Selected Contribution: Regulatory pathways involved in mechanical induction of c-fos gene expression in bone cells

2000 ◽  
Vol 89 (6) ◽  
pp. 2498-2507 ◽  
Author(s):  
M. A. Peake ◽  
L. M. Cooling ◽  
J. L. Magnay ◽  
P. B. M. Thomas ◽  
A. J. El Haj
Keyword(s):  
Bone Cells ◽  
Mechanical Load ◽  
Mechanical Strain ◽  
Collagen Type I ◽  
Bone Cell ◽  
Type I ◽  
Regulatory Pathways ◽  
Four Point Bending ◽  
Load Response ◽  
Factor C

The regulatory pathways involved in the rapid response of the AP-1 transcription factor, c- fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MG-63 bone cell line were investigated using a four-point bending model. HOB and MG-63 cells showed upregulation of c- fos expression on fibronectin and collagen type I substrates; however, MG-63 cells did not respond on laminin YIGSR substrates. Addition of cytochalasin D and Arg-Gly-Asp peptides during loading did not inhibit the response, whereas addition of β1-integrin antibodies inhibited the load response. The role of Ca2+ signaling has been demonstrated by blocking upregulation with addition of 2 mM EGTA, which chelates extracellular Ca2+, and gadolinium (10 μM), which inhibits stretch-activated channels. Addition of the Ca2+ ionophore A-23187 induced upregulation without loading; however, addition of nifedipine (10 μM), the L-type channel blocker, failed to prevent the load response. Inhibitors of downstream pathways indicated the involvement of protein kinase C. Our results demonstrate a key involvement of Ca2+ signaling pathways and integrin binding in the c- fos response to mechanical strain.

scholarly journals Osteocyte Characterization on Polydimethylsiloxane Substrates for Microsystems Applications

2012 ◽  
Vol 16 ◽  
pp. 27-42 ◽  
Author(s):  
Spencer L. York ◽  
Ahmad R. Arida ◽  
Karan S. Shah ◽  
Palaniappan Sethu ◽  
Marnie M. Saunders
Keyword(s):  
Mechanical Load ◽  
Collagen Type I ◽  
Bone Cell ◽  
Culture Conditions ◽  
Substrate Material ◽  
Research Field ◽  
The Body ◽  
Type I

In the body, osteocytes reside in lacunae, lenticular shaped cavities within mineralized bone. These cells are linked to each other and surface-residing osteoblasts via physical channels known as gap junctions. It has been suggested that osteocytes sense mechanical load applied to bone and relay that signal to osteoclasts and osteoblasts. Currentin vitroandin vivomodels of mechanotransduction face temporal and spatial barriers. Recent advances in polydimethylsiloxane (PDMS) based microfabrication techniques may be able to overcome some of these hurdles. However, before the bone research field can effectively utilize microsystems techniques, fundamental groundwork must be completed. This study characterized the behaviour of osteocytes on PDMS coated with collagen type I (CTI) and provides the framework for bone cell mechanotransduction studies using microsystems. The goal was to determine whether osteocytes were adversely affected by the substrate material by comparing their behaviour to a standard glass substrate. In addition, optimal culture conditions and time points for growing osteocytes on PDMS substrates were determined. Results of this study suggested that use of PDMS does not adversely affect osteocyte behaviour. Furthermore, the results demonstrated that osteocytes should be cultured for no less than 72 hours prior to experimentation to allow the establishment and maintenance of phenotypic characteristics. These results completed essential groundwork necessary for further studies regarding osteocytes in microsystems modelling utilizing PDMS.

scholarly journals Effects of interfacial micromotions on vitality and differentiation of human osteoblasts

2018 ◽  
Vol 7 (2) ◽  
pp. 187-195 ◽  
Author(s):  
J. Ziebart ◽  
S. Fan ◽  
C. Schulze ◽  
P. W. Kämmerer ◽  
R. Bader ◽  
...  
Keyword(s):  
Static Pressure ◽  
Collagen Type ◽  
Bone Cells ◽  
Cell Activity ◽  
Collagen Type I ◽  
Dead Cell ◽  
Type I ◽  
Collagen Scaffolds ◽  
Human Osteoblasts ◽  
Receptor Activator

Objectives Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. Methods Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. Results Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. Conclusion Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered. Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187–195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1.

Murine Osteochondral Stem Cells Express Collagen Type I More Strongly on PDMS Substrates Than on Tissue Culture Plastic

2013 ◽  
Author(s):  
William S. Van Dyke ◽  
Ozan Akkus ◽  
Eric Nauman
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Cells ◽  
Stem Cell Differentiation ◽  
Collagen Type I ◽  
Fiber Formation ◽  
Substrate Stiffness ◽  
Type I ◽  
Differentiation Potential

The discovery of the multipotent lineage of mesenchymal stem cells has dawned a new age in tissue engineering, where an autologous cell-seeded scaffold can be implanted into different therapeutic sites. Mesenchymal stem cells have been reported to differentiate into numerous anchorage-dependent cell phenotypes, including neurons, adipocytes, myoblasts, chondrocytes, tenocytes, and osteoblasts. A seminal work detailing that mesenchymal stem cells can be directed towards differentiation of different cell types by substrate stiffness alone [1] has led to numerous studies attempting to understand how cells can sense the stiffness of their substrate [2–3] Substrate stiffness has been shown to be an inducer of stem cell differentiation. MSCs on extremely soft substrates (250 Pa), similar to the stiffness of bone marrow, became quiescent but still retained their multipotency [4]. Elastic substrates in the stiffness range of 34 kPa revealed MSCs with osteoblast morphology, and osteocalcin along with other osteoblast markers were expressed [1]. However, osteogenesis has been found to increase on much stiffer (20–80 kPa) [5–6] (400 kPa) [7] as well as much softer substrates (75 Pa) [8]. Overall, cells have increased projected cell area and proliferation on stiffer substrates, leading to higher stress fiber formation. This study seeks to understand if the stiffness of the substrate has any effect on the differentiation potential of osteochondral progenitor cells into bone cells, using an in vitro dual fluorescent mouse model.

scholarly journals Isolation of bone cell clones with differences in growth, hormone responses, and extracellular matrix production.

1982 ◽  
Vol 92 (2) ◽  
pp. 452-461 ◽  
Author(s):  
J E Aubin ◽  
J N Heersche ◽  
M J Merrilees ◽  
J Sodek
Keyword(s):  
Extracellular Matrix ◽  
Bone Cells ◽  
Bone Cell ◽  
Population Doubling ◽  
Cell Populations ◽  
Type I ◽  
Mixed Cell ◽  
Extracellular Matrix Components ◽  
Wide Range

Clones of nontransformed hormone-responsive bone cells have been isolated in vitro from mixed cell populations of fetal rat calvaria. In several independent isolations, microscopically visible colonies appeared at plating efficiencies of 5-10% of the starting cell numbers. Of these clones, approximately 10% grew to mass populations which could be assayed for a number of growth and biochemical properties. Although some similarities existed among the clones, they could be distinguished from each other and from the mixed cell populations. Population-doubling times (tDs) and saturation densities varied over a wide range: e.g., tDs of 24-72 h and saturation densities of 0.4-5 x 10(5) cells/cm2. Morphologies varied from roughly polygonal multilayering cells to typically spindle-shaped monolayering cells. Hormone responsiveness, as measured by stimulation of cAMP by hormones, indicated that some clones were responsive to both parathyroid hormone (PTH) and prostaglandin E2 (PGE2), while others responded to PTH only. Analysis of extracellular matrix components revealed that all clones produced type I and type III collagens, though in different proportions. Similarly, although all clones synthesized four glycosaminoglycans (hyaluronic acid, heparan sulfate, chondroitin sulfate, and dermatan sulfate), the quantities of each were distinctive from clone to clone. Further investigation of such clones is continuing to define more precisely the heterogeneity of clonal bone cell populations in vitro. They represent an important step in the study of the endocrinology and differentiation of bone.

Effects of Mechanical Strain on Osteoblastic Precursor Cells in a Three-Dimensional Scaffold

2007 ◽  
Vol 330-332 ◽  
pp. 1181-1184
Author(s):  
Zhi He Zhao ◽  
Jun Wang ◽  
Yu Bo Fan ◽  
Song Jiao Luo ◽  
Ling Yong Jiang
Keyword(s):  
Collagen Type ◽  
Mechanical Strain ◽  
Three Dimensional ◽  
Collagen Type I ◽  
Precursor Cells ◽  
Dynamic Strain ◽  
Type I ◽  
Pcr Assay ◽  
Periodontal Tissues ◽  
Strain Magnitude

It was well recognized that mechanical strain plays a crucial role in periodontal tissues remodeling. The aim of this study was to investigate the effect of mechanical strain on osteoblastic precursor cells in a collagen type I gel scaffold. Rat MSCs were isolated and cultured according to the established method. Cells were induced with osteogenic medium, then seeded in a collagen type I gel and mechanically stretched by application of cyclic biaxial strain 24h later. Strain cycle was set to 1 cycle/min (0.017Hz), and strain magnitude was set to 2%, 5%, 7% elongation. Cells were collected in 0h, 3h, 6h, 9h, 12h, 24h and 48h respectively. ODF and ICAM-1 mRNA were analyzed by RT-PCR assay. The results shown that 2-7% elongation strain, either dynamic or static, inhibited ICAM-1and ODF expression of osteoblastic precursors, and the effects were relative tightly to strain magnitude. The inhibition effects of dynamic strain loading group exceeded the corresponding static strain. This work suggested that appropriate mechanical strech may suppress differentiation of osteoclasts through inhibiting expression of ICAM-1 and ODF. Application of mechanical stress might have a beneficial effect on quantity of generated bone tissue and might be a important factor in tissue engineering of periodontal tissues.

scholarly journals Concerted Action of Androgens and Mechanical Strain Shifts Bone Metabolism from High Turnover into an Osteoanabolic Mode

2002 ◽  
Vol 196 (10) ◽  
pp. 1387-1392 ◽  
Author(s):  
Ute M. Liegibel ◽  
Ulrike Sommer ◽  
Pascal Tomakidi ◽  
Ulrike Hilscher ◽  
Loes van den Heuvel ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Bone Cells ◽  
Mechanical Strain ◽  
Type I ◽  
High Turnover ◽  
Fibronectin Receptor ◽  
Deformation Strain ◽  
Matrix Deformation ◽  
And Function

Adhesion of bone cells to the extracellular matrix is a crucial requirement for osteoblastic development and function. Adhesion receptors connect the extracellular matrix with the cyto-skeleton and convey matrix deformation into the cell. We tested the hypothesis that sex hormones modulate mechanoperception of human osteoblastic cells (HOB) by affecting expression of adhesion molecules like fibronectin and the fibronectin receptor. Only dihydrotestosterone (DHT), but not 17β-estradiol, stimulated fibronectin (137%) and fibronectin receptor (252%) protein expression. The effects of deformation strain on HOB metabolism were investigated in a FlexerCell® strain unit. Cyclically applied strain (2.5% elongation) increased DNA synthesis (125%) and interleukin-6 (IL-6) production (170%) without significantly affecting alkaline phosphatase (AP) activity, type I collagen (PICP), or osteoprotegerin (OPG) secretion. 10 nM DHT pretreatment abolished the mitogenic response of HOB to strain and increased AP activity (119%), PICP (163%), and OPG production (204%). In conclusion, mechanical strain stimulates bone remodeling by increasing HOB mitosis and IL-6 production. DHT enhances the osteoanabolic impact of deformation strain by increasing bone formation via increased AP activity and PICP production. At the same time, bone resorption is inhibited by decreased IL-6 and increased OPG secretion into the bone microenvironment.

scholarly journals Collagen metabolism in cultured osteoblasts from osteogenesis imperfecta patients

1992 ◽  
Vol 286 (1) ◽  
pp. 73-77 ◽  
Author(s):  
M Mörike ◽  
R E Brenner ◽  
G B Bushart ◽  
W M Teller ◽  
U Vetter
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Collagen Type ◽  
Bone Cells ◽  
Collagen Type I ◽  
Collagen Metabolism ◽  
Type I ◽  
Type Iii ◽  
Type Iv

Collagen produced in vitro by bone cells isolated from 19 patients with different forms of osteogenesis imperfecta (OI) was analysed. Clinically, four patients were classified as OI type I, 10 patients as OI type III and five patients as OI type IV. Bone cells of 12 of the 19 OI patients produced structurally abnormal type I collagen. Electrophoretically uniformly slower migrating collagen type I alpha-chains were found in one case of OI type I, in seven cases of OI type III and in one case of OI type IV; two cultures of OI type III produced two different populations of collagen type I alpha-chains, and one culture of OI type IV showed reduction-sensitive dimer formation of alpha 1(I) chains, resulting from the inadequate incorporation of a cysteine residue into the triple helical domain of alpha 1(I). Quantitative analysis of collagen metabolism led to the distinction of two groups of cultured OI osteoblasts. In osteoblasts of OI type I, mainly production of collagen was decreased, whereas secretion, processing and pericellular accumulation of (pro)collagen type I was similar to that in control osteoblasts. In contrast, in osteoblasts of OI types III and IV, production as well as secretion, processing and pericellular accumulation of (pro)collagen type I were significantly decreased. Low levels of type I collagen were found irrespective of the presence or absence of structural abnormalities of collagen type I in all OI types.

A Collagen Grafting on Hydroxyapatite to Enhancement of Bone Cell Attachment

2008 ◽  
Vol 396-398 ◽  
pp. 41-45
Author(s):  
D.W. Lee ◽  
E.J. Lee ◽  
Sung Su Chun ◽  
Myun Whan Ahn ◽  
I.W. Song ◽  
...  
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Type I ◽  
Bone Cell ◽  
Major Constituent ◽  
Type I ◽  
Connective Tissues ◽  
Coating Materials ◽  
Attached Cell ◽  
Scanning Electron

A collagen material was chemically grafted on hydroxyapatite (HA) to enhance bone cell attachment because the collagen is a major constituent of connective tissues and has been regarded as one of the most excellent coating materials for bone bonding. First, HA disks were prepared with 12mm diameter and 1mm thickness. And then collagen (type I) was immobilbized on the HA surface using a 3-APTES coupling agent on HA disk surfaces. MC3T3-E1 osteoblasts were seeded on the collagen-grafted and non-grated HA disks and cultured for 4 hrs to evaluate the cell adhesion on the HA discs. The Attached cell morphology on discs was observed with a fluorescent optical microscopy (FOM) and a scanning electron microscopy (SEM). The osteoblasts on the collagen-grafted sample were more spread than those on the non-grafted sample. It is believed that collagen-grafted HA surface provides suitable sites for cell attaching due to the high biocompatibility of collagen.

Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression

2000 ◽  
Vol 278 (5) ◽  
pp. L974-L980 ◽  
Author(s):  
Tomohiko Nakamura ◽  
Mingyao Liu ◽  
Eric Mourgeon ◽  
Art Slutsky ◽  
Martin Post
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Collagen Type ◽  
Mechanical Strain ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Collagen Type I ◽  
Marker Genes ◽  
Type I ◽  
Hormonal Factors

Physical forces derived from fetal breathing movements and hormones such as glucocorticoids are implicated in regulating fetal lung development. To elucidate whether the different signaling pathways activated by physical and hormonal factors are integrated and coordinated at the cellular and transcriptional levels, organotypic cultures of mixed fetal rat lung cells were subjected to static culture or mechanical strain in the presence and absence of dexamethasone. Tropoelastin and collagen type I were used as marker genes for fibroblasts, whereas surfactant protein (SP) A and SP-C were used as marker genes for distal epithelial cells. Mechanical strain, but not dexamethasone, significantly increased SP-C mRNA expression. Tropoelastin mRNA expression was upregulated by both mechanical strain and dexamethasone. No additive or synergistic effect was observed when cells were subjected to mechanical stretch in the presence of dexamethasone. Neither mechanical strain nor dexamethasone alone or in combination had any significant effect on the expression of SP-A mRNA. Dexamethasone decreased collagen type I mRNA expression, whereas mechanical strain had no effect. The increases in tropoelastin and SP-C mRNA levels induced by mechanical strain and/or dexamethasone were accompanied by increases in their heterogeneous nuclear RNA. In addition, the stretch- and glucocorticoid-induced alterations in tropoelastin and SP-C mRNA expression were abrogated with 10 μg/ml actinomycin D. These findings suggest that tropoelastin and SP-C genes are selectively stimulated by physical and/or hormonal factors at the transcriptional level in fetal lung fibroblasts and distal epithelial cells, respectively.

Biocompatibility study of modified injectable hyaluronic acid hydrogel with mannitol/BSA to alveolar bone cells

2020 ◽  
pp. 088532822097174
Author(s):  
Kwanhatai Areevijit ◽  
Nirada Dhanesuan ◽  
Jittima Amie Luckanagul ◽  
Sorasun Rungsiyanont
Keyword(s):  
Alkaline Phosphatase ◽  
Hyaluronic Acid ◽  
Collagen Type ◽  
Alveolar Bone ◽  
Bone Cells ◽  
Collagen Type I ◽  
Control Group ◽  
Type I ◽  
Hyaluronic Acid Hydrogel ◽  
Alveolar Bone Cells

The quality and quantity of bone are crucial to the success of dental implant treatment. Recently, bone grafting materials have reached some limitations. This study aimed to evaluate the biocompatibility of novel drug delivery material, injectable methacrylated hyaluronic acid hydrogel incorporated with different ratios of mannitol and BSA (Man/BSA MeHA), to human alveolar bone cells. The three-dimensionally encapsulated cell culture was evaluated with the resazurin cell viability test, alkaline phosphatase activity assay, immunohistochemistry test for collagen type-I synthesis, and cell morphology. The results showed that the encapsulated cells were viable in all four ratios of Man/BSA MeHA hydrogel and the average metabolic rate was not less than the control group. The morphology test showed round shape cells at the upper portion of the hydrogel and fibroblast-like or polygonal shape at the lower portion of hydrogel next to the culture plate. All four groups could express enzyme alkaline phosphatase and collagen type-I. In conclusion, four ratios of Man/BSA MeHA hydrogel were biocompatible with primary human alveolar bone cells.

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