Monomeric Collagen Research Articles

Flow-Induced Crystallization of Collagen: A Potentially Critical Mechanism in Early Tissue Formation

The type I collagen monomer is one of nature's most exquisite and prevalent structural tools. Its 300 nm triple-helical motifs assemble into tough extracellular fibers that transition seamlessly across tissue boundaries and exceed cell dimensions by up to 4 orders of magnitude. In spite of extensive investigation, no existing model satisfactorily explains how such continuous structures are generated and grown precisely where they are needed (aligned in the path of force) by discrete, microscale cells using materials with nanoscale dimensions. We present a simple fiber drawing experiment, which demonstrates that slightly concentrated type I collagen monomers can be "flow-crystallized" to form highly oriented, continuous, hierarchical fibers at cell-achievable strain rates (<1 s(-1)) and physiologically relevant concentrations (∼50 μM). We also show that application of tension following the drawing process maintains the structural integrity of the fibers. While mechanical tension has been shown to be a critical factor driving collagen fibril formation during tissue morphogenesis in developing animals, the precise role of force in the process of building tissue is not well understood. Our data directly couple mechanical tension, specifically the extensional strain rate, to collagen fibril assembly. We further derive a "growth equation" which predicts that application of extensional strains, either globally by developing muscles or locally by fibroblasts, can rapidly drive the fusion of already formed short fibrils to produce long-range, continuous fibers. The results provide a pathway to scalable connective tissue manufacturing and support a mechano-biological model of collagen fibril deposition and growth in vivo.

Advances Toward Forming Synthetic Mimetic Tendon

ABSTRACTCollagen is the most abundant protein present in the human body and found in connective tissues, bone, and tendon. It is also known as a natural resource for healing damaged skin tissues [1]. In this study, under specific microenvironment conditions, mimetic collagen gels were successfully formed synthetically from reconstituted Bovine type I collagen monomers. This was achieved by controlling ionic strength, temperature and pH, allowing fibrils with native mimetic D periodic banding structure to assemble spontaneously within the gels. In addition, by providing appropriate aging temperatures and times, mature collagen fibril growth is also realized in the gels in vitro. Mimetic gels were subsequently formed into fibers through a wet-spinning process. These spun fibers were found to preserve the native mimetic D periodic banding and fibrillar structure formed in the initial gels. As a result, the synthetic fibers resemble native tendon. Here structural development within the gel samples and fibers as a function of processing was analyzed by scanning electron microscopy (SEM). Results in this study also show a potentially new route for the fabrication of synthetic collagen fibers mimicking tendon, which may find applications as engineered tissues or scaffolding materials.

Modified platelet deposition on matrix metalloproteinase 13 digested collagen I

SummaryBackgroundAtherothrombosis underlies acute coronary syndromes, including unstable angina and acute myocardial infarction. Within the unstable plaque, monocytes express collagenolytic matrix metalloproteinases (MMPs), including MMP‐13, which degrades fibrous collagen. Following rupture, vessel wall components including degraded collagen are exposed to circulating platelets. Platelet receptors then mediate the recruitment and activation of platelets to form a thrombus, blocking blood flow and resulting in myocardial infarction and sudden death.ObjectivesHere we aim to provide information on the effects of collagen degradation on platelet adhesion and thrombus formation.MethodsUsing increasing concentrations of MMP‐13, we induced progressive degradation of fibrous and monomeric collagen I, visualized by electrophoresis, and then investigated the capacity of the resulting fragments to support static platelet adhesion and thrombus formation in whole flowing blood.ResultsBoth integrin and glycoprotein VI‐dependent interactions with fibrous collagen underpin high levels of platelet adhesion under both conditions, with little obvious effect of MMP‐13 treatment. Static platelet adhesion to monomeric collagen was strongly α2β1‐dependent regardless of degradation status. Under flow conditions, partially degraded monomeric collagen supported increased thrombus deposition at 10 μg mL −1 MMP‐13, falling close to background when collagen degradation was complete (100 μg mL −1 MMP‐13).ConclusionsNew binding activities come into play after partial digestion of collagen monomers, and net platelet‐reactivity through all axes is abolished as degradation becomes more complete.

Investigation of the Lag Phase of Collagen Fibrillogenesis Using Fluorescence Anisotropy.

The lag phase of collagen fibrillogenesis (1.0 mg/mL collagen solution) with an L-glutamine-L-arginine mixture (Glu-Arg) was monitored by the fluorescence anisotropy of tyrosine residues in real time. A suitable concentration of Glu-Arg (40 mmol/L) could control the aggregate ingredients in a collagen solution effectively before fibrillogenesis, and the mechanism was found to be similar to that with the monovalent ions. Fluorescence anisotropy analysis in the lag phase for a 1.0 mg/mL collagen solution confirmed the formation of collagen nuclei in multiple steps during the lag phase when the initial state of the collagen molecules was monomeric. A comparison of the fibrillogenesis lag phase for collagen solutions of 0.25, 0.50, and 1.0 mg/mL with 40 mmol/L Glu-Arg suggested that the length of the lag phase is inversely proportional to the increase in collagen concentration. Atomic force microscopy was used to investigate the effect of collagen aggregates on the fiber size. Based on the fluorescence anisotropy and atomic force microscopy results, it was proposed that an equilibrium exists between collagen aggregates and monomers accompanied by a nucleation of collagen monomers. A kinetic analysis for 0.25 and 1.0 mg/mL collagen with 40 mmol/L Glu-Arg at 18-38 °C indicated that collagen nucleation in the lag phase was favored by increasing temperature, and a corresponding activation energy of 76 and 97 kJ/mol was obtained for a collagen fibrillogenesis lag phase of 0.25 and 1.0 mg/mL, respectively.

Activation of phosphatidylinositol 3-kinase β by the platelet collagen receptors integrin α2β1 and GPVI: The role of Pyk2 and c-Cbl

Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kβ upon recruitment of the two main platelet collagen receptors, integrin α2β1 and GPVI. PI3Kβ-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2β1, but occurred normally upon GPVI ligation. Integrin α2β1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kβ activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KβKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kβ activity also hampered GPVI-induced tyrosine–phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kβ inhibitor TGX-221. These results outline important differences in the regulation of PI3Kβ by GPVI and integrin α2β1 and suggest that inhibition of Pyk2 may target PI3Kβ activation in a selective context of platelet stimulation.

Advances in the research of modulation of dermal collagen fibrin assembly by decorin

Formation of dermal collagen fiber is a complicated and sequential process with the progressive assembly of collagen. Collagen monomers form stepped and orderly protofibrils through longitudinal displacement. Subsequently, protofibrils or protofibrils and collagen are bonded by covalent bonds to form orderly lamellar structure of collagen fibers. Then collagen fibers are tightly wound into coarse collagen fiber bundles by covalent crosslinking. Decorin is a multifunctional small leucine-rich proteoglycan. It can prevent the aggregation of protofibrils by binding to the specific site of collagen with its core protein, and adjusting the spacing between the protofibrils with its glycosaminoglycan chain. Thus, by effecting the formation of collagen fibers with regulation of collagen assembly, decorin may help prevent scar formation and even promote regeneration.

Investigating Force-Induced Structural Changes in Single Collagen Molecules

Collagen is a structural protein found in abundance throughout the body, within the extracellular matrix and connective tissues including skin, bone, and tendons. One of the functions of collagen is to maintain tissue structure in the presence of external forces. However the effect of force on the collagen's structure is not clear:1 in the presence of an external force, collagen has been proposed to have one of two opposing structural changes: either overwinding, reducing its cleavage2 or underwinding, increasing its cleavage.3 Using a high-throughput single-molecule stretching instrument, the centrifuge force microscope (CFM),4 we are developing a technique to investigate force-dependent structural changes of collagen under various loading conditions. Here, we report on our progress tethering, enzymatically cleaving and applying a tunable external force to single molecules of collagen. In addition, we present characterization of the instrument and technique accomplished with single DNA molecules.1.Chang, S.W. and Buehler M. (2014) Molecular biomechanics of collagen molecules. Mat. Today. 17, 70-76.2.Camp, R., Liles, M., Beale, J., Saeidi, N., Flynn, BP., Moore, E., Murthy, S., and Ruberti, JW. (2011) Molecular Mechanochemistry: Low Force Switch Slows Enzymatic Cleavage of Human Type I Collagen Monomer. JACS, 133, 4073-8.3.Adhikari, A., Glassey, E. and Dunn, AR. (2012) Conformation Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases. JACS, 134, 13259-65.4.Halvorsen, K. and Wong, W. (2010) Massively Parallel Single-Molecule Manipulation Using Centrifugal Force. Biophys J., 98, L53-L55.

Directing collagen fibers using counter-rotating cone extrusion

The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to −40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10μm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900–2000kPa and 19–35mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine.

Collagen self-assembly on orthopedic magnesium biomaterials surface and subsequent bone cell attachment.

Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials.

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.

The tyrosine sulfate domain of fibromodulin represents a new binding site to collagen enhancing the interaction and fibril formation

Purpose: Fibromodulin is a collagen modulating protein belonging to the family of the small leucine rich repeat proteoglycans. The collagen modulating effect is demonstrated by in vitro fibrillogenesis assay, resulting in retardation of turbidity compared to fibrillogenesis by collagen alone. Fibromodulin binds collagen via the LRR domain, but whether the binding via the LRR has any role in the assembly of the collagen is not clear. Fibromodulin null mice have larger population of thinner collagen fibers in ligaments compared to the wild type mice and develop osteoarthritis due to joint instability, indicating a regulatory role by fibromodulin in vivo as well. The N-terminal domain of fibromodulin has a highly negative charge due to the presence of the posttranslational modification O-sulfation on tyrosine residues that binds heparin binding proteins. The aim of this work is to investigate binding of this domain to collagen and its role in the regulation of collagen fibril assembly. Methods: Different variants of fibromodulin (full length, the N-terminal domain of fibromodulin and N-terminal truncated fibromodulin) were used to study interaction to collagen type I in solid phase assay and surface plasmon resonance assay. Fibrillogenesis assay was used to monitor changes in turbidity over time in the presence of the different variants of fibromodulin. Samples were collected from the fibril formation and visualized by EM negative staining. Specific binding sites for fibromodulin on the collagen monomers by detection with a gold-labeled fibromodulin specific antibody was also identified by EM negative staining. Results: In solid phase assay, all three variants of fibromodulin bind to coated collagen type I, showing a novel binding site located in the N-terminal domain of fibromodulin. By surface plasmon resonance a distinct difference in the dissociation phase is observed. The dissociation of full-length fibromodulin is much slower compared to the N-terminal truncated variant of fibromodulin, indicating contribution of both the N-terminal site and the LRR-domain sites in binding to the collagen molecule. In an in vitro fibrillogenesis assay, full-length and N-terminal truncated fibromodulin delays the fibril formation. Adding the N-terminal tyrosin sulfate rich domain instead accelerates the fibril formation, indicating that this domain binds and facilitates collagen monomer assembly. Samples taken after 24 h of fibril formation in the presence of the different fibromodulin variants or PBS were analysed by electron microscopy. Full-length fibromodulin shows the thinnest fibers, but with the most mature cross-striation. Presence of the N-terminal also results in thinner fibers compared to the control and the N-terminal truncated variant but showed less maturation. Further, all three variants bind to three common sites on the collagen type I molecule, visualized by electron microscopy. An informative observation by the electron microscopy is the finding of full-length fibromodulin binding two collagen molecules at the same time. This shows engagement of the N-terminal and one of the binding sites in the LRR region, concomitantly binding different collagen molecules, which can be important for the assembly. Conclusion: This work shows that the tyrosine sulfate rich domain of fibromodulin contributes to the high affinity binding of the fibromodulin molecule to collagen. It influences and contributes to the arrangement of the collagen molecules, which results in well defined, highly organized collagen fibers.

Induced ablation of Bmp1 and Tll1 produces osteogenesis imperfecta in mice

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.

Reprogramming cellular phenotype by soft collagen gels.

A variety of cell types exhibit phenotype changes in response to the mechanical stiffness of the substrate. Many cells excluding neurons display an increase in the spread area, actin stress fiber formation and larger focal adhesion complexes as substrate stiffness increases in a sparsely populated culture. Cell proliferation is also known to directly correlate with these phenotype changes/changes in substrate stiffness. Augmented spreading and proliferation on stiffer substrates require nuclear transcriptional regulator YAP (Yes associated protein) localization in the cell nucleus and is tightly coupled to larger traction force generation. In this study, we show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrates (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented spreading and other hard substrate cytoskeleton architectures on soft collagen gels are not correlated with the cell proliferation pattern and do not require YAP localization in the cell nucleus. Finally, we examine the response of human colon carcinoma (HCT-8) cells on soft collagen gels. Recent studies show that human colon carcinoma (HCT-8) cells form multicellular clusters by 2-3 days when cultured on soft polyacrylamide (PA) gels with a wide range of stiffness (0.5-50 kPa) and coated with an extracellular matrix, ECM (collagen monomer/fibronectin). These clusters show limited spreading/wetting on PA gels, form 3D structures at the edges, and eventually display a remarkable, dissociative metastasis like phenotype (MLP), i.e., epithelial to rounded morphological transition after a week of culture on PA gels only, but not on collagen monomer coated stiff polystyrene/glass where they exhibit enhanced wetting and form confluent monolayers. Here, we show that HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels and eventually form confluent monolayers as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results suggest that cell-material interactions (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.

Lung Collagens Perpetuate Pulmonary Fibrosis via CD204 and M2 Macrophage Activation

Idiopathic pulmonary fibrosis is characterized by abundant collagen production and accumulation of alternatively activated macrophages (M2) in the lower respiratory tract. Mechanisms as to how alveolar macrophages are activated by collagen breakdown products are unknown. Alveolar macrophages were obtained by bronchoalveolar lavage from 30 patients with idiopathic pulmonary fibrosis (IPF) and 37 healthy donors (HD). Alveolar macrophages were cultured in the presence of collagen type I, III, IV and V monomers w/wo a neutralizing antibody against scavenger receptor I class A (CD204). Culture supernatants were assayed for the M2 markers CCL18, CCL2, and interleukin-1 receptor antagonist (IL-1ra) by ELISA. Furthermore, expression of phospho-Akt was measured using ELISA and expression of CD204 by RT-PCR and flow cytometry. Stimulation with collagen type I and III monomers significantly up-regulated CCL18, IL-1ra production of alveolar macrophages. Furthermore, expression of CCL2 and CD204 were up-regulated by collagen type I exposure. In addition, collagen type I stimulation increased pospho-Akt expression. Collagen type I effects were abrogated by neutralizing antiCD204 and a non-selective Phosphatidylinositide 3-kinase inhibitor (LY294002). Spontaneous CD204 expression of alveolar macrophages was significantly increased in patients with IPF. In conclusion, our findings demonstrate that monomeric collagen type I via CD204 induces phospho-Akt expression shifting alveolar macrophages to the profibrotic M2 type. Innate immune responses induced by collagen monomers might perpetuate pulmonary fibrosis.

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.

A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response

During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs.

Effects of various salts on structural polymorphism of reconstituted type I collagen fibrils

Even though the behavior of collagen monomers self-assembling into fibrils is commonly understood in terms of hydrophobic and electrostatic interactions, the mechanisms that drive their ordered, longitudinal alignment to form a characteristic periodicity are still unclear. By introducing various salts into the collagen fibrillogenesis system, the intermolecular interactions of fibril formation were studied. We found that the pH and ion species play a critical role in forming native fibrils. Turbidity and electron microscopy revealed that collagen self-assembled into fibrils with a native banding pattern in the presence of multivalent ions. The isoelectric point of collagen in 12mM of NaCl is pH 8.9; it shifted to pH 9.4 and pH 7.0 after adding 10mM CaCl2 and Na2SO4, respectively. Native fibrils were reconstituted at pH 7.4 in salts with divalent anions and at pH 9.0 in salts with divalent cations. Circular dichroism spectroscopy showed a loss of helicity in the conditions where fibrillogenesis was unable to be achieved. The multivalent ions not only change the surface charge of collagen, but also facilitate the formation of fibrils with the native D-periodic banding pattern. It is likely that the binding multivalent ions induce the like-charge attraction and facilitate monomers’ longitudinal registration to form fibrils with the native banding.

Thermal Memory in Self-Assembled Collagen Fibril Networks

Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability.

The aggregation behavior of native collagen in dilute solution studied by intrinsic fluorescence and external probing

The aggregation behavior of type I collagen in acid solutions with the concentrations covering a range of 0.06–1.50mg/mL was studied utilizing both of the fluorescence resonance energy transfer (FRET) between the phenylalanine and tyrosine residues and the external probing of 1,8-anilinonaphthalene sulfonate (ANS). FRET at 0.30mg/mL showed the distance among collagen monomers was within 10nm without the obvious aggregates formed. The predominance of tyrosine fluorescence in FRET in the range of 0.45–0.75mg/mL identified the existence of collagen aggregates companied with the formation of hydrophobic microdomains revealed by the change of the fluorescence of ANS. The blue-shift of tyrosine fluorescence from 303 to 293nm for 0.90–1.50mg/mL dedicated the formation of high order aggregates. The results from the two-phase diagrams of the intrinsic fluorescence for the guanidine hydrochloride-induced unfolding of collagen confirmed these conclusions. By the two-dimensional correlation analysis for the intrinsic fluorescence of collagen solutions of 0.45, 0.75 and 1.05mg/mL, the probable characteristic fluorescence peaks for the interactions of proline-aromatic (CH∼π) among the collagen molecules were found at 298 and 316nm.