Edward E. Putnins
University of British Columbia
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Featured researches published by Edward E. Putnins.
Biomaterials | 2010
Yi Yang; Fabio Rossi; Edward E. Putnins
Regeneration of lost periodontium is a challenge in that both hard (alveolar bone, cementum) and soft (periodontal ligament) connective tissues need to be restored to their original architecture. Bone marrow mesenchymal stromal cells (BM-MSCs) appear to be an attractive candidate for connective tissue regeneration. We hypothesized that BM-MSCs are able to sense biological cues from the local microenvironment and organize appropriately to contribute to the regeneration of both soft and hard periodontal connective tissues. To test this hypothesis, we transplanted GFP(+) rat BM-MSCs expanded ex vivo on microcarrier gelatin beads into a surgically created rat periodontal defect. After three weeks, evidence of regeneration of bone, cementum and periodontal ligament was observed in both transplanted and control animals. However, the animals that received BM-MSCs regenerated significantly greater new bone. In addition, the animals that had received the cells and beads transplant had significantly more appropriately orientated periodontal ligament fibers, indicative of functional restoration. Finally, donor-derived BM-MSCs were found integrated in newly formed bone, cementum and periodontal ligament, suggesting that they can directly contribute to the regeneration of cells of these tissues.
Cell Adhesion and Communication | 1999
Edward E. Putnins; James D. Firth; Angie Lohachitranont; Veli-Jukka Uitto; Hannu Larjava
Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.
Infection and Immunity | 2005
Veli-Jukka Uitto; Daniel Baillie; Qiang Wu; Renée Gendron; Daniel Grenier; Edward E. Putnins; Arja Kanervo; James D. Firth
ABSTRACT Fusobacterium nucleatum is closely associated with human periodontal diseases and may also be a causative agent in other infections, such as pericarditis, septic arthritis, and abscesses of tonsils and liver. Initiation and outcome of infective diseases depend critically on the host cell signaling system altered by the microbe. Production of proteinases by infected cells is an important factor in pericellular tissue destruction and cell migration. We studied binding of F. nucleatum to human epithelial cells (HaCaT keratinocyte line) and subsequent cell signaling related to collagenase 3 expression, cell motility, and cell survival, using a scratch wound cell culture model. F. nucleatum increased levels of 12 protein kinases involved in cell migration, proliferation, and cell survival signaling, as assessed by the Kinetworks immunoblotting system. Epithelial cells of the artificial wound margins were clearly preferential targets of F. nucleatum. The bacterium colocalized with lysosomal structures and stimulated migration of these cells. Of the 13 anaerobic oral bacterial species, F. nucleatum and Fusobacterium necrophorum were among the best inducers of collagenase 3 mRNA levels, a powerful matrix metalloproteinase. Production of collagenase 3 was detected in fusobacterium-infected cells and cell culture medium by immunocytochemistry, immunoblotting, and zymography. The proteinase production involved activation of p38 mitogen-activated protein kinase in the infected cells. The study suggests that F. nucleatum may be involved in the pathogenesis of periodontal diseases (and other infections) by activating multiple cell signaling systems that lead to stimulation of collagenase 3 expression and increased migration and survival of the infected epithelial cells.
American Journal of Pathology | 2008
Farzin Ghannad; Daniela Nica; Maria I. Garcia Fulle; Daniel Grenier; Edward E. Putnins; Sarah Johnston; Ameneh Eslami; Leeni Koivisto; Guoqiao Jiang; Marc D. McKee; Lari Häkkinen; Hannu Larjava
Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.
Infection and Immunity | 2002
Edward E. Putnins; Ali-Reza Sanaie; Qiang Wu; James D. Firth
ABSTRACT Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation. Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors. Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide. In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4. Lipopolysaccharides from Escherichia coli and Porphyromonas gingivalis induced membrane expression of CD14 at 1, 3, and 24 h. Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells. Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction. The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression. The transcription factors AP-1 and NF-κB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression. These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.
Matrix Biology | 1996
Edward E. Putnins; James D. Firth; Veli-Jukka Uitto
The role of heparin and heparan sulfate in the control of epithelial collagenase production was investigated utilizing a histiotypic cell culture model. The effect of keratinocyte growth factor (KGF), a heparin-binding growth factor, on collagenase secretion was also examined. Heparin, and, to a lesser extent, heparan sulfate induced release of a 58-kDa, gelatin-degrading enzyme which was subsequently identified as the collagenase, matrix metalloproteinase-1. The increase in collagenase secretion by heparin was further enhanced by the addition of KGF. KGF alone did not have any effect. Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis. Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin. Heparin and heparan sulfate in combination with KGF may thus have important roles in the regulation of epithelial cell collagenase under conditions such as inflammation and wound healing.
Journal of Biomedical Materials Research Part A | 2011
Yi Yang; Benedikt Hallgrímsson; Edward E. Putnins
Large craniofacial bony defects remain a significant clinical challenge. Bone marrow mesenchymal stromal cells (BM-MSCs) constitute a multipotent population. Previously, we developed a novel approach for BM-MSC expansion on 3D CultiSpher-S gelatin microcarrier beads in spin culture with preservation of their multipotentiality, reduction of apoptosis, and enhancement of bone formation in vivo. Here, we hypothesized that such cultured BM-MSCs without exogenous growth factors would respond to the orthopedic microenvironment, thus promoting craniofacial defect regeneration. BM-MSCs isolated from green fluorescent protein (GFP) transgenic rats were ex vivo expanded and transplanted into critical-sized (5-mm diameter) rat calvaria defects. Gelatin beads or defect alone served as controls. By 28 and 42 days, rats were sacrificed for microcomputed tomography (microCT), histologic, and immunohistochemistry examination. MicroCT results demonstrated that BM-MSCs were a statistically significant factor contributing to new bone volume regeneration. Histologic assessment showed that the BM-MSCs group produced more and higher quality new bone compared with beads or defect-alone groups in both osteoinductive and osteoconductive manners. Specifically, immunohistochemical staining identified GFP(+) cells residing in new bone lacunae in conjunction with non-GFP(+) cells. Therefore, ex vivo expanded BM-MSCs at least in part regenerated critical-sized calvaria defects by osteogenic differentiation in vivo.
American Journal of Pathology | 2009
Daisuke Ekuni; James D. Firth; Tarun Nayer; Takaaki Tomofuji; Toshihiro Sanbe; Koichiro Irie; Tatsuo Yamamoto; Takashi Oka; Zhenzi Liu; Juergen R. Vielkind; Edward E. Putnins
Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.
Journal of Biomedical Materials Research Part A | 2010
Yi Yang; K. Kusano; Hanspeter Frei; Fabio Rossi; D. M. Brunette; Edward E. Putnins
Microtopographic features affect diverse cell behaviors. Adult bone marrow progenitor cells (AMPCs) constitute a multipotent heterogeneous population. We hypothesized that microtopographies could direct AMPCs lineage-specific differentiation. AMPCs isolated from Sprague-Dawley rats were CD45 depleted, expanded, and plated at 10(5) cells/cm2 on epoxy-microfabricated: (1) 60-microm-deep grooves with 95-microm pitch (D60P95), (2) 55-microm-wide and 10-microm-deep squares (W55D10), (3) 30-microm-deep grooves with 45-microm pitch (D30P45), (4) 17-microm-wide and 10-microm-deep pillars (W17D10), and (5) smooth control. AMPCs were cultured using expansion, chondrogenesis, or osteogenesis supporting media. Cell cultures were examined by scanning electron microscopy, qRT-PCR, and immunostaining at 2, 9, 16, and 23 days after plating. Expressions of osteogenesis-related genes, such as Runx-2, alkaline phosphatase, osteopontin, osteocalcin, and parathyroid hormone-related protein receptor (PTHr), and chondrogenesis-associated genes, such as Sox-9, type II collagen, and aggrecan, were determined. In expansion medium, W55D10 induced a transient increase of Sox9 expression. Compared with smooth surfaces, type II collagen mRNA and protein expressions in chondrogenic medium were significantly upregulated on W55D10 by day 23. In contrast, osteocalcin and PTHr expressions were significantly increased on D30P45 in osteogenic medium. We have demonstrated that W55D10 and D30P45 enhanced AMPCs chondrogenic and osteogenic terminal differentiation with appropriate culture conditions.
Journal of Dermatological Science | 2009
Min Li; James D. Firth; Edward E. Putnins
BACKGROUND KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood. OBJECTIVES We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism. METHODS Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors. RESULTS Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%). CONCLUSION Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.