Kinds Of Collagen Research Articles

Comparison of collagens extracted from swim bladder and bovine Achilles tendon

Collagen is a type of natural biopolymer material, which is widely used in tissue engineering and medicine owing to its exceptional properties such as biodegradability, biocompatibility, hemostatic properties, and low immunogenicity. Collagens from different sources can differ in type, structure, and function. In this study, collagen was extracted from swim bladder and bovine Achilles tendon by acid-enzyme binding method at low temperature. UV spectrum, Fourier transform infrared spectrum, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, scanning electron microscope, and differential scanning calorimetry were used to characterize these two collagens. The blood compatibility and cytotoxicity of the two kinds of collagen were studied.The results showed that the collagens from the two sources belong to the characteristics of type I collagen and had biological safety. Their differences in structure and thermal stability can provide a theoretical basis for the selection of collagen in practical application.

Determination of 14 metal elements in collagen by inductively coupled plasma mass spectrometry with microwave digestion

Objective To propose an analysis method for determining 14 kinds of metal elements in collagen, including Mn, Cu, Zn, Al, Ti, Pb, Ba, Cd, Co, Cr, Ni, Fe, As and Hg. Method The conditions of collagen microwave digestion, inductively coupled plasma mass spectrometry (ICP-MS) working conditions and the modes used in the determination of various metal elements in ICP-MS were optimized. The content of 14 metal elements in 9 kinds of collagens with different specifications was determined by microwave digestion/ICP-MS, and the tagged recovery verification and method accuracy determination were conducted. Results The detection limit of the instrument was 0.1~5.0 μg/L. The detection limit of the proposed method was 0.001 25~0.625 mg/kg. The relative standard deviation (n=11) was not more than 4.0%, and the recovery rate of the sample was 87.7%~107.4%. Under the same conditions, 14 metal elements in the pig liver standard were measured and the measured values were within the standard range. Conclusions The microwave digestion/ICP-MS method possesses the advantages of rapidness, simplicity, high sensitivity, good stability and high accuracy. It is worthy of reference and promotion. Key words: Microwave digestion; Inductively coupled plasma mass spectrometry; Collagen; Metal elements

The degradation and local tissue effects of collagen hydrogel and sponge implants in muscle

Collagen products will degrade and evoke certain local tissue reaction after being implanted. ISO 10993.6 is the international standard to evaluate local tissue reaction for biomaterials. However, even in the present version, it does not define the specific view fields for scoring local tissue reaction in the sections, which may influence the fair evaluation for the degradable polymer, such as collagen. In this study, 4 kinds of collagens and 2 kinds of bio-inert products were implanted into the rabbit muscle for 26 weeks, and two different scoring methods (uniform distribution scoring method and area locating scoring method) were applied to evaluate the local tissue reaction for each implant. The results showed that the tissue reaction on the interface was active, but the various scores in the implant and surrounding areas could help us to understand the differences of the degradation process. An area locating scoring method which selected the fields in the implant, interface and surroundings respectively showed a higher total score and offered more detailed information compared to the uniform distribution scoring method. Since the degradation and tissue reaction degree in the different areas are ever changing for the degradable implants, this area locating scoring method is sensitive, effective and applicable for the semi-quantitative evaluating system of ISO 10993.6.

Establishment of Caco-2 cell monolayer model with collagen coating 6-well plates for study of traditional Chinese medicine prescription

Caco-2 cell monolayer model is widely utilized in drug absorption study and 12-well transwellTM plates were commonly used to study the absorption of different kinds of natural products. To establish a stable method for the study of traditional Chinese medicine prescription, 6-well plates were chosen because of the larger well volumes than 12-well plates. To study the impacts of collagen kinds, coating density as well as coating time on the cell culture, the transepithelial electrical resistance of Caco-2 cell monolayers grown on different collagen coating transwells was determined, and the permeations of propranolol and atenolol as standard markers were detected with HPLC. The results showed that the kinds of collagen, the different coating densities and coating time of rat tail collagen had no significant influences on the Caco-2 cell monolayer integrality and absorption capacity. 6-well plates coated with 2 micro g Scm-2 rat tail collagen for 1 hour were enough reliable and suitable for the study of traditional Chinese medicine prescription in vitro.

Characterization of acid-soluble collagens from the cartilages of scalloped hammerhead (Sphyrna lewini), red stingray (Dasyatis akajei), and skate (Raja porosa)

Acid-soluble collagen from the cartilages of scalloped hammerhead (Sphyrna lewini, ASC-S), red stingray (Dasyatis akajei, ASC-D), and skate (Raja porosa, ASC-R) were isolated with yields of 5.64±0.41, 8.72±0.31, and 6.74±0.28% on the basis of wet weight, respectively. ASC-S, ASC-D, and ASC-R had glycine as the major amino acid with contents of 220, 231, and 228 residues/1,000 residues, respectively, and contained imino acid of 174, 155, and 142 residues/1,000 residues, respectively. Three kinds of collagens more likely were comprised of 2 types of collagen (type I and II) and had some differences in primary structures. The denaturation temperatures (Tds) of the ASC-S, ASC-D, and ASC-R were 23.8, 15.1, and 12.1°C, respectively. Maximum solubility of the 3 ASCs in 0.5 M acetic acid was observed at pH 2, and a sharp decrease in solubility above 2%(w/v) was observed in the presence of NaCl. The 3 lyophilized collagens displayed loose, fibrous, and porous structures.

Preparation, characterization, and antibacterial activity evaluation of collagen–Zn complex

The different kinds of collagen–Zn complexes were prepared by zinc acetate, zinc chloride, zinc nitrate, and zinc sulfate reacted with collagen protein. Their antibacterial activities have been investigated by MIC method. It was found that the antibacterial activity of collagen–ZnSO4 complex is better than that of others. To obtain a better antibacterial activity, collagen–ZnSO4 complexes with different zinc amount were prepared using zinc sulfate as starting material. These complexes were characterized by FT-IR, XRD, and atomic absorption spectrometry. The results showed that zinc ion could chelate with N–H, C–O, and C=O group in collagen to form the stable complex. Antibacterial activities of collagen–ZnSO4 complexes containing different Zn amount were evaluated against Escherichia coli and Staphylococcus aureus. The results suggested that antibacterial activity increases with the increase of zinc amount.

Study on the effects and mechanisms of bone marrow mesenchymal stem cells on porcine primary hepatocyte culture in vitro

To investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro. Mononuclear cells were isolated from bone marrow of swine by density gradient centrifugation. MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized. Immunocytochemical analysis was performed to monitor the expression and distribution of ECM. The purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function: including albumin secretion and urea synthesis (P < 0.01). The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture (P < 0.01). RNAi experiments verified that there was a correlation between ECM and hepatic functions. ECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.

Detection of V, III and I type collagens of dermal tissues in skin lesions of patients with systemic sclerosis and its implication

This study investigated the contents and distribution of collagen V (Col V) in skin lesions of the patients with systemic sclerosis (SSc) and its roles in the pathogenesis. The contents and distribution for alpha1 chain of collagen type I, III and V [alpha1 (I), alpha1 (III) and alpha1 (V)] in skin lesions of 36 patients with SSc (9 cases of mild fibrosis, 14 moderate, and 13 severe) were detected by using immunohistochemical SP method. Six cases of normal skin tissues served as controls. The results showed that there was diffuse distribution for three kinds of collagens in dermis. The deep staining alpha1 (I) and alpha1 (III) masses or bands were seen in reticular layer, while alpha1 (V) was distributed more homogeneously. From control to weak, moderate and severe fibrosis stages, alpha1 (I), alpha1 (III) and alpha1 (V) showed a gradually increased trend in skin lesions (P<0.05). alpha1 (V) was obviously elevated in skin lesions at early stage and persisted in whole fibrotic process and risen in greater contents, while alpha1 (I) and alpha1 (III) were to go higher late and were apparently elevated at moderate and late stages. Compared with alpha1 (I), alpha1 (V) took leading increase at early stage in skin lesions (P<0.01), and had more elevated contents than alpha1 (III) at moderate and late stages. The fibrotic changes in dermal reticular layer occurred earlier than those in papillary layer, and the abnormalities of alpha1 (V)/alpha1 (I) ratio appeared before alpha1 (III)/alpha1 (I) ratio. It was concluded that a lot of alpha1 (V) began to deposit in greater contents prior to alpha1 (I) and alpha1 (III) at early stage in SSc and persisted in whole fibrotic process. The changes of alpha1 (V) contents in reticular layer occurred earlier than those in papillary layer, and it suggested that the fibrosis in reticular layer appeared earlier.

Isolation and Characterization of Collagen from Skin of Bullfrog, Rana catesbeiana Shaw

In order to utilize skin of bullfrog (Rana catesbeiana Shaw) as an alternative source of collagen, we investigated and compared biochemical and physical properties of collagens isolated from bullfrog skin. Two kinds of collagen (BSASC; bullfrog skin acid-soluble collagen and BSPSC; bullfrog skin pepsin-solubilized collagen) were isolated by subsequent treatments with acetic acid and pepsin. The amounts of skin collagen isolated in the subsequent treatments were 7.3% BSASC and 18.2% BSPSC on the basis of lyophilized bullfrog skin weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the BSASC has the chain composition of <TEX>${\alpha}1{\alpha}2{\alpha}3$</TEX> heterotrimer, and the BSPSC consists of two <TEX>${\alpha}$</TEX> chains of <TEX>${\alpha}1{\alpha}2$</TEX>. In addition, the denaturation temperatures of all collagens tested were ranged from <TEX>$30^{\circ}C\;to\;38^{\circ}C$</TEX>. This study suggests that there is a possibility to use bullfrog skin collagen as an alternative source of collagen for industrial purposes, and subsequently it may increase the economical value of the bullfrog.

コラーゲンスポンジによる外来耳手術

Recently, a new artificial dermis was developed as a permanent material for the reconstruction of the skin or mucosal defects. It is composed of two different kinds of collagen; fibrillar and heat-denatured collagen, which are freeze-dried to form a sponge-like structure. These modifications reduce the inflammatory response and increase cellular affinity.We have been using this artificial dermis in an out patient clinic and in this study, we investigated the effectiveness of this material for repairing tympanic membrane defects and external auditory canal skin defects.The findings show that the rate of successful repair was 90.9% (20/22 ears) for tympanic membrane defects, and 100% (7/7 ears) for external auditory canal skin defects. We consider this artificial dermis to be very useful for middle ear operations in outpatients settings.

Collagen, a common thread in extracellular matrix evolution

Among multicellular animals, collagen is probably one of the most constant extracellular proteins. Collagen fibrils are encountered in sponges. The organization of their genes suggests that they have not varied to a great extent during evolution. However, they are not present in all animals. Basement membrane collagen is certainly the only ubiquitous collagen. The comparisons of collagen in lower animals suggest similarities with several collagen types of vertebrates. It is interesting to try to imagine the fundamental problems for which solutions based on collagen have been adopted. The first one isadhesion. Animals with poorly packed cells need a cement to be attached to the bottom of the sea. In sponges, this function has been fulfilled by microfibrils of spongin, a short-chain collagen. Later on, other organisms, such as mussels, have used a chimeric elastin-collagen protein to obtain both elasticity and strong attachment. The second important problem has been mechanical support, that is askeleton. Here again, spongin has been involved in sponges. The best is the solution combining vertebrate collagen fibrils and calcium phosphate. A third vital problem has beenprotection. It is achieved by different kinds of collagens in invertebrates and lower vertebrates. It could be suggested that in vertebrates, some of the non-fibrillar collagen types are involved in tissue micro- or macro-specialization.

骨移植を行わない二次的顎裂閉鎖 第２報 Ｘ線ＣＴ像による骨形成の評価

We attempted secondary closure of alveolar clefts without using a bone graft in 18 patients (19 alveolar clefts) with narrow alveolar clefts. The surgical procedure to make a recipient bed was the same as that used for bone grafting in our department. Bone formation at the alveolar clefts were evaluated by CT radiographs taken at least 1 year after operation.Good bone bridge formation was found in about 50% of the cases. Good bone bridge formation was apparently associated with the use of Aviten® at the alveolar cleft region or injury of the bone surface of the alveolar cleft region with a chisel to promote osteoplastic activity.In the future, we expect that bone morphogenetic protein (BMP) or new carriers such as some kind of collagen will be developed.Moreover, CT radiographs were useful in evaluating bone formation at alveolar clefts.

Temperature dependence of pH for solubilized collagen solutions

AbstractThe temperature dependence of the pH for an acid‐soluble collagen in solution was measured, so that the pH largely increased in the temperature region including ca. 40°C of the thermal denaturation temperature. As the changes of the optical rotation, αD, and the electrical conductivity, σ, were observed in this temperature region, the change of the pH is due to the thermal denaturation of collagen. The differential curve of pH vs. t gave clear deflection points and a large peak at ca. 40°C. The thermal denaturation temperature, td, could be estimated from the peak temperature. The td obtained by the measurement of the αD, tdαD, decreased with the decrease in the heating rate and with the increase in the concentration of collagen. However, the td obtained through the measurement of the pH, tdpH, was independent of the variations of the heating rate and the concentration of collagen. These measurements were carried out for different kinds of collagens prepared by various methods. Some of them had one kind of peak; others had two kinds of peaks. The tdpH correlated with the tdαD for different kinds of collagen preparations. Therefore, the measurement of the temperature dependence of the pH was useful for the determination of the td. © 1994 John Wiley &amp; Sons, Inc.

Structural and functional changes in lung tissue of mice fed with beta-aminopropionitrile fumarate, L-3-cis-hydroxyproline, and L-4-cis-hydroxyproline.

Structural and functional changes in lung tissue of mice fed with beta-aminopropionitrile fumarate, L-3-cis-hydroxyproline, and L-4-cis-hydroxyproline. We fed 0.1% solutions of 3-cis-hydroxyproline (3cisHP), 4-cis-hydroxyproline (4cisHP) or beta-aminopropionitrile fumarate (beta APN) to 5-week-old mice for 1 month and studied the effect of each of these substances on the lung function and structure. Compared to control animals the compliance of the respiratory system in the mean was increased by 3-15% in the mice fed beta APN or 3cisHP, and decreased by 2-8% in the mice fed 4cisHP. On electron microscopial examination no overt morphological changes were detectable although as proven by biochemical analysis 3cisHP and 4cisHP were incorporated into the collagen of the mice who received these substances. We conclude that the effect of substances interfering with the normal production of collagen molecules depends on the kind of collagen affected and on the rate of collagen turnover.

Constituents of connective tissue around the capillary of chick pecten oculi.

The constituents of the connective tissues around the capillary of the chick pecten oculi were examined electron microscopically by HCl-collagenase and HCl-elastase methods. The basal lamina like membrane below the endothelial cell of the pecten capillary was digested by collagenases I, II and IV and elastase, and may be a false basal lamina. The basal lamina of cells with pigment granules which surround the capillary was digested by collagenase IV and elastase, and contained type IV collagen. Fibrils between the basal lamina like membrane of the pecten capillary endothelium and the basal lamina of the cells with pigment granules were digested by collagenases I, II and IV, and elastase. Thus, these fibrils are composed of many kinds of collagen. Elastase may be responsible for the breakdown of most collagens as well as elastin.

Chemical estimation of interstitial and basement membrane collagen in obstructive nephropathy

Analyses of total 3-hydroxyproline and 4-hydroxyproline in tissue samples can be used to estimate the quantity of two kinds of collagen: that of basement-membrane and that of the several interstitial Types. Applied to an experimental model for obstructive nephropathy in the rabbit, such data show that the large increase in collagen in the renal cortex (at the 32nd day following unilateral ureteral ligation) is primarily of interstitial type. However there is also an appreciable increase in basement membrane collagen. Both findings are in agreement with the qualitative impression from electronmicrographs. The chemical method offers an approach to quantitation of these two kinds of collagen (basement membrane and interestitial) in normal and diseased tissues.

Comparative Immunologic Studies on Fish and Invertebrate Collagens

Abstract Rabbit antisera prepared against collagens from carp and two invertebrate species were fractionated using an immunoadsorption technique. The two antibody fractions, one with and one without affinity for the rabbit collagen immunoadsorbent, were investigated for cross-reaction with various collagens derived from mammalian, fish and invertebrate species by hemagglutination and hemagglutination-inhibition studies. The antibody fractions without affinity for rabbit collagen, representing the main fractions, reacted only with the kind of collagen used for immunization or, to a smaller extent, with collagens from closely related species. The second type of antibody fractions (with affinity for rabbit collagen) exhibited cross-reaction with all the collagens investigated.