Alterations in lumbar spine mechanics due to degenerative disc disease

2010 Fall.Includes bibliographical references.Degenerative disc disease is a major source of low back pain. It is hypothesized to significantly alter the biomechanics of the lumbar spine both at the tissue and motion segment (multi-vertebral) levels. However, explicit correlations between the former and the latter has not been established, and this critical link is only possible through modeling the intervertebral disc tissue behavior within a constitutive framework and implementing it in a finite element model of the lumbar spine. In order to develop a better appreciation of the biomechanics of disc degeneration, the main objectives of this dissertation work were to investigate the degenerative disease related mechanical alterations on lumbar spine through finite element modeling and experimentation, and evaluate the contemporary treatment strategies. To meet this objective, a finite element model of the healthy human lumbar spine was generated based on computed tomography (CT) imagery. Mesh convergence was verified based on strain energy density predictions. Kinematic and mechanical predictions of clinical interest, including range of motion and intradiscal nuclear pressure, were validated under pure moment loading. The mechanical properties of healthy and degenerated annulus fibrosus tissue were quantified using an orthotropic continuum model, with empirical determination of the requisite material coefficients derived from biaxial and uniaxial tension tests. The resultant material models were implemented into the validated finite element model in order to simulate disc degeneration at the L3-L4 level. At the tissue level, degeneration was found to significantly increase the dispersion in the collagen fiber orientation and the nonlinearity of the fiber mechanical behavior. At the motion segment level, degeneration increased the mobility of the spine, with concomitant increases in the local stress predictions in the annulus and facet force transmission. Our results were in good agreement with the clinical findings of instability and injury to the intervertebral disc due to degeneration. Total disc replacement was also considered as a treatment option within the aforementioned finite element framework. The model predictions indicated that single and two-level disc replacement restored motion at the treated levels, while linearizing the kinematic response and increasing the facet force transmission. The data reflect that the successful surgical outcome is most likely obtained when maximum preservation of native disc tissue is achieved during implantation of the prosthetic device

The Unmixing Problem: A Guide to Applying Single‐Cell RNA Sequencing to Bone

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well‐defined populations suitable for further studies. Single‐cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single‐cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single‐cell RNA sequencing (scRNA‐seq) experiments, bone biologists will be ideal leaders in the application of scRNA‐seq to the skeleton. Here we provide an overview of the steps involved with a single‐cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/1/jbmr3802_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/2/jbmr3802.pd

Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG that encodes miR-17-92 microRNAs (type 2). Since miR-17-92 expression is transcriptionally regulated by MYC transcription factors, it has been postulated that Feingold syndrome type 1 and 2 may be caused by a common molecular mechanism. Here we show that Mir17-92 deficiency upregulates TGF-β signaling, whereas Mycn-deficiency downregulates PI3K signaling in limb mesenchymal cells. Genetic or pharmacological inhibition of TGF-β signaling efficiently rescues the skeletal defects caused by Mir17-92 deficiency, suggesting that upregulation of TGF-β signaling is responsible for the skeletal defect of Feingold syndrome type 2. By contrast, the skeletal phenotype of Mycn-deficiency is partially rescued by Pten heterozygosity, but not by TGF-β inhibition. These results strongly suggest that despite the phenotypical similarity, distinct molecular mechanisms underlie the pathoetiology for Feingold syndrome type 1 and 2

SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC) patients causes benign cartilage tumors on the bone surface (exostoses) and within bones (enchondromas). To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the data are consistent with paracrine signaling from SHP2-deficient cells causing SHP2-sufficient cells to be incorporated into the lesions

Volume depletion provided by blood donation alters twist mechanics of the heart: Preload dependency of left ventricular torsion

Objectives: The crucial role of twisting motion on both left ventricular (LV) contraction and relaxation has been clearly identified. However, the reports studying the association between LV torsion and loading conditions have revealed conflicting outcomes. Previously normal saline infusion was shown to increase LV rotation. Our aim was to test this phenomenon after volume depletion in healthy volunteer blood donors. Design: A total of 26 healthy male volunteers were included in the study. LV end-diastolic and end-systolic diameter, LV ejection fraction, LV diastolic parameters, LV apical and basal rotation and peak systolic LV torsion were measured by speckle-tracking echocardiography before and after 450 mL blood donation. Results: Blood donation led to a significant decrease in end-diastolic LV internal diameter (48.7 +/- 0.4 versus 46.4 +/- 0.4mm; p<0.001) and cardiac output (6.2 +/- 1.0 versus 5.1 +/- 0.7 L/min; p<0.001). There was a significant decrease in the magnitude of peak systolic apical rotation (4.4 +/- 1.9 degrees versus 2.9 +/- 1.5 degrees; p<0.001) but no change in basal rotation (2.6 +/- 1.4 degrees versus 2.7 +/- 1.6 degrees; p=0.81). Peak systolic LV Torsion decreased after blood donation (6.9 +/- 1.9 degrees versus 5.7 +/- 2.1 degrees; p=0.028). Conclusions: LV apical rotation and peak systolic LV torsion seem to be preload dependent. Preload reduction provided by 450-mL blood donation decreased LV torsion in healthy male volunteers. Volume dynamics should be taken into account in the evaluation of LV torsion

SHP2 depletion or MEK1/2 inhibition increases the abundance of PZ, pre-HZ and early-HZ transcripts, and decreases the abundance of late-HZ transcripts.

<p>A: Heat map showing the fold change in abundance (treated vs. control pellets at w3) of selected transcripts that are associated with a specific stage of chondrocyte maturation. B: Heat maps showing the fold change in abundance (treated vs. control pellets at w3) for all transcripts that we included in the “upper zones” and “lower zones” transcript sets. The graph below the heat map indicates the percentage of transcripts for which the increase (red) or decrease (blue) in abundance was significant (p<0.05). The number of “upper zones” transcripts that increased in abundance in treated pellets (red), and the number of “lower zones” transcripts that decreased in abundance in treated pellets (blue), is indicated below. For <i>Ptpn11</i> cKO pellets, of the 108 “lower zones” transcripts that significantly decreased in abundance, 54 did not significantly change in wild-type pellets treated with 4-OHT.</p

Lineage tracing of SHP2-depleted chondrocytes in mouse vertebral growth plates following mosaic postnatal <i>Ptpn11</i> inactivation.

<p>Tissue sections of lumbar vertebral growth plates (GP) and intervertebral disks (IVD) from <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl</i>^/<i>fl</i> (Col2a1-cKO) or <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl/+</i> (Ctrl) mice that had been administered tamoxifen for 5 days, starting at w1, and sacrificed at w7. A-C: Tissue sections stained with Alcian Blue and Nuclear Fast Red. In Col2a1-cKO mice, the vertebral growth plates are enlarged and disorganized, with ectopic areas of ossification inside the growth plate (red arrow in B) and enchondroma-like lesions below the growth plate (C). D-G: Merged fluorescent images showing cells in which the <i>mTmG</i> reporter allele has (green fluorescence) or has not (red fluorescence) been recombined by Cre recombinase. Note that approximately 50% of growth plate chondrocytes fluoresce green, and in Col2a1-cKO mice, the expanded regions of the growth plate (E), lateral outgrowths (F) and enchondroma-like lesions (G) contain both green and red fluorescing cells. Dashed-white lines outline the cartilage. Scale bars  =  100 µm.</p

Identifying transcripts that change in abundance following SHP2 depletion or MEK1/2 inhibition in pellet cultures.

<p>A: Immunodetection of SHP2, phospho-ERK1/2, total ERK1/2, and phospho-AKT in chondrocyte pellet cultures. Top: pellets from <i>Ptpn11^fl/–</i> or <i>Tg</i>(<i>CMV-CreERT2); Ptpn11^fl/–</i> mice were treated daily with 4-OHT or vehicle control (EtOH, -) and harvested after 1 and 3 weeks. Bottom: pellets from wild-type mice were treated daily with a MEK1/2 inhibitor (U0126), or vehicle control (DMSO, -), and harvested after 1 and 3 weeks. B: Number of transcripts that had significantly lower (blue) or higher (red) abundance in treated pellets compared to vehicle treated pellets, either at w1 or w3. Transcripts that changed in abundance in <i>Ptpn11</i> cKO pellets were excluded if they exhibited similar changes in wild-type pellets treated with 4-OHT.</p