Thermoplastic and Biocompatible Materials Based on Block Copolymers of Chitosan and Poly(ε-caprolactone)

Abstract

The development of materials based on chitosan and polyesters that possess thermoplastic, biocompatible, and biodegradable properties is a perspective for additive technologies in biomedicine. Research on obtaining such compositions is constrained because the polysaccharide content does not exceed 5 wt.%, which cannot ensure effective tissue regeneration. Herein, we propose a method for obtaining thermoplastic block copolymers based on chitosan and poly(ε-caprolactone) by ultrasonic irradiation of a homogeneous solution of a homopolymer mixture in dimethyl sulfoxide as a common solvent, achieving a yield of 99%. The distinctive feature of the method is the interaction between the components at the molecular level and provides obtaining copolymers at any component ratio. SEM images revealed a homogeneous structure without structural defects in both solvent-cast films and extruded filaments. The block copolymers were characterized by high mechanical property tensile strength of up to 60–70 MPa and elasticity of up to 35% for films and 25–40 MPa and elasticity of up to 50% for filaments. Cell adhesion of composition investigated on fibroblast cells (hTERT BJ-5TA) is at the level of chitosan and demonstrated the absence of cytotoxicity.

1. Introduction

The development of thermoplastic, biocompatible, and biodegradable polymeric materials with high mechanical properties is one of the urgent tasks in the field of polymer materials science [1,2]. Recently, the interest in polymeric materials in contact with living cells has increased manifold [3]. It includes all types of implants, as well as substrates, gels, and cell culture media [4,5]. The potential danger of using biomaterials of animal origin for these purposes has caused a number of legislative bans and, accordingly, increased interest in the use of products from biocompatible natural polymers [6,7]. Long-term experience in the application of polymers in this field has convincingly shown that for successful cell growth and tissue regeneration, along with biocompatibility, the chemical composition, supramolecular structure, physical and chemical properties of materials, and surface quality are important indicators [8,9,10]. One of the most promising biocompatible and biodegradable polymers for use in regenerative medicine is chitosan [11,12,13].

Chitosan, poly-(β-1-4)-2-amino-2-deoxy-D-glucopyranose, is a deacetylated form of the natural biopolymer chitin [14]. It has been reported that none of the chitosan-based products, regardless of the method of preparation, show traces of viral or prion infections [15]. The choice of chitosan as a basis for biodegradable polymeric materials is also associated with the presence of its unique set of properties, which include degradation under the action of enzymes inside the body and amphiphilicity of the macromolecule, which determines the ability to control hydrophilic–hydrophobic interactions with membrane proteins [16,17,18]. This allows cells of various cell types to successfully attach to the surface of chitosan matrices. The advantage of chitosan as glucosamine is the presence of a primary amino group in the structure of the repeating unit of the macromolecule—a nucleophilic center convenient for chemical modification, including a variety of physiologically active agents [19]. Chemical properties and biological activity of chitosan are necessary but not sufficient factors for obtaining functional materials based on it. Thermoplasticity and mechanical characteristics of the materials are equally important to ensure the possibility of obtaining products by conventional methods [20]. This remains an unsolved problem for the majority of polysaccharides, including chitosan [21]. The challenge of processing materials from chitosan significantly limits the possibilities of its application [22,23]. The most promising approach is to obtain chitosan-based materials with biocompatible and biodegradable thermoplastic polyesters, such as polylactide and polycaprolactone [24,25,26].

One of the most promising polyesters in regenerative medicine is poly(ε-caprolactone), which is an aliphatic polyester [27]. It is a partially crystalline (degree of crystallinity 50–69%), linear, hydrophobic, thermoplastic polymer. Polycaprolactone has attracted attention due to its valuable properties, such as biodegradability, high mechanical properties, and ease of recycling [28,29]. It should be noted that the melting point of polycaprolactone is much lower than that of polylactide and is 61 °C, and unlike polylactide, polycaprolactone is produced from petrochemical products, which determines a lower cost compared to polylactide [30].

Despite the approval of polycaprolactone application in medicine, good mechanical properties, and thermoplasticity [31], the polymer is characterized by low bioactivity compared to chitosan, and during biodegradation under the enzymolysis, caproic acid is formed, which can lead to local acidification of tissues and as a consequence the development of inflammatory processes [32,33,34].

Thus, chitosan and polycaprolactone can harmoniously complement each other’s properties and are a promising basis for the creation of composite biocompatible and biodegradable polymer matrices with advanced properties [35]. To obtain composites based on chitosan with polyesters, it is possible to use two approaches—under high shear stress conditions and through solutions. Recently, using these methods, a number of polymeric materials based on chitosan and polyesters in the form of films, porous frameworks, and micro- and nanoparticles have been obtained and studied from the point of view of their use for biomedicine [36,37,38]. There are controversial data in the literature regarding the compatibility of chitosan with polyesters [39,40].

There are several methods for obtaining compositions of chitosan with polylactide and/or polycaprolactone. An example of combining chitosan with polyesters by means of high shear stress is the use of a twin-screw extruder [41,42,43]. The material obtained by this method has good biocompatibility and biodegradability, but since the grafting of polylactide occurs only on the surface of the particles, the efficiency of this method is limited.

Methods of obtaining compositions based on chitosan with polyesters by mixing two solutions of homopolymers have been described. Aqueous solutions of acids are used as a solvent for chitosan, while polyesters are dissolved in chlorinated hydrocarbons [44,45,46] or dioxane [47], which leads to the formation of two-phase systems; as a consequence, the disadvantages of this method are the heterogeneity of the obtained materials. In addition, due to limited compatibility, it is impossible to introduce large amounts of one of the components [48]. The preparation of a thermoplastic blend based on polylactide and chitosan with the addition of glycerol and water has been described [49]. The use of plasticizers leads to the deterioration of cell adhesion and to the hydrolysis of polylactide, and the elevated temperatures (130–150 °C) required for mixing the polymers lead to partial destruction of chitosan. Another option for combining chitosan and polylactide from a solution of a homopolymer blend is the electrospinning method [50,51,52]. As a result, biocompatible and biodegradable nonwoven materials are obtained, but their strength is low enough (σ < 20 MPa) for tissue engineering applications. The method of obtaining grafted chitosan–polylactide copolymer [53] by copolymerization of lactide in chitosan suspension in dimethylformamide solution is known. In this case, copolymerization took place only on the surface of the particles, and the grafting efficiency was about 40%.

Several results of thermoplastic compositions containing chitosan are known from the literature [54,55]. However, in both cases, a third component (polyvinyl alcohol) is used as a compatibilizer. The disadvantages of this approach are the inability to vary the ratio of polysaccharide and polyester within a wide range, as well as the heterogeneous structure of the obtained materials, and the polyvinyl alcohol used may negatively affect the biological properties of the material.

Thus, the problem of obtaining homogeneous compositions based on chitosan and polyesters remains relevant since polyesters are insoluble in water [56], and there are no data on the solubility of pristine chitosan in organic solvents [57].

The aim of the present work is to obtain block copolymers of chitosan with poly(ε-caprolactone) by ultrasonic treatment of a homogeneous solution of homopolymer mixture in dimethyl sulfoxide as a common solvent to study the compositions, structures, and properties of the obtained compositions.

2. Materials and Methods

2.1. Chemicals and Consumables

Chitosan (CTS) (M_W—200 × 10³ g/mol, degree of deacetylation–82%) was obtained from Bioprogress (Moscow, Russia). Polycaprolactone (M_W = 70 × 10³ g/mol, M_w/M_n = 1.7), salicylic acid, dimethyl sulfoxide, and sodium hydroxide were purchased from Acros Organics (Geel, Belgium). All chemical reagents were of analytical grade and were used without additional purification

2.2. Methodology for Preparation of Block Copolymers

Chitosan (1.5 wt.%) was dissolved in 1.5 wt.% salicylic acid solution in DMSO and a calculated amount of polycaprolactone was added. After obtaining a homogeneous solution of the homopolymer mixture, it was ultrasonically irradiated for 30 min on an I100-6/4 unit with an ultrasonic generator i-10-2.0 with a power of 2000 W and an operating frequency of 22 kHz. The block copolymer was precipitated with ethanol and purified from possibly unreacted homopolymers by sequential treatment of the synthesis product with solvents (3% aqueous acetic acid solution, in which chitosan is soluble and polycaprolactone is insoluble, and chloroform, in which polycaprolactone is soluble and chitosan is insoluble). The precipitant stayed in each solvent for 72 h. Films were obtained by solvent casting from the resulting solution of the block copolymer in DMSO, and filaments were manufactured from the precipitated product using an Wellzoom Desktop Filament extruder (Wellzoom, Zhenzen, China), setting a temperature of 75 °C in the dosing and extrusion zone. When the temperature was reached, the material was placed in the dispenser of the extruder, and the drive was started. The extrusion process was carried out at a speed of 1500 mm/min. The molding nozzle diameter was equal to 1.75 mm. Both films and precipitant before extrusion were treated by 5 wt.%NaOH solution to convert chitosan in the base form from the salt form, and then products were washed with distilled water for 30 min until pH neutral (pH = 7).

2.3. Gel Permeation Chromatography

The molecular weight characteristics of chitosan and polycaprolactone were determined on a Shimadzu CTO20A/20AC high-performance liquid chromatograph (Kyoto, Japan) with the LC-Solutions-GPC v.1.21 SP1 software. For chitosan, 0.5 M aqueous acetic acid solution was used as eluent, with calibration by narrowly dispersed dextran samples. For polycaprolactone, tetrahydrofuran was used as eluent, calibrated by narrowly dispersed polystyrene samples.

2.4. Copolymer Characterizations

The microstructures of the films and filament were examined on a JEOL JSM-IT300LV scanning electron microscope (Tokyo, Japan) with a 20 kV X-ray fluorescence attachment.

X-ray phase analysis of the samples was performed on a Bruker D8 Discover X-ray diffractometer (Billerica, MA, USA) using CuKα radiation. The diffractograms were recorded for the angular range of 10–60° at the diffraction angle of 2θ in a symmetric geometry with a 0.6 mm slit on the primary beam and a linear position-sensitive LynxEye detector (Stockholm, Sweden).

FTIR spectroscopy was used to determine the functional groups. The sample powder and potassium bromide were blended and triturated by agate mortar (sample/KBr ratio of 1:40), and tablets were formed using an air-hydraulic press. IR spectra of composites were recorded on an FTIR spectrometer Shimadzu IR Prestige 21 (Kyoto, Japan) with a scanning range of 4000–400 cm^–1 with a resolution of 2 cm⁻¹ and 100 times scanning.

The thermal analysis was performed on a Mettler Toledo TGA/DSC 3+ (Greifensee, Switzerland) in the temperature range of 40–500 °C under a nitrogen flow (50 mL × min⁻¹) at the heating rate 5 °C × min⁻¹.

The contact wetting angle was determined using a Levenhuk DTX 30 digital microscope (Tampa, FL, USA). Samples in the form of squares (2 × 2 cm) were placed on the substrate, a drop of water of 1 μL was applied to the surface, and the wetting angle was measured after 180 s. All measurements were performed on 5 samples at a temperature of 25 °C.

The moisture absorption capacity of the samples was tested by absorption of saline solution (0.9% NaCl solution). For this purpose, the film samples (2 × 2 cm) were sterilized using 70% ethanol, washed with distilled water, and immersed in 15 mL of the saline solution above at 37 °C for 1 h. Before weighing, the surface water of the samples was gently removed with filter paper. The degree of swelling was calculated according to the following formula:

$$\mathsf{\Delta }\mathrm{W}={\displaystyle \frac{{\mathrm{m}}_{\mathrm{w}}-{\mathrm{m}}_{\mathrm{d}}}{{\mathrm{m}}_{\mathrm{d}}}}\times 100\%$$

where m_w—mass of swollen film and m_d—mass of dry film, respectively.

Determination of the thermoplasticity and melt flow index was carried out using the XNR-400AM Melt Flow Indexer (Dongguan Xihua Machinery Technology, Dongguan, China).

Polymer melt flow index (MFI) is calculated by the following formula:

$$\mathrm{M}\mathrm{F}\mathrm{I}=600\times {\displaystyle \frac{\mathrm{m}}{\mathrm{t}}}$$

where 600 is the standard time equal to 600 s; m is the average mass of extruded pieces, g; t is the time interval between two consecutive cuts of the pieces, s. The method was performed according to the ISO 1133 standard.

Mechanical characteristics of the material were determined on a Roell/Zwick Z005 tensile testing machine (Ulm, Germany). The tests were performed at a tensile speed of 10 mm/min on 60 ± 5 µm thick samples in the form of rectangles with a width of 15 mm for films and on samples with a length of 50 ± 5 mm and a width of 1.75 mm for extruded filaments, according to ISO 527-3 and 527-2 standards, respectively.

2.5. Study of Biocompatibility of Films

The adhesion on the film surface was investigated during the cultivation of human fibroblasts of the hTERT BJ-5ta cell line. Films of material after sterilization by ultraviolet irradiation were placed in the wells of a cell culture plate and filled with 500 μL of DMEM medium. Cells were seeded on the surface of the film at a density of 1.6 × 10⁵/cm² and cultured for 24 h. Cell visualization and cell viability were assessed by fluorescence microscopy. A 2 × 10⁻⁴ wt.% solution of acridine orange in phosphate buffer was used as a dye to stain the fibroblasts. This dye, by intercalation or electrostatic attraction, selectively interacts with DNA and RNA located in the cell nucleus and mitochondria, respectively. This allows the assessment of the overall cellular state—activity, proliferation, and apoptosis. Microsampling of films was performed on an Olympus IX71 invertor microscope (Tokyo, Japan) using a “green” filter (emission 510–555 nm, excitation 460–495 nm), which allows visualizing the green color of the nucleus of living cells.

The cytotoxicity of the samples was studied in an extract test using the L929 cell line. Cells were cultured in DMEM. To obtain extracts, the test samples were poured into DMEM medium supplemented with 2% fetal calf serum, L-glutamine, and 1% antibiotics (penicillin–streptomycin) and placed in an incubator for 1 day and 7 days under standard conditions (37 °C, 5% CO₂, humidified atmosphere) The cells were reseeded every 2–4 days. For this purpose, samples were incubated with the culture medium (25 mg per 1 mL of the medium) at 37 °C for 24 h. The cells of the test culture were seeded in wells in the amount of 10 × 10³ cells/cm² and cultured under standard conditions for 24 h. After 24 h, the growth medium over the cells was replaced with 1 day and/or 7 day extract of the test samples at several dilutions (0:1—control; 1:0; 1:1; 1:1; 1:2; 1:3; and 1:4—extract/growth medium, respectively) and cultured for another 72 h under standard conditions. Then, MTT working solution (5 mg/mL) was added to each well of the plate, and after 3 h of incubation, the MTT medium was completely replaced with DMSO. After 30 min, optical density (OD) was recorded on a TECAN tablet reader (Sanrais, Grödig Austria) at a wavelength of 540 nm. Cell growth intensity (V) was determined using the following formula:

$$\mathrm{V}\left(\%\right)={\displaystyle \frac{{\mathrm{O}\mathrm{D}}_{\mathrm{t}}}{{\mathrm{O}\mathrm{D}}_{\mathrm{c}}}}\times 100$$

where OD_t is the optical density in test wells, and OD_c is the optical density in control wells. The cells cultivated in a fresh medium (without the extracts) were used as a control. The obtained results were evaluated according to a scale for assessing cytotoxicity. According to the scale, cytotoxicity ranks 0 and 1 indicate no cytotoxicity, while ranks 2, 3, 4, and 5 correspond to different levels of cytotoxicity, from mild to evident toxicity.

3. Results and Discussion

3.1. Synthesis of Block Copolymers

Synthesis of block copolymers based on chitosan and polycaprolactone using ultrasonic irradiation on homopolymer solutions is a promising and efficient method and essentially combines two known approaches for compatibilization chitosan with polyesters, namely via solutions and mechanochemical method. Applied force, as in the case of mechanochemical synthesis, allows for the breaking and formation of new chemical bonds due to cavitation processes with the advantage that the reaction takes place in solution, unlike that on a surface (not only on the surface but also in the volume). Dimethyl sulfoxide (DMSO), as one of the most polar solvents (ε = 47), was chosen as the solvent for combining chitosan and polycaprolactone. Polyesters are well soluble in DMSO [58], and in addition, due to the biomedical purpose of the developed materials, it was important to use non-toxic substances and solvents. DMSO is one of the few organic solvents approved for use in medicine [59].

After obtaining a homogeneous, transparent solution of chitosan in a 1.5% wt.% solution of salicylic acid in DMSO, the calculated weight of polycaprolactone was added and ultrasonically irradiated. The samples were prepared with ratios of chitosan to polycaprolactone of CTS:PCL (1:1), CTS:PCL (1:2), CTS:PCL (1:3), and CTS:PCL (1:4).

Ultrasonic cavitation is known to be capable of bond scission, which can lead to both depolymerization and block copolymer formation. Samples were obtained by precipitation of solution after 10, 20, and 30 min of ultrasonic irradiation in ethanol at T = 5 °C under constant stirring, and after removing possible unreacted homopolymers, they were dried to constant weight.

The yield of copolymer was determined by the gravimetric method by the change in the mass of the sample before and after sequential extraction of homopolymers; the optimal ultrasonic irradiation time was 30 min because with less time, the process efficiency decreased, which led to lesser yield, and with time greater than 30 min, there was a decrease in the molecular weight of the block copolymer (

Table 1. Copolymerization conversion study.

Initial Composition of Homopolymer Mixture	Copolymer Yield After 10 min of Irradiation, %	Copolymer Yield After 20 min of Irradiation, %	Copolymer Yield After 30 min of Irradiation, %
CTS:PCL (1:1)	24.7 ± 0.4	60.8 ± 0.3	99.0 ± 0.2
CTS:PCL (1:2)	26.1 ± 0.1	58.2 ± 0.2	98.5 ± 0.1
CTS:PCL (1:3)	25.9 ± 0.1	61.7 ± 0.3	98.7 ± 0.2
CTS:PCL (1:4)	24.4 ± 0.2	63.0 ± 0.1	98.0 ± 0.2

).

It can be seen from the results that the decrease in the mass of the precipitate, which was irradiated for 30 min after extraction, does not exceed 2%, which indicates the high efficiency of the method to obtain block copolymers based on chitosan and polycaprolactone by ultrasonic irradiation of their homogeneous mixtures.

3.2. Molecular Weight Studies

Unfortunately, the molecular weight of the block copolymer could not be determined due to its insolubility in eluents for gel permeation chromatography. The M_W of polycaprolactone blocks was determined. For this purpose, the copolymer samples were subjected to enzymatic treatment with chitosanase to decompose chitosan to monosaccharides, after which polycaprolactone was precipitated with ethanol, dried, then dissolved in tetrahydrofuran and the molecular weight of the blocks was determined by gel permeation chromatography. The M_w of chitosan blocks was found by the indirect method based on the results of homopolymer degradation in the presence of TEMPO (

Table 2. Molecular weight characteristics.

Substance	Mw	Mn	Mw/Mn
Initial PCL	84,000	52,000	1.60
PCL after ultrasonic treatment t = 30 min	30,700	20,000	1.57
Initial chitosan	250,000	140,000	1.8
CTS after ultrasonic treatment t = 30 min	60,000	39,000	1.53
PCL from CTS – b – PCL (1:1)	23,500	16,800	1.40
PCL from CTS – b – PCL (1:2)	31,000	20,000	1.55
PCL from CTS – b – PCL (1:3)	30,000	22,000	1.40
PCL from CTS – b – PCL (1:4)	32,000	23,000	1.45

).

As can be seen from the table, the molecular mass of polycaprolactone blocks isolated from the copolymer and samples formed after ultrasonic treatment of pure polycaprolactone in the presence of TEMPO are close and are about 25,000–30,000 g/mol. This suggests that chain fragments formed during ultrasonic irradiation of the homopolymer mixture determine the size of blocks in the structure of copolymer macromolecules. On this basis, the molecular mass of chitosan blocks was determined by the results of ultrasonic treatment of chitosan solution in DMSO in the presence of TEMPO. A decrease in the molecular weight of chitosan from 250,000 g/mol to 60,000 g/mol was observed. Thus, it can be assumed that the macromolecules of the block copolymer consist of sequences of chitosan blocks with M_w of about 60,000 g/mol and PCL blocks with M_w of about 30,000 g/mol.

3.3. XRD Analysis

The structure of block copolymers was studied by XRD. The study was carried out on film samples obtained by pouring on Teflon substrate. The structure of the block copolymer was compared with the initial homopolymers and blended compositions (Figure 1).

The figure shows that the homopolymers are partially crystalline polymers with peaks at 21° and 23.5° degrees, characteristic for polycaprolactone, and 11.7° and 21° degrees for chitosan.

For the homopolymer blend composition, amorphization of chitosan is observed, which can be seen by a broad amorphous peak at 21°; crystallinity of polycaprolactone is preserved, and both peaks related to PCL can be seen. In contrast to the homopolymers blend, the block copolymer is characterized by a broad, amorphous peak, which is due to a decrease in the mobility of PCL blocks separated by chitosan blocks, as well as to the formation of covalent bonds between chitosan and polycaprolactone, which together exclude the crystallization of polycaprolactone.

3.4. FTIR Analysis

The chemical composition of block copolymers was investigated on chitosan – b – PCL (1:2) composition using FTIR analysis, and the spectrum is illustrated in Figure 2.

The spectrum exhibits peaks near 3412 cm⁻¹, which is attributed to N–H and O–H stretching vibrations of chitosan. The peak at 2942 cm⁻¹ is assigned to CH₂ stretching vibrations. A strong peak at 1733 cm⁻¹ can be attributed to the stretching vibration of C=O of polycaprolactone carbonyl groups. A vibrational peak at 1289 cm⁻¹ is assigned to the stretching of C–O and C–C. The peak at 1170 cm⁻¹ is related to the stretching of C–O–C. Some positional deviations of peaks in the FTIR spectrum of block copolymer are caused by structural changes at copolymer formation, which can lead to intramolecular interaction between amino and hydroxyl groups of chitosan blocks with carbonyl groups of polycaprolactone blocks and molecular weight degradation during ultrasonic irradiation.

3.5. Melt Flow Studies

Obtaining block copolymers of chitosan with poly(ε-caprolactone), which exhibit thermoplasticity, determines the possibility of manufacturing thermoplastic compositions. The influence of copolymer composition on melt flowability was investigated (

Table 3. Melt flow indexes of samples at T = 75 °C.

Composition	Melt Flow Rate, g/min
CTS	-
PCL	17 ± 0.7
CTS:PCL (1:1) blend	-
CTS:PCL (1:2) blend	5.5 ± 1.3 (with phase separation)
CTS:PCL (1:3) blend	8.3 ± 1.7 (with phase separation)
CTS:PCL (1:4) blend	11 ± 1.4 (with phase separation)
CTS – b – PCL (1:1)	-
CTS – b – PCL (1:2)	7.2 ± 0.6
CTS – b – PCL (1:3)	9.5 ± 0.3
CTS – b – PCL (1:4)	14.2 ± 1.1

).

The results of the study show that at the mass ratio (1:1) of components, neither the block copolymer nor the blended composition exhibit thermoplasticity. For both types of samples containing a larger share of polycaprolactone, flowability was observed, which increased with increasing content of polyester. But at the same time, it is significant that the block copolymers were not observed at phase separation, which took place for the blended compositions. The absence of phase separation is due to the covalent bonding between the blocks; thus, polycaprolactone “pulls” chitosan blocks during extrusion.

3.6. Scanning Electron Microscopy (SEM) Studies

Despite the visual homogeneity of the films, it was important to evaluate the structure of the composition after extrusion under conditions of applied force on the sample. The surface of thermoplastic samples of composition CTS – b – PCL 1:2 before extrusion and after extrusion were studied by the SEM method. The frontal surface and the end face were studied (Figure 3).

As can be seen from the microscopy results, for the block copolymer, the original homogeneous structure is maintained after extrusion, thus producing a homogeneous filament. SEM microphotographs approve layering of blend compositions in contrast for the block copolymer.

The homogeneity of extruded block copolymer is confirmed by the results of elemental analysis on nitrogen distribution by the SEM method using energy dispersive attachment (Figure 4).

It is essential that in the process of extrusion, there is no destruction of the copolymer, as evidenced by the elemental composition of the sample CTS – b – PCL (1:2) (

Table 4. Elemental composition of samples.

Composition	C (%)	O (%)	N (%)
CTS – b – PCL (1:2) before extrusion	65.62 ± 3.25	31.86 ± 1.63	2.52 ± 0.08
CTS – b – PCL (1:2) after extrusion	65.58 ± 3.43	31.83 ± 1.56	2.59 ± 0.12
CTS:PCL (1:2) theoretically calculated	65.42	31.92	2.66

).

Results of elemental composition for block copolymers before and after extrusion are the same and practically coincide with those theoretically calculated, which shows the preservation of the chemical composition after extrusion in contrast to the polymer blend.

3.7. Mechanical Properties

Materials used in tissue engineering should have mechanical strength and elasticity comparable to the replaced tissue. The results of measuring the mechanical properties of block copolymer films with different component ratios are presented in (

Table 5. Mechanical properties of films.

Composition	Tensile Strength, σ (MPa)	Elongation at Break, ε (%)
CTS	14.4 ± 0.8	1.2 ± 0.1
PCL	32.3 ± 1.5	45.9 ± 2.0
Films by solvent casting method
CTS – b – PCL (1:1)	42.1 ± 2.1	15.2 ± 1.1
CTS – b – PCL (1:2)	67.3 ± 2.8	34.1 ± 2.0
CTS – b – PCL (1:3)	55.3 ± 1.4	38.5 ± 1.9
CTS – b – PCL (1:4)	43.4 ± 2.6	39.3 ± 2.2
Filaments by extrusion
CTS – b – PCL (1:2)	24.5 ± 1.2	38.3 ± 1.5
CTS – b – PCL (1:3)	35.1 ± 3.9	45.1 ± 3.3
CTS – b – PCL (1:4)	41.8 ± 1.5	52.3 ± 2.0

).

The table shows that the mechanical properties of the block copolymers are superior to the original homopolymers. The highest mechanical strength was demonstrated by the samples of the CTS:PCL composition (1:2), and the relative elongation increased linearly with the increase in the proportion of polycaprolactone. The results of all samples are comparable with the values for skin tissue [60].

3.8. Water Contact Angle and Swelling Capacity Test

It is known [61] that to ensure successful tissue regeneration, the initial stage of which is cell adhesion to the surface of polymeric material, the latter should not be highly hydrophilic or hydrophobic. An indicator of this property is the contact angle of wetting [62], which should lie in the range of 80–120 degrees. The swelling capacity is a very significant property for materials in tissue engineering. Swelling is unfavorable in many situations, especially biomedical applications, such as tissue engineering, hemostatics, etc., as the swelling of a polymer leads to a volume expansion, which not only drops the mechanical properties of the material but can bring unnecessary negative effects on the surrounding tissues when being in contact with it [63]. The results of the study of films on water wettability and water uptake are presented in

Table 6. Values of contact angles of wetting of films with water.

Composition	Contact Angle, Deg	Water Uptake, %
CTS	96 ± 1.5	160 ± 11
PCL	131 ± 0.4	0
CTS – b – PCL (1:1)	110 ± 0.7	92 ± 3
CTS – b – PCL (1:2)	118 ± 0.3	62 ± 5
CTS – b – PCL (1:3)	123 ± 1.0	45 ± 7
CTS – b – PCL (1:4)	125 ± 0.5	36 ± 4

.

It can be seen that the surface wettability of the block copolymer films lies in the range required for cell adhesion. Water uptake decreases with higher content of polycaprolactone due to its water-insoluble nature.

3.9. Thermal Properties

The thermogram and DSC curves of the CTS – b – PCL (1:2) copolymer are presented in Figure 5.

The TGA curve does not demonstrate any weight loss up to 200 °C, which is what makes it thermally stable during processing at 75 °C in the extruder for further applications in additive manufacturing. The first decomposition stage, with 9.53% weight loss, is attributed to the dehydration of bound water in chitosan blocks. The second stage, with 75% weight loss, is attributable to depolymerization and decomposition of copolymer. The DSC curve demonstrates the endothermic melting peak of polycaprolactone blocks at about 66 °C, and it deviates from pure PCL, whose melting temperature is 61 °C [64] and is attributed to interactions with chitosan blocks, which increase the melting point. The peak at 310 °C is assigned to the decomposition of amine units in chitosan; it deviates from pure chitosan, whose thermal decomposition temperature is in the range from 300 to 305 °C [65], and the increase in the decomposition temperature is associated with intramolecular interaction between amino groups of chitosan and carbonyl groups of polycaprolactone through the formation of hydrogen bonds.

3.10. Biological Properties

In vitro studies on adhesion and proliferation of fibroblast cells as precursors of connective tissue formation were performed for pure chitosan, chitosan–polycaprolactone (1:2) blend, and CTS – b – PCL (1:2) block copolymer samples (Figure 6).

Figure 6 shows photos of the films after 24 h of cell incubation. Both blend and block copolymer films are non-toxic to cells and have sufficient adhesive properties for cell attachment and development on their surface. The results for block copolymers are insignificantly inferior for the pure chitosan. On the contrary, the blend-based films are characterized by a slightly worse distribution of fibroblasts on the surface. For chitosan, the cell growth density was about 45,000 cells/cm², the surface of blend composition film had a density of about 37,000 cells/cm², and copolymer film surface was characterized by 43,000 cells/cm², which is similar to chitosan and indicates high cell adhesion and, at the same time, possesses positive properties of polycaprolactone like processability and mechanical strength and elasticity.

The results obtained for block copolymer and blend composition show a 0 or 1 rank for both 1-day and 7-day extracts (

Table 7. Assessment of cytotoxicity for 1 day extract.

Series	Parameters	Composition
		Pure CTS	CTS:PCL (1:2) Blend	CTS – b – PCL (1:2)
Control (n = 8)	OD	0.583 ± 0.088	0.479 ± 0.082	0.521 ± 0.084
	V, %	100	100	100
	Cytotoxicity rank	0	0	0
Extract (n = 8)	OD	0.710 ± 0.033	0.454 ± 0.031	0.620 ± 0.049
	V, %	122	95	119
	Cytotoxicity rank	0	1	0
Extract 1:1 (n = 8)	OD	0.630 ± 0.042	0.473 ± 0.084	0.588 ± 0.017
	V, %	109	100	113
	Cytotoxicity rank	0	0	0
Extract 1:2 (n = 8)	OD	0.618 ± 0.016	0.466 ± 0.047	0.575 ± 0.022
	V, %	106	97	110
	Cytotoxicity rank	0	1	0
Extract 1:4 (n = 8)	OD	0.650 ± 0.028	0.440 ± 0.038	0.610 ± 0.087
	V, %	111	91	117
	Cytotoxicity rank	0	1	0

and

Table 8. Assessment of cytotoxicity for 7 day extract.

Series	Parameters	Composition
		Pure CTS	CTS:PCL (1:2) Blend	CTS – b – PCL (1:2)
Control (n = 8)	OD	0.494 ± 0.014	0.405 ± 0.021	0.427 ± 0.011
	V, %	100	100	100
	Cytotoxicity rank	0	0	0
Extract (n = 8)	OD	0.510 ± 0.023	0.340 ± 0.010	0.470 ± 0.034
	V, %	104	84	110
	Cytotoxicity rank	0	1	0
Extract 1:1 (n = 8)	OD	0.507 ± 0.014	0.425 ± 0.029	0.429 ± 0.012
	V, %	103	105	100
	Cytotoxicity rank	0	0	0
Extract 1:2 (n = 8)	OD	0.550 ± 0.035	0.376 ± 0.043	0.447 ± 0.038
	V, %	112	93	105
	Cytotoxicity rank	0	1	0
Extract 1:4 (n = 8)	OD	0.580 ± 0.023	0.383 ± 0.033	0.455 ± 0.022
	V, %	118	95	106
	Cytotoxicity rank	0	1	0

, respectively) corresponding to biocompatibility. The growth of optical density shows cell proliferation. It is seen that extracts of material based on block copolymer show a proliferative activity.

Thus, the summary of results for biological properties demonstrates not only the biocompatibility for both blend and copolymer but also a positive proliferative effect for block copolymer and better cell adhesion compared to blend composition.

4. Conclusions

Thus, this paper presents an innovative method for obtaining biocompatible and thermoplastic material with chitosan content up to 33 wt.% based on its block copolymer with polycaprolactone. The proposed method is based on the synthesis of a chitosan block copolymer with thermoplastic polyester by ultrasonic irradiation on a solution of a homopolymer mixture using DMSO as a common solvent. So far, such an approach has not been identified in publications. Chemical bonds between blocks of chitosan and PCL in macromolecules prove, on the one hand, the homogeneity of the system, and on the other hand, the block structure prevents the formation of crystallites of both chitosan and PCL and, a consequence, the absence of phase separation during processing. The study of melt flow and mechanical properties of copolymers with different compositions allowed us to determine the smallest share of polycaprolactone, providing thermoplasticity and necessary mechanical properties of the composition. The copolymer containing 33 wt.% of chitosan units and 67 wt.% of caprolactone units meets these requirements. The filament based on this copolymer with a tensile strength of 25 MPa and an elongation at break of 38% can be produced by extrusion at a temperature of 75 °C. The results of biological studies showed that cell adhesion, cytotoxicity, and proliferative activity of the material remain at the level of pure chitosan. The summary of the results determines the prospects for the development of biomedical products based on chitosan block copolymer with polycaprolactone by additive technologies.