Keywords
nonsintered - hydroxyapatite - block - hydrothermal - compressive strength
Introduction
Hydroxyapatite (HA) is known as a biocompatible and osteoconductive bone graft material.[1] It has been used for decades with good clinical outcomes.[2] Despite good clinical results, its slow resorption is the main drawback of HA bone
grafts.[3]
[4] Commercial HA bone grafts are often prepared through sintering. This type of HA
has low resorption during implantation. Nonsintered HA bone graft would be more resorbable
compared with sintered one.[5]
Deproteinized bovine bone is one of the most widely used nonsintered HA.[6]
[7]
[8] Bio-Oss is one of the deproteinized bovine bone products that is used clinically.
It is derived from the bovine bone using a chemical reaction to remove organic substances.
However, deproteinized bovine bone HA such as Bio-Oss also has slow resorption.[4] Tadjoedin et al[9] have reported that the resorption rate of Bio-Oss is 10% per year. Another study
reported that Bio-Oss has remained even after 4.5 years of implantation. Several studies
have been reported on the fabrication of nonsintered HA other than the deproteination
method. One method was developed by Suzuki et al to produce an HA block using calcium
sulfate dihydrate (CSD) as a precursor via the dissolution–precipitation technique.[10] The results suggested that HA was formed. However, brushite was also found in the
fabricated block as impurities.
Previously, we successfully fabricated new nonsintered HA blocks through the phase
transformation of gypsum blocks under hydrothermal conditions.[11] The gypsum block with a diameter of 6mm and height of 3mm could be fully transformed
into an HA block at 180°C for 24 hours. However, the compressive strength of the HA
block has not been known. Therefore, this research aimed to evaluate the compressive
strength of the newly developed non-sintered HA block.
Materials and Methods
Sample Preparation
Nonsintered HA blocks were prepared according to the method that was previously reported
with the modification of sample dimension and hydrothermal reaction time. Calcium
sulfate hemihydrate (CSH) was mixed with distilled water at the liquid-to-CSH powder
ratio of 0.5 according to the previous report.[11] The paste was molded in a cylindrical split mold (4 mm in diameter and 8 mm in height)
made of acrylic. Briefly, the paste was taken from the mixing bowl using a spatula
and placed in the mold. Two glass slides were put at the top and the bottom of the
mold and pressed with paper clips to firm it. The paste was left to set for 24 hours
at room temperature. The set gypsum blocks were immersed in a polytetrafluoroethylene
(PTFE)-lined vessel containing 1 mol/L sodium phosphate (Na3PO4).12H2O (Merck, Darmstadt, Germany) solution. The PTFE-lined vessel was then placed in a
hydrothermal vessel consisting of a shell made of stainless steel. The hydrothermal
vessel was put in an oven at 100°C, 140°C, and 180°C for 48 hours. After hydrothermal,
the specimens were washed with distilled water three times and dried at 37°C for 24 hours.
Material Characterization
The gypsum block and the obtained blocks were crushed into a powder. The powders were
characterized using X-ray diffraction (XRD) (PANanalytical-Xpert Pro; CuKα, λ = 1.54Å).
The XRD characterization was performed at a current of 30 mA and voltage of 40 kV
with the step size of 0.0170 from 2θ of 10.0084 to 89.9764°. Rietveld refinement of
the obtained XRD peaks was employed using Xpert Highscore software to determine the
phase composition, lattice parameter, and crystallite size of the HA crystal formed
in each specimen. An automatic Rietveld profile was used (Pseudo-Voigt fitting).
Mechanical Test
For mechanical strength measurement, the gypsum and the obtained blocks were subjected
to compressive strength tests using a universal testing machine (Shimadzu, AGSX-50Kn).
The load cell was 500 N with a crosshead speed of 0.5 mm/minute. The force at which
the block started to break was recorded. Nine specimens were used for each group to
determine their compressive strength. The number of specimens was calculated by the
Federer formula. The average value of compressive strength was then calculated.
Data Analysis
Statistical analysis was done to determine the significance of the mechanical strength
among the samples using SPSS Software. Shapiro–Wilk test was used to determine the
normality of the data, followed by one-way analysis of variance. The significance
of the compressive strength among the group of samples was calculated by Tamhane post-hoc
analysis, in which a p-value of 0.05 was considered significant.
Results
Characterization of HA Blocks
[Fig. 1] shows the photograph of the gypsum block and the obtained block after hydrothermal.
After hydrothermal, the block did not crumble or collapse. [Fig. 2] demonstrated the XRD peaks of gypsum and the obtained blocks. The XRD peaks were
indexed using a crystallography open database (COD). After immersion at 100°C, the
gypsum phase was transformed into HA (COD: 96–230–0274). However, calcium sulphate
(CaSO4) anhydrate (COD: 96–900–4097), gypsum (COD: 96–901–3165), and portlandite (COD: 96–900–0114)
phases were also found as listed in [Table 1]. As the temperature increased to 140°C, the obtained block was composed of 97.5%
HA and 2.5% portlandite (Ca(OH)2). At the temperature of 180°C, the obtained block is considered fully transformed
HA (99.5%) with only a trace amount of portlandite (0.5%). The intensity of HA peaks
was higher with the increasing temperature. Crystallite size increased with the increasing
temperatures ([Table 2]). The a-lattice decreased with the increase in temperature. The c-lattice increased from 100°C to140°C but decreased at 180°C ([Table 2]).
Table 1
Crystal phases of the obtained block after immersion in 1 mol/L Na3PO4 at 100°C, 140°C, and 180°C for 48 hours
|
Group
|
Phases
|
|
HAP-100
|
Hydroxyapatite (63.6%)
CaSO4 anhydrate (28.2%)
Gypsum (4.2%)
Portlandite (4.0%)
|
|
HAP-140
|
Hydroxyapatite (97.5%)
Portlandite (2.5%)
|
|
HAP-180
|
Hydroxyapatite (99.5%)
Portlandite (0.5%)
|
Abbreviations: CaSO4, calcium sulphate; Na3PO4, sodium phosphate.
Table 2
Unit cell parameter and crystallite size of the obtained blocks obtained using Rietveld
analysis
|
Sample name
|
Lattice parameter
|
Crystallite size (nm)
|
|
a (Å)
|
b (Å)
|
c (Å)
|
|
HAP-100
|
9.433
|
9.4335
|
6.8906
|
20.66
|
|
HAP-140
|
9.422
|
9.4226
|
6.8929
|
34.86
|
|
HAP-180
|
9.418
|
9.4187
|
6.8869
|
65.06
|
Fig. 1 Photograph of gypsum block (A) and the obtained block after immersion in sodium phosphate (Na3PO4) at 100°C (B), 140°C (C), and 180°C (D) for 48 hours. CaSO4, calcium sulphate.
Fig. 2 X-ray diffraction peaks of gypsum block, the obtained block after immersion in sodium
phosphate (Na3PO4) at 100°C, 140°C, and 180°C, and hydroxyapatite reference.
Compressive Strength
The compressive strength of the gypsum and the obtained blocks were shown in [Fig. 3]. Gypsum block showed a compressive strength value of 22.11 ± 2.03 MPa. After immersion
in Na3PO4 solution, the compressive strength was decreased to 4.92 ± 0.70 MPa, 5.28 ± 0.49
MPa, and 3.43 ± 0.27 MPa for HAP-100, HAP-140, and HAP-180, respectively ([Table 3]). The compressive values from each specimen per group were subjected to the Shapiro–Wilk
normality test. Normal distribution was obtained from the test. Further, the homogeneity
test was performed and the data showed not homogenous. Thus, Tamhane's post-hoc analysis
was done to see the difference between the group. The difference in compressive strength
value between the gypsum block and the obtained blocks after hydrothermal was statistically
significant. However, within the obtained blocks, only HAP-180 shows a significant
value compared with both HAP-100 and HAP-140. Meanwhile, between HAP-100 and HAP-140,
the values were not significant.
Table 3
List of average compressive strength of gypsum block and the obtained block after
immersion in Na3PO4 at 100°C, 140°C, and 180°C compared with human cancellous bone
|
Group
|
Average compressive strength (MPa)
|
|
Gypsum
|
22.11 ± 2.03
|
|
100°C
|
4.92 ± 0.70
|
|
140°C
|
5.28 ± 0.49
|
|
180°C
|
3.43 ± 0.27
|
|
Cancellous bone
|
0.1–16
|
Abbreviation: Na3PO4, sodium phosphate.
Fig. 3 Compressive strength of gypsum block and the obtained block after immersion in sodium
phosphate (Na3PO4) at 100°C, 140°C, and 180°C (n: 6; *p < 0.05, n.s.: not significant).
Discussion
HA has been widely used as a bone graft in dentistry.[12]
[13]
[14]
[15] Bovine-based HA is among the most applied bone grafts besides sintered HA due to
their biocompatibility and osteoconductivity.[16]
[17]
[18]
[19] The main drawback of bovine HA and sintered HA is their slow resorption during implantation.
This study attempted to fabricate nonsintered HA to improve resorption. Previously,
the newly developed nonsintered HA showed better solubility compared with sintered
HA in an acetate buffer solution that simulates osteoclastic environments.[10] The solubility of bone graft material in acetate buffer was reported to directly
correlate to its resorption during implantation.[20]
[21] Higher solubility was thought due to the carbonate content found in the HA crystal.
It was reported that carbonated HA showed higher solubility in osteoclastic simulation
due to the release of carbonate ions.[21]
This study aimed to evaluate the compressive strength of nonsintered HA obtained via
phase transformation of gypsum block under hydrothermal conditions. Besides, phase
purity, lattice parameter, and crystallite size of the obtained blocks were also evaluated.
In this study, a nonsintered HA block could be fabricated via hydrothermal reaction
using a gypsum block. The obtained blocks were not collapsed after hydrothermal treatment,
which suggests the method preserved the original sample shape. This is important since
it could be used for other complex shapes.
Based on the XRD, hydrothermal reaction at 100°C produced not only the HA phase but
also the CaSO4 anhydrate, gypsum, and portlandite phases. CaSO4 anhydrate and portlandite were most probably intermediate phases that formed before
they transformed completely into the HA phase. The gypsum phase was still detected
at 100°C (4.2%). At 140°C, CaSO4 anhydrate and gypsum phases were transformed completely into the HA phase, and only
2.5% portlandite remained. The gypsum phase was no longer detected at 140°C. Further
increase in hydrothermal temperature to 180°C produced HA blocks with the highest
purity (99.5%) and only a trace amount of portlandite phase (0.5%) was detected. Therefore,
the gypsum block could be considered fully transformed into an HA block. It is known
that HA is the most stable calcium phosphate phase at alkaline pH.[22] In this study, gypsum block was immersed in Na3PO4 solution that has a very basic pH; thus it would create a condition for HA precipitation.
As a result, an HA block could be formed. The lattice parameters of the obtained blocks
were changed with the increasing hydrothermal temperature. Our previous results suggest
that these changes might be due to the crystal growth and the substitution of carbonate
ions into the HA crystal.
Phase transformation from gypsum block into HA block decreased compressive strength
considerably. Based on [Fig. 3], the decrease in compressive strength was up to 75% regardless of the reaction temperatures.
The lowest compressive strength value was shown in HAP-180 where the gypsum block
was considered fully converted to HA phase. The decrease in compressive strength might
be caused by the change in microstructure due to the phase transformation of the gypsum
block into HA. Previously, our research group has found that after phase transformation,
more pores were formed between the interlocked crystals of the HA block compared with
that of the gypsum block precursor.[10] These more pores observed in the obtained blocks might cause a decrease in compressive
strength.
The HA block obtained in this study is intended for nonload-bearing applications.
Nevertheless, obtaining an HA block having compressive strength close to that of human
bone is preferred. It was reported that the compressive strength of human cancellous
bone is ranged between 0.1 and 16 MPa.[23] In this study, the compressive strength of the obtained blocks decreased after phase
transformation due to the formation of HA crystals. The decrease in compressive strength
is expected not to affect the material's performance clinically. The obtained HA-180
has a compressive strength of 3.43 MPa, which is still in the range of human cancellous
bone.
Although the nonsintered HA block has shown promising results as a bone graft candidate
with better resorbability than bovine bone, additional evaluation is necessary before
it can be used in clinical settings. Further evaluations include cytotoxicity and
animal tests to prove the osteoconductivity of the material.
Conclusion
Gypsum block could be fully transformed into HA block via hydrothermal reaction at
180°C for 48 hours. The compressive strength of the obtained blocks decreased significantly
compared with the gypsum block with the increase in the HA phase. The compressive
strength of the obtained HA block is still in the range of that of cancellous bone.
