Collagen Fibrillogenesis In Vitro: A Characterization of Fibril Quality as a Function of Assembly Conditions

McPherson, John M.; Wallace, Donald G.; Sawamura, Steven J.; Conti, A; Condell, Richard A.; Wade, S.; Piez, Karl A.

doi:10.1016/s0174-173x(85)80034-0

Cited by 76 publications

(49 citation statements)

References 33 publications

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“…These results are consistent with literature reports demonstrating that collagen polymerized at different temperatures results in different fiber structures (Wood, 1960). During the self-assembly process, collagen fiber thickness is affected by both pH and temperature, where lower pH and temperature provides a longer nucleation phase to produce thicker fibrils (Wood, 1960;McPherson et al, 1985). Interestingly, the mechanical stiffness of collagen hydrogels is dependent upon collagen fiber microstructure, where thinner fibers result in stiffer gels (Roeder et al, 2002;Raub et al, 2007;Yang et al, 2009).…”

Section: Dicussionsupporting

confidence: 91%

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound

Garvin

VanderBurgh

Hocking

et al. 2013

The Journal of the Acoustical Society of America

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Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.

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Section: Dicussionsupporting

confidence: 91%

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound

Garvin

VanderBurgh

Hocking

et al. 2013

The Journal of the Acoustical Society of America

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“…Trypsin/chymotrypsin digestion were performed as described previously (19), with minor modifications. Prior to addition of the proteases, samples were preincubated at the desired temperatures (20,25,30,35,40, and 45°C) for 15 min. The proteases were added and incubated for an additional 2 min.…”

Section: Methodsmentioning

confidence: 99%

“…Fibril Formation-Fibrils were formed by dialysis against 20 mM sodium phosphate, pH 7.2, at 15°C (20). The suspension of collagen fibrils was diluted with 20 mM sodium phosphate to 0.125, 0.25, 0.5, and 1.0 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Production of Human Type I Collagen in Yeast Reveals Unexpected New Insights into the Molecular Assembly of Collagen Trimers

Olsen¹,

Leigh²,

Chang³

et al. 2001

Journal of Biological Chemistry

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Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N-or C-propeptide, or both propeptide domains, from both pro␣-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N-and C-propeptides from both pro␣-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/ telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.Collagen is the single most abundant protein found in animals. In the human body, it is expressed in most tissues and plays a structural, as well as a signaling, role in the development, maintenance, and repair of tissues and organs. 20 different collagen types are coded by more than 30 genes. Assembly of trimeric collagen intracellularly and formation of collagen fibers in the extracellular matrix is the result of a complex multistep process (1, 2). Within the endoplasmic reticulum, the individual procollagen polypeptides undergo several co-and post-translational modifications, including hydroxylation of specific prolyl and lysyl residues, selection and alignment of three procollagen polypeptides, and disulfide bond formation among the C-propeptides. Experimental evidence suggests folding of the triple helix begins at the C terminus and propagates toward the N terminus. Prior to triple helix formation, prolyl hydroxylase converts proline in the Y position of GXY triplets to hydroxyproline. Hydrogen bonding between the ␣-chains of the triple helix increases the denaturation temperature of the molecule, preventing it from unfolding at the animal's body temperature (3). Triple helical procollagen is secreted from the cell, and the N-and C-terminal propeptides are removed by specific N-and C-proteinases. The resulting collagen monomers, consisting of triple helical and telopeptide regions, undergo a self-assembly process to generate collagen fibril intermediates and then mature collagen fibers. These fibers are further stabilized by covalent cross-links within the triple helix and telopeptide regions, providing strength and support to the surrounding tissue.

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“…High concentration fibrillar collagen gel suspensions (35 mg/ml) were prepared from Vitrogen at 17°C according to the method of McPherson et al (1985). Approximately 0.5-ml aliquots of the fibrillar collagen suspension were placed in each well of a 24-well plate.…”

Section: Preparation Of Collagen Gels and Filmsmentioning

confidence: 99%

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet‐derived growth factor

Rhudy

McPherson

1988

Journal Cellular Physiology

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The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.

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Collagen Fibrillogenesis In Vitro: A Characterization of Fibril Quality as a Function of Assembly Conditions

Cited by 76 publications

References 33 publications

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound

Production of Human Type I Collagen in Yeast Reveals Unexpected New Insights into the Molecular Assembly of Collagen Trimers

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet‐derived growth factor

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