Glutaraldehyde-crosslinking chitosan scaffolds reinforced with calcium phosphate spray-dried granules for bone tissue applications

Journal Pre-proof Glutaraldehyde-crosslinking chitosan scaffolds reinforced with calcium phosphate spray-dried granules for bone tissue applications

Rosana V. Pinto, Pedro S. Gomes, Maria H. Fernandes, Maria E.V. Costa, Maria M. Almeida PII:

S0928-4931(19)33330-2

DOI:

https://doi.org/10.1016/j.msec.2019.110557

Reference:

MSC 110557

To appear in:

Materials Science & Engineering C

Received date:

7 September 2019

Revised date:

11 December 2019

Accepted date:

12 December 2019

Please cite this article as: R.V. Pinto, P.S. Gomes, M.H. Fernandes, et al., Glutaraldehydecrosslinking chitosan scaffolds reinforced with calcium phosphate spray-dried granules for bone tissue applications, Materials Science & Engineering C (2018), https://doi.org/ 10.1016/j.msec.2019.110557

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© 2018 Published by Elsevier.

Journal Pre-proof Glutaraldehyde-crosslinking chitosan scaffolds reinforced with calcium phosphate spray-dried granules for bone tissue applications Rosana V. Pintoa, Pedro S. Gomesb,c, Maria H. Fernandesb,c, Maria E.V. Costaa and Maria M. Almeidaa

a

Department of Materials and Ceramic Engineering, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal. b

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FMDUP, BoneLab - Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine, University of Porto, Porto, Portugal c LAQV/REQUIMTE, Faculty of Dental Medicine, U. Porto, Porto, Portugal.

KEYWORDS. Bone tissue engineering; Scaffold; Glutaraldehyde; spray-dried granules;

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Hydroxyapatite; β-TCP.

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ABSTRACT. The clinical demand for bone scaffolds as an alternative strategy for bone grafting has increased exponentially and, up to date, numerous formulations have been

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proposed to regenerate the bone tissue. However, most of these structures lack at least one of the fundamental/ideal properties of these materials (e.g., mechanical resistance, interconnected

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porosity, bioactivity, biodegradability, etc.). In this work, we developed innovative composite scaffolds, based on crosslinked chitosan with glutaraldehyde (GA), combined with different atomized calcium phosphates (CaP) granules - hydroxyapatite (HA) or biphasic mixtures of HA

biological response.

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and β - tricalcium phosphate (β-TCP), with improved biomechanical behaviour and enhanced

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This innovative combination was designed to improve the scaffolds’ functionality, in which GA improved chitosan mechanical strength and stability, whereas CaP granules enhanced the scaffolds’ bioactivity and osteoblastic response, further reinforcing the scaffolds’ structure. The biological assessment of the composite scaffolds showed that the specimens with 0.2% crosslinking were the ones with the best biological performance. In addition, the inclusion of biphasic granules induced a trend for increase osteogenic activation, as compared to the addition of HA granules. In conclusion, scaffolds produced in the present work, both with HA granules or the biphasic ones, and with low concentrations of GA, have shown adequate properties and enhanced biological performance, being potential candidates for application in bone tissue engineering.

HIGHLIGHTS 1

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Micrometric calcium phosphate (CaP) granules are produced by spray-drying

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Porous chitosan scaffolds loaded with CaP granules are produced by freeze-drying

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Chitosan matrices are crosslinked and mechanically reinforced with glutaraldehyde

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CaP granules enhance bioactivity and osteoblastic response of chitosan scaffolds

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Biphasic CaP granules induce increased osteogenic activation of chitosan scaffolds

1. Introduction

Over the last decades, the development of biomaterials for bone tissue engineering (BTE) has

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grown exponentially, aiming to develop ideal materials able to repair large bone defects, while restoring the biological function of damaged tissue [1–3]. Bone engineering typically relies on

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the implantation of a supporting matrix (i.e., a scaffold) that provides the suitable microenvironment for cell attachment, proliferation and differentiation, allowing enhanced bone

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ingrowth [4–8].

The ideal implanted scaffold should accomplish several criteria: (1) be biocompatible, (2)

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possess controlled biodegradability which rate should be similar to the formation of the native tissue, (3) be osteocompatible/osteoinductive, facilitating bone formation, (4) offer a high

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porosity with interconnected pore structure to allow bone ingrowth, and (5) have adequate biomechanical properties, compatible with the in situ anatomical location, in terms of flexural tensile

strength

and

hardness,

ensuring

structural

integrity

during

the

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strength,

formation/remodeling process [3–5,8–10].

Chitosan, a natural polymer derived from chitin, and its derivatives have been explored on

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several biomedical fronts because of their polyvalent features including biocompatibility, biodegradability, ability to promote cell adhesion and migration, and antimicrobial activity [11– 13]. In addition, chitosan may assume several forms (e.g. microparticles [14,15], CH-based hydrogels [16,17], 3D scaffolds [12,18]) and their chemical nature allow many possibilities for ionic and covalent modifications which afford an extensive adjustment of their mechanical and biological performance [13]. Although chitosan is tough and flexible, it lacks the strength necessary to be used alone in load bearing applications. To overcome this limitation, crosslinking strategies capable to cross chitosan free amine groups have been reported [19,20], with glutaraldehyde being regarded as the most practical and widely used crosslinking agent [21]. Another limitation of chitosan is its low bioactivity, a critical property for bone regeneration purposes [8,22]. Thus, the combination of a chitosan matrix loaded with bioactive ceramic materials has emerged as an attractive strategy, providing a useful synergy, bringing together the mechanical strength and bioactivity of the ceramic and the toughness and flexibility of the polymer. The resulting bioactive and biodegradable composite scaffolds may be furthermore structurally tailored to mimic the native bone and anatomical defect, hence making this a very promising approach in terms of biomaterial design [10,23,24]. In this regard, calcium

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Journal Pre-proof phosphates (CaP) are bioceramics particularly relevant for bone tissue applications, due to their chemically resemblance of bone mineral; they are also bioactive and osteocondutive, and when designed with the appropriate geometry and topography, are able to stimulate bone tissue formation and directly bond with bone, while inducing the formation of a uniquely strong biomaterial-bone interface [5,25–27]. These arguments explain the wide use of CaP in bone related therapies, either as implant coatings, cements constituents, or as part of scaffolds based on polymer-ceramic composites [28–30]. Different CaP phases have been used, wherein hydroxyapatite (HA) and tricalcium phosphate (β-TCP) are the most commonly reported phases. The mixture of both HA and β-TCP, as a biphasic CaP, has demonstrated to result in an increased osteoinduction and bio-resorbability, as comparing to single phase HA or β-TCP [31–33]. Additionally, the biological performance of

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a CaP is also conditioned by its morphological characteristics. CaP are available in various physical forms and sizes – spheres, particles, blocks, either dense or porous, with a wide range

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of particle size - nano to macro structures [34]. CaP porous microspheres, for example, seem to have adequate properties to enhance the biological response, such as large specific surface

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area and good flowability, being optimal candidates for drug delivery strategies and local tissue implantation [35,36]. Among the production methods of CaP granules, the spray drying

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technique is widely used as it allows the preparation of homogeneous granules with controlled size and shape [33,37].

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Composite chitosan/CaP scaffolds with a wide range of attributes have thus been exploited in recent years, aiming to identify the most favorable combination and architecture, for biological enhancement within the clinical setting. For instance, variations on chitosan/CaP ratio [38–41],

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chitosan deacetylation degree [42,43], techniques used on CaP synthesis [8,39], types of CaP phase [44,45] and their physical form [8,41,46] have been frequently reported in the literature.

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However, several drawbacks remain difficult to overcome including insufficient mechanical strength, variable degradation rates and inadequate tissue penetration. Thus, there is still a need for improvement on the processing route towards the fabrication of a truly effective bone tissue engineering scaffold with optimum 3D porous structure and mechanical properties. In this work, we propose a set of new composite scaffolds based on chitosan combined with different concentrations of glutaraldehyde, a crosslinking agent that will reinforce the chitosan’ mechanical properties, further incorporating ceramic granules which, besides their bioactivity, will facilitate cell adhesion by the creation of contact points over the scaffold surface. Two types of granules were developed by spray drying and used to incorporate in the chitosan matrices: (1) HA granules and (2) granules with a biphasic mixture of HA and β-TCP (1:1 wt ratio). These granules provide a high surface area which is expected to improve the biological response and, due to their simple and malleable production technique, allow for the easy incorporation of pharmaceuticals or growth factors that may improve the potentialities of the scaffold. To the best of our knowledge, these complex GA-crosslinked chitosan matrices reinforced with multiphasic calcium phosphate granules produced by spray drying have not been addressed so far. These new 3D solids were evaluated in terms of morphology and mechanical behavior to

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Journal Pre-proof seek the best scaffold attributes that might trigger an adequate biological response. Moreover, preliminary study showing the impact of different glutaraldehyde concentrations (0.2%, 1% and 10% (v/v)) on the chitosan matrices was performed and the concentrations that presented improved potentialities for BTE were selected to the composites’ preparation.

2. Materials and Methods

2.1 Materials and cells Hydroxyapatite (HA) nanoparticles’ suspension (nanoXIM) was purchased from Fluidinova S.A.,

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Portugal. Beta tricalcium phosphate (β-TCP) and acetic acid (100% pure) were obtained from

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Fluka and Merk, respectively. Chitosan (CH) with a deacetylation degree of 80%, glutaraldehyde (Grade II 25% in H2O), penicillin, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), triton, sodium hydroxide and sodium cacodylate were all purchased from Sigma-

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Aldrich. Minimum Essential Medium Alpha (α-MEM), fetal bovine serum (FBS), fungizone,

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trypsin and phosphate buffered saline (PBS) were all obtained from Gibco. AlamarBlue® was supplied by Life Technologies. All the reagents were used as purchased without further

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purification. Cellular assays were performed using MG63 human osteoblastic-like cells (ATCC ®

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CRL-1427).

2.2. Preparation of CaP granules

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HA particles and biphasic (HA + β-TCP; 1:1wt ratio) mixture were suspended in distilled water (∼1 wt.%). The spray drying process was carried on using a laboratory spray dryer (Buchi B191) under specific operating conditions previously optimized including an inlet temperature of the drying air of 180°C, an inlet hot air flow of 80% and an inlet flow of the feeding suspension of 20% [47]. The obtained spray dried granules were identified according to the labeling presented in table 1.

2.3. Preparation of crosslinked CH matrices and composite scaffolds CH matrices were prepared by dissolving CH 2 wt% in acetic acid [0.2M] under stirring at pH 5. Then, different concentrations of GA were introduced (0.2%, 1% and 10% (v/v)). The obtained suspensions were putted into molds, frozen at -20 ºC during 24 hours, transferred to a freeze-3

dryer (Labconco Freeze Dry System) and kept at -52 °C and 12x10

mbar, during 4 days.

Freeze-dried samples were rehydrated and stabilized with ethanol treatment following the procedure described by Madihally et al.[48], frozen again in liquid nitrogen and freeze-dried for more 24 hours and stored in the desiccator until characterization. Composite structures were obtained using the above protocol described for CH matrices, with

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Journal Pre-proof the addition of 1 wt% of CaP granules (i.e, either HA or biphasic) to the CH solution, under stirring, prior to the addition of glutaraldehyde (0.2%, 0.5% or 1% (v/v)). The composite suspensions were frozen and freeze-dried in the same conditions as described above. Table 1 summarizes the compositions of the various scaffolds formulations addressed in this work as well as their specific preparation conditions and labeling.

2.4. Materials characterization Crystalline phase composition of both CaP granules and composite scaffolds was identified by X-ray diffraction (XRD) analysis (Rigaku PMG-VH with CuKa radiation = 1.5405 A°). Specific surface area (SSA) of the CaP granules was determined by N 2 gas absorption using a

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Micromeritics Gemini 2370 V5 equipment and the multipoint Brunauer-Emmett-Teller (BET) adsorption isotherm. Fourier transform infrared spectroscopy (FTIR) (Bruker Tensor 27) in

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Attenuated Total Reflectance (ATR) mode was used to identify the scaffolds’ functional groups -1

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in the range of 400-4000 cm . The morphology of both spray dried granules and 3D structures was evaluated by scanning electron microscopy (SEM, HITACHI S-4100) at 10kV. Samples for SEM analysis were sputtered with a conductive carbon film prior to SEM observation. Scaffolds’

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pore sizes were estimated based on SEM analysis and data processing, using the ImageJ 1.45 software. 300 pores were randomly measured for each condition.

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The compressive mechanical behavior of the CH and composite matrices was evaluated using a universal testing machine (Zwick/Roell Z020). The specimens were cut with a cylindrical

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shape (~15 mm height and ~18 mm diameter). The speed of the testing machine crosshead -1

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was controlled at 1 mm.min , until specimen deformation ~50%.

2.5. Cytocompatibility studies Human osteoblastic-like cells (MG63 cells ATCC ® CRL-1427) were cultured in α-MEM medium supplemented with 10% (v/v) fetal bovine serum, penicillin-streptomycin (100 UI/ml and 100 μg/ml, respectively), fungizone (2,5 mg/ml) and ascorbic acid (50 μg/mL), and incubated at 37 °C in a humidified atmosphere with 5% CO2 in air. Prior to cell seeding, sterile scaffolds were placed in a 48-well culture plate with 1 mL of cell 4

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culture media and incubated for 30 minutes. Following, cell suspension (5 x 10 cells/cm ) was seeded on the top of the scaffolds, allowing cells to distribute throughout the porous structure. Seeded scaffolds were cultured at 37ºC in a humidified incubator with 5% CO 2 for 7 days. Fresh medium was replaced every 2 days until the end of the cultivation period. Five replicates of each scaffold were used for experimental assays. Viability and proliferation were analyzed after 1, 4 and 7 days of culture. After the respective time of incubation, fresh medium with alamarBlue® (10µg/ml) was replaced and incubated for 3h, prior to the quantification. alamarBlue® reduction was quantified by fluorescence (λ ex= 530 nm, λem= 590 nm) in an ELISA reader (Synergy HT, Biotek). For each time, each condition was

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Journal Pre-proof tested in quintuplicates. Alkaline phosphatase (ALP) activity was determined in cell lysates, following the incubation with p-nitrophenyl phosphate - in an alkaline buffer pH 10.3; 30 min, 37 ºC - and evaluation of the conversion to p-nitrophenol, at λ = 400 nm, in a Synergy HT, Biotek, ELISA system. ALP levels were normalized to total protein content, determined by the Bradford’s method. Data on ALP activity were normalized to those attained in control. Cell adhesion and morphology was addressed by SEM microscopy and fluorescence microscopy, following cytoskeletal, mitochondrial and nuclear labelling. For SEM observation, cell cultures were fixed with 1.5% glutaraldehyde during 10 min, washed with PBS and maintained in a buffer solution (pH 7.3) of 0.14M sodium cacodylate. Samples were dehydrated in graded alcohols, critical-point dried, sputter-coated with gold and analyzed in a scanning electron microscope equipped with X-ray energy dispersive spectroscopy (EDS) microanalysis

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capability (Quanta 400FEG ESEM / EDAX Genesis X4M). For fluorescence imaging, live cell cultures were incubated with MitoSpy Red CMXRos (Biolegend, Germany - 150 nM, 15 min),

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prior to fixation (3.7% paraformaldehyde, 15 min), permeabilization (0.1% Triton in PBS, 5 min), and incubation with a solution of 10 mg/ml of bovine serum albumin with 1 μg/ml RNAse in PBS,

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1 h, to reduce unspecific staining. Subsequently, cells were incubated with 5 U/ml Flash Phalloidin™ Green 488 for 20 min, for filamentous actin staining, and with DAPI 0.1 μg/ml, 10

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min, for nucleous counterstaining. Images of fluorescent-labelled cells were obtained with a Selena S digital imaging system (Logos Biosystems).

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Gene expression analysis was conducted through a quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis, aiming to address the expression of relevant osteogenesis-related markers. Total RNA was extracted with TRIZOL® reagent (Invitrogen),

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according to the manufacturer protocol, and reverse transcribed into complementary DNA (cDNA) with iScript™ Adv cDNA Kit (BioRad), according to the manufacturer’s instructions. The

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expression of genes: Runt-related transcription factor 2 (Runx2, qHsaCED0044067), alkaline phosphatase (ALP, qHsaCED0045991), secreted phosphoprotein 1 - ostepontin (OPN, qHsaCED0057074),

bone

gamma-carboxyglutamate

protein

-

osteocalcin

(OC,

qHsaCED0038437), collagen type I - alpha 1 (COL1A1, qHsaCED0043248) and bone morphogenetic protein 2 (BMP2, qHsaCID0015400) was quantitatively determined in a RT-PCR equipment (CFX96, BioRad) using iQTMSYBR®Green Supermix (BioRad). The housekeeping gene beta-actin (ACTB, qHsaCED0036269) was used as a reference for normalization. Quantitative data was presented as mean and standard deviation. Statistical analysis was performed using analysis of variance (ANOVA one-way), with a significance level of p ≤ 0.05.

3.

Results and Discussion

3.1 GA-crosslinked CH matrices

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Journal Pre-proof During the production and evaluation of implantable scaffolds, particular attention should be given to their physic-chemical characteristics, biomechanical behavior and biological functionality [49]. In the present approach, glutaraldehyde was incorporated as a strategy to increase the mechanical behavior and stability of the CH scaffolds, tentatively without interfering with their morphology/porosity and biological response. With that in mind, different concentrations of GA (i.e., 0.2%, 1% and 10%) were used and produced scaffolds thoroughly characterized. Accordingly, Fig. 1 shows SEM micrographs from the obtained CH matrices with the different % GA. SEM micrographs of chitosan scaffolds (Fig. 1 a) and b)) revealed the existence of interconnected pores, with pore sizes between 50 and 200 μm, sustaining a high adequacy for bone tissue applications, allowing for appropriate tissue and vascular ingrowth [50]. As it can be observed in Fig. 1 c) to h), glutaraldehyde addition did not significantly alter

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the morphology or the pore size, maintaining a clear interconnectivity between pores In addition to the morphological assessment conducted by SEM characterization, the

tests. Fig. 2 shows the obtained stress-strain curves.

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mechanical behavior of the different chitosan structures was evaluated through compression

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The addition of GA clearly increased the resistance of the scaffolds to compression. Uncrosslinked chitosan matrix exhibit a compressive strength of approximately 0.08 MPa.

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Comparable values were obtained by Zhang et al. [51], who produced CH scaffolds under conditions comparable to those of the present work (2% CH in 0.2% acetic acid), obtaining

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scaffolds with compressive strength of 0.11 MPa, after 60% deformation. The addition of GA, as reported [41,52], reacts with chitosan, making the structure more rigid and mechanically more resistant. As verified through the obtained strain-strain curves, increasing the concentration of

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GA lead to a rise of scaffolds’ mechanical properties, a process associated with the formation of a higher number of bonds between GA and chitosan [41,53]. Furthermore, a progressive

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increase in the Young’s modulus was also observed in direct relation to the increase of GA content. This translates to an increased rigidity of the scaffolds, of further clinical relevance for orthopedic loadbearing applications.

Despite the fact that higher concentrations of GA are those associated to improved mechanical properties, it is critical that GA content does not impair the biological functionality of the crosslinked biomaterials. Thus, the biological response to human osteoblastic cells was addressed for the different crosslinked CH scaffolds, and data presented on Fig. 3. Assessment of the metabolic activity at day 1 revealed that all the materials allowed the adhesion of viable cells. The addition of glutaraldehyde to the scaffold composition significantly improved the biological performance, justified by the higher values attained with the 0.2, 1 and 10% GA compositions, as compared to control (uncrosslinked CH), at day 1. Crosslinked scaffolds with lower % of GA, particularly the scaffolds with 0.2%, showed an increased culture’s metabolic activity, over the 7 days culture period, which can be correlated with an active cell proliferation. Further, representative SEM images of cultures grown on CH+0.2 GA revealed a high number of adhered cells within the scaffold’s porous structures, displaying an elongated morphology and a flattened shape with numerous cytoplasmic extensions, and

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Journal Pre-proof distinctive intercellular contacts, which is characteristic of the phenotype of this cellular population [54]. Accordingly, it may be suggested that, with 0.2% GA, most of the aldehyde functional groups are crosslinked with the polymer framework, inducing no cytotoxic effects and further enhancing cell functionality, as compared to control [55,56]. Compositions with higher GA levels revealed an inferior biological performance: scaffolds with 1% GA allowed for the viability of adhered cells throughout the culture period, but similar resazurin reduction values were attained for different time points, sustaining no evidence of active cell proliferation; scaffolds with the highest concentration of GA (10%) induced a reduction in the culture’s metabolic activity throughout the culture period, indicating a low biological performance, expectedly due to the potential excess of free dialdehydes in the scaffold structure that compromise the cellular behavior [57].

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In view of these results, although crosslinked scaffolds with 0.2% and 1% of GA displayed inferior biomechanical properties than those with a higher GA content, they revealed an

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improved biological performance, as assessed through direct osteoblastic culture. Thus, crosslinking CH with GA can be advantageous if the GA concentrations remain below to 1%,

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which will be considered to the subsequent development and characterization of the CH

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composite scaffolds.

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3.2 CaP granules characterization

The X-ray diffraction patterns of both initial powders (HA and β-TCP) and of the spray dried HA

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and biphasic granules are presented in Fig. 4. As observed, both powders of HA and β-TCP revealed only the characteristic peaks of a pure crystalline phase according to the JCPDS files number 09-0432and 09-0169 respectively, as no other secondary phases were detected. The XRD results of the spray dried HA and β-TCP granules were found to be identical to those of the initial correspondent particles, indicating that the spray drying process did not affect their crystallinity. Regarding the biphasic granules, XRD results evidence only the presence of the two mixed phases (i.e., HA and -TCP), hence confirming that the spray drying technique did not alter the crystal phase composition of the starting powders mixture. The initial particles used to prepare the initial suspensions of HA and β-TCP are showed in Fig. 5, in which the initial HA particles (Fig.5 a)) present an nanosized and an uniform distribution, whereas β-TCP particles (Fig. 5 d)) display a larger and less regular size. Regarding both CaP granules, SEM images shown in Fig. 5 confirm that the spray-drying process promoted an assembling of the initial particles into granules, for both compositions, being HA granules uniformly shaped as smooth spheres, with an average diameter around 2.73±0.15 µm (Fig. 5 b) and c)), whereas biphasic granules presented a more heterogeneous morphology, with sharp edged irregular shapes coexisting with smooth spheres (Fig. 5 e) and

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Journal Pre-proof f)). The irregular shape found in biphasic granules is due to the incorporation of β-TCP, since initial particles are irregular, both in size and shape. Also, biphasic granules presented an average diameter slightly smaller than HA spray dried granules (2.41±0.38 µm), but with a higher size dispersion. Different approaches have been proposed for the production of HA and/or biphasic granules for example precipitation [58,59], drip casting [36], emulsification [60], and extrusionspheronization [61] and, depending on the final application, a broad range of properties can be obtained, regarding particle diameter, size distribution, morphology, porosity and composition [62]. The spray drying process has numerous advantages that outperform most the other techniques, namely its feasibility to produce free-flowing particles (generally in a spherical shape) with well-defined particle size - that may vary from submicron-to-micron depending on

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the conditions (i.e. volume fraction, feed slurry and/or atomization pressure) [63,64]. Within the context of bone tissue engineering, this technique has attracted considerable interest

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since it may allow an easy incorporation of active pharmaceutical compounds and/or biomodulators, making them useful and suitable for drug delivery [64]. In this regard, our group

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has successfully demonstrated the suitability of spray dried granules to be loaded with 5fluorouracil [37] or dexamethasone [47], for biomedical applications. In addition the possibility to

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produce small granules with enhanced surface area is highly desirable for bone tissue applications, facilitating the bone cell contact and tissue osteoconduction [65] particularly when

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loaded on three-dimensional macroporous scaffolds. Table 2 presents the specific surface areas of initial CaP particles and produced spray-dried granules. Comparing the SSA of the various particles, i.e. before and after spray drying, it is observed that

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the SSA values of the initial particles are slightly larger than those of the corresponding spray dried granules. This indicates that during spray drying the aggregation of the initial powder

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particles takes place without substantial loss of free surface suggesting that porous granules are produced. The large SSA exhibited by these granules is an important attribute that favors cell anchoring and adhesion, as well as enhanced contact with the microenvironment, modulating its composition and favoring tissue infiltration and the bone to implant contact [65– 67].

3.3 Composite scaffolds

3.3.1 Physical and Chemical characterization Composite scaffolds were prepared using GA crosslinked-CH matrices with the incorporation of the above described CaP spray dried granules. For that, low concentrations of GA (0.2%, 0.5% and 1%) were tested with 1 wt% of each developed CaP granules, originating HA/ Chitosan (HA/CH) and Biphasic/Chitosan (Bi/CH) scaffolds, with different GA contents. Incorporation of CaP granules is a strategy to improve the scaffolds’ biomechanical properties and bioactivity

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Journal Pre-proof [68], aiming to further enhance the functional activity of osteoblastic cells. The chemical and crystal phase composition of the synthesized composite scaffolds were analyzed using XRD and FTIR techniques. The obtained XRD patterns for HA/CH and Bi/CH (both with 1%GA) are shown in Fig. 6. In the case of HA/CH scaffolds, the main characteristic lines of hydroxyapatite (JCPDS file no. 09-0432) overlap well with most of the diffraction peaks of the synthesized material, indicating that HA is the dominant crystalline phase. Moreover, a broad peak centred at 20⁰ 2θ is assigned to chitosan, being consistent with the typical X-ray diffraction pattern of chitosan reported in a number of previous studies [69–71]. In addition, one new peak which had not been detected in the XRD pattern of HA granules is now found at 28⁰ 2θ. This peak may be attributed to octacalcium phosphate (OCP) which main peaks overlap with those of HA within the 2θ region under analysis [72]. This result might indicate that the

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acidic conditions used for the preparation of the composite scaffold favoured the dissolution of a small fraction of HA followed by re-crystalization as OCP [73]. It is known that OCP has a large

vertebrates hard tissues. Under

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similarity in crystal structure with HA, behaving possibily as a precursor of carbonated apatite in physiological conditions OCP is converted to HA through

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hydrolysis [73], thus anticipating that OCP will probably revert to HA during the scaffold life time.

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Regarding the Bi/CH scaffold XRD pattern, the broad peak of chitosan at 20⁰ 2θ together with the representative peaks of both hydroxyapatite and β-TCP (JCPDS files 09-0432 and 09-0169,

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respectively) are clearly identified. In this case, the relative intensities of the peaks corresponding to HA and β-TCP are similar to those of biphasic granules, thus suggesting that the (1:1) ratio of (HA + β-TCP) biphasic mixture was likely preserved during the scaffold

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preparation.

The functional groups in HA/CH and Bi/CH composites were investigated using FTIR spectroscopy, as shown on Fig. 7. In line with XRD results, both FTIR spectra also evidence the

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existence of HAp, as confirmed by the bands at 561, 603 and 1020 cm

-1

3-

belonging to PO4

vibration mode [74]. Moreover, in Bi/CH FTIR spectrum, other vibrational bands assigned to β-1

TCP are also detected (1120, 1042, 604 and 433 cm ) [75], although they are superimposed by -1

others more intense groups. The bands observed at 3420, 1650 and 1406 cm in both scaffolds denounce the vibration modes corresponding to CH [69,70]. In addition, the band found at 1556 cm

-1

in both spectra is attributed to amide II (N-H bending) which is a characteristic band

reported for chitosan crosslinked with glutaraldehyde [69,76]. Microstructures of the developed HA/CH (with 0.2% and 1% GA) and Bi/CH scaffolds (with 0.2% and 1% GA) are illustrated in Fig. 8 (a, b, e, f) and (c, d, g, h), respectively. As observed, SEM images of both type of composite scaffolds, i.e. HA/CH and Bi/CH with different amounts of GA showed an identical microstructure, with well-defined and arranged pores, presenting a good interconnection, which is a positive factor for ensuring delivery of cells and nutrients during seeding and subsequent culture/implantation, as well as for the clearance of cells’ metabolic end products [77,78]. Moreover, it was possible to verify that HA and biphasic granules were distributed uniformly all over the scaffolds’ structure, rendering them with thicker walls than those obtained in GA-crosslinked chitosan scaffolds (Fig. 1), contributing to the

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Journal Pre-proof reinforcement of the composite scaffolds’ structure. Regarding pore dimensions (Table 3), composite scaffolds loaded with either HA or biphasic granules exhibited a similar range, with an average size close to 200 µm. As in line with the correspondent SEM micrographs (Fig. 8), and to the previously addressed CH matrices, GA seems to have strong influence on the average pore size, with a direct correlation between the increase of GA amount, and the average pore size. Ma et al. [79] found similar results on porous collagen/chitosan scaffolds treated with glutaraldehyde, observing an increase in the mean pore size from 100 µM to >200 µM, following crosslinking treatment. A similar trend was observed when collagen/chitosan scaffolds were crosslinked with genipin a natural crosslinking agent [80,81]. Overall, the effect of GA crosslinking on composite scaffolds’ morphology results in well-defined

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large pores and thicker pore walls, with an intensification of these characteristics with the increase of the crosslinker amount. In addition, all the produced scaffolds presented pore sizes

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in the range of 100-350 µm, recognized as an appropriate size range for bone tissue applications, known to allow for vascular ingrowth and support osteoblastic cells’ performance,

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improving bone ingrowth [82,83].

The biomechanical properties of developed composites, loaded with either HA or biphasic

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granules, particularly the stress-strain curves and the characteristic Young’s modulus, are presented in Fig. 9.

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As observed, mechanical resistance of the composite scaffolds is strongly influenced by the GA concentration. Moreover, an increase of the Young’s modulus (Fig. 9 C)) is verified with the increase content of the crosslinking agent, being the structures more resistant, with the highest

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concentration of crosslinker (1%). Glutaraldehyde-crosslinked chitosan scaffolds composed with hydroxyapatite nanoparticles developed by Li et al. [41] also demonstrated that appropriate

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crosslinking is beneficial to improve the mechanical properties, showing that when the crosslinking degree reaches 10%, the flexural strength of the composite scaffold reached a maximum value of 20 MPa - a 45% increase when compared with noncrosslinked scaffolds. Other works (Ma et al.[79]; Sharma et al.[84] ) also used GA as crosslinking agent in chitosan matrices, in order to improve the scaffold’s biostability and to lower the rate of biodegradation. Regarding the type of granules incorporate into CH matrices, no significant differences in the mechanical behavior were observed, despite that both HA and biphasic-loaded composite scaffolds with 0.2% and 0.5% GA presented lower values of Young’s modulus than the crosslinked CH matrices with the same concentrations of GA (Fig. 9 C). These results may be due to the difficulty of the chitosan to crosslink in the presence of the added ceramic granules, which considerably decreased the mechanical properties of the structures. This crosslinking deficit was only compensated by increasing the GA concentration (1%), with scaffolds presenting, in these cases, the highest Young's modulus (Fig. 9 C). The mechanical values obtained for these composites are in the range of those reported in the literature by Zhang et al [51], which produced CH scaffolds reinforced with β-TCP with a weight ratio CH/β-TCP [90/10], and achieved compressive strength values of around 0.25 MPa.

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Journal Pre-proof Overall, results indicated that the incorporation of CaP granules into the CH matrices, together with the crosslinker, successfully improved the mechanical resistance and the morphology of the produced composites. In addition, CaP granules are expected to maximize the biological performance, making these new materials ideal for stimulating and guiding the growth of native bone tissue.

3.3.2 Cytocompatibility study The biological response of the developed composite scaffolds was evaluated through a direct contact assay, with human osteoblastic cells. GA scaffolds (0.2% or 0.5%), loaded with either

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HA or biphasic particles were assayed and compared to established control – 0.2% GA scaffolds. Established cultures were characterized regarding metabolic activity, cell adhesion

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and cell/biomaterial interaction through SEM imaging, and gene expression of relevant osteogenic markers.

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Results regarding cultures’ metabolic activity are presented on Fig. 10 A, which showed an increase of the viability/proliferation of seeded cells throughout the seven days culture period,

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for all experimental conditions. Comparatively to control (CH 0.2%GA scaffolds), composites loaded with either HA or biphasic granules and distinct GA concentrations presented increased

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resazurin reduction levels, with significant higher values for both 0.2% GA scaffolds, loaded with either HA or biphasic particles, from day 1 onwards, and for 0.5% GA scaffolds, loaded with either HA or biphasic particles, from day 4 onwards. These results come in line with previous

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works, supporting an increased cell adhesion and proliferation in chitosan substrates loaded with ceramic particles [85,86] sustaining an increased bioactivity of the developed composites.

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The improved adhesion and proliferation, attained with the addition of HA or biphasic granules to CH-GA scaffolds, may relate to the added topographical features of the particles, mimicking more closely the biological architecture of the bone tissue [87]. Additionally, the chemical microenvironmental modulation achieved with the added apatite mineral, may further allow a selective protein adsorption - including general adhesion-enhancing molecules (such as fibronectin or vitronectin) and also osteoblastic-specific molecules – which favor cell recognition, interaction and binding [88]. The increased cellular binding enhances the activation of distinct intracellular signaling pathways that prime cellular functionality, improving adhesion, proliferation and the osteogenic differentiation process [89]. Regarding the ALP activity of established cultures, presented on Fig. 10 B, revealed an increase level for cultures grown on 0.2% GA scaffolds loaded with biphasic granules, both at day 4 and day 7, and regarding 0.2% GA scaffolds loaded with HA granules at day 7. ALP activity has been widely used as a marker of osteogenic differentiation progression in grown osteoblastic cultures [90], thus suggesting an increased osteogenic activation of cultures seeded and grown over 0.2% GA scaffolds. In Fig. 11, representative SEM and fluorescence microscopy images of the cells’ interaction with the developed scaffolds is presented, regarding control scaffolds, and those formulated with

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Journal Pre-proof 0.2% GA loaded with either HA or biphasic granules. The interaction between cells and scaffolds was addressed at day 1 and 7 of the culture. SEM analysis revealed that, at day 1, cells adhered to control scaffolds, presenting essentially a round morphology. In 0.2% GA scaffolds, cells also presented a round morphology, whether some degree of cytoplasmic spreading and filopodial extensions could be observed, particularly in cells adhered to the scaffolds loaded with biphasic granules. After 7 days, in control scaffolds, a cell number similar to that attained at day 1 was identified, with morphology ranging from spherical to fusiform, with minor cytoplasmic spreading. In 0.2% GA scaffolds loaded with granules, a significant higher number of cells was acknowledged, revealing an elongated and flattened structure, increased cytoplasmic spreading, high number of thicker and denser filopodia and extensive cell-to-cell contact.

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Fluoresce microscopy observation, conducted following immunostaining of mitochondria, Factin and nuclei, supported SEM observations. At 7 days, cells grown on control scaffolds

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presented an active mitochondrial activity, sustained by the red staining of the mitochondrial probe, with a predominant perinuclear distribution. Cells grown on 0.2% GA composites loaded

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with granules presented a more intense staining, correlated with a higher mitochondrial membrane potential and metabolic activity [91] as verified in Fig. 10 A. Furthermore, an

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increased polarized mitochondrial distribution was attained around the nucleus, a pattern associated with a cytocompatible response induced by the substrate [92]. Cells grown on

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loaded 0.2% GA composites also revealed an increased F-actin staining, a more elongated and spread morphology, denser stress fibers, more developed cell-to-cell contacts and filopodial extensions, as comparing to cells grown on control scaffolds, as in accordance with SEM

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observations. The increased arrangement of the F-actin cytoskeleton and cell spreading have been associated with a higher activation of predicted differentiation pathways, leading to an

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increased osteogenic differentiation and higher expression of osteogenic-related markers [93,94]. Also, more significant cell-to-cell contacts and filopodial extensions were further associated with an improved osteogenic activation [95], thus supporting an increased osteogenic commitment of cultures grown on 0.2% GA composites loaded with granules. In order to further address the functional activity of grown cultures, a gene expression analysis of relevant osteoblastic-related genes – i.e., Runx-2, alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), collagen type I (COL1A1) and bone morphogenic protein 2 (BMP2), was conducted by quantitative PCR (Fig. 12). Comparatively to cultures grown on control scaffolds, significantly higher levels were attained for cultures grown on GA 0.2% scaffolds loaded with either HA or biphasic granules, regard11ing all the assayed genes. Furthermore, a trend for increased gene expression was also verified for cultures grown on biphasic GA 0.5% scaffolds, and found to be significantly higher regarding the expression of osteocalcin and osteopontin. Cultures grown on Bi/CH scaffold with 0.2% presented significantly higher ALP levels than those grown on HA/CH scaffolds with 0.2% GA. Characterized genes are relevant osteoblastic markers, playing a fundamental role within osteogenesis. Runx2 is a major transcription factor required for the commitment and

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Journal Pre-proof determination of the osteoblastic lineage, being able to regulate the expression of all important bone matrix proteins such as the downstream ALP, OC and OPN [96]. ALP hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization, being regarded as a key enzyme for the initiation of the mineralization process either in vitro or in vivo [97,98]. Osteocalcin and osteopontin are important coordinators of the organic matrix maturation and formation, as well as its subsequent mineralization – osteocalcin has been used routinely as a late osteoblastic marker and believed to act in the regulation of the matrix mineralization process; further, it has been associated with a mechanical function, by tightly binding mineral HA to organic collagen through osteopontin, which also regulates matrix remodeling and tissue calcification [99,100]. Type 1 collagen – the product of COL1A1 and COL1A2 - is by far the most abundant protein synthesized by osteoblasts, greatly determining the biomechanical properties

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of the bone tissue [101]. BMP2 is a potent inducer of osteogenesis, acting through an autocrine or paracrine signaling to induce the expression of osteoblastic phenotypic markers, such as

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ALP or OC, in a Runx2-dependent or independent process [102,103].

Attained results suggest that scaffolds with 0.2% GA, loaded with HA or biphasic granules

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induce an increased osteoblastic phenotypic expression of the seeded osteoblastic populations, with significantly higher levels of expression of all the assayed genes (i.e., Runx2, ALP, OC,

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OPN, COL1A1 and BMP-2). Scaffolds with 0.5% GA, loaded with particles, presented an increased trend, with higher levels of expression of OC, OPN and COL1A1, whether scaffold

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prepared with 1% GA revealed a biological behavior similar to control, further underlining the importance of low amounts of crosslinking agents to improve scaffold’s biological response [104,105]. In addition, scaffolds loaded with biphasic granules presented a trend for enhanced

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osteoblastic activity, with significantly higher ALP and BMP-2 expression, and a trend for higher OC and OPN expression, as comparing to HA-loaded scaffolds, with a similar amount of GA,

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suggesting the tendency for biphasic CaP ceramics to improve the osteogenic response. Accordingly, the chemical properties of ceramics may influence the biological response of seeded osteoblastic populations [106]. Biphasic calcium phosphates, given the possibility to fine tune the ratio of the composition phases may present a more favorable biodegradation profile, and contribute to improved microenvironmental parameters, in terms of Ca and P availability, that increase the cellular activity [107]. In this way, the use of biphasic-based ceramics has been previously found to improve the early stages of bone regeneration through an enhanced ionic release that allows for a greater deposition of collagen [108] and vascular bed [109], further enlightening the subsequent bone healing process.

4. Conclusions Herein we have firstly described the influence of different concentrations of glutaraldehyde in crosslinking chitosan three-dimensional matrices, displaying a significant increase in the mechanical behaviour with the increase of the GA content, and the preference of osteoblastic cells for lower GA concentrations. The spray drying fabrication and characterization of HA and

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Journal Pre-proof biphasic mixtures was also reported for the first time, with potential application in bone regeneration and drug delivery therapies. Finally, we successfully designed freeze-dryed composite scaffolds, with interconnected porous, by GA-crosslinked chitosan (using the adequate GA concentrations previously explored and optimized) reinforced with the produced CaP micro-sized granules. In the production and characterization stage of CaP granules, atomized granules with micrometric size were obtained, revealing a slight agglomeration of the initial particles with loss of specific surface area. Attained granules were spherical, in the case of HA, and presented a more irregular shape with the incorporation of β-TCP (biphasic granules). Although the crystallinity of CaP was not altered by the atomization process, the scaffold preparation process favoured the dissolution-recrystallization of HA leading to the new CaP phase of OCP.

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Regarding the characterization of the composite scaffolds, the GA treatment had an influence on the morphology and the mechanical properties, increasing the pore size and improving the

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mechanical behaviour, with increasing glutaraldehyde content. CaP granules were uniformly distributed within the chitosan matrix, which showed thicker pore walls and enhanced

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mechanical properties, particularly in those with a higher degree of crosslinking. The biological assessment of the composite scaffolds showed that the specimens with 0.2%

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crosslinking were the ones with the best biological performance, regarding metabolic activity, ALP expression, cell morphology, cell/scaffold interaction and gene expression, while scaffolds

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with 1% glutaraldehyde present an inferior performance. In addition, the inclusion of biphasic granules seemed to induce a trend for increase osteogenic activation, as assessed through relevant osteoblastic gene expression analysis, as compared to the addition of HA granules.

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In conclusion, scaffolds produced in the present work, both with HA granules or the biphasic ones, and with low concentrations of GA, have shown adequate properties and enhanced

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biological performance, being potential candidates for application in bone tissue engineering.

Acknowledgements

This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, and by REQUIMTE project UID/QUI/50006/2019, financed by national funds through the FCT/MCTES.

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Author statement Rosana Pinto: Writing - Original Draft, Formal analysis, Investigation, Visualization, Data Curation Pedro Gomes: Conceptualization, Methodology, Supervision, Writing - Review & Editing Maria Fernandes: Conceptualization, Supervision, Methodology, Writing Review & Editing, Funding acquisition, Project administration

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Maria Costa: Conceptualization, Methodology, Visualization, Supervision, Resources, Writing - Review & Editing, Funding acquisition, Project administration

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Maria Almeida: Conceptualization, Supervision, Visualization, Methodology, Resources, Writing - Review & Editing, Funding acquisition, Project administration

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Declarations of interest: none

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Fig. 1 – Scaffolds morphology assessed by SEM analysis: a) and b) pure chitosan scaffolds; c) and d) crosslinked chitosan matrices with 0.2% glutaraldehyde; e) and f) crosslinked chitosan matrices with 1% glutaraldehyde; and g) and h) crosslinked chitosan matrices with 10% glutaraldehyde. Fig. 2 – Mechanical behavior of chitosan matrix and GA-crosslinked chitosan scaffolds; A) Compressive stress-strain curves; and B) Young’s modulus. Fig. 3 –Metabolic activity of human osteoblastic cultures established directly over crosslinked chitosan matrices for 1, 4 and 7 days; SEM micrograph reveals the adhesion/morphology of the cells grown on the crosslinked CH matrix with 0.2% GA, after 4 days. (*) significantly different from CH (p < 0.05).

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Fig. 4 – X-ray diffraction patterns of HA and β-TCP initial powders and of HA, β-TCP and biphasic spray dried granules.

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Fig. 5 – Images of the initial powders and the spray dried granules: a) SEM image of the initial HA nanopowder b) and c) SEM images of spray-dried HA granules; d) SEM image of β-TCP

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initial suspension and e) and f) spray-dried biphasic granules.

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Fig. 6 – X-ray diffraction patterns of HA/CH and Biphasic/CH composite scaffolds. Fig. 7 – FTIR spectra of HA/CH and Biphasic/CH composite scaffolds.

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Fig. 8 – SEM micrographs of the HA scaffolds scaffolds with 0.2% (a, b) and 1% GA (e, f) and of the biphasic composite with 0.2%GA (c, d) and 1% GA (g, h).

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Fig. 9 – Mechanical resistance of of the different ceramic/CH composite scaffolds: A) Stressstrain curves of HA/CH composite scaffolds; B) Stress-strain curves of Bi/CH composite

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scaffolds and C) Young’s modulus of the different CH and composite scaffolds calculated from the stress-strain curves.

Fig. 10 – Biological activity of osteoblastic cultures grown within developed composite scaffolds. A) Metabolic activity of cultures - alamarBlue® assay. B) Phosphatase alkaline activity of the cultures. These results are shown as a percentage of the control (100%). (*) stands for significantly different from control (p < 0.05). Fig. 11 – Representative SEM and fluorescence microscopy images of cell cultures grown on the control scaffolds and on the composite HA or Biphasic scaffolds formulated with 0.2%GA, after 1 and 7 days of culture. In fluorescent microscopy imaging, cells were stained for mitochondria (red), F-actin cytoskeleton (green) and nuclei (blue). Fig. 12 – Gene expression analysis of osteoblastic cultures grown within developed composite scaffolds. Regarding each gene, results were normalized to the expression levels of control (set as 1). (*) stands for significantly different from control (p < 0.05), (**) stands for significantly different from HA + 0.2% GA.

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Table 1 – Composition and labeling of the 3D scaffolds addressed in the present work. Granules

Glutaraldehyde Chitosan 0.2%

CH+1GA

X

CH+10GA HA/CH+0.2GA

X X

HA/CH+0.5GA

X

HA/CH+1GA Bi/CH+0.2GA

X X

Bi/CH+0.5GA

X

Bi/CH+1GA

X

10%

Hydroxyapatite (HA)

Biphasic (HA + β-TCP 1:1wt ratio)

X X X X

X X

X X

X

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X

1%

X X X

X X X

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CH+0.2GA

0.5%

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Scaffold label

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HA β-TCP Biphasic mixture

Specific surface area of the initial particles 2 (m /g)

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Composition of CaP Suspensions

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Table 2 – Specific surface area of starting CaP particles and of spray dried granules.

124 48 65

Specific surface area of the spray dried granules 2 (m /g) 106 17 60

Table 3 – Average pore size of the different HA/CH and Bi/CH composite scaffolds.

GA variation 0.2 % 0.5 % 1.0 %

HA/CH Bi/CH Scaffolds Scaffolds Average pore size (µm ± µm) 151 ± 41 175 ± 53 185 ± 64 205 ± 59 224 ± 58 219 ± 46

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12