Shinshu Med J, 66⑵：139～150, 2018

Mechanical Properties and Histological Evaluation ofBone Grafting Materials Containing Different Ratios of Calcium
Phosphate Cement and Porous β-tricalcium Phosphate Granules
Hirokazu Tanaka1）2）, Shin-ichi Yamada1）＊, Hitoshi Aizawa1）
Kiyonori Hayashi1）, Tetsu Shimane1）, Imahito Karasawa1）
Nobuhiko Yoshimura1）, Fumihiro Nishimaki1）, Yuko Arakawa1） and Hiroshi Kurita1）
1） Department of Dentistry and Oral Surgery, Shinshu University School of Medicine
2） Department of Dentistry and Oral Surgery, Aizawa HospitalObjective : Although calcium phosphate cement (CPC) is a bone substitute material having good formativeness,
biocompatibility, and osteoconductivity, it has very slow biodegradability in vivo. This study aimed to create a
new porous bone cement with enough strength, good formativeness, biodegradation, and osteoconductivity forbone regeneration by adding porous β-tricalcium phosphate (β-TCP) granules to CPC.
Methods : A commercially available CPC and porous β-TCP were used in this study. Three testing materials
with different mixing ratios of CPC and β-TCP (C0, C30 and C50, having mixing ratios of β-TCP of 0, 30, and50 wt%, respectively) were examined. We evaluated the basic materialological properties in vitro. In addition,
biodegradation and osteoconductivity were evaluated in vivo by implantation in rabbit femurs.
Results : The setting times for C30 (15 minutes) and C50 (18 minutes) were thought to be within a clinically
acceptable range. Although the compressive strength decreased with increasing content of β-TCP, that of C50
was the same as for cancellous bone. The porosity and permeability increased with addition of more β-TCP
granules. Histological evaluation revealed that biodegradability of the material increased with the addition of
more β-TCP granules and the material had good bioactivity. Notably, at 36 weeks after implantation, C50 was
almost completely resorbed and replaced by regenerated bone.
Conclusions : The results of this study suggested that the composite created by adding equal weight percent ofporous β-TCP granules to CPC paste had good biodegradability and adequate mechanical strength, and wasadaptable as a bone substitute for bone defect repairs. Shinshu Med J 66 : 139―150, 2018
(Received for publication November 22, 2017 ; accepted in revised form December 28, 2017)
Key words : calcium phosphate cement, β-TCP, bone regeneration, bone replacement
have excellent biocompatibility and osteoconductivi­

Ⅰ Introductionty10）-12）, its very slow biodegradability must be im­

Various calcium phosphate cements (CPC) have

proved to promote bone regeneration. As the reac­

1）
-10）

.

tion of CPC progresses, hydroxyapatite (HA) is

CPC paste has been used in bone reconstruction

precipitated13）. HA is hardly absorbed in vivo. Espe­

because of its ability to be shaped similarly to thecially in oral and maxillofacial surgery, non- or slow

bone defect. Although CPC has been reported tobiodegrading bone materials might carry a higher

been studied for application as bone substitutes

risk of infection. One way to improve bioabsorption＊

Corresponding author : Shin-ichi Yamada
Department of Dentistry and Oral Surgery,
Shinshu University School of Medicine,
3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
E-mail : yshinshin@shinshu-u.ac.jp

No. 2, 2018

and bone replacement is to increase the porosity ofhardened CPC.
β-tricalcium phosphate (β-TCP) has been widelyused as a bone substitutional material for bone re­139

Tanaka・Yamada・Aizawa et al.

generation because of its excellent biocompatibili­
14）
15）

ty

. β-TCP biodegrades more rapidly than crys­
16）
-19）

tively) were prepared. At first, the CPC powder was
kneaded with the malaxation liquid for 60 seconds.

. Porous β-TCP is considered to be a

Thereafter, different wt % of β-TCP granules was20）

more suitable scaffold for bone formation than HA .

added into the CPC paste and was softly mixed for

However, β-TCP has some problems such as poor

30 seconds at room temperature. The CPC powder/mechanical strength and formativeness.

malaxation liquid (P/L) ratio was 3.57 for C0, 3.18 fortalline HA

By taking advantage of the formability and streng­th of CPC and the bioabsorbability and osteoconduc­

C30, and 2.1 for C50 in order to achieve the same
final consistency．

tivity of β-TCP, we tried to create new porous bone
cement composed of porous β-TCP granules andIn vitro study

CPC with sufficient strength, good formativeness, andC

Consistency

biodegradation for bone regeneration. We mixed po­

The consistency of the composite paste was mea­rous β-TCP and CPC paste to create a composite

sured using the following procedures : one gram ofcontaining macro- and micropores. Its mechanicalthe paste-like samples just after kneading wasproperties, micro- and macro-structures were ex­

gently placed on a glass plate, then another 120 g

amined in vitro, and biodegradation, biocompatibility,

glass plate was gently placed on the paste and theand osteogenesis in vivo.

longest and shortest diameters of the spread paste

Ⅱ
A

Materials and Methods

CPC and β-TCP

were measured in mm. The measurements werecarried out five times and the values averaged.
D

Measurement of paste setting time

The CPC used in this study was Cerapaste®(NGK

The mixed composite cement paste was placed in

Spark Plug Co. Ltd, Aichi, Japan). It consists ofa ring (internal diameter : 10 mm, width : 7 mm) andmixed powder and liquid. The powder contains

flattened on its top surface. The paste was thentetracalcium phosphate (TeCP : 67.5wt%) and dibasic

incubated at 37±1 ℃ and 95% to 100% humidity. A

calcium phosphate anhydrous (DCPA : 32.5%), andflat-tipped 2-mm-diameter needle was gently placed

the liquid contains water and dextran sulfate sodium.

on the top surface at different time points. The

The hardening reaction of the CPC is temperature

setting time was defined as the time at which nodependent ; it starts hardening promptly at 37 ℃

indent was formed by the needle (300 g in weight).

(near body temperature), and hardens slowly at

Measurement was carried out 5 times and the val­

25 ℃ (near room temperature), and finally hardens

ues averaged.

as HA.

E

The β-TCP used in this study was Cerabeta®gran­Measurement of the compressive strength of
the hardened bodyules (NGK Spark Plug Co. Ltd, Aichi, Japan) with a

The mixed composite cement paste was injected

diameter of 0.6-1.0 mm. The granule has a hollow in­

into plastic cylindrical containers (internal diameter :terior and porous shell. The porosity of Cerabeta®

7 mm ; height : 14 mm). After setting, the hardened

granules is 35%, the macroporosity is 200 μm, andbody was immediately picked-up and immersed in

the microporosity is one μm in diameter. CPC anddistilled water (DW) and incubated at 37±1 ℃ for

β-TCP were kindly provided by NGK Spark Plugone day, and then used for measurement of com­

Co. Ltd.

pressive strength. Compressive strength was meas­

Bured using a universal testing machine (AG-100kNIS

Fabrication of study samples (composite of CPC
and β-TCP granules with different ratios)MO®, Shimadzu Corporation, Kyoto, Japan) with aThree testing samples with different mixing ratios

crosshead speed of 0.5 mm/min, and the mean valueof β-TCP granules to CPC (C0, C30, and C50, havingof 5 samples was calculated.

mixing ratios of β-TCP of 0, 30, and 50 wt%, respec­

F

140

Density and Porosity
Shinshu Med J. 66

Mechanical properties and biotransformation of bone grafting materials composed of CPC and β-TCP

To estimate the bulk density and total porosityof the hardened body, samples were prepared in
the same way as for measurement of compressive
strength. The total porosity (P) of the specimens wascalculated as follows : dry weight (M) of each speci­
men was divided by volume (V) to obtain density (D).
This was divided by the theoretical density of hy­
droxyapatite (DHAp＝3.16 g/cm3), subtracted from 1,

Fig. 1

Implants (hardened composites) withdifferent CPC/β-TCP ratios
The surface is rough as the mixing ratioof β-TCP increases.

and expressed as a percentage.
D＝M/V (1)P＝100×(1-D/DHAp) (2)
D＝Density
M＝Dry Weightcenter height was cut using a model trimmer (Y-230,

V＝Volume

Yoshida Dental Distribution Co. Ltd., Tokyo, Japan),and the red-stained area was measured using NIH

P＝Porosity
DHAp＝Density of hydroxyapatite＝3.16 g/cm
G

3

SEM micrographs of the cross-section of the

ImageJ software (National Institutes of Health,Bethesda, MD). The average of 5 samples was calcu­

hardened-body

lated. The penetration rate (Pr) was calculated ac­

The surface morphology of the gold coated hard­cording to the following formula :Pr＝red stained area / total area×100%

ened body was analyzed by scanning electron mi­
croscopy (SEM, Stereoscan S360 Cambridge Ltd.) at
an operating voltage of 20 kV and electrical current

In vivo studyof 10 mA.

K

H

X-ray computed microtomography of the hard-

Implantation of the samples
Six male New Zealand white rabbits weighing

ened body

between 3.5 and 4.0 kg were used for this study. All

After one day soaking in DW, the hardened bodyof the surgical procedures were performed under

was scanned using 3D micro-CT (CosmoScan, Riga­

general and local anesthesia by administering 3%

ku Corp., Tokyo, Japan). The X-ray source operates

pentobarbital (30 mg/kg/i.v. Somnopentyl ; Kyoritsu

at 90 kV and 150 μA, scan time 8 min, image range

Seiyaku, Japan) and 2% lidocaine (1 ml i.m. ; Astra­(9.6×9.6 mm) and voxel size (20×20×20 μm). I-

Zeneca, Osaka, Japan). A cylindrical defect throughview-R (J. Morita Mfg. Corp., Kyoto, Japan) was usedthe cortical bone surface into the cancellous boneas the viewer.

was prepared with a trephine drill (4 mm diameterI

X-ray diffraction analysis (XRD)

and 10 mm depth) in the lateral epicondyle of the

The phase composition of each synthesized mate­bilateral femora at a low rotational speed under con­rial after 2 days soaking in DW was checked bytinuous saline cooling. Subsequently, each defect was

XRD (Ultima IV, Rigaku Corp., Tokyo, Japan) withfilled with already-hardened implant materials andCu-kα radiation operated at 40 kV and 40 mA inthen closed with sutures in layers. Each implantcontinuous scanning mode at a step size of 0.02°andmaterial was made by filling a cylinder (cylinder-a scanning rate of 2.00°/min.

shaped ; 4 mm in diameter and 10 mm in height) andJ

incubating at 37±1 ℃ and 95% to 100% humidity for

Penetration test
Each hardened-body (cylinder-shaped ; 7 mm in

30 min (Fig. 1). The left and right femora were ran­diameter and 14 mm in height) was immersed in a

domly assigned to receive study samples (C0, C30,

red-colored propylene glycol solution for 24 hours.

and C50). The samples were placed in 12 femur bones

Thereafter, a horizontal section at the representativeof 6 rabbits. Postoperatively, penicillin was admin­

No. 2, 2018141

Tanaka・Yamada・Aizawa et al.

istered to prevent infection (50000 IU/kg) for 3 days.

tios, L/P ratio), consistency, and setting times for the

The animal protocol was approved by the Committee

composite pastes with different mixing ratios offor Animal Experiments of Shinshu University

β-TCP granules to CPC are shown in Table 1. Us­(Approval Number #280022).

ing the recommended malaxation conditions (L/P

L

ratio : 3.57), the setting time of CPC cement was 10±

Histological evaluation
After 4, 12 and 36 weeks, rabbits were sacrificed

1.09 minutes. To get the same consistency as theby an overdose of pentobarbital. All samples includ­

CPC cement, C30 and C50 required L/P ratios of 3.18

ing surrounding bone tissue were extirpated en bloc,and 2.10, respectively. The setting time increased

fixed by immersion in 10% neutral buffered formalin,with additional β-TCP to 15±1.72 minutes for C30

and subjected to histological assessments. For histo­and 18±1.41 for C50. The setting time of the com­morphometric examination, the thigh bone samples

posite had a direct relation with the mixing ratio of

were dehydrated in a graded series of ethanol, em­

β-TCP.

bedded in light-curing resin (Technovit 7200 VLC,

B

Compressive strength

Kulzer, Wehrheim, Germany), and cut into sections

Fig. 2A shows the compressive strengths of C0,

150 μm in thickness with a diamond saw (Cutting

C30, and C50. There was a significant difference

Grinding System, BS-300CP band system, EXAKT,

among them (Kuruskal-Wallis, p<0.05). The compres­

Apparatebau, KG, Norderstedt, Germany). Sections

sive strength of C0 was 28 MPa (median). As β-TCP

were then ground to a thickness of about 60 μm

content increased, the compressive strength signifi­

with a micro-grinding system (400 CS micro grind­cantly decreased ; the median of compressive streng­

ing system, EXAKT Norderstedt, Germany). The

th of C30 was 22 MPa and that of C50 was 13 MPa.

section used for staining was cut at the center of theC

Total porosity rate

sample. These bone samples were stained with 5%

The total porosity rates of the hardened compos­

toluidine blue, and examined with a light microscope

ites are shown in Fig. 2B. There was a significant(Biorevo BZ-9000, Keyence, Osaka, Japan).

difference among the different mixing ratios of β-

Biodegradation of the implant material (absorption

TCP granules to CPC (Kruskal Wallis test, p<0.05).

rate) was measured using NIH ImageJ software (Na­Porosity rates increased with increasing β-TCP

tional Institutes of Health, Bethesda, MD). Regions ofcontent (median ; 27% in C0, 31% in C30, and 36% ininterest (ROI ; 1.5 mm in length and 1.5 mm in width)

C50).

were set randomly in 3 central and 3 surface areas

D

SEM micrographin each histological section (toluidine blue staining).

The surface morphology of the cross-section of

The absorption rate (Ar) was calculated using thehardened composite was observed by SEM (Fig. 3).

following formula : Ar＝area of ROI - area of residual

C30 and C50 were increasingly rough on the surface

sample / area of ROI×100%

in that order. In addition to the macropores of β-M

TCP granules, CPC cement had other macropores or

Statistical analysis
Statistical analysis was performed using one-way

fissures. In C30 and C50, CPC cement was present be­

ANOVA, Kruskal Wallis test and Wilcoxon test by

tween the β-TCP granules and adhered to them. The

means of SPSS 13.0 computer software (SPSS Inc,

macropores of the β-TCP granules were present

Chicago, IL, USA). P values less than 0.05 were con­without any involvement of CPC cement in both C30

sidered significant.

and C50.
Ⅲ

Results

E

Micro-Computed Tomography (Micro-CT)
Several shapes and sizes of pores were observed

In vitro study

in the hardened composite cements (Fig. 4). In C0,

Aseveral sizes of macropores were observed in the

Setting time
The malaxation conditions (liquid-to-powder ra­
142

CPC cement. In C30, β-TCP granules were scat­
Shinshu Med J. 66

Mechanical properties and biotransformation of bone grafting materials composed of CPC and β-TCP
Table 1

Setting times of composite pastes of CPC and β-TCP
β-TCP mass%in
CPC

Powder/liquid ratio
Setting time (min)Consistency (mm)

CO

C30

C50

3.57
10±1.0920

3.18
15±1.72202.10
18±1.41
20

Ａ

Ｂ

Fig. 2
A : Compressive strength of hardened composites of CPC cement and β-TCP granules (n＝5).
As β-TCP content increased, the compressive strength decreased.
There was a significant correlation between compressive strength and β-TCP contents (Kruskal-Wallis test : p＝0.014).
B : Total porosity of hardened composites of CPC cement and β-TCP granules (n＝5).
Porosity of the cements had a tendency to increase with the increment of β-TCP content
There was a significant relationship between the porosity and β-TCP contents (Kruskal-Wallis test : p＝0.016).

tered among CPC cement, and macropores wereof β-TCP.

mainly observed in the β-TCP granules. In C50,

G

Penetration test

β-TCP granules were in contact with each other in

To examine the penetration rate, cross sections of

CPC cement. Macropores were observed in both

hardened composites were observed after 24 hours’

CPC and β-TCP granules. Macropores tended toimmersion in red solution (Fig. 6A). The area stained

increase with increasing β-TCP content. The mac­with red solution became larger with increasingropores of the β-TCP granules were maintained

weight % of β-TCP granules. In C0, only the outer

without any involvement of CPC cement.

layer was stained. In C30, red solution penetrated up

X-ray diffraction (XRD) analysis

to 2 mm but did not reach the center part. In C50,F

The XRD patterns of C0, C30, and C50 are shown

red solution reached the center of the composite

in Fig. 5. The crystalline phase of CPC cement (C0)cement. Almost all of C50 was dyed with red, and its

contained hydroxyapatite (HA) and tetracalcium

outer surface collapsed and became rough. The cal­

phosphate (TeCP), resulting from the low crystallini­

culated median penetration rates were 20.5% in C0,

ty of CPC cement. The crystalline phases of C30 and

73.3% in C30, and 96.5% in C50. The difference was

C50 contained β-TCP, and they had decreasing

statistically significant (Fig. 6B, Kruskal Wallis test,

amounts of HA and TeCP with increasing weight %p<0.05).

No. 2, 2018143

Tanaka・Yamada・Aizawa et al.

Fig. 3

Cross-section analysis of the hardened composite bodies of CPC cement and β-TCP granules
β-TCP granules were observed diffusely in C30 and C50. β ; β-TCP, ; Pore ; Macropore of β-TCP, C ; CPC.

Fig. 4 Micro-computed tomography of the materials used in this study
C30 and C50 contain β-TCP granules (blue arrows). Blue arrows ; β-TCP, ; P ; Macropore of β-TCP, C ; CPC.
144

Shinshu Med J. 66

Mechanical properties and biotransformation of bone grafting materials composed of CPC and β-TCP

Fig. 5 X-ray diffraction patterns of cements composed of CPC and β-TCP.
As the mixing rate of β-TCP increased, the HA formations were decreased.
○ ; HA, β ; β-TCP, 4 ; TeCP.

Ａ

Ｂ
Fig. 6
A : Cut surfaces of cements composed of CPC and β-TCP in penetration test.
B : Penetration rates of cements composed of CPC and β-TCP (n＝5).
As the mixing rate of β-TCP increased, the penetration rate also increased.
There was a significant difference in penetration rates among C0, C30, andC50 (Kruskal-Wallis test, p＝0.002).

In vivo study

face of β-TCP (Fig. 7A : C30-12W-H, C50-12W-H).

H

Furthermore, in C50, absorption also occurred fromHistological evaluation
Histological photos of each specimen are shown in

Fig. 7A. In all samples, at 4 weeks after implantation,

the inside of the implant material (Fig. 7A : C5012W-L).

living bone was directly in contact with outer andIn C0, the living bone and the implant were close­

inner surfaces (especially in C30 and C50) of thely adherented at 4 weeks after implantation. In ad­

implanted material without any interposing tissue.

dition, the surface of the implant material was cov­

The β-TCP was absorbed with time. In both C50

ered with new bone (Fig. 7A : C0-4W-H). However,and C30, absorption occurred from the exposed sur­even at 36 weeks, CPC absorption was negligible.

No. 2, 2018145

Tanaka・Yamada・Aizawa et al.

Ｂ

Ａ
Fig. 7
A : Histological images of specimens of different composites of CPC and β-TCP cement (C0, C30, and C50) at 4, 12, and36 weeks (H : ×10 magnification, L : ×2 magnification, toluidine blue staining).
Black arrow ; new bone area, β ; β-TCP, C ; CPC.
In C0, the living bone and the implant were closely adherented at 4 weeks after implantation. The surface of theimplant material was covered with new bone. CPC absorption was negligible at 36 weeks. In C30 and C50, new bone
was formed at the surface of the implant material and bone penetrated into it at 4 weeks after implantation. Bone
formation could be confirmed in the interior (macropore or fistula) of the implant material (C50-4W-H). After 12 weeks,
new bone was formed at the sites where β-TCP was absorbed (C50-12W-H). After 36 weeks, in C50, almost all of theimplant material was absorbed, and new bone covered the remainder (C50-36W-H).
B : Biodegradation of implant material (absorption rate)
C30 showed no statistically significant difference in any period compared to C0, while C50 had a statisticallysignificant difference from C0 in all periods (Wilcoxon test, p<0.05).
＊ ; P<0.05, n.s. ; not significant

Moreover, bone covered the entire surface of C0, but

implantation (Fig. 7A : C30-4W-L, C50-4W-L). More­there was little penetration into the implant material

over, bone formation could be confirmed in the inte­(Fig. 7A : C0-36W-H). In contrast, in C30 and C50,

rior (macropore or fistula) of the implant material

new bone was formed at the surface of the implant(Fig. 7A : C50-4W-H). After 12 weeks, new bone wasmaterial and bone penetrated into it at 4 weeks after

formed at the sites where β-TCP was absorbed

146

Shinshu Med J. 66

Mechanical properties and biotransformation of bone grafting materials composed of CPC and β-TCP(Fig. 7A : C50-12W-H). After 36 weeks, in C50, al­

Therefore, in this study, porous β-TCP granules

most all of the implant material was absorbed, andhaving macro- (200μm) and micropores (1μm) were

new bone covered the remainder.

mixed to create an interconnected network of mac­I

ropores in the new bone composite substitute. Our

Biodegradation of implant material (absorption
rate)

results showed that the total porosity of the hard­

In the C50 sample, 83% of material was absorbed

ened composite increased with an increasing mix

after 36 weeks. In C0, little absorption had occurredratio of β-TCP granules to CPC. The penetrationeven after 36 weeks. As the amount of β-TCP in­

test demonstrated higher permeability with a great­creased, the material was absorbed more quickly iner percentage of β-TCP granules. Especially in C50,

vivo. C30 showed no statistically significant differ­

the red solution reached into the center of the mate­ence in any period compared to C0, while C50 had arial. These results suggested that the porosity andstatistically significant difference from C0 in allpermeability were increased by adding more β-TCP

periods (Wilcoxon test, p<0.05).

granules. However, the type of porosity and size of

Ⅳ Discussion

pores are a key for bone substitution because highporosity and large pores enhance biodegradation

In oral and maxillofacial diseases, bone defects

and bone ingrowth into the bone substitute24）.

sometimes occur due to tumors, trauma, bone cysts,

Micro-CT and SEM images of C50 showed that

etc. Bone resorption caused by periodontal disease

β-TCP granules were in contact with each otheroften results in tooth loss. β-TCP has been used

and the macropores of the β-TCP were not filled

widely as a bone substitute for such bone defects,

with CPC cement. The CPC cement did not invade

because it shows good biodegradation and osteocon­

the macropores of the β-TCP granules, probably

ductivity. However, β-TCP has disadvantages such

because the β-TCP granules were added after the

as low mechanical properties and difficulty of forma­

CPC cement was kneaded. These results do nottiveness. CPC has been widely used clinically as adefinitively prove the existence of interconnectingbone filler material. However, CPC has disadvan­

macropores. However, there might be a possibility

tages such as poor bioabsorption and bone replace­that the adjoining β-TCP granules and several sizes

21）

ment . Both materials offer a trade-off between

and shapes of macropores created in the CPC ce­

mechanical and biological performance. Therefore, inment produced an interconnection of macropores. If

this study, composite pastes with different ratios of

the CPC cement invaded the macropores of the β-

CPC and porous β-TCP granules were examined to

TCP and blocked the macropores, it was predicted

develop a new composite bone cement with ade­that the absorption rate in vivo would obviously de­quate strength, formativeness, and good bioactivity

crease. However, this study proved that even when(biodegradation, biocompatibility and osteogenesis).

CPC cement and β-TCP granules were mixed, CPC

Porosity and interconnectivity are important prop­

cement did not invade the pores of β-TCP granules.

erties for bone substitute materials. Interconnecting

Therefore, as shown in the in vivo study, almost all

pores are advantageous for the resorption of boneof C50 (83%) was resorbed and replaced at 36 weeks

substitute and bone formation because they allow

after the implantation, compared with little resorp­

migration and proliferation of bone cells as welltion and replacement in C0 and C30. In the histologi­

as vascularization. CPC is intrinsically nonporous

cal examination, in both C30 and C50, absorption

and does not contain an interconnected network

occurred from the exposed surface of β-TCP. Fur­

22）of micropores . The average pore size of CPC isthermore, in C50, absorption also occurred from thelarge enough to allow the transport of nutrients but

inside of the sample. In C50, β-TCP granules existed

too small to allow the entry of osteoclasts andin contact with each other. Thus, if β-TCP is ab­

osteoblasts to resorb the CPC and form new bone23）.

sorbed, further communication of macropores (holes

No. 2, 2018147

Tanaka・Yamada・Aizawa et al.

where the β-TCP granules come out) would be cre­

CPC by adding sodium bicarbonate, but reported that

ated inside the CPC cement, which would further

the mechanical strength was only 2.1±0.3 Mpa29）.

allow resorption of the CPC cement and migration ofEndo et al. formed macropores by mixing collagen I

osteoblasts and mesenchymal cells. From these re­in CPC, but reported that when mixed with 9.1

sults it was suggested that C50 had sufficient porosi­

mass%, the strength decreased to 3.63±0.20 Mpa30）.

ty and a possibility of existing and/or resulting in­

Compared to these studies of macropores in CPC,

terconnecting macropores which might promote

the composite material of CPC paste and porous β-resorption and replacement by the surrounding host

TCP granules had higher and sufficient strength asbone.

a bone substitute material applicable to non-load-

As shown in this study, the biodegradability in­bearing areas.

creased with the addition of more β-TCP granules.

Our new material with porous β-TCP granules

In vivo biodegradability can be achieved by disso­

added to CPC paste had good bioactivity and osteo­

lution or cell-mediated resorption. Micro- or mac­conductivity. In histological examinations, the newlyroporosity of the bone substitute material plays anformed bone was directly in contact with the sur­important role in this biodegradability. The incre­

face of the implant materials. It was reported thatased porosity of C50 might be responsible for thethe surface topography is important for cell attach­higher absorption of the implant material. However,ment31）32）. As shown in the SEM study, the surface

biodegrability of a bone substitute depends not only

roughness of the studied materials was thought toon porosity but also on material properties including

enhance attachment, proliferation and differentiation

25）

Ca/P ratio, crystallinity, and so on . As the reactionof anchorage-dependent bone-forming cells33）. Osteo­of CPC progresses, HA is precipitated5）. HA is hard­

conductivity is the ability of a material to serve as aly absorbed in vivo, while β-TCP is known to show

scaffold to guide new bone formation. In the results

26）

faster resorption than HA . As revealed by the XRDof this study, histological examination revealed bone

examination, HA as well as tetracalcium phosphate

formation along the surface of the composite of CPC(TeCP) decreased with an increasing amount of β-and β-TCP granules. In addition, new bone forma­

TCP. The rate of absorption of C50 might be enhan­tion was also observed inside the implant material

ced by a decreased crystal change from CPC to HA.

where bioabsorption occurred. These results sug­

Although the increased porosity facilitates biode­gested that the composite material of CPC paste andgrability and bone formation, the result is a reduc­porous β-TCP granules had excellent bioactivity

tion in mechanical strength. The results of this study

and osteoconductivity.

also showed that an increase of β-TCP content result­

The setting time of CPC paste/β-TCP granules

ed in a reduction of compressive strength. Sawa­

increased with increasing ratio of β-TCP content.

mura et al. reported that the compressive strength

However, the setting time of C50 was 18 minutes,

increased with increasing amounts of HA13）. The

which we thought to be within the clinically accept­

decrease of HA as shown by the XRD examination

able range. Setting time correlates with temperature,also was responsible for the lower comprehensive

typically increasing sharply when the temperaturestrength. The median compressive strength of C30

is low. In this study, the setting time was measured

was 22 MPa and that of C50 was 13 MPa. The com­

at body temperature (37 ℃ ). However, the settingpressive strength of human cortical bone ranges

time would be prolonged if hardening took place

between 90 and 230 MPa, whereas the compressive

under conditions of low temperature13）.

strength of cancellous bone ranges between 2 and45 MPa27）28）. The mechanical strengths of C30 and

Ⅴ Conclusion

C50 were almost the same as for cancellous bone.

Based on the results of this study, we believe that

Real et al. succeeded in forming macropores withthe composite created by adding porous β-TCP

148

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Mechanical properties and biotransformation of bone grafting materials composed of CPC and β-TCP

granules to CPC paste had good biodegradabilityⅥ

and adequate mechanical strength adaptable forbone defects as a bone substitute. If porous β-TCP

Conflict of interest

The authors declare no conflict of interest.

granules were added as equal wt% of CPC cement

Ⅶ(C50), the composite material was almost completely

Acknowledgement

bioabsorbed and replaced by newly formed bone at

We are grateful to Dr. Li Yinghui of Hebei Medical

36 weeks after implantation. It is thought that C50 is

University for providing technical advice. We also

suitable for clinical application and we would like towould like to thank Dr. Akari Takeuchi of the De­

conduct a clinical study.

partment of Chemistry, Faculty of Science, Shinshu
University who provided us advice for XRD exam­
ination.
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