Nanomechanical Mapping of Hydrated Rat Tail Tendon Collagen I Fibrils

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10⁵ nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10⁵ nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.     

Comparative study on the thermostability of collagen I of skin and bone: Influence of posttranslational hydroxylation of prolyl and lysyl residues

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and afterin vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circulardichroism measurements and limited trypsin digestion. Melting of fibrils from standardizedin vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (T _m ) at 43.3°C in 0.05% acetic acid. This is 1.9°C above control skin (T _m =41.4°C), most likely, due to a higher degree of prolyl hydroxylation—0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce theT _m slightly (1°C). Underhydroxylated bone collagen has aT _m which is 2°C below control. Melting temperatures ofin vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3°C, exceeds control bone by 1.4°C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4°C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.     

Nanomechanical Mapping of Hydrated Rat Tail Tendon Collagen I Fibrils

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10⁵ nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10⁵ nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.     

Collagen-related gene and protein expression changes in the lung in response to chronic hypoxia

Collagen accumulation likely contributes to increased vascular and airway impedance in hypoxia-induced pulmonary hypertension (HPH). Collagen exists in multiple subtypes and can accumulate via increased synthesis or decreased degradation. To better understand the individual contributions of fibrillar (FB) and basement membrane (BM) collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) to pulmonary vascular and airway remodeling in HPH, we investigated the temporal changes in gene and protein expression in the lungs of mice exposed to hypoxia for 0, 3, 6, 10 and 15 days. The earliest and largest change in gene expression was of type I FB collagen, which was significantly increased over control levels at 6, 10 and 15 days of hypoxia (p  <  0.05). Type III FB and type IV BM collagen were increased at 10 and 15 days of hypoxia (p <  0.05); MMP and TIMP gene expression levels were typically higher but sometimes lower than control levels at various time points. Collagen protein content was increased in whole lungs as early as 6 days of hypoxia and increased monotonically with longer exposures. However, neither qualitative nor semi-quantitative analysis of immunohistochemistry demonstrated accumulation of type I FB collagen in compartments of the lung other than large airways, suggesting that other collagen subtypes may be important contributors to collagen protein accumulation. These results provide insight into the patterns of gene and protein expression relevant to collagen accumulation in the lung in response to chronic hypoxia, through which we can develop a better understanding of the time course of changes in matrix biology and biomechanics that occur in HPH.     

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.     

FT-IR Microspectroscopy of Rat Ear Cartilage

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140–820 cm^-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of –SO₃^- groups at 1064 cm^-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of –SO₃^- groups (1236–1225 cm^-1) overlapped with that of amide III bands, it is not recommended for evaluation of the –SO₃^- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027–1016 cm^-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.     

High-affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a ¹²⁵I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. ¹²⁵I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.     

Fibrillogenesis in dense collagen solutions: a physicochemical study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength = 150 mM, 20 °C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH ∼ 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.     

Implications for Collagen Binding from the Crystallographic Structure of Fibronectin 6FnI1�C2FnII7FnI

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, ⁶FnI^1–2FnII^7–9FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains ^8–9FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains ⁶FnI^1–2FnII⁷FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains ²FnII and ⁷FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the ^8–9FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.     

Structural Analysis of Collagen Type I Interactions with Human Fibronectin Reveals a Cooperative Binding Mode

Despite its biological importance, the interaction between fibronectin (FN) and collagen, two abundant and crucial tissue components, has not been well characterized on a structural level. Here, we analyzed the four interactions formed between epitopes of collagen type I and the collagen-binding fragment (gelatin-binding domain (GBD)) of human FN using solution NMR, fluorescence, and small angle x-ray scattering methods. Collagen association with FN modules ^8–9FnI occurs through a conserved structural mechanism but exhibits a 400-fold disparity in affinity between collagen sites. This disparity is reduced in the full-length GBD, as ⁶FnI^1–2FnII⁷FnI binds a specific collagen epitope next to the weakest ^8–9FnI-binding site. The cooperative engagement of all GBD modules with collagen results in four broadly equipotent FN-collagen interaction sites. Collagen association stabilizes a distinct monomeric GBD conformation in solution, giving further evidence to the view that FN fragments form well defined functional and structural units.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

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.     

Fe2+-catalyzed non-enzymatic glycosylation alters collagen conformation during AGE-collagen formation in vitro

Advanced glycation end-products (AGEs) are one of the major factors of hyperglycemia related complications for diabetic patients. We studied the formation of AGEs in type I collagen after Fe²⁺-catalyzed non-enzymatic glycosylation in vitro. Type I collagen isolated from rat tail tendon was incubated with glucose and increasing concentrations of iron ions Fe²⁺. After 4 weeks incubation, cytotoxity of AGEs was indicated by the cytotoxity assay of primary human umbilical vein endothelial cells and primary human monocytes cultured with glycosylated collagen AGEs. Fourier transform infrared spectroscopy analysis revealed that structural changes of functional groups in glycosylated collagen are accelerated by the catalyst Fe²⁺. Using two-dimensional Fourier-transform infrared correlation spectroscopy analyses, for the first time, we demonstrated that the order of structural changes of these functional groups is -CH- > Amide I > Amide II > Amide III > ν(CO) the carboxylic group of Asn, Gln or polyproline amino acid residue in the course of AGE-collagen formation. Knowing the positions of these functional groups in collagen, this order of changes indicates that during glycation of collagen, the structure of the main chain residues in collagen changed first, and then the side chain changed gradually, which may lead to more carboxylic groups exposed to glucose for further formation of AGE-collagen irreversibly. The findings presented may support the design of new therapeutic strategies to prevent or slow down the Fe²⁺-catalyzed glycosylation of collagen and other matrix proteins.     

Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration through mimicry of the bone extracellular matrix in vivo. The aim of this study was to compare the collagen types I, II, and III with regard to their ability to bind the small leucine-rich proteoglycans (SLRPs) decorin and biglycan during fibrillogenesis in vitro in phosphate buffer. In addition, the influence of SLRPs on the proportion of collagen molecules incorporated into fibrils during fibrillogenesis in vitro at high and low ionic strength was investigated, as were their effects on the morphology of collagen fibrils and the speed of fibrillogenesis. Considerably more biglycan than decorin was bound by all three collagen types. Collagen II bound significantly more SLRPs in fibrils than collagen I and III. Decorin and biglycan decreased the proportion of collagen molecules of all three collagen types incorporated into fibrils in similar fashion. Biglycan affected neither fibril diameter nor the speed of fibrillogenesis. Decorin reduced the fibril diameter of all three collagen types. The differences in SLRP-binding ability between collagen types could be of significance when selecting collagen type and/or SLRPs as scaffold materials for tissue engineering or implant coatings.     

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII α1 chain was characterized as a collagenase sensitive band migrating at ~ 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.     

Formation and structure of scales in the Australian lungfish,neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodusforsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid‐trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear littleresemblance to typical bone. J.Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Fourier transform infrared and Raman‐based biochemical profiling of different grades of pure foetal‐type hepatoblastoma

The biomolecular events resulting from the progression of hepatoblastoma remain to be elucidated. Fourier‐transform infrared (FTIR) and Raman spectroscopies are capable of noninvasively and accurately capturing the biochemical properties of biological tissue from its pathological status. Our aim was to probe critial biomolecular changes of liver accompanying the progression of pure foetal hepatoblastoma (PFH) by FTIR and Raman spectroscopies. Herein, biochemical alterations were both evident in the FTIR spectra (regions of 3100‐2800 cm^?1 and 1800‐900 cm^?1) and the Raman spectra (region of 1800‐400 cm^?1) among normal, borderline and malignant liver tissues. Compared with normal tissues, the ratios of protein‐to‐lipid, α‐helix‐to‐β‐sheet, RNA‐to‐DNA, CH3 methyl‐to‐CH2 methylene, glucose‐to‐phospholipids, and unsaturated‐to‐saturated lipids intensities were significantly higher in malignant tissues, while the ratios of RNA‐to‐Amide II, DNA‐to‐Amide II, glycogen‐to‐cholesterol and Amide I‐to‐Amide II intensities were remarkably lower. These biochemical alterations in the transition from normal to malignant have profound implications not only for cyto‐pathological classification but also for molecular understanding of PFH progression. The successive changes of the spectral characteristics have been shown to be consistent with the development of PFH, indicating that FTIR and Raman spectroscopies are excellent tools to interrogate the biochemical features of different grades of PFH.      

Quantification of collagen organization in the peripheral human cornea at micron-scale resolution

The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1–1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.