Migratory behavior of cardiac cushion tissue cells in a collagen-lattice culture system

A culture system was devised for the study of factors which influence the migration of cardiac cushion tissue cells. Explants of isolated chick atrioventricular canal cushions were placed on hydrated collagen lattices. Cells grew out of the explants from the endocardium and across the surface of the collagen lattices. During further incubation, mesenchyme-type cells seeded from the surface population into the underlying collagen matrix. These cells were morphologically similar to the mesenchymal cushion tissue cells which are derived from the endocardium and which migrate into and through the cardiac jelly matrix in the embryonic heart. The events observed in the culture system mimicked those occurring in the developing chick atrioventricular cushion.     

Neurite growth in 3D collagen gels with gradients of mechanical properties

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.     

Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities.     

Collagen fibrillogenesis in a three-dimensional fibroblast cell culture system

The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system. Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures. Sample meshes were processed for electron microscopy or immuno-electron microscopy. Fibrils 20–30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate. As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter. By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen. No collagen fibril formation occurred in control cultures to which no ascorbate was added. However, large amounts of microfibrils were observed. Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35–54 nm in diameter. We believe that this system, which morphologically resembles a normal dermis, will werve as an excellent model for the study of collagen fibrillogenesis.     

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.     

Measuring collagen fiber orientation: a two-dimensional quantitative macroscopic technique.

This paper describes the design, evaluation, and application of a new system for quantifying two-dimensional collagen fiber orientation in soft tissue. Series of transmitted polarized light images were collected using a custom-designed macroscope. Combined analysis of pixel brightness, and hue from images collected with a compensator plate, permitted the assignment of each pixel into the appropriate orientation band. Experiments were performed to quantify the linearity and noise of the system. Validation was performed on a specimen composed of strain-birefringent plastic strips at various orientations. Preliminary collagen fiber orientation data is presented from a tendon specimen. This study demonstrates the utility of this approach for studying collagen fiber orientation across large areas.     

Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels.

We describe the application of three-dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type I collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site: and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.     

Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.     

Type I collagen reduces lipid accumulation during adipogenesis of preadipocytes 3T3-L1 via the YAP-mTOR-autophagy axis

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss.     

A new constitutive model for multi-layered collagenous tissues

Collagenous tissues such as the aneurysmal wall or the aorta are multi-layered structures with the mean fibre alignments distinguishing one layer from another. A constitutive representation of the multiple collagen layers is not yet developed, and hence the aim of the present study. The proposed model is based on the constitutive theory of finite elasticity and is characterized by an anisotropic strain-energy function which takes the material structure into account. The passive tissue behaviour is modelled and the related mechanical response is assumed to be dominated by elastin and collagen. While elastin is modelled by the neo-Hookean material the constitutive response of collagen is assumed to be transversely isotropic for each individual layer and based on an exponential function. The proposed constitutive function is polyconvex which ensures material stability. The model has five independent material parameters, each of which has a clear physical interpretation: the initial stiffnesses of the collagen fabric in the two principal directions, the shear modulus pertaining to the non-collagenous matrix material, a parameter describing the level of nonlinearity of the collagen fabric, and the angle between the principal directions of the collagen fabric and the reference coordinate system. An extension-inflation test of the adventitia of a human femoral artery is simulated by means of the finite element method and an error function is minimized by adjusting the material parameters yielding a good agreement between the model and the experimental data.     

Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo

We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo.     

Morphogenesis in vitro of dissociated fetal rat small intestinal cells upon an open surface and subsequent to collagen gel overlay

The morphogenesis of cells dissociated from fetal rat intestine at 18 days of gestation was compared in vitro on two different substrates, tissue culture plastic and collagen gel. During the first 2 days in culture growth on both substrates was similar, resulting in the formation of layers of epithelial cells and of small lumina within them. On plastic, the cell layers contracted over time, resulting in the formation of small mounds of cells bearing on their surface small protrusions covered with epithelial cells which had densely packed microvilli on their apical surfaces. When the cultures on collagen gels were overlaid with more collagen gel, vesicles lined with epithelial cells developed. These cells were joined by junctional complexes and displayed an apical brush border which was periodic acid-Schiff and alkaline phosphatase positive. After 1 week in culture, when the vesicles reached their maximum extent, numerous epithelial cells were actively incorporating labeled thymidine and cells were being extruded into the lumen. These results demonstrate that the dissociated intestinal cells have the capacity to form intestine-like organoids in vitro and that surrounding the cells with the collagen gel allows expression of this potential. The collagen gel cultures thus undergo morphogenetic processes which can be modified by the surrounding environment and should provide a useful in vitro model system for the study of intestinal development.     

Contributions of Glycosaminoglycans to Collagen Fiber Recruitment in Constitutive Modeling of Arterial Mechanics

The contribution of glycosaminoglycans (GAGs) to the biological and mechanical functions of biological tissue has emerged as an important area of research. GAGs provide structural basis for the organization and assembly of extracellular matrix (ECM). The mechanics of tissue with low GAG content can be indirectly affected by the interaction of GAGs with collagen fibers, which have long been known to be one of the primary contributors to soft tissue mechanics. Our earlier study showed that enzymatic GAG depletion results in straighter collagen fibers that are recruited at lower levels of stretch, and a corresponding shift in earlier arterial stiffening (Mattson et al., 2016). In this study, the effect of GAGs on collagen fiber recruitment was studied through a structure-based constitutive model. The model incorporates structural information, such as fiber orientation distribution, content, and recruitment of medial elastin, medial collagen, and adventitial collagen fibers. The model was first used to study planar biaxial tensile stress-stretch behavior of porcine descending thoracic aorta. Changes in elastin and collagen fiber orientation distribution, and collagen fiber recruitment were then incorporated into the model in order to predict the stress-stretch behavior of GAG depleted tissue. Our study shows that incorporating early collagen fiber recruitment into the model predicts the stress-stretch response of GAG depleted tissue reasonably well (rms = 0.141); considering further changes of fiber orientation distribution does not improve the predicting capability (rms = 0.149). Our study suggests an important role of GAGs in arterial mechanics that should be considered in developing constitutive models.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

A multi-station culture force monitor system to study cellular contractility

Cellular contraction contributes to the formation of scar tissue, which is characterized by an over-produced, disorganized collagen matrix. To study the contractility of cells in vitro and its potential contribution to scar tissue formation, we have developed a multi-station culture force monitor (CFM) system. This system consists of four vertical cantilever beams with semiconductor strain gages and a computerized data acquisition unit to monitor contractile forces of the cells in a collagen gel. Calibration showed that this system has a highly linear voltage-force relationship (R(2) > 0.99). Further, to demonstrate the applicability of this system, contractile forces of human skin fibroblasts in a collagen gel were measured. These fibroblasts were found to produce an average force of 0.2 nN/cell, which is consistent with the data in literature. The significant advantage of this CFM system is its ability to test multiple samples simultaneously. Therefore, the system can facilitate statistical design and analysis of experiments to study the effects of growth factors (e.g., TGF-betas) on cellular contraction and their potential role in scar tissue formation.     

In vitro carcinogenesis of mammary epithelial cells byN-nitroso-N-methylurea using a collagen gel matrix culture

Summary Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of “microtumors” in collagen gels, induced by 7,12-dimethylbenz(a)anthracene. In the present study the direct acting water soluble, mammary carcinogen,N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiated cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these “tumors” was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.     

Preferential usage of glycyl-tRNA isoaccepting species in collagen synthesis.

Chick embryo tRNA charged with [3H]glycine was incubated in an in vitro protein-synthesizing system using polysomes isolated from either chick embryo liver or calvaria. Using collagenase digestion to measure the fraction of protein synthesized which was collagenous, the results indicate that in the calvaria system approximately 65% of the incorporated [3H]glycine was in collagen. The incorporation of [3H]glycine into protein from individual isoaccepting species was determined by chromatography on a reversed phase system of the charged tRNA before and after incubation in the polysome systems. In the calvaria system, a single tRNAGly species cognate to GGU and GGC and which is found in unusually large amounts in collagen-synthesizing tissues was used preferentially in collagen-synthesizing tissues was used preferentially in collagen synthesis. In the liver system, the rate of incorporation was similar to the calvaria, but no collagen synthesis was detected and only a relatively small preferential usage of any of the four major isoaccepting species was observed. These results support the notion that the complement of tRNA found in a cell may be adapted to the synthesis of a particular protein. It is also possible that under certain circumstances, collagen synthesis may be controlled in vivo at the translational level by the concentration of particular tRNA species.     

Lymphocyte migration into three-dimensional collagen matrices: a quantitative study

Lymphocytes have been plated onto the surface of three-dimensional gels of native collagen fibers, and their distribution throughout the three- dimensional collagen matrix has been determined in a quantitative fashion at various times thereafter. Information regarding the total number of applied cells may be obtained by this means. Lymphocyte penetration into the collagen gel does not appear to involve the expression of collagenolytic activity, nor does it require the presence of serum. Analysis of the kinetics of lymphocyte penetration into the gel matrix indicates that lymphocytes are migrating in a "random-walk" fashion. Our objective has been to establish a model system for studying the cell-matrix and cell-cell interactions which influence the pattern of lymphocyte recirculation in vivo and the results presented here are discussed in this context.     

Three-dimensional structure of type IV collagen in the mammalian lens capsule.

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.