Maria Helena Fernandes

Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, β-glycerophosphate and dexamethasone on osteoblastic differentiation

This work analyses the proliferation/differentiation behaviour of human bone marrow cells cultured in alpha-minimum essential medium supplemented with 10% foetal bovine serum (standard medium) and in the presence of ascorbic acid (AA, 50 microg ml(-1)), beta-glycerophosphate (betaGP, 10 mmol) and dexamethasone (Dex, 10 nmol) under selected experimental conditions. Cultures were compared concerning cell morphology, cell growth, ALP activity and ability to form calcium phosphate deposits. Cells growing in the various experimental conditions proliferated gradually with the incubation time and presented high ALP activity. Cultures grown in standard medium and in the presence of either AA or Dex failed to form calcium phosphate deposits. Cultures grown in the presence of betaGP, betaGP + AA and betaGP + AA + Dex, i.e. in the presence of a source of phosphate ions, showed the formation of a mineralised extracellular matrix. The presence of Dex resulted in a significant induction in the ALP activity and ability to form mineral deposits. The behaviour of the various cell cultures is in agreement with previous studies stating a reciprocal and functionally coupled relationship between proliferation and differentiation, i.e. cultures grown in a medium containing betaGP presented a less proliferative but more differentiated osteoblastic cell population, as compared to cultures lacking the mineralisation process.

Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in α-MEM and in DMEM

Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic differentiation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modification (alpha-MEM) and Dulbeccos modified Eagles medium (DMEM). Cultures were grown for 35 d and compared concerning morphologic appearance on scanning electron microscopy (SEM), cell viability/proliferation, total protein content, activity of alkaline phosphatase (ALP) and ability to form calcium phosphate deposits. Results showed that cell proliferation was similar in cultures grown in the two media but ALP activity and ability to form mineralised deposits were lower in DMEM cultures. In both experimental situations, osteoblastic parameters were strongly reduced on cell passage, particularly from the first to the second subculture. In the experimental conditions used (presence of ascorbic acid, sodium beta-glycerophosphate and dexamethasone in the primary and secondary cultures), osteoblastic differentiation was observed in the first and second subcultures grown in alpha-MEM and in the first subculture grown in DMEM. These results underline the importance of the definition of the experimental conditions in studies involving bone cell cultures.

Physical, chemical and in vitro biological profile of chitosan hybrid membrane as a function of organosiloxane concentration

We attempted to prepare chitosan-silicate hybrid for use in a medical application and evaluated the physico-chemical properties and osteocompatibility of the hybrids as a function of gamma-glycidoxypropyltrimethoxysilane (GPTMS) concentration. Chitosan-silicate hybrids were synthesized using GPTMS as the reagent for cross-linking of the chitosan chains. Fourier transform infrared spectroscopy, (29)Si CP-MAS NMR spectroscopy and the ninhydrin assay were used to analyze the structures of the hybrids, and stress-strain curves were recorded to estimate their Youngs modulus. The swelling ability, contact angle and cytocompatibility of the hybrids were investigated as a function of the GPTMS concentration. A certain fraction of GPTMS in each hybrid was linked at the epoxy group to the amino group of chitosan, which was associated with the change in the methoxysilane group of GPTMS due to hybridization. The cross-linking density was around 80% regardless of the volume of GPTMS. As the content of GPTMS increased, the water uptake decreased and the hydrophilicity of the hybrids increased except when the content exceeded amolar ratio of 1.5, when it caused a decrease. The values of the mechanical parameters assessed indicated that significant stiffening of the hybrids was obtained by the addition of GPTMS. The adhesion and proliferation of the MG63 osteoblast cells cultured on the chitosan-GPTMS hybrid surface were improved compared to those on the chitosan membrane, regardless of the GPTMS concentration. Moreover, human bone marrow osteoblast cells proliferated on the chitosan-GPTMS hybrid surface and formed a fibrillar extracellular matrix with numerous calcium phosphate globular structures, both in the presence and in the absence of dexamethasone. Therefore, the chitosan-GPTMS hybrids are promising candidates for basic materials that can promote bone regeneration because of their controllable composition (chitosan/GPTMS ratio).

Si3N4-bioglass composites stimulate the proliferation of MG63 osteoblast-like cells and support the osteogenic differentiation of human bone marrow cells☆

The invitro osteocompatibility of a n ovel Si 3N4-bioglass composite (70–30% weight proportion) with improved mechanical properties (fracture toughness=4.4 MPa m 1/2 ; bending strength=383747 MPa) is reported. Immersionof the composite samples in culture medium (30 min to 7 days) resulted in rapid protein adsorption to the surface and, also, dissolution of the intergranular phase of bioglass (time-dependent process) with the formation of different size cavities. ‘‘As-received’’ and pre-treated material samples presented a similar behaviour concerning the proliferation of MG63 osteoblast-like cells, evaluated during a 5-day culture period. Seeded materials showed a higher cell growth rate as compared to cultures performed onthe standard plastic culture plates. To assess the osteogenic potential of the composite, ‘‘as-received’’ material samples were seeded with human bone marrow cells and cultured for 35 days in experimental conditions that favour the development of the osteoblastic phenotype. The cell adhesion process was similar to that observed in control cultures. Cells successfully adapted to the irregularities of the surface and were able to grow towards inside the cavities; in addition, osteogenic differentiation occurred with the formation of abundant cell-mediated mineralised deposits. Results suggest that this Si3N4-bioglass composite seems to be a promising candidate for high-stress medical applications.

Preparation and characterization of collagen‐nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications

Recent efforts of bone repair focus on development of porous scaffolds for cell adhesion and proliferation. Collagen-nanohydroxyapatite (HA) scaffolds (70:30; 50:50; and 30:70 mass percentage) were produced by cryogelation technique using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide as crosslinking agents. A pure collagen scaffold was used as control. Morphology analysis revealed that all cryogels had highly porous structure with interconnective porosity and the nanoHA aggregates were randomly dispersed throughout the scaffold structure. Chemical analysis showed the presence of all major peaks related to collagen and HA in the biocomposites and indicated possible interaction between nanoHA aggregates and collagen molecules. Porosity analysis revealed an enhancement in the surface area as the nanoHA percentage increased in the collagen structure. The biocomposites showed improved mechanical properties as the nanoHA content increased in the scaffold. As expected, the swelling capacity decreased with the increase of nanoHA content. In vitro studies with osteoblasts cells showed that they were able to attach and spread in all cryogels surfaces. The presence of collagen-nanoHA biocomposites resulted in higher overall cellular proliferation compared to pure collagen scaffold. A statistically significant difference between collagen and collagen-nanoHA cryogels was observed after 21 day of cell culture. These innovative collagen-nanoHA cryogels could have potentially appealing application as scaffolds for bone regeneration.

Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosis

Marine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.

Effects of AISI 316L corrosion products in in vitro bone formation

Rat bone marrow cells were cultured in experimental conditions that favour the proliferation and differentiation of osteoblastic cells (i.e., 2.52 x 10(-4) mol l(-1) ascorbic acid, 10(-2) mol l(-1) beta-glycerophosphate and 10(-8) mol l(-1) dexamethasone) in the absence and in the presence of stainless-steel corrosion products, for a period of 18 days. An AISI 316L stainless-steel slurry (SS) was obtained by electrochemical means and the concentrations of the major metal ions, determined by atomic absorption spectrometry, were 8.78 x 10(-3) mol l(-1) of Fe, 4.31 x 10(-3) mol l(-1) of Cr and 2.56 x 10(-3) mol l(-1) of Ni. Bone marrow cells were exposed to 0.01, 0.1 and 1% of the SS and at the end of the incubation period, control and treated cultures were evaluated by histochemical assays for the identification of the presence of alkaline phosphatase and also calcium and phosphate deposition. Cultures were further observed by scanning electron microscopy. Levels of total and ionised calcium and phosphorus in the culture media collected from control and metal exposed cell cultures were also quantified. Histochemical staining showed that control cultures presented a strong reaction for the presence of alkaline phosphatase and exhibited formation of calcium and phosphates deposits. The presence of 0.01% SS caused no detectable biological effects in these cultures, 0.1% SS impaired osteoblastic behaviour and, 1% SS resulted in cell death. In the absence of bone cells, levels of total and ionised calcium and phosphorus in the control and metal added culture medium were similar throughout the incubation period. A significant decrease in the levels of ionised calcium and phosphorus were observed in the culture medium of control cultures and also in cultures exposed to 0.01% SS after two weeks of incubation, an event related with the formation of mineral calcium phosphate deposits in these cultures. In cultures grown in the presence of 0.1 and 1% SS corrosion products, levels of calcium and phosphorus were similar to those observed in the absence of cells. Results showed that stainless-steel corrosion products above certain concentrations may disturb the normal behaviour of osteoblast-like rat bone marrow cell cultures.

In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions

For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells. The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation. Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy. Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni). The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression. However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect. Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed. Fe did not affect the progression of the mineralization phase. Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells. Quantification of metal ions in the culture media showed that Cr (originated from AISI 316L corrosion products but from not Cr3+ salt) and Ni (originated from AISI 316L corrosion products and Ni salt) appear to be retained by the bone marrow cultures.

Cytotoxicity evaluation of nanocrystalline diamond coatings by fibroblast cell cultures

The cytotoxicity profile of nanocrystalline diamond (NCD) coatings on a Si(3)N(4) ceramic was investigated. This material is envisaged to have biomedical dental applications such as burrs and surgical instruments. Two fibroblast cell culture systems were used to address the cytotoxicity of NCD-coated samples: L929 cells (a mouse permanent cell line) and human gingival fibroblasts. Cell behavior was evaluated in terms of cell adhesion, cell viability/proliferation (mitochondrial function, MTT assay) and the pattern of cell growth. Fibroblast cell behavior on standard polystyrene culture plates was used as control, as Si(3)N(4) substrates have previously been shown to be biocompatible. NCD coatings provided a suitable surface for cell attachment, spreading and proliferation. Human gingival cells showed a homogeneous cytoplasm spreading, a flattened elongated morphology and a typical parallel alignment on confluent cultures. In comparison, L929 cells denoted a lower cytoplasm expansion, a heterogeneous spreading but a higher proliferation rate. For both cells, after few days, the NCD coating was completely covered with continuous cell layers. As compared to standard polystyrene culture plates, no deleterious or cytotoxic responses were observed with L929 and human fibroblast cell cultures, and in both a slight enhancement in cell proliferation was observed. In addition, the seeded NCD film allowed reproduction of the typical features of the two cell culture systems tested, further suggesting the lack of cytotoxicity of this coating.

Hydroxyapatite surface roughness: Complex modulation of the osteoclastogenesis of human precursor cells

It is recognized that the surface roughness affects osteoblastic differentiation, but little information is available regarding its effect on osteoclastogenesis. With this work, the osteoclastogenic behaviour of human peripheral blood mononuclear cells (PBMCs), cultured isolated (1.5×10(6)cellscm(-2)) or co-cultured with human bone marrow cells (hBMCs; 10(3)cellscm(-2)), was assessed on surface-abraded hydroxyapatite disks with three different surface roughnesses (R(a) 0.0437-0.582 μm). Monocultures and co-cultures were performed for 21 days in the absence or presence of recombinant M-CSF and RANKL. Results showed that PBMCs supplemented with M-CSF and RANKL or co-cultured with hBMCs displayed typical osteoclastic features, i.e. multinucleated cells with actin rings, vitronectin and calcitonin receptors, gene expression of TRAP, cathepsin K, carbonic anhydrase 2, c-myc and c-src, TRAP activity and resorbing activity. The osteoclastogenic response increased with surface roughness in PBMCs cultured with M-CSF and RANKL but decreased in PBMCs co-cultured with hBMCs. However, co-cultures supplemented with the osteoclastogenic inducers displayed high and similar levels of osteoclast differentiation in the three tested surfaces. In conclusion, modulation of osteoclast differentiation by surface roughness seemed to be dependent on the mechanisms subjacent to the osteoclastogenic stimulus, i.e. the presence of soluble factors or direct cell-to-cell contacts between osteoblastic and osteoclastic cells.