Differential osteogenicity of multiple donor-derived human mesenchymal stem cells and osteoblasts in monolayer, scaffold-based 3D culture and in vivo

2016 ◽  
Vol 61 (3) ◽  
pp. 253-266 ◽  
Author(s):  
Verena M.C. Quent ◽  
Christina Theodoropoulos ◽  
Dietmar W. Hutmacher ◽  
Johannes C. Reichert
Keyword(s):  
Matrix Protein ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Matrix Mineralization ◽  
Dentin Matrix Protein ◽  
Cellular Models ◽  
Parathyroid Hormone Related Protein ◽  
Study Results

Abstract We set out to compare the osteogenicity of human mesenchymal stem (hMSCs) and osteoblasts (hOBs). Upon osteogenic induction in monolayer, hMSCs showed superior matrix mineralization expressing characteristic bone-related genes. For scaffold cultures, both cell types presented spindle-shaped, osteoblast-like morphologies forming a dense, interconnected network of high viability. On the scaffolds, hOBs proliferated faster. A general upregulation of parathyroid hormone-related protein (PTHrP), osteoprotegrin (OPG), receptor activator of NF-κB ligand (RANKL), sclerostin (SOST), and dentin matrix protein 1 (DMP1) was observed for both cell types. Simultaneously, PTHrP, RANKL and DMP-1 expression decreased under osteogenic stimulation, while OPG and SOST increased significantly. Following transplantation into NOD/SCID mice, μCT and histology showed increased bone deposition with hOBs. The bone was vascularized, and amounts further increased for both cell types after recombinant human bone morphogenic protein 7 (rhBMP-7) addition also stimulating osteoclastogenesis. Complete bone organogenesis was evidenced by the presence of osteocytes and hematopoietic precursors. Our study results support the asking to develop 3D cellular models closely mimicking the functions of living tissues suitable for in vivo translation.

Expression of SIBLINGs and Their Partner MMPs in Salivary Glands

2004 ◽  
Vol 83 (9) ◽  
pp. 664-670 ◽  
Author(s):  
K.U.E. Ogbureke ◽  
L.W. Fisher
Keyword(s):  
Salivary Glands ◽  
Matrix Protein ◽  
Epithelial Tissue ◽  
Matrix Mineralization ◽  
Dentin Matrix Protein ◽  
Dentin Matrix ◽  
Matrix Extracellular Phosphoglycoprotein ◽  
Sibling Family

Three members of the SIBLING family of integrin-binding phosphoglycoproteins (bone sialoprotein, BSP; osteopontin, OPN; and dentin matrix protein-1, DMP1) were recently shown to bind with high affinity (nM) and to activate 3 different matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively) in vitro. The current study was designed to document the possible biological relevance of the SIBLING-MMP activation pathway in vivo by showing that these 3 SIBLINGs and their known MMP partners are co-expressed in normal adult tissue. BSP, OPN, and DMP1 were invariably co-expressed with their partner MMPs in salivary glands of humans and mice. The 2 SIBLING proteins without known MMP partners, dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE), were also expressed in salivary glands. Expression of all SIBLINGs in this normal, non-mineralizing epithelial tissue suggests that they serve at least one function in vivo other than directly promoting matrix mineralization—a function we hypothesize involves local activation of MMPs.

scholarly journals Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3042
Author(s):  
Eun Ju Lee ◽  
Khurshid Ahmad ◽  
Shiva Pathak ◽  
SunJu Lee ◽  
Mohammad Hassan Baig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cell Adhesion ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Primary Cells ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

scholarly journals The influence of peptide bioregulators on skin aging in 3D culture model

2016 ◽  
Vol 19 (1) ◽  
pp. 58-63 ◽  
Author(s):  
K. V Kozhina ◽  
E. N Volkova ◽  
I. N Saburina ◽  
Sergey G. Morozov ◽  
I. M Zurina ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
3D Culture ◽  
Dermal Fibroblasts ◽  
Extracellular Matrix Protein ◽  
Cytokeratin 19 ◽  
Collagen Type Iv ◽  
Cultured Fibroblasts ◽  
Study Results ◽  
Extracellular Matrix Components

He effect of mesotherapy injection (Meso-Wharton R199TM) on the dermal fibroblasts culture, simulating condition of (mature) aging skin cells are studied. Material and methods. The culture of 4th passage fibroblasts (P4), that corresponds to young skin fibroblasts (control) and the culture of 18th passage fibroblasts (P18), that has all the signs of aging dermal fibroblasts (predominance of large cells, slow cell division) were used. Bioactivity was assessed by cell morphology, epithelium-mesenchyme plasticity and expression of fibroblasts markers: cytokeratin 19, elastin, a-smooth muscle actin (aSMA), PCNA (proliferation marker), collagen types I, III, IV and fibronectin. The formation of spheroids occur when fibroblasts P18 are cultivating with the injection medication, on terms comparable to the formation of spheroids from P4 young fibroblasts. From culture of fibroblasts P18, that was cultured without medication, does not form the full spheroid, but aggregation of cells and their gradual destruction with necrotic masses within the unit are observed. The presence of the medication stimulates the “rejuvenation” of cells and subsequent recovery of the mesenchyme-epithelial plasticity of cultured fibroblasts due to the reduced ability to synthesize sufficient to establish the amount of intercellular contacts the extracellular matrix components (fibronectin and collagen), which affects the ability to form spheroids. Culturing spheroids formed with the medication stimulates expression of elastin, collagen type IV, fibronectin extracellular matrix protein that supports the skin elasticity and superficial cells actively express cytokeratin 19. The study results clearly demonstrate the effectiveness of mesotherapeutic treatment for skin rejuvenation.

scholarly journals FiloQuant reveals increased filopodia density during DCIS progression

2017 ◽  
Author(s):  
Guillaume Jacquemet ◽  
Ilkka Paatero ◽  
Alexandre F. Carisey ◽  
Artur Padzik ◽  
Jordan S. Orange ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Ductal Carcinoma ◽  
Biological Significance ◽  
Cell Types ◽  
Neuronal Cultures ◽  
Cellular Models ◽  
Tumour Spheroids ◽  
Microscopy Data

AbstractFilopodia are commonly observed cellular protrusions in vitro and in vivo. Defective filopodia formation is linked to several pathologies including cancer, wherein actively protruding filopodia, at the invasive front, and filopodia-mediated probing of the microenvironment accompanies cancer cell dissemination. Despite wide biological significance, delineating the function of these finger-like protrusions in more complex systems remains technically challenging, particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia and filopodia-like protrusions in both fixed and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information including protrusion dynamics, density and length from multiple cell types and in a range of microenvironments, such as during collective or single cancer cell migration in 2D and 3D, in fixed neuronal cultures, in activated natural killer cells and in sprouting endothelial cells in vivo. In cellular models of breast ductal carcinoma in situ (DCIS) we reveal a link between filopodia formation at the cell-matrix interface, during collective invasion and in 3D tumour spheroids, with the previously reported local invasive potential of these breast cancer models in vivo. Finally, using intravital microscopy, we observed that tumour spheroids display prominent filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers

2021 ◽  
pp. 088532822110377
Author(s):  
Dganit Stern-Tal ◽  
Shmulik Ittah ◽  
Ella Sklan
Keyword(s):  
Spider Silk ◽  
Cultured Cells ◽  
Human Mesenchymal Stem Cells ◽  
Extended Period ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Cell Spheroids ◽  
Culture Cells

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing.

Transglutaminase Crosslinking of SIBLING Proteins in Teeth

2005 ◽  
Vol 84 (7) ◽  
pp. 607-612 ◽  
Author(s):  
M.T. Kaartinen ◽  
W. Sun ◽  
N. Kaipatur ◽  
M.D. McKee
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Transglutaminase 2 ◽  
Protein Crosslinking ◽  
Dentin Matrix Protein ◽  
Lysine Residues ◽  
Dentin Phosphoprotein ◽  
Matrix Maturation

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).

scholarly journals Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation

2017 ◽  
Vol 22 (5) ◽  
pp. 583-601 ◽  
Author(s):  
P. Marc D. Watson ◽  
Edel Kavanagh ◽  
Gary Allenby ◽  
Matthew Vassey
Keyword(s):  
Cell Culture ◽  
Drug Discovery ◽  
Glial Cell ◽  
Critical Role ◽  
3D Culture ◽  
Cell Types ◽  
Culture Systems ◽  
Cellular Environment

Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer’s and Parkinson’s disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell–cell and cell–matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

Utilization of M06-G3 Immortalized Odontoblast Cells in Studies Regarding Dentinogenesis

2001 ◽  
Vol 15 (1) ◽  
pp. 25-29 ◽  
Author(s):  
Mary MacDougall ◽  
Aaron Unterbrink ◽  
David Carnes ◽  
Sheela Rani ◽  
Xianhong Luan ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Line ◽  
Transforming Growth Factor Beta ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Cell Types ◽  
Oral Epithelium ◽  
Specific Gene ◽  
Dentin Matrix Protein

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized M06-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line M06-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major noncollagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing

scholarly journals Growth Plate Chondrocytes: Skeletal Development, Growth and Beyond

2019 ◽  
Vol 20 (23) ◽  
pp. 6009 ◽  
Author(s):  
Shawn A. Hallett ◽  
Wanida Ono ◽  
Noriaki Ono
Keyword(s):  
Growth Plate ◽  
Bone Marrow Stromal Cells ◽  
Skeletal Development ◽  
Cell Types ◽  
Postnatal Growth ◽  
Lineage Tracing ◽  
Molecular Characteristics ◽  
Growth Plate Chondrocytes ◽  
Parathyroid Hormone Related Protein

Growth plate chondrocytes play central roles in the proper development and growth of endochondral bones. Particularly, a population of chondrocytes in the resting zone expressing parathyroid hormone-related protein (PTHrP) is now recognized as skeletal stem cells, defined by their ability to undergo self-renewal and clonally give rise to columnar chondrocytes in the postnatal growth plate. These chondrocytes also possess the ability to differentiate into a multitude of cell types including osteoblasts and bone marrow stromal cells during skeletal development. Using single-cell transcriptomic approaches and in vivo lineage tracing technology, it is now possible to further elucidate their molecular properties and cellular fate changes. By discovering the fundamental molecular characteristics of these cells, it may be possible to harness their functional characteristics for skeletal growth and regeneration. Here, we discuss our current understanding of the molecular signatures defining growth plate chondrocytes.

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