Stability Of Collagen Fibrils Research Articles

A comparative investigation of anionic polysaccharides on the structure and gastrointestinal digestion of collagen fibrils

Collagen, the most abundant and widely distributed functional protein in mammals, typically assembles into collagen fibrils through side-by-side packing. The purpose of this study was to comparatively investigate the fate of sea cucumber collagen fibrils in the gastrointestinal tract when interacting with different anionic polysaccharides (fucoidan (FUC), Kappa-carrageenan (K-car), sodium alginate (SA)). Results revealed that the gel properties and viscosity values of collagen fibrils were notably enhanced, and the rate of structural alteration in collagen fibrils was reduced when K-car and SA were introduced. Conversely, in the presence of FUC, collagen fibril viscosity decreased, and the secondary structure of collagen fibrils underwent changes. FUC was found to diminish the structural stability of collagen fibrils and accelerate the gastric digestion rate, which was further exacerbated by thermal treatment. All these anionic polysaccharides were observed to facilitate the formation of collagen peptide aggregates by binding to polysaccharides during intestinal digestion. This study bridged the knowledge gap regarding the impact of anionic polysaccharides on the gastrointestinal digestion of collagen fibrils, potentially paving the way for broader applications of collagen in the food industry.

Control of the extracellular matrix by hypoxic lymphatic endothelial cells: Impact on the progression of lymphedema?

Initial lymphatic vessels do not have a continuous basement membrane. Therefore, the ability of lymphatic endothelial cells (LECs) to produce extracellular matrix (ECM) has received little attention. Untreated lymphedema is a chronic disease that progresses to massive fibrosclerosis in advanced stages. Expansion of the intercellular space and fibrosclerosis cause hypoxia, which also affects the LECs. We studied the expression of genes in human LECs in vitro by RNA sequencing, analyzed the effects of hypoxia (1% O2 ) vs. normoxia (21% O2 ), and focused on ECM genes. LECs express fibrillin-1 and many typical components of a basement membrane such as type IV, VIII, and XVIII collagen, laminin β1, β2, and α4, perlecan, and fibronectin. Under hypoxia, we found significant upregulation of expression of genes controlling hydroxylation of procollagen (PLOD2, P4HA1), and also cross-linking, bundling, and stabilization of collagen fibrils and fibers. Also striking was the highly significant downregulation of elastin expression, whereas fibulin-5, which controls the assembly of tropoelastin monomers, was upregulated under hypoxia. In the dermis from genital lymphedema, we observed significant PLOD2 expression in initial lymphatics. Overall, hypoxia results in the picture of a dysregulated ECM production of LECs, which might be partly responsible for the progression of fibrosclerosis in lymphedema.

Role of epigallocatechin gallate in collagen hydrogels modification based on physicochemical characterization and molecular docking

Collagen Type I derived from fish is mainly limited by its poor physicochemical properties for further applications. In this study, we developed epigallocatechin gallate (EGCG) cross-linked collagen hydrogels (EC hydrogels) to realize physicochemical improvements, basing on the interaction mechanism between collagen and EGCG. The integrity of collagen framework with slight secondary structure change in the presence of EGCG was confirmed. The stronger stability of collagen fibrils was proved by slower swelling ratio, declined enzymatic degradation, improved thermal analysis and mechanical test due to EGCG modification. To illustrate the potential mechanism between collagen and EGCG, molecular docking was used to identify both covalent (CN bond, between lysine of collagen and C2-ring B of EGCG) and non-covalent bonds (hydrogen bond and hydrophobic interaction) within in EC hydrogel. Taken together, this work would offer some insights into the further study about the interaction between EGCG and collagen.

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2−/−) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2−/− mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

The influences of 3,4-dihydroxybenzaldehyde on the microstructure and stability of collagen fibrils

3,4-dihydroxybenzaldehyde (DB), a derivative of catechol, were applied to improve the physicochemical properties of collagen fibrils, prepared via the self-assembly of collagen molecules. The aldehyde group of DB interacted with ε-amino group of collagen, and then the catechol group of DB was oxidized and self-polymerized, resulting in the formation of cross-links among collagen. The results of spectral experiment showed that the triple helix structure of collagen were integrated and the Tyr residues of collagen might react after cross-linking. Collagen fibrils became dark brown and the yellowness (Δb*) ascended from 0 to 3.24 when the [CHO]/[NH2] ratio rose from 0:1 to 40:1. Due to the effects of DB, the adjacent collagen fibrils agglomerated without particular orientation and the pore size of collagen fibril network decreased. The thermal stability of collagen fibrils had been improved about 15 °C while the elastic modulus obviously increased from 19 to 834 Pa after cross-linking. Moreover, the presence of DB also caused the dramatically enhancement in the enzymatic resistance of collagen fibrils while the biocompatibility of collagen fibrils was still desirable. These promising data suggested biomedical materials based on collagen fibrils with requisite properties can be obtained via adjusting the dose of DB.

Modulation of the Self-Assembly of Collagen by Phytic Acid: An In Vitro Study

Phytic acid, containing a myoinositol ring coupled with six phosphate groups, can react with the amino groups of collagen to regulate their self-assembly behavior. The aim of this research is to evaluate the effects of phytic acid on the selfassembly behavior of collagen, the structures and properties of the resulting fibrils and hydrogels. Turbidity and chloramine T assay suggested that phytic acid could improve the self-assembly kinetics and degree of collagen, and the optimal ratio of phytic acid/collagen was 1/1 (w/w). Scanning electron microscopy (SEM) analysis indicated that co-fibrils of collagen with phytic acid are more slender than that of pure collagen, and transmission electron microscopy (TEM) reveals that the characteristic D-periodicity of collagen fibrils is not affected by phytic acid. Besides, differential scanning calorimetry (DSC) and rheology revealed that the thermal stability of collagen fibrils and the viscoelasticity of collagen hydrogels could be improved by phytic acid and the optimal ratio of phytic acid/collagen is 1/1 (w/w).

Effect of Ionic Liquids on the Fibril-Formation and Gel Properties of Grass Carp (Ctenopharyngodon idellus) Skin Collagen

Self-assembled environment of collagen is one of the important factors for improving and regulating the properties of collagen-based biomaterials. This study aimed to investigate the effect of ionic liquids (ILs) on the fibril-formation and gel properties of grass carp (Ctenopharyngodon idellus) skin collagen. Fibrillogenic kinetics analysis showed that the collagen self-assembly can be suppressed by the introduction of ILs, and the inhibitory effect is influenced by concentration and types of ILs. Scanning electron microscopy test indicated that the assembled collagen fibrils in the presence of ILs had bigger diameters than that in the conventional buffer. Differential scanning calorimetry analysis revealed that the thermal stability of collagen fibrils can be significantly increased when self-assembly is performed in the presence of ILs. Moreover, the introduction of ILs enhanced the mechanical strength of collagen gels. Finding from this work provides a new idea for improving the performance of fish-sourced collagen biomaterials.

Effects of collagen cross-linking on the keratoconus metabolic network.

Keratoconus (KC) is a multifactorial, ectatic corneal disease. Metabolic changes in the corneal stroma with alterations in collagen fibril stability, oxidative stress, and urea cycle, have previously been reported as key players in KC pathobiology. Recently, corneal collagen cross-linking (CXL) has been introduced as a treatment that can address the progressive nature of KC. While the treatment has been successful in the early days, it is not without clinical ramifications. In this study, we investigated the alterations in KC metabolic profiles due to CXL. Primary human corneal fibroblasts (HCFs) from healthy donors and human KC fibroblasts (HKCs) from KC donor patients were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added to the cultures followed by UVA irradiation (365 nm). Using mass spectrometry, we measured the major differences in metabolites in HKCs compared to HCFs pre- and post CXL. The analysis of 276 metabolites in HCFs and HKCs revealed that the most affected metabolites due to CXL were glutathione disulfide, ascorbic acid, proline, and lysine. A significant decrease in the pro-inflammatory biomarkers (myo-inositol and histidine) was also observed. Furthermore, a significant downregulation of many amino acids, lactate levels, and other water-soluble metabolites was noted in HKCs following CXL. CXL is a KC treatment available to patients within certain criteria. Surprisingly, the cellular and molecular mechanisms are considerably understudied limiting our ability for more precise and targeted CXL treatments. In this study, for the first time, we report the effects of CXL on KC metabolism.

Use of polyphenols as a strategy to prevent bond degradation in the dentin-resin interface.

This study evaluated the effect of dentin pretreatment with the polyphenols quercetin and resveratrol on the resin-dentin microtensile bonding strength (μTBS) and collagen fibrils stability of the adhesive interface. Different concentrations (100, 250, 500, or 1,000μgml-1 ) of quercetin or resveratrol, or a mixture of quercetin and resveratrol (3:1, 1:1, 1:3; vol:vol), as well as distilled water or 2% chlorhexidine digluconate, were applied to etched dentin. Then, a two-step etch-and-rinse adhesive was applied followed by composite restoration. Measurements of resin-dentin μTBS were made after 1 and 120d. The stability of collagen fibrils in the hybrid layer was evaluated using transmission electron microscopy. The Student's t-test and two-way factorial anova with Tukey's test were used to analyze the effects of dentin pretreatment and storage time on μTBS values. Comparisons between μTBS measurements made on 1 and 120d showed that resveratrol had the best performance, with significantly higher μTBS values after 120d for all concentrations of resveratrol tested. Quercetin pretreatment resulted in a significant rise of μTBS when used at concentrations of 100 and 500μgml-1 . Quercetin+resveratrol at the ratio of 1:1 performed better than when used at ratios of either 3:1 or 1:3. Resveratrol might represent a potential approach to achieve desirable bonding stability and reduce the frequent replacement of composite restorations.

Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases

Collagen and its denatured form, gelatin, are biopolymers of fundamental interest in numerous fields ranging from living tissues to biomaterials, food, and cosmetics. This study aims at characterizing mixtures of those biopolymers at high concentrations (up to 100 mg·mL-1) at which collagen has mesogenic properties. We use a structural approach combining polarization-resolved multiphoton microscopy, polarized light microscopy, magnetic resonance imaging, and transmission electron microscopy to analyze gelatin and collagen/gelatin dense phases in their sol and gel states from the macroscopic to the microscopic scale. We first report the formation of a lyotropic crystal phase of gelatin A and show that gelatin must structure itself in particles to become mesogenic. We demonstrate that mixtures of collagen and gelatin phase segregate, preserving the setting of the pure collagen mesophase at a gelatin ratio of up to 20% and generating a biphasic fractal sample at all tested ratios. Moreover, differential scanning calorimetric analysis shows that each protein separates into two populations. Both populations of gelatins are stabilized by the presence of collagen, whereas only one population of collagen molecules is stabilized by the presence of gelatin, most probably those at the interface of the fibrillated microdomains and of the gelatin phase. Although further studies are needed to fully understand the involved mechanism, these new data should have a direct impact on the bioengineering of those two biopolymers.

Urea, Uric Acid, Prolactin and fT4 Concentrations in Aqueous Humor of Keratoconus Patients

ABSTRACTPurpose: Keratoconus is a noninflammatory disease of the cornea associated with progressive thinning and conical shape. Metabolic alterations in the urea cycle, with changes in collagen fibril stability, oxidative stress, thyroid hormones and prolactin with regulatory effect on biosynthesis and biomechanical stability of corneal stroma, may all play a role in keratoconus etiology. Our purpose was to determine urea, uric acid, prolactin and free thyroxin (fT4) concentrations in human aqueous humor (hAH) of keratoconus and cataract patients.Methods: hAH was collected from 100 keratoconus (penetrating keratoplasty) (41.9 ± 14.9 years, 69 males) and 100 cataract patients (cataract surgery) (71.2 ± 12.4 years, 58 males). Urea, uric acid, prolactin and fT4 concentrations were measured by Siemens clinical chemistry or immunoassay system. For statistical analysis, a generalized linear model (GLM) was used.Results: Urea concentration was 11.88 ± 3.03 mg/dl in keratoconus and 16.44 ± 6.40 mg/dl in cataract patients, uric acid 2.04 ± 0.59 mg/dl in keratoconus and 2.18 ± 0.73 mg/dl in cataract groups. Prolactin concentration was 3.18 ± 0.34 ng/ml in keratoconus and 3.33 ± 0.32 ng/ml in cataract patients, fT4 20.57 ± 4.76 pmol/l in KC and 19.06 ± 3.86 pmol/l in cataract group. Urea concentration was effected through gender (p = 0.039), age (p = 0.001) and diagnosis (p = 0.025). Uric acid concentration was not effected through any of the analyzed parameters (p > 0.056). Prolactin and fT4 concentration were effected only through diagnosis (p = 0.009 and p = 0.006).Conclusions: Urea and prolactin concentrations are decreased, fT4 concentration is increased in aqueous humor of keratoconus patients, and uric acid concentration remains unchanged. Urea concentration in aqueous humor is also increased in older and male patients. Therefore, metabolic disorder and hormonal balance may both have an impact on keratoconus development. Further studies are necessary to assess the specific impact.

An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

PurposeCurrent literature contains scant information regarding the extent of enzymatic collagen cross-linking in the keratoconus (KC) cornea. The aim of the present study was to examine levels of enzymatic lysyl oxidase–derived cross-links in stromal collagen in KC tissue, and to correlate the cross-link levels with collagen fibril stability as determined by thermal denaturation temperature (Tm).MethodsSurgical KC samples (n = 17) and Eye-Bank control (n = 11) corneas of age 18 to 68 years were analyzed. The samples were defatted, reduced (NaBH4), hydrolyzed (6N HCl at 110°C for 18 hours), and cellulose enriched before analysis by C8 high-performance liquid chromatography equipped with parallel fluorescent and mass detectors in selective ion monitoring mode (20 mM heptafluorobutyric acid/methanol 70:30 isocratic at 1 mL/min). Nine different cross-links were measured, and the cross-link density was determined relative to collagen content (determined colorimetrically). The Tm was determined by differential scanning calorimetry.ResultsCross-links detected were dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine, lysinonorleucine (LNL), and histidinohydroxylysinonorleucine in both control and KC samples. Higher DHLNL levels were detected in KC, whereas the dominant cross-link, LNL, was decreased in KC samples. Decreased LNL levels were observed among KC ≤ 40 corneas. There was no difference in total cross-link density between KC samples and the controls. Pyridinolines, desmosines, and pentosidine were not detected. There was no notable correlation between cross-link levels with fibril instability as determined by Tm.ConclusionsLower levels of LNL in the KC cornea suggest that there might be a cross-linking defect either in fibrillar collagen or the microfibrillar elastic network composed of fibrillin.

Chlorhexidine-containing Acid Conditioner Preserves the Longevity of Resin-dentin Bonds

The current study evaluated the effect of 2% chlorhexidine digluconate (CHX) on the immediate and six-month resin-dentin bond strength (BS) and nanoleakage pattern (NL) of etch-and-rinse adhesives when applied in aqueous or associated to the phosphoric acid conditioner. The occlusal enamel of 42 caries-free extracted molars was removed in order to expose a flat dentin surface. In groups 1 and 2 (control-C), the surfaces were acid etched with conventional phosphoric acid, and the adhesives Prime&Bond NT (PB) and Adper Single Bond 2 (SB) were applied after rinsing, drying and rewetting with water. In groups 3 and 4 (Ac/CHX), the adhesives were applied in a similar manner, however, a 2% CHX-containing acid was previously applied. In groups 5 and 6 (CHX), the adhesives were applied according to the control group; however, the rewetting procedure was performed with an aqueous solution of 2% CHX for 60 seconds. Composite buildups (Opallis, FGM) were constructed incrementally, and the specimens were longitudinally sectioned in the "x" and "y" directions to obtain bonded sticks (0.8 mm2) to be tested in tension at 0.5 mm/minute immediately or after six months of water storage. For NL, two bonded sticks from each tooth were coated with nail varnish, placed in silver nitrate and polished down with SiC paper. Resin-dentin interfaces were analyzed by EDX-SEM. The BS and NL data from each adhesive was submitted to two-way repeated measures ANOVA and Tukey's test (alpha=0.05). After six months of water storage, significant reductions in BS were observed for both adhesives in the control group (p<0.05). When Ac/CHX or CHX was used, no significant reductions in BS were observed for both systems. Nanoleakage was more evident in the control group than in the experimental groups (p<0.05), even after six months. The use of CHX in an aqueous solution or associated with the acid conditioner was effective for reducing degradation of resin-dentin bonds after six months of water storage.

Acceleration of fibril formation and thermal stabilization of collagen fibrils in the presence of taxifolin (dihydroquercetin)

We studied the effect of flavonoid taxifolin (dihydriquercetin) on the structure and thermal stability of collagen I fibrils. Taxifolin accelerated fibril formation with reconstruction of periodical cross-striation characteristic of these fibrils. Differential scanning calorimetry showed elevation of melting temperature of collagen fibrils formed in neutral or weakly alkaline media, but not of individual tropocollagen molecules in acid medium. Taxifolin capacity to stimulate fibril formation and promote stabilization of fibrillar forms of collagen can be used in medicine.

Distinct effects of gonadectomy in male and female mice on collagen fibrillogenesis in the skin

Collagen biosynthesis and deposition is a complex, multistep process, which is tightly regulated to maintain proper tissue homeostasis. Sex steroid hormones have been implicated in regulating collagen synthesis; however the specific mechanisms regulating the process remain largely unknown. To investigate the role of estrogens and androgens in the regulation of genes involved in collagen synthesis and fibrillogenesis using gonadectomized C57/B6 mice. Collagen content was assessed by hydroxyproline measurement and acetic acid extraction of collagen with or without the addition of pepsin. The mRNA levels of fibrillar collagens and enzymes involved in fibrillogenesis were determined by QPCR analysis. The protein expression of decorin, lumican and fibromodulin was confirmed by immunostaining. We have shown that castration resulted in a markedly decreased skin thickness and collagen content without affecting collagen solubility. Furthermore, the mRNA levels of fibrillar collagen genes including types I, III, and V were decreased, suggesting that androgens positively regulate the rate of collagen gene transcription. Conversely, ovariectomy mainly affected collagen solubility. The absence of estrogens resulted in decreased expression levels of several of the small leucine-rich repeat proteins and proteoglycans (SLRPs) including decorin, fibromodulin and lumican. Estrogens may not be directly involved in the regulation of collagen synthesis; however, they may play a critical role in regulating organization and stability of collagen fibrils. Androgens play a positive role in the regulation of collagen biosynthesis. In summary, our data demonstrate that androgens and estrogens regulate distinct aspects of collagen fibrillogenesis in mouse skin.

Effects of Copper and Cross-Linking on the Extracellular Matrix of Tissue-Engineered Arteries

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.

TIMP-1 alters susceptibility to carcinogenesis.

Tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins known to possess a broad range of biological activities, including inhibition of metalloproteinase activity, regulation of proliferation and apoptosis of a variety of cell types, and, depending on the context, differential regulation of angiogenic and inflammatory responses. Elevated mRNA expression of TIMP family members correlates with malignancy and clinical outcome in many human cancer types; however, a protective role for TIMPs also has been observed in various mouse models of human cancer. In the current study, we found distinct spatial-temporal expression patterns for the mRNA of TIMP family members in a mouse model of epithelial carcinogenesis [i.e., keratin 14-human papillomavirus 16 (K14-HPV16) transgenic mice]. To test the hypothesis that elevated expression of TIMP-1 functionally regulates epithelial carcinogenesis, we introduced a human TIMP-1 transgene into K14-HPV16 transgenic mice and assessed neoplastic progression. Results from these studies suggest that TIMP-1 enhances tumorgenicity by potentiating keratinocyte hyperproliferation and appearance of chromosomal aberrations in premalignant cells, thereby increasing their risk to undergo malignant conversion. In addition, TIMP-1 inhibits tissue gelatinolytic activity in tumor stroma, affects stabilization of collagen fibrils, but does not inhibit malignant conversion of dysplasias into carcinomas or development of metastases. The combined implications of these studies suggest that TIMP-1 is an important contributor to epithelial neoplastic progression and supports the concept that TIMP-1 exerts differential regulation on tissues in a stage-dependent manner.

Decorin Binds Near the C Terminus of Type I Collagen

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment alpha1(I) CB6 and is approximately 25 nm from the C terminus, in a zone that coincides with the c(1) band of the collagen fibril d-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

Independent modulation of collagen fibrillogenesis by decorin and lumican.

The leucine-rich proteoglycans (also known as "small, leucine-rich proteoglycans," or SLRPs) lumican and decorin are thought to be involved in the regulation of collagen fibril assembly. Preparation of these proteoglycans in chemical amounts without exposure to denaturants has recently been achieved by infecting HT-1080 cells with vaccinia virus that contains an expression cassette for these molecules. Addition of lumican and decorin to a collagen fibrillogenesis assay based on turbidity demonstrated that lumican accelerated initial fibril formation while decorin retarded initial fibril formation. At the end of fibrillogenesis, both proteoglycans resulted in an overall reduced turbidity, suggesting that fibril diameter was lower. The presence of both proteoglycans had a synergistic effect, retarding fibril formation to a greater degree than either proteoglycan individually. Competitive binding studies showed that lumican did not compete for decorin-binding sites on collagen fibrils. Both proteoglycans increased the stability of fibrils to thermal denaturation to approximately the same degree. These studies show that lumican does not compete for decorin-binding sites on collagen, that decorin and lumican modulate collagen fibrillogenesis, and that, in the process, they also enhance collagen fibril stability.

The relative contribution of electrostatic interactions to stabilization of collagen fibrils

Electrostatic energies of interaction between type I collagen molecules were calculated, using models developed by Timasheff and Hill. These energies, along with a contribution from hydrophobic forces, were then incorporated into an equation due to Flory that described phase equilibria of rod-like polymers. The Flory formalism in turn permitted a calculation of the overall free energy of fibril formation (delta Ff), and an assessment of the relative contribution of electrostatic and hydrophobic forces to delta Ff. Lastly, delta Ff was used in a nucleation-growth model relating halftimes of fibril formation (t1/2) to ionic strength (I) and temperature. Because the theory provided no basis for setting absolute levels of the energetic contributions, five parameters in the model had to be derived from experimental data. Based on the fit of theory to experimental results both for intact and pepsinized collagen, it was found that very low electrostatic energies (about -1 kcal/mole per collagen molecule) were sufficient to explain experimental t1/2 vs I relationships. This energy is equivalent to 1 close charge-pair interaction per molecule and appears to be lower than the energy assignable to hydrophobic interactions.