journal of the mechanical behavior of biomedical materials 103 (2020) 103572

Available online 30 November 20191751-6161/© 2019 Elsevier Ltd. All rights reserved.

Strategy to improve the mechanical properties of bioabsorbable materials based on chitosan for orthopedic fixation applications

Lígia Figueiredo a,b,*, Rita Fonseca c, Luís F.V. Pinto b,d, Frederico Castelo Ferreira e, Am�elia Almeida f, Alexandra Rodrigues g

a IDMEC – Instituto Superior T�ecnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal b Bioceramed S.A., 2660-360 S~ao Juli~ao do Tojal, Loures, Portugal c CDRSP – Center for Rapid and Sustainable Product Development, Instituto Polit�ecnico de Leiria, 2430-028, Marinha Grande, Portugal d CENIMAT/I3N, Faculdade de Ciencias e Tecnologia – Universidade Nova de Lisboa, 2829-516 Caparica, Portugal e Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior T�ecnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal f CeFEMA – Center of Physics and Engineering of Advanced Materials and Department of Chemical Engineering, Instituto Superior T�ecnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal g CI-MOSM/ISEL, Instituto Superior de Engenharia de Lisboa, 1959-007 Lisboa, Portugal

A R T I C L E I N F O

Keywords: Bioabsorbable polymers Chitosan Plasticizers Ceramics Mechanical properties Microstructure

A B S T R A C T

Bioabsorbable polymeric fixation devices have been used as an alternative to metallic implants in orthopedics, preventing the stress shielding effect and avoiding a second surgery for implant removal. However, several problems are still associated with current bioabsorbable implants, including the limited mechanical stiffness and strength, and the adverse tissue reactions generated. To minimize or even eliminate the problems associated with these implants, strategies have been developed to synthesize new implant materials based on chitosan. To overcome the brittle behavior of most 3D chitosan-based structures, glycerol and sorbitol were blended to chi-tosan and the effect of these plasticizers in the produced specimens was analyzed by flexural tests, Berkovich tests, scanning electron microscopy (SEM) and micro-CT analyzes. The improvement of the mechanical prop-erties was also tested by adding ceramics, namely hydroxyapatite powder and biphasic mixtures of hydroxy-apatite (HA) and beta-tricalcium phosphate (β-TCP). In the plasticizers group, the best combination of the measured properties was obtained for chitosan with 10% glycerol (flexural strength of 53.8 MPa and indentation hardness of 19.4 kgf/mm2), while in the ceramics group the best mechanical behavior was obtained for chitosan with 10% HAþβ-TCP powder (flexural strength of 67.5 MPa and indentation hardness 28.2 kgf/mm2). All the tested material compositions were dense and homogeneous, fundamental condition for a good implant perfor-mance. These are encouraging results, which support the continued development of chitosan-based materials for orthopedic fixation applications.

1. Introduction

Musculoskeletal diseases are characterized by reduced physical function and pain. These include back and neck pain, osteoarthritis, rheumatoid arthritis and bone and soft tissue injuries caused by sports, road and workplace accidents (March et al., 2014; Vos et al., 2015). These conditions represent a higher cost for the individuals and the society through the associated disability and the healthcare needs and they have been growing due to the aging of population and changes in lifestyle (Bevan, 2015). The rise in orthopedic disorders and the

emergence of advanced solutions and technologies will promote the continuous growth of the orthopedic implants market (Briggs et al., 2018). This market was valued at $47,261 million in 2016, and is ex-pected to garner $74,796 million by 2023, registering a compound annual growth rate (CAGR) of 6.8% during the forecast period 2017–2023 (Radhakrishnan, 2018).

Traditionally, the use of metallic implants made of stainless steel or titanium is still the gold standard method for the majority of the ortho-pedic treatments due to their outstanding mechanical properties (Eglin and Alini, 2008; Suchenski et al., 2010). Although strong and cost

* Corresponding author. IDMEC – Instituto Superior T�ecnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal. E-mail address: ligia.figueiredo@tecnico.ulisboa.pt (L. Figueiredo).

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Journal of the Mechanical Behavior of Biomedical Materials

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https://doi.org/10.1016/j.jmbbm.2019.103572 Received 13 May 2019; Received in revised form 14 August 2019; Accepted 29 November 2019

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effective, these materials present several disadvantages. When the metallic devices are in contact with bone, they will bear most of the load due to their high modulus of elasticity. The consequence is the pro-duction of a stress shielding effect in the adjacent bone, which may induce local bone resorption that will lead to the eventual failure and loosening of the implant (Ambrose and Clanton, 2004; Mukherjee and Pietrzak, 2011; Suchenski et al., 2010). After the fracture healing, a second operation is often necessary to remove the metallic implant, especially in pediatric procedures (Eglin and Alini, 2008). Removing surgeries involve further costs since they require hospital admission for a new surgery with the inherent risks associated for the patient, loss of productivity and increasing costs.

In recent years, the research trends of medical devices have been directed to the use of bioabsorbable implants, particularly in applica-tions that only require a transient existence of the implant (Ciccone et al., 2001; Mukherjee and Pietrzak, 2011; Ramsay et al., 2010). These implants are susceptible to be eliminated by the body once the healing process has occurred. Its biodegradability should occur at the same rate as the patient’s own tissue is regenerated and into harmless, non-toxic, by-products, which are eliminated by the body via natural pathways (Mukherjee and Pietrzak, 2011; Pietrzak, 2008). The use of biopolymers for such purposes is becoming increasingly popular as materials derived from nature are expected to exhibit greater compatibility with humans (Mukherjee and Pietrzak, 2011; Pietrzak, 2008). Such polymeric devices have elastic constants lower than cortical bone, thus preventing the stress shielding effect (Ramsay et al., 2010). However, usually they are not stiff enough to be used in major load-bearing applications (Ramsay et al., 2010). Despite the described advantages, several drawbacks are still associated with bioabsorbable implants, including insufficient me-chanical properties, premature material breakdown, sterile fluid accu-mulation and sinus formation over the implantation site (Givissis et al., 2010; Mukherjee and Pietrzak, 2011). In order to eliminate the reported problems associated with current biodegradable implants, new implants based on natural polymers have been studied, with chitosan being one of the most recently studied biomaterials (Mano et al., 2007; Oliveira et al., 2014).

The potential of chitin and chitosan as biomaterials, namely their good biological properties and tunable biodegradability, related with their degree of acetylation and/or molecular weight (Anitha et al., 2014; Cheung et al., 2015), has been reported in scientific literature for more than 40 years, but practical applications have been severely limited in processes involving three-dimensional (3D) geometries. The major application of chitosan and its derivatives in three dimensional forms are very limited because chitosan has a degradation temperature lower than its melting temperature, limiting the use of conventional manufacturing processes (Sarasam and Madihally, 2005). To overcome such limitation, a new processing method has been recently developed (Oliveira et al., 2013) which allows the production of 3D dense chitosan specimens with high strength and stiffness (Oliveira et al., 2014). However, due to the brittle behavior of most 3D dense chitosan-based structures, several strategies have been proposed to improve their me-chanical properties (Correlo et al., 2005; Croisier and J�erome, 2013). In this area, strategies may include blending chitosan with hydrophilic polymeric compounds such as polyvinylpyrrolidone (PVP), phospho-molybdic acid (PMA) and cellulose, and/or adding other biodegradable polymers, such as polycaprolactone (PCL), poly(butylene succinate) (PBS), poly(lactic acid) (PLA) and poly(butylene terephthalate adipate) (PBTA) (Bhat et al., 2011; Chen and Hwa, 1996; Hsieh et al., 2005; Lavorgna et al., 2010).

To produce 3D dense chitosan-based structures for orthopedic implant applications, it is essential that their final composition have adequate stiffness, strength and hardness, able to comply with the bone tissue requirements. Also, it is important to prevent brittle fracture if overload occurs. With this goal in mind, a study was developed to improve the mechanical behavior of chitosan-based specimens, by blending chitosan with different types of materials (plasticizers and

ceramics). The microstructures obtained were evaluated by scanning electron microscopy (SEM) and computerized microtomography (micro- CT). The mechanical evaluation was performed through flexural and nanoindentation tests.

2. Materials

Medical grade chitosan from Bioceramed S.A. (Loures, Portugal), with high molecular weight and viscosity (700 kDa; 90% deacetylation degree, D.D.; viscosity 800 cP) was used. The glacial acetic acid was purchased from Carlo Erba Reagents and the sodium hydroxide solution (50% w/v) from Liqual, Lda. The pharmaceutical grade glycerol (purity degree � 99.5%) was purchased from Labchem and sorbitol (no crys-tallizable solution, 70%) from Carlo Erba Reagents. All reagents were used without further purification, except otherwise stated.

The calcium phosphates were provided by Bioceramed S.A, with three different materials being used: hydroxyapatite (HA) powder, a biphasic powder mixture of 70% HA with 30% β-tricalcium phosphate (HA-TCP powder) and a biphasic granules mixture of 70% HA and 30% β-TCP (HA-TCP granules). The powders had a particle size distribution of 7 μm (d.0,9) whereas the granules size ranged from 75 to 125 μm.

3. Methods

3.1. Production of 3D dense chitosan-based specimens

Chitosan (3% w/v) was dissolved in an aqueous solution of acetic acid (2% v/v) for 2 h, using mechanical stirring. After this time, the resultant homogeneous solution was poured in rectangular prismatic molds, with a predefined geometry (117 � 47 � 37 mm), and it was left at rest until confirmation by carefully visual inspection that all the air bubbles disappear, prior to be frozen at � 20 �C for 24 h. Subsequently, the frozen solutions were removed from the molds and plunged into a sodium hydroxide (NaOH) aqueous solution (10% w/v) for 48 h. The precipitation in NaOH occurred quickly, without thawing the solutions. The resulting hydrogel specimens were abundantly washed with deionized water, until pH~7 and then were dried in an oven at 40 � 2 �C. During the drying stage, the specimens shrunk and densified giving rise to solid rectangular blocks with the average dimensions of 30 � 17 � 12 mm. These blocks were machined using a conventional milling machine from Optimum (model MF 4 Vario). A 16 mm end mill cutting tool of high speed steel (HSS) was used to thin out each block into specimens with rectangular cross-section with average dimensions of 25 � 12 � 4 mm.

To increase the ductility of the material and to prevent brittle frac-ture of the chitosan-based specimens, glycerol and sorbitol were inde-pendently added in the production process, according to the concentration levels defined in Table 1.

All the ceramic-based specimens were produced from chitosan so-lutions with 10% of glycerol (w/v). Each ceramic concentration was added to the formulation after total dissolution of the chitosan in acetic acid aqueous solutions. For complete dispersion of the ceramics in the solution, the stirring continued for further 2–3 h. The concentrations of HA, HA-TCP powder and HA-TCP granules are presented in Table 2.

For a qualitative confirmation of the dispersion of the ceramics in the final machined specimens, blocks of chitosan blended with 15% HA-TCP granules and 15% HA (i.e., the highest concentration level) were cut with a saw in two distinct zones: near the bottom and near the top. SEM analyses were performed in the resultant cross sections, and the ceramic

Table 1 Concentrations of plasticizers (sorbitol and glycerol) used to produce 3D dense chitosan specimens.

Concentrations (w/v)

5% 10% 15%

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particles were counted using the image analysis software ImageJ.

3.2. Flexural tests

Three-point bending tests were performed according to ASTM stan-dard D790-15 (ASTM, 2016), using a universal testing machine from Instron (model 5566) equipped with a load cell of 10 kN and extension control. The tests were performed with a crosshead speed of 0.1 mm/min at room temperature. The results were processed using the Bluehill® 2 Materials Testing Software.

Four specimens were tested for each plasticizer and for each ceramic. The results presented are the average of those measurements.

3.3. Hardness tests – nanoindentation

The tests were performed using a Dynamic Ultra Microhardness Tester from Shimadzu (model DUH 211S), using a Berkovich indenter. The results were processed in the DUH-211S software, according to the International Standard ISO 14577 (Fischer-Cripps, 2011), using a load-creep-unload cycle. After loading up to a maximum force of 200 mN at a speed of 5 mN/s, a creep stage of 15 s was done before unloading. The hardness value was obtained by dividing the indentation load by the projected area of the contact. The elastic modulus was calculated from the slope of the initial 30% of the unloading curve (Fischer-Cripps, 2011).

The specimens subjected to the nanoindentation hardness tests were first smoothed with water sandpaper (P2000). On average, 8 in-dentations were made for each specimen from each composition. The results presented are the average of those measurements.

3.4. SEM

SEM analyses were performed on the top surface and on cross- sections of specimens from each plasticizer and ceramic composition. The analyses were made using a high resolution field emission gun scanning electron microscope (FEG-SEM) from JEOL (model JSM- 7001F), using beam voltages of 5 or 15 kV. The samples were previ-ously coated with a thin film of a gold-palladium alloy and they were analyzed using a magnification of 400X. To analyze the ceramic parti-cles of the blocks that were cut, the magnification level was 30X.

3.5. Microtomography

Morphologic studies and internal microstructure images were ob-tained by microtomography using a micro-CT equipment from Brucker (model SkyScan 1174v2). This equipment produces high resolution 3D images and applies differences in X-ray attenuation properties of the materials to reconstruct their 3D structure (Hanke et al., 2016). These studies were performed without sample preparation or chemical fixation and to one specimen of each plasticizer and ceramic composition.

The scan acquisition parameters were: image pixel size of 7.76 μm, source voltage of 50 kV, exposure time of 4500 ms and rotation step of 0.9�. At each step, three new projection images were taken. After scanning, the sample’s reconstruction procedure was performed using the NRecon Software, while the CTAn® software was used to analyze and quantify the fractions of the different components in the specimens.

CTVox® program was used to obtain the 3D realistic visualization of

the scanned specimens.

3.6. Statistics

Independent sample t-tests were performed for statistical comparison between the groups. In all statistical evaluations, statistical significance was set to a p-value � 0.05, under the assumption of the null hypothesis (H0: the means of the two groups are equal). The statistical evaluations with p-value � 0.05 are visually labelled with an asterisk (*)

4. Results and discussion

Chitosan-based structures were produced according to the concen-tration levels indicated in Tables 1 and 2 Concentrations above 15%, of both plasticizers and ceramics, were not considered in this study to not impair the expected biological behavior of chitosan. This restriction is in line with the polymer-based compositions of bioabsorbable implants currently in the market, such as BioComposite Corkscrew FT from Arthrex (85% PLLA, 15% β-TCP) (Figueiredo et al., 2018).

For the HA-TCP material, two distinct particle sizes were considered in this study: a small size (powder, ~10 μm) and a large size (granules, ~100 μm). Therefore, the impact of this ceramic material on the me-chanical properties of the chitosan-based specimens was assessed considering both the concentration levels and the size of the particles used.

The production process was successfully repeated for all the com-positions. However, for the 15% ceramic compositions, the stirring was more difficult and took a total of 5 h to guarantee the complete dispersion of particles in the chitosan-glycerol mixture.

Figs. 1 and 2 show examples of 3D dense chitosan-based blocks ob-tained after the drying stage of the production process.

The chitosan blocks with plasticizers had the same appearance, regardless of the concentration level. The color changed from brown to white for the blocks produced by adding the ceramics to the 10% glycerol composition. This plasticizer concentration was selected for the ceramics study knowing in advance the results of the flexural tests. The goal was to continue to take advantage of the effect of this plasticizer on the chitosan’s polymeric chain.

The blocks produced with granules were slightly rougher, and a uniform distribution of these particles on their surface was clearly visible. The uniform dispersion of the ceramic particles was analyzed with the ImageJ software. Fig. 3a and b show the images of the particles counted, respectively, on the top and bottom cross-sections of a block produced with 15% HA-TCP granules, after the respective cuts. In this case, 309 circular particles were counted on the bottom cross-section, while 323 particles were counted on the top cross-section of the block.

The previous results confirm a uniform dispersion of the ceramic granules in the blocks and consequently in the specimens that were machined from these blocks. Even though the images are not shown, the same observations were made when the counting was performed on the top and bottom cross-sections of the blocks of chitosan blended with 15% HA, which allows to generalize the uniformity of dispersion for all the other ceramic particles.

Figs. 4 and 5 show examples of specimens obtained after the machining stage.

4.1. Flexural tests

Fig. 6 shows examples of stress-strain curves obtained after the three- point bending tests for the chitosan-based specimens produced with plasticizers and ceramics. Additionally, Fig. 7 shows the flexural modulus, the flexural strength and the maximum flexural strain results obtained for each group of specimens.

The stress-strain curves of Fig. 6 show that both the specimens containing plasticizer and ceramic, when subjected to transverse loading, fracture with practically no plastic deformation. The same

Table 2 Concentrations of ceramics (HA, HA-TCP powder and HA-TCP granules) with 10% (w/v) glycerol used to produce 3D dense chitosan specimens.

Concentrations (w/w)

5% 10% 15%

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behavior is noted for the HA-TCP granules composition, although the curve is not represented in the image. During the tests, crack initiation was observed at the bottom of the specimens, where higher tensile stresses develop. Crack propagation quickly occurred, giving rise to fractures with a brittle surface. Although these observations result from testing the samples in their dry state, under in vivo conditions this behavior is expected to be different. According to the literature, chitosan is a hygroscopic material, which means that the water molecules in contact with chitosan will eventually bind to its chain, acting as a

plasticizer as it permeates into the structure (Gonz�alez-Campos et al., 2009; Rodríguez-V�azquez et al., 2015). This may promote polymer chain re-orientations which change its mechanical behavior, namely the elongation at break (Gonz�alez-Campos et al., 2009). This potential behavior can be determined if the flexural tests are performed on wet samples.

The results of Fig. 7a, b and c show that the mean flexural modulus decreases with the increasing concentration of plasticizers, but the same is not observed for the flexural strength and maximum flexural strain results. This phenomenon is more evident in the sorbitol formulations, though the dispersion of results is relatively high. Comparing each plasticizer individually, the highest values for the flexural modulus, strength and strain are obtained, respectively, for the 5% glycerol, 10% glycerol and 15% sorbitol compositions. Despite these results, it can be stated that the 10% glycerol composition shows a lower dispersion of results, especially in the flexural strength (53.8 MPa).

For all the assessed properties, the statistical tests (t-test) revealed weak evidence that the means of the two groups of plasticizers, with the same concentration, are statistically different.

Several studies report that the strength of chitosan films decreases as the content of glycerol increases, contrary to what happens with the elongation at break (Nor et al., 2013; Thakhiew et al., 2010; Ziani et al., 2008). This occurs because glycerol penetrates through the polymer

Fig. 1. Examples of blocks produced by blending chitosan with a) 15% glycerol and b) 15% sorbitol.

Fig. 2. Examples of blocks produced by blending chitosan with a) 10% HA and b) 10% HA-TCP granules.

Fig. 3. Counting of ceramic particles dispersed in the block of chitosan blended with 15% HA-TCP granules, with the ImageJ software: a) bottom part cross-section and b) top part cross-section.

Fig. 4. Specimen produced by blending chitosan with 5% sorbitol, after machining.

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matrix, interfering with the polymer chains and promoting the polymer mobility (Gabriela et al., 2015; Thakhiew et al., 2010). In this study, the addition of plasticizers was not always accompanied by an increase of the elongation at break. This may be related with variability associated with the manual machining process inhere used, which did not guar-antee exactly the same finishing and dimensions between all the pro-duced specimens. For this reason, some outliers are associated to the observations, causing the results dispersion in the flexural tests. The impact of this machining method on the mechanical properties can be reduced by using computer numerical control (CNC) machining.

The results of Fig. 7d, e and f indicate that the highest mean flexural modulus and flexural strength values were obtained for the 10% HA-TCP powder blend. This may indicate an optimal homogenization of this powder concentration in the specimens. On the other hand, the maximum mean flexural strain value is obtained for the 15% HA-TCP powder blends. However, the dispersion of results is high, as confirmed by the statistical tests that revealed weak evidence that the average values obtained for 10% and 15% groups are different, for the three mechanical properties assessed.

The results were expected to be similar for the HA and HA-TCP powder blends. However, the values obtained are only similar for the 5% and 15% concentrations (in this latter case, only for the flexural

strength). Once again, the better homogenization of the HA-TCP powder in the specimens could explain such observations. The results also indicated that it is difficult to benefit from ceramic particles with larger sizes, since the increased concentration of the granules led to the dispersion of results, which is more evident in the flexural modulus and flexural strength measurements. Analyzing the graphics in Fig. 7, it is difficult to associate specific trends on the mechanical behavior of the specimens of chitosan blended with ceramics simply to the concentra-tion of these materials. These specimens behave as composite materials and therefore can have distinct mechanical behaviors when performing a flexural test (Ahmed and Jones, 1990; Feng et al., 2016), which for example depend on the particle size and the interrelation established between the ceramic particles, the chitosan polymer chain and the re-sidual pores that may exist. Also, the manual machining process could have caused variability and produced outliers that affect the flexural tests.

Overall, the specimens produced with 10% HA-TCP powder present better results for the properties assessed: a flexural modulus of 2679 MPa, a flexural strength of 67.5 MPa and a maximum flexural strain of 5.7%. Comparing these results with those obtained for the specimens produced with 10% glycerol, there is an increase of 60% in the flexural modulus and of 25% in the flexural strength.

Fig. 5. Specimens produced by blending chitosan with a) 10% HA and b) HA-TCP granules, after machining.

Fig. 6. Stress-strain curves for specimens produced with a) 10% glycerol, b) 10% sorbitol, c) 10% HA and d) 10% HA-TCP powder.

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The mechanical properties obtained for the chitosan-based speci-mens are comparable to the properties of PLDLA and PLA, namely the compositions obtained by adding 10% HA-TCP powder to the chitosan, as reported by Figueiredo et al. (2018). In addition, the flexural modulus and the flexural strength values also approach the elastic modulus and strength associated to bone (Figueiredo et al., 2018).

4.2. Hardness tests – nanoindentation

The indentation hardness and indentation modulus results are pre-sented in Fig. 8a and b for the chitosan-based specimens with plasticizers and in Fig. 8c and d for the chitosan-based specimens with ceramics.

The results in Fig. 8a show that the indentation hardness of chitosan-

Fig. 7. Mechanical properties of specimens of chitosan blended with plasticizers – a) flexural modulus, b) flexural strength and c) maximum flexural strain – and blended with ceramics – d) flexural modulus, e) flexural strength and f) maximum flexural strain.

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based specimens increases with an increasing concentration of glycerol. Similar results are obtained for the sorbitol specimens, although the 10% and 15% concentrations of this plasticizer show practically the same hardness value. The highest value for the indentation hardness is ob-tained for chitosan with 15% glycerol, which stands out from the cor-responding concentration of sorbitol. In this case, the nanoindentation hardness is 21.3 kgf/mm2. Comparing the results of Fig. 8b for the different specimens, the highest mean indentation modulus value was obtained for chitosan with 10% sorbitol. If the analysis is only restricted to the values obtained for glycerol, the highest elastic modulus (4093 MPa) is obtained for the 10% glycerol composition. The t-tests per-formed to all concentration pairs of glycerol and to sorbitol reveal strong evidence that the mean values of both 10% concentrations are statisti-cally different from the other compositions (5% and 15%).

The nanoindentation results in Fig. 8c show that the highest mean values are obtained for the 10% and 15% concentrations of HA-TCP powder. The indentation hardness of specimens increases with increasing concentrations of this powder, remaining practically the same for the 10% and 15% concentrations (~28 kgf/mm2). The opposite trend is observed for the HA-containing specimens, for which increasing the HA content promotes a decrease in the measured hardness. For the HA-TCP granules specimens, the indentation hardness presents a maximum hardness at 10% concentration (~23 kgf/mm2).

The indentation modulus results of Fig. 8d indicate higher values for increased content of powder and granules in the specimens. Once again,

an opposite trend is observed for the specimens with increasing content of HA. Like in the hardness evaluation, the highest elastic modulus value (4896 MPa) is obtained for the concentration of 15% HA-TCP powder, followed by the concentration of 10% (4550 MPa).

Comparing the nanoindentation results of the 10% HA-TCP powder blend with the results obtained for the chitosan blends with 10% glyc-erol, it is observed that the mean hardness value increased by 44% and the mean indentation modulus by 11%.

Danilchenko et al. (2011) studied non-porous formulations of chi-tosan/HA and found that when the load of 0.2 N was applied (the same used in this study), the Vickers microhardness (HV) varied from 0.12 to 0.22 GPa according to the HA content. Comparing the results obtained for the 10% HA-TCP powder blend (indentation hardness of 0.28 GPa and indentation modulus of 4.6 GPa) with values from the literature (Danilchenko et al., 2011), highest values were obtained for the speci-mens obtained in this work. This result may indicate better densification and homogenization of this ceramic composition. The difference in hardness values between the two methods may also be explained by the specific characteristics associated with each one. In both methods, the hardness values are obtained considering the calculated area of inden-tation. However, in nanoindentation this area is measured from the projected area of contact that is estimated from the depth of penetration, whereas in microindentation this area is obtained by measuring the indent diagonals resulted from the indent shape impressed on the specimen (Fischer-Cripps, 2011). Therefore, nanoindentation provides

Fig. 8. Variation of the indentation hardness and indentation modulus, for specimens of chitosan blended with plasticizers – a) and b); and blended with ceramics – c) and d).

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the very localized hardness of the material surface whereas micro-indentation gives the average hardness over a large area, thus for the same specimen the former provides results that are higher in magnitude when compared to the latter, according to the literature (Fischer-Cripps, 2011).

Overall, the main advantage of nanoindentation tests, comparing to microindentation, is to obtain the elastic and plastic properties of the materials simultaneously, in a single test. Additionally, homogenous areas can be selected for nanoindentation tests. As a result, this method helps to understand the potential of the different material compositions, if they are fully densified.

Since it is desirable to produce orthopedic bioabsorbable implants that are resistant to wear but not harder than bone, the hardness values of the chitosan-based specimens should be compared with values asso-ciated to the bone (the ideal hardness). However, this is valid only if the same experimental conditions are ensured (e.g. maximum load, loading speed and holding time) (Broitman, 2017; Milman et al., 2011). In fact, each author uses his own experimental conditions, which does not facilitate the discussion of the direct comparisons between hardness results among different studies. Lin et al. (2010) performed nano-indentation tests in bone specimens collected in femoral heads from living donors using loading to a maximum force of 2 mN in 5 s, holding for a period of 5 s and releasing the load in 5 s. In these conditions, they obtained indentation hardness values ranging from 0.53 GPa to 0.63 GPa, which are higher values when comparing with the indentation hardness of 0.28 GPa obtained in this study.

4.3. SEM

Fig. 9 shows representative images of the surface topography of chitosan-based specimens blended with plasticizer and ceramic.

The SEM analyses show that the specimens are mostly dense and do not present significant pores. This observation is valid not only for these specific compositions but for all the specimens from the remaining compositions.

4.4. Microtomography

Tables 3 and 4 present the quantitative (percentage) evaluation of closed pores (non-connected cavities) that were detected in the chitosan- based specimens with plasticizers and ceramics, respectively. Addi-tionally, Table 4 indicates the percentage of ceramic material dispersed in the specimens.

Fig. 10 and Fig. 11 show images (frontal plane, x0z) from the interior of the 10% chitosan-based blends, obtained after applying the volume rendering reconstruction software (CTVox).

The preliminary tests reveal that the chitosan specimens produced by adding either glycerol or sorbitol are dense, showing a maximum porosity level of 1.20% (Table 3). Fig. 10 shows the inner structure of the specimens, revealing a continuous material with few black areas (pores).

The results of Table 4 show that, globally, the porosity increases with the amount of ceramic material added to the specimens and the granules specimens present higher porosity compared with the HA specimens. The initial percentage of ceramic dispersed in the specimens is only confirmed for the composition with HA-TCP granules. Larger black areas are perceptible in the granules image of Fig. 11c, confirming the higher level of porosity of these specimens when compared to the remaining. In this case, it was easier to identify the ceramic material added, which corresponds to a second grey threshold.

Although the results of Tables 3 and 4 are limited by the resolution of the technique and the operator conditionings, namely in the definition of the material threshold, the results indicate that ceramic-containing specimens have higher porosity, mainly the specimens produced with HA-TCP granules. This may be related with air retention during the dissolution stage, a consequence of the higher rotation speed employed to uniformly disperse the ceramic materials in the solution.

5. Conclusions

Chitosan has been widely used in the biomedical field especially due to its good biological properties. However, to make this material a strong candidate for the development of orthopedic bioabsorbable implants, it is necessary to improve its mechanical properties, to bring them closer to the necessary bone tissue requirements. With this purpose, blends of chitosan with plasticizers and ceramics were tested to improve the

Fig. 9. SEM images of chitosan specimens blended with a) 10% glycerol and b) 10% HA-TCP powder.

Table 3 Percentage of closed pores present in the two types of plasticized specimens.

Content (w/v) of plasticizer material in the specimen (%) Closed pores (%)

Glycerol 5 0.31 10 0.75 15 1.20

Sorbitol 5 0.21 10 0.63 15 0.53

Table 4 Percentage of closed pores and dispersion of ceramic material in the specimens produced by blending chitosan with ceramics.

Content (w/w) of ceramic material in the specimen (%)

Ceramic dispersed (%) Closed pores (%)

HA 5 0.34 0.36 10 0.07 1.63 15 0.13 1.57

HA-TCP powder 5 0.68 2.17 10 0.49 0.08 15 1.41 3.09

HA-TCP granules 5 4.50 1.65 10 11.08 2.99 15 15.73 6.90

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mechanical properties of 3D dense chitosan-based specimens, especially the stiffness, strength and hardness. Therefore, the goal of the current study is to report the development and optimization of such chitosan blends, and more important to select the best combinations on the basis of mechanical behavior assessment. The knowledge gained on the cur-rent study is crucial to allow further investigation concerning biological and degradation profiles of the selected compositions.

The mechanical performance of three different concentrations of glycerol and sorbitol was tested under flexure, and the hardness and elastic modulus were determined by nanoindentation. The results ob-tained show that the chitosan blended with 10% glycerol presented the best combination of all measured properties: flexural modulus, flexural strength, flexural strain, indentation hardness and indentation modulus. A set of mechanical properties was considered to select the specimens with the best mechanical results, instead of making such a selection only according to one specific mechanical property. This strategy ensures more confidence in the experimental results. Additionally, the selection of the two chitosan-based compositions was validated in the SEM and micro-CT analyses, also contributing to the confidence of the results.

The mechanical properties of the chitosan-based specimens were also assessed by adding different concentrations of HA, HA-TCP powder and HA-TCP granules to the 10% glycerol composition, thus continuing to take advantage of the plasticizer effect on chitosan. The goal was to further improve the mechanical properties of these specimens, espe-cially the flexural modulus, strength and hardness. According to the results of the flexural and nanoindentation tests, the composite material with the best mechanical behavior was obtained by adding 10% HA-TCP powder to the chitosan-glycerol mixture. In this case, the highest mean values for the flexural modulus and flexural strength were, respectively, 2679 MPa and 67.5 MPa, representing increases of 60% and 25%, relatively to the specimens containing 10% glycerol only. These results agree with the mechanical properties associated to the current bio-absorbable implants and approach bone values, which reveal the po-tential of this chitosan blends in the development of new chitosan-based product implants for orthopedics.

As expected, plasticizers increase chitosan’s ductility and strength, whereas ceramics its modulus, strength and hardness. As an example, when HA-TCP powder was added to the chitosan blends with 10% glycerol, the measured hardness was 28.2 kgf/mm2, an increase of 44%.

The specimens are mostly dense and homogeneous as confirmed by the SEM and micro-CT analyses. These characteristics are important to achieve implant candidate materials with an isotropic behavior, fundamental for their intended performance and final properties. These characteristics were achieved during the production process, due to an effective dissolution of chitosan and dispersion of ceramic particles, and the quick precipitation of the frozen solutions in NaOH, not allowing them to thaw. Another important property that was assured in this study was the development of chitosan-based structures that can be trans-formed into any geometry by machining. The ability to withstand the forces that result from the machining process is an important feature for the use of these materials as future custom fixation orthopedic devices. Nevertheless, to decrease the specimens’ mechanical behavior vari-ability caused by the manual machining, its substitution by CNC machining should be considered in the following studies. Future studies should also include larger sample sizes to increase its statistical soundness.

The specimens of chitosan blended with 10% glycerol (plasticizers group) and the specimens of chitosan blended with 10% HA-TCP powder (ceramics group) were the ones selected for further studies. Therefore, those specimens were assessed with negative results for cytotoxicity (i.e. specimens are not cytotoxic), following the ISO-10993 guidelines - see supplementary information. The mechanical results obtained for these two blends, as for the other conditions tested, support the design of future studies in the development and optimization of chitosan-based structures for orthopedic implant applications.

Acknowledgment

The authors thank Bioceramed S.A. for providing the materials used in the present study and Fundaç~ao para a Ciencia e Tecnologia (FCT) for

Fig. 10. Frontal (x0z) images of Micro-CT of specimens produced by blending chitosan with a) 10% glycerol and b) 10% sorbitol.

Fig. 11. Frontal (x0z) images of Micro-CT of specimens produced by blending chitosan with a) 10% HA, b) 10% HA-TCP powder and c) 10% HA-TCP granules.

L. Figueiredo et al.

Journal of the Mechanical Behavior of Biomedical Materials 103 (2020) 103572

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the PhD grant SFRH/BD/51949/2012. Funding received from FCT (UID/BIO/04565/2019), from Programa Operacional Regional de Lis-boa 2020 (Project N. 007317) and PAC (Project 016394) is also acknowledged.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jmbbm.2019.103572.

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