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. Author manuscript; available in PMC: 2020 Aug 18.
Published in final edited form as: Carbohydr Polym. 2018 Jul 19;199:426–436. doi: 10.1016/j.carbpol.2018.07.044

Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate

Janitha M Unagolla a, Turki E Alahmadi a, Ambalangodage C Jayasuriya a,b,*
PMCID: PMC7433903  NIHMSID: NIHMS1613760  PMID: 30143148

Abstract

Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after leaching out) scaffolds were measured according to the ASTM standards. The highest compressive strength of 27 MPa was reported on 10% CaP scaffolds while 20% CaP scaffolds showed the lowest. The increasing CaP content reduces the compressive strength of the scaffolds. The highest wet state compressive strength was reported on 0% CaP scaffolds with 0.36 MPs and 0.40 MPa at day 1 and day 3 respectively. In vitro cell culture studies showed good cell adhesion and cell proliferation on 10% CaP scaffolds.

Keywords: Chitosan, Carboxymethyl cellulose, Scaffold, Compressive strength, Cell proliferation

1. Introduction

In bone tissue engineering, biodegradable scaffolds play an important role, since it serves as a temporary skeleton for the lost bone or site of defective bone to support and stimulate the tissue growth and bone regeneration while the scaffold gradually degrades and replaced by the new bone tissue (Li, Ramay, Hauch, Xiao, & Zhang, 2005; Persidis, 1999; Petite et al., 2000; Vacanti & Langer, 1999). Bone is a heterogeneous composite which consists of inorganic phase, organic phase, and water in decreasing order. Inorganic phase mainly contains hydroxyapatite, and organic phase contains type I collagen, non-collagenous protein, and lipids (Boskey, 2013). Therefore, a combination of both ceramic and polymer provides a better-suited scaffold for bone tissue engineering. Moreover, the porous materials are most suited with bone tissue applications, as porosity allows osteogenesis into the pores, which strengthen the union between the host bone and the implant (Langer & Vacanti, 1993).

Biopolymers have been used over the decades as scaffolding materials for bone grafts due to its favorable biological properties, such as biodegradability and biocompatibility. Natural polymers have shown more favorable biological properties compared with the synthetic polymers. Synthetic polymers demonstrate lower cell adhesion due to their hydrophobic nature and lack of functional groups for further surface modification (Cai et al., 2009; Li et al., 2005). Chitosan, a natural cationic copolymer of β-[1→4]-linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose, is one of the most studied natural polymers in the field of tissue engineering due to its appealing intrinsic properties, such as biodegradability, bioactivity, non-toxicity. At physiological pH, chitosan is positively charged and hence susceptible to surface modifications and proper cell adhesions (Berger, Reist, Mayer, Felt, & Gurny, 2004). Also, the biodegradability of chitosan is due to the enzymes in the human body, such as lysozyme (Berger et al., 2004; Seda Tiǧli, Karakeçili, & Gumusderelioglu, 2007). The degradation rate of chitosan by lysozyme is inversely related to the molecular weight, and degree of crystallinity of the chitosan (Nwe, Furuike, & Tamura, 2009). Also, chitosan with a higher degree of deacetylation (DD) shows higher degradation (Thein-Han & Kitiyanant, 2007; VandeVord et al., 2002). Because of the N-acetylglucosamine repeating units, chitosan has some similarity to the glycosaminoglycan (GAG), the major component of the extracellular matrix of bone and cartilage (Khor & Lim, 2003), and thus binds to the growth factors (Muzzarelli et al., 1994). Also, chitosan scaffolds are osteoconductive, and enhance osteogenesis has been shown in both in vitro and in vivo conditions.

Carboxymethylcellulose (CMC), an anionic hydrophilic polymer derived from cellulose, is readily soluble in water due to the presence of carboxymethyl group. This negatively charged carboxymethyl group allows making complexation with positively charged polymers, such as chitosan (Gaihre & Jayasuriya, 2016; Kassem, Negm, Shukry, & El-Kalyouby, 2014; Rosca, Popa, Lisa, & Chitanu, 2005). Several authors have studied this strong polyelectrolyte complex due to its improved biological functions (Chen & Fan, 2007; Kawasaki, Nakaji-Hirabayashi, Masuyama, Fujita, & Kitano, 2016; Sainitya et al., 2015). The primary concern associated with the biopolymer scaffold is their low mechanical strength and shape retention problems due to excessive swelling. The polyelectrolyte complex formation reduces the swelling. Adding bioactive ceramics is one of the standard methods to increase the mechanical properties of the polymer scaffolds. Calcium phosphate (CaP) bioceramics, including hydroxyapatite (HA), are the favored bioceramic types used in bone tissue engineering due to its chemical and crystallographic similarities to the human bone. These CaP ceramics showed good osteoconductivity, and hence these are heavily used in numerous craniofacial and orthopedic procedures (Xu & Simon, 2005).

Number of attempts have been taken to improve the mechanical properties of the chitosan-based scaffolds by incorporating bioceramics, such as nano-HA (nHA) (Cai et al., 2009; Oliveira et al., 2006; Thein-Han & Misra, 2009), calcium phosphate (Sendemir-Urkmez & Jamison, 2007; Xu & Simon, 2005), and β-tricalcium phosphate (Yin et al., 2003). These studies indicated a significant improvement in the compressive strength of chitosan-based scaffolds. Jiang et al. (2008) reported 3.54 MPa of compressive strength in nHA/chitosan/CMC scaffolds. Also, it was reported that the highest tensile strength of 40 MPa in the dry state and 12 MPa in the wet state (Liuyun, Yubao, & Chengdong, 2009). Moreau and Xu (2009) reported that chitosan-calcium phosphate scaffolds with the flexural strength of 10 MPa in the dry state. All of these reported mechanical strength values are higher than the polymer scaffolds without bioceramics (Chung et al., 2002; Li et al., 2005). Wan, Wu, Cao, and Dalai (2008) reported that the incorporation of synthetic polymer, poly(caprolactone) into chitosan was increased the mechanical properties.

The main objective of this research work was to develop a natural polymer-based scaffold which can exhibit better mechanical properties in both dry and wet state. According to the published literature, the mechanical stability of the polymer-based scaffolds in the wet state is not in the acceptable level for using as bone grafts. Therefore, we investigated a method to enhance the mechanical stability by incorporating chitosan microparticles (MPs). Even though chitosan MPs were extensively studied as a drug delivery system, insufficient studies have been carried out to incorporate MPs into the scaffolds. In this study, commonly used salt leaching method was used to fabricate the porous scaffolds with chitosan MPs. CS solution and CMC solution were used as a binder to aggregate chitosan MPs. Also, Calcium phosphate (CaP) only scaffolds were previously studied in our lab, and low mechanical properties and poor stability in wet conditions were reported (Aryaei, Liu, Jayatissa, & Champa Jayasuriya, 2015). CaP was added as an osteoconductive material to enhance the cell attachment and proliferation. So, we examined the compressive strength and mechanical stability of Chitosan MPs based scaffold with the addition of the CaP and further, the cytotoxicity of the scaffolds were checked on murine pre-osteoblast cells.

2. Materials and methods

2.1. Materials

Low MW chitosan (MW: 50,000–190,000 kDa) with a DD of 85%, potassium chloride (ACS reagent 99%), sodium tripolyphosphate (TPP), acetic acid (99.7%), sodium carboxymethylcellulose (CMC - MW: 90,000), hexamethyldisilazane (HMDS), and cell proliferation reagent WST-1 (Roche diagnostic) were all purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA). Calcium phosphate tribasic (CaP) and calcium chloride dehydrate were obtained from Fisher Scientific (USA). Alpha minimum essential media (α-MEM), Fetal Bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco’s phosphate buffered saline (DPBS), and penicillin/streptomycin were purchased from Gibco, life technologies (Thermo Fisher Scientific, USA). Live/Dead cell viability/cytotoxicity kit was purchased from the Invitrogen (USA). 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer solution was purchased from electron microscopy sciences (Hatfield, PA, USA).

2.2. Fabrication of microparticles and scaffolds

2.2.1. Preparation of microparticles (MPs)

Simple coacervation method was used to prepare the chitosan MPs. 20 ml of 2% (w/v) chitosan solution was made using 2% (v/v) acetic acid and the solution was filtered through a nylon mesh with 52 μm pores to remove any undissolved chitosan. Then the solution was added dropwise to the 300 ml of 1% (w/v) TPP solution while under continuous stirring at 300 rpm. The mixture was kept for 4 h under stirring for proper crosslinking between chitosan and TPP. After 4 h, MPs were washed with deionized (DI) water and air-dried overnight.

2.2.2. Preparation of scaffold

Scaffolds were prepared using three different compositions of the CaP, 0%, 10%, and 20% (w/w). All the scaffolds contained varying amount of chitosan MPs according to the CaP content and fixed 20% (w/w) KCl salt to make pores after leaching out. The weight percentages were calculated according to the solid portion of the scaffold. All the solid portions were mixed well and then 2% (w/v) CMC solution and 2% (w/v) chitosan solution with 2% (w/v) CaCl2 were added to the mixture. For 1 g of solid portion, 1 ml of chitosan and 1 ml of CMC solutions were used. For 1 g of solid portion, 800 mg of MPs and 200 mg of KCL was added to make the 0%CaP scaffolds. 100 mg of CaP was added to 10% CaP scaffolds while reducing the MPs content to 700 mg, and 200 mg of CaP and 600 mg of MPs were added to make the 20% CaP scaffolds. After proper mixing, the mixture was added to the stainless steel cylindrical molds, and the molds were properly closed using two glass slides, as shown in the Fig. 1, and air-dried for two days. After drying, the scaffolds were immersed in PBS solution for three days to leach out the KCl salt. To distinguish the effect of MPs on the mechanical stability, CaP only scaffolds were prepared by using 80%(w/w) of CaP and 20%(w/w) of KCl and mixing with same amount of CS and CMC solutions.

Fig. 1.

Fig. 1.

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Schematic representation of preparation of scaffolds (A) 0% CaP, (B) 10% CaP, (C) 20% CaP.

2.3. Porosity of scaffolds

The porosity of scaffolds was calculated using the following equation with a measured volume of scaffolds.

Porosity=(Volume of PoresTotal Volume)×100%=(Total Volume−Volume of SolidsTotal Volume)×100% (1)

2.4. Testing of mechanical properties

Cylindrical test samples were used for all the mechanical tests according to the ASTM standard. Four types of scaffold conditions were used to understand the mechanical behavior under various conditions: (i) non-porous dry scaffolds before leaching out of the KCl salt, (ii) porous dry scaffolds after leaching out of the KCl salt, (iii) wet scaffolds at day 1- immersed in 200% (w/w) PBS for one day, and (iv) wet scaffolds at day 3 – immersed in 200% (w/w) PBS for three days. All the tests were performed using the ADMET eXpert 2600 series Universal testing machine, and constant rate of 0.01 mm/s was used. The compressive strength and compressive modulus of the scaffolds were calculated using the 0.2% offset method and the linear portion of the stress-strain curve respectively.

2.5. Swelling study

The swelling behavior of the scaffolds was investigated by immersing them in a vial which contained 2 ml of PBS (pH 7.4) at 37°C under continuous shaking (50 rpm) and the swelling ratio was calculated, at different time points ranging from 1 h to 3 days, according to the weight change. At each time point, wet scaffolds were taken out from the vial and placed on a blotting paper to absorb excess water and wet weight was immediately measured. The swelling ratio was calculated according to the formula given in Eq. (2).

Swelling ratio(SR)(%)=Ws−WiWi×100 (2)

Where Ws is the weight of the swollen scaffold, and Wi is the initial dry weight of the scaffold.

2.6. X-ray diffraction (XRD) analysis

X-ray diffractometer (Ultima III, Rigaku, Japan) employing mono-chromated Cu Kα radiation at 40 kV was used to investigate crystallinity and the phase content of the scaffolds on powder form. Data was collected from 5° to 80° 2θ values, with a step size of 0.03° and a counting time of 1 s per step.

2.7. Morphological analysis and energy dispersive X-ray (EDX) analysis for elemental mapping

The surface morphology of both porous and non-porous scaffolds was examined using the secondary electron detector of scanning electron microscopy (SEM) (FEI Quanta 3D FEG dual beam ESEM, USA) followed by copper sputter-coating at accelerating voltage of 5 keV. EDX analysis was done for elemental mapping of scaffolds using backscattered electron detector of SEM followed by gold-palladium sputter-coating at accelerating voltage of 20 keV.

2.8. In vitro cell culture

Murine osteoblast cell line (OB-6) was cultured in α-MEM containing 15% FBS, and 1% penicillin-streptomycin. The culture medium was replaced every 3 days and culture was maintained in a humidified incubator at 37°C with 5% CO2. After 80% of confluency, the cells were digested and sub cultured using 0.25% trypsin EDTA. Then, the cell suspension, which contains required number of cells, was seeded to the each scaffold that had been sterilized under UV light for 30 min. In this study, 20,000 cells per 1 ml were added to the each scaffold in 24 well plate. For proper adhesion of the cells, scaffolds were incubated for 2 h in complete α-MEM media at 37 °C and 5% CO2 before adding the cell suspension. Also, the required cell suspension was directly added to the top surface of the scaffold and then half of the media was added to barely cover the scaffold and incubated for 3 h for proper cell adhesion. After 3 h, remaining amount of α-MEM media was added. This procedure was carried out for all the cell culture studies.

2.8.1. Live and dead cell viability/cytotoxicity assay

Live/dead cell cytotoxicity assay was carried out for cell seeded scaffolds at three-time points; day 1, day 4, and day 7; according to the manufacturer protocol. The live cells were indicated by the green fluorescent of calcine and dead cells were indicated by the red fluorescent of ethidium homodimer-1. Top surfaces of the scaffolds were observed using cell imaging microscopy (Cytation 5, BioTek Inc, USA). Both live and dead cell images were processed and combined using Gen 3.03 software.

2.8.2. Cell proliferation

Cell proliferation study was conducted using WST-1 assay, a calorimetric assay for quantification of cell proliferation. In this assay, cell enzyme activity is directly proportional to the number of metabolically active cells, and it leads to the formation of formazan dye. All three types of scaffolds in triplicates were seeded with 20,000 cells per well as mentioned previously and WST-1 assay was carried out at four different time points; day 1, day 4, day 7, and day 14. After a predetermined time, medium was removed, and scaffolds were moved to new 24 well plate after washing with PBS. Then, new α-MEM medium was added and later 10%(v/v) WST-1 reagent was added to each well. Blank samples that contain only scaffolds with medium and WST-1 reagent were also prepared. Then, scaffolds with cells and blank scaffolds were incubated for 4 h at 37 °C and 5% CO2. After 4 h, 100 μl from each well was transferred to 96 well plate and the level of dye formed was measured using spectrophotometer (Molecular Devices, SpectraMax 190) at a wavelength of 440 nm. The optical density values from scaffolds without cells were subtracted from each experimental values as a background (Helen, Merry, Blaker, & Gough, 2007).

2.8.3. Morphology and attachment of cells

For morphological analysis of the cell attached scaffolds, the samples were washed with PBS after removing the medium and fixed with 0.6 ml of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer solution for 30 min at 4 °C. After thorough washing twice with PBS, scaffolds were dehydrated through a series of graded ethanol concentrations (30%, 50%, 70%, 90% and 100%) and finally dried in HMDS. After proper drying, scaffolds were coated with gold and palladium using a sputter coater (Cressington 108 Auto) and were then observed using an SEM (FEI Quanta 3D FEG dual beam ESEM, USA) operated at an accelerating voltage of 5 kV.

2.9. Statistical analysis

All data values in the graphs are presented as a mean ± standard error of the mean for each group of samples. For mechanical properties, seven samples were tested per group (0% CaP, 10% CaP, and 20% CaP), and for swelling and cell culture studies, three samples were tested per group. One-way Analysis of Variance (ANOVA) was performed using IBM SPSS statistical software (version 21, IBM Company, Armonk, NY, USA) to determine the statistical difference between three groups. For mechanical properties, non-porous and porous state statistical difference between the same group was also obtained in addition to the statistical difference between three groups. Post hoc Tukey’s test was performed to determine the statistical difference between the groups. All tests were conducted with 95% confidence intervals (p value < 0.05).

3. Results and discussion

3.1. Mechanical properties

The mechanical stability of the scaffolds were depend on the amount of the CaP in the each scaffolds. The CaP only scaffolds showed the very low mechanical stability and the data is given as a supplementary material. The estimated amounts of CaP, CS MPs, and KCl are given in the Table 1. The CaP only scaffolds were prepared by using the same liquid to solid ratio which has used to prepare MPs contained scaffolds. The stability of the CaP only scaffolds can be enhanced by changing the liquid to solid ratio. This study was done in our lab and reported compressive modulus values are significantly lower than the MPs contained scaffolds (Aryaei et al., 2015). Further, the CaP only scaffolds were not stable in wet conditions and therefore, not suitable for cell studies. So, CaP only scaffolds were not investigated further. From the preliminary studies, 40% CaP contained scaffolds were also disregarded due to the poor mechanical stability in wet conditions and inferior compressive strength (around 1 MPa, data is not shown). The highest CaP content for this studies was selected as 20% (w/w) according to the wet state mechanical stability from our preliminary studies.

Table 1.

Estimated solid constituents of each scaffolds.

Sample CaP (mg) MPs (mg) KCl (mg)
CaP only 160 0 40
20% CaP 33 100 33
10% CaP 17 117 33
0% CaP (MPs only) 113 33
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The CS MPs contained scaffolds showed very high compressive strength and compressive modulus at dry state. Porous scaffolds compressive strength were increased compare to the non-porous scaffolds except for 10% CaP contained scaffolds, but the difference is not statistically significant. The highest compressive strength of 26.86 MPa, was obtained by the 10% CaP contained scaffold. According to the Fig. 2, scaffolds without CaP showed less compressive strength (12.01 MPa), but further addition of the CaP reduces the mechanical strength as 20% CaP scaffolds showed lower compressive strength value (4.71 MPa) compare to MPs only scaffolds. The binding strength of the MPs was coming from the formation of complexes between CS, CMC, and Ca2+. The addition of the CaP powder initially increases the strength of the scaffold by reducing the pores and making the rigid structure as indicated by the high compressive strength value of 10% CaP scaffolds. However, further addition of the CaP may reduce the complexation and interlocking between CS and CMC and result in lower compressive strength (Liuyun, Yubao, Li, & Jianguo, 2008). The porous scaffolds mechanical strength was significantly increased in both 0% CaP and 20% CaP scaffolds, but in10% CaP, it was reduced without statistical significance. One possible reason for this observation is that the swollen state reduces the stress concentrations of the polymer chains and this may lead to reducing the brittleness of the structure. Also, the crystalline structure of KCl particles could be another reason for the low compressive strength in the non-porous state as it increases the brittleness of the scaffolds. However, further studies are required to study the mechanism of this behavior.

Fig. 2.

Fig. 2.

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Dry state mechanical properties of non-porous and porous scaffolds, (A) Compressive strength, (B) Compressive modulus, * significance p < 0.05.

The compressive strength of human cortical bone and cancellous bone are around 167–193 MPa, and 1.9–10 MPa respectively. So, according to the experimental values of all the scaffold types, both porous and non-porous dry state compressive strength values are above or within the range of cancellous bone. Also, the compressive modulus of cancellous bone was reported around 50–150 MPa (Eliaz & Metoki, 2017; Murugan & Ramakrishna, 2005). As shown in Fig. 3, the maximum compressive modulus was shown by the 10% CaP scaffold, which had an average compressive modulus of 427 (± 13.73) MPa at non-porous dry state. 0% CaP scaffold and 20% Cap scaffold showed 289 (± 16.48) MPa and 178 (± 13.73) MPa of compressive modulus respectively. The reduction of compressive modulus was observed for the non-porous scaffolds except 20% CaP scaffold, which had shown a slight increase. There is a significant difference between the groups, but the difference between the non-porous and porous groups are not statistically significant except for 10% CaP scaffold as shown in the Fig. 2. So, the average compressive modulus for all three sample groups is within the acceptable range of compressive modulus for human cancellous bone. Further, we developed these scaffolds to be used in craniofacial bone defects, such as defects on maxilla, mandible. According to the Misch, Qu, and Bidez (1999), the ultimate compressive strength (UCS) of the cancellous bone in the human mandible is ranged from 0.22 to 10.44 MPa with a mean value of 3.9 MPa. All above reported compressive strength values were calculated using 0.02% offset method at yield point, and hence all the compressive strength values are less than the scaffolds UCS value. Therefore, the dry state scaffolds showed superior compressive strength compare to the cancellous bone in human mandible.

Fig. 3.

Fig. 3.

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Wet state mechanical properties at day 1 and day 3, (A) Compressive strength, (B) Compressive modulus.

The actual condition of the bone is not a complete dry situation, and bone contains 10–20% of water according to its dry weight. Also, bone is always in contact with blood and body fluids. Therefore, mechanical properties of the wet condition are essential when developing a scaffold for bone defects. In this research work, 200% (w/w) of PBS, roughly ten times higher than the actual the water content of the bone was added to the scaffolds. The wet compressive strength and compressive modulus of the scaffolds were checked at two different time points, day 1 and day 3, to check the stability of the scaffold concerning the swelling time. Day 1 was selected to check the initial mechanical stability of the scaffolds, and day 3 was selected according to the swelling study since scaffolds were in equilibrium swelling on day 3. As shown in the Fig. 3, both compressive strength and compressive modulus are significantly reduced at the hydrated state of the scaffolds. In contrast to the dry state compressive strength values, wet state compressive strength values for all three groups are very close without statistical significance, and the highest compressive strength was reported on the 0% CaP scaffold, 0.36 (± 0.066) MPa at day 1 and 0.40 (± 0.085) MPa at day 3. These values are still in the previously mentioned range of UCS values. The 10% CaP scaffold showed a mean compressive strength of 0.26 (± 0.089) MPa and 0.39 (± 0.045) MPa at day 1 and day 3, respectively. The lowest mean compressive strength values were shown by the 20% CaP scaffolds, 0.21 (± 0.025) MPa and 0.28 (± 0.032) MPa at day 1 and day 3 respectively. The high compressive strength of 0% CaP scaffolds at wet condition can be explained by the homogeneity of the scaffold as it is entirely organic material based scaffold, but 10% CaP, and 20% CaP scaffolds contained an inorganic part. The interfacial interaction between the inorganic/organic content and the ratio of the inorganic/organic content are the main parameters for the mechanical stability of these scaffolds (Cai et al., 2009). When polymer network was swollen in the wet state, the interaction between the organic and inorganic components reduces and resulted in lower compressive strength. The mean UCS values of the wet scaffolds are given in Table 2. All the UCS values are above the 0.22 MPa, which is the reported lowest compressive strength of the mandible bone (Misch et al., 1999).

Table 2.

The ultimate compressive strength of wet state scaffolds.

Sample Mean ultimate compressive strength (UTS) ± SD
Day 1 Day 3
0% CaP 0.39 ± 0.192 0.46 ± 0.303
10% CaP 0.27 ± 0.091 0.44 ± 0.136
20% CaP 0.22 ± 0.069 0.31 ± 0.096
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It was reported that freeze-dried scaffolds showed lower compressive strength values compared to the air-dried scaffolds and therefore, in this study, salt leaching method was used to make porous scaffolds. Several authors reported high compressive strength values of CS-CMC-polyelectrolyte complex scaffold with the addition of nHA (Cai et al., 2009; Liuyun et al., 2008). However, they used more than 50% of the inorganic component, for these studies they have used nHA, and different process conditions. Further, the formation of polyelectrolyte complexes was used to improve the mechanical properties of CS, such as CS-alginate polyelectrolyte scaffolds (Chung et al., 2002; Li et al., 2005) and CS-CMC with the addition of a low amount of nHA (Jiang et al., 2008). Since most of the authors did not report the wet condition mechanical properties, comparison of the wet state results is difficult. However, according to our knowledge, this is the first time that higher compressive strength of both dry and wet state scaffolds was reported with more than 70% of the natural polymer contained scaffolds. Also, this is the first time that MPs were used as a structural constituent to provide better mechanical stability for the scaffold rather than using as a delivery vehicle. This study showed that chitosan MPs could be incorporated into three-dimensional scaffolds with excellent mechanical stability at both dry and wet conditions.

3.2. Swelling study

According to the Fig. 4, all three types of scaffolds exhibited same swelling behavior. There is no significant difference between the groups, and swelling ratio gradually increased with time. The only significant difference was observed between the 1 h swelling and all other time points of 10%

Fig. 4.

Fig. 4.

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Swelling ratio of the scaffolds at different time points, * indicates the significant difference with respect to day1 of 10% CaP scaffolds.

CaP scaffolds. After 24 h, no significant increase in the swelling ratio was observed. Swelling ratio was reduced with the increasing CaP amount as shown in Fig. 4. CS and CMC are both hydrophilic polymers (Liuyun et al., 2009) and diffusion of the liquid phase into the polymer matrix is facilitated by the mobility of the polymer chains and the free-volume between the polymer backbone and cross-links (Farag & Mohamed, 2012). However, the addition of CaP reduces the swelling behavior because of the less hydrophilicity of the CaP particles, and further, it reduces the free volume of cross-linked polymer chains. Secondly, all the scaffolds contain CS MPs, which are hydrophilic at physiological pH of 7.4. The amount of CS MPs was reduced with the increasing amount of CaP as 0% CaP scaffolds contained the highest amount of MPs and 20% CaP scaffolds contained the lowest amount of MPs. So, the scaffolds with a higher amount of MPs showed higher swelling ration and vice versa. Those are the two major reasons for the reduction of the swelling ratio with the increasing amount of CaP. Cross-linking degree is also effective on the swelling behavior of CS/CMC/CaP scaffolds as higher crosslinking degree reduces the hydrophilic groups in CS and CMC (Ma et al., 2003). For in vitro studies, the initial swelling behavior is desirable as it facilitates the cell attachment and three-dimensional growth pattern due to the increment of the pore sizes (Li et al., 2005). However, continuous swelling results in loss of mechanical integrity of the scaffolds. During this swelling study period of three days, all the scaffolds were stable and remained intact. After two weeks, reduction of the mechanical integrity of the 20% CaP scaffolds was observed. The main reason for this behavior was the reduction of crosslinking degree due to the high amount of CaP, as it reduces the complexation between CS and CMC.

3.3. XRD analysis

Fig. 5 represents the XRD spectrums of scaffolds with different composition CaP and before and after leaching out of KCl. Reduction of CaP composition was indicated in the spectrum with reducing the intensity of the characteristics CaP peaks. Removal of the KCl after leaching out was also visible in the spectrums. Another main observation could be made is that the peak at 2θ = 20°, which represents the chitosan-CMC polymer crystallinity, increases with the reducing CaP content as shown in Fig. 5. Also, porous 20% CaP scaffold and non-porous 20% CaP scaffold showed a variation in the intensity of chitosan-CMC peak; porous scaffold has shown higher crystallinity. High crystallinity may be a reason for the increase in the compressive strength of after leaching out (porous) scaffolds.

Fig. 5.

Fig. 5.

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XRD spectrums of scaffolds with different CaP content and different conditions, * indicates the characteristic peaks of CaP, + indicates the characteristics peaks of KCl, and # indicates the characteristics peaks of chitosan/CMC polymer.

3.4. Porosity and morphological analysis

The calculated porosity of 0% CaP, 10% CaP, and 20% CaP scaffolds after KCl leaching out was 22%, 22%, and 20%, respectively. This porosity values are approximately similar to the incorporated KCl amount in the scaffolds. KCl does not effect to the surface roughness of the scaffolds, as it leaches out, but the surface pores created due to leaching out of KCl increases the surface irregularities of the scaffolds as shown in the Fig. 6. Porosity of the scaffold is an important parameter since it effects to the cellular colonization rates, angiogenesis, and degradation rate (Lee, Kim, Chong, Hong, & Lee, 2005; Madihally & Matthew, 1999). Fig. 6A represents the SEM micrograph of CS MPs, and Fig. 6B represents the SEM micrograph of the scaffolds before and after leaching out the KCl salt. According to the Fig. 6A, MPs show the spherical shape and a rough surface with surface irregularities and perforations. There are flake-like structures on the surface of the scaffolds as shown in Fig. 6BI (A, 0% CaP) images, which are attributed to the KCl salt. Those flake-like structures are not visible on the KCl leached out scaffolds in Fig. 6BII. Instead of flakes, pores are visible on the surface, and this further confirmed the complete leaching out of KCl. MPs in the 0% CaP scaffolds are clearly visible, but MPs in the 10% CaP and 20% CaP scaffolds are not clearly visible as it covered with CaP particles. The surface roughness of the scaffolds increases with the increase of the amount of CaP as shown in the Fig. 6B. This type of rough and porous surface is ideal for the growth and proliferation of the cells, and diffusion of nutrients while facilitating ascularization (Oliveira et al., 2006). Also, higher porosity enhances the osteogenesis in vivo (Karageorgiou & Kaplan, 2005).

Fig. 6.

Fig. 6.

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A- SEM micrograph of chitosan MPs; A- Magnification (X) −50; B- X250; C- X2000; B- SEM micrograph of three types of scaffolds at magnification of 65; BI- before leaching; BII- after leaching.

Elemental mapping was done using backscattered electron detector at accelerating voltage of 20 keV and calcium, phosphorous, and potassium elements were mapped. Fig. 7 shows the images of element mapping and EDX spectrum for 10% CaP scaffold before leaching out of KCl. As shown in the Fig. 7A, KCl shows in light color due to its crystalline phase and CaP and polymer phases are shown in dark color. This behavior can be further observed in the element mapping images in Fig. 7B–D. Homogeneous element distribution was observed.

Fig. 7.

Fig. 7.

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Element mapping images and EDX Spectrum; A- SEM Image; B- Ca; C–P; D–K; magnification – 65; E- EDX spectrum.

3.5. In vitro cell culture

Fig. 8 shows the live and dead cell assay images of scaffolds at day 1, day 4, and day 7. Proper cell attachment is visible on CaP contained scaffolds compare to the 0% CaP scaffolds. Enhanced cell attachment is mainly due to the presence of CaP because it has an ability to absorb ECM proteins such as fibronectin (Eliaz & Metoki, 2017). 0% CaP scaffolds showed low cell adhesion compare to the 10% and 20% CaP scaffolds at day 1 and day 4. No dead cells were visible on day 1 images, but there are few red dots, which represent dead cells, at day 4.d more dead cells are visible on 10% and 20% CaP scaffolds at day 4 and day 7. Also, the number of viable cells were reduced on 20% CaP scaffolds at day 7 compared to both 0% CaP and 10% CaP scaffolds, which showed a gradual increase of cell proliferation with the increasing culture time. However, because of the 3D nature of the scaffolds and non-homogeneous cell attachment, the cell proliferation and cell viability cannot be quantified by using live/dead cell assay. WST-1 cell proliferation assay was used to overcome this problem.

Fig. 8.

Fig. 8.

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Live/dead cell assay fluorescence images of three type of scaffolds as indicated in the image, green-live cells, red-dead cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

According to the Fig. 9, similar cell proliferation behavior to the live/dead cell assay images was observed. At day 1, there was a significant difference between 0% CaP scaffold and 10% and 20% CaP scaffolds, while 10% CaP showed the highest cell proliferation, 20% showed the lowest. The cell proliferation increases with the time as expected and a significant difference was observed at the time point for each group, except for day 4 and day 7. According to these results, 10% CaP scaffold showed the highest cell proliferation except for day 4. The reduction in the cell proliferation of 20% CaP scaffolds compare to the 10% CaP scaffolds is confirmed the low cell viability observed on live/dead cell assay images. At day 14, cell proliferation on 20% scaffolds was lower than for both 0% and 10% CaP scaffolds, and there was a significant difference of cell proliferation between 10% and 20% scaffolds. This result suggested that the amount of CaP effects on the proliferation of murine pre-osteoblasts cells. In this study tribasic calcium phosphate, which is also known as tricalcium phosphate (TCP), was used. Liu et al., reported that the high α-TCP concentration inhibits the proliferation and differentiation of rat bone marrow mesenchymal stem cells. According to the results, it can be concluded that the CaP increases the cell adhesion and proliferation up to some extent, but further addition of the CaP inhibits the cell adhesion and proliferation.

Fig. 9.

Fig. 9.

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WST-1assay results at day1, day 4, day 7, and day 14, *significance of p < 0.05.

SEM analysis of cell attachment and morphology showed that the cells were proliferating on the surface of the scaffolds. According to the Fig. 10, 0% CaP scaffold showed proper cell attachment and cells could be clearly differentiated from the scaffold surface because of the relatively smooth surface compared to the 10% and 20% CaP scaffolds, which had a very rough surface as shown in the Fig. 10. The main difference in cell morphology on three surfaces is their shape and the spreading. Cells on the 0% CaP scaffolds showed more rounded shape while cells on 10% and 20% CaP scaffolds showed more elongated and flat surface with extensive filopodia, indicating the strong cellular adhesion and the growth (Thein-Han & Misra, 2009). For this type of CaP contained scaffolds, integrin-mediated cell binding was suggested to be the possible mechanism (Kilpadi, Chang, & Bellis, 2001). In general, CaP (in this study TCP) compounds absorb fibronectin and vitronectin and enhance the protein adsorption, which later facilitates the binding of integrin and osteoblast precursors to CaP.

Fig. 10.

Fig. 10.

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SEM micrographs of cell attachment at day 14 of cell culture, rows- magnification (X), columns- scaffold type.

In this study, chitosan MPs were used as a structural component of the scaffold to provide better mechanical stability and strength. Therefore, the higher cross-linking degree of chitosan MPs was obtained by allowing cross-linking for 4 h with TPP. Since CS was dissolved in acetic acid, the final dried scaffold had acidity, and after drying, scaffolds were immersed in PBS for three days not only to remove KCl but also to reduce the acidity of the scaffolds. The high compressive strength and better cell attachment enhance the significance of this type of MP based scaffolds. The in vivo behavior of these scaffolds by using a rat model and release of drug and growth factors as a controlled delivery system will be further investigated in our future research works.

4. Conclusions

Chitosan MPs were successfully incorporated into chitosan-CMC polyelectrolyte complex, and superior mechanical stability was observed. Addition of the CaP increases the compressive strength and compressive modulus of the scaffolds, but further addition of the CaP reduces the stability and compressive strength of the scaffolds at both dry and wet conditions. The function of KCl was shown on SEM micrograph by making pores on the surface after leaching out. XRD spectrum further confirmed this removal of KCl from the XRD spectrum of leached out scaffolds. The in vitro cell studies showed good biocompatibility of scaffolds with murine pre-osteoblast cells. Altogether, 10% CaP scaffold showed the best mechanical properties and better cell proliferation.

Supplementary Material

Supplement Figure

Acknowledgment

This work was supported by the National Institutes of Health (NIH) grant number: R01DE023356.

Footnotes

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.07.044.

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