Protein levels of WT and db-/db- mice 3dpO were not significantly different (two sample t-test)impaired bone tissue regeneration in db-/db- mice, which could be confirmed by RUNX-2 immunohistochemical stainings 3dpO as well as aniline blue histomorphometry 7dpO

Tissue has been mechanically homogenized and tissue fragments have been collected in PBS supplemented with full protease inhibitor cocktail (Roche).VR23 Non-operated WT and db-/db- tibiae served as controls. Two tibiae have been pooled for examination. Proteome profiler mouse angiogenesis array (R&D Programs, ARY015) was performed according to manufactures’ guidelines. Equivalent overall protein amounts were employed for every single sample. Protein concentration willpower was carried out with BCA protein assay package (Pierce) according to manufacturesinstructions and ELISA reader ELx800 (Biotek). For quantification, designed x-ray films had been scanned, and pixel density of every spot of the array was established with Image J. For information evaluation, common background signal (damaging manage spots) was subtracted from duplicate location signal depth which was subsequently normalized to constructive manage spots and connected to indicators from WT mice.Outcomes of the examine are introduced as imply regular error of the mean (SEM) of at the very least a few independent experiment. P-values have been calculated by student’s t-test evaluating two groups and ANOVA if comparing a lot more than two groups. Statistical significances had been set at a p-price < 0.05.At first, we aimed to analyze defined stages of T2DM bone regeneration in an established unicortical tibia defect model. To analyze the regenerative potential, we compared osteoid formation in injured tibiae between WT and db-/db- mice 5 and 7 days post operation (dpO) (Fig. 1). Therefore, 1 mm unicortical tibial defects were created and histomorphometry of aniline blue stained sections was performed. 5dpO, bone formation was reduced by 94% in db-/db- mice as compared to WT mice. Additionally, bone formation remained significantly decreased 7dpO in db-/db- mice by 80% compared to WT mice. Additionally, tibial defects were stained with impaired bone regeneration in a T2DM mice tibia defect model. (A) Aniline blue staining of tibial defects 5 and 7 days post operation (dpO). db-/dbmice showed impairments in new bone formation at both time points compared to WT mice. (B) Quantification of aniline blue positive pixels revealed that bone formation is significantly reduced in db-/db- mice compared to tibiae of WT mice. Results are shown as SEM. P-value: < 0.001 (two sample ttest). Scale bar: 200 m. Cb indicates cortical bone.Safranin-O to exclude endochondral ossification (S1 Fig.). Both groups showed no evidence for endochondral ossification.In order to further characterize impaired bone healing in db-/db- mice, immunohistochemical stainings of PCNA, RUNX-2 and Osteocalcin were performed at 3, 5 and 7dpO in db-/db- and WT mice. At early and intermediate bone regeneration stages less proliferating PCNA-positive cells could be detected in defects of db-/db- compared to WT mice (Fig. 2A). Moreover, decreased RUNX-2 and Osteocalcin levels could be observed 3 and 7dpO, respectively. Quantifications of immunohistochemical stainings confirmed that proliferation is significantly reduced in db-/db- mice 3 and 5dpO compared to WT mice (Fig. 2B,C). Moreover, WT mice showed significantly more RUNX-2 positive cells at early stages of bone regeneration (Fig. 2B) as well as significantly higher Osteocalcin levels at intermediate and late stages of bone regeneration compared to db-/db- mice (Fig. 2C,D)it is well known that angiogenesis is a crucial step in early bone healing. In order to investigate the potential impact of T2DM on angiogenesis in bony defects, immunohistochemical stainings for PECAM-1 were performed. We could show that angiogenesis in cortical bony defects is impaired in db-/db- mice 3dpO compared to WT mice as indicated by less blood vessels (Fig. 3A,B). Quantification revealed a significant reduction of blood vessel formation by 91% in the defect area of db-/db- mice could be confirmed by quantification of PECAM-1 positive pixels (Fig. 3C).Altered proliferation and differentiation patterns in tibia defects of db-/db- mice. (A) Immunohistochemistry for PCNA, RUNX-2, and Osteocalcin was performed at 3, 5 and 7dpO. Compared to WT, db-/db- mice showed less osteoblasts and proliferating cells. Moreover, Osteocalcin levels were decreased in db-/db- mice at 7dpO. Quantification of Nova Red positive pixel revealed that cell proliferation is significantly reduced in bony defects of db-/dbmice compared to tibiae of WT mice in early (B) and intermediate (C) bone regeneration. At early stage, RUNX-2 levels are significantly enhanced in WT mice (B), while Osteocalcin levels are enhanced in intermediate and late bone regeneration (D). Results are shown as SEM. P-value: < 0.05 < 0.01 < 0.001 not significant, (two sample t-test). Scale bar: 200 m.In order to investigate osteoclast invasion in db-/db- mice, we performed staining for TRAP, which is highly expressed in osteoclasts. Comparison of TRAP stainings in db-/db- and WT mice 7dpO revealed that db-/db- mice showed significantly less osteoclast invasion into damaged bone areas (Fig. 4A), which was confirmed by quantification (Fig. 4B).Our data indicated that both angiogenesis and osteogenesis were markedly reduced in db-/dbmice. Given their pro-angiogenic and pro-osteogenic properties, we applied FGF-9 and VEGFA proteins, or PBS (control) soaked collagen sponges to tibial defects in order to reconstitute bone regeneration. We analyzed the effect of VEGFA and FGF-9 application on angiogenesis in bony defects of db-/db- and WT mice at early (Fig. 5A,B) and late stages (Fig. 5C,D) of bone regeneration. We found significantly enhanced angiogenesis in db-/db- defects treated with FGF-9 at both analyzed time points reaching expression levels equivalent to WT mice.Reduced angiogenesis in db-/db- mice tibia defects. (A) Blood vessels and endothelial cells were detected by fluorescence immunohistochemistry for PECAM-1 (3dpO). db-/db- mice showed less angiogenesis compared to WT mice. (B) Higher magnifications from (A) (white box). (C) Quantification of PECAM-1 positive pixels (arbitrary units, a.u.) revealed that angiogenesis is significantly reduced in db-/db- mice compared to tibiae of WT mice. Results are shown as SEM. P-value: < 0.01 (two sample t-test). Scale bars: A: 200 m, B: 45 m.Moreover, VEGFA application led to significantly enhanced angiogenesis at late bone regeneration (7dpO) in db-/db- mice.After having observed an increase in angiogenesis, we next assessed bone regeneration and remodeling. To quantify bone regeneration, we harvested tissue at postoperative day 7 and performed histomorphometry of aniline blue stained sections as indicated. Upon local application of recombinant VEGFA, bone formation was significantly increased in db-/db- mice 7dpO (Fig. 6 A,B,D). Importantly, bone regeneration could be further increased by local application of FGF-9 to levels comparable to WT mice. While local application of FGF-9 into WT defects had no effect on bone formation, VEGFA reduced bone formation. Moreover, application of either VEGFA or FGF-9 further enhanced osteoclasts invasion into defects of db-/db- mice as indicated by TRAP staining 7dpO (Fig. 6 C,E) which was markedly reduced in db-/db- control mice. Of note, application of growth factors had no effect on ostoclastogenesis in WT mice.The experiments performed led to the conclusion that impaired angiogenesis played an important role for the decreased bone regeneration as seen in db-/db- mice. In order to investigate angiogenesis upon bone regeneration in further detail, we performed protein array analyses of decreased osteoclasts invasion into bony defects of db-/db- mice. (A) TRAP stainings of db-/dband WT mice 7dpO revealed that osteoclasts invasion is decreased in db-/db- mice (arrows indicate TRAPpositive pixels) which could be confirmed by quantification of TRAP-positive pixel in the defect area (B). Results are shown as SEM. P-value: < 0.001 (two sample t-test). Scale bar: 45 m db-/db- and WT mice 3dpO in comparison to uninjured control tibiae from db-/db- and WT mice. Equal protein amounts have been used for each sample. Signal intensity of spots were normalized to positive controls and related to signals from WT mice. Data were not statistically significant due to inter-experimental variability, however they do show trends. For instance, our experiments revealed that Pentraxin-3 (PTX3) which is a well-known inhibitor of angiogenesis is upregulated in db-/db- mice 3dpO compared to native and operated WT mice (Fig. 7). Moreover, Serpin E1 (Plasminogen activator inhibitor-1, PAI-1) level has been found to be higher in db-/db- mice 3dpO as well as MCP-1 (monocyte chemotactic protein-1), MMP3 (matrix metallopeptidase-3) and TIMP-1 (metallopeptidase inhibitor-1). These proteins are related to angiogenesis but could also play essential roles in osteogenesis and bone remodeling. Angiopoietin-1, CD26, Endoglin (CD105), Endostatin, FGF acidic (FGF-1), hepatocyte growth factor, IGFBP-1, IGFBP-3, MMP-8, MMP-9, IGFBP-9, Osteopontin, CXCL4, SDF-1, Serpin F1 and Thrombonspondin-2 have been found to be expressed in all tissues but without obvious differences (data not shown). All other spots on the array had only background signals.In the present study, we investigated different stages of bone regeneration in an unicortical tibia defect model comparing db-/db- and WT mice. Our experiments revealed that bone regeneration is impaired in db-/db- mice due to compromised angiogenesis and osteogenesis. This was accompanied by reduced cell proliferation. Moreover, we showed that osteogenesis-related angiogenesis is enhanced after FGF-9 treatment in db-/db- mice. Blood vessels were detected by immunohistochemical staining for PECAM-1 at early (A,B) and late stages (C,D) of bone regeneration. FGF-9 (2 g) and VEGF (2 g) treated db-/db- mice show enhanced angiogenesis compared to control db-/db- mice (PBS). (B and D) higher magnifications of boxed areas from (A) and (C), respectively. Arrowheads indicate PECAM-1 enriched areas identified as vessels. (E) Quantification of PECAM-1 positive pixels revealed that angiogenesis is significantly increased in FGF-9 treated db-/db- mice compared to tibiae of control mice whereas VEGF application showed no significant (n.s.) increase in vascular formation (PBS) in early bone regeneration (3dpO). (F) Quantification of PECAM-1 positive pixels in late bone regeneration (7dpO) revealed highly significant enhanced angiogenesis in treated db-/dbmice. WT animals treated with recombinant proteins showed no enhanced vascular formation compared to control in early (3dpO) and late bone regeneration (7dpO). Results are shown as SEM. P-value < 0.05 < 0.01 < 0.001 (two sample t-test). Scale bars: A,C 200 m B,D 45 m proteins RUNX-2 and Osteocalcin are downregulated in db-/db- mice and that osteoid formation is impaired in T2DM tibial bone defects. In addition, impaired bone regeneration was accompanied by decreased angiogenesis which could in turn lead to a decrease in osteogenesis. Likewise, osteoclastogenesis seems to be impaired in db-/db- mice indicating an imbalanced bone remodeling. Altered protein levels of angiogenesis and bone remodeling related proteins application of recombinant FGF-9 and VEGFA enhances bone formation and bone resorption in db-/db- mice. (A) Aniline blue staining of tibial defects 7dpO of control treated (PBS), FGF-9 and VEGFA treated tibiae of WT and db-/db- mice. Application of FGF-9 (2 g) and VEGFA (2 g) lead to more osteoid formation compared to the control group (PBS) in db-/db- mice. (B) Extracted osteoid formation area. (C) TRAP staining of FGF-9 and VEGFA treated WT and db-/db- mice 7dpO revealed that osteoclast invasion into the defect area is enhanced. (D) Quantification of aniline blue positive pixels revealed that bone formation is significantly enhanced by locally applied FGF-9 and VEGF in db-/db- mice compared PBS treated diabetic mice. We observed a significant reduction of osteoid formation in VEGF treated WT mice while FGF-9 had no effect on osteoid formation in WT mice. There was no significant difference in osteoid formation of db-/db- mice treated with FGF-9 compared to WT mice. (E) Quantification of TRAP-positive pixel resulted in significantly enhanced osteoclast invasion after treatment compared to untreated db-/db- mice. Results are shown as SEM. P-value: < 0.05 < 0.01 < 0.001 not significant (two sample t-test). Scale bars: A: 200 m, C: 45 m. Cb indicates cortical bone, yellow dashed line indicates collagen sponge (not traceable in WT PBS)in db-/db- mice was further suggested by protein array analysis. Importantly, these pathological conditions could be reversed. As such, FGF-9 application led to an almost complete rescue of bone repair accompanied by significantly increased angiogenesis and bone remodeling. Insight in cell proliferation is essential for understanding of fracture repair [35]. PCNA, which is synthesized in the late G1 and S phase of DNA replication, is downregulated in the callus in db-/db- mice during fracture healing at early and intermediate stages of bone healing compared to WT mice. There are several studies revealing that combination of PCNA and RUNX-2 is indicative for osteoprogenitor cells [27,36,37]. Less proliferating cells indicate array analysis of db-/db- and WT mice 3dpO. (A) Protein lysates derived from WT and db-/db- mice 3dpO and from non-operated native tibiae were run on each array experiment in parallel. (B) Quantification of relative protein levels. Pixel density was normalized to spots of native WT mice after subtraction of background signal. Mean values SEM from three independent experiments are given for MCP-1, MMP-3, Pentraxin-3, Serpin E1 and TIMP-1 which are upregulated in operated db-/db- mice.21964340 Dashed line serve as reference for relative protein levels. Protein levels of WT and db-/db- mice 3dpO were not significantly different (two sample t-test)impaired bone tissue regeneration in db-/db- mice, which could be confirmed by RUNX-2 immunohistochemical stainings 3dpO as well as aniline blue histomorphometry 7dpO. Most studies investigating bone regeneration in diabetes were previously performed in T1DM models. Although human diabetes is rarely caused by single trigger, monogenic models are commonly used to investigate T2DM. For the purpose of this study, we utilized the well-established leptin receptor knock-out model, mimicking human T2DM conditions including features of the metabolic syndrome [38,39]. In accordance to our data in T2DM, recent studies in animal models of T1DM have demonstrated reduced proliferation in the callus and impaired fracture healing [9]. Moreover, RUNX-2 expression was downregulated followed by decreased Osteocalcin levels in T1DM models [40].