Osseointegrative effect of rhBMP-2 covalently bound on a titan-plasma-spray-surface after modification with chromosulfuric acid in a large animal bone gap-healing model with the Göttingen minipig

Background: Bone morphogenetic proteins play an important role as osseointegrative factors. It is used widely in orthopedic research and surgery to enhance the osseointegrative potential of implants, e.g., in spinal fusion or alveolar socket augmentation. The aim of the present study was to investigate the benefit of rhBMP-2 on a titan plasma spray (TPS) layer after a special modification with chromosulfuric acid (CSA) at different postoperative times, regarding osseoconduction and osseoinduction. Methods: We allocated 27 Göttinger minipigs into three groups consisting of nine animals each. They received four dumbbell-shaped implants in the metaphyseal parts of the femora. The implants had a TPS surface with (CSA group) and without a CSA treatment (TPS group). The former received an additional layer of BMP-2 (BMP-2 group). For the assessment of osseointegration after healing periods of 4, 8, and 12 weeks, histomorphometry was applied to undecalcified specimens after staining according to Masson-Goldner. An intravital labeling with different fluorochromes was used in the gap model. A multivariable analysis with repeated measurement design was performed for statistical evaluation. Results: We observed several statistical differences in a three-way ANOVA. The comparison between the BMP-2 and the TPS group (two-way ANOVA) showed statistically significant differences in terms of the osseoinduction (osteoid volume), and pronounced for the osseoconduction (bone and osteoid ongrowth), in favor of the BMP-2 group. In the pairwise comparison between BMP-2 and CSA (two-way ANOVA), no statistical significance occurred. The intravital staining with tetracycline, calcein green, and xylenol orange revealed no considerable differences between the groups. Conclusion: BMP-2, covalently bound on a CSA-treated TPS surface, has positive effects on the osseointegration in the large animal bone gap-healing model over the observation period of 12 weeks

Analysis of the osseointegrative force of a hyperhydrophilic and nanostructured surface refinement for TPS surfaces in a gap healing model with the Göttingen minipig

Background: A lot of advantages can result in a high wettability as well as a nanostructure at a titanium surface on bone implants. Thus, the aim of this study was to evaluate the osseointegrative potential of a titan plasma-sprayed (TPS) surface refinement by acid-etching with chromosulfuric acid. This results in a hyperhydrophilic surface with a nanostructure and an extreme high wetting rate. Methods: In total, 72 dumbbell shape titan implants were inserted in the spongy bone of the femora of 18 Göttingen minipigs in a conservative gap model. Thirty-six titan implants were coated with a standard TPS surface and 36 with the hyperhydrophilic chromosulfuric acid (CSA) surface. After a healing period of 4, 8, and 12 weeks, the animals were killed. The chronological healing process was histomorphometrically analyzed. Results: The de novo bone formation, represented by the bone area (BA), is increased by approximately 1.5 times after 12 weeks with little additional benefit by use of the CSA surface. The bone-to-implant contact (BIC), which represents osseoconductive forces, shows results with a highly increased osteoid production in the CSA implants beginning at 8 and 12 weeks compared to TPS. This culminates in a 17-fold increase in BIC after a healing period of 12 weeks. After 4 weeks, significantly more osteoid was seen in the gap as de novo formation in the CSA group (p = 0.0062). Osteoid was also found more frequently after 12 weeks at the CSA-treated surface (p = 0.0355). The site of implantation, intertrochanteric or intercondylar, may influence on the de novo bone formation in the gap. Conclusions: There is a benefit by the CSA surface treatment of the TPS layer for osseointegration over an observation time up to 12 weeks. Significant differences were able to be shown in two direct comparisons between the CSA and the TPS surface for osteoid formation in the gap model. Further trials may reveal the benefit of the CSA treatment of the TPS layer involving mechanical tests if possible

MiR144/451 expression is repressed by RUNX1 during megakaryopoiesis and disturbed by RUNX1/ETO

Abstract: A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product. Author Summary: The regulatory network between transcription factors, epigenetic cofactors and microRNAs is decisive for normal hematopoiesis. The transcription factor RUNX1 is important for the establishment of a megakaryocytic gene expression program and the concomitant repression of erythroid genes during megakaryocytic differentiation. Gene regulation by RUNX1 is frequently disturbed by mutations and chromosomal translocations, such as the t(8;21) translocation, which gives rise to the leukemogenic RUNX1/ETO fusion protein. We found that RUNX1 regulates microRNAs, which are of importance at the megakaryocytic/erythroid branching point. Specifically, RUNX1 down-regulates expression of the microRNA cluster miR144/451 during megakaryocytic differentiation by changing the epigenetic histone modification pattern at the locus. We could show that miR451, one of the micorRNAs of the miR144/451 cluster, supports erythroid differentiation. We found that expression of miR451 is repressed by the RUNX1/ETO fusion protein, resulting in up regulation of miR451 target genes. Our data support the notion that RUNX1 suppresses the erythroid gene expression program including the erythroid microRNA miR451 and that the RUNX1/ETO fusion protein interferes with normal gene regulation by RUNX1

Inhibition of RUNX1/ETO increases miR144/451 expression.

<p><b>(A)</b> Expression of endogenous RUNX1/ETO in Kasumi1, SKNO1 and two RUNX/ETO positive patient samples. Expression of RUNX1/ETO in Kasumi1 cells was set as 1 and the expression levels of the SKNO1 cells and two patient samples are shown as fold compared to Kasumi1 cells. HEK293 and hCD34+ serve as RUNX1/ETO negative controls. Values were gathered by q-RT-PCR and normalised to GAPDH. <b>(B)</b> Expression of miR144/451 in Kasumi1, SKNO1 and two different patient samples. HEK293 and hCD34+ served as miR144/451 negative and positive controls, respectively. Q-RT-PCR values were normalised to GAPDH expression and are shown as fold relative to values gathered with Kasumi1 cells. <b>(C)</b> Knock-down of RUNX1/ETO in Kasumi1 cells. Knock-down of RUNX1/ETO by an shRNA targeting the R/E fusion site leads to increased miR144/451 expression. <b>(D)</b> Treatment of Kasumi1 cells with trichostatin-A (TSA) leads to degradation of RUNX1/ETO. Kasumi1 cells were treated with the indicated concentrations of TSA for 24 hours. Protein expression was determined by Western blot using an anti-ETO antibody. R/E runs at about 100 kD. As loading control (l.c.) a protein band running at 55 kD visible upon Ponceau S staining of the membrane is shown. <b>(E)</b> MiR144/451 expression is up-regulated in Kasumi1 cells upon TSA treatment. MiR144/451 expression upon treatment with TSA was measured by q-RT-PCR. Values were normalised to GAPDH expression. <b>(F)</b> MiR144/451 expression is up-regulated in patient samples upon TSA treatment. Cells were treated with 1 uM TSA for 24 hours. Q-RT-PCR values were normalised to GAPDH expression. Q-RT-PCR against RUNX/ETO was performed with specific primers detecting the fusion protein. Error bars give the standard deviation from at least four independent determinations. The P-values were calculated using Student’s t test. **P <0.01, ***P <0.001.</p

Regulation of miR144/451 expression.

<p><b>(A)</b> Schematic representation of the miR144/451 locus. The first 4700 bp of the 5’-region of miR144/451 are shown. Binding sites for hematopoietic transcription factors are found about 4500 bp upstream (enhancer) and in the proximal promoter region (promoter). A less conserved region (not conserved, n.c.) separates these regions. <b>(B)</b> Luciferase reporter gene experiment with constructs harbouring the enhancer or the promoter region of the miR144/451 cluster. Reporter constructs were transfected into HEK293 cells or K562 cells, respectively. Relative light units (RLU) are given as fold induction compared to values gathered with empty luciferase vector. Luciferase values were normalised for transfection variations by measuring the activity of a cotransfected beta-galactosidase expression vector. Error bars indicate the standard deviation from six independent transfections and measurements. <b>(C)</b> ChIP assay in primary hCD34+ cells shows binding of TAL1 predominantly to the enhancer region (enh.) as opposed to the promoter region (prom.). <b>(D)</b> ChIP assay in hCD34+ cells shows binding of RUNX1 to the promoter region (prom.) but only little to the enhancer region (enh.). <b>(E)</b> The human miR144/451 promoter contains a conserved binding site for RUNX1. The RUNX1 binding site was mutated by changing two base pairs in the core RUNX1 site. <b>(F)</b> Luciferase reporter assay using the wild type (wt prom) and the mutated (RUNXmut) miR144/451 promoters, respectively. Transfection of RUNX1, the RUNX1/ETO (R/E) fusion protein or the truncated RUNX1/ETO (R/Etr) repressed wild type (wt prom), but not the mutated promoter (RUNX1mut prom). Values are presented as fold change compared to the relative light units gathered upon transfection of the reporter gene and empty expression vector. Luciferase values were normalised for transfection variations by measuring the activity of a cotransfected beta-galactosidase expression vector. Error bars represent the standard deviation from six independent transfections and measurements. The P-value was calculated using Student’s t test. **P <0.01.</p

Identification of RUNX1 regulated microRNAs.

<p><b>(A)</b> Schematic representation of the experimental setup. K562 cells were transduced with RUNX1 expression vector or empty vector control. The transductions were performed in independent triplicates and differentially expressed microRNAs were determined by small-RNA sequencing. <b>(B)</b> 588 small RNAs were differentially expressed upon RUNX1 expression in K562 cells. 31 were snRNAs (small nuclear RNA), 45 rRNAs (ribosomal RNA), 142 snoRNAs (small nucleolar RNA) and 370 microRNAs. RNAs were included if they displayed an at least -0.5 or +0.5 log2-fold change and a P-value <0.05. <b>(C)</b> Of the 370 identified microRNAs, 237 were up-regulated and 133 down-regulated upon RUNX1 expression. <b>(D)</b> Schematic representation of hematopoiesis from the stem cell throughout the myeloid lineage. Those microRNAs are shown, which were altered upon RUNX1 over-expression. The green arrow indicates a positive role and the red blunted arrow indicates a negative role in differentiation according to published work. HSC: hematopoietic stem cell, MPP: multipotent progenitor, CMP: common myeloid progenitor, GMP: granulocyte monocyte progenitor, MEP: megakaryocyte erythrocyte progenitor, MkP: megakaryocyte progenitor, EP: erythrocyte progenitor. <b>(E)</b> Independent evaluation of microRNA expression. A subset of mature microRNAs influenced by RUNX1 and with a role in myeloid differentiation identified by RNA-sequencing, was tested by q-RT-PCR. Q-RT-PCR values are given as relative expression of RUNX1 transduced K562 cells, compared to K562 cells transduced with empty vector. Values were normalised to RNU6-2 expression. The error bars give the standard deviation from four independent determinations. All values were significantly different from the control according to Student’s t test P <0.05. <b>(F)</b> RUNX1 over-expression leads to a decrease of miR144/451 (pri-microRNA) transcript. Q-RT-PCR values are shown as fold expression compared to empty vector transduced K562 cells. Error bars represent the standard deviation from four independent determinations. The P-value was calculated using Student’s t test. **P <0.01.</p

RUNX1/ETO inhibits miR144/451 expression during erythroid differentiation.

<p><b>(A)</b> Colony formation assay with sorted GFP-positive transduced hCD34+ cells shows that full-length RUNX1/ETO (R/E) and RUNX1/ETOtr (R/Etr) inhibit colony formation. The average number of colonies per experiment is given. The error bars give the standard deviation from four independent experiments. <b>(B)</b> The percentage of erythroid colonies is reduced in a colony formation assay upon transduction of hCD34+ cells with R/E or R/Etr. Shown is the relative abundance of erythroid colonies in percent. <b>(C)</b> Expression of the erythroid marker gene GYPA is reduced on the mRNA level upon transduction of hCD34+ cell with R/E or R/Etr, as measured by q-RT-PCR. <b>(D)</b> Expression of the pri-miR144/451 is reduced upon transduction of hCD34+ cells with R/E or R/Etr, as shown by q-RT-PCR. <b>(E)</b> Expression of full length RUNX1/ETO (R/E) in transduced K562 cells. Western blot was performed with anti-HA-antibody, which detects the transduced R/E. Endogenous RUNX1 was detected with anti-RUNX1 antibody. Tubulin served as a loading control. <b>(F)</b> Expression of truncated RUNX1/ETO (R/Etr) in transduced K562 cells. Western blot was performed with anti-HA-antibody, which detects the transduced R/E. The endogenous RUNX1 was detected with an anti-RUNX1 antibody. Tubulin served as a loading control. <b>(G)</b> Expression of pri-miR144/451 is reduced upon transduction of K562 cells with R/E or R/Etr as measured by q-RT-PCR. <b>(H)</b> Induction of the miR144/451 cluster during erythroid differentiation is impaired upon transduction of K562 cells with R/E or R/Etr. Transduced K562 cells were treated with 30 μM hemin. <b>(I-J)</b> Knock-down of R/Etr expression in R/Etr-K562 cells with an shRNA targeting R/E. <b>(I)</b> The knock-down of R/Etr is shown on the mRNA level by q-RT-PCR. <b>(J)</b> The knock-down of R/Etr is shown on the protein level by Western blot using an HA-tag antibody, which detects the transduced R/Etr. An antibody against RUNX1 detected the endogenous RUNX1. Tubulin served as a loading control. <b>(K)</b> Knock-down of R/E by shRNA increases miR144/451 expression measured by q-RT-PCR. <b>(C-K)</b> Expression data were gathered by q-RT-PCR with gene specific primer pairs, values were normalised to GAPDH expression. Shown is the relative expression compared to cells transduced with empty vector or expression vectors harbouring a no-targeting shRNA, respectively. Error bars represent the standard deviation from at least four determinations. <b>(L)</b> ChIP assay in K562 cells transduced with HA-tagged RUNX1/ETO (R/E-HA) shows binding of R/E-HA to the miR144/451 promoter. For ChIP an anti-HA-tag antibody was used. <b>(M)</b> ChIP assay in Kasumi1 cells provides evidence that endogenous RUNX1/ETO binds to the miR144/451 promoter. For ChIP an anti-ETO antibody was used. <b>(N)</b> ChIP assay with K562 cells transduced with R/Etr using an anti-H3K4me3 antibody reveals reduced H3K4 methylation. <b>(O)</b> ChIP assay with K562 cells transduced with R/Etr using an anti-RNApol-II antibody reveals reduced RNA polymerase II occupancy. <b>(L-O)</b> Q-PCR was performed with primers in the promoter region of miR144/451. The P- values were calculated using Student’s t test. **P <0.01, ***P <0.001.</p

MiR451 increases erythroid differentiation.

<p><b>(A)</b> Schematic representation of miR144/451 expression constructs. The genomic sequence of human miR144/451 was cloned into the LEGO vector [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005946#pgen.1005946.ref048" target="_blank">48</a>] behind the GFP expression cassette, which is driven by a spleen focus-forming virus promoter. The wild type and mutant constructs are shown, with the X marking the mutational site. <b>(B)</b> The mutations introduced in the seed sequences of miR144 and miR451 are indicated. <b>(C)</b> A colony formation assay was performed with sorted GFP-positive transduced hCD34+ cells. <b>(D)</b> The total number of colonies in the colony formation assay is shown. Colonies were counted on day 12. <b>E)</b> The frequency (%) of erythroid colonies among total colonies in the colony formation assay is shown. Error bars represent the standard deviation of four independent determinations. *P-value <0.05, ***P-value <0.001 according to Student’s t test.</p

MiR144/451 expression is epigenetically repressed during megakaryocytic differentiation.

<p><b>(A)</b> MiR144/451 expression is decreased upon megakaryocytic differentiation and increased upon erythroid expression of primary hCD34+ cells. Differentiation was done for 6 days (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005946#pgen.1005946.s007" target="_blank">S7 Fig</a>). Expression of the endogenous pri-microRNA was measured by q-RT-PCR. <b>(B)</b> ChIP assay analysis of RUNX1 binding to the miR144/451 promoter during erythroid (CD34-E) or megakaryocytic (CD34-M) differentiation of hCD34+ (CD34) cells. <b>(C)</b> Analysis of RUNX1 protein abundance in hCD34+ cells and upon megakaryocytic differentiation (CD34-M) by Western blot using a RUNX1 antibody and a lamin antibody as loading control. <b>(D-I)</b> Cofactor and histone modification changes at the promoter of miR144/451 during megakaryocytic differentiation (CD34-M) using ChIP analysis in hCD34+ cells. <b>(D-F)</b> ChIP analysis reveals altered binding of PRMT6, p300 and WDR5 to the miR144/451 promoter upon megakaryocytic differentiation. <b>(G-I)</b> ChIP reveals altered H3R2me2, H3K9ac and H3K4me3 at the miR144/451 promoter upon megakaryocytic differentiation. <b>(D-I)</b> Q-PCR values are given as percent input. Histone modification ChIP values were corrected by a Histone 3 ChIP for nucleosome density. The error bars represent the standard deviation from at least four independent determinations. The P-values were calculated using Student’s t test. **P <0.01, ***P <0.001.</p