o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 9

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/odw

Research paper

Evaluation of bone regeneration by porous alpha-tricalciumphosphate/atelocollagen sponge composite in rat calvarialdefects

Yoshiyuki Arima a,*, Naoya Uemura b, Yoshiya Hashimoto c, Shunsuke Baba b,Naoyuki Matsumoto d

aDepartment of Orthodontics, Graduate School of Dentistry, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata,

Osaka 573-1121, JapanbDepartment of Oral Implantology, Osaka Dental University, 1-5-17 Otemae, Chuo-ku, Osaka 540-0008, JapancDepartment of Biomaterials, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, JapandDepartment of Orthodontics, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan

a r t i c l e i n f o

Article history:

Received 28 June 2012

Received in revised form

17 October 2012

Accepted 16 November 2012

Published on line 14 December 2012

Keywords:

Porous a-tricalcium phosphate

Collagen sponge composite

Bone tissue engineering

Rat calvarial defects

a b s t r a c t

Purpose: Both atelocollagen and alpha-tricalcium phosphate (a-TCP) particles are widely

applied as bone graft materials. In this study, we combined atelocollagen and porous a-TCP

particles to form an a-TCP/atelocollagen composite sponge (a-TCP/CS), and we evaluated its

effects on bone regeneration in rat calvarial defects.

Materials and methods: a-TCP granules were mixed with a collagen solution. The mixture was

poured into plastic molds and then frozen to �80 8C and freeze-dried for 24 h. The compo-

sites were then cross-linked in vacuo at 140 8C for 24 h. The obtained composites were

characterized by XRD and SEM and observed using micro-CT and histological analysis.

Results: In vivo micro-CT images at 2, 4, and 6 weeks after surgery showed that in the a-TCP/

CS group, the bone volume, bone mineral density, and bone mineral content were higher

than those in the groups with only atelocollagen and without scaffolds at 6 weeks after

surgery. Histological analysis showed that a-TCP was completely absorbed, and new bone

that was continuous along the original bone was observed.

Conclusion: This study demonstrated that composite sponges created using porous a-TCP

lagen were sufficiently adaptable for treating bone defects.

sevier Ltd and the Japanese Orthodontic Society. All rights reserved.

# 2012 El

particles and atelocol

1. Introduction

Cleft lip and palate is a frequently occurring congenital

malformation that is caused by alveolar bone defects [1,2]. A

cleft can lead to problems with feeding and speech, among

others, and therefore, surgical closure is strongly recom-

mended [3,4]. Among various surgical treatments, autologous

* Corresponding author. Tel.: +81 72 864 3078; fax: +81 72 864 3178.E-mail addresses: arima-y@cc.osaka-dent.ac.jp, arima_dental@ybb

1344-0241/$ – see front matter # 2012 Elsevier Ltd and the Japanesehttp://dx.doi.org/10.1016/j.odw.2012.11.001

bone grafting has become a well-accepted treatment for

restoring the function and structure of the maxillary arch at

the cleft site.

Autologous bone is widely regarded as the gold standard

graft material [5,6]. However, a limited amount of autologous

bone can be harvested from an individual, and the procedure

is invasive. Therefore, scaffold materials should be bioabsorb-

able and be ultimately replaced by autologous tissue [7].

.ne.jp, arima.dental@gmail.com (Y. Arima).

Orthodontic Society. All rights reserved.

o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 924

Furthermore, they should have outstanding handling char-

acteristics for surgical applications, because conventional

granular materials tend to leak from pores and are difficult to

apply to bone defects [8].

Owing to such issues, various materials have been

developed as substitutes to autologous bone. Popular among

these are hydroxyapatite (HA), tricalcium phosphate (TCP),

and tetracalcium phosphate, which are widely used in

orthopedic and maxillofacial surgery, because they show high

biocompatibility and osteocompatibility [9]. Among various

calcium phosphate materials, studies have previously

highlighted the promise of a-TCP particles as bone rebuilding

materials, because they gradually biodegrade while bone

regenerates around them [10]. For example, Kitamura et al. [11]

developed an a-TCP porous body with a continuous pore

structure via an easy processing route by using a conventional

sintering procedure—they used a slurry of b-TCP and potato

starch to produce a-TCP that was in a thermodynamically

stable phase at temperatures above 1100 8C.

Separately, collagen has been widely applied clinically in

various forms, such as gels or sponges, because it shows good

biodegradability, biocompatibility, and absorbability [12,13].

For example, collagen sponges are considered to be a useful

scaffold matrix for several types of tissues. Our previous study

evaluated the effects on bone formation of combining

synthesized collagen model polypeptides consisting of a

Pro-Hyp-Gly [poly(PHG)] sequence and a-TCP in a skull defect

model in mini-pigs [8].

In light of these advantageous characteristics, in this study,

we fabricated a porous a-TCP/collagen sponge (aTCP/CS)

composite for the regeneration of alveolar bone defects by

bonding collagen fibrils and a-TCP granules by a dehydrother-

mal cross-linking method. Then, we evaluated the effects of a-

TCP/CS on bone regeneration in surgically created rat calvarial

defects.

2. Materials and methods

2.1. X-ray diffraction measurements of a-TCP

X-ray diffraction (XRD) measurements were performed using a

Geigerflex diffractometer (Ultima IV; Rigaku, Tokyo, Japan)

with Cu Ka radiation generated at 40 kV and 40 mA. The scan

rate was 48/min with a step size of 0.028 over a 2u range of

10–808.

2.2. Preparation of a-TCP/collagen sponge composites

Porous a-TCP granules were kindly supplied by Taihei

Chemical Ind. Co., Ltd., Osaka, Japan. The granules were

prepared by the pulverization of an a-TCP block with total

pores of 80% having continuous pore structures of both 10 and

200 mm diameters. The collagen used was extracted from

porcine skin by enzymatic treatment with pepsin (Nippon

Meat Packers, Osaka, Japan). Next, a-TCP particles were mixed

with the homogenized collagen solution at a ratio of 150 mg/

mL (a-TCP particles/collagen solution). The mixture was then

poured into plastic molds, following which all samples were

immediately frozen to �80 8C and freeze-dried for 24 h. The

freeze-dried a-TCP/collagen composites resembled sponge-

like structures, and they were subsequently cross-linked in

vacuo at 140 8C for 24 h. The microstructures of the obtained

scaffolds were then observed using an SEM (S-450, Hitachi Ltd.,

Tokyo, Japan).

2.3. Surgical procedure

The protocol for this experiment was approved by the Osaka

Dental University Animal Ethics Committee (Approval Num-

ber 11-03043). We used the rat calvarial defect model to test

our hypothesis, as it is a convenient model for the study of

bone regenerative materials because of its lack of fixation

requirements. A total of 27 male Sprague-Dawley rats (age: 12

weeks, weight: 350–370 g) were used for transplantation. The

rats were anesthetized with a 30 mg/kg intraperitoneal

injection of pentobarbital sodium (Somnopentyl1; Kyoritsu

Seiyaku, Tokyo, Japan); in addition, 0.2 mL of local anesthetic

(1% xylocaine/epinephrine 1:100,000, AstraZeneca, Tokyo,

Japan) was injected into the surgical sites before the start of

surgery. The surgical areas were shaved and disinfected with

povidone-iodine (Isodine Surgical Scrub, Meiji, Tokyo, Japan).

Then, skin incision and subperiosteal dissection were carried

out. A skin flap was raised, and bone defect (diameter: 5 mm,

depth: 1.0 mm) was created at the center of the skull using a

bone trephine bur (external diameter: 5 mm) under running

water. Three groups were prepared for the experiments. One is

filled with a-TCP/CS; another is filled with only CS; and the

control group was not filled with any material. In all groups,

the defects were covered using a GORE-TEX1 GTR membrane

(Japan Gore-Tex Co., Tokyo, Japan). Finally, the flap was

repositioned and sutured. The skin was sutured by 3-0 silk

(Natsume Seisakusho Co., Tokyo, Japan). The animals were

euthanized at 2, 4, and 6 weeks after surgery (n = 3).

2.4. Micro-CT and computer analysis

After euthanization, the calvarial bone was scanned by micro-

CT (65 kV, 90 mA) (SMX-130CT; Shimadzu, Kyoto, Japan). The

linearity of the micro-CT scanner was established by scanning

a phantom containing several densities of a standard

calibration material. The 3D calvarial bone was measured

and its structural indices were calculated using a morpho-

metric program (TRI/3D-BON; Ratoc System Engineering,

Tokyo, Japan). For bone density measurements, an HA

calibration curve was prepared from the images of phantoms

(Ha content: 200, 300, 400, 500, 600, 700, and 800 mg/cm3). New

bone was measured using the TRI/3D-BON 3D analysis routine

using the obtained CT values. In the 3D analysis, the total

volume (TV, cm3), bone volume (BV, cm3), and bone mineral

content (BMC, mg) were measured directly. The volumetric

density was calculated as BV/TV (%), and the bone mineral

density (BMD) was calculated as BMC/BV (mg/cm3); these

indices respectively evaluated the quantity and density of new

bone.

2.5. Histological analysis

Histological examination was carried out to observe the tissue

structures and thicknesses of the new bone. Samples were

[(Fig._1)TD$FIG]

Fig. 1 – XRD patterns of a-TCP particles/CS (A) and a-TCP

particles (B).

o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 9 25

fixed with 10% phosphoric acid in neutral buffered formalin

for 2 weeks. Subsequently, they were processed for histologi-

cal examination according to standard procedures—the

samples were decalcified, embedded in paraffin, sectioned

at 4-mm thickness, and subjected to hematoxylin and eosin

(HE) staining. All the stained sections were observed by light

microscopy (BZ-9000; Keyence Co., Osaka, Japan).

2.6. Statistical analysis

All data were expressed as the mean and standard error.

Differences were evaluated by analysis of variance (ANOVA)

with Scheffe’s F-test. Differences were considered significant

at p < 0.05.

3. Results

3.1. X-ray diffraction measurements of a-TCP particles

Fig. 1 shows XRD patterns of a-TCP/CS and the original a-TCP.

The diffraction peaks of a-TCP/CS (Fig. 1A) were reduced

compared to those of the original a-TCP (Fig. 1B). However, a

comparison of the scatter plot data of the synthesized a-TCP

particles against a-TCP data registered with the Joint Com-

mittee on Powder Diffraction Standards (JCPDS) confirmed

that these peaks appeared at the same angles.

3.2. SEM observation of a-TCP/collagen sponge

Fig. 2A shows the appearance of CS, and Fig. 2B shows the

microstructure of a-TCP/CS. The composites were composed

of a-TCP granules (diameter: 0.2 mm) and a three-dimensional

porous structure (with an anatomizing network).

3.3. Three-dimensional color mapping (3D map) andmicro-CT examination

Fig. 3 shows a representative BMD distribution map of the 3D

map and micro-CT images of bone regeneration. Each model

was analyzed at three time points: 2, 4, and 6 weeks after

surgery. The 3D map shows the BMD at these time points. The

[(Fig._2)TD$FIG]

Fig. 2 – SEM photographs of CS (A) and a-TCP/CS (B), where the a

porous a-TCP granules. Original magnification is 200T.

BMD color scale is as follows: red and orange = high BMD,

yellow and green = medium BMD, and light blue and pur-

ple = low BMD.

3.3.1. Results 2 weeks after surgeryA low BMD value (purple color) was observed in the control and

collagen groups. Micro-CT images showed that defects were

unfilled with new bone in both groups. In contrast, a low-to-

medium BMD value (green color) was observed in the a-TCP/

CS group. Micro-CT images showed that defects were filled

with a small amount of new bone.

3.3.2. Results 4 weeks after surgeryA low BMD value (purple color) was observed in the control

and collagen groups. In the control group, micro-CT images

showed that defects were unfilled with new bone. In the

CS group, more bone repair was observed compared

with the control group. In contrast, a medium BMD value

(yellow color) was observed in the a-TCP/CS group. Micro-CT

images showed that defects were filled with many hard

tissues.

3.3.3. Results 6 weeks after surgeryA low BMD value (purple color) was observed in the control and

collagen groups. Micro-CT images showed that defects were

rrow and asterisk respectively indicate collagen fibrils and

[(Fig._3)TD$FIG]

Fig. 3 – Micro-CT images and the BMD distribution map of the 3D map at 2, 4, and 6 weeks after surgery. (For interpretation of

the references to color in the text, the reader is referred to the web version of this article.)

o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 926

filled with a small amount of new bone. In contrast, a high

BMD value (orange color) was partly observed in the a-TCP/CS

group. Micro-CT images showed that defects were completely

filled with new bone, and clearly, the amount of new bone in

this group increased considerably with time.

3.4. BV/TV, BMC, and BMD analysis

Fig. 4 shows BV/TV of each group at each time. At 2 weeks post-

transplant, there was a difference between BV/TV in both

transplantation groups and the control group. At 4 weeks post-

transplant, BV/TV in the a-TCP/CS group was greater than

those in the other groups. At 6 weeks post-transplant, BV/TV

[(Fig._4)TD$FIG]

Fig. 4 – New bone volume to total volume ratio (BV/TV, %) of

each group to evaluate the quantity of new bone at 2, 4,

and 6 weeks after surgery. *p < 0.05.

in the a-TCP/CS group was the highest among the three

groups.

Fig. 5 shows BMC of each group at each time point. At 2 and

6 weeks post-transplant, BMC in the a-TCP/CS group was the

highest among the three groups. At 4 weeks post-transplant,

there was a difference between the BMC in both transplanta-

tion groups and the control group.

Fig. 6 shows BMD of each group at each time point. At 2 and

6 weeks post-transplant, there was a statistically significant

difference between BMD in the a-TCP/CS group and the other

two groups. At 4 weeks post-transplant, no significant

difference was found among the three groups.

3.5. Histological analysis (H-E staining)

3.5.1. Results 2 weeks after surgeryIn the control group, the bone defects were filled with thin,

loose, connective tissue; however, no new bone was observed

(Fig. 7A). In the collagen group, the outside of the bone defect

was filled with sparse connective tissue and the inside, with

the remaining collagen sponge; furthermore, new bone was

slightly observed (Fig. 7B). In the a-TCP/CS group, the bone

defects were filled with a-TCP; furthermore, new bone linked

to the parental area of the floor bone was observed at the area

of the bone defect (Fig. 7C).

3.5.2. Results 4 weeks after surgeryIn the control group, the bone defects were filled with sparse

connective tissue (Fig. 7D). In the collagen group, the

bone defects were filled with dense connective tissue

and the remaining collagen sponge (Fig. 7E). In the a-TCP/CS

[(Fig._5)TD$FIG]

Fig. 5 – BMC (mg) of each group to evaluate the quantity of

new bone at 2, 4, and 6 weeks after surgery. *p < 0.05.[(Fig._6)TD$FIG]

Fig. 6 – BMD (mg/cm3) of each group to evaluate the

strength of new bone at 2, 4, and 6 weeks after surgery.

*p < 0.05.

o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 9 27

group, a-TCP inside the bone defect was decreased compared

with that in week 2. Furthermore, the remaining a-TCP was

found, and new bone was formed surrounding the a-TCP

(Fig. 7F).[(Fig._7)TD$FIG]

Fig. 7 – Histological micrographs of HE-stained sections from ea

magnification: 100T. Scale bars: 200 mm. NB, new bone; CT, conn

3.5.3. Results 6 weeks after surgeryIn the control group, most parts of the bone defects were filled

with connective tissue; a small amount of new bone was

observed (Fig. 7G). In the collagen group, new bone that was

not continuous along the original bone was observed (Fig. 7H).

In the a-TCP/CS group, a-TCP was completely substituted by

bone and matured bone was recognized over the entire bone

defect (Fig. 7I).

4. Discussion

At present, autologous bone is the preferred material used for

the closure of osseous defects in the alveolar process [14,15].

However, harvesting an autologous bone graft involves several

disadvantages. Donor site morbidity after bone graft harvest

remains a recognized limitation in the reconstruction of the

cleft primary palate [16]. In this study, we fabricated a

biodegradable sponge composite for bone regeneration by

combining porous a-TCP and collagen sponge. a-TCP/CS can

be easily cut with scissors or a sharp knife, and therefore, it

can be easily molded for use for alveolar bone augmentation.

We evaluated its effects on bone regeneration after implanta-

tion into rat calvarial defects.

Many studies have investigated the use of calcium

phosphate as a bone graft material because of its good

biocompatibility [9]. The in vivo degradation rate of TCP is

higher than that of HA. a-TCP dissolves more easily in water

than b-TCP even though they have exactly the same

chemical composition [17]. Furthermore, a-TCP has a higher

dissolution rate in solutions with low pH values [18]. The

collagen used in this study was dissolved in HCl aqueous

solution at pH 3. In addition, porous a-TCP was used

to fabricate the composite scaffold. However, the XRD

pattern of a-TCP/CS was not particularly different from that

of a-TCP.

Collagen sponge works well as a scaffold matrix for tissue

regeneration [19]. Collagen is the most abundant macromole-

cule in the body, and its molecular structures are very similar

across animal species. Collagen exists as an extracellular

ch group at 2, 4, and 6 weeks after surgery. Original

ective tissue; TCP, a-TCP particles/CS; CS, collagen sponge.

o r t h o d o n t i c w a v e s 7 2 ( 2 0 1 3 ) 2 3 – 2 928

matrix in various tissues. During development and regenera-

tion processes [20–24], the highly ordered structure of collagen

plays an important role in various biological events in the

body. One example is the calcification process in bone tissue

[25].

In our previous study [12], we have shown that the stress

values of b-TCP/CS (0.2 g/mL) were significantly greater than

those of b-TCP/CS (0.1, 0.05, and 0.02 g/mL) over the entire

strain range through compression testing. In particular, the

initial Young’s modulus of b-TCP/CS (0.2 g/mL) differed

significantly from those of the other three composites. In this

study, a composite sponge was not molded because there were

too many granules at a 0.2 g/mL mixing concentration of a-

TCP and the atelocollagen solution. Thus, the optimal mixing

concentration of a-TCP and the atelocollagen solution was

decided as 0.15 g/mL.

The TCP’s porous body gradually dissolves away from bone

defects during bone repair, allowing for its substitution by

regenerated bone [26]. Such a bioabsorbable ceramic with

macropores is attractive for use as a bone filler in medical

applications because its porous structure allows cell invasion

[26]. However, the solubility of porous a-TCP might be higher

than that of the original a-TCP, making it liable to be

completely resorbed in the body before a bone defect can be

repaired by bone regeneration. Kitamura et al. [11] demon-

strated that the dissolution rate of porous a-TCP could be

reduced by coating it with an organic polymer; their in vivo

evaluation showed that such a coating is effective for

controlling the degradation rate of implanted a-TCP. In this

study, we expect that the mixture of porous a-TCP and

collagen will be similarly effective.

Micro-CT has a high resolution and uses very thin slices. A

larger number of slices means that the spatial differences

within areas of the specimen become negligible [27]. The

geometrical properties of the healing bone can be acquired

along with the spatial distribution of BMD by simultaneous

scans of a calibration phantom using micro-CT [27]. The BMD

distribution map may be useful for evaluating the regenera-

tion of new bone. We selected the BV/TV, BMD, and BMC of

mineralized tissue comprising the bone quality as the

denominator along with the bone volume. In our study,

micro-CT analysis showed that BV/TV and BMD in the a-TCP/

CS group was highest at 2–6 weeks and BMC was the highest

among the three groups at 4–6 weeks post-transplant. On the

other hand, BMD in the CS group showed no significant

difference compared with the control group at all weeks post-

transplant. The bone quantity and quality of the experimental

group had increased considerably compared with that of the

CS group. These results suggested that porous a-TCP promot-

ed new bone formation, and the use of only CS might not

promote an increase in bone quantity.

With regard to the biodegradability of the atelocollagen,

collagen sponge was almost completely degraded at 4 weeks

after implantation in the CS and a-TCP/CS groups. However,

CS was replaced only by soft connective tissue and not by

completely newly formed bone. In contrast, a-TCP/CS was

replaced by a network of newly formed bone. Furthermore, the

remaining a-TCP granules observed after 4 weeks were in

direct contact with the newly formed bone. Osteogenic

progenitor cells migrated from the adjacent bone edge or

bottom, adhered to the surface of the a-TCP granules, and

subsequently formed new bone [12]. In addition, porous a-TCP

often shows early absorption before adequate bone replace-

ment has occurred. The atelocollagen composite should

effectively control the degradation rate of the porous a-TCP

implant. A continuous, thick, lamellar structural bone was

formed at the top of the defect 4–6 weeks after operation in the

a-TCP/CS group. These observations indicated that the a-TCP/

CS composites supported bone regeneration, including lamel-

lar and trabecular bone formation and remodeling.

The reconstruction of alveolar cleft defects is a well-

established procedure, with the most widely applied approach

being secondary alveolar cleft osteoplasty. Successful grafting

allows eruption of teeth into the former cleft area and further

orthodontic expansion of a typically collapsed maxilla [28].

Biomaterials for bone grafting are required to possess

sufficient mechanical strength to sustain their shape until

they are replaced by newly formed bone. The morphology of

bony defects in cleft lip and palate is usually complicated, and

therefore, it is typically difficult to adapt the shape that is to be

fitted into a bone defect. However, the proposed a-TCP/CS

composite can be easily cut with scissors or a sharp knife, and

therefore, it can be easily molded for use such as alveolar bone

augmentation.

In conclusion, this study demonstrated that composites

created using porous a-TCP granules and collagen sponges

were sufficiently adaptable for treating bone defects.

Furthermore, the biodegradable a-TCP/CS was replaced by

newly formed bone without any adverse responses. There-

fore, this newly developed type of sponge composite may be

useful for bone tissue engineering. The clinical application

of a-TCP/CS as bone graft materials for alveolar cleft defects

should be successful. Further studies of bone regeneration

will be required in an alveolar cleft defect model in a larger

animal.

Acknowledgments

The authors express their sincere gratitude to Taihei Chemical

Industrial Co., Ltd. (Osaka, Japan), for providing the a-TCP

particles. The authors also thank Ratoc System Engineering

Co., Ltd. (Tokyo, Japan).

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