# How Sensitive Is the Elasticity of Hydroxyapatite-Nanoparticle-Reinforced Chitosan Composite to Changes in Particle Concentration and Crystallization Temperature?

^{1}

^{2}

^{3}

^{4}

^{5}

^{*}

## Abstract

**:**

## 1. Introduction

_{10}(PO

_{4})

_{6}(OH)

_{2}; HA) which occurs naturally in vertebrate tissues such as bones and teeth [1]. Thus, we can expect it to possess excellent biocompatibility, osteoconduction and osteointegration [2]. Additionally, we note that there are two key characteristics of HA which make it an ideal candidate for blending into and reinforcing the chitosan matrix for applications in tissue engineering [3,4,5]: (1) mechanical stability (when synthesized into the form of nanoparticles with size of 100 nm or less) [6,7] and (2) an affinity to biopolymers [8]. To this end, HA-reinforced chitosan composites in the form of fibres—especially with hierarchical architectures [9] synthesized for consistency with the natural hierarchy of biological tissues [10]—find potential applications in tissue engineering. These findings are well-reported in the literature. For instance, Bhattarai et al. [11] demonstrated that chitosan-based nanofibers with an average fiber diameter controllable from a few microns down to 40 nm and that a narrow size distribution could be fabricated by electrospinning. The nanofibers were deposited as a nonwoven mat or as an aligned bundle of controllable size; in either case, the nanofibrous structure demonstrated the capacity to promote the attachment of human osteoblasts and chondrocytes and to maintain the characteristic cell morphology and viability [11]. Zhang et al. [12] found that HA/chitosan composite fibre could encourage the growth of cells residing on it; the population of cells increased by 43% (10 days) and 110% (15 days) in scaffolds made from electrospun HA/chitosan fibres. Lai et al. [13] demonstrated that the osteogenic differentiation of human mesenchyma stem cells (hMSCs) increased with HA concentration in an HA-reinforced chitosan fibrous scaffold. The study of Lai et al. [13] reveals that seeding hMSCs in an HA-reinforced chitosan fibrous scaffold has potential for bone regeneration, and such a scaffold may be used for bone tissue engineering.

## 2. Results

**Table 1.**The mechanical properties of the hydroxyapatite (HA)-reinforced chitosan fibres. A total of eight different combinations of crystallization temperature (two levels) and HA concentration (four levels) were explored. The values of the respective mechanical properties are shown as mean ± SEM for informational purposes (SEM: standard error of the mean).

Crystallization Temperature (°C) | HA Concentration (mass fraction) | No. of Specimens | Stiffness (MPa) | Strength (MPa) | Toughness (MPa) |
---|---|---|---|---|---|

40 | 0.05 | 8 | 2612.6 ± 131.7 | 34.2 ± 2.2 | 1.6 ± 0.3 |

40 | 0.10–0.15 | 16 | 1330.8 ± 87.9 | 19.7 ± 0.9 | 1.1 ± 0.2 |

40 | 0.20–0.30 | 16 | 1213.8 ± 48.3 | 16.9 ± 0.7 | 0.7 ± 0.1 |

40 | 0.40–0.60 | 8 | 1205.3 ± 199.0 | 20.1 ± 3.1 | 0.4 ± 0.1 |

100 | 0.05 | 8 | 1123.5 ± 100.3 | 19.2 ± 1.3 | 1.4 ± 0.2 |

100 | 0.10–0.15 | 8 | 970.1 ± 51.3 | 16.4 ± 0.9 | 0.6 ± 0.1 |

100 | 0.20–0.30 | 16 | 1384.8 ± 90.9 | 23.9 ± 1.9 | 0.7 ± 0.1 |

100 | 0.40–0.60 | 16 | 2899.5 ± 194.6 | 36.3 ± 2.4 | 1.5 ± 0.2 |

**Figure 1.**Main effect plots. (

**a**) Plot of the mean value of the respective mechanical properties (averaged over the values of the respective specimens for the different crystallization temperatures) versus hydroxyapatite (HA) concentration; (

**b**) Plot of the mean value of the respective mechanical properties (averaged over the values of the respective specimens for the different HA concentrations) versus crystallization temperature.

**Figure 2.**Interaction plots. (

**a**) Plot of the mean value of the respective mechanical properties (for each crystallization temperature) versus hydroxyapatite (HA) concentration; (

**b**) Plot of the mean value of the respective mechanical properties (calculated for each HA concentration) versus crystallization temperature. The P values for the overall interaction effects test are indicated above each row of graphs. In the right panel, the P values to the right of the legends are associated with respective HA concentration.

_{0}, of the chitosan fibre reinforced by HA nanoparticles. It is observed that β increases with increase in HA concentration, peaking at 0.10–0.15 (for both 40 °C and 100 °C levels). Thereafter, β decreases somewhat with an increase in HA concentration. With regards to the 40 °C crystallization temperature, σ

_{0}is high at low HA concentration (i.e., 0.05); σ

_{0}decreases when the HA concentration increases to 0.10–0.15 and fluctuates with further increase in HA concentration. With regard to the 100 °C crystallization temperature, while the σ

_{0}is high at a low particle concentration (i.e., 0.05), the magnitude decreases when the HA concentration increases to 0.10–0.15; thereafter, the σ

_{0}increases dramatically with increasing HA concentration. Figure 3 illustrates how these parameters influence the reliability of the fibres using a plot of the reliability function, R(σ), versus fracture stress in the fibre, σ, for all the levels of HA concentration and crystallization temperature considered in this study. Thus the form (parameterized by β) of the curves corresponding to 40 °C (0.10–0.15, 0.20–0.30, 0.40–0.60 HA concentration) levels and 100 °C (0.05, 0.10–0.15, 0.20–0.30 HA concentration) levels features a very narrow spreads of strength variability as compared to 40 °C (0.05 HA concentration) levels and 100 °C (0.40–0.60 HA concentration) levels. Correspondingly, the σ

_{0}for the 40 °C (0.05 HA concentration) and 100 °C (0.40–0.60 HA concentration) levels are dramatically larger than those associated with the other levels.

**Table 2.**Analysis of the Weibull modulus, β, and the characteristic strength, σ

_{0}, of chitosan fibres reinforced by hydroxyapatite (HA) nanoparticles.

Crystallization Temperature (°C) | HA Concentration (mass fraction) | β | σ_{0} (MPa) |
---|---|---|---|

40 | 0.05 | 5.6 | 36.9 |

40 | 0.10–0.15 | 6.3 | 21.2 |

40 | 0.20–0.30 | 6.1 | 18.2 |

40 | 0.40–0.60 | 2.6 | 23.0 |

100 | 0.05 | 5.3 | 20.8 |

100 | 0.10–0.15 | 6.6 | 17.6 |

100 | 0.20–0.30 | 3.2 | 26.8 |

100 | 0.40–0.60 | 3.9 | 40.1 |

**Figure 3.**Plot of reliability function, R(σ), versus fracture stress in the fibre, σ, at varying hydroxyapatite (HA) concentrations and crystallization temperatures. Note: R(σ) = exp(−(σ/σ

_{0})

^{β}), where σ

_{0}and β are the characteristic strength and the Weibull modulus, respectively.

## 3. Discussion

**Figure 4.**Schematic of HA-nanoparticle-reinforced chitosan composite fibre. (

**a**,

**c**) and (

**b**,

**d**) depict HA in the chitosan matrix at low and high concentrations, respectively; (

**a**,

**b**) and (

**c**,

**d**) depict HA particles corresponding to crystallization temperature at 40 and 100 °C, respectively. Overlaying one of the particles in each panel is a sketch of the graph of axial stress (σ

_{z}) in the particle versus distance (z) along the particle axis.

## 4. Experimental Methods

_{3}PO

_{4}to 250 mL of 0.2M Ca(OH)

_{2}. HA nanocrystals were precipitated at 40 °C and 100 °C respectively; the reaction in the medium was terminated at pH = 7.4. The white precipitates were collected after sedimentation. The chitosan solution (10 mL, 3% w/v) was prepared by stirring chitosan in acetic acid (1% w/v). A suspension (5 mL), containing precipitates of HA, was mixed with the chitosan solution (10 mL). With the help of a coagulant (NaOH 10% w/v) solution a predrawn wet spun fiber extrusion process [30] was used to extrude fibres from the chitosan-HA mixture (at a rate of 3.1 cm/min). At the exit, the fibres were collected into a coagulant solution (NaOH 10% w/v) and subsequently removed from the coagulant solution. The fibres possessed diameters ranging 174.1 to 240.3 μm. The fibres were sectioned to lengths of 150 mm; each test specimen was stretched to rupture using a tensile machine (E1000, Instron, Norwood, MA, USA) at a displacement rate of 0.04 mm/s.

_{0}]

^{β}). Here, β and σ

_{0}represent the Weibull modulus and the characteristic strength, respectively. In particular, β parameterizes the variability of σ; low β values correspond to high variability and vice versa. σ

_{0}is the stress value at which 63% of the fibres have fractured. In strength definition, we have the reliability function R(σ) = exp(−{σ/σ

_{0}}

^{β}) which refers to the proportion of the population of specimens sampled which will have a survived at fracture stress σ. To evalue β and σ

_{0}for the different treatment groups, we first determined the median rank position (MR, to order of magnitude, this is identified with R) for each experimentally derived value of σ. This was carried out by ranking the σ data in ascending magnitude. The corresponding estimates of MR was evaluated using the equation MR = {i − 0.3}{n + 0.4}

^{−1}, where n represents the size of the treatment group and i is the position of the corresponding σ. We then fitted straight lines to the Weibull plot of log(log(1/{1–MR(σ)})) versus log(σ) for each group. Here we note that the value of β was identified with the slope of the respective straight lines while the value of σ

_{0}was found by equating −βlog(σ

_{0}) to the y-intercept of the straight line.

## 5. Conclusions

- The mechanical properties of the chitosan composite fibre are sensitive to HA concentration and crystallization temperature;
- However, owing to interactions between the two factors, the changes in the mechanical properties due to varying HA concentration also depend on the crystallization temperature (and vice versa);
- There exists a critical HA concentration level at which the magnitude of the respective mechanical properties is a minimum.

## Acknowledgements

## Author Contributions

## Conflicts of Interest

## References

- Sadat-Shojai, M.; Atai, M.; Nodehi, A.; Khanlar, L.N. Hydroxyapatite nanorods as novel fillers for improving the properties of dental adhesives: Synthesis and application. Dent. Mater.
**2010**, 26, 471–482. [Google Scholar] [CrossRef] [PubMed] - Petit, R. The use of hydroxyapatite in orthopaedic surgery: A ten-year review. Eur. J. Orthop. Surg. Traumatol.
**1999**, 9, 71–74. [Google Scholar] [CrossRef] - Wang, Z.; Hu, Q. Preparation and properties of three-dimensional hydroxyapatite/chitosan nanocomposite rods. Biomdical Mater.
**2010**, 5. [Google Scholar] [CrossRef] [PubMed] - Li, J.; Zhu, D.; Yin, J.; Liu, Y.; Yao, F.; Yao, K. Formation of nano-hydroxyapatite crystal in situ in chitosan–pectin polyelectrolyte complex network. Mater. Sci. Eng. C
**2010**, 30, 795–803. [Google Scholar] [CrossRef] - Xie, J.Z.; Hein, S.; Wang, K.; Liao, K.; Goh, K.L. Influence of hydroxyapatite crystallization temperature and concentration on stress transfer in wet-spun nanohydroxyapatite- chitosan composite fibres. Biomed. Mater.
**2008**, 3, 2–6. [Google Scholar] [CrossRef] [PubMed] - Sadat-Shojai, M.; Khorasani, M.-T.; Dinpanah-Khoshdargi, E.; Jamshidi, A. Synthesis methods for nanosized hydroxyapatite with diverse structures. Acta Biomater.
**2013**, 9, 7591–7621. [Google Scholar] [CrossRef] [PubMed] - Kumar, R.; Prakash, K.H.; Cheang, P.; Khor, K.A. Temperature driven morphological changes of chemically precipitated hydroxyapatite nanoparticles. Langmuir
**2004**, 8, 5196–5200. [Google Scholar] [CrossRef] - Yamaguchi, I.; Tokuchi, K.; Fukuzaki, H.; Koyama, Y.; Takakuda, K.; Monma, H.; Tanaka, J. Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J. Biomed. Mater. Res.
**2001**, 55, 20–27. [Google Scholar] [CrossRef] - Kureshi, A.; Cheema, U.; Alekseeva, T.; Cambrey, A.; Brown, R. Alignment hierarchies: Engineering architecture from the nanometre to the micrometre scale. J. R. Soc. Interface
**2010**, 7, S707–S716. [Google Scholar] [CrossRef] [PubMed] - Goh, K.L.; Listrat, A.; Béchet, D. Hierarchical mechanics of connective tissues: Integrating insights from nano to macroscopic studies. J. Biomed. Nanotechnol.
**2014**, 10, 2464–2507. [Google Scholar] [CrossRef] - Bhattarai, N.; Edmondson, D.; Veiseh, O.; Matsen, F.A.; Zhang, M. Electrospun chitosan-based nanofibers and their cellular compatibility. Biomaterials
**2005**, 26, 6176–6184. [Google Scholar] [CrossRef] [PubMed] - Zhang, Y.; Reddy, J.; El-turki, A.; Ramakrishna, S.; Su, B.; Lim, C.T. Biomaterials electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering. Biomaterials
**2008**, 29, 4314–4322. [Google Scholar] [CrossRef] [PubMed] - Lai, G.; Shalumon, K.T.; Chen, J. Response of human mesenchymal stem cells to intrafibrillar nanohydroxyapatite content and extrafibrillar nanohydroxyapatite in biomimetic chitosan/silk fibroin/nanohydroxyapatite nanofibrous membrane scaffolds. Int. J. Nanomed.
**2015**, 10, 567–584. [Google Scholar] - Rao, R.R.; Roopa, H.N.; Kanan, T.S. Solid state synthesis and thermal stability of HAP and HAP–β-TCP composite ceramic powders. J. Mater. Sci. Mater. Med.
**1997**, 8, 511–518. [Google Scholar] [CrossRef] [PubMed] - Deptula, A.; Lada, W.; Olczak, T.; Borello, A.; Alvani, C.; Dibartolomeo, A. Preparation of spherical powders of hydroxyapatite by sol-gel process. J. Non. Cryst. Solids
**1992**, 147, 537–541. [Google Scholar] [CrossRef] - Valet-Regi, M.; Gutierrez-Rios, M.T.; Alonso, M.P.; De Frutos, M.I.; Nicolopoulos, S. Hydroxyapatite particles synthesized by pyrolysis of an aerosol. J. Solid State Chem.
**1994**, 112, 58–64. [Google Scholar] [CrossRef] - Sadat-Shojai, M.; Khorasani, M.-T.; Jamshidi, A. Hydrothermal processing of hydroxyapatite nanoparticles—A Taguchi experimental design approach. J. Cryst. Growth
**2012**, 361, 73–84. [Google Scholar] [CrossRef] - Lim, G.K.; Wang, J.; Ng, S.C.; Chew, C.H.; Gan, L.M. Processing of hydroxyapatite via microemulsion and emulsion routes. Biomaterials
**1997**, 18, 1433–1439. [Google Scholar] [CrossRef] - Chen, C.; Li, J.; Huang, Z.; Cheng, X.; Yu, J.; Wang, H.; Chi, R.; Hu, Y. Phase transformation process and step growth mechanism of hydroxyapatite whiskers under constant impulsion system. J. Cryst. Growth
**2011**, 327, 154–160. [Google Scholar] [CrossRef] - Zhang, Y.; Lu, J. The transformation of single-crystal calcium phosphate ribbon-like fibres to hydroxyapatite spheres assembled from nanorods. Nanotechnology
**2008**, 19, 155608:1–155608:4. [Google Scholar] [CrossRef] [PubMed] - Fu, S.Y.; Feng, X.Q.; Lauke, B.; Mai, Y.W. Effects of particle size, particle/matrix interface adhesion and particle loading on mechanical properties of particulate-polymer composites. Compos. Part B Eng.
**2008**, 39, 933–961. [Google Scholar] [CrossRef] - Goh, K.L.; Aspden, R.M.; Mathias, K.J.; Hukins, D.W.L. Effect of fibre shape on the stresses within fibres in fibre-reinforced composite materials. Proc. R. Soc. London A
**1999**, 455, 3351–3361. [Google Scholar] [CrossRef] - Goh, K.L.; Mathias, K.J.; Aspden, R.M.; Hukins, D.W.L. Finite element analysis of the effect of fibre shape on stresses in an elastic fibre surrounded. J. Mater. Sci.
**2000**, 5, 2493–2497. [Google Scholar] [CrossRef] - Ng, X.W.; Hukins, D.W.L.; Goh, K.L. Influence of fibre taper on the work of fibre pull-out in short fibre composite fracture. J. Mater. Sci.
**2010**, 45, 1086–1090. [Google Scholar] [CrossRef] - De Silva, R.; Pasbakhsh, P.; Qureshi, A.J.; Gibson, A.G.; Goh, K.L. Stress transfer and fracture in nanostructured particulate-reinforced chitosan biopolymer composites: Influence of interfacial shear stress and particle slenderness. Compos. Interfaces
**2014**, 9, 807–818. [Google Scholar] [CrossRef] - Goh, K.L.; Meakin, J.M.; Hukins, D.W.L. Influence of fibre taper on the interfacial shear stress in fibre-reinforced composite materials during elastic stress transfer. Compos. Interfaces
**2010**, 17, 75–81. [Google Scholar] [CrossRef] - Goh, K.L.; Huq, A.M.A.; Aspden, R.M.; Hukins, D.W.L. Nano-fibre critical length depends on shape. Adv. Compos. Lett.
**2008**, 17, 131–133. [Google Scholar] - Goh, K.L.; Aspden, R.M.; Mathias, K.J.; Hukins, D.W.L. Finite-element analysis of the effect of material properties and fibre shape on stresses in an elastic fibre embedded in an elastic matrix in a fibre-composite material. Proc. R. Soc. London A
**2004**, 460, 2339–2352. [Google Scholar] [CrossRef] - Ghosh, S.K.; Pal, S.; Roy, S.K.; Pal, S.K.; Basu, D. Modelling of flame temperature of solution combustion synthesis of nanocrystalline calcium hydroxyapatite material and its parametric optimization. Bull. Mater. Sci.
**2010**, 33, 339–350. [Google Scholar] [CrossRef] - Dalton, A.B.; Collins, S.; Munoz, E.; Razal, J.M.; Ebron, V.H.; Ferraris, J.P.; Coleman, J.N.; Kim, B.G.; Baughman, R.H. Super-tough carbon-nanotube fibres. Nature
**2003**, 423. [Google Scholar] [CrossRef] [PubMed] - Chew, S.L.; Wang, K.; Chai, S.P.; Goh, K.L. Elasticity, thermal stability and bioactivity of polyhedral oligomeric silsesquioxanes reinforced chitosan-based microfibres. J. Mater. Sci. Mater. Med.
**2011**, 22, 1365–1374. [Google Scholar] [CrossRef] [PubMed]

© 2015 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Wang, K.; Liao, K.; Goh, K.L. How Sensitive Is the Elasticity of Hydroxyapatite-Nanoparticle-Reinforced Chitosan Composite to Changes in Particle Concentration and Crystallization Temperature? *J. Funct. Biomater.* **2015**, *6*, 986-998.
https://doi.org/10.3390/jfb6040986

**AMA Style**

Wang K, Liao K, Goh KL. How Sensitive Is the Elasticity of Hydroxyapatite-Nanoparticle-Reinforced Chitosan Composite to Changes in Particle Concentration and Crystallization Temperature? *Journal of Functional Biomaterials*. 2015; 6(4):986-998.
https://doi.org/10.3390/jfb6040986

**Chicago/Turabian Style**

Wang, Kean, Kin Liao, and Kheng Lim Goh. 2015. "How Sensitive Is the Elasticity of Hydroxyapatite-Nanoparticle-Reinforced Chitosan Composite to Changes in Particle Concentration and Crystallization Temperature?" *Journal of Functional Biomaterials* 6, no. 4: 986-998.
https://doi.org/10.3390/jfb6040986