Use Of Collagen Gel Research Articles

Mechanical performance of collagen gels is dependent on purity, α1/α2 ratio, and telopeptides.

This article describes the compositional, mechanical, and structural differences between collagen gels fabricated from different sources and processing methods. Despite extensive use of collagen in the manufacturing of biomaterials and implants, there is little information as to the variation in properties based on collagen source or processing methods. As such, differences in purity and composition may affect gel structure and mechanical performance. Using mass spectrometry, we assessed protein composition of collagen from seven different sources. The mechanics and gelation kinetics of each gel were assessed through oscillatory shear rheology. Scanning electron microscopy enabled visualization of distinct differences in fiber morphology. Mechanics and gelation kinetics differed with source and processing method and were found to correlate with differences in composition. Gels fabricated from telopeptide-containing collagens had higher storage modulus (144 vs. 54 Pa) and faster gelation (251 vs. 734 s) compared to atelocollagens, despite having lower purity (93.4 vs. 99.8%). For telopeptide-containing collagens, as collagen purity increased, storage modulus increased and fiber diameter decreased. As α1/α2 chain ratio increased, fiber diameter increased and gelation slowed. As such, this study provides an examination of the effects of collagen processing on key quality attributes for use of collagen gels in biomedical contexts.

Effect of storage time on the quality of chicken sausages produced with fat replacement by collagen gel extracted from chicken feet

The objective of this study was to evaluate the use of collagen gel extracted from chicken feet on chicken sausages during 42 d of refrigerated storage. Three chicken sausages were processed: standard (SS); replacing 50% fat with commercial collagen powder (SC); replacing 50% fat with chicken foot collagen gel (SG). Sausages were stored at 4°C and analyzed every 14 d, for proximate composition, fatty acid profile, thiobarbituric acid reactive substances (TBARS) number, antioxidant activity, electrophoresis, instrumental color, water holding capacity (WHC), texture profile analysis, and quantitative descriptive analysis. Sausages SC and SG had similar behavior to the standard in the sensorial parameters of appearance and color over 28 d of refrigerated storage. SG had the highest WHC (81.05%), the lowest TBARS value (0.38 mg MDA/kg), and the highest antioxidant activity in addition to having the best atherogenicity and thrombogenicity index compared with SC treatment, making collagen gel viable to replace fat and control the effects of lipid oxidation.

РАЗРАБОТКА КОЛБАСНЫХ ИЗДЕЛИЙ С ИСПОЛЬЗОВАНИЕМ ПРЕБИОТИКА И КОЛЛАГЕНОВОГО ГЕЛЯ

Currently, the food industry faces challenges that are not solved by a simple quantitative increase in production, but require qualitatively new approaches. The article discusses the possibility of using a prebiotic and collagen gel in the sausage production technology. The main task in the development of a new recipe for sausages was to use the best combination of prebiotic, collagen gel, raw meat and to expand the range of sausages, as well as to reduce the cost of the finished product. Fat reduction and prebiotic fortification are the main goals of the functional nutrition concept. Increased consumer interest in healthy eating contributes to an increase in demand for meat products with a reduced fat content, which poses a challenge for meat industry specialists associated with the important role of adipose tissue in forming consumer characteristics. Particular attention is paid to the substances that can replace adipose tissue in meat products. In this regard, it is important to use inulin, which, on the one hand, is a prebiotic, and on the other, it has technological properties that allow imitating fat in a product. The use of collagen gel improves economic performance of production by reducing production costs and losses during heat treatment and by rationalizing the use of low-grade meat raw materials. Experimental studies were carried out in the laboratory of the Department of Food Technologies of the Donskoy State Agrarian University. Basing on the research carried out, a recipe and a technological scheme for sausages production have been developed. Evaluation of the minced meat composition quality parameters indicates that the minced meat composition containing 80% of poultry meat, 15% collagen gel and 5% inulin, has a high degree of protein and fat balance. On the basis of organoleptic and physicochemical studies, a rational formulation of the product is proposed. An improvement in organoleptic and rheological characteristics has been established. The optimal dose of prebiotic and collagen gel introduction into the developed sausages has been substantiated. Studies have shown that the production of cooked sausages using a prebiotic and collagen gel is a promising direction.

The effect of basic fibroblast growth factor on regeneration in a surgical wound model of rat submandibular glands.

This study developed an animal model of surgically wounded submandibular glands (SMGs) and investigated the effects of collagen gel with basic fibroblast growth factor (bFGF) on tissue regeneration of surgically wounded SMGs in vivo. The animal model was produced by creating a surgical wound using a 3-mm diameter biopsy punch in SMGs. The wound was filled with collagen gel with bFGF (bFGF group) or without bFGF (control group). In the animal model of surgically wounded SMGs, salivary glands without scar tissue around the wound area were observed with smaller areas of collagen gel. Small round and spindle-shape cells invaded the collagen gel in both groups after operation day (AOD) 5, and this invasion dramatically increased at AOD 7. Host tissue completely replaced the collagen gel at AOD 21. The invading immune cells in the group treated with collagen gel with bFGF were positive for vimentin, α-smooth muscle actin (αSMA), CD49f, c-kit and AQP5 at AOD 7. Similarly, the mRNA expression of vimentin, αSMA, CD49f, keratin19 and AQP5 was also increased. This study suggests that the use of collagen gels with bFGF improves salivary gland regeneration.

Behavior of Bone Marrow Mesenchymal Stem Cells on Collagen Scaffolds in a Dynamic Culture System

Aim: BMSCs cultivation in vitro under three-dimensional (3D) dynamic conditions. Methods: a novel bioreactor system without shear stress acting on cells was developed by the combined use of collagen gel, hollow fiber membranes (HFMs) and air-lift loop bioreactor (ALB). During the expansion period, the cell growth curves were measured and after 7 days, the cell viability and morphology were assayed by fluorescence staining microscopically. Moreover, the BMSC characters of expanded cells were identified by specific expressions of CD29, CD34, CD44 and CD45 and by multilineage differentiation into osteoblasts, chondrocytes and adipocytes. Results: the cultured BMSCs expanded about 17-fold under dynamic condition, 13-fold under static condition with the initial density of 5×105cells·mL-1; after 7 days, the cells maintained high viability of about 92% and spread with a spindle appearance and long cell protrusions in gel; the expanded cells were CD29 and CD44 positive, CD34 and CD45 negative, and they could differentiate into osteoblasts, chondrocytes and adipocytes; the cells expanded under dynamic condition spread more and showed stronger multi-differentiation potentials.

Development of a novel smart scaffold for human skeletal muscle regeneration

Skeletal muscle defects are notoriously difficult to manage and the current methods used are associated with many limitations. Engineered skeletal muscle tissue has the potential to provide a solution that circumvents these disadvantages. Our previous work has identified a novel three-dimensionally aligned degradable phosphate glass fibre scaffold that can support myoblast differentiation and maturation. This current study has further developed the scaffold by encasing the fibres within a collagen gel to produce a smart composite scaffold that provides key biomimetic cues and supports the formation of a tissue that may be implanted in vivo. The constructs formed were approximately 30 mm long and microscopic examination confirmed favourable unidirectional cell alignment. There was good cell survival, and gene expression studies demonstrated upregulation of the myogenic regulatory factors and developmental and adult myosin heavy chain isoforms indicating myofibre formation and maturation respectively. Compared with the three-dimensional glass fibre scaffolds, the composite scaffolds had later gene upregulation, however, the use of collagen gels reinforced with degradable aligned glass fibres offers the opportunity to create a tissue analogue that can be easily manipulated. Furthermore, the glass fibre ends could support tendon/bone formation, and the channels formed as the fibres degrade could allow for vascular ingrowth.

Use of collagen gel as an alternative extracellular matrix for the in vitro and in vivo growth of murine small intestinal epithelium.

Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel(™), a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP+) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono- and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction demonstrated evidence of Cdx2, villin 1, mucin 2, chromogranin A, lysozyme 1, and Lgr5 expression, suggesting a fully elaborated intestinal epithelium. Additionally, collagen-based enteroids co-cultured with myofibroblasts were successfully recovered after 5 weeks of in vivo implantation, with a preserved immunophenotype. These results indicate that collagen-based techniques have the capacity to eliminate the need for Matrigel in intestinal stem cell culture. This is a critical step towards producing neo-mucosa using good manufacturing practices for clinical applications in the future.

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.

Three-Dimensional Culture of Hepatocytes on Spongy Polyethersulfone Membrane Developed for Cell Transplantation

Implantable bioartificial liver has been investigated for patients suffering from liver insufficiency after mass liver resection or acute liver failure. Liver cells are implanted as free cell suspension, in microencaplsulation systems or using microcarriers. To exhibit their typical functions, hepatic cells need a three-dimensional environment that is much more physiological than a flat one. The aim of our study was in vivo evaluation of spongy polyethersulfone membranes as a synthetic support for hepatic cells grown three dimensionally and transplanted to SCID/NOD mice. Spongy membranes were prepared using phase inversion from membrane-forming mixtures containing the following: polyethersulfone (based polymer), dimethylformamide (solvent), polyvinylpyrrolidone MW 10000 (small pore precursor), and cellulose (large pore precursor). We observed that polyethersulfone membranes were well tolerated by C3A cells and we did not observe any toxic effect, resulting in viability of cells >95%. Use of collagen gel as a support for cells on the scaffold gives the opportunity to increase 10 times the number of cells seeded on the membrane. Heparin addition to collagen gel did not influence albumin production in SCID/NOD mice. We observed an increase of albumin production after 7 and 14 days after implantation. Use of collagen gels in combination with polymer scaffolds allows preparation of bioartificial organs possessing high cell concentration for transplantation purposes.

Collagen gel delivery of Tgf‐β3 non‐viral plasmid DNA in rat osteoblast and calvarial culture

Different forms of collagen as a carrier for naked plasmid DNA have shown potential as vehicles for therapeutic gene delivery and tissue engineering. The objective of this study was to determine the suitability of a dense collagen gel as a vehicle for sustained delivery of plasmid DNA in cell and organ culture. Plasmid DNA encoding Tgf-beta(3) was combined with collagen gel. DNA released into the media was measured by Pico-Green spectrophotometry. Results showed that DNA was released from the collagen gel at a gradual rate for up to 14 days. To evaluate collagen-mediated transfection in tissue, calvariae were exposed to collagen containing plasmid encoding GFP or DsRed. Transfection was visualized by fluorescence localized to tissue adjacent to the vehicle. To evaluate protein production, fetal rat calvarial osteoblasts were cultured with a collagen/Tgf-beta(3) plasmid mixture or in media containing plasmid alone. Media was collected at various time points to measure Tgf-beta(3) protein production. ELISA assays showed that collagen-transfected osteoblasts demonstrated an elevated Tgf-beta(3) protein production for up to 14 days. Therefore, collagen delivery of viable plasmid DNA created a sustained transient transfection of calvarial osteoblasts resulting in prolonged and elevated growth factor production. Together, these results suggest that use of collagen gel as a vehicle may provide a strategy to achieve localized and controlled, non-viral gene delivery in vivo.

Investigation of the percutaneous penetration of prostaglandin E 1 and its ethyl ester

The optimization of percutaneous delivery of PGE 1 and its ethyl ester in alcoholic buffer solution through hairless mouse skin was investigated. A reversed-phase HPLC system with a photodiode array detector was used to differentiate the UV spectra of the penetration products. By comparison of the UV spectrum for each chromatographic peak, the conversion of PGE 1 ethyl ester to PGE 1 by enzymatic hydrolysis was found to be the predominant degradation pathway during the in vitro penetration. The quantification of ethyl ester was developed based on the same principle as that for PGE 1. It was then applied to monitor the penetration of prostaglandins through hairless mouse skin from the vehicles containing various fractions of alcohol. Results demonstrated that the alkyl group promoted the penetration mainly as a result of enhancing the drug partitioning into the stratum corneum at its maximal thermodynamic activity. The alcohol fraction around 20% seemed to be optimal for the percutaneous delivery of the ethyl ester. The use of collagen gel to carry PGE 1 ethyl ester for percutaneous application was included for comparison. The effect of adding alcohol in the collagen gel on the penetration of PGE 1 ethyl ester was found to be slightly lower than that from the same vehicle without collagen.

Techniques for purification of rabbit osteoclasts and analysis of their functions

In the process of bone remodeling or modeling, the balance between bone formation and bone resorption maintains normality of function and structures of bone. Major bone-resorbing cells, osteoclasts, are terminally differentiated from hematopoietic stem cells and multinucleate cells. However, direct effects of osteotropic factors on osteoclast function have been unclear. Attempts to obtain isolated mammalian osteoclasts of high purity have been unsuccessful so far. Initially we succeeded in isolating osteoclasts of high purity using tissue culture dishes because of their high affinity for the tissue culture dishes, but the shortcoming of this method is that it was impossible to detach osteoclasts from the dishes. Therefore, we could not estimate bone-resorbing activity of mature osteoclasts on mineralizing substratum without any influence of bone-cells other than osteoclasts. Recently we developed a method to avoid the defect in the above-described method by the use of collagen gel. Our new method may shed new light on our understanding of the cellular and molecular mechanisms of osteoclasts.

Human breast cancers respond to growth factors in vivo but not in vitro

Growth response of human breast cancer cells to epidermal growth factor (EGF) and transforming growth factoralpha (TGF-α) was tested both in culture and in vivo in nude mice. Human breast cancer cells were obtained from palpable tumors resulting from xenografted primary breast cancers in nude mice. In collagen gel culture, the breast cancer cells grew autonomously as expanding spherical masses of loosely adherent cells in the basal medium and the supplementation of growth factors had no additional stimulatory effect. To determine whether this in vitro response is reflected in vivo, the collagen gel embedded human breast cancer cells were transplanted into athymic nude mice and the growth response to EGF was studied in vivo. In contrast to the situation in vitro, exogenous EGF was growth promoting in vivo. Our results demonstrate the importance of the combined in vitro-in vivo approach in studying physiologically relevant growth regulation. In addition, the use of collagen gel embedded human breast cancer cells for transplantation studies may more closely model the clinical situation in view of the close histopathological resemblance of the recovered gels to the surgical breast specimens.

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis. Roberts, R. A., and Soames, A. R. (1993). Fundam. Appl. Toxicol. 21, 149-158. To explore peroxisome proliferator-perturbed hepatocyte growth regulation, robust in vitro models of liver are required. This has always posed a problem since isolated hepatocytes show a rapid loss of viability and differentiation status and cease to be useful after 3-4 days in culture. We now describe a model system in which rat hepatocytes are maintained as three-dimensional spheroids. The maintenance of hepatocyte viability and morphology in these cultures is considerably prolonged over that seen in monolayer culture and is comparable to that obtained by the use of collagen gels or dimethyl sulfoxide. The spheroid system is, however, free of any additives that may lead to artifact and free of excessive exogenous protein that may compromise subsequent analyses. Ultrastructural examination reveals extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile cannalicular structures. Furthermore, hepatocytes maintained as spheroids retain expression of liver markers such as albumin and also retain their ability to respond to peroxisome proliferators: even after 12 days in culture, treatment with the peroxisome proliferator nafenopin causes a 4.5-fold increase in cytoplasmic volume fraction of peroxisomes. There is a concomitant induction of peroxisomal bifunctional enzyme and cytochrome P4504A, the enzyme markers associated with peroxisome proliferation. The spheroids also maintain expression of the peroxisome proliferator-activated receptor and preliminary data indicate that they are able to undergo replicative DNA synthesis in response to nafenopin. Hepatocyte spheroids will provide us with a model system for studying the early changes in rodent liver nongenotoxic carcinogenesis.

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis

To explore peroxisome proliferator-perturbed hepatocyte growth regulation, robust in vitro models of liver are required. This has always posed a problem since isolated hepatocytes show a rapid loss of viability and differentiation status and cease to be useful after 3–4 days in culture. We now describe a model system in which rat hepatocytes are maintained as three-dimensional spheroids. The maintenance of hepatocyte viability and morphology in these cultures is considerably prolonged over that seen in monolayer culture and is comparable to that obtained by the use of collagen gels or dimethyl sulfoxide. The spheroid system is, however, free of any additives that may lead to artifact and free of excessive exogenous protein that may compromise subsequent analyses. Ultrastructural examination reveals extensive interhepatocyte junctional complexes and in terdigitation of adjacent membranes together with the presence of bile cannalicular structures. Furthermore, hepatocytes maintained as spheroids retain expression of liver markers such as albumin and also retain their ability to respond to peroxisome proliferators: even after 12 days in culture, treatment with the peroxisome proliferator nafenopin causes a 4.5-fold increase in cytoplasmic volume fraction of peroxisomes. There is a concomitant induction of peroxisomal bifunctional enzyme and cytochrome P4504A, the enzyme markers associated with peroxisome proliferation. The spheroids also maintain expression of the peroxisome proliferator-activated receptor and preliminary data indicate that they are able to undergo replicative DNA synthesis in response to nafenopin. Hepatocyte spheroids will provide us with a model system for studying the early changes in rodent liver nongenotoxic carcinogenesis.

Methods for Cultivation of Keratinocytes with an Air-Liquid Interface

In ordinary cultures, cells are grown on artificial substrates and immersed in culture medium. In vivo, interfollicular epidermal cells grow on the basement membrane and are exposed to air. In a first effort to render the culture of these cells more physiological it seems legitimate to raise the cultured cells to the air-medium interface. Epidermal cells can be raised by the use of collagen gels maintained on a rigid support. They can also be grown on nitrocellulose filters coated with collagen or coated with a basement-membrane equivalent (BME) previously deposited by bovine corneal endothelial cells. By raising the cultures to the air-medium interface there is some evidence of a more complete differentiation, as evaluated by morphologic criteria. However, biochemically, the raising of the cultures does not seem to induce the synthesis of those keratin polypeptides which are not expressed in immersed cultures. Epidermal cells can also be raised by culturing them on dermal substrates or dermal equivalents. When they were cultured on inverted dead pig skin, epidermal cells synthesized membrane-coating granules (MCG). MCG were not found in immersed controls. By culturing epidermal-cell suspensions on dead deepidermized dermis (DED), all morphologic markers of differentiation were seen except the keratin pattern. In addition, partial reexpression of high-molecular-weight keratin polypeptides occurred. However, the complete expression of keratins by cultured cells depends on the filtering action of the dermal substrate (the cultures are fed from underneath) more than on exposure to the air-liquid interface. In summary, several methods are available to culture epidermal cells at the air-liquid interface that are of interest in an investigation of the response of these cells to epigenetic influences.