day 4 of culture were equal to 79 +/- 14 and 8.5 +/- 0.6 micrograms/10(5) cells for RFL and NRFL, respectively; tPA levels were equal to 5.8 +/- 0.6 (RFL) and 2.1 +/- 0.3 ng/10(5) cells (NRFL) and differences were still evident at day 7. The kinetics of tPA production were identical in either retracting fibrin or collagen lattices. On the contrary, uPA and proMMP2 productions were similar in RFL and NRFL. Isometric forces, but not the matrix support, were responsible for accelerated tPA production and fibrinolysis in HGF populated lattices.

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures.

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen of fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by epsilon-amino-caproic acid (epsilon ACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva.

The influence of tissue pretreatment on the immunohistochemical demonstration of type I and III collagens and tenascin in fetal human tooth germs.

The influence of tissue pretreatment on the PAP immunostaining for type I and III collagens and tenascin was studied in formalin-fixed and paraffin-embedded human tooth germs at the 24th and 25th weeks of fetal life. Three variables were considered: the type of buffer used (PBS or Tris), pepsin digestion and the use of normal serum as a blocking agent prior to immunostaining. All three proteins needed an enzymatic digestion to be intensely revealed. Pepsin promoted, even at low concentrations, an intracellular staining of type I collagen in the secretory odontoblasts and in the pulpal fibroblasts. Normal serum partially blocked unspecific immunoreaction when polyclonal rabbit antibodies were used. The Tris buffer increased the staining intensity of the three macromolecules and revealed an unusual tenascin-like immunoreactivity in the ameloblasts. This study demonstrated that pepsin digestion and the use of normal serum and different buffers may influence the immunoreactivity of ECM proteins.

[The deciduous tooth. Diagnosis in pedodontics and its problems. The role of histopathology]. / La dent temporaire: le diagnostic en pédodontie et ses difficultés. Apport de l'histopathologie.

Accurate diagnosis of pulpal and periodontal pathology in children is an intricate question, in fact of mixed dentures and physiological and symptomatological dissimilarity between permanent and deciduous teeth. Some figures of most frequently encountered tissue modifications of deciduous teeth are proposed in order to be considered in the way of diagnosis and therapeutics in pedodontics.