Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation

Journal Pre-proof Tailoring the collagen film structural properties via direct laser crosslinking of starshaped polylactide for robust scaffold formation K.N. Bardakova, E.A. Grebenik, N.V. Minaev, S.N. Churbanov, Z. Moldagazyeva, G.E. Krupinov, S.V. Kostjuk, P.S. Timashev PII:

S0928-4931(18)34011-6

DOI:

https://doi.org/10.1016/j.msec.2019.110300

Reference:

MSC 110300

To appear in:

Materials Science & Engineering C

Received Date: 31 December 2018 Revised Date:

12 September 2019

Accepted Date: 9 October 2019

Please cite this article as: K.N. Bardakova, E.A. Grebenik, N.V. Minaev, S.N. Churbanov, Z. Moldagazyeva, G.E. Krupinov, S.V. Kostjuk, P.S. Timashev, Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110300. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1.Extraction of Collagen from Bovine 1.NaOH (2.5 M), Na2SO4 (0.85 M) 2. Na2SO4 (0.85 M) 3. H3BO3 (4%) 4. CH3COOH (0.5 M, 0.25 M)

2.Рreparation of Photo-Crosslinked Collagen film casting/ evaporation

UV exposure

collagen solution

3.Pattern Formation via Laser Crosslinking

washing with

photoactive laser material* crosslinking

1. CH2Cl2 2. C2H5OH

FMN solution (5mM)

collagen film cross-linked collagen film

365 nm 30 min

Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation K.N. Bardakovaa,b,*, E.A. Grebenika, N.V. Minaevb, S.N. Churbanova,b, Z. Moldagazyevac, G.E. Krupinovd, S.V. Kostjuka,e,f, P.S. Timasheva,b,g a

Institute for Regenerative Medicine, Sechenov University, 8-2 Trubetskaya st., Moscow 119991, Russia b Institute of Photonic Technologies, Research center “Crystallography and Photonics”, Russian Academy of Sciences, 2 Pionerskaya st., Troitsk, Moscow 108840, Russia c Almaty technological university Chemistry, chemical technology and ecology department Baytursynov Street, Almaty 050000, Kazakhstan d Institute for Urology and Reproductive Health, Sechenov University, 2-1 Bolshaya Pirogovskaya st., Moscow 119435, Russia; e Research Institute for Physical Chemical Problems of the Belarusian State University, 14 Leningradskaya st., Minsk 220030, Belarus f Department of Chemistry, Belarusian State University, 14 Leningradskaya st., Minsk 220006, Belarus g Semenov Institute of Chemical Physics, Russian Academy of Sciences, 4 Kosygina st., Moscow 119991, Russia Corresponding author; E-mail address: [email protected] Abstract Application of restructured collagen-based biomaterials is generally restricted by their poor mechanical properties, which ideally must be close to those of a tissue being repaired. Here, we present an approach to the formation of a robust biomaterial using laser-induced curing of a photosensitive star-shaped polylactide. The created collagen-based structures demonstrated an increase in the Young’s modulus by more than an order of magnitude with introduction of reinforcing patterns (from 0.15±0.02 MPa for the untreated collagen to 51.2±5.6 MPa for the reinforced collagen). It was shown that the geometrical configuration of the created reinforcing pattern affected the scaffold’s mechanical properties only in the case of a relatively high laser radiation power density, when the effect of accumulated thermomechanical stresses in the photocured regions was significant. Photo-crosslinking of polylactide did not compromise the scaffold’s cytotoxicity and provided fluorescent regions in the collagen matrix, that create a potential for noninvasive monitoring of such materials’ biodegradation kinetics in vivo.

Keywords: collagen; mechanical properties; photopolymerization; biocompatible polymers; reinforcements; riboflavin

1. INTRODUCTION Collagen is a fibrous natural protein which is a dominating substance in all the biological tissues (the basic component of skin, bones, cartilage), it is extremely cell-friendly, hydrophilic and undergoes enzymatic biodegradation [1–3]. After collagen separation and reconstruction, a significant reduction of the mechanical and wear properties is observed for the created materials in comparison with the intact tissues [4]. Therefore, collagen-based 3D matrices undergo enzymatic and hydrolytic degradation, while addition of cells may lead to spontaneous collagen gel formation [5]. Disordering of the collagen structure also results in the worsening of the manipulative properties of collagenbased materials. Thus, it is of importance to develop different approaches for the improvement of the manipulative and mechanical properties (as well as of the enzymatic resistance) of collagen matrices. For example, collagen materials may be modified with various fillers. There are approaches which involve addition of bioactive glass nanoparticles [6], silk fibroin [7], different biomolecules, such as chitosan [8,9] and elastin [10,11], combination of collagen with synthetic networks [12]. The protocols on the reinforcement of collagen materials via plastic deformation or using various external loads (compression or extension) were also reported [13–15]. Such approaches generally initiate orientation of fibrils which leads to the mechanical anisotropy of a matrix. The most wide-spread approach for modifying collagen matrices is their crosslinking via different techniques. The crosslinking techniques are divided into physical, chemical and enzymatic [16]. Physical or photochemical crosslinking is an efficient way of collagen strengthening and has a reduced toxicity compared to other methods [13]. UV-irradiation forms bonds through aromatic tyrosine and phenylalanine residues, which are less important for the biological activity of collagen than, for example, cross-links through carboxylate anions forming during chemical crosslinking with carbodiimide [17]. In this article, we present an original scheme for tailoring the collagen film, which is realized via the combination of photochemical crosslinking of collagen in the presence of flavin mononucleotide (FMN) as a photoinitiator and directed laser-induced coating with

reinforcing structures based on star-shaped polylactide possessing reactive methacrylate groups (Fig.1).

Fig.1. Basic stages of the suggested approach to the robust scaffold formation. *The photoactive material (step 3) contains the photosensitive tetrafunctional poly(D,L-lactide) as the basic component and 4,4'-bis(diethylamino)benzophenone as a photoinitiator for polymerization.

FMN is known to absorb in the region of 330-470 nm [18], being a nontoxic photoinitiator, and, in particular, when excited by the light, it may initiate formation of superoxide radicals (O2•−) which subsequently induce a number of specific reactions [19]. As shown in [13], riboflavin-crosslinked collagen demonstrates a higher affinity to water, higher fibril thickness, as compared to non-crosslinked control samples, that leads to higher values of elastic moduli. However, such an approach does not allow regulation of the obtained matrix’s mechanical properties in a wide range. As an additional treatment, we suggest a technique of laser-induced formation of orientation patterns based on a biocompatible photosensitive starshape polylactide. In our previous study [20], laser-induced coating with polylactide allowed increasing the Young’s modulus of a porous collagen sponge and achieving an oriented growth of fibroblasts. Note, that such a technique creates regions with not only different mechanical properties, but also different chemical composition, that may be used in the future for directed stem cell differentiation [21,22]. Based on the above, the general hypothesis of this study was the following: laserinduced photocuring of photosensitive polymer compositions placed onto the surface of collagen materials allows creation of materials with specifically distributed surface and mechanical properties. To verify this hypothesis, we performed photocuring of collagen films

in the presence of flavin mononucleotide to obtain a relatively stable collagen construction for the subsequent laser reinforcement. After the photocuring, we placed a mixture of photosensitive polylactide onto the film surface and selected the parameters of laser patterning for patterns of different geometry (see Fig.1). Then, the physico-chemical and mechanical properties of the prepared samples were studied to determine the influence of the created patterns’ geometry on these properties. Besides, a complex in vitro analysis was performed for the cytotoxicity evaluation. 2. MATERIALS AND METHODS 2.1. Separation of collagen and preparation of a collagen film The technique of collagen separation is presented in detail in [20]. Briefly, fragments of dermis (a medium layer of the cattle dermis was used) with the size of 5×5 cm were placed in a solution containing NaOH (2.5 М) and Na2SO4 (0.85 М) for 48 hrs at 20оС and periodic stirring. Further, the fragments of dermis were rinsed in a 0.85 М Na2SO4 solution for 6 hrs, with the following storage in a 4% boric acid solution until the complete alkali’s neutralization at the cross-section. The degree of neutralization was determined quantitatively by the reaction with phenolphthalein. The neutralized samples were rinsed with distilled water until a negative test for the sulfate presence, then they were placed into a 0.5 M acetic acid solution until the complete dissolution. The obtained solution was purified by the collagen deposition with a NaCl solution (12 wt%), the precipitate was separated by centrifugation (20 min, 3000 rpm) and again dissolved in an acetic acid solution (0.25 М). To remove the salts, the prepared solution was dialyzed against a 0.25 M acetic acid solution for 24 hrs. The collagen solution (1 wt%) was poured in polymeric (polystyrene) cuvettes to the layer thickness of 8-10 mm and dried at a temperature of 22-24 ºС to the residual humidity of 10-12%. The prepared film thickness was equal to 100 µm. 2.2. Photocrosslinking of a collagen film Square samples with the side width of 15 mm were cut out from a collagen film. Each square was placed into a 5 mМ aqueous FMN solution for 40 min. Then, the samples were washed for a day in distilled water. The quantity of impregnated FMN was estimated by means of spectrophotometry, measuring the optical density at the wavelength of 444 nm (the FMN absorption band) with a Cary-50 spectrophotometer (Varian Optical Spectroscopy Instruments). Photocrosslinking of collagen films was conducted using a LED source with a wavelength of a 365 nm (Epileds, Taiwan) at the intensity of 3.9 mW/cm2. The samples were

irradiated from both sides, the total duration of irradiation was 5, 7.5, 10, 20 and 30 min, for the selection of the optimal mechanical properties of a sample. The local mechanical characteristics for the initial collagen film and for the irradiated samples were measured with a Piuma Nanoindenter (Optics11). A cantilever with a spring constant of 0.45 N/m and the tip radius of 26.5 µm was used. The measurements were performed according to [23] in distilled water at 37 оС, the scan size was 300×300 µm with a 5 µm step by the X and Y axes. Using the Hertzian model, we calculated the effective Young’s modulus and created distribution maps of the Young’s moduli over the surface. 2.3. Selection of the laser irradiation speed During the first stage, a photosensitive composition was prepared by the following technique. A charge of tetrafunctional branched poly(D,L-lactide) (Мntheor=2880 g/mol; Mn(NMR)=2400 g/mol), obtained according to [24], was dissolved in dichloromethane until a 5wt% polymer solution, then the 4,4'-bis(diethylamino)benzophenone photoinitiator (1wt% in the final composition) was added. The composition was left under stirring for the whole night. After that, the collagen samples were covered with the polylactide-based photosensitive composition (about 150 µL) and dried at room temperature for 6 min. The reinforcement parameters selection was performed at a setup for laser stereolithography designed at the Institute of Photonic Technologies, Research center “Crystallography and Photonics” (Troitsk, Russia), see a detailed description in [25]. The irradiation was arranged from below, using a MDL-III-405 laser (CNI-Laser) as a source, with the wavelength of 405 nm and the maximum radiation power of 100 mW. The irradiation was focused by means of an F-theta objective SL-405-100-160, providing a spot with a diameter of 50 µm in the focal plane. The laser spot movement was performed by a galvano scanner. On a collagen film, a set of separate lines of the crosslinked polylactide was created at the laser speeds of 1, 2, 3, 4, 5, 10, 15, 20 mm/s, thus presetting a various power density. The laser power was constant and equal to 70 mW. Lines with the length of 12 mm, at a distance of 1.5 mm from each other were formed. To wash a sample from uncrosslinked polymer, it was placed into dichloromethane for 4 hours, then – into ethanol for 2 days. To estimate the width of the created lines after the 3D printing, a KOZO XJF900 (KOZO OPTICS, China) fluorescent microscope in combination with a UCMOS14000KPA (ToupCam, China) digital camera was used. The topography of individual created lines was estimated by SEM with a Phenom Pro X. (Phenom-World BV., Netherlands) instrument. The width of the formed lines and their 3D relief after washing were evaluated with a Huvitz HRM (Huvitz, South Korea) 3D microscope.

2.4. Pattern creation Collagen films (15×15 mm; in addition, the rectangular films of 24×8 mm were prepared for the subsequent stretching tests) were coated with the photosensitive composition similarly to the procedure described in Section 2.3. For the laser irradiation speed of 15 mm/s we used the reinforcing lines density of 8 lines/mm, for the laser speed of 3 mm/s the density was 3 lines/mm. Two configurations were created, using one-axis scanning (striped samples) and two-axes scanning (gridded samples). The pattern configuration was preset with the galvano scanner control software LDesiner 5.0. After the pattern creation, the samples were washed in dichloromethane for 2 days and in ethanol for 4 days. 2.5. Pattern characterization To visualize the created patterns after the 3D printing, a KOZO XJF900 (KOZO OPTICS, China) fluorescent microscope in combination with a UCMOS14000KPA (ToupCam, China) digital camera was used. The topography of the created patterns and their 3D relief were estimated by SEM with a Phenom Pro X. (Phenom-World BV., Netherlands) instrument and by a Huvitz HRM (Huvitz, South Korea) 3D microscope. To determine the water swelling, the created patterns were soaked in water and sandwiched between two pieces of filter paper to remove the excess of moisture. The weight of the samples was recorded prior to and after drying at 60 °C for 20 h. The swelling was expressed as a relative change (in percent) in the sample weights (wet and dry). IR spectra were acquired with a FT-IR Spectrum Two (PerkinElmer, Inc., United Kingdom) spectrometer in the ATR mode with a resolution of 4 cm-1 and averaged over 8 scans. Each collagen spectrum was calibrated by the intensity of the С-Н vibration (2932 cm1

) and underwent the Fourier-transform smoothing procedure by 4 points. A Spotlight 200i (PerkinElmer, Inc., United Kingdom) FT-IR Microscope with a MCT

(mercury cadmium telluride) MIR detector and PerkinElmer SpectrumIMAGE software were used. The reflection spectra of the patterns were performed with a 18×18 measurement-grid, each grid cell with an individual aperture of 50×50 µm. The images were reconstructed at the wavenumber of 1547 cm-1. The stretching tests of the initial collagen films and the created patterns were performed in air at room temperature with the speed of 5 mm/min using a Shimadzu EZTest EZ-SX (Japan) universal testing machine. The effective specimen size studied was 12×8 mm. The initial collagen films and the created patterns after washing from uncrosslinked polymer

were hydrated with phosphate buffer saline (PBS) for 2 days. The samples were fixed in the grips using abrasive paper. All stretching tests were performed in a direction parallel to the created reinforcing lines. Three specimens of each group of samples were tested. 2.6. Cytotoxicity evaluation In order to evaluate the influence of the reinforcement procedure on the collagen film cytocompatibility, we performed a complex in vitro analysis. Extraction test was adopted from ISO 10993-5 for estimation of the cytotoxicity of soluble compounds of the intact and reinforced films. The contact cytotoxicity analysis was based on the measurement of lactate dehydrogenase (LDH) release from 3T3 murine fibroblasts cultured on the reinforced films. Directed cellular growth on the films with the orientation patterns was demonstrated using fluorescence microscopy assisted with a vital dye set. 2.6.1. MTT-test The extraction test was based on the measurement of MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide]-reductase activity of 3T3 murine fibroblasts exposed to the extracts of the intact and reinforced films. The extracts were prepared by 24-h incubation of the ethanol-sterilized films (total surface area = 6 cm2) in 1 ml of DMEM/F12 culture medium supplemented with 100 U/mL streptomycin, 100 g/mL penicillin, 1 Vol.% GlutaMAX (Gibco) and 5 Vol.% fetal bovine serum (FBS, HyClone). 3T3 murine fibroblasts were seeded in 96-well plates at the concentration of 5×103 cells per well and cultured in a humidified 5% CO2 incubator at 37 ºC in the culture medium supplemented with 10 Vol.% FBS for 24 h. After that, the culture medium was discarded and serial dilutions of the extracts were added to the wells for 24 h. Then MTT-reductase activity of the cells was estimated in relation to untreated cells (negative control). Sodium dodecyl sulphate (SDS) solution was used as a positive control in the concentration range from 5 to 200 µg/ml. For the MTT-test, the extracts and control media were replaced with 100 µL of the MTT solution (0.5 mg/mL in the medium without supplements) followed by 3-h incubation in a CO2-incubator at 37 °C. After discarding the MTT solution, 100 µL aliquots of dimethyl sulfoxide were added to all the wells and mixed. The colour developed was quantified by measuring absorbance at 550 nm using the Multiscan FC microplate photometer (reference, 650 nm). 2.6.2. LDH-test The cellular membrane integrity of 3T3 murine fibroblasts cultured on the reinforced collagen films was determined by measurement of LDH release indicating cell damage. 3T3

murine fibroblasts were seeded on the films with orientation patterns in a 96-well plate at a concentration of 1×105 per well and cultured for 24 h in the culture medium with 10 Vol.% FBS. The released LDH activity was measured in the supernatants using an LDH enzymatic assay kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The optical densities at the wavelengths of 490 nm and 640 nm were measured using a VICTOR Nivo Multimode Microplate Reader (Perkin Elmer). The lysis buffer was added to the control wells to estimate total LDH activity of the cells (max value). The results are expressed as percent of the max value. The cells alone were cultured on tissue culture polystyrene (TCPS) to estimate spontaneous LDH release. 2.6.3. Directed cell growth Directed cell growth was visualized using a primary culture of murine fibroblasts isolated from 13-day-old embryos of murine line C57BL/6-Tg(ACTbEGFP)1Osb/J. At the passage 5, the fibroblasts were seeded on the ethanol-sterilized intact and reinforced films in the culture medium with 10 Vol.% FBS and cultured at 37 оC in a humidified atmosphere with 5% СО2. After 72 h in culture, the cells were stained with propidium iodide and SYTO 9 (Invitrogen, USA) and visualized using fluorescence microscope Axiovert 200 (Karl Zeiss, Germany) in order to record the green living cells and the red dead cells. 2.7. Statistical Analysis The obtained results were reported as mean ± standard deviation (SD). Data were analyzed by Student t-test with significance level p-value less than 0.05. SPSS19.0 software (USA) was used for statistical analysis. 3. RESULTS AND DISCUSSION 3.1. Photochemical structuring of collagen The photoinitiator concentration and the dose of laser radiation absorbed by the material determine both the mechanical properties of the formed matrix [25] and the viability of cells cultured at its surface [26]. As shown by spectrophotometry, the collagen film storage in the FMN solution for 40 min leads to the photoinitiator concentration in the film of about 0.26 mM. As demonstrated in [27], low concentrations of riboflavin (0.03-0.1 mM) suffice for photocuring of collagen hydrogels in the successful injection therapy for annulus fibrosus repair. The authors in [2] relate the decrease in the cell viability to the high riboflavin concentration (0.5 mM) they used and note the necessity of applying lower photoinitiator

concentrations. Thus, the FMN concentration utilized in our experiments is sufficient for the collagen sample photocuring at the next stage. At the same time, such an FMN concentration (0.26 mM) is not supposed to diminish the photocured collagen biocompatibility. Using nanoindentation, we measured the change in the elastic modulus of collagen films resulting from the UV irradiation. Photocuring in the presence of FMN as a photoinitiator allowed an increase in the elastic modulus of collagen by 2.6 times: the elastic modulus grew from 64.5±14.1 kPa (initial collagen) to 156.8±40.4 kPa (irradiated collagen) after 20 min of irradiation (Fig.2a). According to [13], one may obtain a similar increase in the elastic modulus of collagen constructs using a combination of photochemical curing and the plastic compression method. The studies [29,30] report also the growth of elastic modulus by 2.5 times following irradiation of enucleated porcine eyes and rabbit sclera, respectively, in similar conditions. In our experiment, the collagen samples receive the dose of approx. 4.7 J×cm-2 for 20 min of irradiation. As shown in [31,32], such a dose is sufficient for obtaining crosslinked hydrogels at the chosen photoinitiator concentration. Note, the studies on the keratoconus treatment with riboflavin and UV irradiation report spatial non-uniformity of the corneal collagen photocuring, with the crosslinking maximum observed within 300 µm of the top surface [32]. In our experiment, a relatively uniform distribution of the elastic modulus over the surface of photocured collagen films was found (Fig.2b).

Fig.2. (a): The Young's modulus of collagen films at different UV irradiation times. (b): An example of the Young's modulus distribution over the surface of a collagen film irradiated for 7.5 min.

The uniform distribution is provided by the initial transparency and low thickness of the collagen film, as well as bilateral photocuring of the collagen sample. For the further experiments, the samples were irradiated for 30 min.

3.2. Selection of the laser radiation speed In laser curing of the photosensitive polylactide and collagen film (at a constant power), the laser speed determines the power density. Hence, by regulating the speed of the laser spot movement over the matrix surface, one can select optimal geometric dimensions of the reinforcing pattern, as well as the laser radiation parameters, in order to reduce or eliminate a potential destructive effect on collagen. We performed the selection of the laser radiation speed based on the shape and geometric dimensions of the created lines. Besides, we took into account the stability (preservation) of the created reinforcing lines after their washing from the uncured composition.

Fig.3. SEM micrograph after the laser treatment for polylactide structured lines on a collagen film at the laser speed of 1 mm/s (а) and 2 mm/s (b). In (a) the following is additionally marked: 1 – a cured polylactide line; 2 – collagen film; 3 – substrate. The appearance of the created lines at the laser speed of 4 mm/s (с) and 10 mm/s (b). The lateral (X,Y) grid dimension is 150 µm, the vertical (Z) grid dimension is 50 µm.

In Fig. 3a, a depression and a crack in the structured band of polylactide are clearly visible for the lowest laser speed of 1 mm/s, the crack is also observed for the speed of 2 mm/s in Fig. 3b. The depression decreases when the laser speed is increased, at the laser speed of more than 3 mm/s it disappears (Fig.3c, d). The crack appearance and destruction of

the photocured line is most probably related to the duration of the laser irradiation or the high radiation power density. Application of the photosensitive composition of tetrafunctional polylactide as the basic component results finally in the formation of crosslinked polymers with both linear and star-shaped macromolecules. Thus obtained crosslinked polymers not only lose their ability to dissolve, but also become excessively fragile due to reduction of their molecular lability, that leads to the formation of microcracks after the additional laser treatment [33]. Among the causes for the superficial layer shrinking and microcrack formation in polymer samples, the following factors are also mentioned: accumulation of thermomechanical stresses and changes in the conformations of macromolecules [34], formation of gaseous products resulting from breaking carbon-carbon and carbon-oxygen bonds in polymers [35,36]. Note, that the laser treatment of a high intensity may lead not only to destruction of the cured polylactide, but also to creation of a rough surface during bubble formation [37]. When the speed of the laser radiation is increased (for the speeds of 10-20 mm/s), the photocured line becomes less uniform in its height. At the speed of 20 mm/s the trace of the laser movement is almost absent visually, as the line is very heterogeneous, thin, absent or barely visible at some sites of the collagen film. A shorter and, hence, less intensive laser treatment results in the formation of looser sparsely crosslinked polymer networks. Their formation occurs mainly due to the “rapid” polymerization stage [38], when diffusion and the growth of “small” labile radicals prevail. During washing of the created structures, the solvent molecules may destroy weakly polymerized lines, that is clearly illustrated in Fig.4.

Fig.4. The created lines’ width immediately after the laser treatment (the results are obtained from the 3D microscope, based on fluorescence photographs) and after washing from the uncured polylactide (the results are obtained using 3D microscopy), depending on the laser speed (1, 2, 3, 4, 5, 10, 15, 20 mm/s).

The width of the created polylactide lines after washing from the uncured photosensitive composition decreases by about 2 times for the laser radiation speeds of 10 – 20 mm/s. A minimal washing out of the photocured lines is observed for the laser radiation speeds of 1 – 5 mm/s. In the latter case, washing out occurs most probably due to the presence of microcracks in the lines, as discussed above. For the further experiments on the creation of reinforcing patterns at the collagen surface, we selected two speeds of the laser treatment – 15 mm/s and 3 mm/s. In the first case, a reinforcing pattern consisting of densely located thin polymerized lines would be created with a high productivity, while in the second case the pattern would consist of wide, high and sparse lines. 3.3. Formation of reinforcing patterns

Fig.5. Macrophotographs of reinforced matrix created at the laser speed of 3 mm/s: (a) pattern in the form of a grid immediately after the laser-induced process (the aluminum window was used for the sample fixation); (b) pattern in the form of a strip after the washing from the uncured polymer composition.

As seen in Fig.5a, the created photocured polylactide lines are clearly visible on the matrix immediately after the laser-induced process. In contrast to the initial collagen film and collagen film coated with the polymer composition, the sites of laser treatment are opaque, of the light-grey color. After photocuring, the collagen film loses plasticity, becomes stiff and fragile. Such properties of the reinforced matrix are determined by the uncured polymer composition solidified on the collagen surface. However, after the washing from the uncured polymer composition the properties of the reinforced matrix are close to the properties of the initial collagen film. Water swelling of the reinforced matrix is found to be 95±2%. The reinforced matrix restores its shape after various twisting and manipulations with tweezers,

may acquire both valley-like and tubular shapes. The macrophotograph (Fig.5b) also clearly demonstrates the 3D character of the created pattern.

Fig.6. Microphotographs after the reinforcement and SEM images of patterns at the laser speed of 15 mm/s (a, b) and 3 mm/s (c, d)

At the micrographs displayed in Fig.6, one can see the brightest regions at the loci undergone the laser treatment and, hence, polylactide photocuring. The fluorescence is most likely induced by incomplete photobleaching of the 4,4'-bis(diethylamino)benzophenone photoinitiator, as well as the formation of the crosslinked and fluorescent polymer network, but this does not affect the cytotoxicity of the samples as shown below. Formation of such fluorescent regions on top of the collagen material in an advantage of the suggested treatment of biopolymer matrices, since allows studying their further biodegradation in vivo without the need of developing complicated histological procedures and sacrificing experimental animals. One may note a high heterogeneity and coarseness of the created polylactide lines for both speeds of the laser spot movement. This coarseness remains after washing of the reinforced matrices, as well (Fig.6b, d), the SEM-micrographs demonstrating the more pronounced porous relief for the laser speed of 3 mm/s. Such a relief may be formed not only

due to bubble formation [37] at the high intensity of the laser treatment, but also due to destruction of the crosslinked structure during its washing. In the case of the 3 mm/s speed, a possible cause may be related to the thermomechanical stresses.

Fig. 7. Reinforced matrices, created at the laser speeds of 15 mm/s (a,b) and 3 mm/s (c,d). The grid size is 500 µm by the X,Y-axes.

3D images of created reinforced matrices (Fig.7) demonstrate a good reproducibility of the preset relief. The reinforcement is performed uniformly over almost the entire surface of collagen films. The obtained geometric parameters of the patterns are presented in Table 1.

Laser speed (mm/s) 3 15

Table 1. 3D-microscope-derived dimensions. Line width Distance between lines (µm) (µm) 97±23 175±23 23±12

70±12

The obtained width of the cured lines depends on the power density of the laser radiation, the distribution of which is well approximated by the Gauss function. At low speeds the effective profile of the cured region is wider, due to the more intense laser action causing the polymerization process in the photosensitive composition.

3.4. IR spectra and IR microscopy

Fig.8. (a): IR spectra: 1 – cured photosensitive polylactide-based composition; 2 – initial collagen film; 3 – photocured collagen film; 4 – reinforced pattern on collagen. (b) and (c): IR maps of reinforced matrices (laser speed of 3 mm/s), reconstructed at the wavenumber of 1547 cm-1.

The IR spectrum 1 of the photocured polylactide (Fig.8а) contains absorption bands typical for lactide chains, namely: 1746 cm-1 – stretching vibration of the carbonyl group С=О, 1456 and 1385 cm-1 – bending vibrations of СН3, 1185 cm-1 – asymmetrical stretching of С-О-С, 1083 cm-1 – symmetrical stretching of СН3, 1045 cm-1– С-СН3 stretching [39]. The spectra (2), (3), (4) in Fig.8а, as was expected, were obtained with the bands of peptide bonds characteristic of collagen [13], namely, Amide I at ̴ 1630 cm-1 (C=O stretching), Amide II at ̴ 1547 cm-1 (in-plane N-H bending and C-N stretching) and a triplet of weaker Amide III

bands with the maximum at ̴ 1236 cm-1 (C-N stretching and in-plane bending of C-N-H). Note that, in the spectrum 3 for the photocured collagen film and in the spectrum 4 for the reinforced matrix (Fig.8а), the position of Amide I band increases to 1633 cm-1. This fact may indicate collagen denaturation after irradiation and laser treatment. However, there is no change in the intensity of Amide I band, and the band positions of Amide III, which are very sensitive to denaturation and conformational transitions of collagen molecules. Hence, the Amide I band shift most probably takes place as a result of hydrogen bonds strengthening with the hydration of collagen macromolecules. In the spectra 3 and 4 of irradiated collagen film and reinforced matrix, respectively, the peaks at 1086, 1046 and 879 cm-1 are present, which are not observed in the initial collagen film. Note also that the peak at 1046 cm-1 for the pattern on collagen is more intense than that for the irradiated film, which may be attributed to the addition of polylactide into the system. We also relate the appearance of a peak at 1727 cm-1 assigned to carbonyl groups according to our previous study [24] to polylactide. The clearly visible changes observed after collagen irradiation in the spectra 3 and 4 at 1086 and 879 cm-1 most probably may be assigned to vibrations of ester and epoxy groups. The bright regions at the IR maps of reinforced matrices (Fig. 8b, c) show the locations of the collagen material, hence, the dark regions point where collagen is covered with the photosensitive polylactide. As can be seen, for the striped patterns the photosensitive material is washed out rather well. For the gridded patterns, on the contrary, the washing was shown not to result in a thorough removal of the uncured polymer. It is also possible that the IR-microscope visualizes the residual cross-linked polylactide which was washed out from the polymerized regions due to their fragility. 3.5. Mechanical properties of reinforced matrices

Fig.9. Typical stress-strain curves of reinforced matrices created at the laser speeds of 15 mm/s (a) and 3 mm/s (b) compared to the initial irradiated collagen film. Inset: the schematization of the behavior of the matrices for each region.

Table 2. The results of the mechanical testing of reinforced matrices created at the laser speeds of 15 mm/s and 3 mm/s. Data are represented as the mean ± SD (n = 3, p<0.05) Elastic modulus Strain at fracture (MPa) (%) Initial irradiated collagen film 0.15 ± 0.02 22.6 ± 2.5 V = 15 mm/s grid 49.5 ± 5.4 16.5 ± 1.8 stripes 51.2 ± 5.6 15.8 ± 1.7 V = 3 mm/s grid 2.1 ± 0.2 55 ± 6.1 stripes 13.5 ± 1.5 33.4 ± 3.7

As has been shown by the mechanical testing (Fig.9a, b), the elastic modulus of the reinforced matrices exceeds that for the photocured collagen sample (Table 2). This finding testify the ability of the created reinforced matrices withstand significantly high stresses, both for reversible and irreversible deformation, in comparison with the samples which underwent only photocuring. Note some observed tendencies for the different laser speeds and configurations of the reinforcing pattern. For the laser speed of 15 mm/s the strength of the reinforced matrix does not depend on the geometric configuration of the pattern. For the lower speed of 3 mm/s, in contrast, the elastic modulus increases by 6 times: from 2.1±0.2 MPa to 13.5±1.5 MPa for the gridded and striped samples, correspondingly. The difference in the strength for the laser speed of 3 mm/s for the different geometric configurations of the pattern is most likely determined by the influence of arising mechanical stresses during the prolonged laser action. The larger the area of the matrix which underwent the laser treatment, the more microcracks are to be present in the sample structure. Indeed, as follows from the calculations based on Table 1, at a given laser speed, for the gridded pattern configuration approximately 63% of the matrix surface area is occupied by the photocured polylactide. For the striped configuration this value is about 39% of the surface area. The value of the matrices’ strain at fracture also depends on the laser treatment parameters, and for the speed 3 mm/s it depends also on the configuration of the reinforcing pattern (Table 2). For the laser speed of 3 mm/s, large non-diffusion radicals most probably begin to participate in the polymerization [38], more bonds forms with the participation of long polymer chains. Polylactide in our matrix plays a role of a distinctive plasticizer when the increase of the carbon chain length of a plasticizer leads to a significant growth of the strain. As in most cases with adding plasticizers to polymeric materials, one has to accept an essential reduction in the strength at a significant elongation of samples.

3.6. Cytocompatibility evaluation MTT-test for the cytotoxicity evaluation of soluble compounds of the collagen films before and after the reinforcement did not reveal any dose-dependent differences between the sample extracts. The decrease in the cell proliferation rate with the increasing extract concentration is accounted for the presence of acetic acid traces. SDS was used as a positive control to validate the method and caused remarkable cell death (IC50 ~ 0.03 mg/ml).

Fig.10. Cytocompatibility evaluation of the intact and reinforced collagen films. a and b – Cell survival curves following exposure of 3T3 murine fibroblasts to the extracts from the films based on MTT-reductase activity measurements (a). Sodium dodecyl sulphate (SDS) is used as a positive (cytotoxic) control (b). c and d – Visualisation of living (c) and dead (d) primary murine fibroblasts on the reinforced collagen films with orientation patterns. Scale bar = 100 µm.

The release of LDH from the 3T3 murine fibroblasts cultured on the reinforced films (6.1±0.8 %max) was close to that of the cells cultured on TCPS (3.5±0.7 %max). The results indicate low level of LDH leakage from the cells after 24 h in culture on the film. The slight LDH signal in the extracellular medium is accounted to the spontaneous release due to lysis of non-adhered cells. Directed cellular growth on the films with the orientation patterns is illustrated in Fig.10 c, d. Living cells are stained green indicating high cytocompatibility of the films. To conclude, the proposed method for reinforcement of collagen films takes a benefit of the increased stability, cytocompatibility and directed cell growth.

4. CONCLUSION In this study, we demonstrate a principal possibility of the laser treatment application for the selection of mechanical and surface properties of collagen-based biopolymer matrices. Varying the power density of the laser radiation and the speed of the laser spot movement over the sample surface, one may regulate the parameters of the photo-crosslinked regions from which the reinforcing pattern is created, as well as change their topology and stability during washing. The geometric configuration of the created reinforcing pattern affects the mechanical properties of the whole matrix only in the case of low speeds of laser radiation (high power density), when the influence of the accumulated thermomechanical stresses is essential in the photocured regions. At high laser speeds (low power density) the strength of the reinforced matrices does not depend on the geometric pattern configuration and significantly exceeds the strength of matrices created at low laser speeds. The developed technique of laser-induced curing allowed also creation of fluorescent regions from crosslinked polylactide that will allow noninvasive studies of biodegradation in vivo in the future. The orientation patterns enabled directed cell growth without affecting cytotoxicity of the collagen matrices. Funding This study was supported partially by the Russian Foundation for Basic Research (Grant 17-02-00445, biodegradable scaffold formation and Grant 18-32-00222, the laser and ultraviolet radiation treatment of hydrogel scaffolds), partially by the Russian Science Foundation (Grant 15-15-00132, the collagen processing), partially by the Russian academic excellence project ‘5-100’ (pattern characterization), and partially by the Ministry of Science and Higher Education within the State assignment FSRC «Crystallography and Photonics» RAS (3D laser printing). Acknowledgement The authors thank Kotova S.L. (Sechenov University, Russia) for providing language help, Prof. Istranov L.P. and Istranova E.V. (Sechenov University, Russia) for providing the collagen solution and Selezneva I. I. (Institute for Theoretical and Experimental Biophysics, Russian Academy of Sciences) for primary fibroblast cultures.

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Highlights •

Developing an approach to the formation of robust collagen-based scaffolds using laser-induced curing of a photosensitive polylactide;

•

Improvement of the elastic mechanical properties of the reinforced collagen-based scaffolds by more than an order of magnitude (from 0.15±0.02 MPa for the untreated collagen to 51.2±5.6 MPa for the reinforced collagen);

•

The fluorescent regions in the collagen scaffold were fabricated, that creates a potential for noninvasive monitoring of such materials’ biodegradation kinetics in vivo.

Kseniia Bardakova is the junior researcher of the Institute for Regenerative Medicine of Sechenov University and also the junior researcher of the Institute of Photonic Technologies (Russian Academy of Sciences). Bardakova graduated from the Dmitry Mendeleev University of Chemical Technology of Russia in 2016. Her research interests are mainly focused on biopolymers, their characterization and applications in regenerative medicine.

Dr. Ekaterina Grebenik obtained her PhD in Biophysics at Macquarie University in Australia, followed by post-doctoral research in tissue engineering at Sechenov University in Russia. Her research interests are mainly focused on engineering nervous, bone and epithelial tissues.

Minaev Nikita has received a specialist degree in quantum electronic and solid materials physics in Moscow Engineering-Physical Institute (State University) in 2003-2008. After that, since 2008 he has been working at the Institute of Photonic Technologies of the Federal Research Center "Crystallography and Photonics" of the Russian Academy of Sciences. In 2015 he has received his Ph.D. degree in quantum electronic. Currently, he is head of the Laboratory of Laser Nanoengineering, in which research is being conducted on the development of new laser technologies for photonics and biomedicine.

Churbanov Semyon obtained a specialist degree in physical chemistry (2016) in Dmitry Mendeleev University of Chemical Technology of Russia, Institute of Modern Energetics and Nanomaterials. Current research interests include: (1) polymer functionalization with biomedical and biotechnological interest including smart stimulus-responsive biomaterials (2) characterization of biomaterials and medical devices at the nano- and micro-scale level; (3) biomaterials processing in supercritical CO2.

Moldagazyyeva Zhanar Ph.D of Chemical Sciences, Associate Professor of the Department of Chemistry, Chemical Technology and Ecology of the Almaty Technological University, graduated from the Kazakh State University. Al-Farabi, (bachelor degree-1998, magistracy-2000) on specialty “Highmolecular compounds”, defended her thesis under the guidance of academician Yergozhin EE, on the topic: “The study of radical homo-and copolymerization of new allyl redox molimers”. She studies the problems of polymer chemistry, ecology, the author of more than 60 works in scientific and pedagogical fields.

Dr. German Krupinov graduated from the Sechenov Moscow Medical Academy in 1997. In 1999, he graduated from residency in urology. In 1999, he gained his PhD degree, and in 2010 DSc degree. He was engaged in scientific, medical and educational activities at the Department of Urology. In 2010, he became a professor of the Department of Urology. Since 2018, he has been working as a professor of the Institute for urology and reproductive health. His research interests cover the diagnostics and minimally invasive treatment for prostate diseases.

Sergei Kostjuk has received his Ph.D. degree in polymer chemistry in 2002 from Belarusian State University. In 2002, he joined Research Institute for Physical Chemical Problems and since 2008 he is heading the laboratory of catalysis of polymerization processes in the same Institute. During last years in cooperation with BASF SE he has developed a new process for making of highly reactive polyisobutylene, which is used in engine lubricants and fuel additives. His research interests lie in the fields of polymer design and synthesis (cationic and anionic polymerization).

Dr. Peter Timashev graduated from the Moscow State University of Fine Chemical Technologies in 2002. In 2006, he gained his PhD degree at the Karpov Institute of Physical Chemistry, and in 2016 - DSc degree. In 2016, he became a Head of the Department for Advanced Biomaterials at the Institute for Regenerative Medicine (Sechenov University, Russia). Since 2018, he has been working as a Director. His research interests cover the development of novel biodegradable biocompatible materials and approaches to their structurization (inc. laser-based technologies), 3D bioprinting, and clinical translation of tissue engineering.

The authors declare no conflict of interest.