scholarly journals Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Harris Pratsinis ◽  
Dimitris Kletsas
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Intervertebral Disc ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Collagen Type I ◽  
Cell Physiology ◽  
Type I ◽  
Collagen Gels

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD’s extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

scholarly journals In vitro production of human dermal equivalent

2010 ◽  
Vol 63 (7-8) ◽  
pp. 459-464 ◽  
Author(s):  
Zoran Milosavljevic ◽  
Biljana Ljujic
Keyword(s):  
Vitamin C ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Collagen Gels ◽  
In Vitro Production ◽  
Dermal Equivalent ◽  
Enzymatic Dissociation ◽  
Vitro Production

Introduction. Human dermal tissue is composed of loose and dense connective tissue. Main cell populations are fibroblasts and the dominant fibers are built from collagen type I. The aim of our study was to determine the precise method and time frame for the in vitro production of human dermal equivalent and to investigate the effects of ratio of structural elements and vitamin C on characteristics of the engineered tissue. Material and methods. Primary isolation of the foreskin fibroblasts was performed by explant method and enzymatic dissociation. Various collagen gels were obtained by mixing cells (from 25x103 to 200x103/ml) and neutralized collagen type I (from 2 to 4 mg/ml), with or without vitamin C. The routine histological and morphometrical examination was performed. Results. Enzymatic dissociation of the foreskin proved to be a faster method for production of desired number of fibroblasts (7.5x105 for 4 days). The contraction of collagen-gels started from day one through day seven and was dependent on cell and collagen concentration with higher density gels being contracted to a greater extent, except for the lowest/highest values. The best result was achieved with 100x103 cells and 2 mg/ml collagen. Vitamin C at 50 ?g/ml had no effect on speed of tissue formation. Conclusion. A precise approach that mimic the in vivo conditions is needed for the in vitro production of the dermal equivalent suitable for the possible treatment of tissue defects. Nearly ten days are necessary from the donor tissue dissociation to the final product.

scholarly journals Collagen Type I Containing Hybrid Hydrogel Enhances Cardiomyocyte Maturation in a 3D Cardiac Model

Polymers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 687 ◽  
Author(s):  
Sam G. Edalat ◽  
Yongjun Jang ◽  
Jongseong Kim ◽  
Yongdoo Park
Keyword(s):  
Growth Factor ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Embryonic Stem ◽  
Collagen Type I ◽  
Transforming Growth Factor Β1 ◽  
Type I ◽  
Hybrid Hydrogel ◽  
Cardiac Model

In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor β1 (TGF-β1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation.

Bacterial Endotoxin Inhibits Migration, Attachment, and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

1987 ◽  
Vol 66 (9) ◽  
pp. 1449-1455 ◽  
Author(s):  
S. Pitaru ◽  
M. Soldinger ◽  
D. Madgar ◽  
Z. Metzger
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Human Gingival Fibroblasts ◽  
Bacterial Endotoxin ◽  
Type I ◽  
Gingival Fibroblasts ◽  
Collagen Gels

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

scholarly journals Current insights on use of growth factors as therapy for Intervertebral Disc Degeneration

2018 ◽  
Vol 9 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Justin C. Kennon ◽  
Mohamed E. Awad ◽  
Norman Chutkan ◽  
John DeVine ◽  
Sadanand Fulzele
Keyword(s):  
Tissue Engineering ◽  
Low Back Pain ◽  
Back Pain ◽  
Growth Factors ◽  
Growth Factor ◽  
Intervertebral Disc ◽  
Transforming Growth Factor ◽  
Low Back

AbstractChronic low back pain is a critical health problem and a leading cause of disability in aging populations. A major cause of low back pain is considered to be the degeneration of the intervertebral disc (IVD). Recent advances in therapeutics, particularly cell and tissue engineering, offer potential methods for inhibiting or reversing IVD degeneration, which have previously been impossible. The use of growth factors is under serious consideration as a potential therapy to enhance IVD tissue regeneration. We reviewed the role of chosen prototypical growth factors and growth factor combinations that have the capacity to improve IVD restoration. A number of growth factors have demonstrated potential to modulate the anabolic and anticatabolic effects in both in vitro and animal studies of IVD tissue engineering. Members of the transforming growth factor-β superfamily, IGF-1, GDF-5, BMP-2, BMP-7, and platelet-derived growth factor have all been investigated as possible therapeutic options for IVD regeneration. The role of growth factors in IVD tissue engineering appears promising; however, further extensive research is needed at both basic science and clinical levels before its application is appropriate for clinical use.

Modulation of thrombin and thrombin receptor peptide mitogenicity by human lung mast cell tryptase

1994 ◽  
Vol 267 (2) ◽  
pp. L113-L119 ◽  
Author(s):  
T. Hartmann ◽  
S. J. Ruoss ◽  
G. H. Caughey
Keyword(s):  
Mast Cell ◽  
Smooth Muscle ◽  
Growth Factors ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Dna Synthesis ◽  
Human Lung ◽  
Thrombin Receptor ◽  
Mast Cell Tryptase

In previous studies, mast cell tryptase acted as a potent mitogen for fibroblasts from human lung and rodent embryonic tissue but failed to stimulate growth of cultured rat aortic vascular smooth muscle cells (VSMC). The current study shows that tryptase inhibits DNA synthesis in VSMC stimulated by thrombin. However, it does not affect the stimulation of DNA synthesis by the synthetic thrombin receptor peptide Ser-Phe-Phe-Leu-Arg-Asn-Pro (SFFLRNP), which mimics the amino-terminus of thrombin receptor proteolytically activated by thrombin. Nor does tryptase alter the mitogenic response of VSMC to purified growth factors, such as platelet-derived growth factor (PDGF). These data suggest that tryptase inhibits thrombin-induced DNA synthesis without interfering with intracellular mitogenic signaling pathways activated by thrombin or other growth factors. This study further suggests that tryptase neither cleaves nor inactivates thrombin. Therefore, inhibition of thrombin's mitogenic effects by tryptase is not mediated by destruction of thrombin itself. The inhibition by tryptase of thrombin-induced DNA synthesis in VSMC contrasts with the stimulatory effect of tryptase on fibroblasts, in which synergy is observed with thrombin, with thrombin receptor peptide and with other growth factors. These data provide in vitro evidence that mast cell tryptase interferes with thrombin-stimulated vascular smooth muscle growth and suggest that tryptase is a multifunctional growth factor whose actions are cell specific.

scholarly journals B cell growth factor-induced proliferation of hairy cell lymphocytes and inhibition by type I interferon in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 937-942
Author(s):  
KA Paganelli ◽  
SS Evans ◽  
T Han ◽  
H Ozer
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Dna Synthesis ◽  
B Cell ◽  
Clinical Activity ◽  
Type I ◽  
Type I Ifn ◽  
Hairy Cell ◽  
B Cell Growth Factor

Malignant B cells from hairy cell leukemia (HCL) patients are unable to proliferate when stimulated with standard B cell mitogens. Using chromatographically purified B cell growth factor (BCGF), HCL can be stimulated to proliferate as assessed by incorporation of tritiated thymidine [3HTdR] into DNA. Proliferation was found to be time dependent, with no detectable 3H-TdR incorporation in up to three days of culture, and significant stimulation evident at days 6 and 10. The presence of 10% BCGF in culture was an absolute requirement for HCL proliferation; however, this BCGF-induced DNA synthesis could be further augmented by the addition of anti-immunoglobulin heavy chain antibodies. BCGF-induced proliferation was abrogated in six of six patients by addition of 1,000 U/mL of recombinant alpha 2-interferon (IFN) at day 0, although 1,000 U/mL of recombinant gamma-IFN had no inhibitory effect in five of six patients studied. Specific cellular receptors for type I IFN were demonstrated in HCL by inhibition of binding of 125I-alpha 2-IFN by a 40-fold excess of unlabeled alpha 2 or beta IFN with no inhibition by unlabeled gamma-IFN. These data demonstrate that malignant HCL lymphoblasts express specific type I IFN receptors and that type I, but not type II IFN, can inhibit growth factor-induced DNA synthesis by hairy cells in vitro. They further suggest a direct antiproliferative mechanism of action for IFN in HCL and predict equivalent clinical activity by either alpha or beta, but not gamma IFN in this malignancy.

Effects of growth factors and hormones on basal and FSH-stimulated inhibin production by porcine granulosa cells in vitro

1991 ◽  
Vol 3 (2) ◽  
pp. 201 ◽  
Author(s):  
U Michel ◽  
S Ludemann ◽  
H Jarry ◽  
W Wuttke
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Type I ◽  
Tgf Beta ◽  
G Cells ◽  
Porcine Granulosa Cells ◽  
Igf I ◽  
Factor Type

The effect of several growth factors, protein and steroid hormones on follicle stimulating hormone (FSH)-stimulated and basal inhibin secretion by mature porcine granulosa cells (g-cells) in culture was examined in order to elucidate the putative role of growth factors and hormones in the regulation of inhibin secretion by porcine g-cells in vitro. Cells were incubated with the respective hormones over a timespan of 0-144 h and immunoreactive inhibin was measured with a radioimmunoassay against porcine inhibin. Epidermal growth factor (EGF) and human transforming growth factor type beta (TGF-beta) decreased basal and gonadotrophin-stimulated inhibin and progesterone in a dose-dependent manner. In the absence of insulin, insulin-like growth factor type I (IGF-I) caused a 4-fold enhancement of basal inhibin secretion, but inhibin secretion was elevated only to 20% above control in the presence of 500 nM insulin. Porcine platelet-derived growth factor (PDGF) had no significant effect on basal or FSH-induced inhibin secretion by g-cells. In addition, neither gonadotrophin-releasing hormone (GnRH) nor prolactin (PRL), arginine vasopressin (AVP) and oxytocin affected basal or FSH-stimulated inhibin release by porcine g-cells. Oestradiol caused a slight but significant (P less than 0.01) rise of basal inhibin production (158% of control) in the last 2 days of culture (96-144 h) and the effect of androstenedione on basal (158% of control) and FSH-stimulated (140% of control) inhibin release (P less than 0.01) was also only visible on Days 4-6 of culture. In contrast to androstenedione and oestradiol, progesterone did not show any effect during 6 days of culture in a dose range of 10(-5) to 10(-9) M. Like steroids, prostaglandin E2 (PGE2) had a stimulatory effect on basal inhibin production (250% of control) by porcine g-cells, visible on Days 3-6 of culture, but an inhibitory effect on FSH-stimulated release (less than 40% of control). Over all the experiments with different hormones and growth factors, tested in varying doses and over a time span of 0-144 h, there was a strong correlation between progesterone and inhibin secretion by g-cells (0-48 h = 0.78; 48-96 h = 0.92; 96-144 h = 0.92). These results suggest that EGF, TGF-beta, IGF-I, oestradiol and androstendione as well as PGE2 have para- and/or autocrine modulatory effects on basal and FSH-stimulated inhibin secretion by mature porcine g-cells in vitro and further demonstrate that the secretion of the proteohormone inhibin and the steroid progesterone are closely related.

scholarly journals Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells

2018 ◽  
Vol 19 (8) ◽  
pp. 2207 ◽  
Author(s):  
Stefan Stich ◽  
Anke Möller ◽  
Mario Cabraja ◽  
Jan Krüger ◽  
Sylvia Hondke ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Type I ◽  
Anulus Fibrosus ◽  
Tissue Samples ◽  
Disc Regeneration ◽  
Intervertebral Disc Regeneration

Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)β3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFβ3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.

scholarly journals Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

1990 ◽  
Vol 1 (10) ◽  
pp. 747-761 ◽  
Author(s):  
N Nishizawa ◽  
Y Okano ◽  
Y Chatani ◽  
F Amano ◽  
E Tanaka ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Pertussis Toxin ◽  
Guanosine Triphosphate ◽  
Kinase C

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation.

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