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Effect of inorganic and organic bioactive signals decoration on the biological performance of chitosan scaffolds for bone tissue engineering

Effect of inorganic and organic bioactive signals decoration on the biological performance of... The present work is focused on the design of a bioactive chitosan-based scaffold functionalized with organic and inorganic signals to provide the biochemical cues for promoting stem cell osteogenic commitment. The first approach is based on the use of a sequence of 20 amino acids corresponding to a 68–87 sequence in knuckle epitope of BMP-2 that was coupled covalently to the carboxyl group of chitosan scaffold. Meanwhile, the second approach is based on the biomimetic treatment, which allows the formation of hydroxyapatite nuclei on the scaffold surface. Both scaffolds bioactivated with organic and inorganic signals induce higher expression of an early marker of osteogenic differentiation (ALP) than the neat scaffolds after 3 days of cell culture. However, scaffolds decorated with BMP-mimicking peptide show higher values of ALP than the biomineralized one. Nevertheless, the biomineralized scaffolds showed better cellular behaviour than neat scaffolds, demonstrating the good effect of hydroxyapatite deposits on hMSC osteogenic differentiation. At long incubation time no significant difference among the biomineralized and BMP-activated scaffolds was observed. Furthermore, the highest level of Osteocalcin expression (OCN) was observed for scaffold with BMP2 mimic-peptide at day 21. The overall results showed that the presence of bioactive signals on the scaffold surface allows an osteoinductive effect on hMSC in a basal medium, making the modified chitosan scaffolds a promising candidate for bone tissue regeneration. 1 Introduction engineering are represented by primary osteogenic cells (i.e., osteoblasts); while, also mesenchymal stem cells have Nowadays, tissue-engineering approaches are based on the shown a good potential to readily differentiate in osteogenic repair of systemically altered tissue structure by transplan- phenotype [1, 2]. In that case, osteoinductive scaffold bio- tation of cells combined with supportive scaffolds and materials can induce osteogenesis of stem cells by stimu- biomolecules. To this aim, main cell sources for bone tissue lating osteogenic signalling pathways that improve osteogenic differentiation [3, 4]. In this view, several bio- degradable polymers are used as scaffold materials for bone tissue engineering [5–9] and, among them, natural polymers have attracted great interest due to their biological and These authors contributed equally: Alessandra Soriente and Ines chemical similarities to natural tissues [10]. The Chitosan Fasolino. has been found an interesting candidate in a wide range of * Maria Grazia Raucci applications for its important biological properties including mariagrazia.raucci@cnr.it biocompatibility, biodegradability, nontoxicity, notable * Christian Demitri affinity to proteins, antibacterial and fungistatic properties christian.demitri@unisalento.it [11–13]. Chitosan is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by β Institute of Polymers, Composites and Biomaterials – National (1–4) glycosidic bonds. The amount of glucosamine is Research Council (IPCB-CNR), Mostra d’Oltremare Pad.20 - Viale J.F. Kennedy 54, Naples 80125, Italy calculated as the degree of deacetylation (DD). Its mole- cular weight may range from 300 to over 1000 kD with a Department of Engineering for Innovation, University of Salento, Via Monteroni, Lecce 73100, Italy DD from 30 to 95% [14, 15], due to the origin and 1234567890();,: 1234567890();,: 62 Page 2 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 preparation procedure. In its crystalline form, chitosan is covalently to the carboxyl group of chitosan scaffold [21]. It typically insoluble in aqueous solution over pH 7, whereas, is reported that peptide possesses ectopic bone morphoge- in diluted acid solutions as acetic acid (pH 6.0), the proto- netic activity in vivo when covalently linked to alginate nated free amino groups on glucosamine facilitate solubility hydrogel [22], and also showed good properties for skin of the molecule [16]. Thanks to its excellent ability to be wound healing [23] and driving in vitro nerve formation processed into porous structures and the possibility to [24]. control physical and chemical properties under mild con- The second approach is based on inorganic particles ditions [10], Chitosan has been used as a non-protein matrix based on calcium phosphate, in particular hydroxyapatite for 3D tissue growth providing the biological primer for (HA). The HA (Ca (PO ) (OH) ) having a chemical 10 4 6 2 cell-tissue proliferation and reconstruction. In bone tissue composition similar to that of the mineral phase of bone, is engineering, the porous structure of Chitosan provides a 3D used as a load bearing implant [25] for bone repair and scaffold for bone cells to grow and proliferate in order to regeneration for its bioactive and osteoconductive proper- obtain a good osteointegration; in that case the scaffold ties. To improve the bioactivity on the Chitosan scaffold must have optimal micro structure such as pore size, shape surface, the materials were treated by biomimetic approach and good interconnection [17]. Among all the properties, its [26]. In this way, the bonelike apatite shows a composition chemical structure (which includes three types of reactive and structure similar to that of mineralized bone, rather than functional groups, an amino group as well as both primary of sintered stoichiometric apatite. This provides further and secondary hydroxyl groups at the C(2), C(3), and C(6) important characteristics such as low crystallinity and positions respectively) is the feature that allows modifica- nanoscale sizes, similar to the resorption and remodelling tion of chitosan like graft copolymerization for specific properties of bone [27]. Consequently, the apatite formed in applications. Furthermore, these chemical groups can form SBF is believed to exhibit even higher bioactivity and covalent and ionic bonds thus improving their own che- biocompatibility [28, 29] than sintered hydroxyapatite. mical properties useful for biological cues aimed at regen- In this study, the effect of biomimetic cues on cellular erating bone tissue. To this purpose, biomimetic functional behaviour in terms of proliferation and osteogenic differ- scaffolds can be developed by modifying scaffolds with entiation of hMSC in osteoblast phenotype were investi- bioactive components including inorganic particulates and gated. Cell proliferation was analyzed by using standard osteogenic growth factors/peptides and by incorporating procedures such as AlamarBlue assay that allows to study these components into the scaffolds [4, 18]. These types of the effect of chitosan based scaffold on cell metabolic direct and indirect scaffold modifications provide bio- activity as index of cell viability. The capability of these chemical cues for promoting stem cell osteogenic bioactive scaffolds to induce osteogenesis in terms of MSC commitment. osteogenic differentiation was investigated through com- In this work, we propose two different approaches to mercial ELISA and colorimetric kit for testing the expres- obtain bioactive chitosan scaffolds by using biomolecules sion of early and late marker of osteogenesis.Morphological as peptide mimicking growth factors and/or inorganic cal- investigations by Scanning Electron Microscopy (SEM) cium phosphate particles. Generally, bioactive molecules were performed to evaluate cell-material interactions. can be injected locally in the healing area or directly incorporated into an implanted scaffold. Several investiga- tions have reported natural and synthetic components were 2 Materials and methods used as carrier of BMP, including collagen, and biode- gradable artificial polymers, such as polylactic acid and 2.1 Scaffold preparation polyglycolic acid. However, all these polymers released most of the BMP with an initial high burst effect, and soon Chitosan scaffolds were prepared by using a combination of the local concentration of BMP dropped below the ther- physical foaming and microwave curing processes [26]. At apeutic level with a loss of its biological activity [19]. first, chitosan solution CS (Medium molecular weight, DD Indeed, major weaknesses of this approach concern a 75–85%) was prepared by dissolving chitosan in 0.1 M potential loss of bioactivity during the incorporation in the Acetic Acid (AA,99%) solution (1.5%wt). The mixture was polymeric matrix and a release rate depending on scaffold stirred for 1.5 h to obtain a homogeneous viscous polymer degradation which might not match to an optimized use of solution; successively, fixed amounts of Polyethylene gly- the factors. The development of the peptide mimicry of col diacrylate (PEGDA 20 and 40%w/V) (average mole- proteins offers an attractive potential alternative [20]. Here, cular weight of 700 g/mol) were added to the CS solution as a first approach to obtain bioactive chitosan scaffold, a and stirred for 1.5 h. In this step 2,2-Azobis [2-(2-imida- sequence of 20 amino acids corresponding to a zolin-2 yl)propane] dihydrochloride was used as thermo- 68–87 sequence in knuckle epitope of BMP-2 was coupled initiator (TI). A selected physical blowing agent as Pluronic Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 3 of 12 62 Table 1 Scaffold composition NH : COOH 2_CS BMP2 and amount of peptide loaded for bioactivation 1:0.007 1:0.018 80CS20P_BMP 3,6 µg peptide/mg scaffold – 60CS40P_BMP 1,4 µg peptide/mg scaffold *3,6 µg peptide/mg scaffold 60CS40P_BMP* The asterisks indicate the sample 60CS40P_BMP with 3.6ug of peptide (60CS40P_BMP*) F127 (0,4% w/w), was added and mixed at 800 rpm for The cleaved products were characterized by analytical 30 min, so that a homogeneous distribution of air bubbles in HPLC and mass spectrometry (micro-TOF; Burker), before a stable form was obtained [26]. In the second step, the and after purification by preparative HPLC. porous structures of the foaming at different concentrations In our system, peptide immobilization occurs among of PEGDA were chemically stabilized via radical poly- NH _CS and COOH_BMP2 with a molar ratio 1:0.007; in merization induced by heating the sample in a microwave this way an amount of 3.6 and 1.4 µg peptide/mg scaffold (MTS 1200 Mega, Milestone Inc., Shelton, CT 06484). was used for 80CS20P and 60CS40P, respectively. More- Some of the resulting samples were washed in acetone to over, for 60CS40P a molar ratio of 1:0.018 of NH _CS and evaluate the possible removal of unreacted PEGDA and COOH_BMP2 was also used to obtain scaffold with the dried in a vacuum oven at 45 °C. The combination of dif- same amount of peptide used in 80CS20P (3.6 µg peptide/ ferent ratio CS and PEGDA allowed to obtain CS-based mg scaffold) see Table 1. In this study, the peptide amount scaffolds at two percentages: 80CS20P and 60CS40P.The was determined on the basis of preliminary biological samples were analysed without any further modifications. experiments performed on BMP-2 peptide at different concentrations. 2.1.1 Scaffold bioactivation by organic signals 2.1.2 Scaffold bioactivation by inorganic signals The first approach for scaffold bioactivation is based on a covalent immobilization of a bioactive peptide of Bone Furthermore, the semi-interpenetrating CS-PEG scaffolds Morphogenetic Protein (BMP-2) on scaffold surface. The (80CS20P and 60CS40P) were bioactivated using a mod- 67–87 residue of the “knuckle” epitope of hBMP-2 ified method described by Kokubo and co-workers [27, 30]. (NSVNSKIPKACCVPTELSAI) was synthesized by The treatment [31], is based on the use of supersaturated microwave-assisted Fmoc solid phase peptide synthesis SBF solutions to stimulate the nuclei formation and growth. (SPPS). The resin support and all amino acids (aa) were It consists of a nucleation phase obtained by incubation of obtained from Inbios srl (Naples, Italy). Rink amide linker scaffolds in a supersaturated SBF solution (5x SBF1) for (0.4 mmoles; Iris Biotech GmBH) was attached to a 3 days, followed by crystallization and growth of apatite Tentagel-S-NH resin (Iris Biotech GmBH) in a glass vial; nuclei in a modified solution without the inhibitors of 1 mL of 0.45 M HBTU in N,N-dimethylformamide (DMF) crystallization as hydrogen carbonate and magnesium ions and 0.5 mL of 33% DIPEA in DMF were added to the [32, 33]. reaction vessel to activate the exposed amino groups of the In the present study, the pH solution was fixed at 7.4 in resin and after each coupling step to activate the deprotected order to promote interactions between decarboxylated − 2+ group of the coupled aa. The aa coupling was performed groups (COO ) and Ca ions. The SBF volume was cal- after stirring the reaction suspension for 7 min in a Micro- culated with reference to the total material surface using a wave synthesizer (Power 13 W; Temperature 60 °C; stirring specific ratio of the exposed surface to SBF volume, as rate 900 rpm); the solvent was removed and the mixture was reported in the literature [34]. After the incubation time, all washed with DMF (Sigma Aldrich). Fmoc-protecting group materials were gently rinsed in distilled water to remove was removed from the coupled aa by incubating the resin excess ions and, then, dried overnight under laminar flow with 20% v/v piperidine in DMF for 3 and 7 min. hood. Following coupling of the final aa, the resin-bound BMP- 2 peptide was washed several times with dichloromethane, 2.2 Characterization methanol, and diethyl ether. Finally, BMP-2 peptide was cleaved from the resin support by exposure to a solution 2.2.1 Morphological analysis composed by TFA 95%-TIS 2.5%-dH O 2.5% mixture for 3 h and precipitated in a cold diethyl ether by centrifugation The CS scaffold surfaces, before and after biomineraliza- for 5 min at 6000 rpm. tion, were analysed by Scanning Electron Microscopy 62 Page 4 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 (SEM, JEOL 6310). For SEM analysis, the materials were 2.3 Biological investigations mounted by a double adhesive tape to aluminium stubs. The stubs were sputter-coated with gold to a thickness of around 2.3.1 In vitro cell culture 15–20 nm. SEM analysis was performed at different mag- nification at a voltage of 20 keV. X-ray energy dispersive In vitro biological assays were performed on human spectroscopy (EDAX, Genesis 2000i) analysis was used for Mesenchymal Stem Cells line (hMSCs) obtained from a qualitative estimation of the Ca/P ratio. LONZA (Milano, Italy). hMSC were cultured in 75 cm cell culture flask in Eagle’s alpha Minimum Essential Medium (α- 2.2.2 Microcomputed tomography (MicroCT) analysis MEM) supplemented with 10% Foetal Bovine Serum (FBS), antibiotic solution (streptomycin 100 µg/ml and penicillin Microcomputed tomography (Bruker Skyscan 1172, Kon- 100U/ml, Sigma Chem. Co) and 2 mM L-glutamine, without tich, Belgium) was used to compare the 3D structure of both osteogenic factors. For all experimental procedures hMSCs at the native and bimomineralized scaffolds to assess their passage 4 were used. Cells were incubated at 37 °C in a porosity. The following MicroCT settings were identified humidified atmosphere with 5% CO and 95% air. for proper scanning of the native samples, based on their X- ray attenuation capacity: (a) the X-ray source was set at 2.3.2 Cell proliferation 25 kV and 139 μA, whitout filtering; (c) for the detection of porosity, the pixel size was set at 10 μm; (d) the exposure The cell biocompatiblity of BMP-2 solutions at different time was 940 ms, with a 2 × 2 binning; (e) samples were not concentrations starting from 2.70 µM to 270pM was eval- rotated. Due to a limited presence of the CaP phase, bio- uated by seeding hMSCs (5000 cells at passage 4) at dif- mineralized scaffolds samples required the same MicroCT ferent time 1, 3, 7 and 14 days in α-MEM supplemented settings. For this reason all scanning parameters were kept with 10% Fetal Bovine Serum (FBS), antibiotic solution unvaried. After reconstruction with NRecon software, (streptomycin 100 µg/ml and penicillin 100U/ml) and 2 mM DataViewer was used to visualize the 3D view of the L-glutamine. The cell proliferation was determined using an scaffolds and the section on the XY plane. The recon- Alamar blue assay (AbD Serotec, Milano, Italy) based on structed grey scale images of the samples were then ana- the metabolic activity of live cells. lyzed with CTAn software to quantify the porosity, after Furthermore, the biocompatibility of neat (80CS20P and selection of a volume of interest (a cylindrical VOI with 60CS40P), and bioactivated scaffold (80CS20P_bio, 3 mm diameter and 3 mm height) and appropriate thresh- 80CS20P_BMP, 60CS40P_bio, 60CS40P_BMP, olding. CTVol software was finally used for the rendering 60CS40P_BMP*) was analysed. The medium in cell-load of 3D VOI models, for both native and biomineralized scaffolds culture plates was removed after cultured for 3, 7, 14 scaffolds. and21daysand in vitro cell proliferation was evaluated with Alamar blue assay, according to the manufacturer’s protocol. 2.2.3 In vitro release study Finally, absorbance was measured at 570 and 600 nm. Over the culture time, the cell medium was replaced every two days. The peptide release profiles from scaffolds at different compositions (80CS20P, 60CS40P and *60CS40P) were 2.3.3 Osteogenic differentiation: alkaline phosphatase determined in vitro by High Performance Liquid Chroma- expression tography system (HPLC, Agilent). The scaffolds bioacti- vated with BMP-2 peptide were dipped in 200 µL sterile Differentiation of hMSC was tested measuring alkaline Tris-buffer solution (pH = 6.8) and kept in a shaking phophatase (ALP) activity in the cultures of neat and incubator (37 °C, 40 rpm) for various time periods of up to bioactivated CS-based scaffolds at different time points 4 weeks. At specific time points supernatant was collected (SensoLyte pNPP ALP assay kit, ANASPEC, Milano, and an equal amount of fresh medium was added to each Italy). At each time point, cultures were washed gently with sample. A quantity equal to 20 μL of the obtained super- PBS, followed by washing with cold 1× assay buffer (BD natant was injected in a chromatograph equipped with a UV Biosciences, Milano, Italy). The ALP activity was evaluated detector and a reversed phase column (Reprospher C18, onto the cell lysates (50 μl), in that case the cultures were 150 mm × 4.6 mm, DR. MAISCH, GmbH). The mobile treated with 1× lysis buffer with 0.2% of Triton X-100. To phase systems consisted of 90% water (A) and 10% acet- correct the ALP values for the number of cells present on onitrile (B). The flow rate was 1.0 mL/min, and the wave- each scaffold, double stranded DNA (dsDNA), as a marker length was set at 220 nm. All experiments were triplicated for the cell number, was measured using a Pico- for each sample. Green_dsDNA quantification kit (Invitrogen). First, 100 µl Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 5 of 12 62 of diluted Picogreen_dsDNA quantification reagent was 80CS20P_BMP, 60CS40P_bio, 60CS40P_BMP, added to 100 µl of cell lysates in a flat-bottomed, 96-well 60CS40P_BMP*) in basal medium for 21 days of culture plate. Following 10 min incubation, the fluorescence of time. Quantitative levels of OCN secreted into the culture Picogreen was determined at a wavelength of 520 nm after medium were determined using an enzyme-linked immu- excitation at 585 nm using a spectrophotometer (Victor X3, noassay kit following the manufacturer’s instructions. Perkin-Elmer, Italy). dsDNA was quantified according to a calibration curve of l-dsDNA standard in 10 mM Tris, 2.3.5 Statistical analysis 1 mM EDTA, pH 7.5, buffer. Each experiment was per- formed three times in triplicate. The results of ALP activity All quantitative experiments were performed in triplicate were reported as nanograms (ng) of ALP normalized to the and the results were expressed as mean ± standard deviation micrograms (µg) of total DNA content. The alkaline pho- (SD). Statistical analysis of the data was conducted using t- phatase activity of hMSC seeded onto biomimetic and non Student test. Differences between the groups of p < 0.05 biomimetic 80CS20P and 60CS40P scaffolds was were considered statistically significant. determined. 2.3.4 Mineralization analysis: osteocalcin expression 3 Results The effect of CS-based scaffolds on osteogenic differentia- 3.1 Morphological analysis tion of hMSCs was evaluated by measuring a later marker of differentiation, such as osteocalcin (OCN). Osteocalcin Morphological investigations performed by SEM analysis levels were measured using a commercially available kit demonstrated that the foaming process allows to obtain an (Quantikine Human Osteocalcin Immunoassay R&D sys- interconnected and homogeneous structure with pores at tem, Italy) following the manufacturer’s instructions. hMSC different size from 20 to 300 µm. Moreover, CS based- cells were cultured on control, neat scaffolds (80CS20P and scaffolds have high interconnected structure inside the walls 60CS40P) and after biomimetic treatments (80CS20P_bio, of the pore as shown Fig. 1. The presence of macropores Fig. 1 SEM images of CS scaffold before and after biomineralization process. Illustration of ionic interaction among chemical groups of CS polymer and SBF ions 62 Page 6 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 enables the cells to migrate in the internal part of scaffold, while the micropores due to solvent evaporation determine a good nutrient flow and avoid radical oxygen species (ROS) formation that is responsible of cell necrosis. However, the scaffold porosity depends upon the CS concentration. In effect, the porosity decreases by reducing the CS con- centration. Furthermore, an open porosity of 78 and 60% for 80CS20P and 60CS40P was revealed respectively by using a pycnometer method [26]. These differences in open por- osity were due to the presence of different percentages of PEGDA which increased the structure stability by reducing the porosity. Furthermore, SEM images of CS-PEGDA scaffolds prepared by biomimetic methods demonstrated the pre- sence of calcium phosphate (CaP) deposits on the scaffold surface and on the internal pore walls (Fig. 1) after only 7 days of treatment. This behaviour in a short time was due to the use of two specific parameters represented by a supersaturated concentration (5x SBF) and pH value. In fact, CS polymer at physiological value (pH = 7.4) shows a negative charge due to the deprotonation of COOH groups (pKa = 6.5). In this way, interactions among two COO Fig. 2 m-CT analysis of the scaffolds. 3D reconstruction of 80CS20P CS 2+ scaffold before and after biomineralization process and a summary groups and bivalent Ca were favoured. Furthermore, table with the calculation of the porosity EDAX analysis, performed during SEM observation, con- firmed the formation of CaP deposits with a Ca/P ratio of about 1.67 (Fig. 1). 3.1.1 Scaffold bioactivation by organic signals and in vitro As shown in Fig. 2, MicroCT scanning of native and release study biomineralized samples highlighted an higher porosity of both scaffolds. The microwave-assisted Fmoc solid phase peptide synthesis 3D analysis also evidenced an overall porosity of 97.9 allows to obtain a peptide in a shorter time than the con- and 98.2% for 80CS20P and 60CS40P scaffolds respec- ventional synthesis process, with a molecular weight of tively. No significant difference can be detected between the 2074.4 Da and high purity (>98%) as demonstrated by two samples. Conversely, a reduction of the porosity can be chemical characterization through mass spectrometry and observed once the bioactivation treatment has been per- analytical HPLC respectively (Fig. 3a). The pure peptide formed. In particular, an overall porosity of 93.5 and 96.8% was immobilized on CS scaffolds (80CS20P and 60CS40P) can be detected for 80CS20P_bio and 60CS40P_bio scaf- (Fig. 3b) by carbodiimide reaction mechanism, where the fold respectively. The pore size distribution among the VOI –NH groups of CS and –COOH groups of BMP-peptide results to be uniform with no difference between the scaf- were involved. This covalent bond guarantees an in vitro folds. Based on the calculation of the volumetric open pore release for a long time. In particular, the results onto size distributions, the average pore size of 80CS20P and 80CS20P scaffolds demonstrated an initial release of pep- 60CS20P scaffolds was found to be 367 ± 234 μm and 456 tide about 8–10 % (Fig. 3b) due to the scaffold properties ± 313, respectively. The biomineralization process does not (i.e. swelling) [26]; however the release is sustained up to induce any relevant change in the pore size dimension and 4 weeks where 80CS20P has released about 94.5% of distribution. From the 3D reconstruction and the XY section peptide. In the case of 60CS40P_BMP scaffolds, having (Fig. 2) a densification of the structure could be observed. higher amount of BMP2 mimic peptide (3.6 µg/mg scaf- This is addressed to the deposition of CaP phase on the fold), a slower release in the first 48 h is obtained, probably surface of the pores on the nanometric scale. Even in this due to a higher amount of PEGDA compared to the case the spatial distribution of the CaP phase is uniform 80CS20P samples; however up 4 weeks 60CS40P_BMP assessing an effective biomineralization over the overall and 60CS40P_BMP* have released 97.3 and 92.9% of scaffold volume. peptide, respectively. Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 7 of 12 62 Fig. 3 a Mass spectrometry (micro-TOF) and HPLC chromatogram peak of BMP-2 peptide; b kinetic release of peptide from 80CS20P scaffolds and 60CS40P at different BMP concentrations (60CS40P_BMP, 60CS40P_BMP*) Fig. 4 (a) Confocal images of hMSCs incubated with BMP-2 peptide and Alkaline phosphatase activity (ng ALP/ug DNA) results of BMP solutions at different concentration (range 2.7 µM–270 pM) after 24 h solutions at different concentrations (range 2.7μM–270pM) after dif- of incubation time; (b–c) Alamar Blue (% reduction of alamar blue) ferent time points, respectively 3.2 Biological properties different concentrations on hMSCs adhesion was deter- mined after 1 day of cell culture. The images obtained using 3.2.1 BMP-2 mimic peptide: biological activity immunofluorescence analysis (Fig. 4a) demonstrated that the cells in contact with solution at higher concentration The bioactivity of neat peptide was investigated by mea- (2.7 µM) showed a polygonal morphology typical of phe- suring its effect on hMSCs behavior in terms of prolifera- notype like-osteoblast than the control (CTR), while at tion and differentiation. First of all, the effect of peptide at lower concentration (270pM) the cells showed a fibroblast- 62 Page 8 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 like morphology. Furthermore, quantitative analysis of the cell proliferation (Fig. 4b) suggested an increasing of pro- liferation at first 3 days and a decreasing at long times. This behaviour was due to an initial ostegoenic differentiation. In Fig. 4c higher ALP values were observed in presence of BMP-2 at concentration of 2.7 µM. Hence, these pre- liminary studies allowed to perform a screening about the optimal concentration of BMP-2 peptide necessary for scaffold bioactivation that is the 2.7 µM solution. 3.2.2 Biological properties of bioactivated scaffolds The effect of 80CS20P and 60CS40P scaffolds with and w/ o BMP-2 mimic peptide (2.7 µM) on hMSCs was also evaluated. This analysis demonstrated that, at 24 h, there (A) was a good cell attachment on the scaffold surface with higher values for the bioactivated scaffolds at different * # Day 3 Day 14 compositions than neat scaffolds (Fig. 5a). After this time, p≤ ≤0.05 and p≤0.00 01 vs 80CS20P * ° Day 21 60 p p≤0.05 and p≤0.01 vs 60CS40P Day 7 the proliferation increases over culture time confirming that the scaffolds support adhesion and cell migration inner the # structure. In particular scaffold containing BMP-2 sig- nificantly (80CS20P_BMP = p ≤ 0001; 60CS40P_BMP = p ≤ 0,05) increase MSC proliferation at day14 (Fig. 5b) compared to both neat scaffolds (80CS20P and 60CS40P) * where the cells become confluent. Meanwhile, at day 21 a slight decreasing was observed for scaffold with highest amount of BMP peptide (60CS40P_BMP*). This latter decreasing in cell proliferation is related to the increase in cell differentiation confirmed by ALP expression. To evaluate the effect of scaffolds on osteogenic differ- entiation, the expression of alkaline phosphatase (ALP), as (b) early marker of hMSCs differentiation in pre-osteoblast phe- notype, was evaluated. The experiments were performed in Fig. 5 a Quantitative analysis of cell adhesion was quantified using Calcein AM assay and represented as percentage of cells attached after basal medium taking in account the hypothesis concerning that 24 h of incubation time; b cell proliferation at long time (3, 7, 14 and the organic and inorganic bioactive signals act as osteoin- 21days) of hMSCs seeded on 80CS20P scaffolds, before and after ductive factors. The results demonstrated that highest ALP biomineralization (80CS20P, 80CS20P_bio) with and w/o organic values (Fig. 6a) were obtained for cells in contact with BMP-2 signals (80CS2P_BMP, 80CS20P_bio_BMP). Quantitative analyses were performed by using Alamar blue™ assay using manufacturer’s like peptide (CTR+ BMP2, 18 µg) at 3 days of cell culture. protocol Furthermore, the scaffolds bioactivated with BMP-2 like peptide (80CS20P_BMP, p ≤ 0,05; p ≤ 0001) and biominer- alized scaffolds (80CS20P_bio, p ≤ 0,05; p ≤ 0001), induce a the culture time (Fig. 6b). This behaviour was also observed significant increase in ALP levels at long-time than neat for the expression of non-collagenous bone ECM protein (i.e. 80CS20P and 60CS40P materials. The scaffolds with lower osteocalcin) used as later marker of osteogenic differentiation amount of CS bioactivated with BMP-2 like peptide (Fig. 7). Here scaffolds bioactivated with BMP-2 and only the (60CS40P_BMP and 60CS40P_BMP*) showed highest ALP biomineralized scaffold with higer percentage of Chitosan values at day 3, probably due to the lower release profile of (80CS20P_bio) were able to significanly (p ≤ 0,05; p ≤ 0,01; peptide in the first 48 h and an higher scaffold stability (as p ≤ 0001) increase osteocalcin values at day 21 of culture time. demonstrated in the kinetic release study reported in the Fig. 3). However, higher ALP expression was observed for the full set of scaffolds and, in particular, 80CS20P_bio and 4 Discussion 80CS20P_BMP showed a similar behaviour than 60CS40P scaffolds (both 60CS40P_bio and 60CS40_BMP) at day 3. In bone tissue engineering the scaffold should provide Additionally, 60CS40P_BMP* showed the best results over necessary support as an artificial extracellular matrix that 80CS S2 20P 80/ /C CS20P_BIO 80CS20P_B BM MP 60CS40P 60CS40P_BI IO O 60CS S4 40P_BMP CS4 _BMP P* * 60 0P Percent of reduction (%) Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 9 of 12 62 allows cells to proliferate and maintain their differentiated functions. Essentially, scaffold acts as a temporary template to guide the formation of a new tissue. In this context, an ideal scaffold is characterised by excellent biocompatibility, molded biodegradability, cytocompatibility, suitable microstructure (pore size and porosity) and mechanical properties. Additionally, scaffold must be capable of pro- moting cell adhesion and retaining the metabolic functions of the attached cells [34]. For these reasons, the aim of this work was to study the effect of biomimetic functional scaffolds obtained by modifying polymer scaffolds with osteoinductive signals including inorganic components, such as hydroxyapatite deposition and organic signals, such (A) as BMP-2 mimetic peptide covalently immolized on the p≤0 0.05 vs 80CS20 0P Day 7 Da ay 14 * scaffolds. Therefore, this research could help to develop ° # p≤0 0.05 ; p≤0.01 an nd p≤0.001 vs 6 60CS40P Day 21 biodedragable scaffolds which are also carriers of osteo- * genic signals in order to obtain positive cellular responses in ]* ]* terms of osteogenic commitment. By a covalent peptide 1000 # # immobilization on the scaffolds with different chitosan concentrations (80CS20P_BMP and 60CS40P_BMP) a low percentage as burst release of peptide, 10 and 20% in the first 48 h, was observed respectively; whereas at longer intervals, a prolonged sustained release of up to 4 weeks has an important effect on cellular behaviour. The effect of BMP-2 on early ALP expression (Fig. 5a) in the first days of cell culture is related to the burst release of peptide. Meanwhile, the release of peptide over time determines the (B) expression of osteogenic marker (ALP-OCN) at long-term. Fig. 6 a Alkaline phosphatase activity expressed as nanograms of ALP This prolonged BMP-2 delivery was due to the high sta- normalized by amount of DNA (µg) produced by hMSCs after early bility of the scaffold structure (prepared by physical foam- (3 days) and long b times (7, 14 and 21 days). DNA amount was ing combined with microwave curing) [27]. determined by dsDNA picogreen assay Previous studies suggested a similar early burst release profile of BMP on hydroxyapatite (HA) bone grafts which are able to induce osteogenic differentiation of C2C12 cells p≤0.05 vs 80CS20P # ## ### p≤0.05, p≤0.01 and p≤0.001 vs 60CS40P [35]. In the present study, the higher percentage of burst release of 80CS20P_BMP samples compared to ## ### 60CS40P_BMP is probably due to the different porosity of these scaffolds. In the case of 60CS40P_BMP scaffolds a slower release in the first 48 h is probably due to a much more stable structure for the higher amount of PEGDA and a lower porosity compared to 80CS20P samples. In porous structures, fluid dynamics must to be coupled with structural mechanics of the scaffold. In this study, scaffold porosity influences effects of fluid flow (flow of cell culture medium) on BMP release. In effect, samples with higher porosity (80CS20P_BMP) showed an higher percentage of BMP release in the first days. Meanwhile up 4 weeks 60CS40P_BMP and 60CS40P_BMP* have released 97.3 and 92.9% of peptide, respectively. In vitro degradation tests showed a gradual dissolution of the scaffolds over time, while maintaining their 3D morphology and integrity Fig. 7 Osteocalcin marker expressed by hMSCs cultured on the scaffolds (80CS20P, 80CS20P_bio, 80CS20P_BMP, even after 6 weeks of incubation [26] and allowing an 80CS_bio_BMP) after 21days of incubation time in a basal medium extended release of organic bioactive signal. This extended 80CS2 20 0P 80/C CS S20P_BIO 80CS20P_B BM MP 60CS40P 60CS40P_ _B BIO 60C CS S40P_BMP 4 _ _B BM MP* 60CS 0P 80CS20P 80/CS20P_BIO 80CS20P_BMP 60CS40P 60CS40P_ IO 60CS40P_BMP 60CS40P_BMP* Osteocalcin levels (ng/ml) ng ALP/μg DNA 62 Page 10 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 release potentially promises the successful infiltration of could be explained by considering the difference in the ratio cells and new tissue formation. Indeed, biological results between the chitosan free amino groups (–NH ) and the demonstrated that the materials act as support for hMSCs induced carboxylic groups of BMP2 (–COOH). In fact, even adhesion and proliferation. A consistent migration of cells if the cumulative release profiles showed no significant into the scaffold is evident, as demonstrated in cross-section differences between the samples, the effect of the bioacti- SEM images. Moreover, the effect of organic and inorganic vation appears in the early osteogenic differentiation. This bioactive solid signals on osteogenic differentiation was trend suggests that, by increasing the COOH/NH ratio, a evaluated. positive effect on cellular behaviour is achieved. Moreover, Indeed, it is possible to characterise the process in three it could be explained with the specific peptide interactions stages: (a) cell proliferation, (b) matrix maturation, and (c) with the receptor on the cellular membrane which induce matrix mineralization [36]. In vitro, matrix maturation and hMSC differentiation into more mature osteobalst mineralization are usually enhanced by growing the cells to phenotypes. complete confluency and by adding specific osteoinductive Moreover, CS-PEGDA scaffold was a good carrier of factors [37]. After the proliferation, the matrix maturation peptide allowing a prolonged release over time and main- phase is characterised by an expression of alkaline phos- taining the local concentration of BMP2 mimic peptide at a phatase (ALP). ALP is a well-known early marker of good level, thus overcoming the drawbacks of some poly- osteogenic differentiation and plays a key role in the mers used as carriers for the therapeutic agents delivery mineralization of bone. As such it is considered a useful [37]. However, all samples showed significant positive biochemical marker of bone formation. Finally, during effects on hMSC behaviour in terms of proliferation and matrix mineralization (c) other genes for proteins such as osteogenic differentiation in basal medium, as confirmed by OC, BSP, and OPN are expressed. Analysis of bone cell- expression of early (ALP) and later signals (OCN) of specific markers like Procollagen, ALP, OPN and OCN, is osteogenic differentiation in bioactivated scaffolds. In par- used to characterise later osteogenic differentiation phases ticular, results have demonstrated that biomimetic scaffolds in vitro of hMSCs. The mineralization process of osteo- showed higher expression of OCN level, however the best blasts in in vitro culture has also been used as a model for behaviour at long time in terms of prolonged release and testing the effects of peptide loading on bone cell differ- cellular behaviour for scaffolds bioactivated with BMP entiation and bone formation. In this work, the expression mimic-peptide was observed. Furthermore, the present work of ALP and OCN were analysed; in particular, it was suggested that bioactivated scaffolds were able to direct observed that at 3 and 7 days no difference among the group osteoinductive processes by expression of early and later of bioactivated scaffold and related controls (60CS40P and markers of osteogenesis. 80CS20P) was observed. Meanwhile, after 14 days the best ALP expression for 80CS20P_BMP scaffold was observed, where a peptide 5 Conclusions release of approximately 65–75% (12–14 µg) was obtained. In parallel, 60CS40P_BMP* showed the highest ALP Our study has demonstrated the possibility of producing expression at 21 days. On the other hand, biomineralized biomimetic scaffolds with highly interconnected and scaffolds developed through biomineralization treatment homogeneous structure with pores of different sizes from 20 (80CS20P_bio and 60CS40P_bio) showed an increased to 300 µm in agreement with the dimensional features ALP expression compared to non biomineralized chitosan required for bone regeneration. The proposed technique materials after 3 days of incubation. At the same point time, holds strong potential for applications in the field of similar cell differentiation behaviour, between 80CS20P_bio custom-made bone substitute. Moreover, the presence of and 60CS40P_bio, was observed. At longer incubation times bioactive signals on the scaffold surface allows to obtain an (7, 14 and 21 days), no significant difference between two osteoinductive effect on hMSC. Indeed, both bioactivated biomineralized groups and BMP-activated scaffolds was scaffolds showed higher ALP values compared to neat observed. In particular, 80CS20P_BMP showed better ALP materials at short time. In particular, scaffolds decorated expression after 14 days than 60CS40P_BMP/BMP*. After with BMP-mimicking peptide presents sustained ALP 21 days the 60CS40P_BMP reached a plateau compared to values at long times. This behaviour suggested that CS- levels at 14 days. On the contrary, 60CS40P_BMP* sample PEGDA scaffold is an appropriate carrier for peptide at 21 days shows an increased ALP value compared to both allowing to maintain the local peptide concentration at good 60CS40P_BMP and 80CS20P_BMP. The same behaviour level over time. Furthermore, biomineralized scaffolds was also detected for Osteocalcin expression (OCN) where showed a better cellular behaviour than neat scaffolds thus the highest level was observed for scaffold bioactivated with confirming the effect of hydroxyapatite deposits on hMSC BMP2 mimic-peptide at day 21. This different behaviour osteogenic differentiation. Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 11 of 12 62 The comparison between the two different approaches 11. Altiok D,Altiok E,Tihminlioglu F, Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme emphasizes that the presence of bioactive signals on CS- oil for potential wound healing applications. J Mater Sci Mater PEGDA scaffolds plays a pivotal role in osteogenesis pro- Med. 2010;21(7):2227–2236. cess. Indeed, the modifications on scaffolds allow to hMSC 12. 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Crit Rev Oral Biol Med. 1992;3:269–305. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Materials Science: Materials in Medicine Springer Journals

Effect of inorganic and organic bioactive signals decoration on the biological performance of chitosan scaffolds for bone tissue engineering

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Springer Science+Business Media, LLC, part of Springer Nature
Subject
Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
ISSN
0957-4530
eISSN
1573-4838
DOI
10.1007/s10856-018-6072-2
pmid
29736686
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Abstract

The present work is focused on the design of a bioactive chitosan-based scaffold functionalized with organic and inorganic signals to provide the biochemical cues for promoting stem cell osteogenic commitment. The first approach is based on the use of a sequence of 20 amino acids corresponding to a 68–87 sequence in knuckle epitope of BMP-2 that was coupled covalently to the carboxyl group of chitosan scaffold. Meanwhile, the second approach is based on the biomimetic treatment, which allows the formation of hydroxyapatite nuclei on the scaffold surface. Both scaffolds bioactivated with organic and inorganic signals induce higher expression of an early marker of osteogenic differentiation (ALP) than the neat scaffolds after 3 days of cell culture. However, scaffolds decorated with BMP-mimicking peptide show higher values of ALP than the biomineralized one. Nevertheless, the biomineralized scaffolds showed better cellular behaviour than neat scaffolds, demonstrating the good effect of hydroxyapatite deposits on hMSC osteogenic differentiation. At long incubation time no significant difference among the biomineralized and BMP-activated scaffolds was observed. Furthermore, the highest level of Osteocalcin expression (OCN) was observed for scaffold with BMP2 mimic-peptide at day 21. The overall results showed that the presence of bioactive signals on the scaffold surface allows an osteoinductive effect on hMSC in a basal medium, making the modified chitosan scaffolds a promising candidate for bone tissue regeneration. 1 Introduction engineering are represented by primary osteogenic cells (i.e., osteoblasts); while, also mesenchymal stem cells have Nowadays, tissue-engineering approaches are based on the shown a good potential to readily differentiate in osteogenic repair of systemically altered tissue structure by transplan- phenotype [1, 2]. In that case, osteoinductive scaffold bio- tation of cells combined with supportive scaffolds and materials can induce osteogenesis of stem cells by stimu- biomolecules. To this aim, main cell sources for bone tissue lating osteogenic signalling pathways that improve osteogenic differentiation [3, 4]. In this view, several bio- degradable polymers are used as scaffold materials for bone tissue engineering [5–9] and, among them, natural polymers have attracted great interest due to their biological and These authors contributed equally: Alessandra Soriente and Ines chemical similarities to natural tissues [10]. The Chitosan Fasolino. has been found an interesting candidate in a wide range of * Maria Grazia Raucci applications for its important biological properties including mariagrazia.raucci@cnr.it biocompatibility, biodegradability, nontoxicity, notable * Christian Demitri affinity to proteins, antibacterial and fungistatic properties christian.demitri@unisalento.it [11–13]. Chitosan is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by β Institute of Polymers, Composites and Biomaterials – National (1–4) glycosidic bonds. The amount of glucosamine is Research Council (IPCB-CNR), Mostra d’Oltremare Pad.20 - Viale J.F. Kennedy 54, Naples 80125, Italy calculated as the degree of deacetylation (DD). Its mole- cular weight may range from 300 to over 1000 kD with a Department of Engineering for Innovation, University of Salento, Via Monteroni, Lecce 73100, Italy DD from 30 to 95% [14, 15], due to the origin and 1234567890();,: 1234567890();,: 62 Page 2 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 preparation procedure. In its crystalline form, chitosan is covalently to the carboxyl group of chitosan scaffold [21]. It typically insoluble in aqueous solution over pH 7, whereas, is reported that peptide possesses ectopic bone morphoge- in diluted acid solutions as acetic acid (pH 6.0), the proto- netic activity in vivo when covalently linked to alginate nated free amino groups on glucosamine facilitate solubility hydrogel [22], and also showed good properties for skin of the molecule [16]. Thanks to its excellent ability to be wound healing [23] and driving in vitro nerve formation processed into porous structures and the possibility to [24]. control physical and chemical properties under mild con- The second approach is based on inorganic particles ditions [10], Chitosan has been used as a non-protein matrix based on calcium phosphate, in particular hydroxyapatite for 3D tissue growth providing the biological primer for (HA). The HA (Ca (PO ) (OH) ) having a chemical 10 4 6 2 cell-tissue proliferation and reconstruction. In bone tissue composition similar to that of the mineral phase of bone, is engineering, the porous structure of Chitosan provides a 3D used as a load bearing implant [25] for bone repair and scaffold for bone cells to grow and proliferate in order to regeneration for its bioactive and osteoconductive proper- obtain a good osteointegration; in that case the scaffold ties. To improve the bioactivity on the Chitosan scaffold must have optimal micro structure such as pore size, shape surface, the materials were treated by biomimetic approach and good interconnection [17]. Among all the properties, its [26]. In this way, the bonelike apatite shows a composition chemical structure (which includes three types of reactive and structure similar to that of mineralized bone, rather than functional groups, an amino group as well as both primary of sintered stoichiometric apatite. This provides further and secondary hydroxyl groups at the C(2), C(3), and C(6) important characteristics such as low crystallinity and positions respectively) is the feature that allows modifica- nanoscale sizes, similar to the resorption and remodelling tion of chitosan like graft copolymerization for specific properties of bone [27]. Consequently, the apatite formed in applications. Furthermore, these chemical groups can form SBF is believed to exhibit even higher bioactivity and covalent and ionic bonds thus improving their own che- biocompatibility [28, 29] than sintered hydroxyapatite. mical properties useful for biological cues aimed at regen- In this study, the effect of biomimetic cues on cellular erating bone tissue. To this purpose, biomimetic functional behaviour in terms of proliferation and osteogenic differ- scaffolds can be developed by modifying scaffolds with entiation of hMSC in osteoblast phenotype were investi- bioactive components including inorganic particulates and gated. Cell proliferation was analyzed by using standard osteogenic growth factors/peptides and by incorporating procedures such as AlamarBlue assay that allows to study these components into the scaffolds [4, 18]. These types of the effect of chitosan based scaffold on cell metabolic direct and indirect scaffold modifications provide bio- activity as index of cell viability. The capability of these chemical cues for promoting stem cell osteogenic bioactive scaffolds to induce osteogenesis in terms of MSC commitment. osteogenic differentiation was investigated through com- In this work, we propose two different approaches to mercial ELISA and colorimetric kit for testing the expres- obtain bioactive chitosan scaffolds by using biomolecules sion of early and late marker of osteogenesis.Morphological as peptide mimicking growth factors and/or inorganic cal- investigations by Scanning Electron Microscopy (SEM) cium phosphate particles. Generally, bioactive molecules were performed to evaluate cell-material interactions. can be injected locally in the healing area or directly incorporated into an implanted scaffold. Several investiga- tions have reported natural and synthetic components were 2 Materials and methods used as carrier of BMP, including collagen, and biode- gradable artificial polymers, such as polylactic acid and 2.1 Scaffold preparation polyglycolic acid. However, all these polymers released most of the BMP with an initial high burst effect, and soon Chitosan scaffolds were prepared by using a combination of the local concentration of BMP dropped below the ther- physical foaming and microwave curing processes [26]. At apeutic level with a loss of its biological activity [19]. first, chitosan solution CS (Medium molecular weight, DD Indeed, major weaknesses of this approach concern a 75–85%) was prepared by dissolving chitosan in 0.1 M potential loss of bioactivity during the incorporation in the Acetic Acid (AA,99%) solution (1.5%wt). The mixture was polymeric matrix and a release rate depending on scaffold stirred for 1.5 h to obtain a homogeneous viscous polymer degradation which might not match to an optimized use of solution; successively, fixed amounts of Polyethylene gly- the factors. The development of the peptide mimicry of col diacrylate (PEGDA 20 and 40%w/V) (average mole- proteins offers an attractive potential alternative [20]. Here, cular weight of 700 g/mol) were added to the CS solution as a first approach to obtain bioactive chitosan scaffold, a and stirred for 1.5 h. In this step 2,2-Azobis [2-(2-imida- sequence of 20 amino acids corresponding to a zolin-2 yl)propane] dihydrochloride was used as thermo- 68–87 sequence in knuckle epitope of BMP-2 was coupled initiator (TI). A selected physical blowing agent as Pluronic Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 3 of 12 62 Table 1 Scaffold composition NH : COOH 2_CS BMP2 and amount of peptide loaded for bioactivation 1:0.007 1:0.018 80CS20P_BMP 3,6 µg peptide/mg scaffold – 60CS40P_BMP 1,4 µg peptide/mg scaffold *3,6 µg peptide/mg scaffold 60CS40P_BMP* The asterisks indicate the sample 60CS40P_BMP with 3.6ug of peptide (60CS40P_BMP*) F127 (0,4% w/w), was added and mixed at 800 rpm for The cleaved products were characterized by analytical 30 min, so that a homogeneous distribution of air bubbles in HPLC and mass spectrometry (micro-TOF; Burker), before a stable form was obtained [26]. In the second step, the and after purification by preparative HPLC. porous structures of the foaming at different concentrations In our system, peptide immobilization occurs among of PEGDA were chemically stabilized via radical poly- NH _CS and COOH_BMP2 with a molar ratio 1:0.007; in merization induced by heating the sample in a microwave this way an amount of 3.6 and 1.4 µg peptide/mg scaffold (MTS 1200 Mega, Milestone Inc., Shelton, CT 06484). was used for 80CS20P and 60CS40P, respectively. More- Some of the resulting samples were washed in acetone to over, for 60CS40P a molar ratio of 1:0.018 of NH _CS and evaluate the possible removal of unreacted PEGDA and COOH_BMP2 was also used to obtain scaffold with the dried in a vacuum oven at 45 °C. The combination of dif- same amount of peptide used in 80CS20P (3.6 µg peptide/ ferent ratio CS and PEGDA allowed to obtain CS-based mg scaffold) see Table 1. In this study, the peptide amount scaffolds at two percentages: 80CS20P and 60CS40P.The was determined on the basis of preliminary biological samples were analysed without any further modifications. experiments performed on BMP-2 peptide at different concentrations. 2.1.1 Scaffold bioactivation by organic signals 2.1.2 Scaffold bioactivation by inorganic signals The first approach for scaffold bioactivation is based on a covalent immobilization of a bioactive peptide of Bone Furthermore, the semi-interpenetrating CS-PEG scaffolds Morphogenetic Protein (BMP-2) on scaffold surface. The (80CS20P and 60CS40P) were bioactivated using a mod- 67–87 residue of the “knuckle” epitope of hBMP-2 ified method described by Kokubo and co-workers [27, 30]. (NSVNSKIPKACCVPTELSAI) was synthesized by The treatment [31], is based on the use of supersaturated microwave-assisted Fmoc solid phase peptide synthesis SBF solutions to stimulate the nuclei formation and growth. (SPPS). The resin support and all amino acids (aa) were It consists of a nucleation phase obtained by incubation of obtained from Inbios srl (Naples, Italy). Rink amide linker scaffolds in a supersaturated SBF solution (5x SBF1) for (0.4 mmoles; Iris Biotech GmBH) was attached to a 3 days, followed by crystallization and growth of apatite Tentagel-S-NH resin (Iris Biotech GmBH) in a glass vial; nuclei in a modified solution without the inhibitors of 1 mL of 0.45 M HBTU in N,N-dimethylformamide (DMF) crystallization as hydrogen carbonate and magnesium ions and 0.5 mL of 33% DIPEA in DMF were added to the [32, 33]. reaction vessel to activate the exposed amino groups of the In the present study, the pH solution was fixed at 7.4 in resin and after each coupling step to activate the deprotected order to promote interactions between decarboxylated − 2+ group of the coupled aa. The aa coupling was performed groups (COO ) and Ca ions. The SBF volume was cal- after stirring the reaction suspension for 7 min in a Micro- culated with reference to the total material surface using a wave synthesizer (Power 13 W; Temperature 60 °C; stirring specific ratio of the exposed surface to SBF volume, as rate 900 rpm); the solvent was removed and the mixture was reported in the literature [34]. After the incubation time, all washed with DMF (Sigma Aldrich). Fmoc-protecting group materials were gently rinsed in distilled water to remove was removed from the coupled aa by incubating the resin excess ions and, then, dried overnight under laminar flow with 20% v/v piperidine in DMF for 3 and 7 min. hood. Following coupling of the final aa, the resin-bound BMP- 2 peptide was washed several times with dichloromethane, 2.2 Characterization methanol, and diethyl ether. Finally, BMP-2 peptide was cleaved from the resin support by exposure to a solution 2.2.1 Morphological analysis composed by TFA 95%-TIS 2.5%-dH O 2.5% mixture for 3 h and precipitated in a cold diethyl ether by centrifugation The CS scaffold surfaces, before and after biomineraliza- for 5 min at 6000 rpm. tion, were analysed by Scanning Electron Microscopy 62 Page 4 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 (SEM, JEOL 6310). For SEM analysis, the materials were 2.3 Biological investigations mounted by a double adhesive tape to aluminium stubs. The stubs were sputter-coated with gold to a thickness of around 2.3.1 In vitro cell culture 15–20 nm. SEM analysis was performed at different mag- nification at a voltage of 20 keV. X-ray energy dispersive In vitro biological assays were performed on human spectroscopy (EDAX, Genesis 2000i) analysis was used for Mesenchymal Stem Cells line (hMSCs) obtained from a qualitative estimation of the Ca/P ratio. LONZA (Milano, Italy). hMSC were cultured in 75 cm cell culture flask in Eagle’s alpha Minimum Essential Medium (α- 2.2.2 Microcomputed tomography (MicroCT) analysis MEM) supplemented with 10% Foetal Bovine Serum (FBS), antibiotic solution (streptomycin 100 µg/ml and penicillin Microcomputed tomography (Bruker Skyscan 1172, Kon- 100U/ml, Sigma Chem. Co) and 2 mM L-glutamine, without tich, Belgium) was used to compare the 3D structure of both osteogenic factors. For all experimental procedures hMSCs at the native and bimomineralized scaffolds to assess their passage 4 were used. Cells were incubated at 37 °C in a porosity. The following MicroCT settings were identified humidified atmosphere with 5% CO and 95% air. for proper scanning of the native samples, based on their X- ray attenuation capacity: (a) the X-ray source was set at 2.3.2 Cell proliferation 25 kV and 139 μA, whitout filtering; (c) for the detection of porosity, the pixel size was set at 10 μm; (d) the exposure The cell biocompatiblity of BMP-2 solutions at different time was 940 ms, with a 2 × 2 binning; (e) samples were not concentrations starting from 2.70 µM to 270pM was eval- rotated. Due to a limited presence of the CaP phase, bio- uated by seeding hMSCs (5000 cells at passage 4) at dif- mineralized scaffolds samples required the same MicroCT ferent time 1, 3, 7 and 14 days in α-MEM supplemented settings. For this reason all scanning parameters were kept with 10% Fetal Bovine Serum (FBS), antibiotic solution unvaried. After reconstruction with NRecon software, (streptomycin 100 µg/ml and penicillin 100U/ml) and 2 mM DataViewer was used to visualize the 3D view of the L-glutamine. The cell proliferation was determined using an scaffolds and the section on the XY plane. The recon- Alamar blue assay (AbD Serotec, Milano, Italy) based on structed grey scale images of the samples were then ana- the metabolic activity of live cells. lyzed with CTAn software to quantify the porosity, after Furthermore, the biocompatibility of neat (80CS20P and selection of a volume of interest (a cylindrical VOI with 60CS40P), and bioactivated scaffold (80CS20P_bio, 3 mm diameter and 3 mm height) and appropriate thresh- 80CS20P_BMP, 60CS40P_bio, 60CS40P_BMP, olding. CTVol software was finally used for the rendering 60CS40P_BMP*) was analysed. The medium in cell-load of 3D VOI models, for both native and biomineralized scaffolds culture plates was removed after cultured for 3, 7, 14 scaffolds. and21daysand in vitro cell proliferation was evaluated with Alamar blue assay, according to the manufacturer’s protocol. 2.2.3 In vitro release study Finally, absorbance was measured at 570 and 600 nm. Over the culture time, the cell medium was replaced every two days. The peptide release profiles from scaffolds at different compositions (80CS20P, 60CS40P and *60CS40P) were 2.3.3 Osteogenic differentiation: alkaline phosphatase determined in vitro by High Performance Liquid Chroma- expression tography system (HPLC, Agilent). The scaffolds bioacti- vated with BMP-2 peptide were dipped in 200 µL sterile Differentiation of hMSC was tested measuring alkaline Tris-buffer solution (pH = 6.8) and kept in a shaking phophatase (ALP) activity in the cultures of neat and incubator (37 °C, 40 rpm) for various time periods of up to bioactivated CS-based scaffolds at different time points 4 weeks. At specific time points supernatant was collected (SensoLyte pNPP ALP assay kit, ANASPEC, Milano, and an equal amount of fresh medium was added to each Italy). At each time point, cultures were washed gently with sample. A quantity equal to 20 μL of the obtained super- PBS, followed by washing with cold 1× assay buffer (BD natant was injected in a chromatograph equipped with a UV Biosciences, Milano, Italy). The ALP activity was evaluated detector and a reversed phase column (Reprospher C18, onto the cell lysates (50 μl), in that case the cultures were 150 mm × 4.6 mm, DR. MAISCH, GmbH). The mobile treated with 1× lysis buffer with 0.2% of Triton X-100. To phase systems consisted of 90% water (A) and 10% acet- correct the ALP values for the number of cells present on onitrile (B). The flow rate was 1.0 mL/min, and the wave- each scaffold, double stranded DNA (dsDNA), as a marker length was set at 220 nm. All experiments were triplicated for the cell number, was measured using a Pico- for each sample. Green_dsDNA quantification kit (Invitrogen). First, 100 µl Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 5 of 12 62 of diluted Picogreen_dsDNA quantification reagent was 80CS20P_BMP, 60CS40P_bio, 60CS40P_BMP, added to 100 µl of cell lysates in a flat-bottomed, 96-well 60CS40P_BMP*) in basal medium for 21 days of culture plate. Following 10 min incubation, the fluorescence of time. Quantitative levels of OCN secreted into the culture Picogreen was determined at a wavelength of 520 nm after medium were determined using an enzyme-linked immu- excitation at 585 nm using a spectrophotometer (Victor X3, noassay kit following the manufacturer’s instructions. Perkin-Elmer, Italy). dsDNA was quantified according to a calibration curve of l-dsDNA standard in 10 mM Tris, 2.3.5 Statistical analysis 1 mM EDTA, pH 7.5, buffer. Each experiment was per- formed three times in triplicate. The results of ALP activity All quantitative experiments were performed in triplicate were reported as nanograms (ng) of ALP normalized to the and the results were expressed as mean ± standard deviation micrograms (µg) of total DNA content. The alkaline pho- (SD). Statistical analysis of the data was conducted using t- phatase activity of hMSC seeded onto biomimetic and non Student test. Differences between the groups of p < 0.05 biomimetic 80CS20P and 60CS40P scaffolds was were considered statistically significant. determined. 2.3.4 Mineralization analysis: osteocalcin expression 3 Results The effect of CS-based scaffolds on osteogenic differentia- 3.1 Morphological analysis tion of hMSCs was evaluated by measuring a later marker of differentiation, such as osteocalcin (OCN). Osteocalcin Morphological investigations performed by SEM analysis levels were measured using a commercially available kit demonstrated that the foaming process allows to obtain an (Quantikine Human Osteocalcin Immunoassay R&D sys- interconnected and homogeneous structure with pores at tem, Italy) following the manufacturer’s instructions. hMSC different size from 20 to 300 µm. Moreover, CS based- cells were cultured on control, neat scaffolds (80CS20P and scaffolds have high interconnected structure inside the walls 60CS40P) and after biomimetic treatments (80CS20P_bio, of the pore as shown Fig. 1. The presence of macropores Fig. 1 SEM images of CS scaffold before and after biomineralization process. Illustration of ionic interaction among chemical groups of CS polymer and SBF ions 62 Page 6 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 enables the cells to migrate in the internal part of scaffold, while the micropores due to solvent evaporation determine a good nutrient flow and avoid radical oxygen species (ROS) formation that is responsible of cell necrosis. However, the scaffold porosity depends upon the CS concentration. In effect, the porosity decreases by reducing the CS con- centration. Furthermore, an open porosity of 78 and 60% for 80CS20P and 60CS40P was revealed respectively by using a pycnometer method [26]. These differences in open por- osity were due to the presence of different percentages of PEGDA which increased the structure stability by reducing the porosity. Furthermore, SEM images of CS-PEGDA scaffolds prepared by biomimetic methods demonstrated the pre- sence of calcium phosphate (CaP) deposits on the scaffold surface and on the internal pore walls (Fig. 1) after only 7 days of treatment. This behaviour in a short time was due to the use of two specific parameters represented by a supersaturated concentration (5x SBF) and pH value. In fact, CS polymer at physiological value (pH = 7.4) shows a negative charge due to the deprotonation of COOH groups (pKa = 6.5). In this way, interactions among two COO Fig. 2 m-CT analysis of the scaffolds. 3D reconstruction of 80CS20P CS 2+ scaffold before and after biomineralization process and a summary groups and bivalent Ca were favoured. Furthermore, table with the calculation of the porosity EDAX analysis, performed during SEM observation, con- firmed the formation of CaP deposits with a Ca/P ratio of about 1.67 (Fig. 1). 3.1.1 Scaffold bioactivation by organic signals and in vitro As shown in Fig. 2, MicroCT scanning of native and release study biomineralized samples highlighted an higher porosity of both scaffolds. The microwave-assisted Fmoc solid phase peptide synthesis 3D analysis also evidenced an overall porosity of 97.9 allows to obtain a peptide in a shorter time than the con- and 98.2% for 80CS20P and 60CS40P scaffolds respec- ventional synthesis process, with a molecular weight of tively. No significant difference can be detected between the 2074.4 Da and high purity (>98%) as demonstrated by two samples. Conversely, a reduction of the porosity can be chemical characterization through mass spectrometry and observed once the bioactivation treatment has been per- analytical HPLC respectively (Fig. 3a). The pure peptide formed. In particular, an overall porosity of 93.5 and 96.8% was immobilized on CS scaffolds (80CS20P and 60CS40P) can be detected for 80CS20P_bio and 60CS40P_bio scaf- (Fig. 3b) by carbodiimide reaction mechanism, where the fold respectively. The pore size distribution among the VOI –NH groups of CS and –COOH groups of BMP-peptide results to be uniform with no difference between the scaf- were involved. This covalent bond guarantees an in vitro folds. Based on the calculation of the volumetric open pore release for a long time. In particular, the results onto size distributions, the average pore size of 80CS20P and 80CS20P scaffolds demonstrated an initial release of pep- 60CS20P scaffolds was found to be 367 ± 234 μm and 456 tide about 8–10 % (Fig. 3b) due to the scaffold properties ± 313, respectively. The biomineralization process does not (i.e. swelling) [26]; however the release is sustained up to induce any relevant change in the pore size dimension and 4 weeks where 80CS20P has released about 94.5% of distribution. From the 3D reconstruction and the XY section peptide. In the case of 60CS40P_BMP scaffolds, having (Fig. 2) a densification of the structure could be observed. higher amount of BMP2 mimic peptide (3.6 µg/mg scaf- This is addressed to the deposition of CaP phase on the fold), a slower release in the first 48 h is obtained, probably surface of the pores on the nanometric scale. Even in this due to a higher amount of PEGDA compared to the case the spatial distribution of the CaP phase is uniform 80CS20P samples; however up 4 weeks 60CS40P_BMP assessing an effective biomineralization over the overall and 60CS40P_BMP* have released 97.3 and 92.9% of scaffold volume. peptide, respectively. Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 7 of 12 62 Fig. 3 a Mass spectrometry (micro-TOF) and HPLC chromatogram peak of BMP-2 peptide; b kinetic release of peptide from 80CS20P scaffolds and 60CS40P at different BMP concentrations (60CS40P_BMP, 60CS40P_BMP*) Fig. 4 (a) Confocal images of hMSCs incubated with BMP-2 peptide and Alkaline phosphatase activity (ng ALP/ug DNA) results of BMP solutions at different concentration (range 2.7 µM–270 pM) after 24 h solutions at different concentrations (range 2.7μM–270pM) after dif- of incubation time; (b–c) Alamar Blue (% reduction of alamar blue) ferent time points, respectively 3.2 Biological properties different concentrations on hMSCs adhesion was deter- mined after 1 day of cell culture. The images obtained using 3.2.1 BMP-2 mimic peptide: biological activity immunofluorescence analysis (Fig. 4a) demonstrated that the cells in contact with solution at higher concentration The bioactivity of neat peptide was investigated by mea- (2.7 µM) showed a polygonal morphology typical of phe- suring its effect on hMSCs behavior in terms of prolifera- notype like-osteoblast than the control (CTR), while at tion and differentiation. First of all, the effect of peptide at lower concentration (270pM) the cells showed a fibroblast- 62 Page 8 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 like morphology. Furthermore, quantitative analysis of the cell proliferation (Fig. 4b) suggested an increasing of pro- liferation at first 3 days and a decreasing at long times. This behaviour was due to an initial ostegoenic differentiation. In Fig. 4c higher ALP values were observed in presence of BMP-2 at concentration of 2.7 µM. Hence, these pre- liminary studies allowed to perform a screening about the optimal concentration of BMP-2 peptide necessary for scaffold bioactivation that is the 2.7 µM solution. 3.2.2 Biological properties of bioactivated scaffolds The effect of 80CS20P and 60CS40P scaffolds with and w/ o BMP-2 mimic peptide (2.7 µM) on hMSCs was also evaluated. This analysis demonstrated that, at 24 h, there (A) was a good cell attachment on the scaffold surface with higher values for the bioactivated scaffolds at different * # Day 3 Day 14 compositions than neat scaffolds (Fig. 5a). After this time, p≤ ≤0.05 and p≤0.00 01 vs 80CS20P * ° Day 21 60 p p≤0.05 and p≤0.01 vs 60CS40P Day 7 the proliferation increases over culture time confirming that the scaffolds support adhesion and cell migration inner the # structure. In particular scaffold containing BMP-2 sig- nificantly (80CS20P_BMP = p ≤ 0001; 60CS40P_BMP = p ≤ 0,05) increase MSC proliferation at day14 (Fig. 5b) compared to both neat scaffolds (80CS20P and 60CS40P) * where the cells become confluent. Meanwhile, at day 21 a slight decreasing was observed for scaffold with highest amount of BMP peptide (60CS40P_BMP*). This latter decreasing in cell proliferation is related to the increase in cell differentiation confirmed by ALP expression. To evaluate the effect of scaffolds on osteogenic differ- entiation, the expression of alkaline phosphatase (ALP), as (b) early marker of hMSCs differentiation in pre-osteoblast phe- notype, was evaluated. The experiments were performed in Fig. 5 a Quantitative analysis of cell adhesion was quantified using Calcein AM assay and represented as percentage of cells attached after basal medium taking in account the hypothesis concerning that 24 h of incubation time; b cell proliferation at long time (3, 7, 14 and the organic and inorganic bioactive signals act as osteoin- 21days) of hMSCs seeded on 80CS20P scaffolds, before and after ductive factors. The results demonstrated that highest ALP biomineralization (80CS20P, 80CS20P_bio) with and w/o organic values (Fig. 6a) were obtained for cells in contact with BMP-2 signals (80CS2P_BMP, 80CS20P_bio_BMP). Quantitative analyses were performed by using Alamar blue™ assay using manufacturer’s like peptide (CTR+ BMP2, 18 µg) at 3 days of cell culture. protocol Furthermore, the scaffolds bioactivated with BMP-2 like peptide (80CS20P_BMP, p ≤ 0,05; p ≤ 0001) and biominer- alized scaffolds (80CS20P_bio, p ≤ 0,05; p ≤ 0001), induce a the culture time (Fig. 6b). This behaviour was also observed significant increase in ALP levels at long-time than neat for the expression of non-collagenous bone ECM protein (i.e. 80CS20P and 60CS40P materials. The scaffolds with lower osteocalcin) used as later marker of osteogenic differentiation amount of CS bioactivated with BMP-2 like peptide (Fig. 7). Here scaffolds bioactivated with BMP-2 and only the (60CS40P_BMP and 60CS40P_BMP*) showed highest ALP biomineralized scaffold with higer percentage of Chitosan values at day 3, probably due to the lower release profile of (80CS20P_bio) were able to significanly (p ≤ 0,05; p ≤ 0,01; peptide in the first 48 h and an higher scaffold stability (as p ≤ 0001) increase osteocalcin values at day 21 of culture time. demonstrated in the kinetic release study reported in the Fig. 3). However, higher ALP expression was observed for the full set of scaffolds and, in particular, 80CS20P_bio and 4 Discussion 80CS20P_BMP showed a similar behaviour than 60CS40P scaffolds (both 60CS40P_bio and 60CS40_BMP) at day 3. In bone tissue engineering the scaffold should provide Additionally, 60CS40P_BMP* showed the best results over necessary support as an artificial extracellular matrix that 80CS S2 20P 80/ /C CS20P_BIO 80CS20P_B BM MP 60CS40P 60CS40P_BI IO O 60CS S4 40P_BMP CS4 _BMP P* * 60 0P Percent of reduction (%) Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 9 of 12 62 allows cells to proliferate and maintain their differentiated functions. Essentially, scaffold acts as a temporary template to guide the formation of a new tissue. In this context, an ideal scaffold is characterised by excellent biocompatibility, molded biodegradability, cytocompatibility, suitable microstructure (pore size and porosity) and mechanical properties. Additionally, scaffold must be capable of pro- moting cell adhesion and retaining the metabolic functions of the attached cells [34]. For these reasons, the aim of this work was to study the effect of biomimetic functional scaffolds obtained by modifying polymer scaffolds with osteoinductive signals including inorganic components, such as hydroxyapatite deposition and organic signals, such (A) as BMP-2 mimetic peptide covalently immolized on the p≤0 0.05 vs 80CS20 0P Day 7 Da ay 14 * scaffolds. Therefore, this research could help to develop ° # p≤0 0.05 ; p≤0.01 an nd p≤0.001 vs 6 60CS40P Day 21 biodedragable scaffolds which are also carriers of osteo- * genic signals in order to obtain positive cellular responses in ]* ]* terms of osteogenic commitment. By a covalent peptide 1000 # # immobilization on the scaffolds with different chitosan concentrations (80CS20P_BMP and 60CS40P_BMP) a low percentage as burst release of peptide, 10 and 20% in the first 48 h, was observed respectively; whereas at longer intervals, a prolonged sustained release of up to 4 weeks has an important effect on cellular behaviour. The effect of BMP-2 on early ALP expression (Fig. 5a) in the first days of cell culture is related to the burst release of peptide. Meanwhile, the release of peptide over time determines the (B) expression of osteogenic marker (ALP-OCN) at long-term. Fig. 6 a Alkaline phosphatase activity expressed as nanograms of ALP This prolonged BMP-2 delivery was due to the high sta- normalized by amount of DNA (µg) produced by hMSCs after early bility of the scaffold structure (prepared by physical foam- (3 days) and long b times (7, 14 and 21 days). DNA amount was ing combined with microwave curing) [27]. determined by dsDNA picogreen assay Previous studies suggested a similar early burst release profile of BMP on hydroxyapatite (HA) bone grafts which are able to induce osteogenic differentiation of C2C12 cells p≤0.05 vs 80CS20P # ## ### p≤0.05, p≤0.01 and p≤0.001 vs 60CS40P [35]. In the present study, the higher percentage of burst release of 80CS20P_BMP samples compared to ## ### 60CS40P_BMP is probably due to the different porosity of these scaffolds. In the case of 60CS40P_BMP scaffolds a slower release in the first 48 h is probably due to a much more stable structure for the higher amount of PEGDA and a lower porosity compared to 80CS20P samples. In porous structures, fluid dynamics must to be coupled with structural mechanics of the scaffold. In this study, scaffold porosity influences effects of fluid flow (flow of cell culture medium) on BMP release. In effect, samples with higher porosity (80CS20P_BMP) showed an higher percentage of BMP release in the first days. Meanwhile up 4 weeks 60CS40P_BMP and 60CS40P_BMP* have released 97.3 and 92.9% of peptide, respectively. In vitro degradation tests showed a gradual dissolution of the scaffolds over time, while maintaining their 3D morphology and integrity Fig. 7 Osteocalcin marker expressed by hMSCs cultured on the scaffolds (80CS20P, 80CS20P_bio, 80CS20P_BMP, even after 6 weeks of incubation [26] and allowing an 80CS_bio_BMP) after 21days of incubation time in a basal medium extended release of organic bioactive signal. This extended 80CS2 20 0P 80/C CS S20P_BIO 80CS20P_B BM MP 60CS40P 60CS40P_ _B BIO 60C CS S40P_BMP 4 _ _B BM MP* 60CS 0P 80CS20P 80/CS20P_BIO 80CS20P_BMP 60CS40P 60CS40P_ IO 60CS40P_BMP 60CS40P_BMP* Osteocalcin levels (ng/ml) ng ALP/μg DNA 62 Page 10 of 12 Journal of Materials Science: Materials in Medicine (2018) 29:62 release potentially promises the successful infiltration of could be explained by considering the difference in the ratio cells and new tissue formation. Indeed, biological results between the chitosan free amino groups (–NH ) and the demonstrated that the materials act as support for hMSCs induced carboxylic groups of BMP2 (–COOH). In fact, even adhesion and proliferation. A consistent migration of cells if the cumulative release profiles showed no significant into the scaffold is evident, as demonstrated in cross-section differences between the samples, the effect of the bioacti- SEM images. Moreover, the effect of organic and inorganic vation appears in the early osteogenic differentiation. This bioactive solid signals on osteogenic differentiation was trend suggests that, by increasing the COOH/NH ratio, a evaluated. positive effect on cellular behaviour is achieved. Moreover, Indeed, it is possible to characterise the process in three it could be explained with the specific peptide interactions stages: (a) cell proliferation, (b) matrix maturation, and (c) with the receptor on the cellular membrane which induce matrix mineralization [36]. In vitro, matrix maturation and hMSC differentiation into more mature osteobalst mineralization are usually enhanced by growing the cells to phenotypes. complete confluency and by adding specific osteoinductive Moreover, CS-PEGDA scaffold was a good carrier of factors [37]. After the proliferation, the matrix maturation peptide allowing a prolonged release over time and main- phase is characterised by an expression of alkaline phos- taining the local concentration of BMP2 mimic peptide at a phatase (ALP). ALP is a well-known early marker of good level, thus overcoming the drawbacks of some poly- osteogenic differentiation and plays a key role in the mers used as carriers for the therapeutic agents delivery mineralization of bone. As such it is considered a useful [37]. However, all samples showed significant positive biochemical marker of bone formation. Finally, during effects on hMSC behaviour in terms of proliferation and matrix mineralization (c) other genes for proteins such as osteogenic differentiation in basal medium, as confirmed by OC, BSP, and OPN are expressed. Analysis of bone cell- expression of early (ALP) and later signals (OCN) of specific markers like Procollagen, ALP, OPN and OCN, is osteogenic differentiation in bioactivated scaffolds. In par- used to characterise later osteogenic differentiation phases ticular, results have demonstrated that biomimetic scaffolds in vitro of hMSCs. The mineralization process of osteo- showed higher expression of OCN level, however the best blasts in in vitro culture has also been used as a model for behaviour at long time in terms of prolonged release and testing the effects of peptide loading on bone cell differ- cellular behaviour for scaffolds bioactivated with BMP entiation and bone formation. In this work, the expression mimic-peptide was observed. Furthermore, the present work of ALP and OCN were analysed; in particular, it was suggested that bioactivated scaffolds were able to direct observed that at 3 and 7 days no difference among the group osteoinductive processes by expression of early and later of bioactivated scaffold and related controls (60CS40P and markers of osteogenesis. 80CS20P) was observed. Meanwhile, after 14 days the best ALP expression for 80CS20P_BMP scaffold was observed, where a peptide 5 Conclusions release of approximately 65–75% (12–14 µg) was obtained. In parallel, 60CS40P_BMP* showed the highest ALP Our study has demonstrated the possibility of producing expression at 21 days. On the other hand, biomineralized biomimetic scaffolds with highly interconnected and scaffolds developed through biomineralization treatment homogeneous structure with pores of different sizes from 20 (80CS20P_bio and 60CS40P_bio) showed an increased to 300 µm in agreement with the dimensional features ALP expression compared to non biomineralized chitosan required for bone regeneration. The proposed technique materials after 3 days of incubation. At the same point time, holds strong potential for applications in the field of similar cell differentiation behaviour, between 80CS20P_bio custom-made bone substitute. Moreover, the presence of and 60CS40P_bio, was observed. At longer incubation times bioactive signals on the scaffold surface allows to obtain an (7, 14 and 21 days), no significant difference between two osteoinductive effect on hMSC. Indeed, both bioactivated biomineralized groups and BMP-activated scaffolds was scaffolds showed higher ALP values compared to neat observed. In particular, 80CS20P_BMP showed better ALP materials at short time. In particular, scaffolds decorated expression after 14 days than 60CS40P_BMP/BMP*. After with BMP-mimicking peptide presents sustained ALP 21 days the 60CS40P_BMP reached a plateau compared to values at long times. This behaviour suggested that CS- levels at 14 days. On the contrary, 60CS40P_BMP* sample PEGDA scaffold is an appropriate carrier for peptide at 21 days shows an increased ALP value compared to both allowing to maintain the local peptide concentration at good 60CS40P_BMP and 80CS20P_BMP. The same behaviour level over time. Furthermore, biomineralized scaffolds was also detected for Osteocalcin expression (OCN) where showed a better cellular behaviour than neat scaffolds thus the highest level was observed for scaffold bioactivated with confirming the effect of hydroxyapatite deposits on hMSC BMP2 mimic-peptide at day 21. This different behaviour osteogenic differentiation. Journal of Materials Science: Materials in Medicine (2018) 29:62 Page 11 of 12 62 The comparison between the two different approaches 11. Altiok D,Altiok E,Tihminlioglu F, Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme emphasizes that the presence of bioactive signals on CS- oil for potential wound healing applications. J Mater Sci Mater PEGDA scaffolds plays a pivotal role in osteogenesis pro- Med. 2010;21(7):2227–2236. cess. Indeed, the modifications on scaffolds allow to hMSC 12. 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Published: May 7, 2018

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