Low Molecular Weight Collagen Research Articles

Study of chemical properties and evaluation of collagen in mantle, epidermal connective tissue and tentacle of Indian Squid, Loligo duvauceli Orbigny.

The chemical composition and evaluation of Indian squid (Loligo duvauceli) mantle, epidermal connective tissue and tentacle is investigated in this current study. It is observed that squid mantle contains 22.2% total protein; 63.5% of the total protein is myofibrillar protein. The unique property of squid myofibrillar protein is its water solubility. Squid mantle contains 12.0% total collagen. Epidermal connective tissue has highest amounts of total collagen (17.8%). SDS-PAGE of total collagen identified high molecular weight α-, β- and γ- sub-chains. Amino acid profile analysis indicates that mantle and tentacle contain essential amino acids. Arginine forms a major portion of mantle collagen (272.5g/100g N). Isoleucine, glutamic acid and lysine are other amino acids that are found in significantly high amounts in the mantle. Sulphur containing cystine is deficit in mantle collagen. Papain digest of mantle and epidermal connective tissue is rich in uronic acid, while papain digest, collagenase digest and urea digest of epidermal connective tissue has significant amounts of sialic acid (25.2, 33.2 and 99.8μmol /100g, respectively). PAS staining of papain digest, collagenase digest and urea digest also identify the association of hexoses with low molecular weight collagen fragments. Histochemical sectioning also emphasized the localized distribution of collagen in epidermal and dermal region and very sparse fibres traverse the myotome bundles.

Different Molecular Weight Chitosan-Based Membranes for Tissue Regeneration.

Natural polymers, such as chitosan, obtained from chitin, are been widely studied for use in the tissue regeneration field. This study established a protocol to attain membranes made from this biopolymer, consisting of high or low molecular weight chitosan. The biocompatibility of these membranes was histologically evaluated, comparing them to collagen membrane surgically implanted in rat subcutaneous tissue. Fifteen Holtzmann rats were divided in three experimental groups: High and Low Molecular Weight Chitosan membranes (HMWC and LMWC) and Collagen membranes (C—control group); each of them with three experimental periods: 7, 15 and 30 days. As a result, after the seven days evaluation, the membranes were present and associated with a variable degree of inflammation, and after the 15 and 30 days evaluations, the membranes were absent in all groups. It is concluded that the chitosan-based membranes were successfully attained and presented comparable resorption times to collagen membranes.

Absorption and Effectiveness of Orally Administered Low Molecular Weight Collagen Hydrolysate in Rats

Collagen, a major extracellular matrix macromolecule, is widely used for biomedical purposes. We investigated the absorption mechanism of low molecular weight collagen hydrolysate (LMW-CH) and its effects on osteoporosis in rats. When administered to Wistar rats with either [(14)C]proline (Pro group) or glycyl-[(14)C]prolyl-hydroxyproline (CTp group), LMW-CH rapidly increased plasma radioactivity. LMW-CH was absorbed into the blood of Wistar rats in the peptide form. Glycyl-prolyl-hydroxyproline tripeptide remained in the plasma and accumulated in the kidney. In both groups, radioactivity was retained at a high level in the skin until 14 days after administration. Additionally, the administration of LMW-CH to ovariectomized stroke-prone spontaneously hypertensive rats increased the organic substance content and decreased the water content of the left femur. Our findings show that LMW-CH exerts a beneficial effect on osteoporosis by increasing the organic substance content of bone.

AMS 14C Dating of Human Bones Using Sequential Pyrolysis and Combustion of Collagen

The Radiocarbon Dating Laboratory at the University of Illinois has been using the pyrolysis-combustion technique to separate pyrolysis-volatile (Py-V) or low molecular weight and pyrolysis-residue (Py-R) or high molecular weight compounds for 14C dating of organic remains since 2003. We have applied this method to human collagen dating to examine the 14C age difference between low and high molecular weight organic compounds. Results show that both fractions of late prehistoric period human bones from Illinois archaeological sites yield identical 14C dates but that Py-V or low molecular weight fractions of Archaic period human bones appear to be slightly contaminated. In this case, Py-V components or low molecular weight collagen fraction yield older 14C dates, which could result from contamination from old organic-rich sediments. The pyrolysis-combustion technique provides an economical alternative method to date bones that have not been satisfactorily dated using conventional purification techniques.

Ferrous Ferric Chloride Stimulates the Proliferation of Human Skin Keratinocytes, Melanocytes, and Fibroblasts in Culture

Ferrous ferric chloride (FFC®) is a distinct form of aqueous iron made up of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions. Although FFC is known to stimulate cellular function, its precise mechanism of action is largely unknown. My previous study showed that FFC stimulated the proliferation of cultured epidermal keratinocytes and melanocytes derived from newborn mice, suggesting that FFC stimulates their function of mice. However, it is not known whether FFC stimulates the proliferation of keratinocytes and melanocytes derived from human skin. Moreover, it is unknown whether FFC stimulates the proliferation of human skin fibroblasts. Pairogen® (Akatsuka Co., Tsu, Japan) is a drink comprising FFC, vinegars, and vitamins, Pairogen Gold® (Akatsuka) contains herbal medicines in addition to FFC, vinegars, and vitamins, while Noncalorie Pairogen® (Akatsuka) contains low molecular weight collagen and hyaluronic acid in addition to FFC, vinegars and vitamins. To clarify whether FFC in addition to the natural factors included in these drinks stimulates the proliferation of human skin cells, FFC and these different drinks were added to culture media and tested for their proliferation-stimulating activity on skin cells. FFC, Pairogen, and Pairogen Gold equally doubled the growth rate of cultured human keratinocytes, melanocytes, and fibroblasts. Non-calorie Pairogen tripled the growth rate of human fibroblasts, but Non-calorie Pairogen only doubled the growth rate of human keratinocytes and melanocytes. FFC plus low molecular weight collagen tripled the growth rate of human fibroblasts, whereas FFC plus low molecular weight hyaluronic acid only doubled the growth rate of human fibroblasts. Low molecular weight collagen, with or without hyaluronic acid failed to stimulate the proliferation of fibroblasts. These results suggest that FFC stimulates the proliferation of human skin keratinocytes and melanocytes. Moreover, FFC markedly stimulates the proliferation of human skin fibroblasts synergistically with low molecular weight collagen.

Radiation-induced crosslinking of collagen gelatin into a stable hydrogel

Gelatin, the low molecular weight collagen derivative from porcine skin was transformed into a stable permanent hydrogel by γ-radiation. A series of samples with 3% gelatin solution in water were irradiated at doses of 12, 25, 50, 100, 150, 200 kGy at room temperature in the absence of air with a dose rate of 2.2 kGy/h. At low dose gelatin hydrogels incorporating all the available water were obtained. At higher doses above 50 kGy, the gelatin hydrogel samples show a curious shrinking phenomenon due to the relatively high crosslinking density level achieved, so part of the available water is squeezed out from the gel cage. The gelatin hydrogel samples were studied by mass fractionation analysis, by spectrophotometric and polarimetric analysis. Further characterization was made by FT-IR spectroscopy and by thermal analysis (DSC, DTA and TGA) of the dried gelatin samples after irradiation in comparison to a reference untreated sample.

Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma

Background: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. Methods: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. Results: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. Conclusion: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

Regulation of neutrophil functions during inflammation by low molecular weight collagen peptides

Regulation of neutrophil functions during inflammation by low molecular weight collagen peptides

Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides

Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides

Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides

Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides

Regulation of neutrophil functions during inflammation by low molecular weight collagen peptides

Regulation of neutrophil functions during inflammation by low molecular weight collagen peptides

Gene structure for a novel human low molecular weight collagen : L. Tikka a,b, T. Pihlajaniemi c, D.J. Prockop b and K. Tryggvason a. Departments of aBiochemistry and cMedical Biochemistry, University of Oulu, Oulu, Finland, bJefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, USA

Gene structure for a novel human low molecular weight collagen : L. Tikka a,b, T. Pihlajaniemi c, D.J. Prockop b and K. Tryggvason a. Departments of aBiochemistry and cMedical Biochemistry, University of Oulu, Oulu, Finland, bJefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, USA

Extracellular matrix-induced synthesis of a low molecular weight collagen by fetal calf ligament fibroblasts.

Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J. Cell Biol. 97:1933-1938). Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specifically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham, R.P., Madaras, J.G., and Senior, R.M., 1984. J. Cell Biol. 98:1804-1812). To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 85 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes. FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control. Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM. For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50%. FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively. This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen). As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%). The presence of the ligament ECM also altered the levels and distribution of secreted proteins between the culture medium and the cell layer. These studies provide evidence for differential expression of the novel collagen FCL-1 by FCL fibroblasts during development and suggest that such expression is affected, at least in part, by interaction of the cell with a ligament ECM.

New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament.

Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight fibroblast collagen: structure, secretion, and differential expression as a function of fetal and cellular age.

A unique low molecular weight collagen that was highly resistant to proteolytic degradation was originally isolated from fetal calf ligamentum nuchae fibroblasts and hence termed FCL-1 [Sage, H., Mecham, R., Johnson, C., & Bornstein, P. (1983) J. Cell Biol. 97, 1933-1938]. The differential expression of this protein was studied as a function both of fetal (donor) age and of subcultivation in vitro. Concomitant isolation, subculture, and metabolic radiolabeling experiments performed on cell strains from fetal calf ligament (FCL) and fetal bovine skin (FBS) representing different gestational ages (85-270 days in utero) showed that (a) FCL-1 was synthesized preferentially by fibroblasts from younger animals and (b) expression of FCL-1 diminished as a function of increased passage in culture. Levels of FCL-1, measured as percent of total radiolabeled culture medium protein that precipitated in a concentration range of 20-50% ammonium sulfate, ranged from 22% in FCL 85 cells to 7.7% in FCL 270 (term) cells. FBS fibroblasts at passages 6-10 secreted from 13% to 6% FCL-1, respectively. When cells from an 85-day fetal ligament were allowed to accumulate copious extracellular matrix in vitro, the production of FCL-1 was increased to 32%. FCL-1 was not immunoreactive with polyclonal antibodies directed toward most of the sequences of the interstitial type I and type III procollagens. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of FCL-1 was 13 000 (on the basis of collagen peptide standards) and approximately 30 000 (on the basis of globular protein standards). Incubation with bacterial collagenase produced a stable cleavage product of Mr 8000 (by collagen standards) or 17 000 (by globular standards). In contrast, pepsin removed a small peptide of approximately 1000-2000 in molecular weight from FCL-1, and a gradual but progressive proteolysis of the collagen was observed over a period of 1-6 h. Pulse-chase studies revealed a secretion time of approximately 60 min for FCL-1, without the appearance of any processed, intermediate forms. These studies confirm that FCL-1 represents a novel member of the collagen gene family that manifests differential expression as a function of development.

Synthesis of a low molecular weight collagen by chondrocytes from the presumptive calcification region of the embryonic chick sterna: the influence of culture with collagen gels.

The mature chick sternum is divisible almost equally into cephalic calcified and caudal cartilagenous regions. Isolation and culture of cells derived from embryonic precursors of these regions has revealed two discrete populations of cells with distinct morphological features and synthetic capabilities. Both cell populations grew well in culture within or upon collagen gels or upon plastic and maintained morphologies similar to those observed in the parent tissue. Polyacrylamide gel electrophoresis of radiolabeled proteins synthesized by the cells in culture demonstrated large differences in the types of collagens synthesized. Both chondrocyte populations synthesized type II and minor cartilage collagens but only chondrocytes isolated from the presumptive calcification region synthesized the previously identified, low molecular weight collagen, termed G collagen. Synthesis of G collagen was stimulated by culture within or upon collagen gels such that it represented an average of 65% of the total collagen synthesized by presumptive calcification region chondrocytes after 7 d of culture within collagen gels. Light and scanning electron microscopy demonstrated that the two chondrocyte types exhibited distinct morphological features and accumulated different extracellular matrices in culture.

The low molecular weight collagen synthesized by chick tibial chondrocytes is deposited in the extracellular matrix both in culture and in vivo.

The low mol. wt. collagen (64 K) synthesized by chick embryo chondrocytes in culture is deposited in the extracellular matrix; its deposition is strictly dependent upon a correct hydroxylation. In vivo the 64 K collagen has been isolated from the cartilage of tibiae obtained from 17-day-old chick embryos. The turnover of this collagen in the extracellular matrix is very rapid: within a few hours it is matured into a 30-K fragment released in the medium. Also this maturation is dependent upon a correct hydroxylation of the molecule. The underhydroxylated form, synthesized in the absence of ascorbic acid or in the presence of alpha-alpha' dipyridyl, is not deposited in the extracellular matrix and is directly secreted as 64 K collagen in the culture medium.

Biosynthesis and processing of collagens in different cartilaginous tissues.

The distribution, structure, and biosynthesis of various collagen types have been studied in growth and structural cartilage from young rabbits. The major collagen of cartilage is alpha 1(II); however, all cartilage matrix also contains 1 alpha, 2 alpha, 3 alpha (Type Cm), as well as a high molecular weight disulfide-linked collagen (Type M). Cartilage fragments in organ culture demonstrate synthesis of precursors of collagen alpha chains and processing to their final forms. Although Type Cm collagen is present in the same proportion in the matrix of growth and structural cartilage, in vitro radiolabeling of rabbit cartilage showed that only growth cartilage is capable of actively synthesizing Type Cm, except in the newborn period when synthesis of Type Cm does occur in structural cartilage. A low molecular weight collagen (designated G collagen) is synthesized in vitro by growth cartilage but not by structural or articular cartilage. Preferential distribution of these minor collagens in growth cartilage suggests a role in development during normal cartilage growth.

Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation.

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.

Biosynthesis of a Low Molecular Weight Collagen by Rabbit Growth Plate Cartilage Organ Cultures

Analysis of [14C]-proline labeled proteins synthesized by organ cultures of rabbit growth plate cartilage slices revealed that this tissue synthesized a low molecular weight collagen in addition to Type II, 1α, 2α, and 3α collagens. This collagen appears to be a distinct molecular species on the basis of electrophoretic mobility, hydroxyproline content and cyanogen bromide peptide mapping. In subsequent studies, a non-disulfide-bonded, higher molecular weight form of the same molecule was identified.