Osseointegration of zirconia implants: an SEM observation of the bone-implant interface

Background The successful use of zirconia ceramics in orthopedic surgery led to a demand for dental zirconium-based implant systems. Because of its excellent biomechanical characteristics, biocompatibility, and bright tooth-like color, zirconia (zirconium dioxide, ZrO2) has the potential to become a substitute for titanium as dental implant material. The present study aimed at investigating the osseointegration of zirconia implants with modified ablative surface at an ultrastructural level. Methods A total of 24 zirconia implants with modified ablative surfaces and 24 titanium implants all of similar shape and surface structure were inserted into the tibia of 12 Gottinger minipigs. Block biopsies were harvested 1 week, 4 weeks or 12 weeks (four animals each) after surgery. Scanning electron microscopy (SEM) analysis was performed at the bone implant interface. Results Remarkable bone attachment was already seen after 1 week which increased further to intimate bone contact after 4 weeks, observed on both zirconia and titanium implant surfaces. After 12 weeks, osseointegration without interposition of an interfacial layer was detected. At the ultrastructural level, there was no obvious difference between the osseointegration of zirconia implants with modified ablative surfaces and titanium implants with a similar surface topography. Conclusion The results of this study indicate similar osseointegration of zirconia and titanium implants at the ultrastructural level

Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton

BACKGROUND: Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear.METHODS: In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs), whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I.RESULTS: It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2.CONCLUSION: It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.Author has checked copyrightDG 16/11/1

Semi-quantitative RT-PCR on amplified cDNA from sorted cells validates I-cells enriched mRNA transcripts.

<p>Semi-quantitative RT-PCR analysis using amplified cDNA from eGFP+ and eGFP− cells. Left panel: eGFP+ cells were highly enriched in mRNA transcripts encoding eGFP and CCK whereas these transcripts were not detected in eGFP− cells. eGFP− cells were enriched in mRNA transcripts encoding AKP3 and MUC2, markers of enterocytes and goblet cells respectively. Right panel: Validation of Gpr40/Ffar1, Gpr120/O3far1, Gpr41/Ffar3, Gpr43/Ffar2, Gpr119 and CB1 mRNA transcripts enrichment in I-cells. 18S rRNA was used as a loading control. PCR products were amplified simultaneously, using different number of cycles (low-for lower number of cycles and high-for higher number of cycles). Equal volumes of each reaction were run on the same 2% agarose gel. L declares Hyperladder V (50–250 bp, Bioline, UK). Asterisk indicates primer dimers.</p

eGFP+ sorted cells represent a highly enriched population of I-cells.

<p>A: Analysis of RNA extracted from eGFP+ and eGFP− cells. RNA was analyzed using a RNA 6000 PicoChip kit. Intact 28S and 18S ribosomal bands (gels, left panel) or distinct 28S and 18S RNA ribosomal peaks (electropherograms, right panel) confirmed integrity of extracted RNA from sorted cells. L indicates Pico 6000 RNA ladder that spans 0.2–6.0 kb. B: eGFP+ cells were highly enriched in Cck mRNA transcripts. Semi-quantitative PCR for Cck mRNA demonstrated that only eGFP+ cells contain Cck mRNA transcript, a marker of I-cells. Akp3 mRNA transcript, a marker of enterocytes, was mainly detected on eGFP− cells, showing little contamination of eGFP+ cells (I-cells) with enterocytes. 18S rRNA was used as a loading control. L declares Hyperladder IV (100–1000 bp, Bioline, UK).</p

I-cells contain GPR40, GPR120, GPR41, GPR43, GPR119 and CB1 mRNA transcripts.

<p>Semi-quantitative RT-PCR analysis revealed that eGFP+ cells (I-cells) were enriched in mRNA transcripts encoding Gpr40/Ffar1, Gpr41/Ffar3, Gpr43/FFAR2, Gpr119 and CB1 whereas Gpr120/O3far1 was enriched in eGFP+ cells, but was also present eGFP− cells; RT-PCR of 18S rRNA confirms that equal amount of cDNA template from eGFP+ and eGFP− cells was used for the analysis. L declares Hyperladder IV (100–1000 bp, Bioline, UK). Asterisk indicates bands representing primer dimers.</p

FACS-sorting of eGFP+ and eGFP− cells.

<p> A: Schematic overview of the methods employed to isolate I-cells and perform transcript analysis. B: Dissociated duodenal cells from eGFP-CCK mice were sorted using a BD FACS Aria cell sorter (lower panels). Dissociated duodenal cells from a WT/CD-1 mouse were used as a control to set gating parameters for analysis and sorting (upper panels). Cells were first stained with PI and analyzed by eGFP (X-axis) and PI (Y-axis) fluorescence intensity (left panels). PI- positive cells (dead cells, P0) were excluded from further analysis. Live cells (Blue, P1) from CCK-eGFP mice displayed a population of highly fluorescent cells (purple rectangle) that were not present in cell from WT mice. Live cells were also analyzed based on eGFP fluorescence intensity (X-axis) and side scatter (Y-axis). Cells that expressed eGFP were gated as eGFP+ cells, representing 0.5% of total cell population (P2, right lower panel). This population was not present in cells from WT mice (P2, right upper panel). All other cells (P3) were gated as eGFP−. eGFP+ cells and approximately equal number of eGFP− cells were sorted.</p

eGFP expressing cells are strongly immunopositive for CCK.

<p>Representive images of transverse sections of duodenum from CCK-eGFP mice. A: eGFP-tagged cells (green) were flask-shaped with a diffuse distribution, characteristic of ‘open-type’ enteroendocrine cell. B: Immunostaining with anti-CCK antibody (red) showed colocalization of CCK and eGFP in ∼ 90% of eGFP-labelled cells. C: High magnification micrograph (2.5X digital zoom) revealed I-cells had a narrow apical membrane (arrow) that was in contact with gut lumen and a broad basolateral membrane (asterisk) with adjacent strong CCK staining. In all images, nuclei were counter-stained with Hoechst 33342 (blue).</p