Krystyna Pietrucha

Effect of fish collagen modification on its thermal and rheological properties

This report describes the effects of different methods of silver carp collagen crosslinking on its properties, particularly their thermal, mechanical viscoelastic and biological behavior. Enzymatic analyses and determination of the degree of crosslinking showed the stabilizing effect of both dehydrothermal (DHT) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) treatments on fish collagen. The results of the thermal (DSC) measurements demonstrated that collagen crosslinked by EDC/NHS ensured a high thermal stability compared with collagen crosslinked dehydrothermally. The denaturation temperature (T(d)) of unmodified collagen samples increased from 77 to 80°C and 88°C for DHT- and EDC/NHS-treated collagen, respectively. The influence of DHT or EDC/NHS crosslinking on the viscoelastic behavior of fish collagen was elaborated by a shift of the tan δ(max) peak toward higher temperatures resulting in higher thermostability of the modified collagen samples.

Physicochemical properties of 3D collagen-CS scaffolds for potential use in neural tissue engineering

Collagen-based composite scaffolds have considerable potential due to their well-known ability to regenerate skin, bone and cartilage. However, the precise composition and structure of scaffolds that optimize their interaction with neural cells remains incompletely understood and yet to be explored. In the present study, a new family of bi-component 3D scaffolds consisting of collagen (Col) and chondroitin sulphate (CS) were synthesized using a two-stage process: multiple freeze-drying followed by carbodiimide modification. Col-CS matrices had an average pore diameter of 31 μm and a relatively high surface area to pore volume ratio. Importantly, the FTIR data indicated that the ratio between the intensity of amide III and 1452 cm(-1) for Col-CS scaffold was 0.87, which indicates that the Col triple helix was preserved during the formation of the bond between Col and CS. All experiments also clearly showed that the Col-CS matrices have a lower enzyme sensitivity and higher thermal resistance than Col alone. These differences are likely due to the relatively large amount of CS in the collagen sponges, which hinders access for attack at specific active sites of the Col triple helix. Improved binary composite scaffolds were designed for neural tissue engineering applications.

Pore structure and dielectric behaviour of the 3D collagen-DAC scaffolds designed for nerve tissue repair

The design and selection of a suitable scaffold with well-defined pores size distribution and dielectric properties are critical features for neural tissue engineering. In this study we use mercury porosimetry and the dielectric spectroscopy in the alpha-dispersion region of the electric field to determine the microarchitecture and activation energy of collagen (Col) modified by 2,3 dialdehyde cellulose (DAC). The scaffold was synthesized in three steps: (i) preparation of DAC by oxidation of cellulose, (ii) construction of a 3D Col sponge-shape or film, (iii) cross-linkage of the Col samples using DAC. The activation energy needed to break the bonds formed by water in the Col-DAC composite is approximately 2 times lower than that in the unmodified Col. In addition, the magnitude of conductivity for modified Col at 70°C is approximately 40% lower than that recorded for the unmodified Col. The largest fraction, of which at least 70% of the total pore volume comprises the sponge, is occupied by pores ranging from 20 to 100μm in size. The knowledge on the dielectric behaviour and microstructure of the Col-DAC scaffold may prove relevant to neural tissue engineering focused on the regeneration of the nervous system.

Development of Collagen Cross-Linked with Dialdehyde Cellulose as a Potential 3D Scaffold for Neural Tissue Engineering

Until now, failed to receive ideal scaffold for applications in nerve tissue engineering. In this study the synthesis and assessment of 3D collagen sponges modified using 2,3-dialdehyde cellulose (DAC) as the cross-linker is presented. FTIR results showed that cross-linking reactions occur between the amino groups of collagen and the aldehyde groups in DAC without affecting the collagen triple helix. Besides, the DSC results reveal that cross-linking of collagen by DAC significantly increase its temperature denaturation (Td). This agrees well with results of DMTA analysis. The viscoelastic behavior of cross-linked collagen and improved mechanical properties was manifested by a shift of the tan δ peak towards higher temperatures. The results suggest that the utilization of Col-DAC scaffolds may be considered as a potential strategy for neural tissue regeneration.

The Behavior of Embryonic Neural Cells within the 3D Micro-structured Collagen-Based Scaffolds

Synthesis of scaffolds providing mechanical support for the growing cells is important in reconstruction of the tissue. Addition of chondroitin sulfate (CS) to collagen scaffolds was proved to regulate the neural cells growth and differentiation. The aim of the study is to test whether collagen-CS cross-linked scaffolds could be used for embryonic neuronal cell culture. Embryonic neuronal cells were isolated from rat foetus brains and their differentiation into neurons or astrocytes was characterized by flow cytometry. Then, the entrapment and distribution of cells within collagen alone or composite collagen with CS scaffolds was studied. The cells were applied to the scaffold and stained with bisbenzimide and then counted. Finally the MTT test was performed. The results suggest that embryonic neuronal cells were differentiated into neurons (MAP2 positive cells) or astrocytes (GFAP positive cells). The cells entered into the two tested 3D scaffolds; however their distribution in the tested scaffolds was not homogenous. The MTT test showed more intensive metabolism of the cells in collagen with CS scaffolds comparing with controls (cells seeded on laminin) or cells growing in scaffolds composed only from cross-linked collagen. Conclusion: the both collagen and collagen with CS scaffolds are the good carriers for the embryonic neuronal cells. However, the sample composed of collagen with CS constitutes better conditions for embryonic neuronal cells culture.

Efficacy evaluation of electric field frequency and temperature on dielectric properties of collagen cross-linked by glutaraldehyde

Solid-state dielectric properties are reported for unmodified collagen (Col) and glutaraldehyde-modified collagen (Col-GA) over the frequency range from 100Hz to 100kHz and at temperatures from 25 to 145°C. In the full temperature and frequency range the average values of the relative permittivity and dielectric loss for Col samples are higher than those recorded for Col-GA samples. The peak temperature of these both parameters associated with the release of loosely bound water is around 73 and 77°C for Col and Col-GA samples, respectively. The activation energy for the reorientation and breaking of hydrogen bonds takes the values 32kJmol-1 for Col and 23kJmol-1 for Col-GA. The relative permittivity decrement and conductivity increment of Col-GA samples fall by 40 and 30% on average in the temperature range 25-75°C, as compared to Col samples. Dielectric properties of Col-GA may be helpful in designing scaffolds for tissue engineering.

Comparison of various types of collagenous scaffolds applied for embryonic nerve cell culture

The purpose of the study was to confirm whether collagen-based scaffolds using different cross-linking methods are suitable elaborate environments for embryonic nerve cell culture. Three 3D sponge-shaped porous scaffolds were composed using collagen alone, collagen with chondroitin sulphate modified by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, and collagen cross-linked by 2,3-dialdehyde cellulose (DAC). Embryonic nerve cells from rats were applied to the scaffolds and stained with bisbenzimide to study cell entrapment within the scaffolds. The metabolic activity of the cells cultured in the scaffolds was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The majority of cells were differentiated into neurocytes or oligodendrocytes. Collagen and collagen-chondroitin sulphate scaffolds entrapped a low number of cells. The highest cell density was found in the collagen-DAC scaffold. Moreover, in collagen-DAC scaffolds, the metabolic activity was markedly higher than in the other samples. Although all used scaffolds are suitable for the culture of embryonic nerve cells, the collagen-DAC scaffold properties are the most favorable. This scaffold entraps the highest number of cells and constitutes a favorable environment for their culture. Hence, the Col-DAC scaffold is recommended as an effective carrier for embryonic nerve cells.

Collagenous scaffolds supplemented with hyaluronic acid and chondroitin sulfate used for wound fibroblast and embryonic nerve cell culture

BACKGROUND Tissue engineering is a strategy aimed at improving the regeneration of injured tissues. OBJECTIVES The aim of the present study was to determine whether a tri-copolymer composed of crosslinked collagen, chondroitin sulfate and hyaluronic acid (Col + CS + HA) provides a better environment for fibroblast and embryonic nerve cell culture than a collagenous scaffold (Col). MATERIAL AND METHODS The porosity of each of the matrices was characterized with a scanning electron microscope. Fibroblasts were isolated from rat wound granulation tissue (polypropylene net implanted subcutaneously). Embryonic nerve cells were obtained from the brains of rat embryos. The cells were applied to scaffolds and then stained with bisbenzimide to calculate cell entrapment within the material. The metabolic activity of the cells cultured within the scaffolds was tested using the 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS The Col scaffolds had a homogenously porous structure with a pore diameter of 50 μm for 70% of pores. The pore diameter in the tri-copolymer (Col + HA + CS) ranged from 24 to 160 μm (95% of total pore volume). Four times more cells (fibroblasts and embryonic nerve cells) were trapped within the superficial part of the collagenous scaffold than that of the tri-copolymer. On the third day of culture the metabolic activity of the fibroblasts within the 2 tested scaffolds was significantly higher than in the control conditions (cell culture on a laminin-coated surface). Also, the embryonic nerve cells demonstrated increased metabolic activity in Col + CS + HA scaffolds than the Col scaffolds. CONCLUSIONS Both fibroblasts and embryonic nerve cells could be seeded within the 2 tested scaffolds. Both the scaffolds provide good conditions for fibroblast culture. However, the Col + CS + HA tri-copolymer is preferable for embryonic nerve cell engineering.

A NEW METHOD OF DETERMINATION OF COLLAGEN CONJUGATED WITH KERATIN

Abstract The paper describes the possibility of using Sirius red dye for the determination of collagen conjugated with keratin of wool. Sirius red assay was shown to be feasible for collagen detection, which was enzymatically coupled onto wool fibers and woven fabric. The effectiveness of combination of keratin protein with collagen was evaluated .

Selecting the correct scaffold model for assessing of the dielectric response of collagen-based biomaterials

Fish collagen (Col) was cross-linked using two methods: dehydrothermal treatment (DHT) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). For the samples marked Col, Col-DHT and Col-EDC/NHS, dielectric properties were measured in the frequency range from 100 Hz to 100 kHz and temperatures from 25 to 145 °C. In the full temperature range, the average values of relative permittivity and dielectric losses for Col samples are lower than those recorded for Col-DHT and Col-EDC/NHS samples. The peak temperature of the dielectric parameters attributed to the denaturation temperature for Col, Col-DHT and Col-EDC/NHS, respectively, is about 75 °C, 83 °C and 89 °C. In addition, the values of these parameters are much higher in Col-EDC/NHS than in Col-DHT at the same temperature and frequency. The permittivity decrement and conductivity increment, respectively, for Col-EDC/NHS are about 62 and 32 times greater than those given by Col-DHT, which is a consequence of the EDC/NHS crosslinking action. Our electrical and dielectric studies of fish Col cross-linked by EDC/NHS or DHT provide deeper insight into the structure of collagen materials and help improve the synthesis of Col-based scaffolds for tissue engineering.