Collagen Dispersion Research Articles

Automated Estimation of Collagen Fibre Dispersion in the Dermis and its Contribution to the Anisotropic Behaviour of Skin

Collagen fibres play an important role in the mechanical behaviour of many soft tissues. Modelling of such tissues now often incorporates a collagen fibre distribution. However, the availability of accurate structural data has so far lagged behind the progress of anisotropic constitutive modelling. Here, an automated process is developed to identify the orientation of collagen fibres using inexpensive and relatively simple techniques. The method uses established histological techniques and an algorithm implemented in the MATLAB image processing toolbox. It takes an average of 15 s to evaluate one image, compared to several hours if assessed visually. The technique was applied to histological sections of human skin with different Langer line orientations and a definite correlation between the orientation of Langer lines and the preferred orientation of collagen fibres in the dermis (p < 0.001, R(2) = 0.95) was observed. The structural parameters of the Gasser-Ogden-Holzapfel (GOH) model were all successfully evaluated. The mean dispersion factor for the dermis was κ = 0.1404±0.0028. The constitutive parameters μ, k(1) and k(2) were evaluated through physically-based, least squares curve-fitting of experimental test data. The values found for μ, k(1) and k(2) were 0.2014 MPa, 243.6 and 0.1327, respectively. Finally, the above model was implemented in ABAQUS/Standard and a finite element (FE) computation was performed of uniaxial extension tests on human skin. It is expected that the results of this study will assist those wishing to model skin, and that the algorithm described will be of benefit to those who wish to evaluate the collagen dispersion of other soft tissues.

Obstruction-Induced Pulmonary Vascular Remodeling

Pulmonary obstruction occurs in many common forms of congenital heart disease. In this study, pulmonary artery (PA) banding is used as a model for pulmonary stenosis. Significant remodeling of the vascular bed occurs as a result of a prolonged narrowing of the PAs, and here we quantify the biophysical and molecular changes proximal and distal to the obstruction. Main and branch PAs are harvested from banded and sham rabbits and their mechanical properties are assessed using a biaxial tensile tester. Measurements defined as initial and stiff slopes are taken, assuming a linear region at the start and end of the J-shaped stress-strain curves, along with a transitional knee point. Collagen, elastin assays, Movat's pentachrome staining, and Doppler protocols are used to quantify biochemical, structural, and physiological differences. The banded main PAs have significantly greater initial slopes while banded branch PAs have lower initial slopes; however, this change in mechanical behavior cannot be explained by the assay results as the elastin content in both main and branch PAs is not significantly different. The stiff slopes of the banded main PAs are higher, which is attributed to the significantly greater amounts of insoluble collagen. Shifting of the knee points reveals a decreased toe region in the main PAs but an opposite trend in the branch PAs. The histology results show a loss of integrity of the media, increase in ground substance, and dispersion of collagen in the banded tissue samples. This indicates other structural changes could have led to the mechanical differences in banded and normal tissue.

Formation of calcium deficient HAp/collagen composites by hydrolysis of α-TCP

Bone-like composites containing calcium deficient hydroxyapatite (CDHAp) were formed by the hydrolysis of alpha-tricalcium phosphate (alpha-TCP) in the presence of type I collagen. CDHAp-collagen composites were synthesized using two techniques. In one technique alpha-TCP was mixed with non-milled (as-received) collagen prior to the addition of the aqueous solution. In the second, the collagen was milled with alpha-TCP in heptane at room temperature prior to its conversion to CDHAp. The effect of milling strongly facilitates the formation of CDHAp at physiological temperature. The proportion of milled collagen between 5 and 20 wt% present in the alpha-TCP/collagen composites has no significant effect on the rate of CDHAp formation. Variations in pH and in calcium and phosphate concentrations were determined as a function of collagen processing and variations specific to the presence of collagen were discerned. Compared to CDHAp or to composites containing non-milled collagen, diametrical and compressive strengths of CDHAp increased in the presence of milled collagen. Lack of collagen dispersion and incomplete formation of CDHAp during 48 h were the bases for reduced strengths of composites containing non-milled collagen.

Collagen fibres by thermoplastic and wet spinning

Collagen threads of high yardage were manufactured by two different techniques being wet spinning of collagen dispersions and melt spinning of thermoplastic collagen. The fibres were characterised according to their structural, textile physical and biochemical properties. The wet spun fibres showed higher physical stability than the thermoplastic spun ones. Both fibre types were cross linked with different agents (formaldehyde, glutaraldehyde, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC)) to increase mechanical stability as well as to lower susceptibility against enzymatic attack. Fibres cross linked by 0.1% glutaraldehyde as well as non cross linked fibres showed no cytotoxic effect against mouse fibroblasts.

NHERF-1: Modulator of Glioblastoma Cell Migration and Invasion

The invasive nature of malignant gliomas is a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) is a multifunctional adaptor protein, recruiting cytoplasmic signaling proteins and membrane receptors/transporters into functional complexes. This study revealed that NHERF-1 expression is increased in highly invasive cells that reside in the rim of glioblastoma multiforme (GBM) tumors and that NHERF-1 sustains glioma migration and invasion. Gene expression profiles were evaluated from laser capture-microdissected human GBM cells isolated from patient tumor cores and corresponding invaded white matter regions. The role of NHERF-1 in the migration and dispersion of GBM cell lines was examined by reducing its expression with small-interfering RNA followed by radial migration, three-dimensional collagen dispersion, immunofluorescence, and survival assays. The in situ expression of NHERF-1 protein was restricted to glioma cells and the vascular endothelium, with minimal to no detection in adjacent normal brain tissue. Depletion of NHERF-1 arrested migration and dispersion of glioma cell lines and caused an increase in cell-cell cohesiveness. Glioblastoma multiforme cells with depleted NHERF-1 evidenced a marked decrease in stress fibers, a larger cell size, and a more rounded shape with fewer cellular processes. When NHERF-1 expression was reduced, glioma cells became sensitized to temozolomide treatment resulting in increased apoptosis. Taken together, these results provide the first evidence for NHERF-1 as a participant in the highly invasive phenotype of malignant gliomas and implicate NHERF-1 as a possible therapeutic target for treatment of GBM.

Aqueous dispersions of gelled collagen particles. Part 2: numerical technique for tribological properties

PurposeTo propose a new numerical elastohydrodynamic (EHD) analysis method suited to cinematic pairs lubricated with aqueous dispersions of collagen gelled media.Design/methodology/approachIn comparison with traditional lubricants, these media are characterized by an apparent viscosity that increases with film thickness. Hence, the Reynolds equation has been numerically solved taking into account the new rheological law. The apparent viscosity of 4 percent collagen gel dispersions, as measured at a fixed shear rate of 100 l/s, is in the range 2‐5 Pa s, when film thickness varies between 20 and 200 μm. These experimental data have been fitted using a power law. The proposed analysis method has been applied to a 180° partial bearing, which rigid journal rotates in a flexible sleeve, made up by a resin shell.FindingsThe results of this tribological analysis have been compared with those concerning traditional isoviscous fluids which viscosity has been set equal to the viscosity of the gelled media when the film thickness is constant, i.e. in the unloaded bearing. The minimum film thickness calculated has been turned out equal to 66.8 μm in the former case, and 27.6 μm in the latter case. Furthermore, when the cinematic pair is lubricated with the aqueous gel media, film thickness distribution over the bearing surface is more uniform, active film region is wider and, consequently, peak pressure is lower than when the isoviscous lubricant is employed.Research limitations/implicationsFor lightly loaded journal bearings, the dependence between apparent viscosity and the shear rate in the lubricant film has been neglected, so that the apparent viscosity has been considered changing only with the film thickness.Originality/valueThe paper introduces a new numerical EHD analysis method for use with cinematic pairs lubricated with aqueous dispersions of collagen gelled media.

Aqueous dispersions of gelled collagen particles. Part 1: experimental characterization of stiffness and rheological properties

PurposeTo study the rheological behavior of aqueous dispersions of collagen gelled media and to bring it into relation with the gel stiffness and microstructure.Design/methodology/approachCollagen gelled media were prepared at different concentrations and their stiffness was measured with dynamic elastic modulus (DEM) technique. The gelled media were reduced into dispersions of micelles and gel liquid phase and the apparent viscosity of these systems was investigated as a function of the gap thickness, the shear rate and the collagen concentration. The gap thickness was varied between 20 and 200 μm. The microstructure of the broken gelled media was finally explored with optical microscopy.FindingsDEM analysis has given values of the collagen gel stiffness ranging between 3 × 10−4 and 3.3 × 10−3 MPa. It has been shown that apparent viscosity grows with the increase of the gap thickness from 2 to 5 Pa s at constant collagen concentration and applied shear rate (4 percent, 100 s−1). A rise in the collagen concentration has produced an increase in the apparent viscosity of gelled media dispersions. Nevertheless, the phenomenological law does not obey previously proposed equations but follows a sigmoid modal. Shear‐thinning phenomenon has been observed when the shear rate is varied between 10 and 2,000 s−1.Research limitations/implicationsThe gel breaking process, which may cause changes in the microstructure of the micelles and hence in the rheological behavior of the dispersions, should be characterized measuring the forces acting on the samples during the rupture.Originality/valueThe paper provides useful information on the rheological behavior of aqueous dispersions of collagen gelled media.

Collagen Scaffolds Reinforced with Biomimetic Composite Nano-Sized Carbonate-Substituted Hydroxyapatite Crystals and Shaped by Rapid Prototyping to Contain Internal Microchannels

The next generation of tissue engineering scaffolds will be made to accommodate blood vessels and nutrient channels to support cell survival deep in the interior of the scaffolds. To this end, we have developed a method that incorporates microchannels to permit the flow of nutrient-rich media through collagen-based scaffolds. The scaffold matrix comprises nano-sized carbonate-substituted hydroxyapatite (HA) crystals internally precipitated in collagen fibers. The scaffold therefore mimics many of the features found in bone. A biomimetic precipitation technique is used whereby a collagen membrane separates reservoirs of calcium and phosphate solutions. The collision of calcium and phosphate ions diffusing from opposite directions results in the precipitation of mineral within the collagen membrane. Transmission electron microscopy analysis showed the dimension of the mineral crystals to be approximately 180 x 80 x 20 nm, indicating that the crystals reside in the intermicrofibril gaps. Electron diffraction indicated that the mineral was in the HA phase, and infrared spectroscopy confirmed type A carbonate substitution. The collagen-HA membrane is then used to make 3-dimensional (3D) scaffolds: the membrane is shredded and mixed in an aqueous-based collagen dispersion and processed using the critical point drying method. Adjusting the pH of the dispersion to 5.0 before mixing the composite component preserved the nano-sized carbonate-substituted HA crystals. Branching and interconnecting microchannels in the interior of the scaffolds are made with a sacrificial mold manufactured by using a 3D wax printer. The 3D wax printer has been modified to print the mold from biocompatible materials. Appropriately sized microchannels within collagen-HA scaffolds brings us closer to fulfilling the mass transport requirements for osteogenic cells living deep within the scaffold.

Microtensile testing of collagen fibril for cardiovascular tissue engineering

As a candidate scaffold material for cardiac tissue engineering, Type I collagen material shows different mechanical properties due to the differences in the structural features such as orientation of fibril alignment and the environmental factors such as ultraviolet (UV) irradiation. In this article one-dimensional tensile stress tests were performed with the use of a nanoscale tensile tester on four groups of Type I collagen samples that were differentiated in terms of fibril orientation and/or UV irradiation. The collagen surface structure was studied with the use of an atomic force microscope (AFM). Effects of fibril orientation and UV irradiation on their mechanical properties were investigated. The mechanical properties and deformation mechanisms are discussed in conjunction with collagen structure, fibril orientation, and dispersion of the generated crosslinking by UV irradiation.

Changes in mechanical properties and cellularity during long‐term culture of collagen fiber ACL reconstruction scaffolds

Resorbable scaffolds for anterior cruciate ligament (ACL) reconstruction should provide temporary mechanical function then gradually breakdown while promoting matrix synthesis by local cells. Crosslinking influences collagen's mechanical properties, degradation rate, and interactions with cells. Our objective was to compare the effects of different crosslinkers on cellularity and mechanical properties during long-term (8 week) culture of collagen fiber scaffolds. Fibers were fabricated from an acid-insoluble dispersion of bovine dermal collagen and crosslinked with either ultraviolet irradiation (UV; a physical crosslinker) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; a chemical crosslinker). Scaffolds consisted of 50 fibers bundled in parallel. Initial attachment of fibroblasts was similar on both scaffolds; however, from 1 to 8 weeks in culture, UV-crosslinked scaffolds had significantly more cells attached than EDC-crosslinked scaffolds. The initial breaking load (3.50 N) and stiffness (2.23 N/mm) of EDC-crosslinked scaffolds were significantly greater than those of UV-crosslinked scaffolds (2.32 N; 1.21 N/mm) and were unaffected by long-term fibroblast culture. In contrast, the load-bearing capacity of fibroblast-seeded UV-crosslinked scaffolds decreased 60% to 0.91 N after 8 weeks in culture. EDC-crosslinked scaffolds maintained strength and moderate cellularity; UV-crosslinked scaffolds, in contrast, were highly cellular, but had poor mechanical properties that decreased during culture. These in vitro results suggest that collagen fiber scaffolds crosslinked with EDC may be more suitable for ACL reconstruction than those crosslinked with UV.

Collagen/PLGA Microparticle Composites for Local Controlled Delivery of Gentamicin

To preserve the positive effect of collagen on tissue regeneration and to locally deliver low molecular weight compounds for an extended time period, a composite for parenteral application was devised based on a collagen sponge with gentamicin‐loaded PLGA microparticles incorporated. Antibiotic liberation from the particles was sustained over 1 week by blending two PLGA polymers. Homogenous incorporation of the microspheres in the porous carrier could be realized by lyophilization of a particle suspension in the aqueous collagen preparation. Particle loss upon incubation was reduced with higher collagen concentration enabling local particle retention after application. Lower freezing rate and longer exposure of the PLGA microparticles to the acidic collagen dispersion at temperatures below the glass transition temperature resulted in an increase of the gentamicin burst. The final implant containing both nonencapsulated gentamicin and an equivalent amount incorporated in the microparticles reflected the microbiological demands and exhibited liberation of a high gentamicin dose initially and subsequently extended antibiotic liberation for about one week. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2145–2151, 2003

Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication

Novel collagen scaffolds possessing predefined and reproducible internal channels with widths of 135 μm and greater have been produced. The process employed to make the collagen scaffold utilises a sacrificial mould, manufactured using solid freeform fabrication technology, and critical point drying technique. A computer aided design (CAD) file of the mould to be produced is created. This mould is manufactured using a phase change ink-jet printer. A dispersion of collagen is then cast into the mould and frozen. The mould is dissolved away with ethanol and the collagen scaffold is then critical point dried with liquid carbon dioxide. The effect of processing on the tertiary structure of collagen is assessed by monitoring the wavenumber of the N–H stretching vibration peak using Fourier transform infra-red spectroscopy and it is found that processing does not denature the collagen. Ultraviolet-visual spectroscopy was used to detect the presence of any contamination from the sacrificial mould on the collagen. The ability to use computer aided design and manufacture (CAD/CAM) provides a route to optimise scaffold designs using collagen in tissue engineering applications.

Characterisation of Collagen Scaffolds using X-ray Microtomography

ABSTRACTCollagen scaffolds have been produced that incorporate predefined internal channels. The scaffolds were obtained with the aid of sacrificial moulds that have been manufactured using a rapid prototyping technique. A computer aided design file of the mould was created and then realised using an ink-jet printer. A dispersion of collagen was then cast into the mould and frozen. Ethanol was used to dissolve the mould leaving the collagen, which was then freeze dried to produce the final product. The scaffold was then analysed using X-ray microtomography (XMT) to determine whether the desired internal structure was obtained. It was found necessary to add saturated potassium iodide (KI) solution to the scaffold in order to analyse it satisfactorily by XMT. The resultant images indicate that the desired internal structure was obtained.

Marine sponge collagen: isolation, characterization and effects on the skin parameters surface-pH, moisture and sebum

A previously described isolation procedure for collagen of the marine sponge Chondrosia reniformis Nardo was modified for scaling-up reasons yielding 30% of collagen (freeze-dried collagen in relation to freeze-dried sponge). Light microscope observations showed fibrous structures. Transmission electron microscopy studies proved the collagenous nature of this material: high magnifications showed the typical periodic banding-pattern of collagen fibres. However, the results of the amino acid analysis differed from most publications, presumably due to impurities that still were present. In agreement with earlier studies, sponge collagen was insoluble in dilute acid mediums and all solvents investigated. Dispersion of collagen was facilitated when dilute basic mediums were employed. The acid–base properties of the material were investigated by titration. Furthermore, a sponge extract was incorporated in two different formulations and compared with their extract-free analogues and a commercially available collagen containing product with respect to their effects on biophysical skin parameters. None of the preparations had a noticeable influence on the physiological skin surface pH. Skin hydration increased only slightly. However, all tested formulations showed a significant increase of lipids measured by sebumetry.

Effects of processing conditions on the rheological behavior of collagen dispersions

Biomedical collagen preparations are mainly based on liquid aqueous preparations either used directly as injectables or transferred for example into solid implants or porous devices. In all cases the viscosity of the dispersion or solution has to be controlled or adjusted by pH, temperature, collagen concentration or crosslinking. We tested these effects on the rheological and structural properties of collagen fiber dispersions focusing on oscillatory rheometry. With increasing difference between pH and pI viscosity increased and went through a maximum with less rigidity of the fibers. The dispersions acted like a predominantly permanently linked network. This character changed to only partially linked at low collagen concentration due to isolation of the individual fibers. Up to 40°C, temperature is a tool suitable for adjusting viscosity without changes in the network structure. At 50°C, fourier transform – infrared spectroscopy (FT–IR) spectroscopy indicated the transition of the helical into random coil structure. Renaturation of the helices was found upon cooling but atomic force microscopy (AFM) indicated severe temperature induced damages of the fibers. Crosslinking with glutaraldehyde (GTA) leads to an increase in viscosity and the effect on the network structure depends on the processing conditions.

Anatomy and physiology of the uterine cervix.

The human uterine cervix undergoes extensive changes during pregnancy. Collagen is reorganized and consolidated early in gestation with proliferation and hyperplasia of the cellular component. As term approaches, multiple factors work together in complex interactions that cause collagen dispersion and the cervix to ripen (clinically become softer). Increases in decorin levels, hyaluronic acid, and physiologic cell death are in part responsible for this remodeling process. As the collagen bundles disperse and lose strength, cytokines, hyaluronic acid, collagenases, and elastase possibly work together to allow effacement. Then, the mechanical forces of uterine contractions extend the elastin and allow dilatation. During dilation, levels of cytokines and hyaluronic acid begin to decrease, which may serve to decrease collagenolytic activity and allow the cervix to begin the process of repairing itself. Despite this advance knowledge of cervical ripening, the signals responsible for the initiation of these changes remain to be elucidated. If we can understand the exact mechanisms that affect these changes, then we may be better able to address such complex issues as cervical incompetence, preterm delivery, postterm delivery, and proper "ripening" of the cervix to avoid surgical delivery for arrest disorders of the active phase.

Cryotropic structuring of aqueous dispersions of fibrous collagen: influence of the initial pH values

The formation of macroporous anisotropic non-covalent cryogels stabilised by H-bonds and hydrophobic interactions as a result of the freezing–thawing of aqueous dispersions of fibrous collagen (protein concentration >4%) possessing pH values of 1.5–4.6 or 10–11.9 was observed. Collagen cryogels formed at pH 12–12.5 were additionally stabilised by covalent links. It was shown that the stability of cryogels in aqueous medium was determined by the changes in supramolecular packing of collagen during freezing–thawing. As this took place the swelling extent of the dispersed particles must be no more than 5–6 g of bound solvent per gram of protein. It was established that for the formation of spongy structures of cryogels the portion of solvent bound by protein should not exceed two thirds of the total amount of solvent in the system and be equal to 0.35–0.65 by weight. One may control the degree of solvent binding in the dispersed system by varying the pH value and ionic composition of the solvent.

Autologous collagen dispersion (Autologen) as a dermal filler: clinical observations and histologic findings.

To assess the histologic behavior and clinical efficacy of autologous collagen dispersion (Autologen) in augmenting human dermis. Adult patients of the Facial Plastic Surgery Clinic at The New York Eye and Ear Infirmary who were undergoing facial aesthetic surgery with skin excision. Five patients were injected intradermally with Autologen in one postauricular area and bovine cross-linked collagen (Zyplast) on the contralateral side. Patients were examined clinically for signs of infection, skin necrosis, or implant rejection/allergy 2, 4, and 12 weeks postinjection. Impressions and photographs of all implant sites were taken at all follow-up visits. Biopsy specimens of each implant were taken 4 and 12 weeks after injection and examined histologically for signs of integration, rejection, and resorption. All implants were well tolerated. No identifiable differences were noted in the clinical persistence of Zyplast vs Autologen. Histologically, there was more variability in the degree of fibroblast infiltration of Autologen vs Zyplast deposits. Our trial suggests that autologous collagen dispersion may represent a viable alternative to bovine collagen. Clinical persistence and histologic behavior of Autologen appear to be at least as favorable as those of Zyplast, and Autologen obviates the need for allergy testing and eliminates the possibility of disease transmission. Arch Facial Plast Surg. 2000;2:48-52

Parameters affecting the isolation of collagen from squid ( Illex argentinus) skins

The solubility of collagen from squid ( Illex argentinus) skin in salt solutions and the efficiency of removal of skin chromatophores were determined. Homogenization of minced squid skin in 5–15% NaCl solution at 0°C solubilized 35–24% of total amount of crude protein and caused 2–5% loss of collagen but was not effective in removing pigments from the skin. The treatment of whole squid skins in 5–15% NaCl solutions at room temperature led to separation of the chromatophores, but the loss of soluble collagen was 57–16%. Collagen, soluble in dilute acid solutions was isolated from squid skins by 24 h soaking in 10% NaCl solution at room temperature, washing with water and bleaching for 48 h in 1% H 2O 2 in 0.01 M NaOH. The yield of collagen was 53%. It could be increased to 90% by using NaOH solution at pH 11.5 instead of 10% NaCl but the isolate was less soluble in dilute acid and the viscosity of 0.5% dispersion of collagen was four times lower. The rancid off-odour could be prevented by adding 0.5% of a non-ionic detergent to all solution used in the procedure. ©

Influence of the preliminary swelling of the dispersions of fibrous collagen on their ability of non-covalent gel-formation

It was established that as a result of the maximum swelling (HCl, pH 3) the dispersed particles of fibrous collagen isolated from limed bovine derma irreversibly change their morphology. This results in changing the nature of supramolecular packing of protein - the transition of swollen bunches of fibrils into separate fibrils. Using method of adsorption of hydrophobic probe sodium dodecylsulphate it was found that under these conditions the significant increase in number of non-polar protein groups, able to participate in intermolecular interactions, took place. Subsequent lowering of the swelling capacity of the dispersed particles (from 19 to 2.2-2.5 g of tightly bound solvent per I g of dry collagen) resulted in the formation ofthe elastic macroisotropic gels. Hydrophobic nature of knots of gel network was confirmed by their stability in water until 62°C and destruction in concentrated solutions of urea and guanidine hydrochloride.