Abstracts Canadian Connective Tissue Meeting London, Ontario, Canada, June 8-10, 2014

Abstracts 20th Annual CCTC 2014 London, ON, Canada

June 8–10, 2014

Oral abstracts

Session 1

Defective bone repair in mast cell deficient mice with c-Kit loss of function

D. A. Behrends^1,2, L. Cheng^1,4, M. B. Sullivan^1,5, M. Wang^1, G. B. Roby^1, N. Zayed¹, C. Gao^1,3, J. E. Henderson^1,2,3, P. A. Martineau^1,2

¹ Bone Engineering Labs, Research Institute-McGill University Health Centre, ² Experimental Surgery, ³ McGill University;⁴ University of British Columbia,⁵ University of Calgary

Presenting author: Dominique Behrends

Category: Graduate Research

If other please specify:

Field of interest: Connective tissue remodelling

An estimated 25 % of fractures in young Canadians fail to heal adequately as a consequence of infection or other underlying factors. The incidence of mal-union and failed joint replacements in the elderly is even higher, as a result of inadequate endogenous healing mechanisms. There is a pressing need for innovative approaches to promote bone repair in cases where healing is predicted to be compromised. This will reduce the number of failed procedures and significantly reduce the economic burden associated with musculoskeletal disease in Canada. Although the primary role ascribed to mast cells is that of protagonist in allergic reactions, there is growing evidence that they also play pivotal roles in normal physiology. A number of early studies noted accumulation of mast cells in and around fracture callus. We tested the hypothesis that mast cells are involved in all phases of fracture repair. We tested the hypothesis that mast cells are involved in fracture repair by comparing bone repair in uni-cortical femoral window defects in mast cell deficient C57BL/6-KitW-sh/W-shmice and their wild type littermates. Cohorts of mice were euthanized at timed intervals (2, 4, 6 and 12 weeks) after surgery and bone healing was quantified using micro CT and histological analyses. Bone mineral content was analyzed with von Kossa stain, and alkaline phosphatase and tartrate resistant acid phosphatase was used to assess osteoblasts and osteoclasts activity respectively. Acidic toluidine blue staining identified mast cells with a high degree of specificity and safranin O and alizarin red stain was used to assess their stage of maturity. Bone regeneration in the mast cell deficient mice is significantly delayed and associated with an abnormal spatial distribution of the new bone, which led to non-unions in half of the cases. Less bone was found at the level of the cortex and more bone inside the medullary cavity. The quality of the regenerating cortical bone was lower in the mast cell deficient mice, as indicated by higher porosity, less osteocytes, lower connectivity and impaired trabecular characteristics. The histological analyses confirmed the poor quality bone shown by micro CT and suggested it may be due to uncoupling of osteoblast from osteoclast activity. The changing maturity, number and distribution of mast cells throughout the process of healing indicate that the role of mast cells depends on the stage of bone regeneration.

Identification of β-catenin gene targets by ChIP sequencing in Dupuytren’s disease

Christina Raykha, Bing Siang Gan, Greg Gloor and David B. O’Gorman

Lawson Health Research Institute, Western University, Canada

Presenting author: Christina Raykha

Category: Graduate Research

If other please specify:

Field of interest: Genetics of Connective Tissue

Dupuytren’s disease (DD) is a fibroproliferative and contractile fibrosis of the palmar fascia that typically results in permanent finger contractures. Total cellular levels of β-catenin, an intracellular trans-activator of gene transcription, are increased in DD tissues. The identities of the genes that interact with β-catenin during DD development have not been previously reported. We have performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses using the Illumina HiSeq platform with an antibody specific to β-catenin to identify these target genes. Specifically, we performed ChIP-seq analyses on primary fibroblasts derived from normal palmar fascia obtained during carpal tunnel release surgeries (CT cells, N = 3), and fibrotic palmar fascia from patients undergoing palmar fasciectomies for DD (DD cells, N = 3). Additional samples of phenotypically unaffected palmar fascia adjacent to fibrotic disease tissue were collected from the latter patients to derive genetically matched control fibroblasts (PF cells, N = 3). Regions of β-catenin association were identified using the peak caller: Model-based Analysis of ChIP Sequencing with a model fold enrichment of 5 and p < 0.01. The results demonstrated that β-catenin associated with 317 genes in DD cells, 209 genes in PF cells and only 10 in CT cell lines based on the independent patient analysis. β-catenin associated with several novel genes in DD including ACTN2, RELN and SOX5. While these genes have been previously implicated in cancer development, their roles in DD have not been reported. As β-catenin is associating with a distinct subset of genes in each of the three cell types, it may be possible to identify genetic markers that distinguish patients predisposed to develop DD from the non-predisposed population. Additionally, the identification of novel genes associated with β-catenin during the development of this fibrosis will allow us to assess their potential as therapeutic targets.

Rejuvenating fracture repair

Gurpreet S. Baht, Puviindran Nadesan, Qingxia Wei, Heather Whetstone, Benjamin A. Alman

University of Toronto, Department of Developmental & Stem Cell Biology, Hospital for Sick Children, Toronto, Canada

Presenting author: Gurpreet S Baht

Category: Post-doctoral Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

The capacity for tissues to repair and regenerate diminishes with age. Likewise, in vivo bone repair and in vitro differentiation of bone marrow stromal cells to osteoblasts is more efficient in young animals than in old animals. We sought to determine the effect of animal age, cell age, and matrix age on bone regeneration and osteoblast differentiation. Fracture repair and osteoblast differentiation were more efficient and more robust in young mice (4 months) than in old mice (18 months). To determine if exposure to youthful circulation is able to rejuvenate aged fracture repair and osteoblast differentiation, a heterochronic parabiosis model was developed. When osteoblasts were ablated in the old heterochronic host, bone regeneration capacity and osteoblastogenesis were completely lost. Conversely, removal of osteoblasts from young partners did not affect rejuvenation of old, heterochronic mice. Engraftment of young hematopoietic cells into old animals rescued fracture repair and osteogenic potential in a mechanism which again required osteoblasts from the host animal but not osteoblasts from the donor animal. Using tissue culture methods, culture media pre-conditioned by young cells was shown to contain a youth factor able to rejuvenate the age-dependent decrease in osteogenic potential. In vivo, β-catenin was found to be elevated upon fracture; however, β-catenin levels were higher in old calluses relative to young calluses and remained elevated throughout repair. Western blot analysis identified a decrease in β-catenin during rejuvenation of old, heterochronic, parabiotic fractures in vivo and old BMSCs in rescue cultures in vitro. Because β-catenin regulation is critical during mesenchymal commitment to an osteochondral lineage, its dysregulation in old bone may contribute to the impaired fracture healing observed. Pharmacologically-induced reduction of β-catenin during early fracture repair, using Dkk-1, improved bone regeneration in old mice. These data demonstrate that youthful circulation carries within it a “youth factor” which originates, at least in part, from hematopoietic cells (bone marrow). This youth factor is able to rejuvenate bone repair and osteoblast differentiation through modulation of β-catenin. Furthermore, this work indicates that agents which modulate β-catenin could be used to improve the quality of fracture repair and osseous integration of implants in the aging population.

Cholesterol homeostasis mediates Hedgehog signaling in chondrocytes and osteoarthritis

Shabana Amanda Ali, Mushriq Al-Jazrawe, Heather Whetstone, Benjamin Alman

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON

Presenting author: Shabana Amanda Ali

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Current evidence suggests that mechanical, genetic, and metabolic factors likely play a role in the pathogenesis of osteoarthritis (OA). Hedgehog (Hh) signaling is known to regulate chondrocyte differentiation, and to be activated in human and murine OA. Since Hh signaling regulates Gli-mediated gene expression, we identified Hh targets genes that are expressed in chondrocytes. Microarray analyses were performed to detect changes in gene expression when the Hh pathway was modulated in human OA cartilage samples. Results from the Affymetrix Human Gene 1.0 ST microarray were analyzed for differentially expressed genes from five patient samples. Using Ingenuity® Pathway analysis, several genes known to be involved in sterol homeostasis were found to be upregulated with Hh inhibition. To investigate the function of these genes in cartilage, mice with chondrocyte-specific cholesterol accumulation were generated. This was achieved by excising Insig1 and Insig2, major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements. With aging or surgically induced joint instability, mice with chondrocyte-specific cholesterol accumulation developed more severe OA than control littermates. They expressed markers of chondrocyte hypertrophy in the articular cartilage, including type X collagen. Statin treatment to inhibit cholesterol production rescued this phenotype and reduced the severity of OA. Genetic manipulation of Hedgehog signaling in these mice suggests that Hh signaling is modulating the phenotype by regulating sterol homeostasis. Here we identified novel Hh target genes in chondrocytes that regulate intracellular cholesterol levels, and found that cholesterol dysregulation in the chondrocytes predisposes to OA. These data suggest that pharmacologic correction of intra-articular sterol imbalance can be used as a treatment for osteoarthritis.

Non-invasive assessment of therapeutic approaches in a murine model of Duchenne muscular dystrophy

Gutpell K 1,2, Faubert S 1, Hoffman L 1,2,3

(1- Department of Anatomy and Cell Biology, Western University 2- Lawson Health Research Institute, London 3- Department of Medical Biophysics, Western University)

Presenting author: Kelly Gutpell

Category: Graduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder that occurs almost exclusively in young boys. The prevalence of DMD in males is 1 in 3,500, making it the most commonly diagnosed fatal childhood disorder. There is no known cure. While clinical trials involving transplantation of muscle stem cells have been occurring for decades, no significant effect has been reported. This inefficacy may be attributed to two factors that create a hostile transplantation environment for stem cells. First is the poorly vascularized nature of the dystrophic skeletal muscle and second is the excessive production of extracellular matrix resulting in fibrosis. It has been proposed that treating dystrophic skeletal muscle with pro-angiogenic growth factors and anti-fibrotic drugs may improve cell survival and engraftment. The current standard for assessing growth factor therapy is muscle biopsy; an invasive process that may contribute to the overall progression of DMD. The objective of the current study is to determine whether dynamic contrast-enhanced computed tomography (DCE-CT) can non-invasively assess muscle perfusion following angiogenic therapy in an mdx mouse, a well-accepted model of DMD.//METHODS: DCE-CT was used to determine if an increase in CD31 and αSMA staining (via immunohistochemical analysis) correlated to a functional increase in muscle perfusion following treatment with vascular endothelial growth factor (VEGF) exclusively, or combined with angiopoietin-1.//RESULTS: Treatment with VEGF, a potent inducer of angiogenesis, is insufficient to induce formation of mature, functional vessels as indicated by DCE-CT measurements (n = 3, p > 0.05). VEGF in combination with angiopoietin-1, however, enhanced the maturation index of newly formed blood vessels, indicated by increased blood flow compared to VEGF alone (n = 3, p = 0.0081).//SIGNIFICANCE: This study directly addresses two fundamental problems associated with current cell therapy for the treatment of DMD. Enhancing the microenvironment of transplanted cells may address the lack of survival and engraftment observed with current myoblast transplants in clinical trials. The use of DCE-CT to measure muscle perfusion may surpass the current gold standard for assessing therapeutic intervention strategies. These studies in preclinical animal models are essential for the rapid translation of methodology to the clinical setting.

Delineating the role of bone sialoprotein in bone development and mineralization

Erik Holm1, Jane E. Aubin3, Graeme K. Hunter1, Frank Beier2, Harvey A. Goldberg1

1Department of Biochemistry, 2Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario. 3Molecular and Medical Genetics, University of Toronto, Toronto, Ontario

Presenting author: Erik Holm

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Background: Development and maintenance of the skeleton is a complex process contributing to mineral balance and overall health. Many mechanistic aspects of normal bone development are well known, but regulation of mineral deposition remains poorly understood. Bone sialoprotein (BSP) is a potential regulatory element for bone development. BSP contains three functional regions including: a collagen-binding domain, poly-Glu regions involved in hydroxyapatite nucleation and binding, and the integrin-binding RGD sequence. In this study we sought to characterize the role of BSP in bone development and matrix mineralization.\\ Methods: Embryonic and neonatal tibiae from wild-type and Bsp−/−mice were dissected and assayed for markers of normal bone development. Mineralization and growth plate development were characterized using von Kossa and Safranin O stains, respectively. Quantitative real-time PCR, immunohistochemistry, and immunofluorescence were performed to assay markers of chondrocyte and osteoblast development. Results: The tibia of the Bsp−/−mouse shows delayed and diminished mineralization during development. The growth plate is also perturbed in the tibia of the Bsp−/−mouse. The resting and proliferative zones of the Bsp−/−tibiae are increased and decreased 20 % in length, respectively. Furthermore, Ki-67 expression shows a decrease in S-phase cells in the proliferative zone, whereas there is a decrease in TUNEL-positive cells in the hypertrophic zone of the Bsp−/−tibia. These results suggest that loss of BSP results in chondrocytes with delayed characteristics of maturation. The neonatal Bsp−/−bone shows a decrease in expression of Col1a1, Sp7, Runx2, and Bglap, suggesting loss of BSP results in impairment of osteogenic maturation. In contrast, the neonatal growth plate showed no change in expression of markers of chondrocyte development.\\ Conclusions: In this study, we demonstrate an aberrant growth plate, decreased mineralization, and decreased osteogenic marker expression in neonatal tibiae from Bsp−/−mice. The neonatal growth plate defects suggest that BSP is required to promote normal cell cycle progression of chondrocytes. The decreased mineralization and osteogenic marker expression confirm that BSP promotes osteoblastic cell development and mineralization. This advocates that BSP is an important regulatory element in normal skeletal development. This work was supported by the CIHR.

Osteoclasts from osteopetrotic Tcirg1R740S mice have impaired autophagy and increased mTORC1 activity

L. Lacroix1, Y. Hu2, C. Owen3, I. Voronov1

1Faculty of Dentistry, University of Toronto, 2Institute of Dental Medicine, Qilu Hospital, Shandong University, China, 3Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, ON, Canada

Presenting author: Dr. Luciene Lacroix

Category: Research Associate

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Osteoclasts are the bone resorbing multinucleated cells formed by fusion of hematopoietic precursors. To resorb bone, osteoclasts pump protons onto the surface of the bone to dissolve mineral and release enzymes necessary for degradation of the extracellular matrix. The proton pump responsible for this acidification is vacuolar H + −ATPase (V-ATPase), a multisubunit enzyme. We have a mouse model with a point mutation (R740S) in the V-ATPase a3 subunit (Tcirg1). These mice have defective V-ATPase activity resulting in mild osteopetrosis in heterozygous (+/R740S) and severe osteopetrosis in homozygous (R740S/R740S) animals. Osteoclasts with R740S mutation have increased lysosomal pH and impaired in vitro osteoclast differentiation. To find a mechanism linking osteoclastogenesis and lysosomal pH, we looked at autophagy, a protein degradation process dependent on lysosomal acidification.\\ To assess the state of autophagy, we looked at protein expression of two key molecules, microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (p62/SQSTM1). LC3 is cytosolic and is necessary for autophagosomal membrane formation; upon induction of autophagy, it becomes lipidated, associates with the autophagosomal membrane and acquires a characteristic punctate expression pattern. p62 is a scaffold protein that targets ubiquitinated proteins for degradation; it is degraded within an autophagosome and is used as an indicator of autophagic protein degradation. Autophagy is activated in response to nutrient (amino acid) deprivation. In the presence of amino acids and growth factors, autophagy activation is inhibited by a complex containing Ser/Thr protein kinase mammalian target of rapamycin (mTOR) called mTORC1.\\ To assess a state of autophagic flux, bone marrow-derived osteoclasts were differentiated in the presence of M-CSF and RANKL for 4 to 6 days, and then incubated with HBSS (amino acid starvation) for 30, 60 and 120 min. To investigate activation of mTORC1, the cells were incubated with HBSS for 60 min and then in fully supplemented media (recovery) for additional 30 min. Immunoblotting results showed that LC3 and p62 protein levels at all time points were consistently increased in +/R740S cells, a finding consistent with blocked autophagy. Immunofluoresence confirmed higher LC3 expression in +/R740S osteoclasts. Unexpectedly, mTORC1 activity was increased in +/R740S cells compared to +/+. To elucidate the mechanism of increased mTORC1 activity, we looked at mTOR protein levels and mTOR localization in osteoclasts. Our preliminary results suggest that mTOR activity as part of mTORC1 complex is regulated by lysosomal degradation. Future experiments will look at the precise molecular mechanisms of mTORC1 activation and degradation in osteoclasts.

PPARdelta promotes the progression of secondary osteoarthritis

Ratneswaran, A (1), LeBlanc, E.A.(1), Welch, I.(1,2), Mort, J.S. (3), Borradaile, N.(1), and Beier, F.(1,4)

(1) Department of Physiology and Pharmacology, Western University, London, Ontario, Canada (2) Department of Animal Care and Veterinary Services, Western University, London, Ontario, Canada (3) Shriners Hospital for Children and McGill University, Montreal, Quebec, Canada (4) Children’s Health Research Institute, London, Ontario, Canada

Presenting author: Anusha Ratneswaran

Category: Graduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Osteoarthritis (OA) is a degenerative joint disorder, characterized by the breakdown of articular cartilage, subchondral bone thickening, osteophyte formation and synovial inflammation. The human burden of this disease influences both the independence and quality of life of those afflicted. Recent findings from our laboratory indicate that activation of the transcription factor PPARdelta induces the expression of enzymes involved in proteoglycan breakdown and can lead to cartilage degeneration in OA, prompting us to speculate whether inhibition of PPARdelta, can protect from cartilage breakdown in OA. We aim to characterize the role of PPARdelta in OA through all encompassing in-vitro, ex-vivo, and in-vivo studies to determine whether it is a feasible target for OA therapy. We hypothesize that inhibition of PPARdelta will slow the progression of OA in animal models. Primary mouse chondrocytes, human chondrocytes and mouse cartilage explants were treated with pharmacological agonist (GW501516) to evaluate changes in gene expression (qPCR), and histology (Safranin-O, immunohistochemistry), and quantitative assays for aggrecan content in the cartilage consistent with OA development. In order to examine the role of PPARdelta in-vivo, cartilage specific knockout mice of PPARdelta were compared to wild-type littermate controls after surgical induction of OA through destabilization of medial meniscus surgery (DMM) at 20 weeks of age. Eight weeks post-surgery mice were evaluated through classical histological, biochemical and functional measures of OA progression including Safranin-O staining with OARSI scoring, immunohistochemistry for cartilage matrix breakdown products, picrosirius red staining and gait analysis. In vitro, PPARdelta agonism (by GW501516) results in upregulation of the expression of proteases implicated in cartilage degeneration (including MMPs, and ADAMTS genes) in chondrocytes. We have also seen that activation of PPARdelta results in significant proteoglycan breakdown in the cartilage matrix of an explant culture system, and quantitative increases in aggrecan breakdown products. Mice with cartilage specific deletion of PPARdelta do not demonstrate any abnormalities in skeletal growth or development, and show significant chondroprotection in comparison to wild-type littermate controls after surgical induction of OA. OARSI scoring and immunohistochemistry confirm strong protection against cartilage matrix breakdown, and decreased breakdown products in the cartilage of PPARdelta knockout mice. This study provides considerable evidence for catabolic role of endogenous PPARdelta in post-traumatic OA and suggests that pharmacological inhibition of PPARdelta is a promising therapeutic strategy.

Impact of inelastic behavior of collagen gels on cell mechanosensation

Mohammadi H, Esmail H, Janmey PA, McCulloch CA

University of Toronto

Presenting author: Mohammadi H

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

The mechanical properties of extracellular matrix proteins strongly influence cell-induced tension in the matrix, which in turn influences cell function. Despite progress on the impact of elastic behavior of matrix proteins on cell-matrix interactions, little is known about the influence of inelastic behavior, especially at the large and slow deformations that characterize cell-induced matrix remodeling. We found that collagen matrices exhibit deformation rate-dependent behavior, which leads to a transition from pronounced elastic behavior at fast deformations to substantially inelastic behavior at slow deformations (1 μm/min; similar to cell-mediated deformation). With slow deformations, the inelastic behavior of floating gels was sensitive to collagen concentration, whereas attached gels exhibited similar inelastic behavior independent of collagen concentration. The presence of an underlying rigid support had a similar effect on cell-matrix interactions: cell-induced deformation and remodeling were similar on 1 or 3 mg/ml attached collagen gels while deformations were 2–4 fold smaller in floating gels of high compared to low collagen concentration. These data indicate that at the slow rates of collagen compaction generated by fibroblasts, the inelastic responses of collagen gels, which are influenced by collagen concentration and the presence of an underlying rigid foundation, are important determinants of cell-matrix interactions and mechanosensation.

Tissue-engineered biological dressings for skin wound healing in a murine model of splinted full-thickness defects

Pascal Morissette Martin, Amandine Maux and Julie Fradette

Centre LOEX de l’Université Laval, Centre de recherche du CHU de Québec - Axe Médecine Régénératrice, Département de Chirurgie, Faculté de Médecine, Université Laval, Québec City, QC, Canada

Presenting author: Pascal Morissette Martin

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Efforts are being made by several teams worldwide to investigate the use of mesenchymal stem cells to stimulate various types of wound healing. Previous work of our team has shown that the self-assembly approach of tissue engineering can be used to produce manipulable cell sheets using human adipose-derived stem cells (ASC). In that model, the mesenchymal stem cells themselves produce their collagen-based extracellular matrix without the use of any exogenous or synthetic biomaterials. During that process, the ASC contained into those cell-sheets can be differentiated into adipocytes; therefore two types of reconstructed tissue may be produced: connective tissues containing ASC and adipose tissues containing differentiated adipocytes. We postulate that those two kind of reconstructed tissues could be used as biological dressings and would have enhancing effects on skin healing using a murine model.

To validate our hypothesis, an animal protocol of silicone-splinted full-thickness excisional wound was developed and adapted. The monitoring of reepithelialisation kinetics and differentiation of circular 8 mm wide wounds was achieved by the use of a mouse strain featuring a fluorescent epidermis (data obtained from a non invasive LuminaIVIS imagery system). Three experimental groups were evaluated: (i) untreated wounds, (ii) ASC-based and (iii) differentiated adipocytes-based dressings (both placed onto wound beds and changed every three days).//

Results of two experiments showed that the kinetics of wound reepithelialisation did not differ between the three experimental groups (between 12 and 18 wounds per group). The extent of epidermal differentiation, assayed by epidermal luminous intensity measurements, revealed globally no significant differences between groups. In addition to imagery data, profiles of bioactive molecules produced by the dressings were determined by Duosets ELISA kits (R&D systems) and normalised with total protein contents. Before being applied to wounds, levels of leptin, angiopoietin-1 and HGF were slightly higher (490 %, 200 % and 190 % respectively; p-values).

Session 2

Limb-specific deletion of Adamtsl2 in mice leads to brachydactyly and joint malformations: a model system for human acromelic dysplasias?

Dirk Hubmacher and Suneel S. Apte

Cleveland Clinic Foundation, Lerner Research Institute, Department of Biomedical Engineering

Presenting author: Dirk Hubmacher

Category: Project Scientist Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Acromelic dysplasias are rare connective tissue disorders characterized by short stature, brachydactyly, stiff joints, and a “pseudo-muscular” build. The distal limb bones are typically affected the most. They include Weill-Marchesani syndrome (caused by FBN1 or ADAMTS10 mutations), acromicric dysplasia (FBN1 mutations), and geleophysic dysplasia (GD) (FBN1 or ADAMTSL2 mutations). In GD, high morbidity, and frequently, juvenile mortality results from cardiac valvular and tracheo-pulmonary anomalies. Fibrillins are the major components of extracellular microfibrils, and in GD, domain-specific mutations in FBN1 are phenocopied by mutations in ADAMTSL2. This strongly suggests a functional relationship between the two proteins, which is substantiated by their direct interaction in vitro. In tissues, microfibrils modulate extracellular TGFb and BMP signaling and provide a scaffold on which other components, such as LTBPs, MAGP, or fibulins are assembled. The Adamtsl2 gene was inactivated in mice by insertion of an IRES-lacZ-neomycin cassette, which provided a gene expression reporter (LacZ) and also allowed Cre mediated tissue specific -gal staining showed specific Adamtsl2 expression in articularΒdeletion. Cartilage, tendons, and skeletal muscle. Global deletion of Adamtsl2 resulted in neonatal lethality due to abnormal lung and heart development. To analyze the role of Adamtsl2 in postnatal limb development, we deleted Adamtsl2 (Adamtsl2-Del) in the limb buds using a Prx1-Cre deleter strain. Successful deletion was confirmed by PCR with template DNA isolated from toe or tail tissue. Adamtsl2-Del mice were fertile and had a normal life span. Deletion of Adamtsl2 resulted in a statistically significant reduction in absolute bone length of all limb segments with greater effect on distal elements, e.g., humerus, radius, ulna, femur, fibula, and tibia were reduced by 5–7 %, but metacarpals and metatarsals by 9–11 %. Anomalies of bone sculpting were found in Adamtsl2-Del mice, including a wider diaphysis and cone-shaped epiphyses. Currently, we are analyzing the distribution of extracellular matrix components in the limbs from Adamtsl2-Del mice with a focus on fibrillin microfibrils and related structures (FBN1, FBN2, MAGP1, elastin, and fibronectin). In summary, we developed a mouse model that recapitulates the skeletal phenotype observed in GD. This model could be used to define pathogenetic mechanisms of GD and advance understanding of mechanisms that regulate bone length and shape.

Conditional β1 integrin-deficient mice display impaired pancreatic function and architecture

Matthew Riopel^1,2, Mansa Krishnamurthy^1,2, Jinming Li^1,3, Shangxi Liu³, Andrew Leask³, and Rennian Wang^1,3

¹Children’s Health Research Institute, ²Departments of Pathology and ³Physiology and Pharmacology, Western University

Presenting author: Matthew Riopel

Category: Graduate Research

If other please specify:

Field of interest: Integration of the Connective Tissue with other Tissues - Intercellular Crosstalk

Integrin receptors are responsible for integrating the extracellular matrix (ECM) environment to the inside of the cell. The most prominent integrin receptor, β1 integrin, has a major role in promoting cell function, survival, and differentiation. It has been established that β1 integrin stimulation promotes beta cell insulin secretion and differentiation as well as maintaining pancreatic tissue integrity. However, the in vivo role of β1 integrin on the adult murine endocrine and exocrine pancreas has not been understood. The aim of the present study was to examine whether knockout of β1 integrin in collagen I producing cells would have physiological and functional implications in pancreatic exocrine and endocrine cells in vivo.

At 3 weeks of age, tamoxifen injection led to β1 integrin removal in collagen I producing cells of the pancreas. The affects of β1 integrin deficiency on glucose metabolism and pancreatic exocrine and endocrine tissues were examined by qRT-PCR, western blot, immunofluorescence staining and other techniques. Male β1 integrin-deficient mice displayed onset of diabetes with significantly impaired glucose tolerance and reduced pancreatic insulin content.

Session 3

Bone growth is still active following removal of static compression on the growth plates

Ménard AL^1,2, Grimard G^1,3 Londono I¹, Moldovan F^1,3, Villemure I^1,2

¹Ecole Polytechnique de Montréal; ²Centre de recherche du CHU Sainte-Justine; ³Université de Montréal

Presenting author: Ménard AL

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Bone growth occurs in the growth plates, at both ends of long bones and vertebrae. Bone growth modulation refers to growth rate reduction under an increased pressure on the plates and to growth rate increase under a reduced pressure. This principle is the basis for novel treatment approaches of progressive musculoskeletal deformities. Following removal of a compressive loading, it is however not known whether the growth plate remains active to proceed with residual bone growth potential. This in vivo study aims at evaluating bone growth rates, following the application and subsequent removal of a finely controlled static compression in an immature rat model. Young Sprague–Dawley rats (28 d.o.) were divided into three groups: control (n = 4), sham (n = 4) and static (n = 4). A micro-mechanical device applied a 0.2 MPa static compression on the 7th caudal vertebra for 15 days. Loading was then removed for 10 days. Sham rats were operated but no loading was applied. Controls underwent no operation. All rats were euthanized at 53 d.o. Growth rates were evaluated using calcein labeling (injected at 5 and 2 days prior to sacrifice) and compared with one-way ANOVA followed by Tukey’s post hoc comparison (p < 0.05). Bone growth rates (μm/day) of both static (28.3) and sham (32.0) rats were significantly lower than growth rates of control rats (36.2). Moreover, growth rate of the static group was lower than shams although this decrease was not significant. The comparison between control and sham rats suggests that the surgical procedure slows down bone growth rate. However, the growth plate remains active after the application and subsequent removal of a 0.2 MPa sustained compression. These results suggest that bone growth could still occur following removal of static-based growth modulation implants in young patients with progressive bone deformities.

Clinical Significance Investigating the effects of loading on bone growth rate has important clinical applications in pediatric orthopedics. These studies provide relevant knowledge for the development of novel growth modulation implants to correct growth asymmetry or progressive bone disorders such as adolescent idiopathic scoliosis. Along with the correction of the deformities, it is implied that these implants would allow preserving the young patients’ growth potential.

Development of a microfluidic system to investigate bone cell signaling

Kevin Middleton, Lidan You

University of Toronto

Presenting author: Kevin Middleton

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Introduction: Most bone diseases are characterized by a disparity between bone loss and bone formation due to an imbalance in osteoclast and osteoblast activity. Bone has long been known to be able to remodel, a process that involves both osteoclasts and osteoblasts, to accommodate the loads under which it is placed; however, the underlying mechanisms that link mechanical stress and bone remodeling have yet to be fully elucidated. A third cell, the osteocyte, is an ideal candidate for driving mechanically stimulated bone remodeling because it is a known mechanotransducer and is thoroughly distributed and well connected throughout the bone matrix. However, current in vitro studies fail to elucidate the direct and dynamic effects of bone cell signaling in terms of remodeling. Because of this, we have developed a microfluidic system to test the hypothesis that mechanically unloaded osteocytes promote osteoclast precursor migration and differentiation.

Methods: A 2-channel microfluidic device was fabricated using PDMS. The channel dimensions are 60 μm high, 1 mm wide and 16 mm long and are separated by 200 μm long and 50 μm wide high resistance side channels to prevent cross-channel fluid flow while allowing for signal diffusion. To test the device, RAW 264.7 cells were seeded into one of the channels at a cell density of 300 k cells/ml while the other channel contained either a control RAW cell media or media supplemented with 50 ng/ml of RANKL. Over the course of 1 week the cells were exposed to a perfusion flow of 1 μl/min and were imaged daily to investigate cell migration and differentiation. At the end of the week, the cells were TRAP stained to validate osteoclastogenesis.

Results: COMSOL modeling we performed on our system suggested that large proteins would be able to diffuse through the side channel. This was validated when it was observed that precursor cell densities increased preferentially closer to the RANKL channel. Similarly, multinucleated osteoclasts formed preferentially within the vicinity of the RANKL channel as opposed to further along the width of the channel. For future studies we will use a modified 3-channel system and seed in RAW cells between MLO-Y4 cells that are exposed to non-stimulatory perfusion flow on one side, and 1 Pa fluid flow on the other side. This will cause a mechanically induced chemical gradient across the central channel so we can directly observe the dynamic effects of osteocyte-osteoclast cross-talk.

Effects of type, magnitude and frequency of in vitro compression on the viability of growth plate chondrocytes

Rosa Kaviani^1,2; Irene Londono ²; Stefan Parent ^2,3; Florina Moldovan ^2,3; Isabelle Villemure^1,2

¹Ecole Polytechnique de Montréal; ²Centre de recherche du CHU Sainte-Justine; ³Université de Montréal

Presenting author: Rosa Kaviani

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

The mechanical modulation of bone growth is one of the underlying reasons of infantile and juvenile angular skeletal deformities progression such as scoliosis. This concept is also exploited in the development of less invasive fusionless treatments for such deformities. To date, effects of the mechanical modulation of bone growth have been investigated in vivo on the metabolic/synthetic activity and histomorphology of growth plates as well as on bone growth rates. However, few studies have investigated the effects of mechanical modulation parameters such as compression magnitude and frequency. The aim of this study was to characterize the effects of compression type, magnitude and frequency on chondrocytes viability in growth plate explants in culture. Full thickness swine ulnar growth plate explants were aseptically extracted and randomly distributed among four groups; baseline (n = 6), control (n = 6), static (n = 2 × 6) and dynamic (3 × 6). The loaded groups were subjected to either static compression (0.1 MPa or 0.2 MPa) or dynamic compression (0.1 MPa or 0.2 MPa combined with 0.1 Hz or 1.0 Hz frequency) for a duration of 12 h over 48 h in culture (DMEM supplemented with 20 % FBS). Following mechanical modulation, a 1 mm thick section was cut from the central part of each explant and labeled for viability assessment. Confocal images spanning the three growth plate zones (reserve, proliferative, hypertrophic) were taken and the number of live/dead cells per zone was quantified using an automatic counting method. Statistical analysis was carried out on viability data using one-way ANOVA followed by Tukey’s post hoc comparison (P < 0.01). Results showed that 48 h of culture decreased chondrocytes viability by 27 % in the hypertrophic zone and 10 % in the proliferative zone, while it did not affect cell viability in the reserve zone. Static and dynamic (0.1 Hz) modulation with 0.1 MPa compression did not change the viability pattern compared to control group while for modulation with 1.0 Hz frequency, chondrocytes viability in the hypertrophic zone decreased by 30 % and no significant changes were observed in the two other zones. Using a 0.2 MPa compression led to 100 % loss of viability in all three zones, both in static and dynamic groups. The viability of mechanically modulated growth plate chondrocytes depends on the magnitude and frequency of the applied compressive stress. No difference was observed between static and dynamic modulation with low frequency while for the higher frequency the viability decreased in dynamically modulated samples. This study provides relevant knowledge for future studies on the effects of mechanical modulation on biomechanical and synthetic responses of growth plate explants in vitro.

Subcellular calcium signalling encodes directional preference during osteoclast migration

Benajmin Wheal, S. Jeffrey Dixon and Stephen M. Sims

Presenting author: Benjamin Wheal

Category: Graduate Research

If other please specify:

Field of interest: other

Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues during development, physiological remodeling and pathological bone loss. Lamellipods are dynamic membrane protrusions at the perimeter of the osteoclast that advance the cell front during migration and are responsible for steering during chemotaxis. The purpose of this study was to investigate the subcellular Ca2+ signaling events underlying lamellipod dynamics, and how this signaling orchestrates turning in response to a chemotactic stimulus. We used ratiometric live-cell imaging to map [Ca2+]i in real time within isolated rat osteoclasts. Vigilant regional analysis of multiple areas of interest revealed subcellular changes in [Ca2+]i. Osteoclasts that were not translating were still motile, as exemplified by the continuous ebb and flow of the lamellipod due to localized retraction and outgrowth. Ca2+ flickers − microdomains of elevated [Ca2+]i lasting 25.5 ± 1.7 s – were observed sporadically within lamellipods. In most cases, Ca2+ flickers were followed promptly by local retraction of the lamellipod (72 %) as opposed to outgrowth (20 %, 25 different regions from 10 cells, p < 0.05). In keeping with this observation, regions of lamellipods exhibiting outgrowth most often did NOT exhibit Ca2+ flicker activity (79 %, 24 different regions from 9 cells, p < 0.05). Furthermore, many lamellipod regions showed outgrowth until a flicker promptly triggered localized retraction. Attenuation of flickers by the intracellular Ca2+ chelator BAPTA promoted unchecked lamellipod outgrowth accompanied by impaired lamellipod retraction, creating highly elongated osteoclasts with a dendritic appearance. The role of Ca2+ flickers in steering osteoclasts during chemotaxis was investigated by creating a localized gradient of M-CSF. Directed migration induced by M-CSF promoted asymmetric Ca2+ flicker activity in lamellipods across the cell. Flicker activity was predominantly on the side of the lamellipod distant from the source of M-CSF, causing regional retraction (pruning). Thus, lamellipod retraction at distant sites underlies turning of the osteoclast towards the M-CSF. Here we show, for the first time, a role for exquisite spatiotemporal regulation of subcellular Ca2+ in the steering of migrating osteoclasts. Ca2+ flickers orchestrate osteoclast steering through lamellipod pruning, thereby encoding directional preference in response to a chemical cue.

Characterization of the bone sialoprotein null (Bsp−/−) mice mandibular phenotype: the influence of mechanical force in inducing mandibular defects

Y. Soenjaya^1,4, B. L. Foster⁶, F. H. Nociti, Jr⁶, K. R. Kantovitz⁶, J. E. Aubin⁵, D. W. Holdsworth ^1,2,4, G. K. Hunter^,3,4, M. J. Somerman⁶, H. A. Goldberg^1,3,4

¹Biomedical Engineering Program, ²Imaging Research Lab, Robarts Research Institute, ³Deparment of Biochemistry, ⁴Schulich School of Medicine & Dentistry, University of Western Ontario ⁵Department Medical Genetics, University of Toronto, and ⁶NIAMS, National Institutes of Health, Bethesda, USA

Presenting author: Yohannes Soenjaya

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Bone sialoprotein (BSP) is an acidic phosphoprotein with collagen-binding, cell- attachment and hydroxyapatite nucleating properties. BSP localizes primarily to mineralized tissues where its expression is upregulated at the onset of mineralization. Studies on Bsp−/−mice have shown decreased bone mineral density, reduced formation rates and impaired bone healing. Recently, our studies on the Bsp−/−mice have also shown a periodontal tissue phenotype with loss of functional acellular cementum, which results in a disturbance in periodontal ligament (PDL) integrity, detachment from the tooth and loss of alveolar bone.

Purpose: To determine whether mastication exacerbates the Bsp−/−mouse periodontal phenotype.

Methods: Bsp−/−mice were maintained on a mixed 129/CD1 background and fed diets that differed only in consistency (hard-pellets vs soft-powdered) to reduce mechanical force. Tissues from Bsp−/−mice and wild-type (WT) littermates were studied using histology, immunohistochemistry, and micro-computed tomography.

Results: At 12 weeks, Bsp−/−mice on hard diet were of smaller size and lower weight than age-matched WT controls, similar to published studies by Malaval et al. in 2008. When fed a soft diet, however, the weight and long-bone lengths of adult Bsp−/−mice did not differ significantly from WT controls. Malocclusion was observed in ~30 % of Bsp−/−mice fed with hard diet but only 3 % when fed soft diet. However, equivalent alveolar bone loss and tooth pitting were evident in Bsp−/−mice regardless of diet type, and these anomalies became more pronounced with age. Independent of diet, the Bsp−/−incisors at 1st and 3rd molar position had increased enamel density (40 % and 230 %, respectively), dentin thickness (50 % and 40 %, respectively), and diminished pulp area (40 % and 30 %, respectively).

Conclusions: The decrease in size of Bsp−/−mice on hard diet compared to WT mice may be due in part to a nutritional deficiency due to reduced tooth function. In support of this concept, the change to soft diet reduced the incidence of malocclusion, likely due to the decrease in mechanical force during mastication. However, regardless of diet, anomalies such as tooth pitting, alveolar bone loss, incisor enamel hypermineralization, reduction in pulp volume and thickening of dentin were still observed. This phenotype is likely induced by constant gnawing, which aggravates the weakened tooth structure noted in Bsp−/−mice due to limited acellular cementum formation and subsequently decreased PDL attachment.

This research was supported by CIHR and NIAMS/NIH. YS was supported by JuMP, a CIHR funded STIHR and the AO Foundation.

Mechanical regulation of osteocyte responses to microdamage in vitro

Liu, Chao; Zhang, Xiaoqing; Wu, Michael; You, Lidan

University of Toronto

Presenting author: Liu, Chao

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

IMicrodamage in bone triggers remodeling with resorption and bone formation along the damage site. However the cellular mechanism of the sensing of microdamage as well as the signaling to guide the repair process is not well understood. Osteocytes are cells distributed throughout the bone. They have been observed to be physically damaged by microdamage. Interestingly, microdamage-induced remodeling requires mechanical loading in vivo. Since osteocytes are the mechanosensors of bone, they could react to mechanical loading at the same time to microdamage. We hypothesize that osteocyte damaged by physical trauma similar to microdamage would produce increased levels of remodeling-related molecules; and this response is mechanically regulated. Osteocyte cell model: MLO-Y4 cells cultured (2.5 % FBS/CS, 1 % Pen/Strap) on glass slides. Microdamage model: Cells were damaged by tungsten needles (1 μm tip) mounted on a frame with horizontally travel, and adjustment of vertical position. The cell culture was damage to have ~15 % dead cells. Cell damage was assessed with Trypan Blue and Live/Dead assay. Fluid shear stress treatment: parallel flow chambers were used to apply fluid shear stress to the cells. The flow was oscillatory with sinusoidal profile at frequency of 1 Hz, and applied peak shear stress of 2 Pa to the cells for 1 h. Gene quantification: Total RNA was isolated from MLO-Y4 cells immediately after flow treatment. Reverse transcription and real-time PCR was used to measure IL-6, TNFα, COX-2, RANKL, and OPG mRNA levels, normalized to 18 s. Significant number of dead cells was observed near the damage sites, ranges from 1 to 10 μm. COX-2 and VEGF mRNA levels increased 24 h after cell damage. Other measured mRNA levels was not changed. Concurrent fluid shear stress and physical damage induced higher COX-2 and VEGF mRNA level than the effect from each individual stimulus.

DISCUSSION Using the cell damage system described here, the dead cell percentage relative to the distance to damage site resembles fatigue loading in vivo. Microdamage has a time-dependent effect in osteocyte expression of inflammatory and angiogenic genes. But osteocytes are not responsible for the changes in RANKL, OPG, IL-6, TNF-α that were observed in fatigue damaged bone in vivo. This study introduced a system that is able to induce sub-cellular damage. This study showed that cellular mechanism in targeted bone remodeling to microdamage includes altered inflammatory and angiogenic gene expression levels in osteocytes; and this response is sensitive to mechanical loading.

Session 4

A microfluidic chamber for live-cell imaging during fluid shear

Lorusso, D., Nikolov, H., Milner, J.S., Sims, S.M., Dixon, S.J., Holdsworth, D.W.

Presenting author: Lorusso, D.

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Introduction: Mechanotransduction is the process by which cells sense—and respond to—the local mechanical environment. This ability to react to external loads and forces is a critical component of mammalian physiology; yet, the underlying mechanisms are still poorly understood. Shear stress due to fluid flow is thought to be a key form of mechanical stimulation. Moreover, fluid shear has been implicated in a wide range of connective tissue diseases including: idiopathic fibrosis, osteoarthritis, and osteoporosis. Our goal is to observe the immediate responses of cells to fluid shear. Here, we describe the development of a microfluidic chamber for live cells, which is compatible with real-time optical microscopy.

Methods and Results: A microfluidic chamber was designed and fabricated from polydimethylsiloxane (PDMS) using replica molding techniques. Thin PDMS membranes were applied to a cell-culture dish with a thin glass-bottom window. Chambers were then cast and sealed to this base membrane, creating a microfluidic chamber with channels 350-μm wide by 80-μm tall. Chambers were imaged non-destructively using micro-CT to analyze channel dimensions. Computational fluid dynamics (CFD) was then used to generate velocity and resultant wall shear profiles at a range of flow rates. A saline solution containing 6-μm diameter polystyrene beads was pumped through the chambers. Imaging of the channels under flow was performed using an inverted microscope and high-speed digital camera (1,200 frames per second). Flow parameters were calculated by micro-particle imaging velocimetry, and compared to the CFD model. Chambers were disinfected and treated with fibronectin to enhance cell attachment. MC3T3-E1 osteoblast-like cells were then seeded into the chamber and imaged. Prototype microfluidic flow chambers were successfully fabricated in a reproducible manner. Steady flow rates up to 75 μL/min were introduced into the chambers, generating shear stresses of up to 3 Pa. In addition, MC3T3-E1 cells adhered and survived within the microfluidic chamber for at least 24 h.

Conclusions: We have developed and tested a microfluidic system capable of delivering physiologically relevant fluid shear stresses. Live cells can be imaged while experiencing controlled flow-induced shear. This system will enable future studies involving microscopic imaging of cellular responses to fluid shear, potentially helping us to understand mechanical factors involved in the pathogenesis of connective tissue diseases.

Painful degenerating intervertebral discs induce neuroplasticity by actively releasing nociceptive factors

Emerson Krock^1,3, Derek H. Rosenzweig^1,3, Anne-Julie Chabot-Doré^2,4, Peter Jarzem³, Michael H. Weber³, Jean A. Ouellet^2,3, Laura S. Stone^2–7, Lisbet Haglund^1,3

¹Orthopeadic Research Laboratory, Division of Orthopedic Surgery ²Alan Edwards Centre for Research on Pain, ³McGill Scoliosis and Spine Research ⁵Anesthesiology, ⁶Pharmacology and Therapeutics ⁷Faculty of Dentistry, McGill University

Presenting author: Emerson Krock

Category: Graduate Research

If other please specify:

Field of interest: Integration of the Connective Tissue with other Tissues - Intercellular Crosstalk

Intervertebral disc degeneration is the main cause of chronic low back pain. Healthy discs are mostly aneural, with nerve fibers only penetrating the peripheral edges of the outer annulus fibrosus. Conversely, evidence suggests the inner portions of the disc become innervated as disc degeneration and low back pain develop, thus linking disc innervation and pain. Furthermore, increased levels of inflammatory and nociceptive factors, such as IL-1β, TNF-α, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) have been found in animal and cell culture models of disc degeneration. Such factors could induce chronic neuronal sensitization and contribute to the development of chronic low back pain, but whether they are released from human discs is unknown. Moreover, the mechanisms of disc innervation and neuronal sensitization are largely unknown. In this study we hypothesized that degenerating discs from low back pain patients secrete increased inflammatory and nociceptive factors and that these factors induce nociceptor plasticity. Eight degenerating discs were surgically removed from patients suffering from chronic low back pain and 11 healthy discs were isolated from pain free human organ donors. Discs were cultured for 48 h using a previously validated ex vivo organ culture system. Conditioned culture media was collected and analyzed by ELISA and protein array. The neuron-like PC12 cell line and mouse dorsal root ganglia sensory neurons were cultured in either media conditioned by degenerating, painful discs, healthy pain-free discs or NGF (positive control). The ability of conditioned media to induce neurite growth was assessed in PC12 cells. Changes in calcitonin-gene related peptide (CGRP) expression, a neurotransmitter involved in pain, were assessed by immunofluorescence in neuron cultures. Anti-body blockades were used to determine factors responsible for neurite growth and CGRP changes. Degenerating discs from patients with back pain released increased levels of 22 inflammatory and nociceptive factors including NGF, BDNF, IL-1β, TNF-α, IFN-γ, IL-6, CCL-2 and CXCL-1. Degenerating disc media induced significantly greater neurite growth in PC12 cells and significantly increased CGRP compared to healthy disc media. Anti-NGF antibody treatment blocked both of these effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, and many other inflammatory and nociceptive factors ex vivo. These factors could induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. This work also supports the further development of anti-NGF therapeutics for low back pain.

Assessing the effect of notochord-specific CCN2 deletion on intervertebral disc degeneration and behaviour associated with back pain

Jake Bedore¹, Sunny Jang¹, Matthew Veras², Amanda Sauvé¹, Andrew Leask^1,3, Cheryle A. Séguin¹

Department of ¹Physiology and Pharmacology ²Biology and ³Dentistry, The University of Western Ontario

Presenting author: Jake Bedore

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of the pathways that regulate disc aging as well as the factors contributing to symptomatic vs. asymptomatic disc degeneration. The specific function of matricellular proteins such as CCN2 during these processes remains enigmatic. Recent research by our group reported a mouse strain with notochord-specific deletion of CCN2, and demonstrated the central role of this protein in regulating the extracellular matrix content of the IVD during both development and aging. Building on these findings, the current study was aimed at further elucidating the role of CCN2 during age-associated disc degeneration. Intervertebral disc health was assessed in notochord-specific Ccn2-null mice and wild-type littermate controls at 6, 9 13 & 19 months of age, using a combination of histological and immunohistochemical evaluation and magnetic resonance imaging (MRI). In addition, measuring stretch-induced axial discomfort using grip force, tail suspension and locomotor assays assessed behaviours associated with disc-specific pain. Loss of Ccn2 in notochord-derived cells accelerated age-associated IVD degeneration, as determined by MRI signal intensity and standard deviation analysis, as well as histological grading of tissue appearance. Immunolocalization of matrix degradation fragments was carried out to determine whether the loss of IVD tissue structure in the notochord-specific Ccn2-null mice was associated with increased MMP and/or aggrecanase activity. Finally, behavioural assessment of mice at 13 and 19 months of age demonstrated reduced ability to tolerate stretch-induced axial discomfort compared to wild-type littermate controls. Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD age-associated tissue maintenance. Given the disconnect between the manifestations of back pain and the radiographic appearance of disc degeneration in human patients, our findings suggesting that Ccn2-null mice demonstrate symptomatic disc degeneration provides extremely valuable information regarding the utility of our mouse model. The ability of CCN2 to regulate the composition of the intervertebral disc suggests it may represent an intriguing clinical target for treatment of disc degeneration.

Acute mechanical injury to the human intervertebral disc initiates events implicated in disc degeneration

Bashar Alkhatib; Derek Rosenzweig,; Emerson Krock,; Peter Roughley,; Lorne Beckman, Orthopaedic Reserach Lab, Montreal, QC; Thomas Steffen, Orthopaedic Research Lab, Montreal, QC, McGill University; Michael Weber, McGill Scoliosis and Spine Center; Jean Ouellet, McGill Scoliosis and Spine Center; Lisbet Haglund, McGill University

Presenting author: Bashar Alkhatib

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Introduction—Low back pain is a prevalent chronic disorder among individuals world-wide. Disc degeneration is a major cause of low back pain in adults. Among other factors, initiation of disc degeneration is thought to occur through mechanical overload. The aim of this study is to investigate cellular responses and matrix changes in whole-organ human intervertebral disc cultures following acute mechanical injury.\\Materials and Methods—Lumbar spine segments were harvested from consenting donors via the Transplant Quebec organ donation program in Montreal, Quebec. Isolated intervertebral discs were mechanically loaded using an MTS material testing system with either 5 % or 30 % strain at a rate of 30 % per second. 30 % strain consistently cracked cartilage endplates allowing visual confirmation of acute trauma. Discs were immediately cultured in low-serum (1 %) media after loading and media was collected at days 3 and 7. Cultured discs were harvested at days 7 and 14 for viability, proteoglycan and histological analysis. Conditioned media from Day 3 post-loading was assessed for released cytokines by array blots, and NGF, BDNF and TNFa were assessed by ELISA. Conditioned media was also applied to PC12 cells, and neurite outgrowth was assessed.\\Results—Mechanical injury of IVDs caused significant NP and AF cell death, while control discs maintained about 85 % viability. Mechanical injury induced a considerable release of GAG from the tissue into the culture media compared to uninjured controls, which was confirmed with Safranin-O staining of tissue sections. Injured discs also displayed increased MMP and aggrecanase-induced cleavage of aggrecan. Cytokine arrays revealed significantly elevated levels of IL-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG in conditioned media from injured discs, yet decreased levels of IL-1α, MCP-1, TGF-ß1, and TNF-α, compared to controls. Conditioned media from injured discs caused significant neurite outgrowth in PC12 cells compared to those cultured with conditioned media from uninjured or no NGF. ELISA analysis confirmed elevated levels of NGF, BDNF, and TNF-α protein levels in culture media from injured versus uninjured discs.\\Conclusion—Acute mechanical injury of the human intervertebral disc ex vivo can initiate release of factors that are associated with disc degeneration and low back pain in vivo. These findings give insights to novel diagnostics and therapeutics for degenerative disc disease and pain.

Parathyroid hormone-related peptide (PTHrP) pellet as a preventative treatment for scoliosis

C Gao^1,2, B.P. Chen^1,3, J. Hui^1,5, H. Wang¹, J.A. Ouellet⁴, N. Saran⁴ and J.E. Henderson^1,2,4

¹Bone Engineering Laboratories, Research Institute-McGill University Health Centre; ²Experimental Medicine, ³Kinesiology & Physical Education, ⁴Orthopaedic Surgery, McGill University, Quebec Canada; ⁵Biotechnology Program, University of British Columbia, Burnaby, BC, Canada

Presenting author: Chan Gao

Category: Graduate Research

Background: Idiopathic scoliosis with typical onset at puberty results in variable degrees of three-dimensional curvature of the spine. A genetic susceptibility hypothesis is supported by the phenotype of mice homozygous for targeted deletion of the gene encoding fibroblast growth factor receptor 3 (FGFR3−/−), which develop kypho-scoliosis by 8 weeks. Amino-terminal fragments of the parathyroid hormone (PTH) related proteins (PTHrP) promote bone growth and PTH is FDA-approved for the treatment of severe osteoporosis. The current study was built on the hypothesis that local delivery of amino terminal PTHrP to skeletally immature FGFR3−/−might reduce curve progression as the skeleton matures.

Method: Pellets releasing 1 mg PTHrP (1–34) or placebo control over 60 days were implanted subcutaneously adjacent to the thoracic spine of 4-week-old FGFR3−/−mice. Radiographic images were taken every month after implantation until 16 weeks when mice were euthanized and the spines scanned on a Skyscan1172 microCT instrument. Analyses included measurement of Cobb angle, axial rotation of the apical vertebra, lengths of concave and convex sides of the apical vertebra and bone volume/tissue volume (BV/TV).

Results: Serial x-rays of the spines of FGFR3−/−mice compared with age matched FGFR3+/+mice between 4 and 16 weeks of age revealed progressive kypho-scoliosis in the mutant mice (p < 0.03). Treatment of FGFR3−/−mice with PTHrP (1–34) pellets resulted in a smaller coronal Cobb angle (p < 0.03) and a reduction in rotational deformity (p < 0.06) compared with mice treated with placebo pellets. The ratio of concave/convex side of the apical vertebra was 0.89 in placebo treated controls compared with 0.94 in PTHrP (1–34) treated mice (p < 0.03). BV/TV of L5, outside of the curve, was marginally increased in treated compared with control FGFR3−/−mice and remained significantly less than in FGFR3+/+mice. When BV/TV of L5 was compared with that in the apical vertebra, it was the same as in the convex aspect and significantly less than in the concave aspect, which was comparable to that in L5 and T13 of wild type mice.

Conclusion: Kypho-scoliosis is apparent in 8 week old skeletally immature FGFR3−/−mice and gets progressively worse as they mature. Slow release of a bone anabolic peptide adjacent to the growing thoracic spine dampened the progression of scoliosis. Additional histological studies will be required to determine if treatment with PTHrP (1–34) had its primary influence on the activity of cells in the vertebral growth plates or in the intervertebral disc.

The RGD cell binding site in fibrillin-1 regulates miRNA expression

Karina Zeyer, Heena Kumra, Amani Hassan, Dieter P. Reinhardt

McGill University

Presenting author: Karina Zeyer

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Background and Aim: Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and nonelastic tissues. They are multi-domain proteins, containing mainly calcium-binding epidermal growth factor (cbEGF)-like domains and 8-cysteine/TB domains. Fibrillin-1 contains one evolutionary conserved Arg-Gly-Asp (RGD) sequence which is located in the fourth TB domain and is required to mediate cell-matrix interactions by binding to cell-surface integrins. Mutations in the domain harboring the RGD sequence lead to a heritable disorder, stiff skin syndrome, which is characterized by diffuse skin fibrosis. This study aims to determine the cell signaling function of the fibrillin-1 RGD sequence in human skin fibroblasts (HSFs).

Experimental Procedures: Two recombinant fibrillin-1 fragments were produced, one wild-type RGD-containing fragment and one fragment containing a mutant RGA sequence, which has been previously shown to abolish interactions with integrins. The loss of cell adhesion property of the RGA-containing fragment was validated by “Electric cell-substrate impedance sensing” (ECIS). The different characteristics of interactions between HSFs and the two fragments were analyzed by light and fluorescence microscopy and monitoring experiments of the cell shape over 24 h. To determine the differential regulation of signaling pathways, microarray analysis of microRNA expression was conducted. MicroRNAs play a key role in the regulation of gene expression. The microRNA expression levels were compared after 24 h of interaction between HSFs and the RGD- and RGA-containing fibrillin-1 fragments.

Results: After 24 h, HSFs were fully attached to plates coated with the wild-type fibrillin-1 fragment and displayed fibroblast-characteristic spindle shapes. In contrast, HSFs weakly attached to plates coated with the RGA-containing fibrillin-1 fragment and formed clusters of cells with untypical cell shapes. The interaction of HSFs with the RGD sequence of fibrillin-1 showed proliferative potential analyzed by immunofluorescence assays. Surprisingly, the microarray displayed differential expression of a large number of microRNAs between the two conditions. The majority of differentially regulated microRNAs were downregulated, thus leading to upregulation of their target mRNAs and activation of different pathways. Among the upregulated microRNAs, miR-29b was most highly upregulated. This is a promising candidate for future experiments as miR-29b has been shown as a potent post-transcriptional repressor of collagens (COL1A1, COL1A2, and COL3A1) and fibrillin-1.

Heparin/heparan sulfate controls fibrillin-1, -2 and -3 self-interactions in microfibril assembly

Laetitia Sabatier1*, Jelena Djokic1*, Dirk Hubmacher1, Dzaner Dzafik1, Valentin Nelea2, Dieter P. Reinhardt1,2

McGill University, 1Faculty of Medicine and 2Faculty of Dentistry, Montreal, Quebec, Canada *co-first authors

Presenting author: Jelena Djokic

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

The fibrillin family consists of three extracellular matrix glycoproteins: fibrillin-1, -2 and -3. Fibrillins are the main components of microfibrils, which fulfill a number of crucial physiological functions in the vasculature, eyes and skin, among other tissues. Functional microfibril formation is dependent on correct fibrillin expression, assembly and homeostasis. Microfibril deficiencies compromise tissue function and integrity, resulting in several severe connective tissue disorders, including Marfan syndrome. The present work investigates the role of heparan sulfate proteoglycan in the assembly of fibrillins into microfibrils. Recombinant N- and C-terminal halves of human fibrillin-1 and -2, as well as the C-terminal half of fibrillin-3, were expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. In solid phase binding assays (SPBA), all fibrillin fragments bound strongly to heparan sulfate and heparin. In addition, the N-terminal protein halves moderately bound dermatan sulfate, whereas no binding to chondroitin sulfate was detected. Monomers, intermediates and multimers of the fibrillin C-terminal halves were separated by size-exclusion chromatography and their interactions with heparin were tested. In SPBA, monomers did not interact with heparin, while intermediates interacted moderately and multimers interacted very strongly. Surface plasmon resonance spectroscopy measured weak monomer binding (KD = 17–39 nM) and strong multimer binding (KD = 0.12–1.37 nM) to heparin. The results from both techniques show that fibrillin multimerization increases its avidity for heparin. Fibrillin-1 forms homotypic N-to-C-terminal interactions and heterotypic interactions with fibrillin-2. Heparin inhibited both the homo- and heterotypic N-to-C-terminal interactions of fibrillin-1 and -2, but did not have any effect on the interaction of the fibrillin halves with fibronectin. Similarly, in cell culture, heparin addition inhibited fibrillin-1 and -2 network formation, but not fibronectin network formation. Based on these results, we propose a novel model where heparan sulfate controls microfibril assembly at the bead interaction stage.

Estrogen receptor status and transcriptome profiling of genes regulated by 17-beta estradiol in human osteoblasts derived from idiopathic scoliosis patients

Hassan A, Patten S, Baggu E, Lebeouf D, Smiljkovic M, Fendri K, Kaufman G, Zauter C, Parent S. and Moldovan F

Ecole Polytechnique de Montréal; Université de Montréal

Presenting author: Amani Hassan

Category: Graduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity affecting up to 4 % of schoolchildren worldwide. The etiology and molecular mechanisms of AIS are not clear; currently the consensus on AIS is of a multifactorial etiology, but the involvement of genetic factors is widely accepted. Several physiopathological, clinical and molecular observations suggest that hormones such as melatonin, estrogens and growth hormones play a critical role in bone mass acquisition and consequently in the progression of AIS, but the precise mechanisms involved are yet unclear. The role for estrogen seems possible due to its interaction with many factors that influence the development and progression of this spinal deformity. Additionally, estrogens impact bone remodeling and growth as well as bone acquisition, all of which are affected in AIS. To study the role of estrogen in AIS, six unrelated individuals with AIS and 6 controls (non-AIS individuals), all French Canadian females from Quebec were studied. Gene expression profiling was preliminarily investigated by microarray analysis in RNA samples from Osteoblasts derived from control (non AIS) and AIS patients. Osteoblasts were cultured for 16 h without or with increasing amounts of Estradiol. Data analysis was performed in R version 2.10.1 (Bioconductor packages oligo and limma). Selected genes with change fold greater than 1.5 were further investigated by RT-qPCR. Microarray analysis revealed several genes that are differentially regulated in AIS osteoblasts compared to control. Many of these genes are involved in different physiological signaling pathways. When we compared the transcriptome profiling of estrogen–regulated genes between non-AIS and AIS osteoblasts, several genes were up-and down-regulated in response to estrogen. W e considered five genes for further anaylsis. These genes showed the most modification upon estrogen treatment and three of these genes were previously associated with AIS. In silico analysis, show that these genes have several estrogen reponse elements in their promoters which confirm the fact that these genes are estrogen regulated. We successfully cloned these genes in PGL3 vector which includes a luciferase coding sequence. The luciferase activity will be measured in the presence or absence of estrogen. More than one gene is likely responsible for AIS, and some of these genes are estrogen–regulated. In the absence of specific causative gene(s) for AIS, our study of gene expression by microarray pointed out putative biological pathways and genes to be carefully investigated.

Investigating effects of novel conjugate drugs for the treatment of osteoporosis

Sally Hu(1)(2), Tom Willet(1)(3), Robert N Young(4), Marc Grynpas(1)(2)

Affiliations:(1)Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto; (2)Laboratory Medicine and Pathobiology, University of Toronto;(3)Institute of Biomaterials and Biomedical Engineering, University of Toronto; (4)Department of Chemistry, Simon Fraser University, Burnaby, BC

Presenting author: Sally Hu

Category: Graduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Most treatments for post-menopausal osteoporosis including bisphosphonates (BPs) are anti-resorptive but show little bone-building effects. Prostaglandin E2 (PGE2) has bone-anabolic effects in vivo through the EP4 receptor but its clinical utility is hindered by gastrointestinal side effects upon systemic administration. To avoid these side effects, synthetic EP4 agonists (EP4a) were covalently linked to the BP alendronate (ALN) to create two ALN-EP4a conjugate drugs, C1 and C2. When administered systemically, C1 and C2 will selectively bind to bone through ALN binding to bone mineral, where local hydrolytic enzymes liberate EP4a from ALN to exert anabolic effects on bone. Although C1 and C2 both have the same ALN and EP4a components, C1 has a short linker between the two components while C2 does not, making C1 more labile in vitro. Previously, we studied the effects of C1 conjugate in a 3 month curative study in rats. Now we seek to characterize and compare effects of both C1 and C2 in a longer term in vivo study. We hypothesize that C1 and C2 show differential levels of bone anabolic effects due to the absence or presence of the linker: Three month old female Sprague–Dawley rats were subjected to bilateral ovariectomy (OVX) or sham procedure and allowed to lose bones for 3 months. They were then treated via tail vein injections before sacrifice at 9 months (n = 10/group, 7 groups). Treatment groups consisted of C1 low (5 mg/kg biweekly), C1 high (5 mg/kg weekly), C2 low (15 mg/kg monthly), C2 high (15 mg/kg biweekly), vehicle for OVX (PBS biweekly), sham control (PBS biweekly), as well C2 compound mixture without conjugation (EP4A and ALN, 0.75 mg/kg each, biweekly: MicroCT showed C1 significantly increased vertebral volumetric BMD and trabecular bone volume compared to OVX controls but not in C2 treated animals. Mechanical testing of C1 vertebrates and femurs revealed significant improvement in load bearing abilities compared to OVX but not in C2 treated animals. Undecalcified histomorphometry of proximal tibial metaphysis showed C2 treatment had no effect while C1 treatment significantly increased bone formation compared to sham.\ The C1 form of ALN-EP4a conjugate drug showed significant bone anabolic effects in treated rats while the C2 conjugate did not, most likely due to slow cleavage of EP4a from ALN of the C2 conjugate in the local bone environment.

Interactions of osteoclasts with collagen type I

Boraschi, Iris; Komarova, Svetlana V.

McGill University

Presenting author: Iris Boraschi

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Abstract:

Introduction: Bone destruction by specialized multinucleated cells, osteoclasts, is important for physiological processes of bone remodeling and tooth eruption. Osteoclasts are also critical contributors to numerous diseases such as rheumatoid arthritis, periodontitis and cancer metastasis to bone. Bone matrix is composed of hydroxyapatite and organic matrix, major component of which is collagen type I. Osteoclasts destroy bone by lowering the extracellular pH to dissolve hydroxyapatite and releasing proteolytic enzymes, such as cathepsin K to digest the organic matrix.

Objective: We hypothesized that while osteoclasts mediate collagen destruction, the resulting degradation products may in turn regulate osteoclast formation and function.

Methods: The effect of full length collagen and its degradation products in formation of osteoclasts from mouse bone marrow was examined.

Results: We characterized the degradation of mouse and human collagen type I by active recombinant cathepsin K. We have found that addition of 0.2 μM of cathepsin K for a period of 30–60 min at a temperature of 32 °C, resulted in a fading of the full length collagen type I and formation of reproducible fragmentation pattern. Most of the prominent fragments with sizes between 90 and 37 kDa. The fragments of collagen were dialyzed prior to their testing in vitro against TBS. FVB mouse bone marrow cells were treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) for 6–7 days to induce osteoclast formation. Full length collagen type I (5–30 μg/ml), the dialyzed collagen type I degradation fragments (5–30 μg/ml) or control solution were added to the osteoclast differentiation media for the duration of an experiment. No significant change in the numbers of osteoclasts formed was observed in the presence of full length collagen type I or the control solution. In contrast, addition of collagen type I degradation fragments significantly and dose-dependently inhibited osteoclast formation.

Conclusions: This study demonstrates that collagen degradation fragments have a inhibitory effect during osteoclastogenesis, providing a novel mechanism for the physiological control of osteoclast formation.

The role of fibronectin as a master organizer of tissue in vivo

Heena Kumra*, Laetitia Sabatier*, Marian Chen*, Amani Hassan*, Takao Sakai**, Pierre Chambon#, Dieter P. Reinhardt*##

*Faculty of Medicine and ##Faculty of Dentistry, McGill University, Montreal, Canada, **Lerner Research Institute, Cleveland Clinic Cleveland, OH, USA, #Institut de Génétique et de Biologie Moléculaire et Cellulaire, France

Presenting author: Heena Kumra

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Fibronectin is an abundant and ubiquitously expressed protein in the extracellular matrix (ECM) of various connective tissues. It plays a major role in cell adhesion, growth, migration and differentiation, and is essential for embryonic development. The importance of fibronectin is demonstrated by the early embryonic lethality (E8.5) of the fibronectin null mouse. Evidence from cell culture systems demonstrates that fibronectin is a “master organizer and stabilizer” for the assembly of various connective tissue proteins, including fibrillin-1, collagen I and III, thrombospondin-1, fibulin-1, LTBP-1 and tenascin-C. Experimental in vitro evidence supports the concept of fibronectin as a master organizer, it is not clear whether this is the case in vivo. In the present project, we aim to determine the role of fibronectin in the formation, homeostasis and functioning of fibrillin-containing microfibrils and other extracellular matrix protein assemblies at the molecular and cellular level in vivo. This information is essential to understand the role of fibronectin in ECM biology, and the pathogenesis of connective tissue disorders caused by ECM components which are dependent on fibronectin. We hypothesize that fibronectin is a master organizer of various extracellular matrix protein assemblies in vivo. We generated a conditional (smooth muscle specific) and tamoxifen-inducible fibronectin knockout mouse. Deleting the fibronectin gene in the FN (fl/fl); SMA-Cre-ERT2/+mouse strain is achieved by injecting tamoxifen at different time points (at E14.5, P1, and P6). We have observed promising and exciting phenotypes in blood vessels and lungs upon fibronectin gene deletion. The aortic wall of the experimental mice was disorganized, featuring numerous breaks in the elastic lamellae as well as visible detachment of the cells from these elastic layers. There was a decrease in collagen deposition in the aorta and in the lung in the experimental mice as compared to the controls. Since the deletion of the fibronectin gene showed regional differences, we needed to correlate the regions of fibronectin gene deletion with protein expression and phenotypic changes. We have incorporated reporter mice, which allow for the detection of gene deletion upon activation of the Cre recombinase by tamoxifen. Soluble plasma fibronectin may be partially able to rescue the loss of tissue fibronectin produced by smooth muscle cells. To eliminate this possibility, we are generating plasma fibronectin-devoid experimental mice by crossing our experimental strain with a mouse strain having a liver specific Cre recombinase gene expression.

Mouse fibrillins are differently organized in microfibrils compared to human fibrillins

Nguyen Hong Phuc Pham, Moqing Wang, Laetitia Sabatier, Valentin Nelea, Dieter P. Reinhardt

McGill University

Presenting author: Nguyen Hong Phuc Pham

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Background and Hypothesis: Fibrillin proteins are the major components of microfibrils in elastic and non-elastic extracellular matrices and are associated with regulation of TGF-beta signaling. Mutations in fibrillin-1 lead to heritable diseases such as Marfan syndrome and Weill-Marchesani syndrome. While different studies have been performed to characterize human fibrillins in detail, mouse fibrillins have not yet been studied in depth. This study aims to generate and characterize mouse fibrillin-1 and -2 recombinant proteins, study potential functional differences compared to their human counterparts, and to generate specific antisera against mouse fibrillin-1 and -2 for subsequent studies in mouse models.

Experimental Procedures: We recombinantly expressed the C-terminal half of mouse fibrillin-1 (mrFbn1-C) and mouse fibrillin-2 (mrFbn2-C) similar to available constructs for human fibrillin-1 and -2. The recombinant proteins were expressed in human embryonic kidney cells, and subsequently purified by immobilized metal ion affinity chromatography. N-terminal sequencing validated the correct sequence of the recombinant proteins. The characteristics and functional aspects of the purified mouse proteins were analyzed using gel filtration chromatography, solid phase binding assay, and indirect immunofluorescence. In addition, the purified mrFbn1-C and 2-C proteins were used to generate polyclonal antibodies in rabbits, which were extensively characterized.

Results: Both purified mouse fibrillin fragments consistently presented in monomeric, intermediate and multimeric forms. Similar to human fibrillins, the mouse fibrillin-1 and -2 fragments showed strong interaction with heparin in solid phase binding assays and in surface plasmon resonance spectroscopy. Addition of the recombinant mouse fibrillin-1 C-terminal fragment to mouse fibroblasts inhibited mouse microfibril assembly. Both antisera had very high titers against the mouse fibrillin fragments as determined by ELISA. As expected, based on the >95 % homology on the amino acid level, the antisera showed high cross-reactivity between the mouse and human fibrillin-1 and -2 fragments. However, the antiserum against mouse fibrillin-1 reacted much more strongly with microfibrils produced from mouse cells and with mouse tissue compared to the antiserum against human fibrillin-1. This indicates that mouse fibrillins is differently organized in microfibrils as compared to human fibrillins.

Session 5

Expression Of α-SMA governs the stemness and fibrotic fate of mesenchymal stromal cells

N. Talele, J. Fradette, J. Davies, B. Hinz

U of Toronto

Presenting author: N. Talele*

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Introduction: Delivery of mesenchymal stromal cells (MSCs) to the chemical and mechanical microenvironment of fibrotic or cancerous tissues bears the risk that MSs will turn into fibrogenic contractile myofibroblasts that worsen rather than resolve the disease. The factors determining the balance of fibrotic and stemness characteristics of MSCs are unclear.

Hypothesis: We hypothesize that expression of α-smooth muscle actin (α-SMA), the hallmark protein of myofibroblasts, results in altered clonogenic and multi-potential properties (stemness) of MSCs.

Methods: To modulate myofibroblast differentiation, we plated human bone marrow-derived MSCs on physiological-soft and fibrosis-stiff elastomeric substrates with and without pro-fibrotic TGF-β1. To test reversibility of the myofibroblast phenotype, α-SMA-positive and α-SMA-negative MSC populations were first enriched by FACS and then subjected to fibrosis-inducing and -suppressing culture conditions. To test the impact of the myofibroblast phenotype on MSC stemness, we performed multi-color flow cytometry, clonal and lineage differentiation assays. To assess the role of α-SMA protein expression in MSC stemness control, we over-expressed α-SMA in α-SMA-negative and down-regulated α-SMA in α-SMA-positive MSC populations.

Results: Culture on soft substrates prevented spontaneous myofibroblast differentiation of MSCs, indicated by low expression of α-SMA and a low profile of fibrotic gene transcript expression in qRT-PCR arrays. High levels of α-SMA expression in MSCs always correlated with reduced clonogenicity and multi-lineage differentiation potential, despite preservation of a typical MSC surface marker repertoire. Overexpression of α-SMA resulted in loss of clonal and multi-lineage differentiation potential. α-SMA-positive MSCs plated on soft substrates lost myofibroblast characteristics and restored multilineage differentiation potential. Preliminary results indicate that expression of α-SMA affects nuclear translocation of YAP/TAZ transcription factors that regulate both, myofibroblast differentiation and MSC stemness.

Conclusion: MSCs, which acquire α-SMA-positive myofibroblast phenotype, represent stromal cells with no clonal potential, and altered multilineage differentiation potential, possibly mediated via YAP/TAZ.

Nanoparticle conjugated anti-microRNA-378a enhances angiogenesis and wound healing in a murine model

Haoran Li, Burton B Yang

U of Toronto

Presenting author: Haoran Li

Category: Graduate Research

If other please specify:

Field of interest: Connective tissue remodelling

Delayed or impaired wound healing is a major public health issue worldwide, especially in patients with diabetes mellitus and vascular atherosclerosis. Wound healing is achieved by complex physiological processes, including hemostasis, inflammation, re-epithelization, vascularization, and tissue remolding. Among many factors that affect these processes, microRNA has emerged as a key regulator of wound healing. We previously reported that microRNA-378a (miR-378a) plays a role in modulating wound healing. In this study, we developed an miR-Pirate378a construct sponge expressing multiple tandem microRNA binding sites. With highly matched sequence, this homological antisense transcript sufficiently blocked the processing of precursor microRNA. CD1 mice along with control group were subject to skin biopsy, which resulted in a pair of full-thickness, excisional wounds on the back of neck. By loading anti-miRNA oligo into PEG conjugated gold nanoparticles, we were able to administrate a single dose of miR-Pirate378a to adjacent wound area by intradermal injection. Wound sizes were measured thereafter, and tissue samples were collected for immunohistochemistry examination. Meanwhile, mouse fibroblast cell line NIH/3 T3 was transfected with miR-Pirate378a and subject to migration, differentiation and angiogenesis assays. MiR-Pirate378a sponge was able to block mature miR-378a functions in vitro and in vivo. We found that nanoparticle treatment significantly reduced open wound area over the course of 2–4 day treatments. Compared to the wounds on the left of the neck which were treated with blank vector loaded nanoparticles, miR-Pirate378a treatment showed a narrowed size and better healing course. In addition, we found that levels of vimentin and integrin beta-3, two modulators that are important in wound healing process, elevated remarkably in the treated mice. Wound scratch and transwell migration assays showed a greater mobility in the miR-Pirate378a-transfected cells, which was due to up-regulation of vimentin and integrin beta-3. Both molecules were confirmed as targets of miR-378a, and thus their expression could be rescued by miR-Pirate378a. Overexpression of vimentin also contributed to fibroblast differentiation, and up-regulation of integrin beta-3 by miR-Pirate378a was responsible for vigorous angiogenesis. Conclusion: We demonstrated that knockdown of miR-378a by endogenous and exogenous integrated antisense fragments could increase the expression of its target proteins, vimentin and integrin beta-3, which accelerated fibroblast migration and differentiation in vitro and enhanced wound healing in vivo. These results add a new layer of knowledge in tissue repair regulated by microRNA.

Whole body vibration results in degeneration of the intervertebral disc and knee joint in a mouse model: is fibrocartilage the culprit?

Matthew R. McCann1+, Priya Patel1+, Michael A. Pest1, Anusha Ratneswaran1, Gurkeet Lalli1, Kim L. Beaucage1, Garth Backler1, Meg P. Kamphuis1, Ziana Esmail2, Jimin Lee1, Michael Barbalinardo2, John Mort3, David W. Holdsworth4,5, Frank Beier1, S. Jeffrey Dixon1, Cheryle A. Séguin1

1Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada. 2Department of Dentistry, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada. 3Genetics Unit, Shriners Hospital for Children and Department of Surgery, McGill University, Montreal, Quebec, Canada. 4Imaging Laboratories, Robarts Research Institute, London, Ontario, Canada 5Department of Medical Biophysics, and Department of Surgery, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada. + Authors contributed equally to work.

Presenting author: Matthew R. McCann

Category: Graduate Research

Field of interest: Biomechanics Mechanobiology and Biomaterials

Objective: High-frequency, low-amplitude whole-body vibration (WBV) has been adopted for the treatment of a range of musculoskeletal disorders; however, there is limited knowledge of the direct effects of vibration on joint tissues. We examined the effects of repeated exposure to daily vibration on mouse skeletal tissues, including intervertebral discs, knee joints and bones. Methods: An in vivo mouse models was used to study changes induced by chronic vibration using custom-designed WBV platform. 10-week-old wild-type mice were subjected to vibration (45 Hz, peak acceleration 0.3 g; 30 min/day, 5 days/week) and skeletal tissues were examined after 2 or 4 weeks. Gene expression was quantified using real-time PCR and tissues were examined by histology, immunohistochemistry and micro-computed tomography (micro CT). Results: The cartilaginous intervertebral disc displayed signs of degeneration after 4 weeks of vibration when compared to sham controls. This was demonstrated by increased glycosaminoglycans throughout the annulus fibrosus and quantified using the Thompson score. Evaluation of collagen fibre architecture in the annulus fibrosus using polarized light microscopy showed tissue damage following 4 weeks vibration. Neoepitope staining for MMP-cleaved collagen and aggrecan displayed higher intensity after 4 weeks of vibration, specifically in the other annulus fibrosus. This was confirmed as MMP3 gene expression was increased both at 2 and 4 weeks of vibration. To further assess the effects of exposure to WVB, TUNEL quantification exhibited a significant increase in cell death within the disc following 4 weeks vibration, notably in the annulus fibrosus. Examination of the articular cartilage revealed the induction of meniscal tears following 2 and 4 weeks of vibration, changes that were not detected in sham controls. Furthermore, we detected focal defects in the articular cartilage tissues following 4 weeks of vibration (2/5 mice), changes that resemble severe osteoarthritis. Gene expression indicates that vibration result in increased expression of Mmp13 in articular cartilage and this is confirmed with increased staining of MMP-cleaved collagen and aggrecan in the knee joint. Assessment of the trabecular bone revealed no changes in microarchitecture, bone density or mineral content. Conclusions: The goal of this study was to elucidate the effects of repeated exposure to WBV on a range of musculoskeletal tissues. These experiments suggest potential negative effects of WBV on joint tissues. As such, these studies suggest that WBV platforms currently being used both in the clinic and personal exercise regimens should be used with caution, pending further studies effects on joint tissues.

Poster abstracts

1. Are antioxidants a possible therapy for fibrotic disorders?

Hannah Murphy-Marshman, Katherine Thompson and Andrew Leask

University of Western Ontario

Presenting author: Hannah Murphy-Marshman

Category: Undergraduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Fibrotic disease can be characterized by an excess amount of extracellular matrix depositions and can result in organ failure or death. Currently there are no therapies for fibrotic disease. A major characteristic of fibrotic disease is an increase in the cytokine transforming growth factor beta (TGFβ). This protein can differentiate a cell type called fibroblasts into a specialized form called myofibroblasts. TGFβ induces expression of the matricellular protein CCN2 (connective tissue growth factor, CTGF) which is not normally found in tissue. CCN2 is a known marker of fibrosis and targeting its overexpression in fibrotic disorders is a possible therapeutic approach. There is evidence to suggest that TGFβ may be signaling through NAD(P)H oxidase (NOX) homolog Nox4 and reactive oxygen species (ROS). ROS molecules are chemically reactive and produced naturally by our body. Antioxidants are used to reduce effects of ROS production. It has been shown that the antioxidant, N-acetylcysteine (NAC), has some benefits in treating idiopathic pulmonary fibrosis as well as reducing CCN2 overexpression in dermal systemic sclerosis fibroblasts. \\Through western immunoblotting and quantitative PCR I have shown that CCN2 protein and mRNA levels are induced by exogenous TGFβ, and this induction is reduced by N-actetylcysteine treatment. Inhibiting ROS production with NAC in both dermal and gingival fibroblasts prevents TGFβ from inducing CCN2 expression. These data provide further evidence that TGFβ can induce CCN2 through NOX and ROS. NAC has been used in the clinical practice for many decades and is readily available as a dietary supplement. Inhibition of ROS with antioxidants is a novel approach to targeting the well-known fibrotic marker, CCN2, and thus, NAC may be a useful antifibrotic drug.

2 Cartilage specific deletion of MIG-6 in the mouse elbow and ankle alters articular cartilage homeostasis

B. Russell(1), M. Pest (1), JW. Jeong(2), and F. Beier(1)

1- Dept. of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, Western University; 2- Dept. of Obstetrics Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University

Presenting author: Bailey Russell

Category: Undergraduate Research

If other please specify:

Field of interest: Genetics of Connective Tissue

Osteoarthritis (OA) is a degenerative joint disease which diminishes the quality of life of the many individuals who suffer from it. OA is characterized by the degradation of healthy articular cartilage, a process in which numerous genes have been implicated. Studies in our lab have associated increased transforming growth factor alpha (TGFα), an activator of the epidermal growth factor receptor (EGFR), with OA (Appleton et al., 2007). Mitogen inducible gene 6 (MIG-6) is a negative regulator of EGFR and its ubiquitous deletion has been shown to cause joint abnormalities and cartilage degeneration (Zhang et al., 2005; Jin et al., 2007). Conditional deletion of MIG-6 in the limb mesenchyme causes similar deformities (Shepard et al., 2013). Recent studies in our lab have characterized the chondrocyte specific deletion of MIG-6, which results in irregularities and ectopic formations, particularly surrounding the knee joint. To evaluate the effect of cartilage specific increases in EGFR signaling in the mouse elbow and ankle joints by conditionally deleting MIG-6 in cartilage. The conditional deletion of Mig6 was achieved by breeding Mig6fl/fl transgenic mice to the cartilage specific Col2a1-Cre+/− to generate Mig6 null, heterozygous, and control mice. Mice were aged to 12 and 36 weeks of age and joint morphology was evaluated histologically. Joints were embedded sagittaly in paraffin wax and serially sectioned at a thickness of 5 μm. Safranin-O/fast green staining was used to assess general changes in the articular surface of elbow and ankle joints and for measurement of articular cartilage thickness by two blinded observers. Protein localization and expression were assessed by immunohistochemistry. Results: In female 12 week old MIG-6 deleted animals, there was a significant increase in articular cartilage thickness in both the elbow and ankle joints owing to an increase in cell density and extracellular matrix production. Increased numbers of chondrocytes in these tissues stained positively for the proliferation marker PCNA, and Sox 9 over control littermates. Additionally, knockout cartilage exhibited an increase in phosphorylated EGFR, which is consistent with our model. In male and female mice aged 36 weeks, both articular cartilage thickness and anabolic markers were increased over controls, though to a lesser extent than in their younger counterparts. Though the anabolic effect of EGFR dysregulation in cartilage is less severe in the elbow and ankle, its attenuation by MIG-6 plays an important role in the maintenance of cartilage homeostasis in numerous joints of the appendicular skeleton.

3. Effect of chronic exposure to whole-body vibration on skeletal tissues

Gurkeet Lalli, Priya Patel, Matthew McCann, Michael Pest, Anusha Ratneswaran, Kim Beaucage, David W. Holdsworth, Frank Beier, S. Jeffrey Dixon, Cheryle Séguin

Presenting author: Gurkeet Lalli

Category: Undergraduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Background: Whole-body vibration platforms have recently become popular in the fitness industry owing to claims that 10 min of vibration training is equivalent to 1 h of conventional resistance training. Whole-body vibration platforms provide low-amplitude, high frequency mechanical stimulation and clinical studies have demonstrated their ability to increase bone mass and muscle strength in humans. Whole-body vibration is also being implemented clinically as a treatment for a multitude of musculoskeletal disorders, including osteoporosis, osteoarthritis and back pain. Despite its popularity, the effects of whole-body vibration on joint health have yet to be determined. The current study aimed to examine the effects of vibration on multiple skeletal tissues. Based on our previous studies, we hypothesize that chronic exposure to whole-body vibration will have a beneficial effect on bone tissues but induce degenerative changes in the knee and intervertebral disc (IVD).

Methods: Ten week old wild-type CD-1 mice were exposed to whole-body vibration using protocols designed to mirror whole-body vibration training in humans (45 Hz, 0.3 g, 30 min/day, 5 days/week) for 2 or 4 weeks. Age and sex-matched control mice were housed on a sham platform to maintain handling and environmental conditions. 24 h following the last vibration, tibia, articular cartilage and IVD tissues were harvested for gene expression analysis.\\

Results: Repeated exposure of mice to whole-body vibration induced beneficial changes in bone, including increased expression of osteoblast markers BSP and Dmp1, as well as Wnt10B. In contrast, repeated exposure to whole-body vibration induced a significant increase in expression of the pro-inflammatory cytokine IL-1β in IVD tissues following 2 weeks. This was accompanied by increased expression of aggrecan and Sox9 at both the 2 and 4 week time points. Significant increase in MMP-3 expression was induced; however no change was observed in ADAMTS gene expression. The response of articular cartilage was similar to that of the IVD, demonstrating increased expression of the matrix-degrading enzyme, MMP-13, as well as Sox9 and type II collagen following 4 weeks of whole-body vibration.

Conclusions: These studies demonstrate the distinct responses of skeletal tissues to whole-body vibration, highlighting the need to simultaneously assess the effects of mechanical stimuli in weight-bearing tissues. Given the widespread use of vibration platforms, further studies are required to fully characterize the changes elicited by whole-body vibration in skeletal tissues as well as the biomechanical parameters that govern these responses.

4. CCN1 a potential anti-fibrotic target?

Katherine Thompson, Shangxi Liu and Andrew Leask

University of Western Ontario

Presenting author: Katherine Thompson

Category: Undergraduate Research

If other please specify:

Field of interest: Connective tissue remodelling

Excessive extracellular matrix deposition and remodeling can lead to chronic fibrosis in the skin. Communication between cells and their extracellular environment is regulated and remodeled by members of the CCN family of proteins. More specifically members CCN1 and CCN2 have been shown to be up-regulated in fibrosis and in response to tissue injury. Loss of CCN2 has been shown to have no effect on tissue repair; however CCN2 is required for fibrosis in the bleomycin-induced mouse model. This strongly suggests that CCN2 is a specific target for fibrotic intervention. CCN1 and CCN2 have been shown to work similarly in vitro, and are both up-regulated in fibrosis and tissue repair. More investigation should be done to see whether CCN1 is also a potential target; or if targeting both CCN1 and CCN2 is a more effective approach in fibrotic disease. We wanted to find out what the role of CCN1 is in skin homeostasis to get a better idea of the role of this protein. A fibroblast-specific deletion of CCN1 in male adult mice was created and skin sections were studied from these mice. Loss of CCN1 leads to a significant decrease in skin thickness. A count of nuclei show that there are more cells per unit area in the CCN1−/− compared to CCN1f/f, suggesting there are the same number of cells but there is less extracellular matrix being produced in the absence of CCN1. CCN1−/−mice show a decrease in total collagen without a change in production of the collagen transcript. Amount of insoluble collagen significantly decreases with loss of CCN1. This may be due to a decrease in PLOD2, P4ha1 and LOX which are proteins associated with cross-linking of collagen and hence collagen stability. This would result in decreasing the stability of insoluble collagen and creating a high turnover of collagen in the skin. Since excessive collagen crosslinks and stability are a hallmark of fibrotic tissue, these data suggest that CCN1 must therefore be necessary for fibrogenesis in skin.

5. Effects of normal calcium diet and low sodium on stone formation and bone resorption

Elise Post(1), Sally Hu(1)(2), Tom Willet(1)(3), Nancy Krieger(4), Marc Grynpas(1)(2), David A. Bushinsky(4)

(1)Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto; (2)Department of Laboratory Medicine and Pathobiology, University of Toronto; (3)Department of Orthapaedic Surgery, University of Toronto; (4)Division of Nephrology, Department of Medicine, University of Rochester School of Medicine and Dentistry

Presenting author: Elise Post

Category: Undergraduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Hypercalciuria is defined as a condition where a patient is excreting more calcium than they are absorbing resulting in the formation of calcium containing kidney stones. The calcium in these stones is likely resorbed from bone leading to lower bone mineral density and bone strength in the individual. Genetic hypercalciuric stone-forming (GHS) rats are a useful model in studies on this topic as they mimic the effects on bone in patients with hypercalciuria. In the kidneys, a secondary active transporter—sodium-calcium exchanger—moves two sodium ions into the kidney cell and one calcium ion out using the sodium gradient across the cell membrane. According to this exchanger, calcium excretion requires the intake of sodium. The hypothesis is that with lower sodium intake, hypercalciuria in the animals will decrease leading to improved bone quality and strength. Twenty-four three-month old male GHS rats were separated into two random groups (n = 12). For 18 weeks, each rat was fed 13 g per day of food with a normal level of calcium (1.2 % Ca) and varying sodium (Na) levels. One group was fed a normal Na diet (NCD) at 0.4 % Na while the other was fed a low Na diet (LND) at 0.05 % Na. Effects of the diet on the bone were analyzed using Dual-Energy X-ray Absorptiometry (DEXA), Micro-Computed Tomography (Micro-CT) and Three-Point Bending Mechanical Testing of femurs. MicroCT analysis showed that the LND group femurs had a significantly lower femoral closed percent porosity than the NCD group (p < 0.05), as well as a higher cortical volumetric Bone Mineral Density (vBMD) compared to the NCD group, although not to a significant level. Three-Point Bending tests of the femurs showed no statistically significant differences between the intrinsic and extrinsic properties of the two groups. However consistent with increased vBMD as shown by microCT analysis, LND femurs did have a higher mean ultimate load compared to NCD. Furthermore, LND femurs also had a lower Young’s modulus (p = 0.077) compared to NCD. Considering that Young’s modulus is dictated by the interactions between the bone’s organic phase and inorganic mineral phase, this may indicate a less stable mineral-collagen interaction in the NCD group. Although there appear to be no structural changes in the L₆ vertebrae or femurs of the two groups, there is an indication of a change in mineral composition and interactions within the bone.

6. CTCF regulates Sox9 expression during cartilage development

Jason R. Bush 1, Katie Rabicki 1, Niels J. Galjart 2, Nathalie G. Berube 3, Frank Beier 1

1 Department of Physiology and Pharmacology, University of Western Ontario, 2 Department of Cell Biology and Genetics, Erasmus University, 3 Department of Biochemistry, University of Western Ontario

Presenting author: Jason R. Bush

Category: Post-doctoral Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Expression of SOX9, the master transcriptional regulator of cartilage development, is thought to require genomic looping: Mutations in or genomic rearrangements affecting non-coding sequences adjacent to SOX9 cause Campomelic Dysplasia (CD), a disorder characterized by dwarfism, severe skeletal defects, craniofacial abnormalities and perinatal lethality. In a speculative model, these non-coding regions containing enhancers are thought to loop over distances greater than 1 Mb, making contact with the SOX9 promoter to achieve robust expression. Genomic lesions affecting these regulatory sequences thus abolish enhancer activity and reduce SOX9 expression. Several proteins are known to mediate chromatin contacts between enhancers and promoters to control gene expression, including the epigenetic regulator CTCF. Interestingly, mutations in CTCF lead to a syndromic disorder that includes dwarfism and skeletal defects, suggesting that CTCF may play an important role in skeletal development. To address the role of CTCF in regulating looping and higher order chromatin conformation at Sox9 and other loci in cartilage, we generated mice with cartilage-specific deletion of Ctcf (Col2-Ctcf-null). Col2-Ctcf-null mice die at birth and display generalized chondrodysplasia, including dwarfism, craniofacial defects and campomelia. These defects are strikingly similar to patients with CD and to mice haploinsufficient for Sox9 in cartilage. Genome-wide ChIP-Seq analysis of CTCF binding in cartilage reveals a number of binding sites in the Sox9 regulatory region. Expression of Sox9 and several Sox9 target genes are significantly decreased in cartilage from Col2-Ctcf-null mice. These results are consistent with the hypothesis that dysregulation of Sox9 is a primary cause of chondrodysplasia in Col2-Ctcf-null mice and suggest that CTCF is a critical regulator of Sox9 in cartilage.

7. Characterization of the skeletal phenotype of transgenic mice over-expressing the G-protein Gαs in osteoblasts

Lucia Zhang, University of Toronto, CANADA; Kim S. Sugamori, University of Toronto, CANADA; Colin Claridge, University of Toronto, CANADA; Ariana dela Cruz, University of Toronto, CANADA; Marc Grynpas, Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, CANADA; Jane Mitchell, University of Toronto, CANADA

Presenting author: Lucia Zhang

Category: Graduate Research

If other please specify:

Field of interest: Other

Gαs is a heterotrimeric G-protein that transduces signals from activated G-protein coupled receptors on the cell surface to stimulate adenylyl cyclase-dependent signaling cascades within the cell. Gαs is found ubiquitously in all cells of the body and in bone cells it is known to play a central role in mediating processes that regulate bone health, maintenance, and repair. Our lab has previously demonstrated that Gαs protein levels are induced in an osteogenic cell line, UMR 106 cells, in response to the potent synthetic glucocorticoid dexamethasone. Moreover, we established that this over-expression translates to an increase in downstream adenylyl cyclase activity. To examine the effects of increased osteoblastic Gαs expression on bone in vivo, our lab has created a transgenic mouse model of Gαs over-expression in osteoblasts (Gs-Tg mice) driven by the 3.6 kb Col1A1 promoter. At the RNA level, we found that expression of total Gαs was increased by two- to seven- fold specifically in collagen-expressing tissues, namely skin, tendon, teeth, and long bone, of Gs-Tg mice compared to wild-type control mice. Analysis by microCT of cortical bone from 9-week old Gs-Tg mice showed a reduced volumetric bone mineral density (BMD) compared to age-matched wild-type control mice. Conversely, trabecular bone showed an increased volumetric BMD in Gs-Tg mice compared to wild-type mice. In terms of microarchitecture, Gs-Tg mice displayed increased trabecular bone and decreased spacing between trabeculae compared to wild-type control mice. Moreover, Gs-Tg mice displayed increased cortical bone perimeter as well as cortical bone area in comparison to wild-type mice, despite having decreased volumetric BMD in the cortical bone. Taken together, our data suggests that Gαs over-expression in osteoblasts results in a skeletal phenotype in which cortical bone and trabecular bone are differentially affected. Clinical studies have found that the bone disorders McCune-Albright syndrome and fibrous dysplasia result from increased activity of Gαs although the specific mechanisms involved in the pathogenesis of these disorders have yet to be elucidated. The bones of our Gs-Tg mice recapitulate several features of McCune-Albright syndrome and fibrous dysplasia bone. As such, Gs-Tg mice present a unique tool for investigating the cellular basis through which these common features present.

8. Characterization of cartilage-specific glucocorticoid receptor KO mice

Dawn Bryce(1), Holly Dupuis(1), Claudine James(1), Jan Tuckermann(2) and Frank Beier(1)

1=Department of Physiology and Pharmacology Western University London, ON, Canada 2=University of Ulm, Germany

Presenting author: Dawn Bryce

Category: Other

If other please specify: Research Technician

Field of interest: Bone Cartilage and tooth development

Glucocorticoid receptor (GR) is an ubiquitously expressed member of the nuclear receptor family of transcription factors. Glucocorticoid signalling plays essential roles in gluconeogenesis, inflammatory responses and during physiological and psychological stress. The synthetic glucocorticoid, dexamethasone, is used to treat inflammatory disorders and in organ transplantation. However, long-term use leads to growth retardation in children and osteoporosis, suggesting that glucocorticoid signalling plays an important role in bone health. The cellular actions of GC on growth plate cartilage are poorly understood, and the function of endogenous GC-GR signaling in vivo has not been examined. We generated a cartilage-specific deletion of the gene encoding GR, Nr3C1, in mice to characterize the role of GR during bone development and in both spontaneous and surgical models of osteoarthritis (OA). Purpose To characterize the effects of cartilage-specific deletion of GR on the developing growth plate and on articular cartilage in OA models. Methods Col2a1cre mice were mated with floxed Nr3C1 mice to generate mice with cartilage-specific deletion of GR. Untreated animals were harvested at Post-natal day 0 (P0) for Western analysis and at P7 and P21 for histology. Dexamethasone (20 mg/day) or PBS/BSA (vehicle) was administered daily to knockout and control littermates by subcutaneous injection for 4 weeks, commencing at P21. Weights were recorded daily and bones were harvested at endpoint for measurement and histology. Transection of the medial meniscal ligament or sham surgery was performed on 14 week old male knockout and control littermates. Joints were harvested 7 weeks post-surgery for histology. Spontaneous OA in 18 month old knockout and control animals was assessed by histology. Results Cartilage-specific deletion of Nr3C1 resulted in transient growth retardation and disorganization of the columnar arrangement of proliferative and hypertrophic chondrocytes. Western analysis of P0 epiphyseal protein showed increased abundance of Sox9, pS6, pFAK and ROCK1 and reduced abundance of p57. Immunohistochemical staining of P21 tibiae showed an extended zone of Sox9 expression and expression of p57 seemed less intense. Growth restriction was more pronounced in dexamethasone-treated controls compared to knockout mice. Histopathological grading did not show any significant differences in cartilage degeneration between genotypes both after DMM surgery and during aging. Growth plate closure was observed in knockout mice but not control littermates at 18 months.

Conclusions Disruption of endogenous GC-GR signalling in chondrocytes alters the expression of genes regulating hypertrophic differentiation. The deleterious skeletal effects of exogenous glucocorticoid act preferentially through chondrocyte GR.

9. Trabecular bone loss in Gα11 transgenic mice is characterized by decreased bone formation and increased osteoclastogenesis

Ariana Dela Cruz, University of Toronto, CANADA; Michael Mattocks, University of Toronto, Canada; Kim S. Sugamori, University of Toronto, Canada; Marc Grynpas, Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, CANADA; Jane Mitchell, University of Toronto, CANADA

Presenting author: Ariana Dela Cruz

Category: Graduate Research

If other please specify:

Field of interest: Other

Gα11 is a member of the heterotrimeric Gq protein family that mediates phospholipase C-dependent signaling downstream of G protein-coupled receptors including the parathyroid hormone 1 receptor (PTH1R). We previously demonstrated that the potent glucocorticoid dexamethasone increases Gα11 protein expression in osteoblasts and can therefore enhance parathyroid hormone (PTH) signaling through the phospholipase C pathway. To identify how increased osteoblastic expression of Gα11 affects bone, we generated transgenic mice that overexpress Gα11 in osteoblasts (G11-Tg) driven by the 3.6 kb Col1A1 promoter.

We found that G11-Tg mice overexpressed Gα11 by 2–6 fold selectively in calvaria, vertebrae, long bone, and bone marrow stromal cells (BMSCs) compared to WT controls. DEXA scans revealed significant reductions in BMD in male and female G11-Tg mice aged 3- to 20-weeks. Furthermore, microCT scans showed that bone volume and microarchitecture parameters were decreased in 8-week old G11-Tg mice trabecular bone, while cortical bone was not changed. The functional consequence of decreased G11-Tg trabecular bone was assessed by vertebral compression testing, showing that G11-Tg vertebrae exhibited reduced biomechanical strength. Consistent with our microCT data, three-point bending tests displayed no differences in normalized cortical bone strength from G11-Tg mice compared to WT controls. These data indicate that Gα11 over-expression preferentially affects the trabecular bone compartment.

To explore the cellular basis for the trabecular bone loss seen in G11-Tg mice, static and dynamic histomorphometry were performed. Bone resorption parameters including osteoclast number and surfaces were significantly increased, whereas osteoblast numbers were not altered. However, we found that G11-Tg rates of mineral apposition and bone formation were significantly decreased. In an in vitro assay to assess osteoblast differentiation, we assessed osteoblasts derived from BMSC cultures of G11-Tg mice and found increased expression of mRNA encoding osteoclast inducting proteins, M-CSF and RANKL, as well as the gene encoding MMP-13, a collagen degrading enzyme. In summary, our results suggest that osteoblastic over-expression of Gα11 results in a dual mechanism of trabecular bone loss by promoting induction of bone-resorbing osteoclasts and decreasing bone formation rate.

10. Role of bone sialoprotein in tendon-bone insertion

Ryan Marinovich, Yohannes Soenjaya, Erik Holm, Andrey Zuskov, Andrew A. Dunkman, Graeme Hunter, David W. Holdsworth, Frank Beier, Louis J. Soslowsky, Harvey A. Goldberg

U of Western Ontario

Presenting author: Ryan Marinovich

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Background: The site at which tendons and ligaments insert into bone, the enthesis, is important for musculoskeletal stability and locomotion. A common injury is the separation of tendons and ligaments from bone. Current restorative surgical techniques are inadequate, as they exhibit high failure rates. Bone sialoprotein (BSP) is an intrinsically disordered phosphoprotein that has been shown to promote the mineralization of bone. In the Bsp−/−mouse, in addition to a bone phenotype, a loss of acellular cementum was observed. This defect leads to disorganization of the collagen of the periodontal ligament (PDL) and poor attachment to the tooth. We hypothesize that BSP also promotes the mineralization of entheses, facilitating the attachment of ligaments and tendons to bone.

Methods: Experiments were performed on male Bsp−/− and wild type (WT) mice, age 15 weeks. Patella-patellar tendon-tibia complexes were isolated and prepared for mechanical testing as described (Miller et al., J Biomech Eng 134, 2012). The cross-sectional areas of tendons were measured using a custom laser-based device. Localization of BSP in the enthesis was determined by immunohistochemistry. Mineralization was detected using the von Kossa method and micro computed tomography. Sections were stained with picrosirius red and collagen organization was visualized using plane-polarized light microscopy.

Results: Immunohistochemical analysis shows that BSP is present in the calcified fibrocartilage and bone of quadriceps tendon-patella enthesis. The entheses of Bsp−/−mice appear to have reduced mineral deposition in the calcified fibrocartilage. Picrosirius red staining viewed under plane polarized light suggests less mature collagen in the Bsp−/−mouse as it inserts into bone when compared to the WT mouse. The patellar tendons of Bsp−/−mice fail at lower loads than WT patellar ligaments. Interestingly, the tendons of Bsp−/−mice have a larger cross-sectional than WT mice.

Conclusion: BSP is present in the mineralized tissues of the enthesis. The loss of BSP results in reduced mineralization of the calcified fibrocartilage as well as altered collagen fibril structure. The tendons of Bsp−/−mice are mechanically inferior yet display a larger cross-sectional area. Our study suggests that BSP mediate the attachment of tendons to bone. Funded by CIHR.

11. Development of a novel collagen-based muscle growth factor delivery system for the treatment of Duchenne muscular dystrophy

Michael Brynkus, Lisa Hoffman (Medical Biophysics), David O’Gorman (Biochemistry), and Wankei Wan

(Biomedical Engineering) Western University

Presenting author: Michael Brynkus

Category: Graduate Research

If other please specify:

Field of interest: Controlled Release for Tissue Engineering

Duchenne Muscular Dystrophy (DMD) is a fatal disease of boys characterized by progressive muscle wasting due to mutations in the dystrophin gene. Resident satellite cells can differentiate into new muscle cells to aid muscle repair, however these cells fail to keep pace with the progressive loss of muscle over time. Therapies such as growth factor (GF) delivery that enhance the regeneration of muscle cells are needed in these patients. Insulin-like growth factor-1(IGF-I) treatment has recently demonstrated enhanced muscle regeneration in mouse models of Duchenne Muscular Dystrophy (DMD). However, limitations exist in previous approaches, such as: quick degradation of GF, the absence of a concentration gradient, and systemic side effects. No previous research has tested the efficacy of a biomaterial-based carrier for controlled release of GF for DMD. Such a delivery system may overcome previous limitations. The objective of this study is to develop collagen-based GF delivery systems to enhance muscle growth in mouse models of DMD. Two controlled release GF delivery systems will be assessed and compared: collagen microbeads and nanofibers with core-shells containing GFs. We hypothesize that sustained GF delivery will regenerate muscle in mouse models of DMD. Collagen nanofibers and microbeads incorporating IGF-1 will be prepared by coaxial electrospinning and electrospraying respectively then cross linked with 0.03 M genipin in aqueous ethanol (5 %) for 3–10 days. Differences in IGF-1 release kinetics and the period of time in which IGF-1 release can be sustained in vitro will be compared between nanofibers and microbeads. In parallel, IGF-I bioactivity assays will be performed using the NIH3T3 mouse preadipocyte cell line. The microbeads/nanofibres will then be implanted into the hind limb of both Mdx and Mdx:Utrn−/− dystrophic mice at ~5–7 weeks of age. The contralateral limb and implantation into healthy wild-type mice will serve as controls. To characterize the ability of microbeads/nanofibres +/−IGF-1 to aid in muscle repair, the muscles from each of these groups will be harvested at 1, 6 and 12 weeks post-implantation and fixed for immunohistochemistry. Such findings may provide support for using core-shell collagen nanofibres and microbeads preloaded with GFs as novel and effective therapeutic to enhance muscle repair in patients with DMD.

12. The effects of the combined use of glucocorticoids and bisphosphonates on musculoskeletal system in a mouse model of Duchenne muscular dystrophy

Sung-Hee Seanna Yoon, Jinghan Chen, Ariana dela Cruz, Kim S Sugamori, Marc D. Grynpas, and Jane Mitchell

Department of Pharmacology and Toxicology, University of Toronto and Department of Laboratory medicine and Pathobiology, University of Toronto. Samuel Lunenfeld Research Institute, Toronto, ON

Presenting author: Sung-Hee Seanna Yoon

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Duchenne Muscular Dystrophy (DMD) is a X-linked recessive genetic disorder occurring in 1 in 3,500 boys. Due to mutations on the gene encoding dystrophin, a sarcolemmal protein critical in stabilizing the link between muscle fibers and dystrophin-associated glycoprotein complex in surrounding extracellular matrix, DMD patients with truncated or no dystrophin progressively show serious muscle membrane fragility and muscular necrosis. Glucocorticoids (GC) are the only currently available treatment for DMD patients for its anti-inflammatory effect on muscles, despite of numerous side effects including GC-induced osteoporosis (GIO), resulting in increased bone fragility and high prevalence of vertebral bone fractures in DMD patients. We hypothesized that the use of bisphosphonates (BP), anti-resorptive agent, at the initiation of GC treatment will ameliorate GIO development during growth, without any harmful effects on dystrophic muscles.\In this study, C57BL10ScSn-mdx mice (mdx), the commonly used animal model for DMD, and wild-type (WT) mice received 8 week GC treatment from 5 weeks of age, and the effects of GC, BP, and combination therapy of GC and BP (GCB), on bones and muscles were examined. The 8-week GC treatment significantly decreased femur length.

13. Targeting P19-derived cardiomyocytes for treatment of Duchenne muscular dystrophy-related cardiomyopathy using PET/MRI

Linshan Liu1,2,4, Rebecca McGirr1, Andrew Bondoc1,2,4, Terry Thompson1,2, Frank Prato1,2, Donna Goldhawk1,2,4, and Lisa Hoffman1,2,3,4

1Imaging program, The Lawson Health Research Institute; 2Medical Biophysics, Western University; 3Anatomy & Cell biology, Western University; 4Collaborative Graduate Program in Molecular Imaging, Western University, London, Ontario

Presenting author: Linshan Liu

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Duchenne Muscular Dystrophy (DMD) is a severe, X-linked form of muscular dystrophy characterized by progressive degeneration of both skeletal and cardiac muscle. Recent advances in cardiovascular research suggest that transplantation of cardiomyocytes into patients’ hearts may be able to repair damaged heart muscle and prevent heart failure (Blake et al. Physiol Rev 2002). However, a limitation to the use of cell replacement therapy is the lack of reliable, non-invasive technology to monitor implanted cells over time. We have generated P19-derived cardiomyocyte cell lines, engineered to stably co-express MagA and sr39tk (mutant herpes simplex virus type 1 thymidine kinase), for magnetic resonance imaging (MRI, Rohani et al. Mol Imaging Bio 2013) and positron emission tomography (PET, Pritha Ray et al. Cancer Res 2004), respectively. The stem cell features of P19 cells (Marcel et al. Cardiovasc Res 2003), together with contrast gene expression, will provide a novel and non-invasive means of MRI/PET imaging of cardiogenic differentiation. P19 cells were transfected with MagA, alone and in combination with a trifusion reporter gene that contains sr39tk under the control of a cardiac-specific promoter (α-myosin heavy chain). MagA was genetically engineered to express the hemaglutinin (HA) epitope to enable protein detection by immunoblotting and immunocytochemistry (ICC). These cells are chemically differentiated into cardiomyocytes by supplementing culture medium with 0.8 % dimethyl sulfoxide for 18 days, MagA-derived contrast was measured in cells using a spherical gelatin phantom. Transverse relaxation rates were acquired on a 3 T Biograph mMR. Results: Generation of the tagged MagA-HA construct has been completed and stable overexpression confirmed by Western blot and ICC using a mouse anti-HA monoclonal antibody. Co-transfection of this cell line with the sr39tk construct has been completed and evidence of cardiomyocyte differentiation in co-transfected cells has been confirmed. Preliminary data shows that MagA-derived contrast can be detected by MRI using a gelatin phantom to measure transverse relaxation rates in undifferentiated cells. As observed in other cell lines, the contrast signal depends on the presence of extracellular iron. We have generated P19 cell lines expressing MagA alone and in combination with the PET reporter gene sr39tk. We have also demonstrated the feasibility of assessing MagA-derived MR contrast in undifferentiated cells. Optimization of these tools, will provide a means of non-invasively assessing cardiac regeneration in a mouse model of DMD using PET/MRI.

14. Substrate stiffness controls collagen endocytosis in fibroblasts

Alice M. Green and Boris Hinz

Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, U of Toronto

Presenting author: Alice Green

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Background: Collagen type I is a major constituent of connective tissues. During fibrosis, the balance of collagen secretion and degradation by fibroblasts is disturbed resulting in collagen accumulation and tissue stiffening. Fibroblasts are sensitive to the mechanical stress associated with stiffened fibrotic tissue and undergo phenotypic change into pro-fibrotic and highly contractile myofibroblasts. Hypothesis: We test the hypothesis that mechanical stress regulates the endocytosis of collagen by myofibroblasts in a caveolae-dependent process. Objective: Determine if mechanics alter collagen internalisation in fibroblastic cells. Methods: Rat lung fibroblasts were cultured on elastic silicone substrates produced to match the stiffness of normal (Elastic modulus E of 3 kPa) and fibrotic (E = 65 kPa) lung. As another means of controlling intracellular stress, cell spreading areas were restricted by growth on microcontact printed gelatin islands. To establish whether different fibroblast activation states lead to different rates of endocytosis, myofibroblast maturation was altered by inhibiting TGF-β signaling. Collagen type I-coated microspheres were then seeded onto the cells and the number of spheres internalized per cell after 1 h was quantified using differential immunofluorescence. To address whether endocytosis was clathrin- or caveolin-mediated, internalized collagen-coated spheres and associated proteins were isolated from fibroblasts using magnetic separation and analysed by Western blotting. Results: Our data demonstrate a decrease in collagen internalisation in lung fibroblasts with increasing substrate stiffness and changes upon fibroblast size restriction and activation state. Caveolin-1 was found to be differentially expressed in fibroblasts and myofibroblasts and was implicated in the uptake of collagen-coated microspheres. Preliminary results indicate that association of caveolin-1 with endocytosed collagen is stress-dependent. Conclusions: The mechanical stress that fibroblasts encounter in fibrotic tissues possibly reduces collagen uptake and degradation contributing to the progression of fibrosis.

15. Cadherin-11 mediated macrophage adhesion promotes myofibroblast persistence

Elizabet Cambridge, Monika Lodyga, Michael Glogauer, Boris Hinz

*Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto

Presenting author: Monika Lodyga

Category: Other

If other please specify: Research Associate

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Background: A variety of fibrotic diseases are characterized by the chronic co-existence of macrophages, producing pro-fibrotic transforming growth factor beta-1 (TGF-β1) and myofibroblasts, excessively secreting and contracting extracellular matrix in response to TGF-β1. The spatial relationship between both cell types remains elusive.

Objective: Understanding how direct interaction with macrophages promotes myofibroblast activation in pro-fibrotic conditions.

Methods: We used primary mouse cells in culture. Distinct populations of fibroblasts and myofibroblasts were obtained by treating fibroblasts from lung explants with the TGF-β1 receptor inhibitor SB431542 (fibroblasts) and with TGF-β1 (myofibroblasts). Bone marrow-derived macrophages were treated with LPS (Mϕ1), IL-4(Mϕ2a), IL-10(Mϕ2c) to obtain desired macrophage polarizations. The polarized macrophages were characterized for fibrotic potential based on TGF-β1 production. Attachment of macrophages to fibroblasts and myofibroblasts was tested by: 1) seeding onto fibroblastic cell monolayers to quantify attachment and spreading kinetics, and 2) in suspension culture to assess cell surface affinity-mediated aggregation and sorting.

Results: Among the different polarization types, MΦ2a produce highest amounts of latent TGF-β1, supporting their pro-fibrotic character. MΦ2a macrophages adhere faster and stronger to myofibroblasts than to fibroblasts, indicating specific interaction of pro-fibrotic cell populations. Direct co-culture of macrophages and fibroblasts increased active TGF-β1 production compared to segregated cultures demonstrating the importance of close proximity of these two cell types in cultivating a profibrotic environment. Macrophage-fibroblast interaction is calcium dependent and the adherens junction protein cadherin-11 was identified as a potential mediator of macrophage-to-myofibroblast attachment by immunofluorescence and Western blotting.

Conclusion: We propose that cadherin-11 junctions keep macrophages and myofibroblasts in close proximity to generate a microenvironment rich in active TGF-β1.

16. Kindlin-2 is a mechanosensitive regulator of the fibroblast to myofibroblast differentiation in cardiac repair

Elena Zimina, Stephanie Hume, Sophie Clement, Andras Kapus, Leena Bruckner-Tuderman, Cristina Has, Boris Hinz

U of Toronto

Presenting author: Elena Zimina

Category: Post-doctoral Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Cardiac fibroblasts are able to respond to the mechanical stress and to differentiate into myofibroblasts. Myofibroblasts are highly contractile cells that are responsible for the tissue repair after the myocardial infarction. In a pathological situation excessive differentiation of the cardiac fibroblasts results in cardiac fibrosis. Mechanical signals in fibrosis result from the local ECM stiffening. Integrin receptors are involved at the initial sensing step at the site of ECM binding. Integrin adaptors are the main candidates for an intracellular transmission of extracellular cues. Kindlin-2 is the adaptor for b1 and b3-integrins. Our findings suggest that cardiac fibroblasts differentiation is controlled by kindlin-2 in mechanosensitive way. We demonstrated that kindlin-2 is upregulated in cardiac fibroblasts upon myofibroblast activation in vivo and in vitro. Kindlin-2 accumulates in focal adhesions of primary human cardiac myofibroblasts. We subjected primary human cardiac fibroblasts to different mechanical conditions to model the long-term and short-term mechanical challenges of fibroblasts upon cardiac repair and fibrosis. Kindlin-2 levels were increased in primary human cardiac fibroblasts cultured on fibrosis-stiff silicone substrates in compare with fibroblasts cultured on soft substrates; moreover the levels of kindlin-2 and differentiation marker aSMA were co-regulated. The short term mechanical stimulation of the cardiac fibroblasts was performed by fibronectin-coated ferromagnetic microbeads or by stretching the cells on deformable silicone membranes. It results in a portion of kindlin-2 translocating from the focal adhesions to the nucleus. Downregulation kindlin-2 in primary cardiac fibroblasts regulates the myofibroblast differentiation by controlling the level of aSMA promoter activity. Taken together our observations show that mechanical stress controls the expression and localization of kindlin-2 in cardiac fibroblasts. Hence kindlin-2 is novel mechanosensor within the myocardium that contributes to the myofibroblast differentiation.

17. Osteocytes’ response to mechanical loading supports breast cancer cell growth and migration

Yu-Heng Vivian Ma, Lidan You

U of Toronto

Presenting author: Yu-Heng Vivian Ma

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Bone metastases are common and severe complications of cancers. It is estimated that 69 % of patients died from breast cancer have developed bone metastases [1]. Cancer cells have such devastating impact on bones due to their ability to alter bone remodeling [2]. Exercise, often used as an intervention for patients suffering from breast cancer [3], regulates bone remodeling. Yet, information on the relationship between bone metastases and mechanical loadings is limited. Since osteocytes are mechanosensors of the bone that signals to reduce bone resorption in response to mechanical loading, this study focuses on the effect of this response on bone metastasis development. To achieve this, MLO-Y4 osteocyte-like cells were subjected to oscillatory fluid flow (1 Pa 1 Hz for 2 h) and media from flow experiments were extracted (conditioned medium; CM). Controls were MC3T3 osteoblasts and glass slides with no cells. Migration of MDA-MB-231 breast cancer cells towards the CM was assayed with Transwell assay. Viability of MDA-MB-231 cells in the CM was measured with trypan blue, apoptosis with APOPercentage, and proliferation with CyQuant. It was observed that significantly more MDA-MB-231 cells migrated towards the CM from MLO-Y4 cells with exposure to flow in comparison to CM from MLO-Y4 cells not exposed to flow (figure 1). MDA-MB-231 cells apoptosis rate was significantly lower in CM from MLO-Y4 cells exposed to flow (figure 2). The current data (not reported) showed no difference in MDA-MB-231 cells viability and proliferation rate between any two groups. Overall, this study suggests that osteocytes subjected to mechanical loading can promote metastases. This gives insight on the potential effects of exercises on patients suffering from cancers. Further investigation on the mechanisms (figure 3) may provide potential targets for bone metastases.

18. Scaffold free cartilage tissue can be formed in vitro using passaged chondrocytes in 3D agarose molds and serum-free media

Justin Parreno, Vanessa Bianchi, Corey Sermer, Elizabeth Delve, Weiqiang Wu, Rita Kandel

U of Toronto

Presenting author: Justin Parreno

Category: Graduate Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Clinically relevant articular cartilage defect repair using autologous cells requires a sufficient number of cells capable of producing cartilage matrix. Chondrocyte passaging in two-dimensional monolayer (2D) culture results in the cell proliferation required but also in dedifferentiation. Dedifferentiated chondrocytes spread, develop actin stress fibers, and reduce cartilage matrix (aggrecan, collagen II) expression. In addition cells increase fibrotic matrix -smooth muscle actin, α(collagen I, tenascin C) and contractile molecule (transgelin) expression which is problematic as cells have the potential to produce fibrocartilage with integration issues. Pharmacological based actin depolymerization represses fibrotic and contractile expression, although such treatments do not recover cartilage matrix expression. In this study, a scaffold-free strategy for redifferentiation of passaged chondrocytes was examined. We hypothesized that: (A) high density 3D culture of passaged cells results in actin depolymerization and repression of fibrotic and contractile genes; (B) addition of chondrogenic medium to cultures results in enhanced cartilage matrix expression and deposition. To test our hypotheses, 3D molds were created by placing molten agarose within 12 well plates. Following gelation, wells were created within agarose using 8 mm biopsy punches. Cells in media containing serum were then seeded into the wells at high density to generate a 3D cell environment. After 4 hours, actin assay determined that the resultant 3D cultures had a greater proportion of depolymerized (monomeric) actin as compared to cells in 2D. RT-PCR demonstrated that this depolymerized actin state correlated with repressed fibrotic matrix and contractile molecule mRNA levels. 3D culture alone did not recover cartilage matrix expression. Transfer of cells on day 1 to serum-free media supplemented with dexamethasone and insulin-transferin-selenium (ITS) (chondrogenic media) enhanced levels of collagen II and aggrecan by day 10. Histological studies at day 20 showed that cells formed a continuous layer of cartilaginous tissue which contained proteoglycans and collagen II as demonstrated by toluidine blue and immunohistochemical staining. Biochemical analysis revealed that collagen and glycosaminoglycan content increased with time and that more matrix accumulation occurred in the latter 10 days of culture as compared to the first 10 days. This data indicates that redifferentiation is a multistep process. 3D culture represses fibrotic matrix genes via actin depolymerization initially in culture while the chondrogenic media allows for the re-expression of cartilage matrix genes and matrix deposition which occurs later in culture. This approach to generate scaffold-free tissue shows potential for use in joint repair but in vivo studies are required to demonstrate this.

19. Cellular speed-dating: controlling cell-cell junction formation with a novel microfabricated platform

S Gribi*1, M Rother*1, E Zimina1, D Hennig1, A Kapus2, and B Hinz1

1Matrix Dynamic Group, Faculty of Dentistry, 2Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, Canada (*both authors contributed equally to this study)

Presenting author: Sandra Gribi

Category: Not selected

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Background: After establishing matrix homeostasis by the end of normal tissue repair, myofibroblasts commit mass suicide by apoptosis. Failure to clear myofibroblasts results in persistent matrix production and remodelling which are hallmarks of fibrotic diseases. We hypothesize that cell-cell adherens junctions regulate myofibroblast persistence in the highly cellularized environment of fibrotic tissues. Current experimental models to assess adherens junction formation at cell population level involve chemical manipulation or variation of cell density; both approaches interfere with matrix secretion and cell contraction and are thus ill-suited to address myofibroblast biology. Objective: We aim to develop a novel culture device to control cell-cell contact formation simultaneously in all cells of the population.\\ Material and Methods: The principle of the device is to growing cells individually on rigid cubes that are arranged as arrays of islands on a highly elastic and expanded membrane. Relaxing the membrane decreases the spacing between the islands, thus allowing de novo cell-cell junction formation over the now reduced gap. To determine cube dimensions that accommodate full cell coverage of the island surface and spacing that allows contact formation, we seeded myofibroblasts on a silicone substrate prototype. We then used photolithography to construct cubic islands of a stiff epoxy resin SU-8 on a highly extendable silicone membrane and glass surfaces.\\ Results: The optimal spacing between the islands to control cells junction dimension was 5 μm. Cubes with a surface of 50 × 50 μm were fully covered by individual myofibroblasts; surfaces of 20 × 20 μm accommodated smaller epithelial cells. Conclusion: It is possible to use the photolithography epoxy SU-8 on elastic membranes as a culture substrate for different cell types. Substrate topographies with the surface of single spread cells and the resolution of intercellular gaps can be achieved on prototypes, providing proof-of-principle for the new device. Future works will concentrate on improving the fabrication step to achieve high resolution patterns on the elastic membrane in a dynamically controlled setup.

Grants: This work was supported by the Collaborative Health Research Programme (CIHR/NSERC) grant #413783 and CIHR grant#219974.

20. Remodeling response of gingival fibroblasts on smooth versus rough titanium

Shawna Kim and Douglas Hamilton

University of Western Ontario

Presenting author: Shawna Kim

Category: Graduate Research

If other please specify:

Field of interest: Connective tissue remodelling

Successful remodeling of gingival connective tissues around dental implants is critical for a proper peri-implant mucosal seal to prevent bacterial infiltration. Implant substratum topography strongly influences cell adhesion, which triggers intracellular signaling that regulates tissue-remodeling phenotypes. The aim of this study was to evaluate adhesion and the remodeling phenotype of human gingival fibroblasts (HGFs) cultured on smooth and rough titanium surface topographies. HGFs were cultured on smooth polished (PT) and sand-blasted, acid-etched titanium (SLA) disks (Straumann). Immunocytochemistry was performed to assess localization of integrins (β1, β3, β5, α5, αvβ3, and αvβ5), vinculin, phosphorylated-cortactin, and tensin-1, after 6 and 24 h and fibronectin deposition and α-smooth muscle actin-positive stress fibers at 0.25, 1, 7 and 14 days. Periostin, versican, biglycan, and decorin were assessed using immunocytochemistry and Extracellular Matrix RT² Profiler PCR Array was performed at 1 and 7 days. Faster assembly of adhesions was seen on PT at 24 h compared to SLA with αVβ3 the predominant integrin in focal adhesions. Fibronectin deposition was lower on SLA compared to PT at 6 and 24 h, but extensive fibrillar fibronectin deposition was seen on at both PT and SLA at 1 and 2 weeks. Myofibroblast differentiation was only observed on PT. Periostin secretion was lower, whereas biglycan and decorin were higher, on SLA. PCR array demonstrated that of 84 genes, increases in 15 and 16 genes on SLA at 1 and 7 days. The fibrotic mediator connective tissue growth factor was significantly reduced on SLA at 7 days (p1000-fold increases in MMP-7, 8, and 9, PECAM-1, and TIMP3 mRNA levels on SLA were detected. We conclude that rough titanium elicits decreased fibrotic and elevated tissue-remodeling responses compared to smooth titanium, and thus shows a competitive advantage for implant abutments to avoid fibrosis during healing.

21. Do changes in the relative velocity of the joint surfaces following Anterior Cruciate Ligament (ACL) and ACL/Medial Collateral Ligament (MCL) transection relate to changes in the cartilage?

S. M. Mehdi Shekarforoush, MSc, Cyril B. Frank, MD, Nigel Shrive, D. Phil

McCaig Institute for Bone and Joint Health, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4 N1, Canada

Presenting author: S. M. Mehdi Shekarforoush

Category: Graduate Research

If other please specify:

Field of interest: Biomechanics Mechanobiology and Biomaterials

Osteoarthritis (OA) is a degenerative joint disease that features pain, disability, and loss of joint function with typical pathological changes in bone and loss of articular cartilage. Severe injury to the knee joint, such as cruciate ligament tears and/or menisci damage, often results in accelerated development of OA. It is thought that some type of injury-induced, mechanical abnormality of the injured joint may lead to cartilage damage. Hypothesis: ACL/MCL transection in a sheep causes small, but significantly, abnormal variation in tibio-femoral relative surface velocity leading to damage of the cartilage surface. This hypothesis will be evaluated by the following specific objectives: 1) measure the relative motion of the femur with respect to the tibia before and after ACL/MCL transection, 2) determine the 3D shape of the femur and tibia, 3) determine the split line orientation of the cartilage and map this onto the 3D shapes, and 4) determine the loci of focal cartilage damage 52 weeks after injury and map onto 3D shapes. Methods: Fifteen mature female sheep will be allocated into three groups: ACL/MCL transections, sham, and control groups (n = 5 for all groups). For all animals, joint kinematics will be measured at 4, 20, and 52 weeks post ACL/MCL transection. The animals will be sacrificed at 52 weeks after ACL/MCL transection and the relative surface velocity of the joints will be determined for each kinematic data collection. The joints will be imaged with MRI and the 3D shapes retained. Cartilage split line orientations will be determined and plotted on these shapes. The relative velocities of the surfaces throughout gait (femur relative to tibial/meniscal combined) will be plotted on the femoral shape and the direct cartilage-cartilage contact area of the tibia. Locations of damage will be determined and plotted, and then correlations will be assessed. \Significance: These experiments will provide new information about the change in surface velocity of a knee joint and the location of focal lesions in the development of OA following ACL/MCL transection.

22. Mechanical stress regulates the binding of integrin β3 to latency-associated peptide (LAP) in fibroblasts

A Koehler, Y Kwon, S Boo, B Hinz

U of Toronto

Presenting author: A Koehler

Category: Other

If other please specify: Research associate

Field of interest: Biomechanics Mechanobiology and Biomaterials

Background: Fibroblast to myofibroblast differentiation by mechanical stress and active transforming growth factor-β1 (TGF-β1) is a pivotal event in the pathogenesis of fibrosis. Wipff et al. (2007) showed that myofibroblasts can liberate active TGF-β1 by integrin-mediated pulling on the LAP portion of the latent TGF-β1 complex. Recent work in our lab showed that integrin αvβ3 binds to LAP with relatively low affinity in the absence of cellular stress, which would not allow liberation of active TGF-β1 by integrin-mediated pulling. Objective: To test whether mechanical stress alters the affinity of integrin αvβ3 to LAP in fibroblasts. Methods: Recombinant human LAP was generated using a bacculovirus expression system. To test whether extracellular stress enhances integrin recruitment to focal adhesions, stiffness-tuneable silicone substrates and tissue culture plastic (TCP) were coated with purified LAP and rat embryonic fibroblasts (REF) stably transfected with a β3-GFP construct assessed for integrin expression and binding. The effect of alteration of intracellular stress on integrin β3 was assessed by micro-contact printing of LAP and treatment of REF grown on LAP-coated TCP with various contraction-inducing or cytoskeleton-modulating drugs. Integrin recruitment to focal adhesions and expression was determined by immunofluorescence and Western blotting, respectively. Results: We have established that integrin αvβ3 is recruited to focal adhesions in a stress-dependent manner in fibroblasts. Conclusions: These preliminary results suggest that mechanical stress plays a role in the up-regulation of integrin αvβ3 in fibroblasts and its affinity to LAP. Further investigation will provide an explanation for the finding that activation of TGF-β1 by integrin pulling only occurs on sufficiently stiff ECM.

23. ED-A domain in fibronectin: binding site for interaction with the latent TGF-β1 binding protein

F Klingberg, M Chow, G Chau, A Koehler, E White, B Hinz

U of Toronto

Presenting author: A Koehler

Category: Other

If other please specify: Research associate

Field of interest: The Extracellular Matrix in Connective Tissue

Background: The ED-A splice variant of fibronectin is neo-expressed in fibro-contractive disorders. Secretion of ED-A fibronectin and stiff extracellular matrix (ECM) are prerequisites for the differentiation of fibroblasts into fibrogenic myofibroblasts. The mechanism of how ED-A fibronectin contributes to myofibroblast differentiation and fibrosis is unknown. We hypothesize that ED-A fibronectin acts as an extracellular store for the pro-fibrotic cytokine TGFβ1 by binding to the latent TGFβ1 binding protein (LTBP-1).

Objective: To analyze the expression and association of ED-A fibronectin and LTBP-1 under myofibroblast-permissive and -suppressive culture conditions.

Methods: We cultured human dermal myofibroblasts on collagen-coated silicone substrates with stiffness of 5 kPa (normal-soft), 50 kPa (fibrotic-stiff), and 3,000 kPa (tissue culture plastic stiff) for 7 days. To determine the binding of LTBP-1 to ED-A fibronectin, fibroblasts were treated with two antagonists specific to the ED-A domain (IST-9 and recombinant purified ED-A domain peptide). Using confocal microscopy and Western blotting, we analyzed expression and organization of ED-A fibronectin and LTBP-1.

Results: In human dermal myofibroblasts ED-A fibronectin and LTBP-1 expression were higher on stiff than on soft substrates. In these conditions, LTBP-1 and ED-A fibronectin always co-localized. Specific blocking the ED-A domain resulted in the loss of LTBP-1 colocalization with ED-A fibronectin. Western blotting demonstrated that ED-A antagonists translocated LTBP-1 from the ECM to the medium supernatant. Solid phase binding experiments revealed that the ED-A domain in fibronectin is the site of binding with LTBP-1, with the 11th and 12th domain enhancing interactions.

Conclusion: ECM stiffness affects the organization and expression of ED-A fibronectin and LTBP-1. Blocking experiments and binding interaction studies show that ED-A fibronectin can serve as a storage site for LTBP-1 in the ECM.

24. Persepectives for tissue regeneragtion in Densitr—need of the time

Mridula Goswami

Presenting author: Mridula Goswami

Category: Other

If other please specify: Research Review

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Tissue engineering is a novel field of research with which it may be possible to repair damaged tissues or even create replacement organs. Tissue engineering can help in the regeneration of enamel and dentin to restore the lost tooth structure. Dental stem cells are most accessible stem cells, demonstrated in Dental pulp, periodontal ligament and dental follicle. The pulpal connective tissue plays a major role in these newer techniques. Caries and periodontal disease remain two of the highly prevalent disorders involving the mankind. Dental caries result in the loss of the tooth structure. Therefore, the regeneration of enamel, cementum and the dentin-pulp complex is the long term goal of Restorative and Endodontic treatment in dentistry. The modern Restorative Dentistry aims at functionally and cosmetically replacing the tooth structure. Till recently, a variety of synthetic materials were developed to restore the damaged tooth structure. Although these materials have proved to be effective, they do not exhibit the same mechanical and physical properties as naturally formed dentine and enamel. Regenerative Stem cells lead to the regeneration of teeth with periodontal ligament that can remodel with the host. Regenerative therapy is less invasive than surgical implantation. The goal is to regenerate the pulp inside the damaged tooth, preventing the need for endodontic treatment. This presentation describes the current knowledge and recent developments in mesenchymal stem cells and their applications in dental tissue regenerative procedures.

25. Induction of calcification in adventitial aortic fibroblasts by oncostatin M

Karen Kwofie, David Schnittker, Matthew Scott, and Carl D. Richards

McMaster Immunology Research Center, Department of Pathology and Molecular Medicine, McMaster University, Hamilton ON, Canada

Presenting author: Karen Kwofie

Category: Graduate Research

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Introduction: Atherosclerosis is one of the leading causes of mortality and morbidity worldwide. It has been recognized that inflammation plays a key role in the development and progression of atherosclerotic lesions within vascular walls. Several pro-inflammatory cytokines such as IL-1 and IL-6 have been implicated and more recently, the IL-6 family cytokine, Oncostatin M (OSM) has been shown to be present in human and mouse atherosclerotic lesions and to regulate vessel wall cell activity. Advanced lesions of atherosclerosis show calcification and evidence of osteoblast-like cell phenotypes. Osteogenesis is a highly complex process that involves several growth factors and cytokines; OSM has been shown to be involved in bone metabolism and osteoblast differentiation. Furthermore, activated macrophages secrete cytokines that includes OSM, which leads to osteoblastogenesis of mesenchymal stem cells in vitro. However, whether this function is involved in calcification of atherosclerotic lesions is not known.

Methods: Here, we examine the in vitro affects of OSM on cells of vasculature including Human Aortic Adventitial Fibroblasts (HAoAFs), mouse aortic adventitial fibroblasts (MAoAF) and Human aortic smooth muscle cells (HAoSMC). These cells were cultured in osteogenic medium, stimulated with OSM, Bone Morphogenetic Protein-2 (BMP-2) and LPS combinations and assessed qualitatively and quantitatively for calcification using alizarin red staining. HAoAF and HAoSMC were also treated with OSM and LPS combinations and assessed mRNA levels for regulation of various inflammatory proteins.

Results: We observed that OSM could induce calcification independent of BMP-2 or LPS treatments at both 13 and 20-day cultures in HAoAF and MAoAF. Indeed, OSM induced markedly greater calcification than BMP-2 or combinations with LPS. On the other hand, smooth muscle cells (HAoSMC) treated with OSM at 5 ng/ml or 20 ng/ml did not exhibit increased calcification as indicated by alizarin red staining. However, HAoSMC did respond similarly to HAoAF upon OSM stimulation in the regulation of MCP-1 IL-6, VEGF and IL-8 mRNA levels.

Conclusion: The data indicates that OSM may be involved in advanced atherosclerotic lesions through mediating calcification and osteogenic-like phenotypes in aortic adventitial fibroblasts but not smooth muscle cells. Expression of MCP-1, IL-6, VEGF, and IL-8 in response to OSM, by both cell types, may also contribute to atherosclerosis. Supported by CIHR.

26. Association of oncostatin M-mediated regulation of ECM remodelling and tumour growth in the oncogenic K-ras mouse model

Izakelian, L; Wong, S; Lauber, S; Richards, CD

McMaster Immunology Research Center, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario

Presenting author: Laura Izakelian

Category: Graduate Research

If other please specify:

Field of interest: Connective Tissue in Disease - Clinical and Fundamental Aspects

Inflammation-mediated extracellular matrix (ECM) remodeling is a growing theme in cancer research. Though the role of inflammation specifically in lung cancer is not yet clear, other models suggest inflammation influences non-tumour stromal cells such as fibroblasts, and subsequent roles in tumour promotion. Fibroblasts, in turn, have been shown to acquire a modified phenotype, termed cancer-associated fibroblasts (CAFs), that produce increased levels of collagen 1, alpha smooth muscle actin (αSMA), and fibronectin, of which fibronectin contributes to tumour angiogenesis as well as metastasis. Amongst numerous cytokines, members of the gp130 family, including Oncostatin M (OSM) and IL-6, have exhibited various roles in inflammation, and we have shown that elevated OSM induces ECM deposition in mouse lungs. We also show that overexpression of OSM increases tumour burden in an inducible oncogenic K-ras mouse model of lung cancer. Thus the aim of this research is to determine the mechanism by which Oncostatin M affects tumour burden by examining cells comprising the tumour stroma, including fibroblasts. The inducible oncogenic K-ras mouse model has been established, where lung tumours are initiated via endotracheal intubation of an adenovirus vector encoding Cre Recombinase and quantified by digital analysis of histopathology. These in vivo studies were accompanied by in vitro studies of mutant K-ras tumour cell lines, Lewis Lung Carcinoma (LLC) cells and LKR-13 cells, as well as primary mouse lung fibroblasts (MLF). Co-culture experiments are ongoing with macrophages or MLFs and luciferase-labelled LLC cells (LUC-LLC). Overexpression of OSM using Ad vector encoding mouse OSM increased levels of fibronectin, collagen 1 and 3, as well as TIMP1 mRNA in mouse lungs in vivo. AdOSM also elevated tumour burden in the inducibe oncogenic K-ras mouse model. Interestingly, direct rOSM stimulation of tumour cells in vitro did not yield changes in proliferation, nor alter fibronectin mRNA levels. Subsequent preliminary experiments examined indirect regulation by Oncostatin M, utilizing macrophages removed from AdDel70 (control vector) and AdOSM-treated mice (at day 7) then cocultured with LUC-LLC cells. OSM treatment didn’t alter LUC-LLC tumour cell proliferation in coculture. In contrast, conditioned media from mouse lung fibroblasts altered tumour cell proliferation of LLC and LKR cells. Overexpression of Oncostatin M in mouse lungs exhibits enhancing effects on tumour burden in the oncogenic K-ras mouse model of lung cancer, and this is associated with increased ECM and a putative CAF phenotype.

27. V-ATPase a4 subunit is glycosylated but is the glycosylation necessary for assembly, folding and/or targeting?

S. Esmail*(1) R. A. Reithmeier (2), M. F. Manolson (1)

Faculty of Dentistry (1) and the Dept. of Biochemistry (2), University of Toronto,

Presenting author: Sally Esmail

Category: Graduate Research

If other please specify:

Field of interest: Integration of the Connective Tissue with other Tissues - Intercellular Crosstalk

Background: Vacuolar H + −ATPases (V-ATPases) are conserved multi-subunit, ATP-dependent molecular motors that pump protons across membranes from the cytoplasm to luminal compartments. V-ATPases targeted to the plasma membrane of osteoclasts, are responsible for acidifying the surface of bone, essential for bone resorption. V-ATPases on the plasma membrane of metastatic cells acidify the extracellular space to facilitate cellular invasion. The “a” subunit is the largest of 14 different subunits that make up the V-ATPase complex. In mammalian species it has four paralogous isoforms, a1–4 and evidence suggests that these isoforms may be responsible for targeting the multi-subunit complex to specific locations. The a1 and a2 isoforms are primarily localized to intracellular compartments, a3 is localized to the osteoclast membrane of resorptive bone, and the a4 subunit is expressed in the kidney in intercalated cells of the distal nephron. In cancer cells, plasma membrane expression of both a3 and a4 are required for cellular invasion. The protein sequence of the a4 subunit contains one putative glycosylation site. \\Hypothesis: V-ATPase a4 subunit is glycosylated in vivo and this glycosylation is important for assembly, folding and/or targeting the rest of the V-ATPase complex to the plasma membrane \\Overall Objective and significance: Elucidate specific features of the a subunit isoforms which govern structure, function and membrane targeting of V-ATPases, in order to facilitate the development of site-directed therapeutics to prevent bone resorption or cell metastasis. Methods: Mammalian cells were transfected with C-terminal FLAG tagged version of the V-ATPase ‘a4’ subunit with or without mutations that eliminate the putative glycosylation site (N499D and N499Q). Expression and plasma membrane targeting were assessed by immunofluorescence and immunoblotting. Glycosylation was assessed by immunoblotting whole cell extracts with or without treatment with endoglycosidases PNGase and Endo H enzymes. Results: Whole cell extracts from cells transfected with wild type ‘a4-FLAG’ were immunoblotted with anti-FLAG revealing a diffuse 96 kDa band. When treated with Endo-H and PNGaseF, the 96 kDa band decreased to 93 kDa suggesting that the a4 subunit is glycosylated. Immunoblots of a4 bearing either the N499D or N499Q mutation resulted in identical 93 kDa bands with reduced intensity relative to the wild type. These results show that N499 is the site of glycosylation. Unglycosylated mutants a4 N499Q and a4 N499Q show decreased protein levels by 10 and 15 % respectively, relative to the wild type. Confocal microscopy images of MDCK show peri-nuclear localization of a4 bearing the N499Q and N499D mutations suggesting that glycosylation may be required for ER exit. Colocalization of a4 with Calnexin is currently underway to confirm ER localization. \\Conclusions: Human V-ATPase a4 subunit is a glycoprotein with N-linked glycosylation at position N499. Evidence suggests that glycosylation is required for ER exit. Current investigations are underway to determine whether glycosylation is important for assembly, folding and/or targeting of the V-ATPase complex.

28. Protein interaction studies of short fibulins by surface plasmon resonance spectroscopy

Valentin Nelea1, Jelena Djokic1, Dieter P. Reinhardt1,2

1Faculty of Medicine and 2Faculty of Dentistry, McGill University, Montreal, Canada

Presenting author: Valentin Nelea

Category: Other

If other please specify: Research Associate level

Field of interest: The Extracellular Matrix in Connective Tissues

Short fibulins (fibulin-3, -4 and -5) are matricellular glycoproteins of the elastic fiber/microfibril system implicated in formation, homeostasis and physiological remodeling of elastic tissues. To accomplish their functions it is believed that fibulins interact with themselves or with other biomolecules to form oligomers, multimers, or complex structures. We report here on the self-interaction of fibulin 3-, -4 and -5 and on the interaction of these fibulins with fibronectin and heparin assessed by the surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Full-length short fibulins recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography were used. Fibulin-fragments (N-term-cbEGF1, an 18 kDa central cbEGF2-5 fragment, and a C-terminal fragment) were separated by chromatography using a Superose 6 column. The binding assays were performed on a Biacore-X SPR instrument using CM5- or C1-type sensors with immobilized fibulin-3, fibulin-4, fibulin-5, fibronectin, and heparin. Real-time association and dissociation were monitored in duplicates or triplicates by injecting proteins diluted at 0.1–100 μg/mL in TBS/2 mM CaCl2, pH 7.4. Kinetic analyses were performed using the BIAevaluation software by fitting the association and dissociation curves with a 1:1 binding model. AFM measurements were performed to visualize possible self-interaction-driven structured assemblies. Fibulin-3, -4, and -5 showed self-interaction properties, while each short fibulin did not interact with another short fibulin. Fibulin-4 had the highest self-association affinity (dissociation constant KD = 6 nM). The KD for fibulin-3 and fibulin-5 were 88 nM and 200 nM. AFM images showed that Fibulin-4 forms monomers, dimers and multimers, confirming the SPR binding data. Fibulin-3 and -5 also showed formation of structured multimer assemblies, but the distinction of monomers and dimers were less evident. Interaction of fibulins with heparin had high binding affinity with KD = 6.2 nM, 4.6 nM and 9.8 nM for fibulin-3, 4-, and 5-, respectively. For all the three fibulins, interactions with fibronectin showed strong associations. Fitting of the fibulin-fibronectin data with the 1:1 binding model generated chi2 errors larger than those of self-interactions or when interacting with heparin, indicating possible multi-site binding events. As calculated, dissociation constant estimates were in the 150–220 nM range. N-term-cbEGF1 fibulin-4 fragment did not bind to heparin, while with it possessed a very weak affinity (KD = 550 nM) for fibronectin. C-term and cbEGF2-5 bound strongly to heparin (KD = 1.6 nM and 5.5 nM, respectively) and weakly to fibronectin (KD = 165 nM and 145 nM, respectively).

29. Proteomic signature of the murine intervertebral disc

Matthew R. McCann1, Priya Patel1,Yizhi (Cindy) Xiao2, Walter L Siqueira2, Cheryle, A Séguin1

1Department of Physiology and Pharmacology, 2School of Dentistry and Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; N6A 5C1

Presenting author: Matthew R. McCann

Category: Graduate Research

If other please specify:

Field of interest: other

Low back pain is the most common musculoskeletal problem with a lifetime prevalence of 84 % in Canada. A significant proportion of low back pain is attributed to degeneration of the intervertebral disc. The lack of effective treatment for this widespread problem is directly related to our limited understanding of the biological pathways responsible for maintaining disc tissue health. In particular, there is an incomplete understanding of the relative importance of specific transcription factors, secreted peptides and matrix components that constitute the unique microenvironment of the IVD. While transcriptional analysis has permitted some initial insights into the pathways responsible for maintaining disc health, a more complete characterization is required for the development of targeted treatments for disc degeneration. Intervertebral discs were isolated from 10-week-old (skeletally mature) CD-1 wild-type mice and total protein was extracted. Using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) protein/peptide separation and mass spectrometric analyses was performed. The MS/MS spectra was searched against a customized mouse database, using the SEQUEST algorithm with a false discovery rate of 1 % using a decoy search strategy against a reverse database. The inclusion criterion for positive identification of proteins was the presence of 2 or more tryptic peptides from the same protein. A total of 973 proteins were identified and categorized using the PANTHER bioinformatic web-tool that combines gene function, ontology, pathway and statistical analysis to enable a large-scale categorization of proteins found within the intervertebral disc. The cellular components identified were primarily intracellular (63 %), followed by extracellular (21 %) and plasma membrane (9 %). From the initial list of identified proteins, they were further classified by biological process into 17 subgroups. The most abundant subgroups were metabolic process (20 %), cellular process (17 %), and cell communication (11 %). Most proteins categorized by molecular function were grouped into three categories: protein binding (29 %), catalytic activity (27 %) or structural activity (14 %). To validate specificity the proteins identified, 13 IVD specific proteins, including matrix proteins, transcriptional regulators, and secreted proteins were queried against our generated list. We correlated 8 of the 13 proteins, indicating that the proteins identified are a valid proteomic characterization of the IVD. This study is the first to provide a comprehensive proteome identification of healthy mouse intervertebral disc tissue. The identification and characterization of the protein composition of healthy intervertebral disc tissue is an important first step in identifying cellular processes and pathways disrupted during aging or disease progression.

30. Imaging myocardial progenitor cells to treat Duchenne muscular dystrophy-related cardiomyopathy

Andrew Bondoc*†, L Liu*†, K Gutpell*‡, R McGirr*, L Hoffman*†‡

*Imaging Program, Lawson Health Research Institute, London, ON; †Department of Medical Biophysics, Western University, ‡Department of Anatomy and Cell Biology, Western University

Presenting author: Andrew Bondoc

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Duchenne muscular dystrophy (DMD) is an X-linked disorder affecting 1 in 3,500 boys, characterized by progressive cardiac muscle deterioration. Transplant of myocardial progenitor cells (MPCs) is an excellent candidate for therapy; however, there are currently no reliable methods to monitor MPC localization and function in vivo. The purpose of this study is to design a MPC that is traceable non-invasively following implant into the heart of a DMD mouse model via molecular imaging technologies: bioluminescence imaging (BLI) and positron emission tomography (PET). A novel transgenic mouse line will express a cardiac-specific tri-fusion reporter protein (TFR); this will allow MPCs isolated from these mice to be targeted non-invasively following implant into a mouse model of DMD via BLI and PET.1) A plasmid containing TFR (luciferase/RFP/sr39 thymidine kinase) under the control of the cardiac-specific αMHC promoter was constructed (previously performed by Hoffman lab); 2) the plasmid was micro-injected into the pronuclei of C57BL/6 mouse embryos (previously performed by Hoffman lab); 3) immunoblot analysis was performed on adult tissue lysates to confirm cardiac-specific TFR expression; 4) viable transgenic MPCs were isolated by fluorescence-activated cell sorting (FACS); 5) the MPCs were expanded and differentiated in culture, and tested for RFP expression by fluorescence microscopy; 6) immunocytochemistry was performed to assess the expression of various cardiac markers. Immunoblot analysis revealed the expression of TFR exclusively in transgenic mouse myocardia. FACS-isolated MPCs also showed appropriate RFP expression as early as day 4 post-differentiation, and immunocytochemistry revealed expression of cardiac troponin and connexin-43, indicating MPCs differentiate appropriately in vitro. Transgenic MPCs may serve as an important cellular tool to further investigate and characterize the molecular and microvascular environments needed for appropriate stem cell regenerative therapy. As such, MPCs will be used in non-invasive imaging studies where they will be implanted into the dystrophic mouse myocardium. Previous work by Bondoc et al. has shown that minute changes in heart function can be detected by three-dimensional echocardiography, and so this technology will be used to assess the efficacy of stem cell-driven tissue regeneration, as well as angiogenic growth factor-mediated improvement of the local microvascular niche. The results of this research may circumvent a major hindrance to DMD stem cell treatment, and have the potential to unveil a new avenue of clinical therapies.

31. The RXR agonist SR11237 causes disturbed skeletal morphogenesis in a rat model

Holly Dupuis, Michael Pest, Thin Vo, Daniel Hardy, Frank Beier

U of Western Ontario

Presenting author: Holly Dupuis

Category: Other

If other please specify: Research Technician

Field of interest: Bone Cartilage and tooth development

Introduction: Retinoid X Receptor (RXR) is a type 2 nuclear receptor with important roles in cell death, development, metabolism and cell differentiation. RXR can form homodimers or heterodimers with other nuclear receptors (PPARs, VDR, LXL and others) in permissive or non-permissive ways. In mice, non-permissive binding of RXR to the retinoic acid receptor (RAR) is important in bone development (exposure to retinoic acid during embryonic development causes deletions and truncations in the forelimbs). However, despite the far-reaching impact of RXR, its role in long bone development is poorly understood. We therefore used the RXR agonist SR11237 to identify RXR activation-dependent effects on endochondral ossification, whereby bone replaces cartilage during long bone development.

Purpose: To examine the effects of RXR specific activation on endochondral bone development in the rat.

Results and Conclusion: Sprague–Dawley rats were given an intraperitoneal injection of 25 mg/kg of SR11237 (pan-RXR specific agonist) or DMSO (vehicle) daily from post-natal day 5 (P5) to P15. Animals were harvested on P16 for Micro-computed Tomography (uCT) scanning (males only; 50 um/voxel resolution), histology and weight determination. Histological sections were stained with Safranin O/Fast green, picro-sirius red, Tartrate-resistant acid phosphatase (TRAP) and TUNEL. Further, p57 and SOX9 immunohistochemistry was also completed. RXR activation by SR11237 disturbed ossification and bone morphology in rats. uCT analyses and Safranin O staining of the long bones show premature growth plate closure and ossified tissue infiltration through the central epiphysis. Additionally, the cells surrounding this ossified tissue invasion were pre-hypertrophic in size, shape and p57 immunohistochemical staining. SOX9 (a proliferative marker) was also found in the cells surrounding the ossified tissue. In the SR11237-treated bones, the central epiphysis stained strongly with TRAP (an indicator for osteoclastic activity), and picro-sirius red staining revealed large amounts of collagen. Further, TUNEL staining showed concentrated cell death at the osteo-chondral junction. These morphological defects were observed in all long bones. uCT also revealed less calcified, pitted surfaces on the scapular faces. The smaller fore- and hindlimb bones were also thinner and somewhat osteopenic. Males were more impaired than females, and the tibia was significantly more afflicted than the humerus and femur. SR11237-treated animals of both sexes weighed 30 % less than the controls. In rats, increased RXR activation caused irregular ossification and premature growth plate closure. This may result in long term effects on long bone and joint morphology, lead to additional pathology (ex. Osteoarthritis) through joint mal-alignment or abnormal gait.

32. Adenosine signaling in cells of the intervertebral disc—possible role in ectopic mineralization in mice lacking equilibrative nucleoside transporter 1

Neil Tenn, Sumeeta Warraich, Hisataka Ii, Diana Quinonez, Peter Chidiac, James R. Hammond, S. Jeffrey Dixon, Cheryle A. Séguin

Department of Physiology and Pharmacology, Western University and Department of Pharmacology, University of Alberta

Presenting author: Neil Tenn

Category: Graduate Research

Background: Extracellular purines such as adenosine regulate many cell functions, including the differentiation and mineralization of connective tissues. The four adenosine receptors (A1, A2A, A2B, A3) are G protein-coupled receptors (GPCRs); A1 and A3 couple to Gi, whereas A2A and A2B couple to Gs. The roles of adenosine signaling in the intervertebral disc remain unexplored. We recently reported that mice lacking equilibrative nucleoside transporter 1 (ENT1−/−) exhibit ectopic mineralization of spinal tissues including the annulus fibrosus of intervertebral discs (IVDs). Moreover, the phenotype of ENT1−/−mice resembles diffuse idiopathic skeletal hyperostosis (DISH) in humans. Consistent with loss of ENT1, plasma levels of adenosine were greater in ENT1−/−mice than in wild-type (WT) mice. Thus, it is possible that aberrant mineralization in these mice arises due to activation of adenosine receptors in affected tissues.

Objectives: Our objectives are: i) to characterize adenosine receptor expression and signaling in intervertebral disc cells, and ii) to determine the role of adenosine signaling in mediating ectopic mineralization in ENT1−/−mice.

Methods: IVDs were isolated from ENT1−/− and WT mice by microdissection and either processed for RNA analysis or subjected to enzymatic dissociation for cell isolation and culture. Cellular cyclic AMP levels were quantified by competitive enzyme-linked immunosorbent assay.

Results: We first assessed expression of adenosine receptors. Quantitative RT-PCR of RNA isolated from intact WT IVDs demonstrated greater levels of A2B and A3 than of A1 and A2A receptor transcripts. Moreover, there was no significant difference in expression levels of either adenosine receptors or genes associated with adenosine metabolism (Adk, Ada, Pnp, Xdh or Nt5e) in IVDs isolated from ENT1−/− versus WT mice. To elucidate the function of these adenosine receptors, we quantified cyclic AMP levels in cultures of annulus fibrosus cells following acute treatment with the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) or adenosine in the presence and absence of the adenylyl cyclase activator forskolin. Preliminary results indicate that NECA and adenosine suppress cyclic AMP levels in the presence of forskolin in annulus fibrosus cells from both ENT1−/− and WT mice. Thus, it appears that adenosine receptors signal predominantly through Gi in these cells.

Conclusions: We show for the first time that cells of the intervertebral disc express adenosine receptors functionally coupled to inhibition of adenylyl cyclase. In the long term, these studies may elucidate pathways underlying ectopic mineralization in ENT1−/−mice.

33. The regulatory role of microRNA-17 in osteogenic differentiation

Shaan Gupta (1,2), Burton B Yang (1,2)

(1) Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto. (2) Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto

Presenting author: Shaan Gupta

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

BACKGROUND: The body maintains bone density by achieving equilibrium between bone formation and resorption, but failure to maintain this equilibrium can lead to degenerative bone diseases such as osteoporosis, which is characterized by excessive bone resorption and inadequate formation of new bone. Emerging research is showing that microRNAs are important regulators of osteogenic differentiation and bone formation both in vitro and in vivo, and previous research has shown that microRNA-17 (miR-17) is downregulated during osteogenic differentiation of induced pluripotent stem cells, suggesting a possible regulatory role. Elucidating the role of miR-17 in MC3T3-E1 cell differentiation may prove useful for therapeutic management of osteoporosis and bone metastasis. HYPOTHESIS: miR-17 was hypothesized to target janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) to inhibit osteogenesis in pre-osteoblast cells. METHODS/RESULTS: After overexpressing miR-17 in MC3T3-E1 cells via plasmid transfection, we assessed the cells’ osteogenic differentiation capacity compared to plasmid control cells. The differentiation capacity was assessed using alkaline phosphatase staining, and miR-17 cells were found to show lower levels of alkaline phosphatase activity, suggesting that miR-17 overexpression leads to an inhibition of osteogenic differentiation capacity. qRT-PCR was used to assess the levels of several key markers of osteogenic differentiation in the transfected cells. It was found that the markers bone sialoprotein and fibronectin mRNA are significantly down-regulated in miR-17 transfected cells compared to the GFP control. Using in silico analysis, JAK1 and STAT3 were predicted to be direct targets of miR-17 and potentially responsible for the observed differentiation inhibition. Western Blot analysis showed JAK1 and STAT3 protein to be down-regulated in miR-17 cells, which is consistent with the hypothesis. MiR-17 cells with JAK1 and STAT3 levels exogenously restored are currently being assessed for recovery of differentiation potential. Binding of miR-17 to the 3′ untranslated region of STAT3 mRNA has been confirmed through the luciferase reporter assay, and miR-17 binding to the JAK1 3′ UTR is currently being assessed. SiRNA-mediated knockdown of JAK1 and STAT3 in wild-type cells is being conducted to determine if osteogenic inhibition is consistent with previous results. In vivo microCT analysis of bone mineralization in mice overexpressing miR-17 is also currently underway. CONCLUSION: Therefore, it appears that miR-17 is exhibiting an inhibitory effect on osteogenesis in pre-osteoblast cells by down-regulating JAK1 and STAT3 protein levels. Further study into this regulation may elucidate novel therapeutic opportunities for the management of osteoporosis and bone metastasis.

34. Liver X receptor activation delays chondrocyte hypertrophy during endochondral bone growth

Margaret Man-Ger Sun and Frank Beier

(Department of Physiology & Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario)

Presenting author: Margaret Man-Ger Sun

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Activation of the Liver X Receptor (LXR) has recently been identified as a therapeutic strategy for osteoarthritis (OA). LXRβ-null mice display OA-like symptoms and LXR agonist administration to OA articular cartilage explants suppresses proteoglycan degradation. We investigated the effect of LXR activation on chondrocyte differentiation to elucidate the molecular mechanisms behind its protective effects against OA. The specific LXR agonist, GW3965, was used to examine the effect of LXR activation on chondrocyte differentiation in three different chondrocyte model systems. Chondrocyte hypertrophy was suppressed by GW3965 treatment, as shown by decreased hypertrophic zone length in tibia organ culture, decreased alkaline phosphatase staining, and down-regulation of hypertrophic gene expression in micromass culture and differentiating ATDC5 cells. Increased chondrocyte proliferation and up-regulation of Col2a1 expression suggest hypertrophy is suppressed secondary to prolonged proliferation. Our findings regarding LXR’s role in cartilage development suggest that LXR activation prevents ectopic chondrocyte hypertrophy and resulting cartilage breakdown, further solidifying LXR’s potential as a therapeutic target in OA.

35. CTCF is critical for hindlimb development

Katherine Rabicki, Jason Bush, Nathalie Berube, Frank Beier

Department of Physiology & Pharmacology, Schulich School of Medicine & Dentistry, Western University; Department of Pediatrics and Department of Biochemistry, Western University; Children’s Health Research Institute, London, Ontario, Canada

Presenting author: Katherine Rabicki

Category: Graduate Research

If other please specify:

Field of interest: Bone Cartilage and tooth development

Introduction: Mutations in the gene encoding the chromatin organizer CCCTC-binding factor (CTCF) have recently been identified in patients presenting with intellectual disability and skeletal defects. CTCF is well-known for its roles in three-dimensional chromatin architecture, gene insulation, and enhancer function in vertebrates. Studies have additionally demonstrated that deletion of CTCF in murine limb mesenchyme results in massive apoptosis in the forelimb, and stunted growth coupled with oligodactyly (reduced number of digits) in the hindlimb. The role of CTCF specifically in the hindlimb, however, has not yet been ascertained.

Objective: To investigate the role of CTCF in hindlimb development through in-vitro and in-vivo models of skeletal growth.

Methods: Our in-vivo approach involves the use of the Cre-Lox system to conditionally inactivate CTCF in developing limb mesenchyme using the Prx1 promoter. Mutant and control mice are collected at various stages of embryonic development, and histology (Safranin-O, immunohistochemistry) is performed on the long bones to analyze cell proliferation and cell death. In situ hybridization and qPCR for candidate target genes will be performed, and chromatin immunoprecipitation will be done to examine whether CTCF binds to these target genes.

Results: Our results confirm that hindlimbs of limb-specific CTCF KO animals are severely shortened and show oligodactyly. Analyses of target gene expression has been initiated.

Conclusions: Our data suggest that CTCF is a key transcription factor regulating hindlimb development.

36. Loss of mitogen-inducible gene 6 results in disturbed cartilage and joint homeostasis

M. Pest, B. Russell, JW. Jeong, F. Beier

University of Western Ontario

Presenting author: Michael Pest

Category: Graduate Research

If other please specify:

Field of interest: Connective tissue remodelling

Introduction: Osteoarthritis (OA) is a progressive degenerative joint disease. Transforming growth factor alpha (TGFα) is upregulated in a subset of human OA cases. TGFα activates epidermal growth factor receptor (EGFR) which is attenuated by mitogen-inducible gene 6 (Mig-6) binding. Whole body deletion of Mig-6 induces development of joint deformities and cartilage degeneration, however the tissue types involved have not yet been identified.

Purpose: To determine the role of Mig-6 in cartilage degeneration and joint homeostasis through the targeted deletion of Mig-6 in the cartilage of mice.

Methods: Mig-6 was selectively deleted in the cartilage of mice using the Cre-Lox system. MicroCT was used to assess changes in bone morphology. Skeletal stains were used to examine gross skeletal morphology. Histological stains, tartrate resistant acid phosphatase (TRAP) and immunohistochemistry (IHC) were also used to further determine morphological and molecular changes in cartilage and bone.

Results: The overall skeletal morphology of cartilage specific Mig-6 knockout mice (KO, Mig-6 fl/fl;Col2-Cre+/−) was comparable to control animals (Mig-6 fl/fl;Col2-Cre−/− or Mig-6 fl/+;Col2-Cre−/−). Ectopic cartilage and calcified tissues in the knees of KO animals were observed at early time points (12 weeks) and later in the spine as the animals aged (36 weeks). IHC showed increased SOX9 and phosphoEGFR (activated) in both the ectopic tissue growths and articular cartilage of KO animals. At 12 weeks the knee articular cartilage of KO animals showed an increase in proteoglycan staining, and a statistically significant increase in thickness and chondrocyte cellularity, which decreased by 36 weeks. Erosion of bone associated with ligaments was observed by microCT in the knees of KO animals. TRAP staining was increased surrounding eroded bone sites, and in the ectopic growths and trabeculae of KO animal knees.

Conclusions: The negative regulation of EGFR signalling by Mig-6 in Col2a1 expressing tissues is essential for the maintenance of joint morphology and homeostasis.

37. Quantifying whole-body composition of mice during growth and aging using micro-computed tomography

Kim L. Beaucage, Steven I. Pollmann, Stephen M. Sims, S. Jeffrey Dixon, David W. Holdsworth

University of Western Ontario

Presenting author: Kim L. Beaucage

Category: Graduate Research

If other please specify:

Field of interest: other

Micro-computed tomography (micro-CT) is used routinely to quantify radio-opaque tissue mass in small animal models. Our goal was to evaluate repeated in vivo micro-CT imaging for monitoring whole-body composition in studies of growth and aging in mice. Male C57BL/6 mice from 2 to 52 weeks of age were anesthetized and imaged using an eXplore Locus Ultra and/or eXplore speCZT scanner. Images were reconstructed into 3D volumes, signal-intensity thresholds were used to classify each voxel as adipose, lean or skeletal tissue, and tissue masses were calculated from assumed densities. Images revealed specific changes in tissue distribution with growth and aging. Quantification showed biphasic increases in total volumetric body mass, lean and skeletal tissue masses, consisting of rapid increases to 8 weeks of age, followed by slow linear increases to 52 weeks. In contrast, bone mineral density rapidly increased to a stable plateau at ~14 weeks of age; whereas, adipose tissue mass increased exponentially with age. A micro-CT-derived total mass was calculated for each mouse and compared with the gravimetrically measured mass, which differed on average by less than 3 %. Parameters were highly reproducible for mice of the same age, but variability increased slightly with age. There was also good agreement in parameters for the same group of mice scanned on the eXplore Locus Ultra and eXplore speCZT systems. This study provides reference values for normative comparisons; as well, it demonstrates the usefulness of in vivo single-energy micro-CT scans to quantify whole body composition in high-throughput studies of growth and aging in mice. These studies were supported by the Canadian Institutes of Health Research.

38. The Double agent: discrete plasticity in collagen fibrils toughens tendons and cues phagocytic recognition of overload damage

J. Michael Lee [1] and Samuel P. Veres [1,2]

1. School of Biomedical Engineering, Dalhousie University, 2. Division of Engineering, Saint Mary’s University

Presenting author: J. Michael Lee

Category: Other

If other please specify: Faculty member

Field of interest: The Extracellular Matrix in Connective Tissues

After an overload injury to soft tissues, we are told that host cells recognize damaged matrix and initiate repairs so that biomechanical function can be restored. It is by no means clear, though, what “damaged” collagen looks like—nor do we know how cells like macrophages can recognize such damage and respond accordingly. We have been interested in the idea that a structural feature of damaged collagen might—in and of itself—may be an important signal to cellular action: degradation, remodelling, and/or replacement. Using a bovine tail tendon (BTT) model, we have identified a characteristic, nano-scaled kink structure that occurs in collagen fibrils after rupture or sub-rupture overload. BTTs were isolated from tails of 24–36 months adult cattle, and were either decellularized or examined intact. In an MTS testing system, tendon samples were subject to: (i) single pull rupture, or (ii) up to 15 cycles of cyclic overloading into the plastic region of the stress–strain curve. Under SEM at magnifications up to 90,000×, rupture or single overload produced serial kinks along the length of individual collagen fibrils, separated by as little as 300 nm. Repeated overload increased the linear density of the kinks, with interkink distances going as low as 100 nm. [No kinks were seen in unloaded collagen.] Collagen at the kink sites was locally denatured, with site-specific solubilization by serine proteases. These kinks appear to form at previously unidentified sites of inhomogeneity along the collagen fibril. Under overload, biomechanical failure of a subset of sub-fibrils occurs at each site, absorbing energy. A different subset of sub-fibrils remains intact, though, and is able to bear further load. Serial failure of these sites toughens tendon collagen and inhibits complete rupture of the tendon. We have recently used the U937 immortal macrophage-like cell line, directly differentiated on tendon collagen, to examine the cellular response to discrete plasticity damage. Decellularized BTTs were subject to 15 cycles of sub-rupture mechanical overload and matched-pairs (damaged and undamaged) had U937s cultured on them. On damaged collagen, cells clustered less, showed ruffled membranes, and frequently spread: increasing their contact area with the damaged substrate. There was also clear structural evidence of pericellular enzymolysis of damaged collagen—but not of control collagen. It appears that, not only does the discrete plasticity mechanism toughen tendon collagen, but it forms an identifiable structural cue to cellular action.

39. Remodeling of heart valve leaflets in a bovine pregnancy model

Sarah Wells, Caitlin Pierlot

School of Biomedical Engineering, Dalhousie University

Presenting author: Sarah Wells

Category: Not selected

If other please specify:

Field of interest: Connective tissue remodelling

While many cardiovascular tissues have been shown to remodel with chronic changes in hemodynamic conditions, little is known about the capacity of heart valves to remodel in a “non-pathological” state. Profound cardiovascular changes occur during pregnancy including large increases in blood volume with enlargement of the heart and valve orifices. We hypothesized that valve leaflets in the maternal heart remodel as a result of the increased mechanical loading conditions associated with pregnancy. Here, we examined heart valve remodeling using a bovine pregnancy model. Hearts were harvested from heifers (never pregnant), and pregnant cows at a local abattoir. Leaflets were excised from all 4 valves for mechanical and structural studies. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Gross leaflet structure was characterized by leaflet dimensions (area, thickness). Biochemical composition (contents of collagen, elastin, sGAGs) was determined using standard biochemical assays. Histological studies assessed architectural changes in cellular and ECM components including collagen crimp. Finally, collagen thermal stability and crosslinking state was assessed using denaturation and hydrothermal isometric tension tests. We observed remarkable changes in the structure and mechanical properties of heart valve leaflets during pregnancy. Leaflets from all valves showed an increase in extensibility early in pregnancy that reversed to pre-pregnant values by late pregnancy. Leaflets from all valves also increased in surface area during pregnancy (nearly doubling in the aortic valve), while thickness remained unchanged. As the leaflets enlarge during pregnancy, their composition was altered. In all valve leaflets, collagen content was increased (up to 22 %), while GAG content was reduced (by up to 80 %), as was elastin content (reduced by up to 90 %). Leaflets from all valves, except the tricuspid, demonstrated a striking loss of collagen crimp during pregnancy, with crimp wavelength increasing by up to 200 %. All valve leaflets became less cellular during pregnancy, with—surprisingly—no change in the proportion of “activated” α-SMA-positive cells. Finally, thermomechanical tests revealed (i) a decrease in collagen thermal stability across all heart valves during pregnancy, suggesting an increase in collagen turnover, and (ii) an increase in mature collagen crosslinking. This study is the first to demonstrate non-pathological remodeling in heart valve leaflets. Their remodeling associated with pregnancy is strikingly similar across all 4 valves, despite differences in their native mechanical loading conditions and embryological origins. Understanding the triggers and mechanisms of the “physiological” remodeling during pregnancy would be a first step in developing treatments for valve pathologies.

40. CD109 regulates cellular migration, matrix metalloprotease expression and fibrotic parameters in skin cells

Priyanka Sehgal, Alban Bizet, Anie Philip

Presenting author: Priyanka Sehgal

Category: Post-doctoral Research

If other please specify:

Field of interest: The Extracellular Matrix in Connective Tissues

Transforming growth factor beta (TGF-β) is a multifunctional cytokine that regulates many fundamental cellular processes such as cell proliferation and l differentiation. TGF-β is considered to be the most potent profibrotic cytokine known because of its strong effects on extracellular matrix (ECM) production and the expression of matrix metalloproteases (MMPs) which breakdown ECM, both of which are key events in the fibrotic process. Dysregulation of TGF-β action is thought to play a key role in many fibrotic skin disorders including hypertrophic scarring, keloids, and scleroderma. Our group has recently reported the identification of CD109 as a TGF-β co-receptor and potent antagonist of TGF-β signaling and responses such as cell proliferation and ECM synthesis in human skin cells in vitro and in vivo. The aim of the present study was to determine whether CD109 regulates TGF-β induced cellular migration and expression of MMPs and fibrotic parameters in human skin cells.

Methods: To determine the effect of CD109 on TGF-β induced cell migration, MMP expression and ECM synthesis in skin cells, we overexpressed CD109 using a plasmid encoding CD109 or blocked CD109 expression using siiRNA, in HaCaT human keratinocytes and A431 squamous carcinoma cells. We then analyzed for TGF-β -induced MMP2, MMP9, fibronectin and plasminogen activator inhibiot-1 (PAI-1) expression, using qPCR and Western blot. Cellular migration was determined by an in vitro wound healing assay using confluent cells treated with or without TGF-β.

Results: Our results show that CD109 overexpression was able to decrease TGF-β-induced fibronectin, PAI-1 expression and MMP2 and MMP9 in HaCaT cells and squamous cell carcinoma cells. Overexpression of CD109 resulted in a markedly diminished migratory phenotype in TGF-β treated HaCaT and A431 cells, as compared empty vector transfected cells treated with TGF-β. Conversely, knock-down of CD109 in HaCaT and A431 cells with CD109 siRNA increased TGF-β-induced fibronectin, PAI-1, MMP2 and MMP9 expression, as compared to control siRNA transfected cells, suggesting that CD109 inhibits TGF-β induced EMT marker expression. In addition, knock-down of CD109 expression enhances TGF-β-induced “wound” closure, as compared to control siRNA transfected cells indicating that CD109 is able to inhibit TGF-β-induced migration.

Conclusion: Our findings suggest that CD109 is an important regulator of TGF-β induced cellular migration, MMP expression and ECM deposition in human keratinocytes and squamous carcinoma cells, indicating that CD109 may play a key role in the fibrotic process in the skin.

41. Substrate stiffness regulates chondrocyte dedifferentiation through actin cytoskeleton

Mortah Nabavi Niaki, Rita Kandel

(Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada), (CIHR-BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada)

Presenting author: Mortah Nabavi Niaki

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

Articular cartilage is a specialized tissue present at ends of articulating bone, composed of chondrocytes embedded in an extracellular matrix (ECM) rich in type II-collagen (Col2) and the proteoglycan aggrecan. Chondrocytes have a round morphology, cortical actin organization, and greater amounts of the globular form of actin (g-actin) compared to the filamentous form of actin (f-actin). When articular cartilage is damaged by disease (e.g. OA) or trauma it has a limited ability to self-repair. In order to biologically repair damaged cartilage using autologous chondrocytes a sufficient number of cells is required that are able to produce cartilage specific matrix molecules. Currently to expand cell numbers, chondrocytes are grown and passaged in vitro in monolayer on tissue culture plastic, a substrate that is much stiffer than the cell’s natural environment. This results in cellular dedifferentiation characterized by a change in ECM synthesis with an up-regulation of type-I collagen (Col1) and alpha smooth muscle actin (αSMA) and a down-regulation of the chondrogenic genes Col2 and aggrecan. Passaged cells also undergo actin polymerization, develop actin stress fibers and have a lower g-/f-actin ratio compared to primary cells. In other cell types the g-actin modulates the activity of the myocardin-related transcription factor (MRTF-A), a known regulator of Col1 and αSMA. The hypothesis of this study is that soft substrate partially inhibits chondrocyte dedifferentiation through actin and MRTF modulation. Bovine chondrocytes were passaged twice in monolayer culture and then grown on polyacrylamide gels of various stiffness. The chondrocytes were analyzed after 3 days. Passaged cells on soft polyacrylamide gels (0.5 kPa), become round, underwent less proliferation, and had higher levels of g/factin compared to cells grown on a stiff substrate (50 kPa). This correlated with a change in MRTF localization as significantly more MRTF was present in the cytosol than in the nucleus. This was associated with decreases in MRTF regulated gene expression, such as type I collagen, αSMA, transgelin, tenascinC and vinculin. This raises the possibility that loss of some aspects of the chondrocyte phenotype during culture in monolayer is regulated by the ratio of globular to filamentous actin and the change in MRTF localization. Modulating substrate stiffness may be one way to modulate the chondrogenic phenotype during cell number expansion in order to obtain sufficient cells suitable for cartilage tissue engineering.

42. The effect of platelet rich plasma on chondrocytes grown in monolayer or 3D culture

Corey Sermer, Rita Kandel, John Theodoropoulos

Institute of Medical Science, University of Toronto, Department of Orthopedic Surgery, Mt. Sinai Hospital; Department of Pathology and Laboratory Medicine, Lunenfeld-Tannenbaum Research Institute, Mt. Sinai Hospital; Department of Orthopedic Surgery, Mt. Sinai Hospital

Presenting author: Corey Sermer

Category: Graduate Research

If other please specify:

Field of interest: Stem Cells in Tissue Regeneration and Tissue Engineering

INTRODUCTION: Osteoarthritis (OA) is a debilitating disease characterized by degradation of articular cartilage. Current therapies for early or midstage disease range from lifestyle modifications to surgical procedures. These procedures result in fibrocartilage repair, which is biomechanically inferior and degrades with time, ultimately necessitating joint replacement. Recently, there is an increased interest in the use of platelet rich plasma (PRP) injections to treat OA. PRP is an autologous source of many growth factors which may aid in the regeneration of articular cartilage. Current advancements in cartilage tissue engineering may lead to improved repair techniques for patients suffering from OA. Difficulties remain, however, when attempting to maintain a chondrogenic phenotype during cell expansion and in generating articular cartilage constructs rich in collagen type II. The objectives of this study are twofold: 1) to investigate the effect of PRP on maintaining chondrogenic phenotype during monolayer culture and 2) determine if PRP enhances cartilage tissue formation by primary chondrocytes in a 3D culture system.

METHODS: Chondrocytes were isolated from bovine articular cartilage and seeded in monolayer culture on polystyrene (2D) or in 3D culture on porous bone substitutes (calcium polyphosphate (CPP)). Cultures were seeded in 5 % fetal bovine serum (FBS). The next day they were serum starved and then grown in Hams F12 supplemented with various concentrations of PRP (1–40 %). Controls were grown in 5 % FBS. Cultures were harvested after day 5. Gene expression for markers of chondrocyte phenotype and proliferation were evaluated by RT-PCR. Significance was assessed by ANOVA followed by Tukey’s post hoc test and assigned at p < 0.05.

RESULTS: In monolayer culture (2D), there was increased gene expression analysis for collagen type II and Ki67 (cell proliferation marker) with increasing PRP concentrations. Collagen type I gene expression increased with PRP as well, except at the 40 % PRP condition where expression levels were similar to controls (no PRP). In tissue forming conditions (3D), collagen type I significantly increased in 20 % and 40 % PRP conditions and Ki67 showed a 25-fold increase compared to control at 40 % PRP.

DISCUSSION: Growing cells in monolayer in the presence of 40 % PRP may be the better condition as it enhanced collagen type II expression while favouring proliferation. In contrast, high concentrations of PRP (20–40 %) may not be beneficial in 3D cultures, because of elevated collagen type I expression. Further studies are required to examine the effect on other pathways that regulate tissue formation in vitro.