Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Properties of intramuscular connective tissue in pork and poultry with reference to weakening of structure

The most common connective tissue research in meat science has been conducted on the properties of intramuscular connective tissue (IMCT) in connection with eating quality of meat. From the chemical and physical properties of meat, researchers have concluded that meat from animals younger than physiological maturity is the most tender. In pork and poultry, different challenges have been raised: the structure of cooked meat has weakened. In extreme cases raw porcine M. semimembranosus (SM) and in most turkey M. pectoralis superficialis (PS) can be peeled off in strips along the perimysium which surrounds the muscle fibre bundles (destructured meat), and when cooked, the slices disintegrate. Raw chicken meat is generally very soft and when cooked, it can even be mushy. The overall aim of this thesis was to study the thermal properties of IMCT in porcine SM in order to see if these properties were in association with destructured meat in pork and to characterise IMCT in poultry PS. First a 'baseline' study to characterise the thermal stability of IMCT in light coloured (SM and M. longissimus dorsi in pigs and PS in poultry) and dark coloured (M. infraspinatus in pigs and a combination of M. quadriceps femoris and M. iliotibialis lateralis in poultry) muscles was necessary. Thereafter, it was investigated whether the properties of muscle fibres differed in destructured and normal porcine muscles. Collagen content and also solubility of dark coloured muscles were higher than in light coloured muscles in pork and poultry. Collagen solubility was especially high in chicken muscles, approx. 30 %, in comparison to porcine and turkey muscles. However, collagen content and solubility were similar in destructured and normal porcine SM muscles. Thermal shrinkage of IMCT occurred at approximately 65 °C in pork and poultry. It occurred at lower temperature in light coloured muscles than in dark coloured muscles, although the difference was not always significant. The onset and peak temperatures of thermal shrinkage of IMCT were lower in destructured than in normal SM muscles, when the IMCT from SM muscles exhibiting ten lowest and ten highest ultimate pH values were investigated (onset: 59.4 °C vs. 60.7 °C, peak: 64.9 °C vs. 65.7 °C). As the destructured meat was paler than normal meat, the PSE (pale, soft, exudative) phenomenon could not be ruled out. The muscle fibre cross sectional area (CSA), the number of capillaries per muscle fibre CSA and per fibre and sarcomere length were similar in destructured and normal SM muscles. Drip loss was clearly higher in destructured than in normal SM muscles. In conclusion, collagen content and solubility and thermal shrinkage temperature vary between porcine and poultry muscles. One feature in the IMCT could not be directly associated with weakening of the meat structure. Poultry breast meat is very homogenous within the species.

Interactions between Mast Cells, Fibroblasts and Connective Tissue Components

It has long been recognized that mast cells occur throughout connective tissues. Histologic studies have revealed that such cells release their granules into the surrounding environment upon exposure to both immunologic and nonimmunologic stimuli. By microscopy these extracellular granules appeared to be phagocytosed by fibroblasts and by blood-borne phagocytic cells as they entered the site of mast cell degranulation. Such in vivo observations led to the suggestion that mast cells both altered connective tissue components and influenced fibroblast function through these discharged granules. Recent in vitro studies using cultured fibroblasts and isolated mast cells and mast cell granules have confirmed both these hypotheses. In addition, such studies have also documented that fibroblasts degrade ingested mast cell granules. Such studies document that a number of critical interactions may occur between mast cells and connective tissue components.

Molecular evolution of connective tissue growth factor in Cyprinidae (Teleostei : Cypriniformes)

Connective tissue growth factor (CTGF) plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages, respectively, with the high nodal supports. The estimation of the ratio of non-synonymous to synonymous substitution (dN/dS) for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 by indel (insertion/deletion) was found at the 5' end of CTGF gene of Cyprinidae, and this 6 by deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Mechanics of biological networks: from the cell cytoskeleton to connective tissue.

From the cell cytoskeleton to connective tissues, fibrous networks are ubiquitous in metazoan life as the key promoters of mechanical strength, support and integrity. In recent decades, the application of physics to biological systems has made substantial strides in elucidating the striking mechanical phenomena observed in such networks, explaining strain stiffening, power law rheology and cytoskeletal fluidisation - all key to the biological function of individual cells and tissues. In this review we focus on the current progress in the field, with a primer into the basic physics of individual filaments and the networks they form. This is followed by a discussion of biological networks in the context of a broad spread of recent in vitro and in vivo experiments.

Connective tissue growth factor antagonizes transforming growth factor-beta 1/Smad signalling in renal mesangial cells

The critical involvement of TGF-beta 1 (transforming growth factor-beta 1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-beta 1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-beta 1 and its physiological significance. CTGF was determined to bind directly to the T beta RIII (TGF-beta type III receptor) and antagonize TGF-beta 1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-beta 1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-beta 1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-beta 1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF Knockdown of T beta RIII restored TGF-beta 1-mediated Smad signalling and cell contractility, suggesting that T beta RIII is key for CTGF-mediated regulation of TGF-beta 1. Comparison of gene expression profiles from CTGF/TGF-beta 1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-beta 1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Study of chemical properties and evaluation of collagen in mantle, epidermal connective tissue and tentacle of Indian Squid, Loligo duvauceli Orbigny

The chemical composition and evaluation of Indian squid (Loligo duvauceli) mantle, epidermal connective tissue and tentacle is investigated in this current study. It is observed that squid mantle contains 22.2% total protein; 63.5% of the total protein is myofibrillar protein. The unique property of squid myofibrillar protein is its water solubility. Squid mantle contains 12.0% total collagen. Epidermal connective tissue has highest amounts of total collagen (17.8%). SDS-PAGE of total collagen identified high molecular weight α-, β- and γ- sub-chains. Amino acid profile analysis indicates that mantle and tentacle contain essential amino acids. Arginine forms a major portion of mantle collagen (272.5 g/100 g N). Isoleucine, glutamic acid and lysine are other amino acids that are found in significantly high amounts in the mantle. Sulphur containing cystine is deficit in mantle collagen. Papain digest of mantle and epidermal connective tissue is rich in uronic acid, while papain digest, collagenase digest and urea digest of epidermal connective tissue has significant amounts of sialic acid (25.2, 33.2 and 99.8 μmol /100 g, respectively). PAS staining of papain digest, collagenase digest and urea digest also identify the association of hexoses with low molecular weight collagen fragments. Histochemical sectioning also emphasized the localized distribution of collagen in epidermal and dermal region and very sparse fibres traverse the myotome bundles

Characterisation of connective tissue from the hypertrophic skeletal muscle of myostatin null mice

Myostatin is a potent inhibitor of muscle development. Genetic deletion of myostatin in mice results in muscle mass increase, with muscles often weighing three times their normal values. Contracting muscle transfers tension to skeletal elements through an elaborate connective tissue network. Therefore, the connective tissue of skeletal muscle is an integral component of the contractile apparatus. Here we examine the connective tissue architecture in myostatin null muscle. We show that the hypertrophic muscle has decreased connective tissue content compared with wild-type muscle. Secondly, we show that the hypertrophic muscle fails to show the normal increase in muscle connective tissue content during ageing. Therefore, genetic deletion of myostatin results in an increase in contractile elements but a decrease in connective tissue content. We propose a model based on the contractile profile of muscle fibres that reconciles this apparent incompatible tissue composition phenotype.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Subepithelial connective tissue graft for root coverage in smokers and non-smokers: A clinical and histologic controlled study in humans

Background: The aim of this study was to evaluate root coverage of gingival recessions and to compare graft vascularization in smokers and non-smokers. Methods: Thirty subjects, 15 smokers and 15 non-smokers, were selected. Each subject had one Miller Class I or II recession in a non-molar tooth. Clinical measurements of probing depth (PD), relative clinical attachment level (CAL), gingival recession (GR), and width of keratinized tissue (KT) were determined at baseline and 3 and 6 months after surgery. The recessions were treated surgically with a coronally positioned flap associated with a subepithelial connective tissue graft. A small portion of this graft was prepared for immunohistochemistry. Blood vessels were identified and counted by expression of factor VIII-related antigen-stained endothelial cells. Results: Intragroup analysis showed that after 6 months there a was gain in CAL, a decrease in GR, and an increase in KT for both groups (P<0.05), whereas changes in PD were not statistically significant. Smokers had less root coverage than non-smokers (58.02% +/- 19.75% versus 83.35% +/- 18.53%; P<0.05). Furthermore, the smokers had more GR (1.48 +/- 0.79 mm versus 0.52 +/- 0.60 mm) than the nonsmokers (P<0.05). Histomorphometry of the donor tissue revealed a blood vessel density of 49.01 +/- 11.91 vessels/200x field for non-smokers and 36.53 +/- 10.23 vessels/200x field for smokers (P<0.05). Conclusion: Root coverage with subepithelial connective tissue graft was negatively affected by smoking, which limited and jeopardized treatment results.