Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis

Biomechanics of the canine mandible during bone transport distraction osteogenesis

This study compared biomechanical patterns between finite element models (FEMs) and a fresh dog mandible tested under molar and incisal physiological loads in order to clarify the effect of the bone transport distraction osteogenesis (BTDO) surgical process. Three FEMs of dog mandibles were built in order to evaluate the effects of BTDO. The first model evaluated the mandibular response under two physiological loads resembling bite processes. In the second model, a 5.0 cm bone defect was bridged with a bone transport reconstruction plate (BTRP). In the third model, new regenerated bony tissue was incorporated within the defect to mimic the surgical process without the presence of the device. Complementarily, a mandible of a male American foxhound dog was mechanically tested in the laboratory both in the presence and absence of a BTRP, and mechanical responses were measured by attaching rosettes to the bone surface of the mandible to validate the FEM predictions. The relationship between real and predicted values indicates that the stress patterns calculated using FEM are a valid predictor of the biomechanics of the BTDO procedures. The present study provides an interesting correlation between the stiffness of the device and the biomechanical response of the mandible affected for bone transport. Copyright © 2014 by ASME

In vitro mechanical evaluation of mandibular bone transport devices

Bone transport distraction osteogenesis (BTDO) is a surgical procedure that has been used over the last 30 years for the correction of segmental defects produced mainly by trauma and oncological resections. Application of BTDO has several clinical advantages over traditional surgical techniques. Over the past few years, several BTDO devices have been introduced to reconstruct mandibular bone defects. Based on the location and outline of the defect, each device requires a uniquely shaped reconstruction plate. To date, no biomechanical evaluations of mandibular BTDO devices have been reported in the literature. The present study evaluated the mechanical behavior of three different shaped prototypes of a novel mandibular bone transport reconstruction plate and its transport unit for the reconstruction of segmental bone defects of the mandible by using numerical models complemented with mechanical laboratory tests to characterize strength, fatigue, and stability. The strength test evaluated device failures under extreme loads and was complemented with optimization procedures to improve the biomechanical behavior of the devices. The responses of the prototypes were characterized to improve their design and identify weak and strong regions in order to avoid posterior device failure in clinical applications. Combinations of the numerical and mechanical laboratory results were used to compare and validate the models. In addition, the results remark the importance of reducing the number of animals used in experimental tests by increasing computational and in vitro trials. © VC 2014 by ASME

Biomechanical characteristics of regenerated cortical bone in the canine mandible

To test the mechanical properties of regenerate cortical bone created using mandibular bone transport (MBT) distraction, five adult male American foxhound dogs underwent unilateral distraction of the mandible with a novel MBT device placed to linearly repair a 30-35 mm bone defect. The animals were sacrificed 12 weeks after the beginning of the consolidation period. Fourteen cylindrical specimens were taken from the inner (lingual) and outer (buccal) plates of the reconstructed mandible and 21 control specimens were removed from the contralateral aspect of the mandible. The mechanical properties of the 35 cylindrical cortical bone specimens were assessed by using a non-destructive pulse ultrasound technique. Results showed that all of the cortical mechanical properties exhibit higher numerical values on the control side than the MBT regenerate side. In addition, both densities and the elastic moduli in the direction of maximum stiffness of the regenerate cortical bone specimens are higher on the lingual side than the buccal side. Interestingly, there is no statistical difference between elastic modulus (E1 and E2) in orthogonal directions throughout the 35 cortical specimens. The data suggest that not only is the regenerate canine cortical bone heterogeneous, but the elastic mechanical properties tend to approximate transverse isotropy at a tissue level, as opposed to control cortical bone, which is orthotropic. In addition, the elastic mechanical properties are higher not only on the control side but also in the lingual anatomical position, suggesting a stress shielding effect from the presence of the reconstruction plate. © 2011 John Wiley & Sons, Ltd

Delayed Versus Immediate Reconstruction of Mandibular Segmental Defects Using Recombinant Human Bone Morphogenetic Protein 2/Absorbable Collagen Sponge

Purpose: To compare the efficiency of recombinant human bone morphogenetic protein 2 (rhBMP2)/absorbable collagen sponge (ACS) in the delayed versus immediate reconstruction of mandibular segmental defects in a canine model. Methods: We randomized 11 dogs into 2 groups: immediate reconstruction (group 1, n = 6) and delayed reconstruction (group 2, n = 5). A 35-mm osteoperiosteal segmental defect was created on the left side of the mandible. Reconstruction with rhBMP2/ACS was carried out in the same setting in group 1 or at 4 weeks postoperatively in group 2. The contralateral side acted as an internal control. Animals were monitored both clinically and radiographically throughout the experiment. Twelve weeks after the application of rhBMP2/ACS, the quantity of bone formation was evaluated using regenerate mapping and histomorphometric analysis. Qualitative evaluation was performed based on bone mineral density and Vickers microhardness (μHV) testing. Results: Postoperative seromas were observed in 83.3% of group 1 dogs only. Group 1 showed significantly larger physical dimensions than group 2 in most regenerate zones. Successful regeneration was achieved in 83.3% of group 1 dogs (discontinuity defect was seen in 1 of 6 dogs in group 1). Meanwhile, none of the 5 dogs in group 2 could be considered to have undergone successful regeneration (3 dogs had discontinuity defects, bony union occurred only in the basal third in the fourth dog, and the last dog showed union with only a shell of bone). The percent bone area and percent defect filling were significantly higher in group 1 than in group 2 (percent bone area, 52.4% ± 5.6% in group 1 and 36.6% ± 11.2% in group 2 [P = .02]; percent defect filling, 56.3% ± 5.5% in group 1 and 38.5% ± 10.8% in group 2 [P = .01]). Group 1 showed higher bone mineral density (0.7 ± 0.3 mg/cm(3) in group 1 and 0.4 ± 0.1 mg/cm(3) in group 2, P = .1). Finally, μHV was significantly higher in group 1 (20.3 ± 2.6 μHV) than in group 2 (13.2 ± 2.4 μHV) (P = .01). Conclusions: Delaying the application of rhBMP2/ACS for 4 weeks attenuated the quantity and quality of regenerated bone in mandibular segmental defects

Effects of <i>In Utero</i> Thyroxine Exposure on Murine Cranial Suture Growth

<div><p>Large scale surveillance studies, case studies, as well as cohort studies have identified the influence of thyroid hormones on calvarial growth and development. Surveillance data suggests maternal thyroid disorders (hyperthyroidism, hypothyroidism with pharmacological replacement, and Maternal Graves Disease) are linked to as much as a 2.5 fold increased risk for craniosynostosis. Craniosynostosis is the premature fusion of one or more calvarial growth sites (sutures) prior to the completion of brain expansion. Thyroid hormones maintain proper bone mineral densities by interacting with growth hormone and aiding in the regulation of insulin like growth factors (IGFs). Disruption of this hormonal control of bone physiology may lead to altered bone dynamics thereby increasing the risk for craniosynostosis. In order to elucidate the effect of exogenous thyroxine exposure on cranial suture growth and morphology, wild type C57BL6 mouse litters were exposed to thyroxine <i>in utero</i> (control = no treatment; low ~167 ng per day; high ~667 ng per day). Thyroxine exposed mice demonstrated craniofacial dysmorphology (brachycranic). High dose exposed mice showed diminished area of the coronal and widening of the sagittal sutures indicative of premature fusion and compensatory growth. Presence of thyroid receptors was confirmed for the murine cranial suture and markers of proliferation and osteogenesis were increased in sutures from exposed mice. Increased <i>Htra1</i> and <i>Igf1</i> gene expression were found in sutures from high dose exposed individuals. Pathways related to the HTRA1/IGF axis, specifically Akt and Wnt, demonstrated evidence of increased activity. Overall our data suggest that maternal exogenous thyroxine exposure can drive calvarial growth alterations and altered suture morphology.</p></div

Effects of <i>in utero</i> thyroxine exposure on post-natal craniofacial morphology.

<p>A-C. Representative 3D μCT skull reconstructions demonstrating dysmorphology in the high dose exposed of 25 day animals (C). D. Cranial vault length measures for control, low and high dose exposed C57BL6 mice at post-natal days 15, 20, and 25 with high dose demonstrating significantly more vault length at 15 (p = 0.046) and 20 days (p = 0.05) than control and low dose, then ceasing to lengthen at 25 days, p = 0.005, having significantly smaller length than both control and low dose respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167805#pone.0167805.t001" target="_blank">Table 1</a> for n). E. Cranial vault width measures for control, low, and high dose exposed mice at post-natal days 15, 20, and 25 with high dose demonstrating significantly more width at 20 days compared to low dose, p = 0.028 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167805#pone.0167805.t001" target="_blank">Table 1</a> for n). F. Cranial vault index for control, low, and high dose exposed mice at post-natal days 15, 20, and 25 with control exhibiting greater values at 20 days compared to high dose, p = 0.016, and high dose having significantly greater values at 25 days compared to both control (p = 0.005) and low dose (p<0.001). All measurements are presented as mean ±SEM. * p = 0.05–0.011.</p

Histological markers of proliferation, bone formation, and apoptosis in control and thyroxine exposed mouse cranial sutures.

<p>A-C. Sagittal (top) and coronal (bottom) sutures for control (right) and high dose exposed (left) 20 day post-natal, C57BL6 mice stained for Proliferating Cell Nuclear Antigen (PCNA) (A), Active Caspase 3 (Caspase) (B), and Alkaline Phosphatase (ALP) (C). Note darker staining for PCNA and ALP in the sutures exposed to thyroxine and the relative lack of Caspase staining, regardless of exposure. Sutures are outlined with periosteum above, osteogenic fronts (OF) on either side, and dura below. D. Quantification of the percent positive staining of PCNA, Caspase, and ALP stained coronal and sagittal sutures from control and high dose exposed 20 day post-natal mice confirms significantly more positive staining for PCNA in the sagittal sutures of mice exposed <i>in utero</i> to high dose thyroxine as compared to control (p = 0.05). Caspase activity was found to be slightly elevated but not statistically significant in both the coronal (p = 0.068) and sagittal (p = 0.295) sutures. Significantly more positive staining for ALP was found in the coronal sutures of mice exposed <i>in utero</i> to high doses of thyroxine as compared to controls (p = 0.024) (n = 3 individuals per exposure x 3 stained sections per suture for each target). E. Western blot analysis of total protein collected from sutures for PCNA suggests increased presence in control samples compared to high dose exposed samples. This was not found to be statistically significant (p = 0.192) F. Gene expression markers of proliferation (<i>Ki67</i>, <i>Ccnd1</i>, <i>Jun</i>), apoptosis (<i>Caspase 3</i>, <i>Bax</i>, <i>Bcl2</i>), and osteogenesis (<i>Runx2</i>, <i>Alp</i>, <i>Bglap</i>) from suture tissue confirm significantly increased expression for a marker of proliferation (<i>Jun</i> p = 0.03), significantly decreased expression for a marker apoptosis (<i>Bax</i> p = 0.026) and significantly increased expression for markers of osteogenesis (<i>Runx2</i> p = 0.023, <i>Alp</i> p = 0.01) with exposure to thyroxine <i>in utero</i> (n = 3 per exposure). All measurements are presented as mean ±SEM. * p = 0.05–0.011; ** p = 0.01–0.001; *** p<0.001.</p

Biomechanical configurations of mandibular transport distraction osteogenesis devices.

Mandibular bone transport (MBT) distraction osteogenesis devices are used for achieving reconstruction of mandibular defects in a predictable way, with few complications, less complexity than other alternative surgical procedures, and minimal tissue morbidity. However, selection of appropriate MBT device characteristics is critical for ensuring both their mechanical soundness and their optimal distraction function for each patient's condition. This article assesses six characteristics of currently available MBT devices to characterize their design and function and to classify them in a way that assists the selection of the best device option for each clinical case. In addition, the present work provides a framework for both the biomechanical conception of new devices and the modification of existing ones