hyaluronic acid injectable hydrogels structurally amended via covalent attachment of surface-modified silica particles

International Journal of Biological Macromolecules 136 (2019) 1196–1208

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Genipin crosslinked bioactive collagen/chitosan/hyaluronic acid injectable hydrogels structurally amended via covalent attachment of surface-modified silica particles Joanna Lewandowska-Łańcucka a,⁎, Adriana Gilarska a,b, Aleksandra Buła a,c, Wojciech Horak d, Anna Łatkiewicz e, Maria Nowakowska a a

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Mickiewicza 30, 30-059 Kraków, Poland c Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, prof. S. Łojasiewicza 11, 30-348 Kraków, Poland d AGH University of Science and Technology, Faculty of Mechanical Engineering and Robotics, Department of Machine Design and Technology, Al. Mickiewicza 30, 30-059 Kraków, Poland e Laboratory of Field Emission Scanning Electron Microscopy and Microanalysis at the Institute of Geological Sciences, Jagiellonian University, Gronostajowa 3a, 30-387 Kraków, Poland b

a r t i c l e

i n f o

Article history: Received 16 May 2019 Received in revised form 13 June 2019 Accepted 24 June 2019 Available online 25 June 2019 Keywords: Biopolymers Injectable hydrogels Surface-modified silica particles

a b s t r a c t Collagen, chitosan and hyaluronic acid based multicomponent injectable and in situ gellating biomimetic hybrid materials for bone tissue engineering applications were prepared in one-step procedure. The bioactive phase in the form of surface-modified silica particles was introduced to the solutions of biopolymers and simultaneously crosslinked with genipin both the biopolymer matrix and dispersed particles at 37 °C. The novel approach presented here involved the use of silica particles which surfaces were priory functionalized with amino groups. That modification makes possible the covalent attachment of silica particles to the polymeric hydrogel network on crosslinking with genipin. That methodology is especially important as it makes possible to obtain the hybrid materials (biopolymer-silica particles) in which the problems related to the potential phase separation of mineral particles, hindering their in vivo application can be eliminated. The hybrids of various compositions were obtained and their physicochemical and biological properties were determined. The in vitro experiments performed under simulated body fluid conditions revealed that the amino-functionalized silica particles covalently attached to the biopolymeric network are still bioactive. Finally, the in vitro cell culture studies shown that the materials developed are biocompatible as they supported MG-63 cells adhesion, proliferation as well as Alkaline phosphatase (ALP) expression. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Tissue engineering (TE), the rapidly developing, interdisciplinary field of research aiming at the regeneration of living tissue has shown a great potential to overcome the limitations of existing treatments [1,2]. It has been shown that the three-dimensional scaffolds that can serve as a temporary extracellular matrix (ECM) mimicking its architecture and providing the mechanical support for cells and simulating the properties of their natural environment, and being bioactive are sought for bone regeneration [3–6]. Fabrication of the scaffold that would meet all demanding conditions for maintaining cell growth and enabling their differentiation presents still a great challenge. It is known that the choice of suitable materials for scaffold designing is a key factor for the success of bioimplantation [7–9]. The biodegradable, injectable hydrogels are very promising ⁎ Corresponding author. E-mail address: [email protected] (J. Lewandowska-Łańcucka).

https://doi.org/10.1016/j.ijbiomac.2019.06.184 0141-8130/© 2019 Elsevier B.V. All rights reserved.

candidates for TE scaffolds, since their mechanical and structural properties make them similar to various tissues and they can be easily design in such a way that can resemble the properties of the natural extracellular matrix (ECM) [10]. It was reported that hydrogels with high swelling ability are of interest in that regard as they create an ideal microenvironment for cell proliferation and differentiation. Moreover, injectable hydrogels can be delivered to the body in a minimally invasive manner and ensure perfect physical integration with the bone defects [11,12]. Furthermore, by supplementation of polymeric sols with additional components the hydrogels of various required therapeutic features can be prepared [13,14]. Biocompatible silica-based materials are considered as excellent candidates for applications in various biomedical fields [15,16]. It was found that silicon plays an important role in bone formation being involved in both collagen synthesis as well as in matrix biomineralization [17,18]. In addition, it was established that silica-based particles exhibit strong biological activities including promotion of the osteoblast adhesion, proliferation and stimulation of the osteogenic differentiation

J. Lewandowska-Łańcucka et al. / International Journal of Biological Macromolecules 136 (2019) 1196–1208

in vivo [19,20]. In our previous papers [21,22] we have focused on studies on the physicochemical and biological properties of the hybrid organic-inorganic materials based on collagen and collagen-chitosan hydrogels supplemented with in situ prepared submicron silica particles. We have demonstrated in in vitro experiments performed under SBF conditions that developed by us hybrids efficiently induced mineralization process. Furthermore, we found that the homogeneous distribution of silica particles (confirmed in microscopic observations) that act as nucleation sites for apatite formation allows for both: the surface and bulk mineralization. Considering the use of the hybrid materials studied as cell scaffolds in bone defects we also evaluated their osteogenic potential in human bone marrow-derived mesenchymal stromal cells (hBMSCs) in vitro culture [23]. We have confirmed that due to the addition of silica particles to collagen/chitosan hydrogels the osteogenic differentiation of hBMSC was significantly enhanced as manifested by increasing RUNX-2, OC and COLI mRNA levels determined after 14 days of culture. Importantly we have found that collagen/chitosan hydrogels modified with silica particles showed high proosteogenic properties without the need of applying any additional osteogenic activation compounds that generally exhibit low stability, limited bioavailability and are rather expensive. One of the drawbacks of hybrid materials is the phase separation; usually the inorganic particles migrate within the structure and tend to aggregate thus worsening the functionality of the material under consideration. The current paper presents the results of our studies addressing that problem. We have developed novel structurally stable injectable hybrid materials in which all components are covalently linked. They were obtained by simultaneous genipin crosslinking of components of collagen/chitosan/hyaluronic acid (Col:Ch:HA) biopolymeric matrix and silica particles modified with amino groups. Collagen, chitosan and hyaluronic acid were chosen for matrix preparation as these biopolymers are used most often as biomaterials. They are characterized by desirable biological properties including biocompatibility, biodegradability and they are non-toxic to the human body [20,24]. Collagen a biopolymer classified as fibrillary proteins is the component of extracellular matrix as well as bones. Although about 29 types of collagen were identified, differing mainly in the conformation of α-chains and their mutual combination in the helix [25], definitely the most popular one is type I collagen, characterized by strong fibers with high extensibility and strength. It can be found in large amounts in the skin, felled, bones and muscles (it is the main component of the fish skin and bones), what resulted in both: its popularity and availability [26]. This protein plays an important role in the proper functioning of the body system; supports skeletal bone function, provides the mechanical protection of the skin, prevents its dehydration, maintains firmness and elasticity and also mediates the organ development and tissue regeneration [27]. Chitosan is a natural polysaccharide, a linear copolymer composed of 2-acetamido-2-deoxy-β-D-glucopyranose and 2-amino-2deoxy-β-glucopyranose units linked by a glycosidic bonds. It is formed as a result of deacetylation of chitin produced on an industrial scale from shellfish shells. Chitosan is soluble in weakly acidic aqueous solution because the primary amine groups are protonated at lower pH (pKa = 6.5). Under these conditions the chitosan behaves as cationic polyelectrolyte because its macromolecules are positively charged [28]. This polymer is biocompatible, biodegradable and bioresorbable, exhibits antibacterial, antifungal properties, as well as accelerates wound healing [29]. These make chitosan an attractive materials for potential applications in tissue engineering (structural similarity to naturally occurring glycosaminoglycans), as well as drug delivery systems (the ability to form hydrogels) [30]. Hyaluronic acid (HA) is a naturally occurring linear polysaccharide belonging to the group of glycosaminoglycans. It is the main carbohydrate component of the extracellular matrix formed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, alternately linked by glycosidic bonds. Under physiological conditions, HA occurs in the form of a hydrophilic polyanion surrounded by water molecules. Its ability to adsorb and accumulate water significantly affects its

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role in the body. The main function of HA in the organism is to maintain the structural coherence of the intercellular spaces in the tissue and to ensure it flexibility and elasticity. Considering its biocompatibility, biodegradability, as well as an excellent predisposition to form gels, HA is an extremely attractive material for biomedical applications [31]. Various types of HA conjugates were fabricated to obtain self-assembled conjugates with the potential application as drug delivery carriers [32]. Furthermore, HA exhibits an affinity to osteoblasts and chondrocytes that allow it to be used in biomaterials for scaffold preparation [33]. The surfaces of silica particles were modified with amino groups prior to addition to the biopolymer mixture. Since genipin is known to react with the primary amino groups, it was expected that the simultaneous reaction with amino groups present in the biopolymers forming matrix and with these present at the surface of the dispersed silica particles will allow to obtain stable hybrid material on crosslinking. This approach seems to be especially important when considering application of scaffolds prepared from hybrid materials under in vivo conditions. It should be also emphasized that conditions of synthesis were optimized in such a way that allow coexistence and surface exposition of amino and silanol groups, Si-OH – crucial for induction of apatite-like mineral formation [34]. To the best of our knowledge that approach has not been presented so far. In our studies, the synthesized amino-functionalized silica particles were dispersed at three concentration in polymeric collagen/chitosan/ hyaluronic acid sols which were subsequently crosslinked with genipin. The swelling, wettability, degradation as well as mechanical features were examined to demonstrate the effectiveness of surface-modified silica particles attachment to polymeric matrix. The rheological studies, a viscoelastic behavior of the sols and elastic nature of the hybrids demonstrated the injectability potential of materials developed. To confirm that amino-functionalized particles possess an ability to induce biomineralization all developed materials were exposed to simulated body fluid (SBF) medium for seven days and next the morphology and the chemical composition of the minerals formed were studied by means of SEM and EDS measurements. Furthermore, cell proliferation, cell morphology and adhesion as well as functional activity of osteoblast MG-63 cells were assessed in in vitro cell culture studies carried out on the surface of materials developed. Obtained hybrids were fully characterized to demonstrate that the proposed herein one-step method of their fabrication deliver the structurally defined injectable materials characterized by desirable physicochemical features while providing a favorable bioactivity and environment for osteoblasts functionality. 2. Materials and methods 2.1. Materials Collagen type I rat tail (3.5 mg/mL solution, BD Biosciences), chitosan (low molecular weight, Sigma-Aldrich), hyaluronic acid (mol wt ~1.5–1.8 × 106 Da, Sigma-Aldrich), genipin (Challenge Bioproducts Co., 98%), acetic acid (Chempur), tetraethoxysilane (TEOS, ≥98%, Fluka), (3-aminopropyl)triethoxysilane (APS, 98%, Sigma Aldrich,) ethanol (99,8%, spectroscopic grade), ammonium hydroxide (25%, pure p. a., Chempur) sodium chloride, NaCl (POCh, p.a.), sodium hydrogen carbonate, NaHCO3 (POCh, p.a.), potassium chloride, KCl (Chempur, p.a.), di potassium hydrogen phosphate trihydrate, K2HPO4·3H2O (Sigma Aldrich, 99%), magnesium chloride hexahydrate, MgCl2·6H2O (POCh, p. a.), calcium chloride, CaCl2 (Sigma Aldrich, 93%), sodium sulfate, Na2SO4 (POCh, p.a.), tris-hydroxymethyl aminomethane, ((HOCH2) 3CNH2) (Tris) (Sigma Aldrich, 99,8%), hydrochloric acid 1 M, HCl (POCh). Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich), penicylin-streptomycin solution (10.00 units/mL), fetal bovine serum (FBS, HyClone), trypsin (HyClone), Alamar Blue reagent (Invitrogen), Cell Digestion Buffer and Cell Assay Buffer compounds: trishydroxymethyl aminomethane, ((HOCH2)3CNH2) (Tris) (Sigma

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Aldrich, 99.8%), zinc chloride, ZnCl2 (POCh), magnesium chloride hexahydrate, MgCl2∙6H2O (POCh), Triton X-100 (POCh)], p-nitrophenyl phosphate (pNPP) (Sigma-Aldrich), glutaraldehyde solution (Sigma-Aldrich), hexamethyldisilazane reagent grade (HMDS, Sigma-Aldrich), Osteoblasts-like culture (Homo sapiens bone osteosarcoma cell line) MG-63 (ATCC® CRL-1427™). 2.2. Sample preparation 2.2.1. Amino-functionalized silica particles preparation Amino-functionalized silica particles were obtained as follows: to the mixture consisting of ethanol (5.1 mL) and water (5 mL) the appropriate amounts of TEOS (1.0 mL) and APTES (0.1 mL) were sequentially added. The solution was stirred for 30 min at room temperature. Resulted particles were centrifuged, washed with distilled water and this procedure was repeated four times. The obtained materials were dried in vacuum chamber at 60 °C. 2.2.2. Hydrogels and hybrids materials preparation The pristine collagen/chitosan/hyaluronic acid hydrogels (ColChHA) were fabricated using the procedure described earlier [12]. Hybrid materials were prepared according to a similar protocol with the difference that before genipin addition the water dispersion of aminofunctionalized silica particles (0.3 mL, 16.6 mg/mL (C1), 8.3 mg/mL (C2) 1.7 mg/mL (C3)) was added to sol (1 mL). The mixture was vigorously vortexed, genipin solution was next added and obtained sols were incubated at 37 °C till gel formation. We have observed that the addition of silica had not effected considerably the gelation time. Employing ColChHA hydrogels supplemented with amino-functionalized silica particles of various concentrations (C1, C2 and C3) the following hybrid materials were obtained: ColChHA C1, ColChHA C2 and ColChHA C3 (see Table 1). 2.3. Methods 2.3.1. Amino-functionalized silica particles characterization Amino-functionalized silica particles were characterized by Fourier transform infrared (FTIR) spectroscopy using a Thermo Nicolet iS10 FTIR spectrometer (Thermo Scientific) with ATR equipment (SMART iTX). The zeta potential and hydrodynamic diameter of the surfacemodified silica particles was measured using a Malvern Nano ZS lightscattering apparatus. Specific surface area (SBET) and pore size distribution were determined from the nitrogen adsorption isotherms obtained at −196 °C using a 3 Flex (Micromeritics, USA) automated gas adsorption system. Prior to the measurements, the samples were degassed under 0.3 mbar at 40 °C for 24 h. Determination of the specific surface area (SBET) was based on the Brunauer-Emmett-Teller (BET) model while the volumes of mesopores (Vmes) were evaluated using the Barrett–Joyner–Halenda (BJH) procedure. X-ray Photoelectron Spectroscopy (XPS) analysis was carried out using a multifunctional ESCA instrument equipped with additional accessories produced by PREVAC. For XPS experiment the silica-based materials were dried at 100 °C for 24 h. For all the XPS measurements proper surface charge neutralization was used. As specified in the experimental part, an electron flood gun (charge neutralizing gun, FS40A-PS, Prevac) was used for that purpose. The voltage and current of the gun was carefully optimized for each

Table 1 Overview of hydrogels developed (Col – collagen, Ch – chitosan, HA – hyaluronic acid). Hydrogel/hybrids

Weight ratio Col:Ch:HA

ColChHA ColChHA C1 ColChHA C2 ColChHA C3

50:40:10 50:40:10 50:40:10 50:40:10

Concentration of silica particles [mg/mL] – 16.6 8.3 1.7

sample. Data analysis was performed using the CasaXPS program. The chemical composition of the developed materials surface was calculated from peak areas normalized on the basis of the acquisition parameters. 2.3.2. Hybrid materials characterization Swelling ability of the hydrogels was investigated under physiological conditions by materials incubation at 37 °C in PBS buffer with gentle shaking for 24 h. After that PBS buffer was removed, the hydrogels were rinsed twice with deionized water and the weighed (Ws). Next materials were dried by lyophilization and weighed again (Wd). The swelling ratio (SR) was calculated using W s −W d the following equation:SR ¼ ∙100% Wettability of obtained Wd hydrogels was analyzed by contact angle measurements carried out using Surftens Universal instrument (OEG GmbH, Frankfurt, Germany). Five contact angle values were measured for each sample of hydrogels and the average value was calculated. The mechanical properties of the tested hydrogels were studied with a Physica MCR-301 rheometer (Anton Paar) equipped with a parallel plate (Ø = 20 mm) made of stainless steel under conditions employed previously [12]. The measurements were carried out in oscillation mode utilizing a frequency of 1 Hz and a strain of 1%, the measuring gap was set at a distance of 1 mm. Process of gel degradation was monitored for 21 days while the hydrogels were incubated at 37 °C in PBS buffer with gentle shaking. At defined period of time the materials were weighted and the fresh portion of PBS buffer was added to the system. For enzymatic degradation evaluation, the materials studied were placed in 24-well plates and exposed to collagenase type I (0.2 mg/mL, 1 mL, ≥125 U/mg) in 1× PBS with 0.36 mM CaCl2 followed by incubation at 37 °C and gentle shaking. At various time intervals, materials were weighted and next the fresh portion of enzyme solution was added to the system. For each sample, the experiments of degradation were carried out in triplicates and the results are presented as the averages. 2.4. In vitro biomineralization in simulated body fluid (SBF) Simulated body fluid was prepared according to Kokubo's method [35]. The prepared hybrids were transferred into 24-well plates and 1 mL of freshly prepared SBF was added to each well. The plates were placed on a shaker table and incubated at 37 °C for 7 days (SBF was renewed every 2 day). Next SBF was removed, the materials were rinsed few times with deionized water and lyophilized for 24 h. The microstructure of the hybrids developed after SBF treatment was evaluated by means of the cold field emission scanning electron microscope (FESEM) HITACHI S-4700 equipped with a NORAN Vantage energy dispersion spectrometer. 2.5. In vitro osteoblasts culture For the biological tests the series of hydrogels (three samples for each type of hydrogels) were prepared in 24-well plate, sterilized with UV radiation and incubated with addition of the cell culture medium without serum for about 1 h at 37 °C. Before cell culture, the medium was removed and the MG-63 cell line was seeded in the plate at a density of 6.7 × 103 cells/well. The medium containing 90 vol% of DMEM (supplemented with 1 vol% of penicylin-streptomycin solution) and 10 vol% of serum was used. After 1, 3 and 7 days of culture the cell viability was studied using Alamar Blue (AB) assay according to procedure employing by us previously [12]. Based on standard curves established with MG-63 cells cultured at various densities the cells number was calculated. ALP activity was studied at culture day 3 and 7 using the protocol described in [23]. The calculation was based on the assumption that 1 mol ALP hydrolyzes 1 mol of pNPP substrate and thus ALP activity was expressed as the amount (nmol) of the product (p-nitrophenyl, pNP). For cells distribution and morphology analysis after 3-days of MG-63

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cells culturing hydrogels were fixed and dehydrated using protocol from [23]. Microscopic observations were performed employing a cold field emission scanning electron microscope (FESEM) HITACHI S-4700.

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in vivo conditions. What is important it is expected that these materials will be bioactive and injectable. 3.1. Silica particles fabrication and physicochemical characterization

2.6. Statistical analysis Experiments were repeated three times and results were expressed as a mean ± standard deviation. Statistical significance was calculated using the one-way Analysis of Variance (ANOVA) with Tukey's honestly significant difference post hoc test (swelling) and Student's t-test (degradation, rheological measurements, proliferation and ALP activity studies, cells spreading). A comparison between two means was analyzed with statistical significance level set at p b 0.05. 3. Results and discussion The primary goal of studies presented in current paper involved fabrication and determination of the physicochemical and biological properties of the structurally stable multicomponent, biomimetic hybrid materials, potentially useful as the bioactive injectable scaffolds for regeneration of the bone tissue. The simultaneous crosslinking of biopolymers and modified silica particles resulted in formation of hybrid materials in which the bioactive inorganic phase was dispersed in biopolymeric matrix. The novelty of the current approach lies in the functionalization of the silica particles with amino groups what makes possible their attachment to the polymeric hydrogel network formed on crosslinking with genipin and thus eliminate the potential phase separation of mineral particles. Therefore, the resulted materials are expected to have well defined and stable nanostructure what is very important when considering the potential application of hybrids under

The conditions for synthesis of amino-functionalized silica particles were optimized to allow the preparation of particles with defined size and to ensure the coexistence of amino and silanol groups (Si-OH), crucial in the context of induction of apatite-like mineral formation, at their surface. To accomplish that we have carried out one-pot synthesis in the system containing: tetraethoxysilane (TEOS)/3aminoprophyltriethoxysilane (APTES)/water/ethanol [36]. As it was previously reported [37] the amino groups in APTES are able to catalyze the hydrolysis and condensation of TEOS. It was demonstrated that APTES introduced to the mixture of ethanol/water/TEOS caused the rapid pH increase owing to the strong protonation of amino groups and production of a large amount of OH– ions that initiated the sol-gel reaction of silica precursors [38]. The FTIR spectrum of the obtained material confirmed the presence of amino and silanol groups (Fig. 1A). The strong, sharp peak at 1027−1 can be assigned to the bending vibration of Si\\O band and that at 779 cm−1 to the Si–O–Si stretching vibration, both characteristic for silica. The weak shoulder peak at 942 cm−1 can be attributed to the Si-OH group, while the signal at 1523 cm−1 is assigned to the NH2\\ groups [39]. The band at 1626 cm−1 can be ascribed to the adsorbed H2O whereas the wide, bulky peak in the range of 2600–3650 cm−1 is an envelope of the bands for adsorbed water molecules, OH– group present at the surface of the samples, and the amino groups [36]. To gain a deeper insight into the chemical composition of the fabricated material XPS measurements were carried out. The peak intensity

Fig. 1. FTIR (A) and XPS (B) spectra of amino-functionalized silica particles.

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Table 2 XPS and theoretical results showing atomic compositions of the amino-functionalized silica particles (silica-NH2) (in %). Atomic composition (%) for silica-NH2 particles

O1s

N1s

C1s

Si2p

XPS results Theoretical resultsa

78 49

3 2

7 5

12 44

a

Calculated from the chemical structure of the amino-functionalized silica particles.

in the XPS spectra is directly related to the atomic distribution on the surface of the material and can be used to quantify the composition of atomic elements [40]. The XPS spectrum is depicted in Fig. 1B while the atomic compositions (%) of the surface of particles developed are presented in Table 2. The peaks observed can be assigned to O 1s (531.1 eV), N 1s (402.6 eV), C 1s (286.5 eV), Si 2s (155.1 eV) and Si 2p (103.5), respectively [41]. N 1s and C1s signals are diagnostic for APTES functionalization since pristine silica particles obtained from TEOS do not contain these elements. The analysis of the surface composition of material developed presented in Table 2 indicated clearly that SiCON structures with the atomic composition of 78%, 3%, 7% and 12% for O, N, C and Si, respectively, were successfully obtained. Theoretical atomic composition was calculated from the chemical structure of the amino-functionalized silica particles considering the TEOS:APTES molar ratio (10:1) employed in the synthesis. The comparison of the experimental and theoretical data suggests that higher oxygen content obtained in XPS experiments resulted from the surface-exposed silanol groups formed in partial polycondensation of precursors used. All data presented above confirmed the successful formation of the aminofunctionalized silica particles under the experimental conditions employed. Moreover, the coexistence of surface-exposed amino and silanol groups was also demonstrated. Fig. 2 presents schematically the formation of resulted silica-based particles. Dynamic light scattering (DLS) and zeta potential measurements were carried out to determine the size and zeta potential values of functionalized silica particles obtained. The values of the mean hydrodynamic diameter (dz), dispersity (DI) and the zeta potential (ζ) are presented in Table 3. The mean hydrodynamic diameter of particles obtained from DLS measurements is about 607 nm with DI = 0.3. As expected, the zeta potential of resulted particle has positive value resulting from protonation of amino groups present in the APTES backbone. Morphology of the material fabricated was visualized employing SEM. In the typical micrographs presented in Fig. 3 particles of two sizes might be observed.

The larger populations are about 400 nm in diameter while the smaller particles are about 100 nm. The smaller particles (marked with arrows on Fig. 3B) have tendency to aggregate around the larger objects. The diameters of the particles found from the DLS measurements were thus greater than those observed on SEM micrographs that might be explained taking into account the fact that the DLS technique determined the mean hydrodynamic diameter of the object, being the size of the particle together with the layer of ions and solvent molecules surrounding it. We have also evaluated the porosity of the material obtained based on the nitrogen adsorption isotherm. Parameters characterizing the porosity of the samples calculated from the adsorption branch of the isotherm are presented in Table 3. The materials studied are characterized by the specific surface area of 24 m2/g with the significant mesoporosity (0.14 cm3/g); the obtained value of SBET is typical for nonor low-porous materials [42]. In our previous paper we have synthesized and characterized the Stöber silica particles with diameters in the colloidal range [39]. Their specific surface area was about 16 m2/g while the volume of mesopores was of 0.04 cm3/g. When comparing the specific surface area values (SBET) for pristine silica particles obtained from TEOS and these for amino-functionalized particles described herein one can notice that their SBET is of the same order of magnitude, however, their mesoporosity differ significantly. 3.2. Hybrid materials fabrications and physicochemical characterization Hybrid materials based on hydrogels with the amino-functionalized silica particles covalently attached to polymeric matrix were obtained. Various concentrations of surface-modified silica particles (C1, C2, C3) incorporated into the hydrogel matrix containing collagen (Col), chitosan (Ch) and hyaluronic acid (HA) were tested. The genipin solution (20 mM) in PBS was used as a crosslinking agent. The physicochemical properties, bioactivity as well as biocompatibility of the novel hybrid materials were evaluated and compared with those obtained for

Fig. 2. Scheme of the formation of the amino-functionalized silica particles.

J. Lewandowska-Łańcucka et al. / International Journal of Biological Macromolecules 136 (2019) 1196–1208 Table 3 Values of the mean hydrodynamic diameter (dz), dispersity (DI), zeta potential (ζ) and parameters measured by nitrogen sorption: specific surface area (SBET), mesopore (Vmes) volume and mesopore diameter (Dmes) for the SiO2-NH2 particles fabricated. dz [nm]

DI

ζ [mV]

SBET [m2/g]

Vmes (BJH) [cm3/g]

Dmes (BJH) [nm]

607 ± 23

0.3

35 ± 1

24

0.14

23

pristine polymeric hydrogels. All types of materials prepared are summarized in Table 1. After crosslinking and formation of hydrogels the colour of materials developed transformed from bright pink to bluish-violet (see Fig. 4). These changes in samples colour and appearance of fluorescence are characteristic for the reaction product of genipin with the primary amino groups [43]. We have observed that colour transformation for both, pristine hydrogel as well as for hybrid materials what confirmed that amino-functionalized silica particles introduced into the system do not hamper the genipin role as crosslinking agent. The swelling behavior of hybrid materials was studied in the presence of PBS. The swelling ratios (SR) of these materials were determined and the results are presented in Fig. 5. The swelling properties of obtained materials were influenced by the presence and content of silica particles in hydrogel matrixes. We have noticed that the swelling ratio (SR) decreases with increase in silica particles concentration. In the case of material with the highest concentration of silica particles (ColChHA C1), the swelling degree is significantly lower compared to pure hydrogel (ColChHA). This result indicates that the silica particles increase the rigidity of the hydrogel structure [22,44]. Another factor that may be responsible for the lower degree of swelling is the functionalization of the silica particles with amino groups. Amino groups are expected to be effectively incorporated into the polymer network and that can increase the density of the network in hybrid materials [45]. However, the silica particles present at lower concentrations like in ColChHA C2 and ColChHA C3 do not change significantly the swelling properties compared to the hydrogel ColChHA. 3.2.1. Wettability of the hydrogels The level of surface wettability influences considerably the cell adhesion efficiency [46]. To determine the wettability of tested materials, measurements of contact angle were performed (data presented in Table 4). For the pure hydrogel ColChHA contact angle is relatively high, its value is close to 90°. According to the literature, contact angle of 90° is a limit value above which the material is considered to be hydrophobic [47]. The addition of amino-functionalized silica particles to the hydrogel matrix causes the surface of the materials to become

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more hydrophilic as demonstrated by the lower values of contact angle compared to the material without silica particles. The material with the highest concentration of surface-modified silica particles (ColChHA C1) is characterized by the most hydrophilic surface while ColChHA C2 and ColChHA C3) have the wettability at the similar level. Improvement in hydrophilicity results from the amino groups present on the surface of amino-functionalized silica particles that are exposed on the surface of hybrid materials as confirmed by XPS findings. It is known that amino groups are hydrophilic and for that reason, they are commonly used for functionalization of hydrophobic surfaces [48,49]. 3.2.2. Hydrogels degradation 3.2.2.1. Degradation in PBS buffer. The degradation of materials obtained was determined by incubating them in PBS buffer at 37 °C for 21 days. The results are depicted in Fig. 6A as the weight of hydrogels (wt%) remaining after defined period of degradation time. The largest weight loss for all tested materials occurs after the first day of degradation experiment. In the following days, only slight fluctuations in weight could be noticed and the mass of the remaining material is almost constant. We have observed a similar kinetic of weight loss on time in our previous study [12]. For ColChHA C1 and ColChHA C2 hybrid materials, the degradation process is similar to that for pristine hydrogel ColChHA throughout the duration of the experiment (the differences are statistically insignificant). That shows that the presence of silica particles at the highest and medium concentration does not affect the rate of hydrogel weight loss. Lack of significant differences in mass losses can also demonstrate the efficient crosslinking between amino-functionalized particles and polymeric network upon genipin treatment since otherwise the silica "leakage" from hydrogel network should be manifested by greater decrease of the hybrid mass. Only the material with the lowest concentration of silica particles (ColChHA C3) shows a relatively larger weight loss compared to the other tested materials for almost all degradation time. It was, however, found that when the experiment was completed (after 21 days), the values of remaining mass of all materials are similar, the degree of degradation for samples ranged from 37 to 48% (the differences are statistically insignificant). 3.2.2.2. Enzymatic degradation. The enzymatic degradation of the materials developed was studied by treating them with collagenase solution for 144 h. As can be seen in Fig. 6B after 4 h of enzymatic degradation, the materials lost about 35–50% of their initial weight. The next substantial change occurred after 24 h when the weight loss for materials tested reached the level of 65–80%. Prolongation of the materials incubation with a collagenase up to 144 h did not induced additional significant fluctuations in their weight. When analyzing the weight loss of all

Fig. 3. SEM micrographs of amino-functionalized silica particles.

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Fig. 4. Optical images of materials developed before and after crosslinking with genipin.

materials studied at a given time it can be noticed that a lower degree of degradation is observed for the hybrid with the highest concentration of amino-functionalized silica particles. After 144 h of experiment, the weight losses of ColChHA C1 and ColChHA C3 accounted for 75 and 85%, respectively. Insignificant differences in the degree of enzymatic degradation between pure hydrogel and hybrids may indicate that the surface-modified silica particles have been effectively immobilized in the hydrogel's network thus their leakage from the structure is eliminated. These results are in line with findings described in previous section.

Hydrogel

Contact angle values [°]

ColChHA ColChHA C1 ColChHA C2 ColChHA C3

87.2 ± 2.5 68.6 ± 1.6 76.9 ± 2.4 76.5 ± 1.7

3.2.3. Rheological evaluation It was established that an ideal scaffold for TE applications should allow for minimally invasive implantation [50]. The hybrids presented in this work were designed to serve as injectable scaffolds. It is assumed that the material in the form of viscous sol will be introduced to the gap in the tissue and will be in situ subsequently gelated there under physiological conditions. Therefore, to prove our concept the rheological measurements were carried out. By the observations of the elastic modulus (G′) changes, it was possible to verify the transition from sol to the gel state. The G′ values measured after 10, 30 and 60 min of experiment are depicted in Fig. 7. As illustrated in Fig. 7 at the beginning of the gelation process (after 10 min) the G′ values for all materials are at the low level (in the range 2–5 Pa, without the statistical significance when compared with each other) confirming their injectable state. G′ values considerably increased after 30 min (statistical significance) and reached the maximum value in 60 min (statistical significance) from the initiation of crosslinking process (gel formation). Thus, rheological findings confirmed that obtained hybrid materials can be prepared in the form of a sol and after the addition of the crosslinking agent, the genipin, and incubation at 37 °C, can be transformed into a gel. That evidences that materials developed can indeed serve as the injectable hydrogels. It can be also concluded that the presence of silica particles functionalized with amine groups, that enables their incorporation by crosslinking into the hydrogel structure, does not hinder the gel formation process. The data obtained demonstrated the injectability potential of the hybrids developed, especially important when considering the ease in their implantation in future TE applications. When comparing the G′ values it can be noticed that the storage modulus for hybrid materials is dependent on the concentration of amino-functionalized silica particles used. The storage modulus

Fig. 5. Swelling ratio for collagen/chitosan/hyaluronic acid hydrogel and hybrid materials based collagen/chitosan/hyaluronic acid hydrogel with amino-functionalized silica particles attached of various concentrations (C1, C2, C3) and incubated in PBS buffer for 24 h. *indicates statistical significance when compared with ColChHA C1 (p b 0.05), # indicates statistical significance when compared with ColChHA C2 (p b 0.05).

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Fig. 6. (A) Changes in the values of remaining weight for the pristine hydrogel and hybrid materials on degradation time monitored during degradation studies in PBS buffer. Statistical analysis was obtained by comparing all types of materials on the same day of degradation. & indicates statistical significance when compared with ColChHA C3 on the 1 day (p b 0.05), % indicates statistical significance when compared with ColChHA C3 on the 3 day (p b 0.05), * indicates statistical significance when compared with ColChHA C3 on the 5 day (p b 0.05), ^ indicates statistical significance when compared with ColChHA C3 on the 7 day (p b 0.05), $ indicates statistical significance when compared with ColChHA C3 on the 9 day (p b 0.05), # indicates statistical significance when compared with ColChHA C3 on the 12 day (p b 0.05), @ indicates statistical significance when compared with ColChHA C3 on the 14 day (p b 0.05), ~ indicates statistical significance when compared with ColChHA C3 on the 16 day (p b 0.05), + indicates statistical significance when compared with ColChHA C3 on the 19 day (p b 0.05). (B) Changes in the values of remaining weight for the pristine hydrogel and hybrid materials on degradation time monitored during enzymatic degradation studies. Statistical analysis was obtained by comparing all types of materials on the same time of degradation. * indicate statistical significance between adequate materials (p b 0.05).

Fig. 7. The values of the storage modulus (G′) measured after 10, 30 and 60 min of experiment are presented in logarithmic scale. Statistical significance was calculated using Student's ttest. A comparison between two means was analyzed with statistical significance level set at p b 0.05; below the black line indicates statistical significance between the results for the same type of material after 10, 30 and 60 min, *indicate statistical significance when compared with ColChHA after 30 min, **indicate statistical significance when compared with ColChHA C3 after 30 min, ***indicate statistical significance when compared with ColChHA C3 after 60 min.

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determined after 60 min of gelation is the highest for ColChHA C3 (statistical significance when compared with both ColChHA C1 and ColCHA C2). However, there are no statistical differences between elastic modulus obtained for ColChHA C3 and pristine hydrogel. It was shown that there are many factors, that may influence the mechanical characteristics of inorganic-organic materials. Alvarez et al. [51] studied the mechanical properties of silica nanoparticle-collagen composite hydrogels and found that at higher particle concentration

the silica shows tendency to form aggregates that induced perturbation in the collagen fibrillary organization. and, as a consequence, deteriorate the elastic properties of the resulted composites. That is also in line with findings of Yanagioka et al. [52]. One can expect that such effects should be also considered in our systems. The amino-functionalized silica particles at highest concentrations (C1 and C2) may create some aggregates during the crosslinking process that affect the spatial organization of the network formed and decrease the elastic modulus values. However, our

Fig. 8. Scanning electron micrographs of the pristine hydrogel and hybrid materials before and after 7-day incubation in SBF.

J. Lewandowska-Łańcucka et al. / International Journal of Biological Macromolecules 136 (2019) 1196–1208 Table 5 Ca/P ratios for mineral structures formed in the studied materials after incubation in SBF. Type of material

Ca/P ratio

ColChHA ColChHA C1 ColChHA C2 ColChHA C3

– 1.29 ± 0.03 1.29 ± 0.08 –

findings clearly show that by playing with the silica particle concentration it is possible to tune the mechanical properties of the resulting organic-inorganic hybrids. 3.3. In vitro biomineralization In view of the potential application of the synthesized hybrids as scaffolds for bone tissue engineering we have performed in vitro biomineralization experiments in SBF medium originally developed by Kokubo et al. It has been demonstrated that the integration of material with the natural bone by formation on its surface the apatite layer in vivo can be simulated in vitro by material incubation in SBF with a mineral composition analogous to human blood plasma. Formation of apatite-like minerals on the surface of scaffolds shows the osteoconductive capability of the materials and it is one of the important parameters of the material to be considered for TE application

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[53]. Since silica particles used for hybrids fabrication were surfacefunctionalized with amine groups it was of great importance to demonstrate that they preserve the ability to act as nucleation sites for apatite formation. To determine the in vitro bioactivity of the hybrids, the process of mineralization was followed by observation of the products formed using SEM and EDS analyses. 3.3.1. SEM and EDS analyses SEM images for the pristine hydrogel and hybrid materials before and after 7-day incubation in SBF are presented in Fig. 8. Addition of silica particles to the hydrogel matrix has a strong impact on the formation of a new mineral phase on the surface of hybrid materials, as observed in the previous work [22]. Microphotographs obtained for all hybrid materials before incubation in SBF clearly show the presence of amino-functionalized silica particles in the polymer network, regardless the particle concentration. After 7-day of incubation in SBF, the significant amount of the mineral phase was formed on the surface of the materials containing higher content of silica particles (ColChHA C1 and ColChHA C2). That indicates that these hybrids are bioactive. To determine the elemental composition of new phases, EDS analysis was performed and in both cases, calcium and phosphorus were identified (see Table 5). That result is important due to the fact that the largest part of minerals in the natural bone are calcium phosphates, in particular in the form of hydroxyapatite crystals [54]. It is known, that the calcium to phosphorus ratio is 1.67 for stoichiometric hydroxyapatite and it is in the range of 1.5–1.7 for the mineral phase in natural bone [21].

Fig. 9. (A) Cell number and (B) Alkaline phosphatase activity (ALP) of MG-63 cells grown on the surface of the materials studied on day 1, 3 and 7 of the culturing. Statistical significance was calculated using Student's t-test. A comparison between two means was analyzed with statistical significance level set at p b 0.05; (A) below the black line indicates statistical significance between the results for the same type of material on the first, the third and the seven day; #indicate statistical significance when compared with ColChHA C1 day 7. (B) The black line indicates statistical significance between the results for the same type of material on the third and the seven day; *indicate statistical significance when compared with control day 3, **indicate statistical significance when compared with control day 7. Cells cultured on the tissue culture plate was considered as a control.

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For both ColChHA C1 and ColChHA C2 the calculated calcium to phosphorus ratio was around 1.29. This value is lower than for bone mineral and closer to that for octacalcium phosphate (OCP, Ca/P ratio of 1.33) what may be indicative for the initial process of formation of mineral apatite [55]. When comparing these results with our previous observation on the bioactivity of silica particles [21] one can conclude that functionalization of silica particles surface with amino groups did not diminish the bioactivity of the particles. The material with the lowest content of silica (ColChHA C3) has not supported considerably the formation of the mineral phase, only traces of new objects were identified. That means that the content of silica particles in this material is too low to enhance the mineralization process. In the case of the hydrogel without of silica particles added, the 7-day incubation in SBF did not initiate the formation of mineral phase and the characteristic structure of the polymer matrix remained intact when compared to the structure before incubation. These findings clearly demonstrate that the aminofunctionalized silica particles in the polymer matrix play a key role in biomineralization occurring only when the particle concentration in hydrogels is higher than 1.7 mg/mL (ColChHA C1 and ColChHA C2).

3.4. Biological evaluation of hybrids developed 3.4.1. Cell proliferation Considering the possible application of developed hybrids in tissue engineering we have quantitatively estimated their ability to support MG-63 cell line proliferation. The viability of cells was monitored using the Alamar Blue assay at different culture time points, namely days 1, 3 and 7. The results of these tests are depicted in Fig. 9A. It was revealed that the cell viability on all types of the hybrids studied exhibit similar alteration tendency with the prolongation of the biological experiment time. The number of viable cells seeded onto the surface of the materials tested substantially increased after 3 days of culturing (statistical significance with one exception when compared ColChHA C2 day 1 with ColChHA C2 day 3) and got the maximum at day 7 (statistical significance). The highest cell number is observed at 7 day for hybrids with the highest concentration of surface-modified silica particles (statistical significance when compared with ColChHA and ColChHA C2). Our findings clearly showed that the addition of aminofunctionalized SiO2-particles to the hydrogels does not deteriorate the

Fig. 10. (A) SEM micrographs of the MG-63 cell line cultured on the pristine hydrogel and hybrid materials. (B) Average spread area of a cell cultured on materials studied. The area of cells was calculated employing ImageJ software.

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biocompatibility of the resulting hybrids that support the proliferation of MG-63. 3.4.2. ALP activity ALP is a bone related protein that activity increases at early stages of osteoblasts differentiation [56]. It was found that the main physiological function of ALP in the process of osteogenesis is the production of phosphate groups essential for the deposition of hydroxyapatite and further matrix mineralization [57]. Therefore, ALP also serves as one of the markers confirming the osteoblastic phenotype and mineralization [58]. In order to evaluate the functions of MG-63 cells cultured on the materials developed, the ALP activity was measured at days 3 and 7, as shown in Fig. 9B. The similar tendency was observed for all materials. We have noticed that the level of ALP increased with statistical significance after 7 days of culturing for all materials studied. Importantly, ALP activity for cells cultured on hydrogel and hybrids was considerably higher compared to the cells cultured on the tissue culture plate at both time points used for observation (on day 3 and 7) (statistical significance). Interestingly, it seems that addition of amino-functionalized silica particles to the polymeric matrix does not enhance ALP activity compared to pristine ColChHA hydrogel. This could be explained considering the biological function of HA present in the hydrogel. It was reported previously that HA exhibits the affinity to osteoblasts and can be employed as biomaterial for ECM preparation since it plays structural function in the ECM [33]. Moreover, HA interacting with cells surface receptors and with binding proteins also regulates cellular responses including proliferation, differentiation, adhesion as well as gene expression [59]. It has been demonstrated that some chemical groups can influence the kinetics and conformation of protein adsorbed from the culture medium and thereby improve cell metabolic activities and maintaining the cell phenotype. Furthermore, it was also revealed that silica based NPs possess strong biological activities including stimulation of osteoblast differentiation [60]. Taking into account above mentioned literature data one may expect that the impact of silica-based particles on ALP expression could be more noticeable in a longer lasted experiments. However, that has to be confirmed in further studies. 3.4.3. MG-63 cells adhesion and morphology studies MG-63 cells adhesion and morphology was evaluated utilizing scanning electron microscopy (Fig. 10A). In our previous work, we have found that the surface of the ColChHA hydrogel (with Col:Ch:HA weight ratio equal to 50:40:10, respectively and crosslinked with 20 mM genipin) provided good environment for the adhesion of MG-63 cells [12]. In this study, we have observed that cells adhere well to both: the hydrogel and hybrid surfaces. This demonstrates that the addition of amino-functionalized silica particles to the hydrogel matrix does not adversely affect cell adhesion. Cells on all surfaces studied have elongated shapes, what may indicate a compact and well-crosslinked structure of tested materials. Lee et al. reported similar shape of MG63 cells for materials with a smaller size of micropores [61]. Moreover, the efficiency of the cell attachment for hybrid materials can also be affected by the functionalization of silica particles with amino groups. According to the literature, positively charged amino groups on a surface can support the cell adhesion and spreading [62], in particular they can promote osteoblast attachment [63]. We have determined the spreading of seeded cells (see Fig. 10B) and found that the cells cultured on all studied materials exhibit similar morphology. 4. Conclusions We have developed the novel multicomponent, bioactive and structurally stable hybrid materials, which can be prepared as sols and crosslinked in situ with formation of hydrogels, as it was confirmed by rheological measurements. Materials prepared are based on collagen/ chitosan/hyaluronic acid biopolymeric hydrogel matrix in which the bioactive phase in the form of amino-functionalized silica particles

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was dispersed. Such modification of silica particles makes possible their attachment to polymeric hydrogel network on crosslinking with genipin, as it was confirmed by degradation and swelling examinations. This approach seems to be especially important as it allows to avoid the occurrence of particle phase separation, the process which would be especially unfavourable for in vivo applications. The effective formation of the spherical amino-functionalized silica particles and the coexistence of surface-exposed amino and silanol groups was demonstrated by means of DLS, SEM, XPS and FTIR analyses. Employing in situ rheological examination, we have confirmed the injectability potential of hybrids developed what is important property since it enables the formation of scaffold in the bone defect area in the minimally invasive way. Moreover, we also proved that the gel formation on genipin crosslinking is not hampered on addition of amino-functionalized silica particles. The in vitro experiments performed under simulated body fluid SBF conditions demonstrated that the amino-functionalized silica particles successfully act as nucleation sites for apatite formation since their presence at the concentration higher than 8.3 mg/mL in the hydrogel matrices indeed ensures the biomineralization. Finally, the in vitro cell culture studies revealed that the materials developed are biocompatible as they supported MG-63 cells proliferation, adhesion as well as ALP expression. Considering all data obtained one can conclude that the facile one-step fabrication as well as the injectability potential of structurally stable hybrid materials developed here make them promising candidates for bioactive scaffolds in bone regeneration procedures.

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