Journal of Biotechnology 182–183 (2014) 46–53
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Chitosan/-1,3-glucan/calcium phosphate ceramics composites—Novel cell scaffolds for bone tissue engineering application Agata Przekora a,∗ , Krzysztof Palka b , Grazyna Ginalska a a b
Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland Department of Materials Engineering, Lublin University of Technology, Nadbystrzycka 36, 20-618 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 7 February 2014 Received in revised form 22 April 2014 Accepted 28 April 2014 Available online 6 May 2014 Keywords: Biomaterials Biocompatibility Cell culture Mechanical properties Young’s modulus Compressive strength
a b s t r a c t Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation. The aim of this work was to fabricate novel tri-component scaffolds composed of chitosan, -1,3glucan, and bioceramics and to evaluate their basic structural, mechanical, and biological properties. It should be noted that we are the first who describe fabrication and characterization of tri-component composites containing -1,3-glucan. Microstructure of novel composites was visualized by computed tomography scanning and SEM. Compressive strength and Young’s modulus of the composites were evaluated by compression testing. The biocompatibility was assessed in vitro by cytotoxicity, cell attachment and cell proliferation tests using human foetal osteoblast cell line. Our results demonstrated that novel composites possess good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are non-toxic, favourable to cell adhesion and promote cell proliferation. However, novel biomaterials revealed relatively low Young’s modulus values. Thus, we infer that fabricated novel composites are promising materials for bone tissue engineering application as cell scaffolds to fill small bone losses rather than as massive bone fillers exposed to mechanical load. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bone tissue engineering is focused on fabrication threedimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation (Chun et al., 2008; Kim et al., 2001; Prabaharan et al., 2007). The goal of tissue engineering is also to create biomaterials possessing optimal structural and mechanical properties similar to the bone tissue and adjusted to the area of implantation (e.g. cortical or trabecular bone). Material similarity to the bone tissue facilitates tissue ingrowth into implanted biomaterial and provides rapid bone regeneration (Hannink and Arts, 2011; Schliephake et al., 1991). Bone tissue consists of organic matrix and mineral part called hydroxyapatite (Liuyun et al., 2009). Thus, vast majority of
∗ Corresponding author. Tel.: +48817423676/+48817423633; fax: +48817423676. E-mail addresses: [email protected], [email protected] (A. Przekora). http://dx.doi.org/10.1016/j.jbiotec.2014.04.022 0168-1656/© 2014 Elsevier B.V. All rights reserved.
biocomposites for bone tissue engineering application are composed of calcium phosphates component such as hydroxyapatite (HAp), -tricalcium phosphate (-TCP), ␣-tricalcium phosphate (␣-TCP) or low-temperature calcium phosphate bone cements (CPCs) (Przekora et al., 2014) and organic component in the form of biodegradable biopolymer e.g. collagen–natural protein of bone extracellular matrix (ECM) (Kim et al., 2001), silk fibroin (Qi et al., 2014), alginate (Jin et al., 2012), amylopectin, chondroitin sulphate-natural component of cartilage (Venkatesan et al., 2012), carboxymethyl cellulose (Liuyun et al., 2009) or chitosan that is structurally similar to glycosaminoglycan (GAG) of bone ECM (Jin et al., 2012; Venkatesan et al., 2012). Because scaffolds should not only have good mechanical but also biological properties providing fast bone regeneration, more and more tri-component biocomposites are now fabricated in order to obtain biocompatible materials that accelerate bone healing. The aim of this work was to modify described in our previous report bi-component chitosan/bioceramics materials (Przekora and Ginalska, 2014; Przekora et al., 2012) by addition of -1,3glucan in order to fabricate novel biocompatible tri-component
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Table 1 Powder and liquid phase compositions of the investigated biocomposites. Biocomposite
Powder phase
Liquid phase
chit/HA
HAp (HA BIOCER, 80 wt.%) mix HAp/-TCP (HT BIOCER, 80 wt.%) HAp (HA BIOCER, 80 wt.%)
4.0 wt.% chitosan solution in 1.0 wt.% acetic acid 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid
chit/HT
chit/glu/HA
chit/glu/HT
mix HAp/-TCP (HT BIOCER, 80 wt.%)
4.0 wt.% chitosan solution in 1.0 wt.% acetic acid (chitosan final conc. 2.0 wt.%), 16.0 wt.% -1,3-glucan solution in distilled water (glucan final conc. 8.0 wt.%) 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid (chitosan final conc. 2.0 wt.%), 16.0 wt.% -1,3-glucan solution in distilled water (glucan final conc. 8.0 wt.%)
composite scaffolds. The -1,3-glucan is a non-toxic homopolymer of D-glucose possessing unique ability to form a firm, resilient gel when heated in an aqueous suspension to above 80 ◦ C (Miwa et al., 1994). The goal of this study was also to evaluate basic structural, mechanical, and biological properties of novel tri-component scaffolds. It should be noted that we are the first who describe fabrication and characterization of tri-component composites composed of chitosan, -1,3-glucan, and bioceramics. 2. Materials and methods 2.1. Biomaterials fabrication Two types of novel tri-component biomaterials marked as chitosan/-1,3-glucan/HA BIOCER (chit/glu/HA) and chitosan/1,3-glucan/HT BIOCER (chit/glu/HT) were fabricated by modification of chitosan/HA BIOCER (chit/HA) and chitosan/HT BIOCER (chit/HT) composites, respectively. Powder phase of chit/glu/HA and chit/glu/HT materials was composed of manufactured calcium phosphate bioceramics: HA BIOCER bioceramics (HAp granules, Ø 0.5–1.6 mm) and HT BIOCER bioceramics (mix of HAp/-TCP granules, Ø 0.5–1.6 mm), respectively (Chema Elektromet Rzeszow, Poland). Solutions of 4.0 wt.% chitosan in 1.0 wt.% acetic acid (POCH Gliwice, Poland) and 16.0 wt.% -1,3-glucan (Wako Pure Chemicals Industries, Japan) in distilled water were used as the liquid phases (Table 1). Applied high molecular weight chitosan (1174 kDa and 73% deacetylated) derived from krill shells (Euphausia superba) was kindly obtained from Sea Fisheries Research Institute in Gdynia (Poland). The first step of biocomposites fabrication was to mix both liquid phases, 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid with 16.0 wt.% -1,3-glucan solution in distilled water. The mixing ratio of liquid phases was 1:1, thus the final concentration of chitosan and glucan in prepared samples was 2.0 wt.% and 8.0 wt.%, respectively. Then the appropriate powder phase–HA BIOCER or HT BIOCER (80 wt.%) was added to prepared biphasic solution. Obtained homogenous paste was moulded into cylinder-shaped samples and heated for 20 min at 90 ◦ C in water bath. After samples cooling materials were neutralized in 1% NaOH solution (POCH Gliwice, Poland) to gel chitosan component of the composite, washed in distilled water and left to air dry for 48 h. Prepared tri-component biocomposite scaffolds in wet condition after soaking in physiological solution are presented in Fig. 1a and b. 2.2. Microstructure visualization The microstructure of novel composites was characterized using computed tomography (Skyscan 1174, Belgium). Scanning electron
Fig. 1. Images of fabricated scaffolds: composites in a wet state (a), elastic deformation of chit/glu/HA composite (b).
microscopy (SEM) was also applied to visualize surface of composite scaffolds (Zeiss ULTRA plus). 2.3. Compression test Composite samples measuring 8 mm in diameter and 15 mm in length was used in order to assess behaviour of the novel material during compression. Composites were applied in wet state after soaking in physiological solution to simulate actual procedure during implantation surgery (biomaterials are soaked in plasma or physiological fluid before implantation). Compression testing was conducted using Zwick Roell Z2.5 testing machine. Pre-load value of 1 N was applied with crosshead moving speed 10 mm/min, followed by basic load rate of 0.5 mm/min. The mechanical compression was carried out till specimen destruction. The obtained data allowed for the stress–strain characteristics and the Young’s modulus determination. The Young’s modulus (E), called also elastic modulus, was determined as the ratio of difference between stresses recorded for strain equal 0.25% and 0.05% () to difference between mentioned strain (ε). The compressive strength of materials was calculated as the ratio of maximum force to the initial cross-sectional area. After compression test, SEM analysis was performed to depict the mechanism of destruction. 2.4. Cell culture in vitro experiments 2.4.1. Materials and reagents for in vitro tests DMEM/Ham F12 culture medium without phenol red, G418 disulfate salt solution, penicillin-streptomycin solution, 0.25% trypsin-EDTA solution, bovine serum albumin, neutral red solution (NRU), cell counting kit-8 (WST-8), lactate dehydrogenase (LDH)
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cytotoxicity kit, Hoechst 33342, and Live/Dead Double Staining Kit were purchased from Sigma-Aldrich Chemicals. Foetal bovine serum (FBS) was supplied by PAA Laboratories and phosphate buffered saline (PBS) was from IMMUNIQ (Poland). Acetic acid and ethanol were purchased from POCH Gliwice (Poland). AlexaFluor635phalloidin fluorescent dye was supplied by Invitrogen. Normal human foetal osteoblast cell line (hFOB 1.19) was obtained from ATCC (American Type Culture Collection, England, UK) and cultured in a 1:1 mixture of DMEM/Ham F12 medium without phenol red supplemented with 10% FBS, 300 g/mL G418, 100 U/mL penicillin, 100 g/mL streptomycin and maintained at 34 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. 2.4.2. Indirect tests for quantitative cytotoxicity assessment Tested biocomposite samples were sterilized by ethylene oxide before all in vitro experiments. The cytotoxicity of novel tricomponent biomaterials was evaluated by indirect method using fluid extracts prepared according to ISO 10993-5 (2009). Preparation of biomaterials extracts was described earlier (Przekora et al., 2014). To assess cytotoxicity, hFOB 1.19 cells were seeded in flat bottom 96-multiwell plates in 100 L complete culture medium at a concentration of 1.5 × 104 cells per well. After 24-h incubation at 34 ◦ C the growth medium was gently discarded and 100 L of the appropriate extracts was added. Culture medium served as negative control of cytotoxicity. After 24 h of incubation, WST-8 and Neutral Red Uptake (NRU) tests were performed to evaluate cell viability. WST-8 test is a very sensitive tetrazolium salt-based method to assess cell metabolic activity via measurement of mitochondrial dehydrogenases activity. Because WST-8 test produces orange water-soluble formazan dye, the detection sensitivity is higher than in common MTT assay. WST-8 test was conducted using Cell Counting kit-8 in accordance with manufacturer procedure. NRU assay allows for cell number evaluation via measurement of neutral red dye incorporation into lysosomes of living cells. NRU test was carried out as described previously (Przekora et al., 2014). The results of WST-8 and NRU tests were expressed as the percentage of OD values obtained with the control cells. The cytotoxicity indirect tests were repeated in three separate experiments. 2.4.3. Direct-contact test for qualitative cytotoxicity assessment To assess cytotoxicity by direct-contact method, hFOB cells were cultured directly on chit/glu/HA and chit/glu/HT biocomposites for 24 h and observed under confocal microscope after live/dead double fluorescent staining. Before the experiment, cylinder-shaped biocomposites samples were cut into discs approximately 1 mm thick and 8 mm in diameter. Prepared specimens were placed in 48-multiwell plate and preincubated overnight in a complete culture medium at 34 ◦ C. Then, hFOB cells were seeded directly on the composites samples in 500 L of the complete culture medium at a concentration of 5 × 104 cells/disc. After 24 h of culture at 34 ◦ C, discs were gently rinsed with PBS buffer and cells were stained using Live/Dead Double Staining Kit in accordance with manufacturer procedure. The kit contains cell membrane permeable calcein-AM dye and the nuclei staining propidium iodide (PI) dye. Only viable cells have active intracellular esterases which remove the acetomethoxy group (AM) from calcein and induce strong green fluorescence. Whereas, PI dye cannot pass through a viable cell membrane and stains nucleic acids of only dead cells giving red fluorescence. Stained live (green) and dead (red) cells were observed under confocal microscope (Olympus Fluoview equipped with FV1000). 2.4.4. Quantitative cell attachment assay LDH total test was performed to assess cell adhesion into tested tri-component biocomposite surfaces. LDH total test evaluates total cell number via cytoplasmic LDH activity measurement after
cell lysis. Before the experiment, composites discs were prepared as described above (Section 2.4.3). Then, hFOB 1.19 cells were seeded directly on biocomposites samples in 500 L of the complete culture medium at a concentration of 5 × 104 cells/disc and incubated for 3 h at 34 ◦ C. Next, the culture medium was removed and biocomposite discs were transferred with great care to the corresponding wells in new 48-multiwell plate, so cells growing on the polystyrene surface did not interfere with the test. Total cell number attached to the tri-component composites surfaces was determined using the absorbance value of calibration curve that was prepared applying known concentration of hFOB 1.19 cells. LDH total assay was conducted using LDH cytotoxicity kit in accordance with manufacturer procedure. LDH total assay was repeated in triplicate. 2.4.5. Qualitative cell proliferation evaluation Cell proliferation on the surface of chit/glu/HA and chit/glu/HT biocomposites was evaluated qualitative by confocal microscope observation after fluorescent staining of osteoblast cytoskeleton and nuclei. Before the experiment, composite discs were prepared as described above (section 2.4.3). Then, hFOB 1.19 cells were seeded directly on biocomposites samples in 500 L of the complete culture medium at a concentration of 2.5 × 104 cells/disc. Osteoblasts were cultured on the tested biomaterials at 34 ◦ C for 9 days. Increase in cell number growing on the composites surfaces was qualitative assessed every third day using double staining with AlexaFluor635phalloidin and Hoechst 33342 fluorescent dyes. Phalloidin is a toxin with high affinity for F-actin isolated from the deadly Amanita phalloides mushroom. Fluorescent phallotoxin conjugates are commonly used in imaging applications to selectively label F-actin of cytoskeleton. AlexaFluor635 dye conjugated to phalloidin provides red fluorescence of cytoskeleton. Hoechst 33342 is cell permeable nucleic acids stain that gives blue fluorescence of nuclei. Before fluorescent staining, hFOB 1.19 cells were fixed in 3.7% formaldehyde solution for 10 min at room temperature and extracted with acetone for 5 min at ≤−20 ◦ C. Then, cytoskeleton and nuclei of hFOB cells were stained for 20 min at room temperature using 300 L per sample staining solution composed of 2 units of phallotoxin conjugate (AlexaFluor635) and 1 g/mL Hoechst 33342. In order to reduce nonspecific background staining, 1% of bovine serum albumin was added to the staining solution. Stained cells growing on the surface of chit/glu/HA and chit/glu/HT biocomposites were observed under confocal microscope. 2.5. Statistical analysis The results were expressed as mean values ± standard deviation (SD). The Kolmogorov–Smirnov test was applied to demonstrate normal distribution of variables, then unpaired t-test was performed to assess statistical differences among groups by two population comparison. Statistical significance was considered at a probability P < 0.05 (GraphPad Prism 5, Version 5.03 Software). 3. Results and discussion 3.1. Microstructure visualization Microstructure analysis revealed that both materials are characterized by low porosity mostly in the spaces between the granules (open porosity). The chit/glu/HA microstructure is characterized by a smooth shape of the ceramic granules that are coated by the thin layer of the chitosan/glucan matrix (Fig. 2a). In the case of chit/glu/HT composite, granules have an irregular shape with tendency to crumbling (Fig. 2b). This may affect chit/glu/HT behaviour under load. Computed tomography demonstrated that both materials shows a uniform distribution of ceramic granules. Each particle
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Fig. 2. Microstructure visualization: SEM images of chit/glu/HA (a) and chit/glu/HT (b); cross section images of chit/glu/HA (c) and chit/glu/HT (d) obtained with computed tomography scanning.
is surrounded by a thin layer of matrix that fills the empty spaces between the granules. Voids (porosity) are visible in the form of dark (black) areas in the interparticulate spaces (Fig. 2c and d).
and test method (e.g. measurements the time of ultrasonic wave transition or mechanical test). We believed that applied in this study mechanical test gives reliable data and is appropriate method for testing multiphasic materials.
3.2. Compression test 3.2.1. Young’s modulus assessment In the first step of compression there was observed linear variation as a function of the strain (Fig. 3). This part of the curve was used to determine the Young’s modulus. Elastic modulus were as follows: chit/glu/HA: E = 0.25 MPa ± 0.03 MPa, chit/glu/HT: E = 0.27 MPa ± 0.11 MPa. Obtained Young’s modulus were relatively low compared to the human trabecular bone Young’s modulus (E in the range 0.18–0.33 GPa). We suggest that the strain was taken by elastic chitosan/glucan matrix whereas the ceramic granules were only slightly deformed. Moreover, compression test was conducted using composites in a wet state. We are convinced that dry composites, that are not so elastic, would reveal significantly higher Young’s modulus values. It should be also noted that elastic modulus can vary somewhat due to differences in sample composition
Fig. 3. Stress–strain curves for tested materials obtained by compression testing.
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Fig. 4. SEM images of composite destruction under compression load: chit/glu/HA (a and b), chit/glu/HT (c and d).
3.2.2. Compressive strength evaluation Considering obtained stress–strain curves for tested materials, it can be observed that the strain up to 20% and up to 40% caused linear elastic deformation of chit/glu/HT and chit/glu/HA respectively. Exceeding these values led to fluctuations due to the destruction of the matrix-ceramic particles connection and the chitosan/glucan matrix itself (Fig. 4a–d). The compressive strength values of tested composites were estimated as follows: chit/glu/HA: 0.26 MPa ± 0.059 MPa, chit/glu/HT: 0.19 MPa ± 0.045 MPa. The moment of decohesion is often dependent on the volume ratio, type and shape of the ceramic powder phase. As both composites had the same volume of ceramic phase (80%), it may be concluded that the strength of novel composites was determined by the type and shape of the ceramic particles. The HT BIOCER particles had sharp edges what resulted in greater concentration of stress and more rapid damage of granules–matrix connections. The HA BIOCER granules had rounded shape what favoured the homogenization of the stress and obtainment of higher strength and greater elastic deformations. Adhesive-cohesive type of the matrix destruction and separation of the granules–matrix connection was observed for both materials. This type of destruction is specific for composites reinforced by particles, where the matrix is characterized by low strength.
tested extracts slightly, but statistically significant, enhanced cell proliferation and cell viability was approx. 120% compared to the control (Fig. 5). Confocal microscope observation confirmed indirect tests and revealed clusters of living green fluorescent hFOB cells on the surface of both biocomposites and only occasional dead cells, that emitted red fluorescence (Fig. 6a and b). It is worth to notice that during microscope observation Nomarski contrast was additionally applied in order to expose composites structure. Furthermore, the images obtained with confocal microscope showed different shape of osteoblast depending on what type of tri-component biocomposite the cells were cultured. Osteoblast cells growing on the chit/glu/HA composite were flattened and had lengthened shape what proves their good adhesion to the composite surface 24 h after
3.3. Cytotoxicity determination Both applied quantitative cytotoxicity tests, WST-8 and NRU, clearly demonstrated that chit/glu/HA and chit/glu/HT composites did not reduce cell viability. WST-8 test revealed that composite extracts were non-toxic and hFOB cell viability was approx. 100% compared to the control. Moreover, NRU assay demonstrated that
Fig. 5. Cytotoxicity evaluation of composite extracts by means of WST-8 and NRU tests. The results were expressed as the percentage of OD values obtained with the control cells. The values for the control cells and the extract-treated cells were significantly different according to unpaired t-test (* P < 0.05).
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Fig. 7. Total cell number attached to the composites surfaces 3 h after cell seeding (LDH total test). The amounts of hFOB cells attached to the chit/glu/HA surface and cells attached to the chit/glu/HT surface were significantly different according to unpaired t-test (* P < 0.05).
cells attached to the surface of chit/glu/HA and chit/glu/HT, respectively. Thus, it may be suggested that HA BIOCER granules have slightly better biocompatibility than HT BIOCER ones. It is in agreement with our previous report that showed better hFOB cells adhesion and growth on the surface of bi-component chit/HA composite than on the chit/HT sample (Przekora and Ginalska, 2014). However, it should be noted that cell adhesion to the chit/HA and chit/glu/HA is only slightly better and chit/HT and chit/glu/HT composites still reveal good biocompatibility. Moreover, this study was conducted using only hFOB 1.19 cell line, other cell lines e.g. MG63, Saos-2, U-2 OS, MC3T3-E1 or primary osteoblast culture may behave different in contact with these biocomposites.
3.5. Cell proliferation assessment
Fig. 6. Qualitative cytotoxicity assessment: confocal microscope images of hFOB cells cultured on the surface of chit/glu/HA (a) and chit/glu/HT (b) after live/dead double staining, magn. 100×. Viable cells give green fluorescence, nuclei of dead cells emit red fluorescence. Composite structure was exposed by additionally applied Nomarski contrast. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cell seeding (Fig. 6a). Cells cultured on the chit/glu/HT sample did not reveal typical lengthened shape of hFOB 1.19 cells, however cells were not spherical and there were visible filopodia what indicates that osteoblasts were attached to the surface but less flattened when compared to the cells growing on the chit/glu/HA composite (Fig. 6b). This observation may suggest that chit/glu/HA surface is more favourable to cell adhesion than chit/glu/HT surface. 3.4. Cell attachment evaluation In order to verify above-mentioned supposition (Section 3.3), LDH total test was carried out to quantitative determine the total number of attached hFOB cells 3 h after cell seeding. LDH total test confirmed confocal microscope observation. The results showed good cell attachment to both composite surfaces. However, the experiment revealed that chit/glu/HA surface is statistically significant more favourable to cell adhesion when compared to the surface of chit/glu/HT composite (Fig. 7).Three hours after cell seeding, there were 2.4 × 104 ± 0.4 × 104 and 1.7 × 104 ± 0.3 × 104 hFOB
Cell proliferation was assessed by confocal microscope observation using fluorescent staining of osteoblast cytoskeleton and nuclei after 3, 6, and 9 days of culture. Microscopic observation showed good osteoblast growth and proliferation on both novel composites. It is worth emphasizing that composite structure was exposed by additionally applied Nomarski contrast and autofluorescence of chitosan component of biomaterials. It should be also noted that it was very hard to obtain very good quality images because of irregular shape of the samples and their porosity. Obtained confocal microscopy images showed slightly more hFOB cells on the surface of chit/glu/HA than on the chit/glu/HT composite 3 days after cell inoculation (Fig. 8a and b). Visualized cells did not have well extensive cytoskeleton and filopodia but showed typical lengthened shape. There were also well visible blue fluorescent nuclei. 6 days after cell seeding, there were similar number of osteoblast cells growing on both biocomposites and there was visible slightly more extensive cytoskeleton (Fig. 8c and d). Whereas, 9 days after cell seeding, both biocomposites surfaces were covered by multilayer of hFOB cells, which revealed extensive network of cytoskeletal filaments and numerous filopodia (Fig. 8e and f). In the case of hFOB cells growing on the chit/glu/HT sample, cell density was so high that observed cytoskeletal filaments of one cell constituted a part of cytoskeletal structure of another cell, so nuclei were not well visible on the image (Fig. 8f). HFOB cells observed on the surface of both composites were well spread, flattened and generated large filamentous structure of the cytoskeleton what indicates that novel composites surfaces are very favourable to cell adhesion, proliferation, and growth. Furthermore, cell number increased with time during the in vitro culture suggesting that novel tri-component scaffolds do not retard cell proliferation.
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Fig. 8. Proliferation evaluation: confocal microscope images of hFOB cells growing on the surface of chit/glu/HA (a, c and e) and chit/glu/HT (b, d and f) composites 3 days (a and b), 6 days (c and d), and 9 days (e and f) after cell seeding, magn. 200×. Cytoskeletal filaments were stained with AlexaFluor635phalloidin (red fluorescence) and the nuclei were stained with Hoechst 33342 (blue fluorescence). Composite structure was exposed by additionally applied Nomarski contrast and blue autofluorescence of chitosan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4. Conclusions
References
Our study led to fabrication of novel chit/glu/HA and chit/glu/HT composites that have good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are nontoxic, and favourable to cell adhesion. Fabricated scaffolds possess total biocompatibility, promote cell growth and proliferation what is very important issue in rapid bone regeneration. Moreover, both tested materials show very large elastic deformations what provides very good adjustment to the implantation area (bone loss or defect) without exerting pressure. However, exceeding the “border” strain (20% for chit/glu/HT and 40% for chit/glu/HA) leads to the destruction of the matrix (loss of coherence) and the materials permanently deform. Therefore, it is necessary to select the right implantation area and to protect properly the site of trauma. Based on the obtained results, we infer that novel composites are promising materials for bone tissue engineering applications as cell scaffolds to fill small bone losses and to accelerate bone regeneration and new bone formation rather than as massive bone fillers exposed to mechanical load.
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Acknowledgments Financial assistance was provided by DS MNd 2 project. The paper was developed using the equipment purchased within the agreement No. PORPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007-2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion. The authors would like to thank Tomasz Piersiak from Department of Biochemistry and Biotechnology, Medical University of Lublin, Poland, for help with confocal microscope analysis.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Chitosan/-1,3-glucan/calcium phosphate ceramics composites—Novel cell scaffolds for bone tissue engineering application Agata Przekora a,∗ , Krzysztof Palka b , Grazyna Ginalska a a b
Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland Department of Materials Engineering, Lublin University of Technology, Nadbystrzycka 36, 20-618 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 7 February 2014 Received in revised form 22 April 2014 Accepted 28 April 2014 Available online 6 May 2014 Keywords: Biomaterials Biocompatibility Cell culture Mechanical properties Young’s modulus Compressive strength
a b s t r a c t Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation. The aim of this work was to fabricate novel tri-component scaffolds composed of chitosan, -1,3glucan, and bioceramics and to evaluate their basic structural, mechanical, and biological properties. It should be noted that we are the first who describe fabrication and characterization of tri-component composites containing -1,3-glucan. Microstructure of novel composites was visualized by computed tomography scanning and SEM. Compressive strength and Young’s modulus of the composites were evaluated by compression testing. The biocompatibility was assessed in vitro by cytotoxicity, cell attachment and cell proliferation tests using human foetal osteoblast cell line. Our results demonstrated that novel composites possess good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are non-toxic, favourable to cell adhesion and promote cell proliferation. However, novel biomaterials revealed relatively low Young’s modulus values. Thus, we infer that fabricated novel composites are promising materials for bone tissue engineering application as cell scaffolds to fill small bone losses rather than as massive bone fillers exposed to mechanical load. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bone tissue engineering is focused on fabrication threedimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation (Chun et al., 2008; Kim et al., 2001; Prabaharan et al., 2007). The goal of tissue engineering is also to create biomaterials possessing optimal structural and mechanical properties similar to the bone tissue and adjusted to the area of implantation (e.g. cortical or trabecular bone). Material similarity to the bone tissue facilitates tissue ingrowth into implanted biomaterial and provides rapid bone regeneration (Hannink and Arts, 2011; Schliephake et al., 1991). Bone tissue consists of organic matrix and mineral part called hydroxyapatite (Liuyun et al., 2009). Thus, vast majority of
∗ Corresponding author. Tel.: +48817423676/+48817423633; fax: +48817423676. E-mail addresses: [email protected], [email protected] (A. Przekora). http://dx.doi.org/10.1016/j.jbiotec.2014.04.022 0168-1656/© 2014 Elsevier B.V. All rights reserved.
biocomposites for bone tissue engineering application are composed of calcium phosphates component such as hydroxyapatite (HAp), -tricalcium phosphate (-TCP), ␣-tricalcium phosphate (␣-TCP) or low-temperature calcium phosphate bone cements (CPCs) (Przekora et al., 2014) and organic component in the form of biodegradable biopolymer e.g. collagen–natural protein of bone extracellular matrix (ECM) (Kim et al., 2001), silk fibroin (Qi et al., 2014), alginate (Jin et al., 2012), amylopectin, chondroitin sulphate-natural component of cartilage (Venkatesan et al., 2012), carboxymethyl cellulose (Liuyun et al., 2009) or chitosan that is structurally similar to glycosaminoglycan (GAG) of bone ECM (Jin et al., 2012; Venkatesan et al., 2012). Because scaffolds should not only have good mechanical but also biological properties providing fast bone regeneration, more and more tri-component biocomposites are now fabricated in order to obtain biocompatible materials that accelerate bone healing. The aim of this work was to modify described in our previous report bi-component chitosan/bioceramics materials (Przekora and Ginalska, 2014; Przekora et al., 2012) by addition of -1,3glucan in order to fabricate novel biocompatible tri-component
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Table 1 Powder and liquid phase compositions of the investigated biocomposites. Biocomposite
Powder phase
Liquid phase
chit/HA
HAp (HA BIOCER, 80 wt.%) mix HAp/-TCP (HT BIOCER, 80 wt.%) HAp (HA BIOCER, 80 wt.%)
4.0 wt.% chitosan solution in 1.0 wt.% acetic acid 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid
chit/HT
chit/glu/HA
chit/glu/HT
mix HAp/-TCP (HT BIOCER, 80 wt.%)
4.0 wt.% chitosan solution in 1.0 wt.% acetic acid (chitosan final conc. 2.0 wt.%), 16.0 wt.% -1,3-glucan solution in distilled water (glucan final conc. 8.0 wt.%) 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid (chitosan final conc. 2.0 wt.%), 16.0 wt.% -1,3-glucan solution in distilled water (glucan final conc. 8.0 wt.%)
composite scaffolds. The -1,3-glucan is a non-toxic homopolymer of D-glucose possessing unique ability to form a firm, resilient gel when heated in an aqueous suspension to above 80 ◦ C (Miwa et al., 1994). The goal of this study was also to evaluate basic structural, mechanical, and biological properties of novel tri-component scaffolds. It should be noted that we are the first who describe fabrication and characterization of tri-component composites composed of chitosan, -1,3-glucan, and bioceramics. 2. Materials and methods 2.1. Biomaterials fabrication Two types of novel tri-component biomaterials marked as chitosan/-1,3-glucan/HA BIOCER (chit/glu/HA) and chitosan/1,3-glucan/HT BIOCER (chit/glu/HT) were fabricated by modification of chitosan/HA BIOCER (chit/HA) and chitosan/HT BIOCER (chit/HT) composites, respectively. Powder phase of chit/glu/HA and chit/glu/HT materials was composed of manufactured calcium phosphate bioceramics: HA BIOCER bioceramics (HAp granules, Ø 0.5–1.6 mm) and HT BIOCER bioceramics (mix of HAp/-TCP granules, Ø 0.5–1.6 mm), respectively (Chema Elektromet Rzeszow, Poland). Solutions of 4.0 wt.% chitosan in 1.0 wt.% acetic acid (POCH Gliwice, Poland) and 16.0 wt.% -1,3-glucan (Wako Pure Chemicals Industries, Japan) in distilled water were used as the liquid phases (Table 1). Applied high molecular weight chitosan (1174 kDa and 73% deacetylated) derived from krill shells (Euphausia superba) was kindly obtained from Sea Fisheries Research Institute in Gdynia (Poland). The first step of biocomposites fabrication was to mix both liquid phases, 4.0 wt.% chitosan solution in 1.0 wt.% acetic acid with 16.0 wt.% -1,3-glucan solution in distilled water. The mixing ratio of liquid phases was 1:1, thus the final concentration of chitosan and glucan in prepared samples was 2.0 wt.% and 8.0 wt.%, respectively. Then the appropriate powder phase–HA BIOCER or HT BIOCER (80 wt.%) was added to prepared biphasic solution. Obtained homogenous paste was moulded into cylinder-shaped samples and heated for 20 min at 90 ◦ C in water bath. After samples cooling materials were neutralized in 1% NaOH solution (POCH Gliwice, Poland) to gel chitosan component of the composite, washed in distilled water and left to air dry for 48 h. Prepared tri-component biocomposite scaffolds in wet condition after soaking in physiological solution are presented in Fig. 1a and b. 2.2. Microstructure visualization The microstructure of novel composites was characterized using computed tomography (Skyscan 1174, Belgium). Scanning electron
Fig. 1. Images of fabricated scaffolds: composites in a wet state (a), elastic deformation of chit/glu/HA composite (b).
microscopy (SEM) was also applied to visualize surface of composite scaffolds (Zeiss ULTRA plus). 2.3. Compression test Composite samples measuring 8 mm in diameter and 15 mm in length was used in order to assess behaviour of the novel material during compression. Composites were applied in wet state after soaking in physiological solution to simulate actual procedure during implantation surgery (biomaterials are soaked in plasma or physiological fluid before implantation). Compression testing was conducted using Zwick Roell Z2.5 testing machine. Pre-load value of 1 N was applied with crosshead moving speed 10 mm/min, followed by basic load rate of 0.5 mm/min. The mechanical compression was carried out till specimen destruction. The obtained data allowed for the stress–strain characteristics and the Young’s modulus determination. The Young’s modulus (E), called also elastic modulus, was determined as the ratio of difference between stresses recorded for strain equal 0.25% and 0.05% () to difference between mentioned strain (ε). The compressive strength of materials was calculated as the ratio of maximum force to the initial cross-sectional area. After compression test, SEM analysis was performed to depict the mechanism of destruction. 2.4. Cell culture in vitro experiments 2.4.1. Materials and reagents for in vitro tests DMEM/Ham F12 culture medium without phenol red, G418 disulfate salt solution, penicillin-streptomycin solution, 0.25% trypsin-EDTA solution, bovine serum albumin, neutral red solution (NRU), cell counting kit-8 (WST-8), lactate dehydrogenase (LDH)
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cytotoxicity kit, Hoechst 33342, and Live/Dead Double Staining Kit were purchased from Sigma-Aldrich Chemicals. Foetal bovine serum (FBS) was supplied by PAA Laboratories and phosphate buffered saline (PBS) was from IMMUNIQ (Poland). Acetic acid and ethanol were purchased from POCH Gliwice (Poland). AlexaFluor635phalloidin fluorescent dye was supplied by Invitrogen. Normal human foetal osteoblast cell line (hFOB 1.19) was obtained from ATCC (American Type Culture Collection, England, UK) and cultured in a 1:1 mixture of DMEM/Ham F12 medium without phenol red supplemented with 10% FBS, 300 g/mL G418, 100 U/mL penicillin, 100 g/mL streptomycin and maintained at 34 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. 2.4.2. Indirect tests for quantitative cytotoxicity assessment Tested biocomposite samples were sterilized by ethylene oxide before all in vitro experiments. The cytotoxicity of novel tricomponent biomaterials was evaluated by indirect method using fluid extracts prepared according to ISO 10993-5 (2009). Preparation of biomaterials extracts was described earlier (Przekora et al., 2014). To assess cytotoxicity, hFOB 1.19 cells were seeded in flat bottom 96-multiwell plates in 100 L complete culture medium at a concentration of 1.5 × 104 cells per well. After 24-h incubation at 34 ◦ C the growth medium was gently discarded and 100 L of the appropriate extracts was added. Culture medium served as negative control of cytotoxicity. After 24 h of incubation, WST-8 and Neutral Red Uptake (NRU) tests were performed to evaluate cell viability. WST-8 test is a very sensitive tetrazolium salt-based method to assess cell metabolic activity via measurement of mitochondrial dehydrogenases activity. Because WST-8 test produces orange water-soluble formazan dye, the detection sensitivity is higher than in common MTT assay. WST-8 test was conducted using Cell Counting kit-8 in accordance with manufacturer procedure. NRU assay allows for cell number evaluation via measurement of neutral red dye incorporation into lysosomes of living cells. NRU test was carried out as described previously (Przekora et al., 2014). The results of WST-8 and NRU tests were expressed as the percentage of OD values obtained with the control cells. The cytotoxicity indirect tests were repeated in three separate experiments. 2.4.3. Direct-contact test for qualitative cytotoxicity assessment To assess cytotoxicity by direct-contact method, hFOB cells were cultured directly on chit/glu/HA and chit/glu/HT biocomposites for 24 h and observed under confocal microscope after live/dead double fluorescent staining. Before the experiment, cylinder-shaped biocomposites samples were cut into discs approximately 1 mm thick and 8 mm in diameter. Prepared specimens were placed in 48-multiwell plate and preincubated overnight in a complete culture medium at 34 ◦ C. Then, hFOB cells were seeded directly on the composites samples in 500 L of the complete culture medium at a concentration of 5 × 104 cells/disc. After 24 h of culture at 34 ◦ C, discs were gently rinsed with PBS buffer and cells were stained using Live/Dead Double Staining Kit in accordance with manufacturer procedure. The kit contains cell membrane permeable calcein-AM dye and the nuclei staining propidium iodide (PI) dye. Only viable cells have active intracellular esterases which remove the acetomethoxy group (AM) from calcein and induce strong green fluorescence. Whereas, PI dye cannot pass through a viable cell membrane and stains nucleic acids of only dead cells giving red fluorescence. Stained live (green) and dead (red) cells were observed under confocal microscope (Olympus Fluoview equipped with FV1000). 2.4.4. Quantitative cell attachment assay LDH total test was performed to assess cell adhesion into tested tri-component biocomposite surfaces. LDH total test evaluates total cell number via cytoplasmic LDH activity measurement after
cell lysis. Before the experiment, composites discs were prepared as described above (Section 2.4.3). Then, hFOB 1.19 cells were seeded directly on biocomposites samples in 500 L of the complete culture medium at a concentration of 5 × 104 cells/disc and incubated for 3 h at 34 ◦ C. Next, the culture medium was removed and biocomposite discs were transferred with great care to the corresponding wells in new 48-multiwell plate, so cells growing on the polystyrene surface did not interfere with the test. Total cell number attached to the tri-component composites surfaces was determined using the absorbance value of calibration curve that was prepared applying known concentration of hFOB 1.19 cells. LDH total assay was conducted using LDH cytotoxicity kit in accordance with manufacturer procedure. LDH total assay was repeated in triplicate. 2.4.5. Qualitative cell proliferation evaluation Cell proliferation on the surface of chit/glu/HA and chit/glu/HT biocomposites was evaluated qualitative by confocal microscope observation after fluorescent staining of osteoblast cytoskeleton and nuclei. Before the experiment, composite discs were prepared as described above (section 2.4.3). Then, hFOB 1.19 cells were seeded directly on biocomposites samples in 500 L of the complete culture medium at a concentration of 2.5 × 104 cells/disc. Osteoblasts were cultured on the tested biomaterials at 34 ◦ C for 9 days. Increase in cell number growing on the composites surfaces was qualitative assessed every third day using double staining with AlexaFluor635phalloidin and Hoechst 33342 fluorescent dyes. Phalloidin is a toxin with high affinity for F-actin isolated from the deadly Amanita phalloides mushroom. Fluorescent phallotoxin conjugates are commonly used in imaging applications to selectively label F-actin of cytoskeleton. AlexaFluor635 dye conjugated to phalloidin provides red fluorescence of cytoskeleton. Hoechst 33342 is cell permeable nucleic acids stain that gives blue fluorescence of nuclei. Before fluorescent staining, hFOB 1.19 cells were fixed in 3.7% formaldehyde solution for 10 min at room temperature and extracted with acetone for 5 min at ≤−20 ◦ C. Then, cytoskeleton and nuclei of hFOB cells were stained for 20 min at room temperature using 300 L per sample staining solution composed of 2 units of phallotoxin conjugate (AlexaFluor635) and 1 g/mL Hoechst 33342. In order to reduce nonspecific background staining, 1% of bovine serum albumin was added to the staining solution. Stained cells growing on the surface of chit/glu/HA and chit/glu/HT biocomposites were observed under confocal microscope. 2.5. Statistical analysis The results were expressed as mean values ± standard deviation (SD). The Kolmogorov–Smirnov test was applied to demonstrate normal distribution of variables, then unpaired t-test was performed to assess statistical differences among groups by two population comparison. Statistical significance was considered at a probability P < 0.05 (GraphPad Prism 5, Version 5.03 Software). 3. Results and discussion 3.1. Microstructure visualization Microstructure analysis revealed that both materials are characterized by low porosity mostly in the spaces between the granules (open porosity). The chit/glu/HA microstructure is characterized by a smooth shape of the ceramic granules that are coated by the thin layer of the chitosan/glucan matrix (Fig. 2a). In the case of chit/glu/HT composite, granules have an irregular shape with tendency to crumbling (Fig. 2b). This may affect chit/glu/HT behaviour under load. Computed tomography demonstrated that both materials shows a uniform distribution of ceramic granules. Each particle
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Fig. 2. Microstructure visualization: SEM images of chit/glu/HA (a) and chit/glu/HT (b); cross section images of chit/glu/HA (c) and chit/glu/HT (d) obtained with computed tomography scanning.
is surrounded by a thin layer of matrix that fills the empty spaces between the granules. Voids (porosity) are visible in the form of dark (black) areas in the interparticulate spaces (Fig. 2c and d).
and test method (e.g. measurements the time of ultrasonic wave transition or mechanical test). We believed that applied in this study mechanical test gives reliable data and is appropriate method for testing multiphasic materials.
3.2. Compression test 3.2.1. Young’s modulus assessment In the first step of compression there was observed linear variation as a function of the strain (Fig. 3). This part of the curve was used to determine the Young’s modulus. Elastic modulus were as follows: chit/glu/HA: E = 0.25 MPa ± 0.03 MPa, chit/glu/HT: E = 0.27 MPa ± 0.11 MPa. Obtained Young’s modulus were relatively low compared to the human trabecular bone Young’s modulus (E in the range 0.18–0.33 GPa). We suggest that the strain was taken by elastic chitosan/glucan matrix whereas the ceramic granules were only slightly deformed. Moreover, compression test was conducted using composites in a wet state. We are convinced that dry composites, that are not so elastic, would reveal significantly higher Young’s modulus values. It should be also noted that elastic modulus can vary somewhat due to differences in sample composition
Fig. 3. Stress–strain curves for tested materials obtained by compression testing.
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Fig. 4. SEM images of composite destruction under compression load: chit/glu/HA (a and b), chit/glu/HT (c and d).
3.2.2. Compressive strength evaluation Considering obtained stress–strain curves for tested materials, it can be observed that the strain up to 20% and up to 40% caused linear elastic deformation of chit/glu/HT and chit/glu/HA respectively. Exceeding these values led to fluctuations due to the destruction of the matrix-ceramic particles connection and the chitosan/glucan matrix itself (Fig. 4a–d). The compressive strength values of tested composites were estimated as follows: chit/glu/HA: 0.26 MPa ± 0.059 MPa, chit/glu/HT: 0.19 MPa ± 0.045 MPa. The moment of decohesion is often dependent on the volume ratio, type and shape of the ceramic powder phase. As both composites had the same volume of ceramic phase (80%), it may be concluded that the strength of novel composites was determined by the type and shape of the ceramic particles. The HT BIOCER particles had sharp edges what resulted in greater concentration of stress and more rapid damage of granules–matrix connections. The HA BIOCER granules had rounded shape what favoured the homogenization of the stress and obtainment of higher strength and greater elastic deformations. Adhesive-cohesive type of the matrix destruction and separation of the granules–matrix connection was observed for both materials. This type of destruction is specific for composites reinforced by particles, where the matrix is characterized by low strength.
tested extracts slightly, but statistically significant, enhanced cell proliferation and cell viability was approx. 120% compared to the control (Fig. 5). Confocal microscope observation confirmed indirect tests and revealed clusters of living green fluorescent hFOB cells on the surface of both biocomposites and only occasional dead cells, that emitted red fluorescence (Fig. 6a and b). It is worth to notice that during microscope observation Nomarski contrast was additionally applied in order to expose composites structure. Furthermore, the images obtained with confocal microscope showed different shape of osteoblast depending on what type of tri-component biocomposite the cells were cultured. Osteoblast cells growing on the chit/glu/HA composite were flattened and had lengthened shape what proves their good adhesion to the composite surface 24 h after
3.3. Cytotoxicity determination Both applied quantitative cytotoxicity tests, WST-8 and NRU, clearly demonstrated that chit/glu/HA and chit/glu/HT composites did not reduce cell viability. WST-8 test revealed that composite extracts were non-toxic and hFOB cell viability was approx. 100% compared to the control. Moreover, NRU assay demonstrated that
Fig. 5. Cytotoxicity evaluation of composite extracts by means of WST-8 and NRU tests. The results were expressed as the percentage of OD values obtained with the control cells. The values for the control cells and the extract-treated cells were significantly different according to unpaired t-test (* P < 0.05).
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Fig. 7. Total cell number attached to the composites surfaces 3 h after cell seeding (LDH total test). The amounts of hFOB cells attached to the chit/glu/HA surface and cells attached to the chit/glu/HT surface were significantly different according to unpaired t-test (* P < 0.05).
cells attached to the surface of chit/glu/HA and chit/glu/HT, respectively. Thus, it may be suggested that HA BIOCER granules have slightly better biocompatibility than HT BIOCER ones. It is in agreement with our previous report that showed better hFOB cells adhesion and growth on the surface of bi-component chit/HA composite than on the chit/HT sample (Przekora and Ginalska, 2014). However, it should be noted that cell adhesion to the chit/HA and chit/glu/HA is only slightly better and chit/HT and chit/glu/HT composites still reveal good biocompatibility. Moreover, this study was conducted using only hFOB 1.19 cell line, other cell lines e.g. MG63, Saos-2, U-2 OS, MC3T3-E1 or primary osteoblast culture may behave different in contact with these biocomposites.
3.5. Cell proliferation assessment
Fig. 6. Qualitative cytotoxicity assessment: confocal microscope images of hFOB cells cultured on the surface of chit/glu/HA (a) and chit/glu/HT (b) after live/dead double staining, magn. 100×. Viable cells give green fluorescence, nuclei of dead cells emit red fluorescence. Composite structure was exposed by additionally applied Nomarski contrast. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cell seeding (Fig. 6a). Cells cultured on the chit/glu/HT sample did not reveal typical lengthened shape of hFOB 1.19 cells, however cells were not spherical and there were visible filopodia what indicates that osteoblasts were attached to the surface but less flattened when compared to the cells growing on the chit/glu/HA composite (Fig. 6b). This observation may suggest that chit/glu/HA surface is more favourable to cell adhesion than chit/glu/HT surface. 3.4. Cell attachment evaluation In order to verify above-mentioned supposition (Section 3.3), LDH total test was carried out to quantitative determine the total number of attached hFOB cells 3 h after cell seeding. LDH total test confirmed confocal microscope observation. The results showed good cell attachment to both composite surfaces. However, the experiment revealed that chit/glu/HA surface is statistically significant more favourable to cell adhesion when compared to the surface of chit/glu/HT composite (Fig. 7).Three hours after cell seeding, there were 2.4 × 104 ± 0.4 × 104 and 1.7 × 104 ± 0.3 × 104 hFOB
Cell proliferation was assessed by confocal microscope observation using fluorescent staining of osteoblast cytoskeleton and nuclei after 3, 6, and 9 days of culture. Microscopic observation showed good osteoblast growth and proliferation on both novel composites. It is worth emphasizing that composite structure was exposed by additionally applied Nomarski contrast and autofluorescence of chitosan component of biomaterials. It should be also noted that it was very hard to obtain very good quality images because of irregular shape of the samples and their porosity. Obtained confocal microscopy images showed slightly more hFOB cells on the surface of chit/glu/HA than on the chit/glu/HT composite 3 days after cell inoculation (Fig. 8a and b). Visualized cells did not have well extensive cytoskeleton and filopodia but showed typical lengthened shape. There were also well visible blue fluorescent nuclei. 6 days after cell seeding, there were similar number of osteoblast cells growing on both biocomposites and there was visible slightly more extensive cytoskeleton (Fig. 8c and d). Whereas, 9 days after cell seeding, both biocomposites surfaces were covered by multilayer of hFOB cells, which revealed extensive network of cytoskeletal filaments and numerous filopodia (Fig. 8e and f). In the case of hFOB cells growing on the chit/glu/HT sample, cell density was so high that observed cytoskeletal filaments of one cell constituted a part of cytoskeletal structure of another cell, so nuclei were not well visible on the image (Fig. 8f). HFOB cells observed on the surface of both composites were well spread, flattened and generated large filamentous structure of the cytoskeleton what indicates that novel composites surfaces are very favourable to cell adhesion, proliferation, and growth. Furthermore, cell number increased with time during the in vitro culture suggesting that novel tri-component scaffolds do not retard cell proliferation.
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Fig. 8. Proliferation evaluation: confocal microscope images of hFOB cells growing on the surface of chit/glu/HA (a, c and e) and chit/glu/HT (b, d and f) composites 3 days (a and b), 6 days (c and d), and 9 days (e and f) after cell seeding, magn. 200×. Cytoskeletal filaments were stained with AlexaFluor635phalloidin (red fluorescence) and the nuclei were stained with Hoechst 33342 (blue fluorescence). Composite structure was exposed by additionally applied Nomarski contrast and blue autofluorescence of chitosan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4. Conclusions
References
Our study led to fabrication of novel chit/glu/HA and chit/glu/HT composites that have good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are nontoxic, and favourable to cell adhesion. Fabricated scaffolds possess total biocompatibility, promote cell growth and proliferation what is very important issue in rapid bone regeneration. Moreover, both tested materials show very large elastic deformations what provides very good adjustment to the implantation area (bone loss or defect) without exerting pressure. However, exceeding the “border” strain (20% for chit/glu/HT and 40% for chit/glu/HA) leads to the destruction of the matrix (loss of coherence) and the materials permanently deform. Therefore, it is necessary to select the right implantation area and to protect properly the site of trauma. Based on the obtained results, we infer that novel composites are promising materials for bone tissue engineering applications as cell scaffolds to fill small bone losses and to accelerate bone regeneration and new bone formation rather than as massive bone fillers exposed to mechanical load.
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Acknowledgments Financial assistance was provided by DS MNd 2 project. The paper was developed using the equipment purchased within the agreement No. PORPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007-2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion. The authors would like to thank Tomasz Piersiak from Department of Biochemistry and Biotechnology, Medical University of Lublin, Poland, for help with confocal microscope analysis.
