Cross-linked chitosan improves the mechanical properties of calcium phosphate-chitosan cement

Abstract

Calcium phosphate (CaP) cements are highly applicable and valuable materials for filling bone defects by minimally invasive procedures. Chitosan (CS) biopolymer is also considered as one of the promising biomaterial candidates in bone tissue engineering. In the present study, some key features of CaP-CS were significantly improved by developing a novel CaP-CS composite. For this purpose, CS was the first cross-linked with tripolyphosphate (TPP) and then mixed with CaP matrix. A group of CaP-CS samples without cross-linking was also prepared. Samples were fabricated and tested based on the known standards. Additionally, the effect of different powder (P) to liquid (L) ratios was also investigated. Both cross-linked and uncross-linked CaP-CS samples showed excellent washout resistance. The most significant effects were observed on Young's modulus and compressive strength in wet condition as well as surface hardness. In dry conditions, the Young's modulus of cross-linked samples were slightly improved. Based on the presented results, cross-linking does not have significant effect on porosity. As expected, by increasing the P/L ratio of sample, ductility and injectabilty were decreased. However, in the most cases, mechanical properties were enhanced. The results have shown that cross-linking can be improved the mechanical properties of CaP-CS and hence it can be used for bone tissue engineering applications.

1. Introduction

In tissue engineering, chitosan (CS), derived from chitin has been explored on several fronts as an organic substance to complex with calcium phosphate (CaP). Part of what makes chitosan a desirable to use substance is its biodegradability, which allows the body to break it down after healing [1, 2], and biocompatibility, which includes minimal foreign body reaction while allowing human cells to grow onto it [3-6].

With such biocompatibility, CS is an ideal substance to complex into a scaffold with other additives [7]. One of the promising candidate for bone paste biomaterials is CaP, the main component of the inorganic portions of bone. CaP cements have gained clinical acceptance as valuable bone replacing biomaterials for almost three decades [8]. These scaffolds would form the basis of a moldable cement intended for bone regeneration applications while also allowing cells such as osteoblasts to grow normally onto them. Porosity, set time and mechanical properties such as, flexural strength, tensile strength, and hardness have been explored in vitro and in vivo. For the sake of clinical applications, traits such as washout resistance and injectability have also been heavily explored [9-13]. These properties have significant effect on the feasibility of injectable cements in bone tissue engineering. CaP cements have drawbacks in terms of graft migration, brittleness, fatigue fracture, and handling difficulties etc. These properties need to be improved for clinical applications. For instance, washout resistance holds significance due to the intended environment of the CaP-CS complexes, which generally involves being inside the human body. The other important property is injectability, which involves the force required to move the CaP-CS cement through a syringe. Injectable CaP-CS cement can be administered to bone defects using a minimally invasive technique.

CaP-CS scaffolds have been created with a variety of different methods as to further explore possible methods for enhancements of their mechanical properties. The more recent methods have involved using nanoparticles or nanofibers [14-17]. Other methods involved precipitating beads of CaP-CS complexes out of a solution and lyophilization freeze drying [18]. Also the fabrication process has been heavily varied by the addition of different additives including organic acids, epoxides, and celluloses [19-21]. The addition of the additives has been enhanced mechanical properties, injectability, and washout. On a chemical level, some of these additives have even induced cross-linking between chitosan.

Nonetheless, cross-linked CS contain CaP-CS scaffolds have not been fully explored. CS contains abundant amino and hydroxyl groups which enable it to form particles via both physical and chemical cross-linking. Ionic cross-linking of CS is a typical non-covalent interaction, which can be realized by association with negatively charged multivalent ions such as tripolyphosphate (TPP) [22]. This paper seeks to focus on the effects of cross-linking of CS in the CaP-CS scaffold and explore its effects on Young's modulus in both dry and wet conditions, porosity, and hardness. Moreover, different powder to liquid ratios (P/L) were examined. Also taken into account were washout resistance and injectability.

2. Materials and Methods

2.1. Materials

All components of CaP cement were purchased from Fisher Scientific. CS (85%, deacetylated), TPP and other chemical materials were purchased from Sigma-Aldrich. Statistical analysis was done using one-way ANOVA and p<0.05 was considered as statistically significant. For each group, three samples were tested.

2.2. Scaffold fabrication

The CaP blend was created with the intention of mimicking the inorganic component of bone. The blend comprised of 55% alpha tricalcium phosphate (α-TCP), 45% dicalcium phosphate anhydrous (DCPA), and 15% calcium carbonate (CaCO₃) similar to as previously reported [23]. The CS solution was mixed at 2 wt% with acetic acid or a ratio of 200 mg per 10 mL of acetic acid. The solution was stirred with a magnetic stirrer for 20 min and then set for 20 min allowing the air bubbles to be disappeared. CaP powder blend was mixed with the CS solution at powder to liquid mass ratios (P/L) of 1.5, 1.7, and 2.0. The mixing was done by manually until a consistent paste-like substance was formed. The paste-like substance was then put into cylindrical molds with dimensions of approximately d = ~6 mm and l = ~12 mm based on the ASTM-C39-05 standard. The molds were placed between two glass slides at 100% relative humidity and stored at 37°C for 4 h. After 4 h, the samples were removed from the molds and placed into a saliva-like solution (SLS) for 20 h at 37°C. SLS comprised 1.2 mM CaCl₂, 0.72 mM KH₂PO₄, 30 mM KCl, and 50 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). This buffer was previousely used to mimic plasma in teeth environment [9]. However, Phosphate Buffered Saline (PBS) is another candidate to replace SLS to accurately mimic bone environment. After 20 h, the samples were removed from the SLS and they were ready for testing. The purpose of fabricating of CaP-CS scaffolds using the molds is to measure the mechanical properties of CaP-CS cement.

2.3. Cross-linked scaffolds

To create cross-linked samples, instead of placing the samples in the molding between glass slides at 100% humidity at 37°C for 4 h, they were only placed in SLS for 2 h. After 2 h, the samples were moved into 80% (wt) TPP solution for 2 h at 37°C to induce the chitosan within the scaffolds to cross-link at a molecular level. Samples had high contact area to the TPP solution and based on the calculated porosity, it is assumed that TPP solution penetrates into the entire sample. This time is fairly enough to form cross-linking between CS molecules [24] Similar to uncross-linked scaffolds, the CaP-CS cross-linked scaffolds were wetted immersing in the SLS solution for 20 h.

2.4. Washout resistance

Ideally, washout resistance test should be done after mixing and preparing paste-like CaP-CS but the test was done after 4 h which was specified for cross-linking CS using TPP. Washout resistance was indicated by the sample maintaining integrity and not being dissolved by the SLS. Samples were carefully moved to a beaker with 20 mL of SLS and kept at room temperature for about 1 h. Then the washout properties were investigated.

2.5. Compressive strength and injectability testing

Using the ADMET 2611 mechanical testing machine with MTestQuatro controller, compressive strength and injectability tests were performed. For the compression test, the crosshead speed was set at 1 mm/min. This speed is suitable for CaP cements reported before [5]. Compressive moduli were tested for both wet and dry samples. Wet samples were the samples that were immediately taken out of the SLS. The dry samples were taken out of the SLS and incubated in a dry environment for 24 h to allow the fluid (mainly SLS) inside to evaporate out. This step was done for both cross-linked and uncross-linked groups. By attaching appropriate custom made grip, injectability test were also performed. A syringe was fitted on the machine and filled to about 5 mL of the CaP-CS paste. The force required for expulsion from the syringe was then tested. Since this test was done immediately after making CaP-CS paste, it was only done with uncross-linked samples. Injection speed was adjusted to be 6 mm/min. CaP cement pastes are usually considered as non-Newtonian fluid. However, Hagen-Poiseuille relationship can be used to determine the viscosity [25] .

2.6. Porosity

For porosity, samples were initially fully dry. They were then massed and had their exact dimensions measured. Each sample was then immersed into 25 mL of deionized H₂O. After 2 h of immersion, the samples were removed and massed again. The mass difference was used to calculate the volume of H₂O absorbed by each sample at 24^°C. The amount of H₂O absorbed was placed in a ratio with the volume of the Ch-CaP scaffold itself $\left(\frac{V_{waterabsorbed}}{V_{Ch-Capscaffold}}\right)$ to derive a dimensionless value that indicated degree of porosity [13].

2.7. Hardness

For hardness test, dried samples were brought before a CM400AT Clark Microhardness tester. To properly prepare the samples, each was sandpapered at the round ends to create a flatter surface. The round ends were also marked with marker to add contrast so as to better view with the microscope. The machine would generate small indentations at various forces. The applied force was 200 g force (gf). The indentations’ dimensions, both horizontal and vertical diameter, would then be measured to generate a hardness value. Final hardness value can be calculated as follows:

$$HV=\frac{1.854F}{d^2}$$

In this equation. HV is the value of vicker's hardness, F is the adjusted load and d is the average of square's diagonals.

3. Results

3.1. Washout resistance

After 4 h, almost all samples were easily removed from the mold and upon placement into the SLS solution, none of the CaP-CS scaffolds washed out. They maintained structural integrity and did not dissolve into solution throughout the incubation time as shown in Figure 1. Particulary, this figure shows the integrity of the samples with P/L= 1.7. For other samples with different P/L ratios, the same behavior was observed. However, washout resistance for just CaP was low and the samples were crashed and disintegrated in SLS after few minutes. This observation shows that having chitosan polymer in CaP enhance the integration of samples.

Figure. 1.

Washout resistance of samples after 1 hour submerging in SLS.

3.2. Compressive strength

Increased P/L typically indicated stronger compressibility [26]. Cross-linking using the TPP solution enhanced mechanical properties, much more noticeably in the wet state of the CaP-CS scaffolds. Cross-linking CaP-CS paste using TPP solution significantly increased the strength and Young's modulus. Figure 2 shows the stress-displacement curve obtained from compression test for samples in wet and dry conditions. As expected, by increasing the P/L ratio, the Young's modulus and compressive strength were increased [27]. Maximum compressive strength for cross-linked CaP-CS samples in wet condition were 0.57, 0.73 and 1.28 MPa for P/L=1.5, 1.7 and 2, respectively. Similarly, this value for uncross-linked CaP-CS samples in wet condition were 0.03, 0.06 and 0.27 MPa for P/L=1.5, 1.7 and 2, respectively. It is worth noticing that significant difference was observed between cross-linked and uncross-linked samples. Adding cross-linked polymer such as CS can drastically increase the Young's modulus and compressive strength. For the dry state, as shown in Figure 3, cross-linking showed mix results with regards to hardness. The Young's modulus of the CaP-CS scaffolds made with the above method typically were in the 10⁶ Pa range. Figure 3 shows the Young's modulus for samples in dry condition. Samples were completely crashed at the end of the test due to the low ductility. Similar to the wet condition, by increasing the P/L ratio, Young's modulus and compressive strength increased. For example, Young's modulus were 39.53, 45.96 and 76.31 MPa for P/L=1.5, 1.7 and 2 in cross-linked samples, respectively. Overall, although the difference is not significant, similar to the wet condition, cross-linking enhanced the Young's modulus.

Figure. 2.

Stress-Displacement curve for (A) Cross-linked samples, (B) Uncross-linked samples. (C) Comparative results of the compression test in wet condition. * shows significant difference between the cross-linked and uncross-linked samples.

Figure. 3.

Stress-Displacement curve for (A) Cross-linked samples, (B) Uncross-linked samples. (C) Comparative results of the compression test in dry condition.

3.3. Injectability

For injectability, only the uncross-linked samples were tested as shown in Figure 4. This was because of the procedure that was followed to make cross-linked samples as explained above. At P/L = 2.0, the test failed. The syringe broke before any amount of the paste could be expelled from the syringe due to the high viscosity of the fabricated paste. Generally, this followed the trend of increased P/L led to decreased injectability [28], which would correspond to a higher injection force. For samples which we could do the injection test, the injected paste was coherent without any disintegration (Figure 4). In addition, it showed great wash out resistance after the injection.

Figure. 4.

(A) Injection test results for uncross-linked samples. (B) Washout resistance after injection.

3.4. Porosity

The results of the porosity are shown in Figure 5. Increased P/L ratios decreased the porosity while CS cross-linking seemed to have made a marginal effect of the porosity based on the results. Porosity variation between all samples were less than 10%. The mass of H₂O absorbed was converted to a volume in mL based on its density at room temperature (24°C) of 0.997 g/mL. These were the values used to generate the dimensionless porosity value.

Figure. 5.

Comparative results of porosity for samples with different P/L ratios.

3.5. Hardness

Increased P/L ratios typically increase the hardness. CS cross-linking also significantly increased the hardness in comparison with uncross-linked samples. For all P/L ratios, after cross-linking the CaP-CS sample, the hardness was at least doubled (Figure 6). The surface hardness for uncross-linked CaP-CS samples were 11, 17.67 and 21 Kgf/mm² for P/L=1.5, 1.7 and 2, respectively. However, after cross-linking, the surface hardness changed to 33.1, 34.2 and 49.9 Kgf/mm² for P/L=1.5, 1.7 and 2, respectively.

Figure. 6.

Comparative results of surface hardness test for samples with different P/L ratios. * shows significant difference between the cross-linked and uncross-linked samples.

4. Discussion

Both CS and CaP are common biomaterials in bone tissue engineering. Different papers have reported enhancement in mechanical properties of the mixture of CaP and CS composite biomaterials [12, 13, 29]. In addition, it has been shown that cross-linking CS has higher elastic modulus and hardness [24]. Our group previously showed that cross-linked CS with certain concentrations of TPP is not cytotoxic for cells [30] and CS mixed with other forms of materials such as carbon nanotubes and Zinc oxide nanoparticles is cytocompatible with osteobalsts [31, 32]. The goal of this investigation was to explore the effect of cross-linking on mechanical properties of CaP-CS composite biomaterial.

In the present study, a new method was applied to cross-link CS in CaP-CS matrix. Samples were made based on available standards. Fabricated samples, showed excellent cohesion (i.e., washout resistance), which was achieved by composition of the CS in CaP. We have shown that Young's modulus of cross-linked CaP-CS samples was significantly higher than that of uncross-linked samples. The different components of CaP-CS also play an important role on the mechanical properties of final CaP-CS cement. As it was observed from Figure 2, the ductility of samples were slightly dereased. This can be interpreted as longer polymer chains in cross-linked samples. When the polymer chains cross-linked, the polymer chains lost the ability of free movement. Compressive strength values of injectable CaP reported in the literature range from few MPa [33] up to 83 MPa [34] and these values are based on the method of testing and CaP compositon.

Cross-linking has shown to enhance mechanical properties of the samples. The Young's modulus of dried samples show higher values for both cross-linked and uncross-linked samples compared to the wet samples. However, the Young's modulus ranges of the samples still do not reach the levels of actual human bone [35]. Better fabrication techniques could make better use of the materials to make a sample that could have a potentially higher Young's modulus. In addition, increasing the cross-linking time and concentration can be beneficial. On the other hand, changing in biological properties and biocompatibility should be monitored and considered.

Higher injectibility could lead to less invasive procedures when uncross-linked CaP-CS cements used in clinical setting. Due to the specific procedure that was followed, we could not do the injection test for cross-linked samples and future investigations needs to be done. Based on the presented results, P/L ratio has great influence on injection force. The injection test was conducted for P/L=1.5 and 1.7 and plastic syringe was collapse during the experiments for samples with P/L=2. However, P/L = 2.0 could have possibly succeeded with a stronger designed syringe. Nonetheless, the large amount of force required for even P/L = 1.7 indicates a need for a method to generate large magnitude of force quickly and consistently. To improve the injectability of CaP-CS, one method is reducing viscosity by adding suitable materials such as biocompatible and biodegradable oils [23]. The porosity results show mixed outcomes of cross-linking CS. The increased porosity of lower P/L ratios is most likely the result of having more space after the fluid has dried out. Maximizing porosity for these scaffolds could be useful as they allow more surface area for cells such as osteocytes to grow into, maximizing the biocompatibility of chitosan in the body. It has been shown that, adding CS does not have significant effect on CaP-CS porosity [13]. In addition to that, we have shown cross-linking does not have meaningful affect on porosity of the samples in different selected P/L ratios.

Another interesting effect of cross-linking on mechanical properties of CaP-CS bone scaffolds is the enhancement of surface hardness. The average Vicker's microhardness of 100 μm-thick sections from bone sample at 25 g applied load was reported to be 50 kg/mm² (490 MPa) [36]. In cross-linked samples, hardness value close to bone tissue was achieved. Furthermore, the hardness is adjustable by changing P/L ratio and cross-linking duration time. The expected trends were followed where increased P/L leads to increased hardness as well as CS cross-linking leading to increased hardness. However, the method indicated in this paper leads room for much inconsistency.

5. Conclusion

In summary, this investigation demonstrated the incorporation of CS into CaP bone cements caused a significant enhancement of the mechanical properties of the biocomposite. In addition to excellent washout resistance, both the compressive modulus and yield strength of the scaffolds were greatly improved, and a reinforced composite cement was obtained. Although cross-linking is not beneficial for the CaP-CS injectability, it has shown that cross-linking of CS in CaP matrix can greatly improve hardness and Young's modulus especially in wet condition. In other words, with better and standardized fabrication techniques, the potential for clinical use of a cross-linking agent could lead to improved bone substitute and tissue engineering applications. However, other important factors such as biodegradability and biocompatibility should also consider carefully.

Research Highlight.

• Chitosan incorporated in calcium phosphate cement improves the washout resistance of the scaffold.

• Cross-linking of the Chitosan improved the mechanical properties of calcium phosphate-Chitosan scaffold.

• Porosity of calcium phosphate- Chitosan samples does not significantly change by cross-linking the Chitosan.

• The effect of Chitosan cross-linking is more significant in wet samples in comparison with dry samples.