Notes

The determination of small amounts of collagen hydroxylysyl glycosides
Glucosylgalactosylhydroxylysine (GlcGalHyl) and galactosylhydroxylysine (GalHyl) in alkaline hydrolysates of insoluble collagens were separated on 130 x 0 cm and 45 x 0 cm columns of Chromobeads A resin (Technicon) and simultaneous analyses with ninhydrin and orcinol-H2SO4 were performed. Ninhydrin, which is the standard reagent in existing methods, gave erroneously high results with both columns due to overlapping, resistant peptides. A rapid and more accurate procedure employing the smaller column, the orcinol-H2SO4 reagent and a new internal standard was developed. The losses of the glycosides in alkaline hydrolysis, especially in the presence of collagen peptides, were much larger than had been reported previously. A shorter alkaline hydrolysis following digestion of the collagen with papain was effective and allowed more reliable corrections to be made. Purging with nitrogen reduced the losses further.

Mature bovine skin collagen contained less GlcGalHyl than embryonic skin collagen whereas the differences in GalHyl were insignificant.Cell-Populated Collagen Lattice Models.Investigation of cell function is often hampered by the complexity of the tissue context. Pharmaceutical intermediates is circumvented by isolating cells from tissues and analyzing their behavior in culture. Most cell types are cultured as monolayers on planar, rigid Petri dishes, an environment that does not reflect the spatial, three-dimensional cellular environment in vivo. Culture in three-dimensional collagen lattices has been devised to optimize in vitro culture conditions and to provide a more physiologic "in vivo-like" environment. Collagen lattices can easily be manipulated to suit diverse cell types and to provide variable mechanical forces.

Cells can be imaged in such surroundings, and gene expression as well as protein production and activity can be monitored.Collagen fibril formation in a wound healing model.Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days.

Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be Type II collagen deficiency in achondrogenesis (Langer-Saldino).Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction.Zhang S, Cao X, Stablow AM, Shenoy VB, Winkelstein BA.


Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. Purchase integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 041; p ≤ 0171) and neuronal aspect ratio (AR) (R2 ≥ 050; p < 0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a "switchlike" fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11% with a 95% confidence interval of 10-13%) for elevating ERK phosphorylation after loading.

As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading.

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