Jani Holopainen

Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid–nanohydroxyapatite fiber scaffolds

The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

Study of amorphous lithium silicate thin films grown by atomic layer deposition

Lithium silicate thin films, which are interesting materials for example in lithium ion batteries, were grown by the atomic layer deposition technique from lithium hexamethyldisilazide [LiHMDS, Li(N(SiMe3)2)] and ozone precursors. Films were obtained at a wide deposition temperature range between 150 and 400 °C. All the films were amorphous except at 400 °C, where partial decomposition of LiHMDS was also observed. The growth behavior was examined in detail at 250 °C, and saturation of growth rates and refractive indices with precursor doses was confirmed, thereby verifying self-limiting surface reactions. Likewise, the linear thickness dependence of the films with the number of deposition cycles was verified. Strong dependence of growth rate and film composition on deposition temperature was also seen. Overall, the amorphous films grown at 250 °C had a stoichiometry close to lithium metasilicate (Li2.0SiO2.9) with 0.7 at. % carbon and 4.6 at. % hydrogen impurities. The corresponding growth rate and refrac...

Osteoclasts in the interface with electrospun hydroxyapatite.

Electrospinning is a method to produce lightweight, resorbable and bioinspired scaffolds for tissue engineering. Here we investigated the influence of electrospun hydroxyapatite fibers (HA) on macrophages and osteoclasts. A mouse macrophage cell line (RAW 264.7) and human bone marrow derived primary osteoclasts (hOC) were cultured with electrospun HA fibers embedded in Matrigel. Cell morphology and the secretion of pro-inflammatory cytokines (IL-6 and TNF-α) were analyzed using macrophages. Both fluorescent microscopy and scanning electron microscopy indicated that the cell morphology differed on the various materials (HA fibers on Matrigel, pure Matrigel and a glass control). Control macrophages were activated with bacterial lipopolysaccharide (LPS) but electrospun HA did not provoke an inflammatory response. Cytokine secretion detected with enzyme-linked immunosorbent assay (ELISA) also supported this observation. LPS, but not HA fibers, stimulated TNF-α and IL-6 secretion by macrophages at the 2 day time point. After 4 days in culture there was an increasing trend in cytokine secretion in the HA fiber samples. Human bone marrow myeloid precursor cells were able to fuse and differentiate on the fibrous mineral scaffold to form functional multinuclear osteoclasts that were able to resorb the HA nanofibers. This indicates that osteoclasts do not necessarily need a continuous bone surface but osteoclast ruffled border membranes can form a resorption interface with a fibrous mineral scaffold.

Preparation and bioactive properties of nanocrystalline hydroxyapatite thin films obtained by conversion of atomic layer deposited calcium carbonate.

Nanocrystalline hydroxyapatite thin films were fabricated on silicon and titanium by atomic layer deposition (ALD) of CaCO3 and its subsequent conversion to hydroxyapatite by diammonium hydrogen phosphate (DAP) solution. The effects of conversion process parameters to crystallinity and morphology of the films were examined. DAP concentration was found to be critical in controlling the crystal size and homogeneity of the films. The hydroxyapatite phase was identified by XRD. ToF-elastic recoil detection analysis studies revealed that the films are calcium deficient in relation to hydroxyapatite with a Ca/P ratio of 1.39 for films converted with 0.2 M DAP at 95 °C. The coatings prepared on titanium conformally follow the rough surface topography of the substrate, verifying that the good step coverage of the ALD method was maintained in the conversion process. The dissolution tests revealed that the coating was nondissolvable in the cell culture medium. Annealing the coated sample at 700 °C for 1 h seemed to enhance its bonding properties to the substrate. Also, the biocompatibility of the coatings was confirmed by human bone marrow derived cells in vitro. The developed method provides a new possibility to produce thin film coatings on titanium implants with bone-type hydroxyapatite that is biocompatible with human osteoblasts and osteoclasts.

Electrospinning of calcium carbonate fibers and their conversion to nanocrystalline hydroxyapatite

Calcium carbonate (CaCO3) fibers were prepared by electrospinning followed by annealing. Solutions consisting of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and polyvinylpyrrolidone (PVP) dissolved in ethanol or 2-methoxyethanol were used for the fiber preparation. By varying the precursor concentrations in the electrospinning solutions CaCO3 fibers with average diameters from 140 to 290 nm were obtained. After calcination the fibers were identified as calcite by X-ray diffraction (XRD). The calcination process was studied in detail with high temperature X-ray diffraction (HTXRD) and thermogravimetric analysis (TGA). The initially weak fiber-to-substrate adhesion was improved by adding a strengthening CaCO3 layer by spin or dip coating Ca(NO3)2/PVP precursor solution on the CaCO3 fibers followed by annealing of the gel formed inside the fiber layer. The CaCO3 fibers were converted to nanocrystalline hydroxyapatite (HA) fibers by treatment in a dilute phosphate solution. The resulting hydroxyapatite had a plate-like crystal structure with resemblance to bone mineral. The calcium carbonate and hydroxyapatite fibers are interesting materials for bone scaffolds and bioactive coatings.

Adhesion and mechanical properties of nanocrystalline hydroxyapatite coating obtained by conversion of atomic layer-deposited calcium carbonate on titanium substrate

AbstractThe purpose of this study was to evaluate the mechanical properties of nanocrystalline hydroxyapatite coating by tensile adhesion testing and scratch testing. The coating was manufactured on titanium substrate by converting atomic layer-deposited (ALD) calcium carbonate thin film in dilute phosphate solution. The tensile adhesion testing was performed with hydraulic testing device in accordance with ISO 4624 and ISO 16276-1. Scratch testing was done according to SFS-EN 13523-12 with spherical 10 µm scratching tip. Characterization of the samples was done with light and electron microscopy after which they were stained with alizarin red and the failure modes and loadings were analyzed. The highest obtained tensile adhesion value was 6.71 MPa produced with 4000 ALD cycles, converted to hydroxyapatite in alkaline solution, and annealed for 30 min in 700 °C. The annealing improved the adhesion values by approximately 0.8 MPa, but examining the samples with electron microscopy showed intact coating in both annealed and non-annealed samples. Samples produced with 4000 cycles performed better in testing than 2000 cycle samples, and better adhesion was also achieved with alkaline conversion solution compared to neutral solution.