Author’s Accepted Manuscript

Effects of Sintering Temperature on the

Compressive Mechanical Properties of
Collagen/Hydroxyapatite Composite Scaffolds for
Bone Tissue Engineering

Md. Shariful Islam, Mitsugu Todo

www.elsevier.com

PII: S0167-577X(16)30348-2
DOI: http://dx.doi.org/10.1016/j.matlet.2016.03.028
Reference: MLBLUE20479
To appear in: Materials Letters
Received date: 29 December 2015
Revised date: 3 March 2016
Accepted date: 5 March 2016
Cite this article as: Md. Shariful Islam and Mitsugu Todo, Effects of Sintering
Temperature on the Compressive Mechanical Properties of
Collagen/Hydroxyapatite Composite Scaffolds for Bone Tissue Engineering,
Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.03.028
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 Effects of Sintering Temperature on the Compressive Mechanical Properties of

Collagen/Hydroxyapatite Composite Scaffolds for Bone Tissue Engineering

Md. Shariful Islam1, 2 and Mitsugu Todo1*

1
Research Institute for Applied Mechanics, Kyushu University, Kasuga, Fukuoka 816-8580,

Japan.

2
Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi-

6205, Bangladesh.

*Corresponding author:

Mitsugu Todo, Associate Professor

Research Institute for Applied Mechanics, Kyushu University, Kasuga, Fukuoka 816-8580,

Japan. Tel & Fax: +81- 092-583-7762, E-mail: todo@riam.kyushu-u.ac.jp

Abstract

Hydroxyapatite (HA) porous scaffolds were prepared by a template method and

fabricated using either collagen (COL) or COL/HA particles. The sintering temperature was

varied to discover the sintering effects on the mechanical properties of the scaffold. It was

found that fabrication of pure HA scaffolds with COL or COL/HA particles introduced a

distinct layer or phase, causing the fabricated scaffolds to be strengthened and their
mechanical properties to be improved. Moreover, it was found that higher sintering

temperatures also enhanced the mechanical properties of fabricated HA porous composite

scaffolds by reducing porosity.

Keywords: Biomaterials; Scaffolds; Mechanical properties; Hydroxyapatite; Tissue

engineering.

1. Introduction

Mechanically robust and biocompatible biomaterials are crucial for successful tissue

engineering. One of their roles is in the construction of scaffolds to act as the matrix for tissue

formation in vitro in bone tissue engineering [1, 2]. When producing a scaffold for bone

tissue engineering, the selection of material is crucially important. The properties of the

material can potentially be of great use in the scaffold [3]. Biomaterials such as bioceramics

and biopolymers have been widely used in biomedical engineering and bone regeneration.

Bioceramics, hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) for instance, have

been introduced to bone tissue engineering for their unique properties of bioactivity,

biocompatibility and osteoconductivity [4, 5]. HA has been introduced in bone tissue

engineering as a scaffold material mainly due to its unique properties, namely excellent

bioactivity, biocompatibility, osteoconductivity and non-toxicity [4-7]. In addition to these

properties, it is a major inorganic component of bone and its use as both filler and porous

bone scaffold has already been documented, where it enhanced mechanical properties, bone

ingrowth and osseointergration [7-9]. However, the low strength and brittleness of HA have

limited its wide applications in hard tissue implants [10]. In order to expand the range of

applications for HA, and to allow for its use effectively in load bearing compartments, the

mechanical properties of the HA porous scaffold must be enhanced [11].

Collagen (COL) is a natural biopolymer and provides good biocompatibility and low

antigenicity as well as responding well to cell attachment and proliferation [12]. The

combination of HA and COL may provide an excellent structure for tissue forming cells and

their composite scaffolds have been expected to be favorable biomaterials for bone tissue

engineering. Most present research is aimed at the optimization of collagen-based

biomaterials for medical applications by enhancing mechanical strength, biodegradability or

delivery characteristics.

However, the effects of HA particles on the physical and biological properties of the

COL/HA composite scaffolds have not yet been investigated. Hence, one of the objectives of

this research was to develop an effective approach that will enhance mechanical properties of

the biomaterials construction from biopolymer collagen scaffold having composite structure.

A second objective was to understand the effects of sintering temperature on the mechanical

behavior and properties of different combinations of COL and HA particles. It is expected

that varying the sintering temperature might affect phase formation on the microstructural

modification of the as-prepared scaffolds for tissue engineering.

2. Experimental Methodology

2.1 Synthesis of HA porous Scaffolds

 HA porous scaffolds were synthesized using the polyurethane (PU) sponge template

method. HA slurry was prepared from commercial micro-HA powder (PS-1, Sangi Co, Ltd.).

HA powder was mixed with poly vinyl alcohol (PVA) (Wako Pure Chemical Industries, Ltd.)

5wt% solution following the ratio of 1:1 (1g HA:1ml PVA) using a centrifuge mixing

machine (Imoto Co, Ltd.). PU sponge templates were cut into cubes and immersed in the

prepared HA slurry. The excess slurry was removed by 95% compression to avoid pore

blocking, and the cubes were dried at room temperature for 24 hours to allow the HA to settle

well on the sponge framework. The calcination was done at 400° C for 6 h at a heating rate of

10° C/min and finally, sintering was done at 1000 °C, 1100 °C and 1200 °C for 3 hours to

solidify the HA porous scaffolds. The dimensions of the prepared scaffolds were measured by

Digital Vernier Caliper (TDC-150P, TRUSCO PRO TOOL, Japan) and the average value

was approximately 8.7×8.2×7.3 mm3.

2.2 Fabrication of HA porous Scaffolds with Collagen and HA particles

The as-prepared HA porous scaffolds were fabricated in two ways, a) with Collagen-1

solution and b) with HA particles in Collagen-1 solution. For type (a), as-prepared HA porous

scaffolds were dipped into the Collagen-1 solution and vacuumed. Excess collagen solution

was removed, and the samples dried at room temperature for at least 48 hours. On the other

hand, for type (b), HA particles were thoroughly mixed with Collagen-1 solution at 30 °C

with magnetic stirring and then treated following the same procedure as type (a). The

concentration of collagen solution was 1 wt%, and the weight ratio of HA particles and

collagen solution was fixed at 1:9 by following the previous study in which the same ratio

was used to mix β-TCP particles and collagen solution [13]. The actual weight ratio of

collagen and HA particles in the scaffolds was approximated as 9:100. These samples were

named ‘HA-Collagen or HA-Collagen/HA particles coated scaffolds’. Further, both types of

coated HA scaffolds were also fabricated in a specific freeze-drying procedure. HA porous

materials were frozen at -20 °C for 24 hours and then freeze-dried at -50 °C for 24 hours.

These specimens were named ‘HA-Collagen or HA-Collagen/HA particles 2-phase scaffolds’.

3. Characterization and analysis

The phase stability of HA scaffolds was studied using X-ray diffraction (XRD) (Rigaku

RINT- TTR III) and microstructures were characterized using FE-SEM (Hitachi, Ltd. S-

4100) according to Islam et al [14]. Compressive modulus and fracture stress were

evaluated by using a Shimadzu Compact Tabletop Testing Machine EZTest (EZ-S Series).

Further, swelling behaviour of each type of scaffold sintering at 1000 °C was evaluated

according to Sang et al [15]. Simply, scaffolds were immersed in PBS (pH =7.4) solution

for 3, 24, and 48 h at 37 °C in a humidified atmosphere of 5% CO2 described by Arahira

and Todo [13].

4. Results and discussion

X-ray powder diffraction was employed to determine the phase stability of HA porous

scaffolds at various sintering temperature with CuKα (α = 1.5405 nm) and a Ni filter. Raw

HA powder and various scaffolds specimens were scanned from 2θ of 10° to 60° with a step

of 0.02°. Typical XRD patterns of raw HA powder and as-prepared HA scaffolds sintering at

1000 °C, 1100 °C and 1200 °C were represented in Fig. 1 (a), (b), (c) and (d), respectively.

Fig. 1 shows that all XRD peak positions and relative intensities of as-prepared HA materials

match well with that of commercial HA powder materials indicating that the synthesis and

sintering of HA materials did not alter the elements. No diffraction peaks from other

crystalline forms are detected, which demonstrates that the as-prepared HA scaffolds have
phase stability and crystallinity (PDF, File No. 01-076-0694, 01-072-1243 and 00-009-0432,

respectively).

Typical FE-SEM micrographs of pure HA scaffolds and fabricated composite scaffolds

were thoroughly examined (see Supplementary materials) which revealed that the HA porous

materials possessed distinguished porous morphology. Further, Fig. 2 shows the

strengthening mechanism of HA scaffolds by introducing COL or COL-HA particles phases.

In Fig. 2A, HA-COL coating, HA-COL 2-phase and their magnifications (10×) are

represented by (a), (b), (a1) and (b1), respectively, whereas in Fig. 2B, HA-COL/HA particles

coating, HA-COL/HA particles 2-phase and their magnifications (10×) are represented by (c),

(d), (c1) and (d1), respectively. It was evidenced that COL or COL-HA particles were

distributed over the pores of the pure HA scaffolds ultimately introducing a layer (red arrows)

which increased strengthening or load bearing ability or reduced the brittleness of the

prepared biomaterials.

Fig.3 shows the relationships between sintering temperature and compressive mechanical

properties of fabricated HA composite scaffolds. Influences of sintering temperature on the

mechanical properties in terms of modulus and fracture stress are represented in Fig. 3 (a) and

(b), respectively. It was clearly noticed that increasing sintering temperature caused an

increment of modulus in almost every specimen, but the highest modulus was observed in

HA-COL/HA particles 2-phase scaffolds sintered at 1000 °C (Fig. 3a). Moreover, a higher

sintering temperature caused a higher fracture stress and a maximum value was observed in

HA-COL 2-phase scaffolds at every sintering temperature. Simultaneously, very slight

increments in fracture stress were also observed in HA-COL/HA particles 2-phase scaffolds

(Fig. 3b). It was noteworthy that introducing COL or COL-HA particles into pure HA

scaffolds facilitated improved mechanical properties or load bearing ability by reducing the

porosity (see Supplementary materials).

 Fig.4. shows that the swelling ratio of the composite scaffolds reached its maximum at

3h for all specimens. The swelling ratio of the HA-COL coated scaffold exhibited a rapid

increase up to 14.1, (Fig.4a) followed by the HA-COL 2-phase scaffold (b), the HA-COL/HA

particles coated scaffold (c) and the HA-COL/HA particles 2-phase scaffold (d). However,

after 24 hours, the HA-COL coated and the HA-COL 2-phase specimens had shown a decline

in swelling ratio, whereas the HA-COL/HA particles coated and the HA-COL/HA particles

2-phase specimens had become constant. Arahira and Todo also reported that purely collagen

scaffolds have shown a much higher swelling ratio, indicating that the deformability of the

collagen scaffold tends to increase the amount of absorbed PBS [13]. Synthetic scaffold

structures for bone grafting can be prepared from degradable or nondegradable polymers,

bioactive glasses or bioceramics. No matter what the material, scaffolds must have sufficient

porosity to allow for cellular infiltration and proper cell function. Additionally, scaffolds

must be osteoconductive so as to promote tissue regeneration and have the appropriate

mechanical properties to withstand in vivo loading. Considering those points, to the best of

our knowledge, this is the first report of fabricating HA particles layered porous Collagen/HA

composite scaffolds with enhanced mechanical properties for tissue engineering. Hopefully,

investigations into the comparative biocompatibility and the osteoblastic cellular responses of

our fabricated biomaterials are in progress, or soon will be.

5. Conclusions

Porous HA composite scaffolds with different sintering temperature were fabricated by

the template method. Compressive modulus and fracture stress increased with increasing

sintering temperature. A distinctive COL or COL-HA particles layer was introduced into the

pure HA scaffolds, causing reduced porosity and improved mechanical properties.

Acknowledgement
The present work was supported by Grant-in-Aid for Scientific Research (No24•02380)

from Japan Society for the Promotion of Science (JSPS) and Grant-in-Aid for JSPS Fellows

(No.P14362).

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Captions

Fig. 1. XRD patterns of raw HA powder (a) and as-prepared HA scaffolds sintering at

1000 °C (b), 1100 °C (c) and 1200 °C (d).

Fig. 2. Strengthening mechanism of HA scaffolds by introducing COL or COL-HA particles

phase (FE-SEM) sintered at 1000 °C. In plate A, HA-COL coating, HA-COL 2-phase and

their magnifications (10×) are represented by (a), (b), (a1) and (b1), respectively, whereas in

plate B, HA-COL/HA particles coating, HA-COL/HA particles 2-phase and their

magnifications (10×) are represented by (c), (d), (c1) and (d1), respectively.

Fig. 3. Effects of sintering temperature on mechanical properties of as-prepared composite

scaffolds. Data were expressed as mean ± SD and n = 6.

Fig. 4. Swelling behaviour of as-prepared composite scaffolds. Here, HA-COL coating, HA-

COL 2-phase, HA-COL/HA particles coating and HA-COL/HA particles 2-phase were

denoted by (a), (b), (c) and (d), respectively.

Fig. 5. FE-SEM porous microstructure of prepared scaffolds at various sintering temperatures.

Fig.6. Sintering effects on porosity of prepared composite scaffolds.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Supplemental materials

Fig. 5
Fig. 6

Highlights

 Hydroxyapatite porous scaffolds were prepared by a template method and fabricated.

 Fabrication of scaffolds with COL or COL/HA particles introduced a distinct layer.

 Fabricated scaffolds shown improved mechanical proprieties.

 Enhanced mechanical properties was reported by reducing porosity.