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Chitosan derivatives 3D structuring via laser stereolithography and two-photon polymerization.

Name
Kseniia
Surname
Bardakova
Scientific organization
FSRC "Crystallography and Photonics" RAS
Academic degree
-
Position
Junior researcher
Scientific discipline
New materials, Manufacturing technologies & Processes
Topic
Chitosan derivatives 3D structuring via laser stereolithography and two-photon polymerization.
Abstract
Three-dimensional structures was formed on the basis of chitosan derivatives by two-photon polymerization, laser stereolithography and micromolding methods. The optimum ratio of the components, methods of preparation of photopolymerizable mixtures, parameters of the laser structuring, method of removing a non-crosslinked material were suggested. We studied cytotoxicity, proliferative activity, the degree and nature of the activation of expression of differentiation marker genes for stem cells, the compatibility of the primary cells of the nervous system.
Keywords
two-photon polymerization, micromolding, laser stereolithography, regenerative therapy, matrices, biocompatible materials
Summary

Synthesis of materials and structures based on biostable and bioresorbable polymers - one of the important directions of modern biomedical materials science. Later these structures can be used for drug delivery or tissue substitution therapy sections or local organ damage. Chitosan is widely used for preparing scaffolds for regenerative medicine due to its ability to enzymatically bioresorption and high affinity to animal cells. One of the key characteristics of tissue-engineering structures are the optimal structures scale and the possibility of varying the 3-D architectonics of such objects. The structures scale could be varied by the method of polymer structuring. As an example, one of the methods of forming of three-dimensional microstructures, providing high spatial resolution is a method of two-photon polymerization or microstereolitography based on the effect of two-photon absorption. The development of a method of two-photon polymerization, which allows to form structures with high spatial resolution is a method of quick stamping or micromolding. Creating structures by traditional one-photon laser stereolithography is also a very promising method for creating three-dimensional structures. The low resolution of the method is compensated by higher productivity

1.Materials

Components of photopolymerizable compositions (PPC) are presented in Table 1.

Table 1. Components used for PPC

 

Name

Structure

Main components of the reactive polymer system

Poly[(1 → 4)-2-amino-2-deoxy-β-D-glucose] (chitosan)

Allylchitosan

Graft copolymers of chitosan with polyvinyl alcohol (chitosan-PVA)

Graft copolymers of chitosan with oligo(D,L)-lactide

Components used as crosslinker

Polyethylene glycol diacrylate (PEG-DA)

Hyaluronic acid-glycidyl methacrylate (HAGM)

 

Photoinitiator

Irgacure 2959

 

 

All chitosan copolymers were obtained by the original solid-state method in Enikolopov Institute of Synthetic Polymer Materials in Prof. T.A. Akopova group.

Chitosan was prepared by the solid-state method from the chitin of crab shells. The molecular weight of chitosan was 40–50 kDa, and the degree of acetylation was 0.30. 

Allylchitosan was prepared using the solid-state method by reacting chitosan with allyl bromide in the extruder [1,2]. In a study  used  chitosan with varying degree of substitution (DS) of its functional fragments allyl groups:CHT-A1 = 0.1, CHT-A2 = 0.15, CHT-A3 = 0.2, CHT-A4 = 0.25, CHT-A5 = 0.5.

For the solid state synthesis of the graft copolymers of chitosan with polyvinyl alcohol, chitin and polyvinyl acetate (PVAc) were used as the reactants, whose deacetylation during the shear deformation of solid reactive mixtures for producing graft copolymers [2].

Graft copolymers of chitosan with oligo(D,L)-lactide. The chemical structure of the main characteristics of the copolymers are shown in Tables 1 and 2 [3].

Table 2.Chitosan-g-oligo(D,L-lactide) copolymer's macromolecular characteristics.

*DA - degree of acetylation; DD-degree of deacetylation; DS - degree of chitosan amino group substitution; PD - average polymerization degree of the side D,L-lactide chains

Sample

DА*

DD*

DS*

PD*

CL_1/0.5

0.1

0.71

0.19

3.0

CL _1/1

0.49

0.41

4.1

CL _1/3

0.53

0.37

9.7

 

Hyaluronic acid-glycidyl methacrylate. The chemical modification of hyaluronic acid was carried out in Hannover Laser Centre in a laboratory under the direction of Prof. B.N.Chichkov. Maximum degree of substitution of the product obtained is 60%[4].

2. Method of preparation and selected PPC compositions.

The optimum ratio of PPC components have been chosen and methods of PPC preparation to create three-dimensional structures by methods of micro-and macrostructuring using laser emmiting have been developed by us.

For photosensitive compositions were prepared 5% solutions of chitosan and its copolymers in 4% acetic acid (or in the case of compositions based on   chitosan-g-oligo(D,L)lactide copolymers solutions were prepared in water). Next, the insoluble fraction was separated by centrifugation, after which the solution was decanted and filtered through a membrane. To form matrices by method of 2PP filtrates were placed in a evaporative weighing bottle where solutions were evaporated to a gel state. For PPC obtaining concentrated solutions (~ 20 wt.%) were mixed with an aqueous solution of Irgacure 2959 and stirred for 24 hours. We also examined the effect of adding in photosensitive composition of PEG-DA (2000 Da) and HAGM.

For the method of stereolithography and micromolding in 4.5wt%  allyl chitosan solutions Irgacure 2959 photoinitiator have been added, mixing the solution is made within 2 hours. After PEG-DA added (700 Da), the composition is allowed to mix for 2 days.

3. Structuring of tissue-engineering scaffold

3.1. Two-photon polymerization (2PP)

For each PPC spectrophotometric analysis was carried out before the structuring. The absorption bands of used copolymers are in the field of up to 500 nm, intensive absorption band with a maximum at a wavelength of 280 nm refers to the photoinitiator, which indicates suitability for microstructuring by two-photon polymerization (Figure 1) [5].

Fig.1. The absorption spectra of: a) 1 - initial allyl chitosan with PEG-DA, 2 - composition of allyl chitosan, PEG-DA and Irgacure 2959; b) 1 - initial chitosan with HAGM, 2 - composition of chitosan, HAGM and Irgacure 2959

Samples of the photosensitive compositions were placed on a glass slide and were limited by spacer of crosslinked polydimethylsiloxane matrix (Figure 2). At the top cover glass was placed, through which the radiation took place. Cover glass prevented the drying of the composition and the crystallization of the photoinitiator.

Fig.2. Schematic illustration of the structuring by 2PP

First, for each sample the selection and optimization of 2PP-structuring parameters were carried out. With a scanner the number of vertical layers and the distance between them (from 5 to 10 microns) were changed, at the other axis the distance between  individual ray passages (5 to 10 microns) was changed. For materials the speed of 2FP process and working laser power were selected: 50 - 150 mW, 5000 - 15 000 mkm/s. Fig. 3 shows the two-dimensional arrays of structural units,  which were structured with various parameters [2].

Fig.3 Photomicrographs of arrays obtained by two-photon polymerization of  chitosan-PVA (a) and allyl chitosan (b).

Since the structures must be the network of macropores and pores, in turn, must be connected to each other to provide the cells ability to migrate through the matrix, to promote tissue growth throughout the scaffold, the size of the cylindrical structural units and their shape were varied while 2PP-structuring. Figure 4 shows two configurations used in works [2,5].

Fig. 4. Photomicrographs of three-dimensional structures of various models

As a result, two-and three-dimensional structures have been formed on the basis of chitosan, allil chitosan, graft copolymer of chitosan and polyvinyl alcohol [2]. In the example copolymers of chitosan with oligo(D,L)-lactide with various grade of polymerization of graft chains was shown that the macromolecular characterization of the synthesized copolymers affect the holding microstructuring process: substitution degree increase in the amino groups of chitosan and the degree of oligolaktide polymerization in copolymers allows the formation of stable three-dimensional crosslinkings upon irradiation [3].

In our study, for each type of matrices developed an algorithm for washing out the non-crosslinked material. It contains a cyclic washing with aqueous ammonia, acetic acid and water. To check the completeness of washing, the hydrogel matrices were studied on an inverted microscope equipped with a confocal laser system (Figure 5)

 

Fig. 5. 3D reconstruction of the washed hydrogel matrices (a, b); obtained scaffold based on allyl chitosan and PEG-DA after washing of the non-crosslinked material (в)

3.2. Micromolding method [5]

In this study the technique of quick-forming matrices of biodegradable allyl chitosan on micromolding technology have been perfected. The method allowed to obtain scaffolds with a simple structure in quantities up to 10 pieces per day. The hydrogel was poured into a 3-dimensional matrix of polydimethylsiloxane fixed to the aluminium base (Figure 6), and then curing of the composition was initiated with a laser at a wavelength of 266 nm, the intensity of 2 mW/cm2 for 5 minutes.

Fig. 6. The substrate for micromolding 1 - convex matrix of polydimethylsiloxane, 2 - aluminum basis.

Model for the matrix has a hexagonal shape, which is a two-tiered array of cylinders, the diameter of a single cylinder ≈280 mkm (Figure 7).

Fig. 7. Scaffolds obtained by micromolding based on allyl chitosan [5].

3.3. Laser stereolithography

All samples were prepared on an experimental model of laser stereolithography apparatus LS-120 . Layer thickness in growing samples was 200 μm. Structurization was performed using a HeCd-laser (wavelength was 325 nm, radiation power was 15 mW). We determined the layer formation rate based on the laser power and the technological parameters of the composition curing deduced from experiments: Ec=50 mJ/cm2 (a parameter characterizing a threshold value of exposure dose for solid polymer film formation start) and Dp=0.15 mm (a parameter to characterize critical thickness of a film)[6].

CHT-A1 and CHT-A5 based matrices produced by laser stereolithography are structurally uniform material. The samples are in the form of crossed helixes (or two superimposed circles with centered beams, and a hole) or in the form of cylinders with slits (Fig.8). Under mild exposure original matrices recover their former shape[6].

Figure 8. Appearance of structured matrices; 1 scale mark = 1 mm

4. Biological research

4.1. Biocompatibility and biological activity of the chitosan-based composition[6]

Biocompatibility for materials based on allyl chitosan was studied in vitro using extractions and cell culture on the surface of the materials themselves. Partial replacement of chitosan amino groups by allyl groups (CТ-А) and the introduction of polyethylene glycol diacrylate (PEG-DA) as a crosslinking agent were found not to reduce the material biocompatibility.

The metabolic activity determination of NCTC L929 cells using MTT assay showed that the samples under study to contain none water-soluble components toxic to mammalian cells (Fig.9).

Figure 9. Metabolic activity of NCTC L929 line cells according to MTТ assay in 48 h incubation of three-day extracts from materials: 1 — allyl chitosan; 2 — allyl chitosan + PEG-DA; 3 — on the cover glass surface

The samples based on CT-A1 and CT-A1 with a crosslinking agent PEG-DA are biocompatible and are able to support adhesion, spreading and proliferative activity of human mesenchymal stromal cells (MSC), but have significant differences in the extent and nature of the expression activation of gene markers for osteogenic differentiation path.

The analysis of morphological traits and viability of human MSC cultured on the surface of CHT-А1 and CHT-А1 with PEG-DA demonstrated the cell death percentage not to exceed 1–2%. Cells spread and proliferated on the surface of both materials under study. The morphology of cells was no different from control, though the density of a cell monolayer on a cover glass on day 7 was significantly higher compared to that on polymer films (Figures 10–12).

Figure 10. Appearance of human mesenchymal stem cells in incubation on the surface of allyl chitosan: incubation day 1 (а), (b); incubation day 7 (c)–(f). Cell staining Syto 9 (а), (с); propidium iodide staining of dead cell nuclei (b), (d); SEM microphotographs (e), (f)

Figure 11. Appearance of human mesenchymal stem cells in incubation on the surface of allyl chitosan film + PEG-DA: incubation day 1 (а), (b); incubation day 7 (c)–(f). Cell staining Syto 9 (а), (с); propidium iodide staining of dead cell nuclei (b), (d); SEM microphotographs (e), (f)

Figure 12. Appearance of human mesenchymal stem cells in incubation on the cover glass surface (control): incubation day 1 (а), (b); incubation day 7 (c)–(f). Cell staining Syto 9 (а), (c); propidium iodide staining of dead cell nuclei (b), (d); SEM microphotographs (e), (f)

To assess the effect of physicochemical characteristics of the materials on differentiating activity of human MSC we determined a phenotypic cell profile at different culture stages. The present study involved real-time PCR to analyze expression of 22 major genetic markers.

The study of the cell cultured on the glass and on the polymers under study showed the differences in expression activation degree of genetic markers of osteogenic differentiation on day 7 (Figure 13, а).

Figure 13. Gene expression levels in human mesenchymal stem cells cultured within 7 (а) and 23 days (b) on the surface of the materials: S and S2 — CHT-А1; T and T2 — CHT-А1 with PEG-DA; control — cover glass

A longer cell culture (23 days) on the test materials slightly changed the gene transcription (Figure 13,b). On allyl chitosan samples only a few genes had a high expression level compared to the cells cultured on the cover glass. A control group, in general, showed the same transcription pattern of marker genes, though the majority of genes enhanced it significantly.

4.2. Compatibility of the nervous cells and structured biodegradable matrices[7]

For hydrogel matrices based on chitosan and HAGM defined  toxic and adhesive properties and showed good compatibility with primary cultures of hippocampal.

It was found an intensive attachment to the hydrogel matrix of viable dissociated hippocampal cells at the end of the first day of culture. 

Next it was observed shoot formation of nerve and glial cells on the surface of the matrix. This is indicative of activation of the processes of formation of the network structure.

Thus, it is shown that the chosen shape of a matrix conducive to the formation of morphologically full neuron-glial networks (Fig. 14).

Fig. 14. Photomicrographs of dissociated hippocampal cells cultured on the hydrogel matrix based on chitosan (a and b DIV 14 and 30 respectively) and control (e, g, on a petri dish coated with polyethyleneimine)

Cytotoxicity of matrices was determined in vitro: uniform growth of cells throughout the matrix surface was maintained throughout the period of observation (Fig. 15).

Fig. 15. Micrographs cultures of dissociated hippocampal cells cultured on a hydrogel based scaffolds for chitosan 14 (a) and 28 days (b) (stained by bis-benzimid)

It was found that the matrix material has a high affinity to cells of the nervous system (neurons and astrocytes). The proportion of dead cells in the culture did not exceed 3.5% for all the test cultivation stages (Fig. 16).

Fig. 16. Change of a share (%) of dead cells in primary cultures of hippocampal for cultured on the glass (1, control) and hydrogel scaffolds based on chitosan (2)

 

 

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