Time-dependent variation of the surface structure of bioceramics in tissue culture medium and the effect on adhesiveness of cells

JOURNALOF FERMENTATIONAND BIOENGWEEIUNG Vol. 81, No. 3, 226-232. 1996

Time-Dependent Variation of the Surface Structure of Bioceramics in Tissue Culture Medium and the Effect on Adhesiveness of Cells TAKAHIRO

SUZUKI,* KAORI NISHIZAWA, YOSHIYUKI YOKOGAWA, YUKARI KAWAMOTO, AND TETSUYA KAMEYAMA

Bioceramics Laboratory,

FUKUE NAGATA,

National Industrial Research Institute of Nagoya, Hirate-cho, Kita-ku, Nagoya 462, Japan Received 5 September 199YAccepted

24 November 1995

Biocompatible ceramics made of ,%tricalcium phosphate (TCP) were found to be actively changing their surface characteristics in tissue culture medium. The time-dependent variation of the zeta potential of TCP ceramics immersed in distilled water and in culture medium with and without addition of fetal bovine serum showed that the surface was unstable with significant changes in the charge being measured. Dry TCP had a zeta potential of - 9.3 mV, which shifted to - 1.8 mV after soaking in water and to - 27.6 mV in culture medium with serum. To clarify the effect of the time-dependent variation of the surface structure on growth and adhesion of anchorage-dependent animal cells, the zeta potential of ceramics in dry state was regulated from - 11.5 mV to + 27.2 mV by means of silane coupling modification. After soaking in distilled water for six days, zeta potential of the mod&d TCPs shifted to between + 7.5 mV and - 7.6 mV, while they were between - 9.9 mV and -23.7 mV in culture medium with serum. Concentrations of calcium and phosphate dissolved in distilled water and in culture medium showed the solubility of the ceramics was considerably high and depended on the pH of the surface layer. The suitable surface property for adhesion of L-929 cells was obtained by the most stable ceramics in the culture condition. In conclusion, the solubility of the thin surface layer of the carrier was considered to be the dominant factor in decreasing the adhesiveness of cells on TCP ceramics. [Key words: cell adhesion, bioactive ceramics, tricalcium Hydroxyapatite (HAP) is the major inorganic component of the hard tissues, such as bone and teeth, in the human body and has high biocompatibility with living

phosphate,

zeta potential,

surface modification]

On the other hand, it has been also suggested that the thin surface layer of TCP-HAP ceramics can be dissolved into the aqueous medium during cultivation (12, 13). Kubo et al. reported that the adhesiveness of fibroblast cells to bioglass which leaks some components into culture medium was weaker than to that of insoluble bioinert soda-lime glass (14). Histochemical observations in animal implant tests have suggested that the slow solubility of calcium phosphate ceramics seems to be necessary for in vivo osteoconductivity and remodeling of the fibrous connective tissues to obtain natural bonding with the implant (15, 16). Furthermore, biocompatibility with no biotoxic reactions has been reported from culturing of osteoblasts on hydroxyapatite ceramics (17). In this study, it was concluded that the surface-reactive bone replacement materials owe their biocompatibility to the ability of their surfaces to interact favorably with the peptides involved in osteomorphogenesis and bone repair (17). These observations suggest that the surface structure of TCP-HAP ceramics changes in culture medium by releasing inorganic components and simultaneously adsorbing solutes from the medium such as serum proteins and minerals. In fact, a long lag phase, consisting of one day, was observed after seeding of the L-929 cells to TCP-HAP carriers. Such growth, however, started immediately on polystyrene dish and soda-lime glass (7). Thus, the adhesiveness and growth of cells on ceramics are considered to be regulated by the time-dependent variation of the surface structure. In this connection, the variation of the surface structure of ceramics in tissue

tissue (l-4). Tricalcium phosphate (TCP) is attracting interest as a bioactive artificial implant material because of its chemical affinity, osteoconductivity, and connectivity with bone tissue (5, 6). In previous studies (7, 8) new effects of the chemical and physical structure of the surface of ceramic material were discovered to have an influence on adhesion and growth of cells using sintered mixtures of HAP and TCP carriers whose chemical compositions were carefully controlled. The adhesiveness of L-929 cells to TCP-HAP sinters was considerably weaker than to polystyrene plates, while the cell division rate slightly increased on the ceramic carriers. Chemical modification of the surface of a TCP carrier by means of silane coupling reagents clarified the effects of the zeta potential (9, 10) and wettability (8, 9, 11) of ceramic carriers on the initial anchoring of cells to the bioceramics. It was also found that the wettability of the ceramic carrier was related to the ionic density within the thin surface layer (11). Thus we arrived at the hypothesis that the negative charge of the surface of TCP-HAP carriers increased the affinity of adherent cells to the ceramic carriers. Cell adhesiveness to TCP-HAP sinters was also enhanced by modifying the surface of the ceramics with each serum protein, fibronectin, and collagen (8). These results suggested that the strong negative charge of the surface’s zeta potential was effective in adsorbing specific proteins and thus for the enhancement of cell adhesiveness.

culture condition was investigated by use of surface modified TCPs to clarify the effects on adhesion and growth of cells.

* Corresponding author. 226

VOL. 81, 1996

CELL

MATERIALS

AND METHODS

Zeta potential of modified TCP B-Tricalcium phosphate (TCP) ceramics were prepared in the same way as described in previous reports (18, 19). A fraction smaller than 5 pm was obtained by grinding the TCP ceramics. Surface modification of TCP was carried out in the same way as described in previous reports (9, 10). Table 1 summarizes the silane coupling reagents used for surface modification of TCP. The initial zeta potential was measured after drying the modified TCP samples at 60°C. The zeta potential of the ceramics was varied from - 11.5 mV to +27.2 mV by introducing organic groups to the surface of TCP by silane coupling reaction. For measurement of the time-dependent variation of the zeta potential, each 400 mg of the dry powder was immersed in 10ml of distilled water, Eagle’s minimum essential medium containing 1.35 g/l of NaHC03 (MEM), and MEM containing 10% of fetal bovine serum and 0.3 g/l of glutamine (lO%FBS/MEM), respectively. They were placed in an incubator at 37°C and shaken at 60 rpm for periods of 1, 3, and 6 d. At the end of each immersion time, the powder was obtained by centrifuging for 10 min at 10,000 g. The centrifuged powder was then washed twice with 10ml of distilled water and dried at 60°C. The zeta potential of the powder was measured at pH 7.0-7.6 using System 3000 (PEN KEM Co. Ltd., Bedfordhills, NY, USA). Calcium, phosphate, and protein The concentration of calcium in the supernatant at the end of each immersion time was measured by flame atomic absorption spectroscopy (Seiko Instruments ISM-70OOS, Tokyo). The concentration of phosphate was measured by calorimetric detection of phosphomolybdate (20). Electric conductivity (Conductivity Meter CM-05, Kyoto Electronics Co. Ltd., Kyoto) and pH of the supernatant were also measured at the end of immersion. Adsorbed protein to the ceramic powder immersed in lO%FBS/MEM was measured as follows. Five ml of 0.1 N NaOH solution was added to each 200mg sample of the dried powder after immersion, and shaken for six hours at 37°C to dissolve the adsorbed protein. The protein concentration in the alkaline solution was assayed using bovine serum albumin as the standard by Lowry’s method (21). All concentrations were measured in triplicate and averaged. Cell culture The surface of the TCP plates, 30mm in diameter and 1.5 mm thick, was polished with a 9micron diamond disk grinder. After autoclaving at 12O”C, the plates were washed twice with culture medium and used for cell culturing experiment. The mousederived cell line L-929 (NCTC Clone 929) was used in this study. The cells were cultured in lO%FBS/MEM. Three ml of cell culture was seeded onto the ceramic plates in polystyrene tissue culture dishes, LUX (Miles Lab. Inc., Naperville, IL, USA), and cultured at 37°C in a 5% CO* gas incubator. Then, the initial anchoring ratio (Ria) and the adhesive strength by the trypsination method (Fa.& were measured in the same way as described previously (8). The definitions for Ri, and F a.enzare as follows: Ri,=Na.cell/(Na.cell+Nf.cell) F a.enz= 1 -N.cell/N.cell

where Nf.cell is the number of floating cells and Na.ceri the number of anchored cells on a carrier plate, which was

TABLE

Sample

ID

TCP 1090-TCP 2190-TCP 2300-TCP 2480-TCP 3150-TCP

1.

ADHESION

Silane coupling reagents of ceramics

ON CERAMICS

for chemical

Reagent

Chemical

LS LS LS LS LS

CFICH2CH2Si(OCH& CHS(CH&SiClj HzC =CHSi(OC2H& H2N(CH2)2NH(CH2)JSi(OCHj)j H2N(CH&Si(OC2H&

1090 2190 2300 2480 3150

structure

227

modification

Initial zeta potential (mV) -9.3 f0.1 -2.0 -11.5 +27.2 f25.2

estimated from the liberation by intensive pipetting after addition of trypsine. Nt.cell and Nr.cell are the total cell number and the cells released in 15 min after addition of trypsine, respectively. Ri, was measured at 1 h after the beginning of cultivation, and F,.,,, after 1 d, 3 d, and 6d, respectively. Cell density was measured by counting the number of cells using a phase-contrast microscope and an eosinophil counter after liberation of anchored cells by trypsination. The total cell number of suspended cells was compatibly measured using a Coulter counter. RESULTS Time-dependent variation of zeta potential Significant changes in the zeta potential were measured. Figure 1 shows the time-dependent variations of zeta potential of TCP and the surface modified ones. The zeta potential of the intact TCP was -9.3 mV. The charge gradually shifted positively with time to reach - 1.8 mV at 6 d by immersing in distilled water. Similarly, zeta potentials of 2190-TCP and 2300-TCP changed from negative charges of -2.0 mV and -- 11.5 mV to +0.9 mV and -7.5 mV in distilled water, respectively. In contrast, the zeta potentials of 1090-TCP, 2480-TCP, and 3150-TCP, which had positive charges by surface modification as f0.1 mV, +27.2mV, and +25.2mV, shifted toward the negative change to -3.3 mV, +7.5 mV, and +5.9 mV, respectively. Similar variation of the zeta potential was obtained when the ceramics were immersed in MEM as shown in Fig. 1 (middle). The zeta potential of TCP shifted toward a positive change in MEM, while they shifted to negative charges in cases of 1090-TCP, 2480-TCP, and 3150-TCP. On the other hand, the zeta potentials shifted toward negative ends except for 2300-TCP when the ceramics were immersed in lO%FBS/MEM as shown in Fig. 1 (bottom). The shifting rates and the amounts of the varied charges were different depending on the initial intensity. In cases of 2480-TCP and 3150-TCP, the charges reached to - 13.7 mV and - 17.6mV at 6d by shifting more than 40mV from each initial potential, while the strongest negative charge of -27.6 mV was obtained in case of TCP. The time-dependent variation of the zeta potential showed that the shifting rates and the amounts of varied charges were considerably high during the first day of immersion. The zeta potentials of intact and modified TCPs in MEM became rather stable during the following immersion period until 6 d. These results suggested that the surface structure of TCP was unstable and soluble in the solutions. Therefore, a part of introduced organic groups were released on day one by accompanied with the dissolution of the surface layer of TCP. In addition, the zeta potentials of various TCPs approached each

228

SUZUKI ET AL.

-301

,

J. FERMENT.BIOENG.,

I

I

I

I

01

“t

20

10 0

_

c Fs4

-10

I

$3 .c

s2 ;1

&l 0 0

1 2 3 4 5 Immersion time (d)

6

0

1 2 3 4 5 Immersion time (d)

6

FIG. 2. Variation of concentrations of calcium and phosphate in distilled water, MEM, and lO%FBS/MEM by immersing intact and modified TCPs. Symbol n shows the concentration in blank solution without immersing ceramics. Other symbols are the same as in Fig. 1.

-10

-20 -301 0

1

2 3 4 Immersion time (d)

5

6

Variation of zeta potential of intact and modified TCPs as a function of immersion time in distilled water (top), MEM (middle), and lO%FBS/MEM (bottom). Symbols are TCP, x; lO%%TCP, 0 ; 2190_TCP, A; 2300.TCP, q ; 2480.TCP, 0 ; and 3150. TCP, A.

other due to immersing the TCPs in MEM as all charges were between -3.3 mV and -9.9mV at 6d, while the final zeta potentials in distilled water were in a wide These results range; between -9.9 mV and -27.6mV. suggested that the surface of TCPs was covered with some precipitates which combined between the ceramic components and solutes in MEM during the immersion. Furthermore, the zeta potentials of TCPs immersed in lO%FBS/MEM shifted toward negative ends even in the period from 3 d to 6 d. Thus, the intensive negative shift in lO%FBS/MEM is believed to be due to the reaction between the ceramics and serum components in the immersing solution. These results suggest that the surface structure of TCPs was dynamically varied by not only dissolving Ca and PO,,, but also adsorbing various components dissolved in MEM and lO%FBS/MEM. Figure 2 shows Calcium, phosphate, and solubility the time-dependent variations of concentrations of calcium and phosphate in each solution. Both concentrations of dissolved Ca and PO4 in distilled water showed that the TCP ceramic powder was highly soluble in distilled water. Furthermore, the solubility was enhanced by

modifying the surface with a silane coupling reagent. In the case of 2190.TCP, both concentrations of Ca and PO4 in distilled water increased up to seven times higher than that of TCP. In the cases of 2480.TCP and 3150. TCP, which had strong positive zeta potentials, the concentration of PO4 was about twice as high as that of TCP, while Ca was scarcely detected in the supernatant. On the other hand, the concentrations of dissolved Ca in MEM and lO%FBS/MEM decreased, with the exception of 2190.TCP. In a similar fashion, the concentration of PO4 at 1 d in lO%FBS/MEM decreased to lower than the initial level, with the exception of 2190.TCP. The concentrations of PO,, in control solutions of MEM and lO%FBS/MEM, in which no ceramics was immersed, maintained values close to the initial concentrations as did the concentrations of Ca. Thus the decreases in concentrations of Ca and PO4 were caused by the immersion of various TCPs. Figure 3 shows the variations in pH and electric conductivity in each solution. The time-dependent profile of the electric conductivity in distilled water was considerably similar to those for concentrations of Ca and PO.+ The conductivity also showed that the solubility of TCPs depended on the surface modification compounds. The dissolved compounds caused a pH shift towards the acidic end in the case of 2190-TCP, and towards the alkaline ends in the cases of 2480.TCP and 3150.TCP, which were introduced with amino groups previously on the surfaces. Each change in pH towards acidic and alkaline ends caused an increase in the electric conductivity as

VOL. 81, 1996

CELL ADHESION ON CERAMICS

5

10 0

1

2

3

4

5

6

cl

Immersion time (d)

1

2

3

4

5

6

Immersion time (d)

FIG. 3. Time-dependent profiles of pH and electric conductivity, o, in distilled water, MEM, and lO%FBS/MEM by immersing intact and modified TCPs. Symbols are the same as in Figs. 1 and 2.

shown in the figure. In contrast, the conductivity at 6 d decreased to values lower than that for 1 d by immersing in lO%FBS/MEM. This suggested that some solutes, such as protein and minerals, were removed from the solution by adsorbing onto the surface of the TCPs in lO%FBS/MEM. In addition, the decreases in electric IO

229

conductivity in the later half period of immersion agree with the decreases in Ca concentration as shown in Fig. 2, thus the dissolved components precipitated via the interaction with Ca. The pH of the control solutions of MEM and lO%FBS/MEM increased gradually day by day. Since the medium contains NaHCOs, increases in pH of control solutions seem to be caused by removal of COZ. Similarly, decreases in dissolved Ca in MEM and lO%FBS/MEM were also considered to be caused by binding with carbonate. It can be assumed that the precipitation of calcium carbonate as well as calcium phosphate were promoted under mild alkaline conditions. Figure 4 shows relations between pH and variations of dissolved Ca and P04. The variations of Ca and PO4 indicate the differences in the concentrations between the initial levels and those of after immersion at 3 d and 6 d. Concentration of Ca changed somewhat linearly depending on the pH of the solution. Although concentration of PO4 in acidic solution changed in the same manner as Ca, it did not depend on pH in alkaline condition. The solubility of PO4 seemed to be enhanced according to the decreases of solubility of Ca under alkaline conditions. The correlation between varied concentrations of Ca and PO., is shown in Fig. 5. The Ca/P04 molar ratio in distilled water, which was approximately 1.67, was higher than the stoichiometric ratio of 1.50 for TCP, while they were very small in MEM and lO%FBS/MEM. These values suggest that Ca was quickly dissociated into ions in solution from TCP, and the dissolved Ca ions precipitated by combining with components of MEM. Because the concentration of dissolved PO4 was considerably higher than Ca compared to the stoichiometric balance, carbonate seemed more likely than phosphate to bind with Ca in both solutions of MEM and lO%FBS/MEM. Figure 6 shows the time-dependent profile of the adsorbed amount of protein. The adsorption progressed gradually as shown in the figure and the higher amount of protein adsorbed to TCP and 2190-TCP at 6 d immersion time. To investigate the effect of the protein on the surface property, the differences of the zeta potential be-

6~ n 6 0

F

g4t

0

8

60

CD

42 E

02

2 0 7

g

, 0 ----- -_---- ----

OE ,: -2 7

3 Li4 -2

8, 5

6

7

6

9

10

-4

PH FIG. 4. Relationships between variations of dissolved calcium and pH, between dissolved phosphate and pH at 3 d and 6 d by immersing intact and modified TCPs in distilled water (Ca: 0, P: 0), MEM (Ca: A, P: A), and lO%FBS/MEM (Ca: n , P: 0).

, I

--L~Q---------_-_---_-__ 49

3L --__&_

50

-2

-1

rc

0

1

c!z!l

2

[PO41 t - [PO410

3

4

5

(mM)

FIG. 5. Relationships between variations of dissolved calcium and phosphate in distilled water, 0 ; MEM, A; lO%FBS/MEM, 0 .

FERMENT. BIOENG.,

J.

230 SUZUKI ET AL.

A

0 0

Ol

I 0

1

2

3

Immersion

4

5

6

20

time (d)

FIG. 6. Time-dependent profiles of adsorbed amount of protein to intact and modified TCPs immersed in lO%FBS/MEM. Symbols are the same as in Fig. 1.

36 30 protein (m&-ceramics)

40

FIG. 7. Relationship between adsorbed amount of protein to ceramics and differences in zeta potential between in lO%FBS/MEM and in MEM by immersing intact and modified TCPs for 3 d, 0 ; and 6d, A.

tween lO%FBS/MEM and MEM each day were calculated during the time of immersion. Figure 7 shows the relation between the changes in the zeta potential and the amount of adsorbed protein to intact and modified TCPs. As clearly shown in the figure, the zeta potential changed more intensively toward the negative charge in agreement with the increase in adsorption of serum protein to the ceramics. It was found that the differences of zeta potential for immersion in lO%FBS/MEM and in MEM in Fig. 1 were correlated to the amount of adsorbed protein on the ceramics. Figure 8 shows depenCeII anchoring and adhesion dencies of the initial anchoring ratio and the adhesiveness on the zeta potential of intact and modified TCPs. The dependencies on the zeta potential of dry ceramics were similar to those previously reported (9). However, it was found that the adhesiveness was higher on weakly charged surfaces than a strong negative charge one by analyzing the relationship between the adhesion strength and zeta potential of 6d-immersion in lO%FBS/MEM. Because the adhesion strength was estimated by averaging the values of 3 d and 6 d culture time, the zeta poten-

tial after immersing in culture medium is considered to be more significant than that of dry state for estimating the effect for adhesion of cells on ceramics. Figure 9 shows the dependencies on the varied amounts of pH, PO4 concentration, and Ca concentration in lO%FBS/MEM at 1 d for the initial anchoring ratio and at 6 d for adhesion strength of cells. The highest values of adhesion strength and the anchoring ratio were both obtained around a 0.25 of pH shift, which was the most smallest change in pH. This suggested that the solubility of the thin surface layer of TCPs reduced the adhesiveness of cells on the carrier by loosening the bonding at the cell-material interface. Similarly, adhesiveness gradually decreased according to increase in the varied amount of PO4 concentration. In the case of the Ca concentration, the highest values of the anchoring ratio and the adhesion strength were obtained at around - 1.5 mM of Ca concentration. The profiles of varied amounts of Ca and PO4 also agree with that of pH, that is the solubility of the surface layer is considered to be the dominant factor for decreasing in the adhesiveness of

‘.OI

‘.OI

0.9

0.9

E.

0.6-

0.6

i

0.7 -

0.7

0.6 -

0.6

0 0 ..

26 Adsorbed

2

n .. '

0.5

-20

-10

10

20

30

5

-30

-I

-20

-10

0

10

cuss)(mV) FIG. 8. Left: effect of zeta potential of intact and modified TCPs in a dry state on the initial anchoring ratio, A; adhesion strength, 0. Right: relations between zeta potential of the TCPs at 1 d in lO%FBS/MEM and the initial anchoring ratio, A; between the zeta potential at 6 d and the adhesion strength, 0.

VOL. 81, 1996

CELL ADHESION

0.0

0.5 [pHlt

1.0

1.5

- [PqJ

FIG. 9. Effects of variations in pH, concentrations variations at 6 d for adhesion strength, 0.

0.0

0.5

1.0

[PO41 t - D’0410

1.5

(mM)

2.0

-2.0

-1.0

ON CERAMICS

0.0

231

1.0

[Cal t - [Cal,, (mM)

of calcium and phosphate at 1 d in lO%FBS/MEM on the initial anchoring ratio, A; the

cells on TCPs. Thus the instability of the surface of carrier material is regarded as the major reason for the weak adhesiveness of cells. DISCUSSION Multiple effects of the instability of bioceramics were found by using the surface modified TCP sinters whose surface zeta potentials were stepwise controlled. The modifications changed the stability of the surface layer of TCP. The series of analyses showed that high solubility caused lower cell adhesiveness levels, although cell growth activities were not damaged (8, 9). In this study the most intensive cell adhesiveness was obtained using a 2300-TCP, which was the most inert in culture medium with small variations of pH shift and dissolved concentration of P04. These findings solve the question relating to why the adhesiveness of cells on TCP ceramics were weaker than LUX, soda-lime glass, and zirconia (8). Stoichiometric analyses on the calcium and phosphate suggested that the chemical structure of the surface of TCP changed dynamically in culture medium through interactive reactions of association and dissociation of Ca and PO4 species. All measurements of zeta potential, pH, electric conductivity, and concentrations of dissolved Ca and PO4 reflected that the TCPs were unstable even in culture medium and so that the surface structure change was dynamic. However, the corrosion of the surface layer of various TCP carriers could not be detected by microscopic observation after culture of L-929 cells for 6d, neither as for damages on cell growth. In addition, the differences in the final pH values of cultures using intact and modified TCPs were small, where all pHs were between 7.25 to 7.65, because the pH of the culture was maintained by controlling CO2 concentration in the gas incubator during cultivation. These results suggest the instability of the thin surface layer of TCP is critical for decreasing in adhesiveness of cells. Both modified ceramics of 3150-TCP and 2480-TCP immersed in lO%FBS/MEM were found to increase pH at the surface layer. The alkaline condition is considered to accelerate calcination at the thin surface layer by trapping the dissolved Ca even in cell culture conditions. Ducheyne et al. reported that Ca was released from hydroxyapatite ceramics by observing the time-dependent

variations of zeta potential, Ca, and PO4 in various pH conditions (12). Our results on TCP are in agreement with their results that the stoichiometric balance of Ca/ PO4 dissolved into distilled water was higher than that of TCP as described above. Furthermore, we found that the surface structure and biological affinity of bioceramits varied dynamically not only by dissociation of components such as Ca and P04, but also association of the medium components of both minerals and proteins. The instability of the bioceramic surface in a biological fluid has an aspect of a preferred effect for changes of the chemical structure to a suitable state for connecting tissues in vivo as well as in vitro. The instability is thought especially to be a factor in accelerating the osteoconductivity and for improving the chemical affinity and connectivity with bone tissue in the body. However, the intensive solubility and high reactivity of bioceramic surfaces may cause damage to adherent cells, which in turn could cause inflammation of surrounding tissues in the body such as synovium connected with artificial joint (22). Our results showed the solubility of biocompatible ceramics including implant materials can be regulated by the chemical modifications of the surface and, therefore, the histochemical affinity is also expected to be improved. Calcium phosphate ceramics have been extensively studied for application as a bioactive artificial implant material because of its biological compatibility in the body. The findings reported in this study will be extremely useful for the development of better implant bioceramits REFERENCES Jar&o, Demus,

M., Bolen, C. H., Bobiek, J. K., Kay, J. F., and R. H.: Hydroxyapatite synthesis and characterization in dense polycrystalline form. J. Mat. Sci., 11, 2027-2035 (1976). Ohtsuka, S.: Basic and clinical study of hydroxyapatite. J.

Jpn. Sot. Biomater., 7, 59-72 (1989). Tsuiji, T., Izutu, T., Motoyasu, S., Obitsu, Y., Osada, K., Tasai, K., Aoki, H., Shin, Y., and Togawa, T.: Implantation

of three patients with percutaneous devices made of sintered hydroxyapatite for enteric hyperalimentation. J. Jpn. Sot. Biomat., 10, 256260 (1992). Nisbihara, K.: Studies on artificial root therapeutics with newly tailored hydroxyapatite root. J. Jpn. Sot. Biomat., 11, 135

232

J. FERMENT.BIOENG.,

SUZUKI ET AL.

152 (1993). 5. Jar&o, M., Salsbury, M. B., Thomas, M. B., and Demus, R.H.: Synthesis and fabrication of p-tricalcium phosphate ceramics for potential prosthetic application. J. Mat. Sci., 14, 142-150 (1979). 6. Akao, M., Aoki, H., and Kato, K.: Dense polycrystalline tricalcium phosphate for prosthetic applications. J. Mat. Sci., 17, 343-346 (1982). 7. Suzuki, T., Toriyama, M., Kawamoto, Y., Yokogawa, Y., and Kawamura, S.: Development of a culture carrier for anchorage-dependent animal cells using calcium phosphate ceramic sinters. J. Ferment. Bioeng., 70, 164-168 (1990). 8. Suzuki, T., Toriyama, M., Kawamoto, Y., Yokogawa, Y., and Kawamura, S.: The adhesiveness and growth of anchorage-dependent animal cells on biocompatible ceramic culture carriers. J. Ferment. Bioeng., 72, 450-456 (1991). 9. Nishizawa, K., Toriyama, M., Suzuki, T., Kawamoto, Y., Yokogawa, Y., and Nagae, H.: Effects of the surface wettability and zeta potential of bioceramics on the adhesiveness of anchorage-dependent animal cells. J. Ferment. Bioeng., 75, 435437 (1993). 10. Nishizawa, K., Torlyama, M., Suzuki, T., Kawamoto, Y., Yokogawa, Y., and Nagata, F.: Surface modification of calcium phosphate ceramics with silane coupling reagents. Nihon Kagaku Kaishi, 1995, 63-67 (1995). (in Japanese) 11. Toriyama, M., Kawamoto, Y., Suzuki, T., Yokogawa, Y., Nishizawa, K., and Nagata, F.: Wettability of calcium phosphate ceramics by water. J. Ceram. Sot. Jpn., 103, 46-49 (1995). 12. Ducheyne, P., Kim, C. S., and Pollack, S. R.: The effect of phase differences on the time-dependent variation of the zeta potential of hydroxyapatite. J. Biomed. Mater. Res., 26, 147168 (1992). 13. Somasundran, P.: Zeta potential of apatite in aqueous solution

14.

15.

16.

17.

18.

19.

20.

21.

22.

and its change during equilibrium. J. Dent. Res., 55, 10671075 (1976). Kubo, K., Kakiioto, T., Tsukasa, N., Nakayama, K., Hiwatashi, K., IzumI, Y., Sueda, T., and Kaneko, N.: Biocompatibility of fibrobiast to bioactive glass. Jpn. Sot. Biomater., 11, 85-89 (1993). Niki, M.: Comparative study of the histological, physical and chemical properties of bone-biomaterial interface. J. Jpn. Sot. Biomater., 12, 5-21 (1994). Ebihara, K., Ohno, A., Fukubayashi, T., Tateishi, T., and Shirasaki, Y.: Biocompatibility of the beagle’s prosthesis coated or reinforced with ceramics. J. Jpn. Sot. Biomater., 10, 303-310 (1992). Bagamhisa, F.B. and Joos, U.: Preliminary studies on the phenomenological behavior of osteoblasts cultured on hydroxyapatite ceramics. Biomaterials, 11, 50-56 (1990). Torlyama, M., Kawamura, S., and Shiba, S.: Bending strength of hydroxyapatite ceramics containing a-tricalcium phosphate. J. Ceram. Sot. Jpn. Intern. Ed., 95, 411-412 (1987). Toriyama, M. and Kawamura, S.: Sintable powder of mechanochemically synthetic ,%tricalcium phosphate. J. Ceram. Sot. Jpn. Intern. Ed., 95, 698-702 (1987). Chrlstoffersen, J.: A new and convenient calorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal. Biochem., 113, 313-317 (1981). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 139, 265-269 (1951). Walsh, D. A., Suzuki, T., Knock, G. A., Blake, D. R., Polak, J. M., and Wharton, J.: AT1 receptor characteristics of angiotensin analogue binding in human synovium. Br. J. Pharmacol., 112, 435-442 (1994).