Intra-cerebral implantation of a variety of collagenous scaffolds with nervous embryonic cells.

Collagenous scaffolds provide good conditions for embryonic nerve cell growth. The aim of the current study was to assess the brains reaction to the implantation of 3D sponge-shaped scaffolds. These scaffolds consisted of collagen (Col) and Col with chondroitin sulphate, which is modified by carbodiimide, or Col crosslinked with dialdehyde cellulose. The current study also evaluated the expression of integrins α2 and ß1 in embryonic nerve cells. Embryonic nerve cells were isolated from the brains of rat embryos. Acellular scaffolds, or scaffolds populated with embryonic nerve cells, were implanted into the rats brain. The fibers of all the implanted scaffolds remained intact and served as a template for cell infiltration. The implants induced minimal to moderate inflammatory responses and minimal glial scar formations. Immunohistochemical studies did not indicate any microtubule-associated protein 2 or glial fibrillary acidic protein-positive cells inside the scaffolds. Acellular and cell-populated scaffolds yielded similar responses in the brain. The expression of integrin α2 and ß1 was observed in embryonic nervous cells. TC-I15, the integrin α2ß1 inhibitor, was not demonstrated to modify cell entrapment within the collagenous scaffolds. All applied scaffolds were well tolerated by the tissue and were indicated to support blood vessel formation. Therefore, all tested biomaterials are recommended for further studies. Additional chemical modifications of the material are suggested to protect the seeded cells from apoptosis after implantation into the brain.

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

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.

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.

Functional properties of different collagen scaffolds to create a biomimetic niche for neurally committed human induced pluripotent stem cells (iPSC).

The biomimetic, standardized conditions for in vitro cultures of human neural progenitors derived from induced pluripotent stem cells (hiPSC-NPs) should meet the requirements to serve as the template and protective environment for therapeutically competent cell population. In this study, two different collagen scaffolds: bi-component consisting of collagen and chondroitin sulphate (Col-CS), and collagen modified by crosslinking agent 2,3-dialdehyde cellulose (Col-DAC) have been used for the first time to encapsulate hiPSC-NPs and compared for the ability to create permissive microenvironment enabling cell survival, growth and differentiation. In our previous report, physicochemical comparison of the scaffolds revealed different elasticity, and diverse size and distribution of the pores within the 3D structure. Binary systems of Col-CS and Col-DAC tested in the current study have the correct balance of properties to serve as a biomimetic niche: they accommodate hiPSC-NPs sustaining their ability to proliferate and differentiate into neural lineages. However, a dense, network structure and rounded in shape pores of the Col-DAC microenvironment resulted in differential cell distributions within the scaffolds, with a tendency for augmented formation of highly proliferating cell aggregates as compared to Col-CS scaffolds. In contrast, Col-CS, which exhibited formation of the network of ellipsoidal and inner interconnected parallel pore channels, promoted enhanced cell viability and neuronal differentiation.

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.

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.

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.

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.

Changes in denaturation and rheological properties of collagen-hyaluronic acid scaffolds as a result of temperature dependencies.

This report describes the effect of temperature on the mechanical viscoelastic properties such as: storage modulus (E'), loss modulus (E''), and loss tangent (tandelta) of the collagen sponges modified with hyaluronic acid (HA). In order to detect collagen-HA copolymer denaturation and to assess its thermal stability, the differential scanning calorimetry (DSC) supplemented by thermogravimetric (TG) measurements was used. The denaturation temperature (T(d)) of unmodified collagen samples increased from 69 to 86 degrees C for cross-linked samples, respectively. These temperature dependencies show remarkable changes in E' and E'' at selected temperature up to 226 degrees C for all samples due to the release of loosely and strongly bound water. The influence of HA on the viscoelastic behavior of collagen is manifested by a shift of the tandelta peak associated with the process of decomposition towards higher temperatures resulting in a higher thermo-stability of the modified scaffolds.