Scaffolds for bone restoration from cuttlefish

doi:10.1016/j.bone.2005.06.018

Bone

Volume 37, Issue 6, December 2005, Pages 850-857

https://doi.org/10.1016/j.bone.2005.06.018 Get rights and content

Abstract

Scaffolds of pure hydroxyapatite suitable for either direct clinical use or tissue-engineering applications were successfully produced via hydrothermal transformation of aragonite, obtained from fresh cuttlefish bones, at 200°C followed by sintering. Beyond low production cost, worldwide availability and natural–biological origin of raw materials, the produced scaffolds have ideal pore size and interconnectivity features suitable for supporting biological activities, such as bone tissue growth and vascularization. Bioactivity in vitro tests were excellent: (a) rapid and pronounced formation of hydroxyapatite occurred when the scaffolds were immersed in simulated body fluid (SBF), and (b) outstanding proliferation of osteoblasts was registered. The produced scaffolds can be machined and shaped very easily at any stage of processing. Therefore, these ceramic scaffolds can satisfy both bioactivity demands and the requirements for shaping of tailor-made individualized implants, especially for randomly damaged bones.

Introduction

Living in the era of life control and prolongation [1], artificial implants of hydroxyapatite (HA), Ca₁₀(PO₄)₆(OH)₂, are very popular for hard tissue (e.g., bone) restorations [2] because they accelerate bone growth around the implant. Biological apatites attract special interest since it is believed that the several substitutions at the Ca²⁺, PO₄³⁻ and OH⁻ sites of HA [3] and the presence of several trace elements play an important role in the overall physiological functioning and in the osseointegration process [4]. The poor mechanical properties of pure biomaterials, such as HA, have directed biomaterials design to tissue-engineering approaches [5]. With regard to materials, the interest is addressed to the production of porous scaffolds, which can host the biological activities in a physiological manner.

Advanced techniques, such as rapid prototyping or the use of foaming agents [6], have been employed to produce porous scaffolds of synthetic HA, but beyond expensive apparatuses needed, there is still poor control of the internal porous architecture. Hydrothermal transformations (HT) of corals from the Pacific Ocean have gained interest since 1970s [7], [8] because the resultant materials are composed of HA and feature similar microstructure to the inorganic mineralized structure of natural bones. Several processing routes, reactors and set-up approaches have been proposed [7], [8], [9], [10], [11]. However, beyond special apparatus and high temperatures needed, these methods require the use of corals, which are not available worldwide or are even species in danger.

The present paper proposes a new approach for developing highly bioactive bone-scaffolds of HA from cuttlefish bones via hydrothermal transformation, aiming at providing a thorough solid documentation to cope with the lack of literature reports on this issue. The new developed HA-scaffolds beneficiate from the following important advantages:

(a)
Worldwide availability and very low cost of raw materials of natural–biological origin.
(b)
Utilization of very simple and inexpensive apparatus.
(c)
Rapid and very efficient transformation of aragonite (CaCO₃) to HA at relatively low temperatures (200°C), minimizing the security risks due to relatively low developed pressures (∼15 atm) in the chamber [12].
(d)
Maintenance of porous structure after sintering and remarkable thermal stability of HA up to 1350°C.
(e)
Optimal pore size and pore interconnectivity features for hosting physiological activities (i.e., tissue growth and vascularization).
(f)
Excellent in vitro bioactivity, as indicated by the rapid and pronounced formation of HA when the sintered scaffolds are immersed in simulated body fluid (SBF) and very good biocompatibility with osteoblasts.
(g)
Excellent machinability for rapid shaping according to the demands.

The formation (i.e., biomineralization) of cuttlefish shells (also usually named as cuttlefish bones) and other similar sea plants (e.g., nacres etc.) is well documented in the literature [13]. The inorganic part of cuttlefish bone is an anisotropically (i.e., lamellar, Fig. 1) mineralized porous structure of aragonite. The hydrothermal transformation (HT) of aragonite to HA should occur according to the following widely reported chemical equation:10CaCO₃ + 6(NH₄)₂HPO₄ + 2H₂O → Ca₁₀(PO₄)₆(OH)₂ + 6(NH₄)₂CO₃ + 4H₂CO₃

Section snippets

Hydrothermal transformation and sintering

Bones of fresh cuttlefishes from the species Sepia officinalis [14] (fished few hours before the experiments) were gently cut into small pieces using a lancet. Differential thermal analysis (DTA/TG, Labsys Setaram TG-DTA/DSC, France, heating rate 5°C/min, in air) was employed to evaluate the exact CaCO₃ content of cuttlefish bone. According to these results, for each batch of cut pieces of fresh cuttlefish bone, the required volume of an aqueous solution of (NH₄)H₂PO₄ was added in order to set

Evolution of phases

The fresh cuttlefish bone comprises of pure aragonite (Fig. 2a). Nevertheless, the relative intensity of the peaks significantly differs from the standard aragonite, plotted at the bottom side of this diagram. The observed differences are plausibly due to the specific layered structure of aragonite (Fig. 1) developed during biomineralization of cuttlefish bone in the sea. A great difference between the intensities of the aragonite peaks was noticed when the X-ray incidence beam was parallel or

Conclusions

Natural aragonite from cuttlefish bone was hydrothermally transformed into hydroxyapatite inside sealed autoclaves at 200°C. The as-obtained hydroxyapatite phase exhibited high thermal stability on sintering up to 1350°C. Transformation to β-TCP occurred upon heat treatment at temperatures ≥1400°C.

The interconnected porous structure of cuttlefish bones was preserved in the produced hydroxyapatite scaffolds. These structural features, associated with the very good in vitro bioactivity in SBF and

Acknowledgments

Thanks are due to the Portuguese Foundation for Science and Technology for the financial support within the project POCTI/CTM/60207/2004 and the fellowship grants of A.F. Lemos (BD/8755/2002), S. Agathopoulos (BDP/1619/2000) and S. Kannan (BPD/18737/2004).

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Scaffolds for bone restoration from cuttlefish

Abstract

Introduction

Section snippets

Hydrothermal transformation and sintering

Evolution of phases

Conclusions

Acknowledgments

Curr. Opin. Solid State Mater. Sci.

Biomaterials

Mater. Sci. Eng. C

Biomaterials

Biomaterials

Biomaterials

Biomaterials

Bone

J. Eur. Ceram. Soc.

Biomaterials

J. Cryst. Growth

Science, faith and ethics

Biological and medical significance of calcium phosphates

Angew Chem.

Dense hydroxyapatite

The influence of sintering temperature on mechanical and microstructural properties of bovine hydroxyapatite

Key Eng. Mater.

Tissue engineering