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J Dent Res 84(9):827-831, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Colloidal ß-Tricalcium Phosphate Prepared by Discharge in a Modified Body Fluid Facilitates Synthesis of Collagen Composites

Y. Shibata*, H. Yamamoto, and Takashi Miyazaki

Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;

* corresponding author, yookun{at}dent.showa-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of hydroxyapatite/collagen composites that are naturally synthesized and need no additional treatment is required for use in bone repair. Since reducing the diameter can increase the specific surface area of calcium phosphate particles that can conjugate collagen molecules, we expected colloidal calcium phosphates of submicron diameter obtained by discharge to be effective in formulating these composites. Additionally, since biodegradable ß-tricalcium phosphate has better osteoconductivity than hydroxyapatite, this study aimed to investigate the synthesis of colloidal hydroxyapatite and ß-tricalcium phosphate/collagen composites. Collagen molecules were tightly polymerized in the ß-tricalcium phosphate/collagen composite by catalysis of the generated -P-O-P- polyphosphate chain. Bonding strength between collagen NH+ amino groups and -P-O-P-, and cross-linking of the Ca++-RCOO– in the collagen were increased compared with those in the hydroxyapatite/collagen composite. These chemical reactions due to colloidal ß-tricalcium phosphate might play a key role in the synthesis of collagen composites.

KEY WORDS: collagen • ß-tricalcium phosphate • biocomposite


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inherent donor-site limitations with respect to tissue rejection and disease transfer are a shortcoming of autografting and allografting. A bio-composite material that induces and promotes new tissue formation at the required site would therefore be desirable for use in craniofacial orthopedics and periodontal repair.

Collagen is currently one of the most popular biomaterials for scaffold production in tissue reconstruction (Matsuda et al., 1993; Grzesiak et al., 1997; Hartgerink et al., 2001). Since collagen, on its own, has poor mechanical properties and experiences loss of biological stability during application (Hubbell, 1995), it is often reinforced with hydroxyapatite particles (Chang and Tanaka, 2002b) or synthetic polymers (Coombes et al., 2002) in a collagen composite. Hydroxyapatite/collagen composites in particular have recently received much attention, because human bone is mainly composed of hydroxyapatite and collagen fibers. However, the use of collagen composites has been limited, because they cannot be synthesized without secondary freeze-drying processes or treatment with toxic chemical agents (Olde et al., 1995; Chang and Tanaka, 2002a,b). Therefore, the development of composites that are naturally synthesized and need no additional treatment is required.

Reducing the diameter can increase the specific surface area of calcium phosphate particles that can conjugate organic molecules (Kurashina et al., 1997). Calcium phosphate particles (average diameter, 3 µm) are generally reduced in size by agate-ball milling, followed by ultrasonic pulverization (Zhao et al., 2002). Since alkaline colloidal calcium phosphate particles with submicron diameters could be prepared by discharge in modified body fluids (Takashima et al., 2004), we expected them to be effective in formulating collagen composites without the need for additional treatment.

ß-tricalcium phosphate (TCP) has been intensively investigated as a possible bone substitute, because of its biodegradable and high osteoconductive properties (Kurashina et al., 1997; Ohsawa et al., 2000; Lee et al., 2001; Ogose et al., 2002). Even though many studies have suggested that biodegradable ß-TCP has better osteoconductivity than hydroxyapatite, synthesis of ß-TCP/collagen composites has yet to be investigated. Thus, this study aimed to investigate the synthesis of colloidal hydroxyapatite and ß-TCP/collagen composites. To investigate the conformational changes between colloidal calcium phosphates and type-I collagen solution (pH 3.0), we used Fourier transformed infrared (FTIR) spectrometry. FTIR is a useful tool for structural investigations because we know the origins of amide bond vibrations, the sensitivity of some of these positions to conformation, and the possibility of predicting band positions for a given helical or extended collagen conformation (Chang and Tanaka, 2002b). Subsequently, to clarify cross-linking between inorganic components and the collagen fibers, we used an x-ray photoelectron spectroscopy (XPS) analyzer.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Electrolytes
Compositions of the electrolytes prepared in our laboratory were described in an earlier study (Takashima et al., 2004). Simulated body fluid, modified by the addition of K2HPO4 without buffer (m•SBF), and Hanks’ balanced salt solution without organic molecules (HBSS) were used. These solutions were buffered at pH 7.4 with adequate HCl.

Preparation of Colloidal Calcium Phosphates
The authors previously proposed that discharging an electrolyte is a controllable method by which various calcium phosphate particles can be prepared (Takashima et al., 2004). Platinum foil of 10 x 30 x 1.0 mm was used as the power supply cathode (developed in our laboratory), and platinum foil of 100 x 50 x 1.0 mm was used as the counter-electrode. Each foil was immersed in 100 mL of m•SBF and HBSS. Subsequently, discharging was maintained at 2.5 A and 100 V for 270 sec. After being processed, colloidal calcium phosphates were prepared by the removal of supernatants. The pH values of the colloidal calcium phosphates were measured with the use of a pH meter. Average values of those used for each composite (hydroxyapatite and ß-TCP/collagen) were expressed as the mean ± standard deviation of 6 specimens (n = 6), respectively, and significant differences were analyzed statistically by Student’s t test. Significant differences were considered when p < 0.01.

Particle Diameter
Dried colloidal calcium phosphates were prepared by gold vaporization with a vacuum evaporation device (IB-2, Eiko Engineering, Tokyo, Japan). Forty colloidal calcium phosphate particles for each composite were sampled randomly, and diameters were measured on a monitor with a scanning electron microscope (S-2360N, HITACHI, Tokyo, Japan). Average diameters were then calculated and expressed as the mean ± standard deviation, and significant differences were analyzed statistically by Student’s t test. Significant differences were considered when p < 0.01.

Preparation of Collagen Composites
A 3-mL quantity of the colloidal calcium phosphates used for each composite was dispersed in the same quantity of collagen solution (8 mg/mL, pH 3.0, Type-I collagen BM; Nitta Gelatin, Osaka, Japan), respectively. Subsequently, each mixture was stirred for 30 sec in a polypropylene centrifuge tube; we found that colloidal ß-TCP/collagen sol changes into a gel phase immediately after being mixed.

FTIR Analysis
Colloidal calcium phosphates were dried and stored for 24 hrs, then prepared as KBr pellets for FTIR analysis. Pure ß-TCP and hydroxyapatite powder (Wako, Osaka, Japan) were used as standard reference materials. In addition, a 10-µL quantity of each composite was immediately placed between ZnSe windows and analyzed with the use of an FTIR analyzer (FT/IR-660, JASCO, Tokyo, Japan) in a vacuum. The conformational changes of each composite over time were monitored for 30 min. At a measuring resolution of 4 cm–1, we performed iterations 200 times within a range of 400–4000 cm–1 to characterize the various functional groups. All data were confirmed with 6 different samples for each composite.

XPS Analysis
Both of the dried composites (hydroxyapatite and ß-TCP/collagen) were analyzed with the use of a XPS device (ESCA-3400, SHIMADZU, Kyoto, Japan). High-resolution spectra of Ca2p, O1s, P2p, C1s, and N1s on both were analyzed with Mg K{alpha} radiation, with an emission current of 20 mA and accelerated voltage of 8 kV. Binding energies for each spectrum were calibrated with a C1s spectrum of 285.0 eV, and average values were expressed as the mean ± standard deviation (SD) of 6 specimens (n = 6). Significant differences in each spectrum between the 2 composites were analyzed statistically by Student’s t test. Significant differences were considered when p < 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Colloidal Calcium Phosphates
FTIR analysis demonstrated PO43– adsorption peaks around 1040, 600, and 580 cm–1 attributable to ß-TCP on the precipitates synthesized in m•SBF (Fig. 1Go). Peaks around 1040, 600, and 3570 cm–1, attributed to OH groups, were observed with precipitation of HBSS (Fig. 1Go); thus, the precipitate of HBSS was determined as hydroxyapatite.



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  		Figure 1. Chemical structure of colloidal calcium phosphates. Pure ß-TCP (1) and hydroxyapatite (2) powder were used as standard reference materials. FTIR analysis demonstrated PO43– adsorption peaks around 1000, 600, and 580 cm–1 attributable to ß-TCP on the precipitate synthesized in m•SBF (3). Peaks around 1000, 600, and 3570 cm–1, attributed to OH groups, were observed with precipitation of HBSS (4); thus, the precipitate of HBSS was determined as hydroxyapatite. All data were confirmed with 6 different samples, respectively, for each colloidal calcium phosphate.

 
pH Values of Colloidal Calcium Phosphates
The pH values of colloidal ß-TCP and hydroxyapatite were 11.31 ± 0.06 and 11.32 ± 0.04, respectively. Significant differences between these values were not detected.

Particle Diameters
The particle diameters of colloidal ß-TCP and hydroxyapatite were 0.20 ± 0.06 and 0.17 ± 0.05 µm, respectively; these differences were not significant (p < 0.01). Since the particle diameters in this study were much smaller than those described in an earlier study (Zhao et al., 2002; average diameter, 3 µm), these colloidal calcium phosphates are thought to be promising for the preparation of collagen composites.

Conformational Changes in the Composites
In the FTIR study, the amide groups of the composites produced several characteristic vibration modes at group frequencies. N-H stretching at 3310 cm–1 for amide A, C-H stretching at 3063 cm–1 for amide B, C=O stretching at 1600–1700 cm–1 for the amide I, N-H deformation at 1500–1550 cm–1 for the amide II, and N-H deformation at 1200–1300 cm–1 for the amide III bands (Chang and Tanaka, 2002b) were detected in the ß-TCP/collagen composite (Fig. 2Go). An isolated OH group around 3600 cm–1 was observed only in the ß-TCP/collagen composite, while amides A and I increased at the beginning of analysis (Fig. 2AGo). After 30 min of processing, the amide A band was clearly detected only in the ß-TCP/collagen composite (Fig. 3AGo); the broad water bands decreased under the vacuum (see MATERIALS & METHODS). The peak height of amide I of the ß-TCP/collagen composite was much higher than that of the hydroxyapatite/collagen composite (Fig. 3BGo). The 1040 cm–1 phosphate vibration mode of ß-TCP shifted to 1025 cm–1, clearly indicating that the -P-O-P- polymerization chain (George, 1994) was produced in the ß-TCP/collagen composite after mixing (Fig. 3CGo).



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  		Figure 2. Conformational changes in ß-TCP/collagen (A) and hydroxyapatite/collagen (B) composites 30 sec, and 10, 20, and 30 min after being mixed. N-H stretching at 3310 cm–1 for amide A, C-H stretching at 3063 cm–1 for amide B, C=O stretching at 1600–1700 cm–1 for the amide I, N-H deformation at 1500–1550 cm–1 for the amide II, and N-H deformation at 1200–1300 cm–1 for the amide III band were detected in the ß-TCP/collagen composite. An isolated OH group around 3600 cm–1was detected only in the ß-TCP/collagen, while amides A and I increased at the beginning of analysis. All data were confirmed with 6 different samples, respectively, for each composite.

 


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  		Figure 3. Narrow-scan FTIR spectra. (A) Narrow-scan FTIR for amide A: The amide A peak of ß-TCP/collagen (1) was clearly detected compared with that of hydroxyapatite/collagen (2). (B) Narrow-scan FTIR for amide I: The amide I peak of ß-TCP/collagen (1) was much higher than that of hydroxyapatite/collagen (2). (C) Phosphate vibration mode of colloidal ß-TCP (1). The phosphate vibration mode of ß-TCP shifted to 1025 cm–1 (2), clearly indicating that a -P-O-P- polymerization chain was produced in the ß-TCP/collagen composite after being mixed. All data were confirmed with 6 different samples, respectively, for each composite.

 
Cross-linking of the Composites
In the ß-TCP/collagen composite, the primary and secondary peaks of P2p were located at 133.2 ± 0.1 and 134.8 ± 0.1 eV, respectively (Fig. 4AGo). The secondary peak indicated NH+-phosphate binding (Renier and Kohn, 1997) in the ß-TCP/collagen composite. Ca2p spectra revealing similar doublets with Ca2p1/2 and Ca2p3/2, typical for the Ca++ state in inorganic calcium phosphate compounds (Charles et al., 2003), were detected in both composites (Fig. 4BGo). Ca2p energy positions were detected at 347.8 ± 0.1 and 348.4 ± 0.1 eV for the hydroxyapatite/collagen and ß-TCP/collagen composites, respectively. The energy positions of Ca2p (Fig. 4BGo) and N1s (Fig. 4CGo) were significantly higher (p < 0.01) with ß-TCP/collagen than with hydroxyapatite/collagen.



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  		Figure 4. XPS high-resolution spectra of each composite. (A) High-resolution spectra of P2p in the ß-TCP/collagen composite; the secondary peak indicates NH+-phosphate binding. High-resolution spectra of Ca2p (B) and N1s (C) in both composites. The energy positions of N1s and Ca2p in the ß-TCP/collagen composite (1) were significantly higher (p < 0.01) than those in the hydroxyapatite/collagen composite (2). Average binding energies are expressed as the mean ± standard deviation (SD) of 6 specimens (n = 6), and significant differences in each spectrum between composites were analyzed statistically by Student’s t test (p < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The quaternary structure of type-I collagen has a triple-helical conformation (Payne and Veis, 1988). The secondary helical structure of collagen is produced by strong hydrogen bonding, which is indicated by its high amide A band, and these reactions are directly associated with the supercoiled triple-helical conformation of collagen fibers (Payne and Veis, 1988). It has been reported that the intensity of amide A is sensitively dependent on the extent of collagen molecule polymerization, which is critically important for bone mineralization (Boskey et al., 1999).

Collagen has several characteristic peptide bands that indicate the conformation of collagen matrices. Particularly, amide I is mainly associated with the stretching C=O vibrations of collagen molecules (Chang and Tanaka, 2002b). The vibration frequency of the C=O bond depends on the strength of the hydrogen bond to carbonyl oxygen and the interaction between each of the amide units, and is very close to the amide A frequency (Doyle, 1975). Since both the amide A and I bands increased significantly in the ß-TCP/collagen composite (Figs. 3AGo, 3BGo), this suggests that many collagen molecules polymerized together to form collagen fibers.

Another large conformational change was indicated in the ß-TCP/collagen composite. FTIR detected an isolated OH group only in the ß-TCP/collagen composite when the amide A and I bands increased at the beginning of analysis (Fig. 2AGo); hydrogen bonding was completed, for the most part, in this composite. Since the internal hydrogen bond of the composite was facilitated as indicated by amides I and A, the setting reaction was mostly completed in the ß-TCP/collagen composite, even after just 30 sec of mixing. Thus, the colloidal ß-TCP/collagen sol changed into a gel phase immediately after mixing (see MATERIALS & METHODS). Surprisingly, the phosphate vibration mode of ß-TCP shifted to 1025 cm–1 (Fig. 3CGo), clearly indicating that a -P-O-P- polymerization chain was produced (George, 1994) in this composite after mixing. Since polyphosphate has been used as a catalyst in organic molecules (Renier and Kohn, 1997; Gough et al., 2003), the -P-O-P- polymerization chain might play an important role in the formation of stable collagen fibers in ß-TCP/collagen composites.

In the XPS study, the secondary peak of P2p, indicating NH+-phosphate binding (Renier and Kohn, 1997), was detected only in the ß-TCP/collagen composite (Fig. 4AGo), suggesting that the -P-O-P- polymerization chain was tightly bonded to the collagen NH+ groups in this composite. In addition, Ca2p spectra revealing similar doublets with Ca2p1/2 and Ca2p3/2, typical for the Ca++ state in inorganic calcium phosphate compounds (Charles et al., 2003), were detected in both composites (Fig. 4BGo). The energy position of a Ca2p doublet increases with cross-linking between calcium phosphate nanocrystals and collagen molecules, because the bonding strength of Ca++-COO- is higher than that of inorganic Ca++-PO43– (Chang and Tanaka, 2002a). The XPS spectra showed the electron-binding states of Ca in the colloidal calcium phosphates (Fig. 4BGo), which were coordinated with both the inorganic PO43– and RCOO– groups of collagen molecules (Hanawa and Ota, 1991; Chang and Tanaka, 2002a). Furthermore, since the energy positions of Ca2p (Fig. 4BGo) and N1s (Fig. 4CGo) in the ß-TCP/collagen composite were significantly higher (p < 0.01) than those in the hydroxyapatite/collagen composite, this suggests that the cross-linking between the collagen RCOO– groups and Ca++ of ß-TCP was also greatly increased in the former (Gough et al., 2003).

Synthesis of hydroxyapatite/collagen composites is recognized as being difficult, because it involves two dissimilar organic and inorganic nanophases that have a specific spatial relationship with one another. In earlier studies, hydroxyapatite/collagen composites have been generally synthesized by the addition of toxic cross-linking agents (Kikuchi et al., 1999; Chang et al., 2001, 2002); because hydroxyapatite is used as the starting material, it is not possible to synthesize collagen composites without such toxic chemical agents. A different approach was described in another study, which showed that a non-cross-linked hydroxyapatite/collagen composite could be manufactured by reinforcement of a synthetic polymer (Coombes et al., 2002). It was also indicated that reinforcement of collagen composites was important in maintaining the biological stability of the composite during application in vivo.

In the present study, collagen molecules were polymerized together to form collagen fibers in the ß-TCP/collagen composite. Furthermore, since bonding strength between the collagen NH+ amino groups and -P-O-P- polymerized chain, and cross-linking between the Ca++ and RCOO– of the collagen fibers, were greatly increased, it can be expected that this composite has high biological stability without the need for polymeric reinforcement. Thus, we believe that colloidal ß-TCP has an important role in the synthesis of spatial structural manipulation of collagen composites.


   ACKNOWLEDGMENTS
 
We thank Dr. Masato Yamamoto for his technical assistance (Department of Chemistry, College of Arts and Sciences, Showa University). This work was supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science, and by a Grant-in-Aid for Encouragement of Young Scientists (B) from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

Received January 28, 2004; Last revision March 29, 2005; Accepted May 1, 2005


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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