Advanced glycation end-products diminish tendon collagen fiber sliding

Connective tissue aging and diabetes related comorbidity are associated with compromised tissue function, increased susceptibility to injury, and reduced healing capacity. This has been partly attributed to collagen cross-linking by advanced glycation end-products (AGEs) that accumulate with both age and disease. While such cross-links are believed to alter the physical properties of collagen structures and tissue behavior, existing data relating AGEs to tendon mechanics is contradictory. In this study, we utilized a rat tail tendon model to quantify the micro-mechanical repercussion of AGEs at the collagen fiber-level. Individual tendon fascicles were incubated with methylglyoxal (MGO), a naturally occurring metabolite known to form AGEs. After incubation in MGO solution or buffer only, tendons were stretched on the stage of a multiphoton confocal microscope and individual collagen fiber stretch and relative fiber sliding were quantified. Treatment by MGO yielded increased fluorescence and elevated denaturation temperatures as found in normally aged tissue, confirming formation of AGEs and related cross-links. No apparent ultrastructural changes were noted in transmission electron micrographs of cross-linked fibrils. MGO treatment strongly reduced tissue stress relaxation (p < 0.01), with concomitantly increased tissue yield stress (p < 0.01) and ultimate failure stress (p = 0.036). MGO did not affect tangential modulus in the linear part of the stress–strain curve (p = 0.46). Microscopic analysis of collagen fiber kinematics yielded striking results, with MGO treatment drastically reducing fiber-sliding (p < 0.01) with a compensatory increase in fiber-stretch (p < 0.01). We thus conclude that the main mechanical effect of AGEs is a loss of tissue viscoelasticity driven by matrix-level loss of fiber–fiber sliding. This has potentially important implications to tissue damage accumulation, mechanically regulated cell signaling, and matrix remodeling. It further highlights the importance of assessing viscoelasticity – not only elastic response – when considering age-related changes in the tendon matrix and connective tissue in general.     

Directional dependence of hydroxyapatite-collagen interactions on mechanics of collagen

Bone is a biological nanocomposite composed primarily of collagen and hydroxyapatite. The collagen molecules self-assemble to from a structure known as a fibril that comprises of about 85–95% of the total bone protein. In a fibril, the molecular level interactions at the interface between molecular collagen and hydroxyapatite nanocrystals have a significant role on its mechanical response. In this study, we have used molecular dynamics and steered molecular dynamics to study directional dependence of deformation response of collagen with respect to the hydroxyapatite surface. We have also studied mechanical response of collagen in the proximity of (0 0 0 1) and (1 0 1¯0) surfaces of hydroxyapatite. Our simulations indicate that the mechanics of collagen pulled in different directions with respect to hydroxyapatite is significantly different. Similar results were obtained for collagen pulled in the proximity of different crystallographic surfaces of hydroxyapatite.     

Effect of d-amino acids on collagen fibrillar assembly and stability: Experimental and modelling studies

The effect of selected d-amino acids (d-AAs) on collagen with 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) initiated crosslinking is evaluated by using experimental and modelling tools. The experimental results suggest that d-Lysine (d-Lys) plays a pivotal role in the self-assembly and conformation of collagen fibrils than d-Alanine (d-Ala) and d-Glutamic acid (d-Glu). The SDS-PAGE, absorption spectrum and viscosity measurements indicate significant differences in the d-Lys crosslinked collagen when compared to other systems. The CD spectra show an increase in the peak intensity at 220 nm in the presence of d-Lys, which could be due to increase in propensity of the structure to form a triple helix. Modelling studies indicated that d-Lys bind with collagen-like peptide (CLP) through multiple H-bonding and hydrophobic interactions. d-Lys has the lowest binding energy (−4.2 kcal/mol, indicating strongest interactions) when compared to d-Ala and d-Glu (−3.6 and −3.7 kcal/mol, respectively). Orientational changes in the collagenase on CLP-d-Lys are observed which may decrease its accessibility to degradation and stabilise CLP against the action of the former. d-Lys has the lowest binding energy and improved fibrillar-assembly and staggered alignment without the undesired structural stiffness and aggregations. The information derived from the present study could help in designing heterochiral collagen-based biomaterial.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro

The formation in vitro of fibrils from type I acid-soluble calf skin collagen has been studied before and after removal of the extrahelical peptides with carboxypeptidase and with pepsin. Turbidimetric studies show that the mechanism of fibril growth in undigested collagen is similar to that in pepsin-digested collagen; following carboxypeptidase digestion, however, a different growth mechanism was apparent. The two mechanisms have been further characterized by electron microscopy. In the course of formation of fibrils from undigested collagen, “early fibrils” (short D-periodic fibrils that have both ends visible) occurred in the lag phase under the precipitating conditions employed here. After pepsin or carboxypeptidase digestion of the collagen no “early fibrils” were seen. In carboxypeptidase-digested collagen, lateral assembly was inhibited; after pepsin digestion, linear assembly was inhibited. Complete removal of the extrahelical peptides prevented fibril formation under the conditions used here. Electron-optical examination of segment-long-spacing (SLS) dimers established a more complete removal of the C-terminal peptide after carboxypeptidase digestion than after pepsin digestion. Analyses of staining patterns of SLS dimers and fibrils from undigested and digested samples showed that the C-terminal peptide in SLS crystallites and fibrils formed from undigested collagen is in a condensed conformation. A proposed conformation, in which condensation occurs predominantly in a hydrophobic region at the proximal end of the C-terminal peptide, is discussed in terms of a dual role for the C-terminal peptide in fibrillogenesis. One role, shared with the N-terminal peptide, is to participate in interactions between the 4D-staggered molecules leading to the formation of linear aggregates; the other is to participate in interactions between these linear aggregates giving rise to D-periodic aggregates and lateral (as well as linear) growth.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

Polymeric collagen fibrils. An example of substrate-mediated steric obstruction of enzymic digestion.

Polymeric collagen fibrils have been reacted with fluorescein and rhodamine isothiocyanates to produce fluorescent dye-labelled fibrils, containing seven dye substituents per molecule of tropocollagen within the polymeric collagen fibrils. Two dye-labelled peptides per molecule of tropocollagen were solubilised by trypsin (EC 3.4.21.4) from the telopeptide regions and four dye-labelled peptides were located in the helical regions solubilised by bacterial collagenase (EC 3.4.24.3). The solubilisation of dye-labelled peptides from these insoluble substrates were employed to measure the kinetics of trypsin and collagenase digestion of the telopeptide and helical regions, respectively, of the insoluble polymeric collagen fibrils. These studies demonstrated an apparent excess of enzyme for the readily available substrate under conditions when it was known that a vast excess of substrate existed in the reaction mixture calculated in terms of a molecular ratio. A point of equivalence was established for both trypsin and bacterial collagenase, approximately one enzyme molecule per 870 substrate molecules. On either side of this point the quantity of products formed was controlled by either the enzyme concentration or the substrate concentration. The results can be explained in terms of the inaccessibility of tropocollagen molecules within the molecular architecture of the polymeric collagen fibrils. The external layer of tropocollagen molecules obstruct collagenolytic enzymes penetrating to, and forming enzyme-substrate complexes with, the bulk of the substrate within the interior of the fibrils.     

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.     

Strain-controlled enzymatic cleavage of collagen in loaded matrix

The purpose of this investigation is to support the novel hypothesis that collagenous matrices are intrinsically "smart" load-adapting biomaterials. This hypothesis is based fundamentally on the postulate that tensile strain directly modulates the susceptibility of collagen molecules to enzymatic degradation (i.e., protects molecules which are under load from cleavage). To test this postulate, collagenase (Clostridiopeptidase A) was applied to a uniaxially loaded, anisotropic, devitalized, collagenous matrix in which a subset of fibrils was loaded in tension while the remaining fibrils carried little or no load. The collagen degradation pattern (as assessed by polarization and transmission electron microscopy) was found to correspond inversely to the tensile stress field such that fibrils under lower tensile load were preferentially cleaved. These results have immediate implications for tissue engineering of load-bearing collagenous matrices in vitro and may contribute significantly to our understanding of synthesis, remodelling, and pathogenesis of collagen matrices in vivo.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

Quantitative phase measurements of tendon collagen fibres

Collagen is the main component of structural mammalian tissues. In tendons, collagen is arranged into fibrils with diameters ranging from 30 nm to 500 nm. These fibrils are further assembled into fibres several micrometers in diameter. Upon excessive thermal or mechanical stress, damage may occur in tendons at all levels of the structural hierarchy. At the fibril level, reported damage includes swelling and the appearance of discrete sites of plastic deformation that are best observed at the nanometer‐scale using, for example, scanning electron microscopy. In this paper, digital in‐line holographic microscopy is used for quantitative phase imaging to measure both the refractive index and diameter of collagen fibres in a water suspension in the native state, after thermal treatments, and after mechanical overload. Fibres extracted from tendons and subsequently exposed to 70 °C for 5, 15, or 30 minutes show a significant decrease in refractive index and an increase in diameter. A significant increase in refractive index is also observed for fibres extracted from tendons that were subjected to five tensile overload cycles.

     

Methylglyoxal-modified collagen promotes myofibroblast differentiation

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p < 0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p < 0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.     

Evidence against proteoglycan mediated collagen fibril load transmission and dynamic viscoelasticity in tendon

The glycosaminoglycan (GAG) dermatan sulfate and chondroitin sulfate side-chains of small leucine-rich proteoglycans have been increasingly posited to act as molecular cross links between adjacent collagen fibrils and to directly contribute to tendon elasticity. GAGs have also been implicated in tendon viscoelasticity, supposedly affecting frictional loss during elongation or fluid flow through the extra cellular matrix. The current study sought to systematically test these theories of tendon structure–function by investigating the mechanical repercussions of enzymatic depletion of GAG complexes by chondroitinase ABC in a reproducible tendon structure–function model (rat tail tendon fascicles). The extent of GAG removal (at least 93%) was verified by relevant spectrophotometric assays and transmission electron microscopy. Dynamic viscoelastic tensile tests on GAG depleted rat tail tendon fascicle were not mechanically different from controls in storage modulus (elastic behavior) over a wide range of strain-rates (0.05, 0.5, and 5% change in length per second) in either the linear or nonlinear regions of the material curve. Loss modulus (viscoelastic behavior) was only affected in the nonlinear region at the highest strain-rate, and even this effect was marginal (19% increased loss modulus, p = 0.035). Thus glycosaminoglycan chains of small leucine-rich proteoglycans do not appear to mediate dynamic elastic behavior nor do they appear to regulate the dynamic viscoelastic properties in rat tail tendon fascicles.     

Ultrashort echo time T21 values decrease in tendons with application of static tensile loads

In early stages of tendon disease, mechanical properties may become altered prior to changes in morphological anatomy. Ultrashort echo time (UTE) magnetic resonance imaging (MRI) can be used to directly detect signal from tissues with very short T2 values, including unique viscoelastic tissues such as tendons. The purpose of this study was to use UTE sequences to measure T2¹, T1 and magnetization transfer ratio (MTR) variations of tendon samples under static tensile loads. Six human peroneal tendons were imaged before and under static loading using UTE sequences on a clinical 3 T MRI scanner. Tendons were divided into two static tensile loading groups: group A that underwent one-step loading (15 N) and group B that underwent two-step loading (15 and 30 N). The T2¹, T1 and MTR variations were investigated in two selected section regions of interest (ROIs), including whole and core sections. Mean T2¹ values for the first step of loading (groups A and B) in both whole section and core section ROIs were significantly decreased by 13 ± 7% (P = 0.028) and 16 ± 5% (P = 0.017), respectively. For the second loading step (group B), there was a consistent, but non-significant reduction in T2¹ value by 9 ± 2% (P = 0.059) and 7 ± 5% (P = 0.121) for whole and core sections, respectively. Mean T1 did not show any consistent changes for either loading steps (P > 0.05). Mean MTR increased slightly, but not significantly for both loading steps (P > 0.05). Significant differences were found only in T2¹ values of tendons by static tensile load application. Therefore, T2¹ monitoring during loading is suggested for quantitative investigation of the tendons biomechanics.     

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.     

Atomistic simulation of nanomechanical properties of Alzheimer’s Aβ(1–40) amyloid fibrils under compressive and tensile loading

In addition to being associated with severe degenerative diseases, amyloids show exceptional mechanical properties including great strength, sturdiness and elasticity. However, thus far physical models that explain these properties remain elusive, and our understanding of molecular deformation and failure mechanisms of individual amyloid fibrils is limited. Here we report a series of molecular dynamics simulations, carried out to analyze the mechanical response of two-fold symmetric Aβ(1–40) amyloid fibrils, twisted protein nanofilaments consisting of a H-bonded layered structure. We find a correlation of the mechanical behavior with chemical and nanostructural rearrangements of the fibril during compressive and tensile deformation, showing that the density of H-bonds varies linearly with the measured strain. Further, we find that both compressive and tensile deformation is coupled with torsional deformation, which is manifested in a strong variation of the interlayer twist angle that is found to be proportional to both the applied stress and measured strain. In both compression and tension we observe an increase of the Young's modulus from 2.34 GPa (for less than 0.1% strain in compression and 0.2% strain in tension), to 12.43 GPa for compression and 18.05 GPa for tension. The moduli at larger deformation are in good agreement with experimental data, where values in the range of 10–20 GPa have been reported. Our studies confirm that amyloids feature a very high stiffness, and elucidate the importance of the chemical and structural rearrangements of the fibrils during deformation.     

Designed to fail: a novel mode of collagen fibril disruption and its relevance to tissue toughness

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules-meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils-were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT–loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72 h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT–loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p < 0.01) and IL-1β (p < 0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p < 0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.     

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.     

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.