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Effect of bone-like layer growth from culture medium on adherence of osteoblast-like cells
Biomaterials 24 (2003) 941–947
Effect of bone-like layer growth from culture medium on adherence of osteoblast-like cells Takashi Kizukia,b,*, Masataka Ohgakia, Mihoko Katsuraa, Satoshi Nakamuraa, Kazuaki Hashimotob, Yoshitomo Todab, Shigekazu Udagawab, Kimihiro Yamashitaa a
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kandasurugadai, Chiyoda, Tokyo 1010062, Japan b Industrial Inorganic Chemistry, Graduate School of Engineering, Chiba Institute of Technology, 2-17-1, Tsudanuma, Narashino City, Chiba 2750016, Japan Received 21 December 2001; accepted 10 June 2002
Abstract The electrical polarization of ceramic HAp had an effect on the acceleration of bone restoration. Cell behavior in the bone-like growth layer was investigated. The deposits on the ceramic HAp was grown and formed layers by soaking in a-minimum essential medium supplemented with 10% fetal bovine serum (a-MEM supplemented with 10% FBS). The shapes of the adhering cells on the grown layer gradually changed from spindle to flat with growth of the layer. On the totally grown layer that was grown on the ceramic HAp by soaking in a-MEM supplemented with 10% FBS for 7 days, all the adhering cells were flat and the surface was filled with the grown cells. From these results, it was revealed that the grown layer on the ceramic HAp is one of the activation factors of cell growth. Consequently, cell growth was reinforced by acceleration of the layer growth on the negatively charged surface of the polarized ceramic HAp. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Hydroxyapatite; Polarization effect; Bone-like layer; Osteoblast-like cells; Cell behavior
1. Introduction A ceramic HAp with a high biocompatibility is used as the bioactive material in the orthopedic and dental fields. When the ceramic HAp is implanted, a bone-like apatite layer is grown between the natural bone and ceramic HAp [1,2]. This phenomenon is a peculiar reaction of the ceramic HAp, while other implantable materials are epidolied by fibroblast cells. Numerous studies using HAp coatings on metals and other ceramics to produce a high biocompatibility have achieved good recovery on a broken part of hard tissue [3–6]. From these results, it was shown that the characteristics of the material surface are very important for contact with the tissues. Recently, it was discovered that the ceramic HAp was electrically polarizable and that the polarized HAp had *Corresponding author. Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kandasurugadai, Chiyoda, Tokyo 1010062, Japan. E-mail address: [email protected] (T. Kizuki).
outstanding effects on the bone-like crystal growth in simulated body fluid (SBF) [7–9]. The bone-like crystal growth was accelerated on the negatively charged surface and decelerated on the positively charged surface of a polarized ceramic HAp. Moreover, the osteoblastlike cell (MC3T3-E1 cell) proliferation was influenced by the polarization effect [10,11]. Control of cell proliferation by the polarization effect was confirmed in not only MC3T3-E1 but also in fibroblastic cells (L-929 cell) and nerve cells (SK-N-SH cell) [11,12]. On the polarized ceramic HAp, the cell proliferation was accelerated on the negatively charged surface and decelerated on the positively charged surface as well as the bone-like layer growth. We postulated that the cell proliferation on the polarized ceramic HAp should have a relationship with the bone-like crystal growth. In this study, the cell behavior on the polarized ceramic HAp was investigated. The relation between the cell proliferation and bone-like layer was estimated by the cell cultivation test on the surface that has a different bone-like layer quantity.
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 4 3 0 - 1
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2. Materials and methods Cell behavior on the polarized ceramic HAp was investigated in Section 2.2 as an investigation of polarization effect. Cell behavior on the bone-like layer that cell growth could be manipulated was investigated in Section 2.3 as elucidation of acceleration of the cell proliferation. 2.1. Materials HAp powder was synthesized by a precipitation reaction from calcium nitrate (Ca(NO3)2) and ammonium dihydrogenphosphate ((NH4)2HPO4). In a stirred aqueous suspension of Ca(NO3)2, an aqueous solution of (NH4)2HPO4 was gradually added. While (NH4)2HPO4 was being added, the pH of the solution was kept at 10.5 by added NH4 solution. The obtained slurry was filtered, and then freeze dried. After ground to under 100 mesh, the HAp powder was calcined at 8001C for 1 h in air. The calcined HAp powder was formed into a pellet using a uni-axial press at 148 MPa. The ceramic HAp was prepared by sintering the HAp pellet at 12501C for 2 h under H2O vapor flow. The obtained ceramic HAp was characterized by X-ray diffractometry (XRD) and infrared spectroscopy (IR). The ceramic HAp was sandwiched between a pair of Pt electrode plates, and electrically polarized in a dc field of 10 kV cm 1 at 4001C for 1 h. The samples were cooled to room temperature under the electric field polarization. Polarizability of ceramic HAp was measured by thermal stimulated depolarization current (TSDC) measurement, as reported in Ref. [7]. The negatively and positively charged surfaces of the polarized specimens were respective classified as the N- and P-surface. The surface of the non-polarized ceramic HAp was denoted as the O-surface. 2.2. Cell cultivation Osteoblast-like cells (MC3T3-E1) were used in this study. MC3T3-E1 cells were cultured in an a-minimum essential medium (a-MEM) supplemented with 10% fetal bovine serum (FBS) in an incubator containing 5% CO2 at 371C. As a separate system, each 2 ml suspension of MC3T3-E1 cells (5.5 104 cells ml 1) was divided into the culture dishes with each specimen. The specimens were separately placed in the culture dish to estimate the effect of each polarized surface. Similarly, a 2 ml suspension of MC3T3-E1 cells (2.4 104 cells ml 1) was divided into a +35 mm culture dish with every specimen to estimate the reciprocal action of the polarized surfaces as a combined system. The cell behavior was investigated by proliferation estimation and scanning electron microscopy (SEM) observations. Adhering cells were fixed by the 2.5% glutalaldehyde
and 1% OsO4 solutions for 1 h. The cells were then dried by a critical point drier after dehydration with ethanol (50–100%). Morphological estimation of the adhering cells was carried out by SEM observations after being coated by Pt–Pd sputtering. 2.3. Formation of the bone-like layer and cell cultivation The ceramic HAp was soaked in 2 ml of culture medium (a-MEM supplemented with 10% FBS) in an incubator containing 5% CO2 in air at 371C to form the bone-like layer. One of the first reactions is adsorption of serum proteins to material surface, when biomaterial is implanted, as reported by many researchers. It is necessary to take account of the adsorption behavior of the serum proteins for more realistic investigation of bone-like apatite formation on the ceramic HAp in this study. Therefore, the culture medium containing serum bovine protein is employed in this study. The medium was changed every 2 or 3 days, and all of the procedures were carried out under a germ-free condition. After soaking in the culture medium for 1, 3, 5, and 7 days, the specimens were collected and washed with 0.1 m PBS (phosphate buffer solution). Formation of the bone-like layer on the ceramic HAp was observed by SEM. MC3T3-E1 cells were cultured in a-MEM supplemented with 10% FBS in an incubator containing 5% CO2 in air at 371C. Each 2 ml suspension of MC3T3-E1 cells (1.1 104 cells ml 1) was placed in a 24-well culture microplate with the ceramic HAp having the overgrown bone-like layer and non-soaked ceramic HAp as the control. The cell cultivation was carried out 2 times. After the cell cultivation for 3 and 7 days, the cell behavior was investigated by proliferation estimation and SEM observations. Cell counts were performed with every three samples by a hemocytometer, and the averaged values were adopted as the number of cells. Adhering cells were fixed by the 2.5% glutalaldehyde and 1% OsO4 solutions for 1 h, and then coated by Pt– Pd sputtering before the SEM observations.
3. Results 3.1. Polarization effect on the cell behavior The ceramic HAp used in this study was identified as pure HAp by XRD (Fig. 1) and IR (Fig. 2). The obtained ceramic HAp had an average relative density of 9671%. The sintered ceramic HAp was used in this study after electrical polarization. Polarization of the ceramic HAp was confirmed by TSDC measurement. The polarization effect was estimated by cell cultivation. It was confirmed that the polarization effect controlled the MC3T3-E1 cell proliferation, from the cell cultivation on the polarized ceramic HAp in the
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adhering cells, and the highest growth site was the grain boundary. Intensity (a.u.)
3.2. Estimation of relation between the bone-like layer and the cell proliferation
0
10
20
30
40
50
CuKα 2θ (deg.) Fig. 1. XRD pattern and IR spectra of the synthesized ceramic HAp obtained by sintering at 12501C in H2O vapor for 2 h.
adsorbed H2O
Transmittance
adsorbed H2O
PO43-
PO43-
OHstr.
OHlib. 4000
3400
2800
2200
1600
1000
400
Wave number (cm-1) Fig. 2. SEM photographs of the ceramic HAp surfaces: (a) before cell cultivation; and (b) after cell cultivation for 4 days.
Table 1 Cell growth rate of MC3T3-E1 cell on polarized and non-polarized HAp ceramics in combined system
Cultivation for 2 days Cultivation for 4 days
O-surface
N-surface
P-surface
100 100
75.7 95.8
145.7 108.5
Values are expressed as a percentage based on the cell numbers cultured on O-surface.
separate system. In the combined system, the P-surface had the highest number of adhering cells compared to the other surfaces in the middle of the cell cultivation (Table 1). In the latter part of the cell cultivation, the number of adhering cells on each surface was not different again. As a result of detailed SEM observations of the cell adhering surface, it was revealed that the overgrown layer existed together with adhering cells. It was discovered that the surface condition of the grown layer was smooth. This layer was found around the
Non-soaked ceramic HAp surface was shown in Fig. 1(a). The ceramic HAp was soaked in a-MEM supplemented with 10% FBS for investigation of the relationship between the bone-like layer and the cell growth. The SEM observations showed that the deposits were already formed on the surface of the ceramic HAp after only 1 day of soaking (Fig. 3(b)). Spherical deposits with a diameter from 100 to 200 nm were uniformly dispersing on the ceramic HAp surface. The surface of the bone-like layer grown by soaking in aMEM supplemented with FBS for 3 days is shown in Fig. 3(c). The deposited particles were gradually grown and formed a thin layer by soaking in the culture medium, and then it completely covered the surface of the specimen. The grain-boundary was not clearly observed when the layer was overgrown by soaking for 5 days (Fig. 3(d)). When soaking from 3 to 7 days, a conspicuous transformation of the surface condition was not discovered against the growth of bone-like layer. The volume of the bone-like layer grown after 7 days of soaking was the largest of the soaked specimens (Fig. 3(e)). As a peculiar case, the bone-like grown layer after soaking in the culture medium for 14 days had a stitch-shaped structure (Fig. 3(f)). It was revealed that the layer included carbon, and the Ca/P ratio of the layer was lower than that of the ceramic HAp based on EDS and EPMA analyses. As a result of the cell cultivation on the grown layer, it was confirmed that the MC3T3-E1 cells grew well on the grown layer. After cell cultivation for 3 days, the cells adhering on the ceramic HAp surface were spindle-like in shape, and then formed a gap junction (Fig. 4(a)). On the grown layer by soaking in the a-MEM supplemented with 10% FBS for 1 day, the morphology of the adhering cells was similar to those on the ceramic HAp (Fig. 4(b)). Flattened MC3T3-E1 cells were observed on the layer grown by soaking in the a-MEM supplemented with 10% FBS for 3 days (Fig. 4(c)). With an increase in the thickness of the grown layer, the shapes of the adhering cells were gradually modified from spindle to flat. Fig. 4(d)–(f) show the adhering cells on the layer grown by soaking in the a-MEM supplemented with 10% FBS for 7 days. The shapes of all the adhered MC3T3-E1 cells were flat (Fig. 4(d)) and formed a tight junction, and few spaces existed among the cells (Fig. 4(e)). Moreover, the surface structure of the ceramic HAp was distinctly visible through the adhering cells (Fig. 4(f)). After cell cultivation on the grown layer that was prepared by soaking in a-MEM supplemented
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Fig. 3. SEM photographs of the ceramic HAp surfaces with bone-like layer overgrown by soaking in culture medium. (a) Non-grown surface of ceramic Hap; (b) bone-like layer grown by soaking for 1 day; (c) bone-like layer grown by soaking for 3 days; (d) bone-like layer grown by soaking for 5 days; (e) bone-like layer grown by soaking for 7 days; and (f) bone-like layer grown by soaking for 14 days.
with 10% FBS for 7 days, the surface was not similar to the grown layer or non-soaked ceramic HAp. The grown layer was leveled by phagocytosis of the cells, and it had nanosized pores. From the adhering cell count after the cultivation for 3 days, the adhered cells were estimated more on the layer grown in the a-MEM supplemented with 10% FBS for 1 day than on the surface of the non-soaked ceramic HAp. The number of adhering cells gradually increased with the soaking time, that is, the amount of the grown layer. This tendency was remarkably confirmed after the cell cultivation for 7 days (Fig. 5). A significant acceleration of the cell proliferation appeared on the bone-like layer that was grown from 3 to 5 days.
4. Discussion It was confirmed that MC3T3-E1 cell proliferation in the separate-system was controlled on the polarized
ceramic HAp by the cell cultivation, and that this result agreed with a previous report [10–12]. On the other hand, MC3T3-E1 cells adhered more on the P-surface than on the O-, and N-surfaces in the combined system. From these results, acceleration of the cell proliferation on the N-surface in the separate system and acceleration of the cell adhesion on the P-surface in the combined system were confirmed. It was considered that the obvious cell proliferation effect on the N-surface could be confirmed for long-term cultivation, because the number of cells on the N-surface compared poorly with the P-surface at the start, but had a similar number of adhering cells in the latter part of the cell cultivation. It is conceivable that the polarization effect on cell proliferation was both directly and indirectly affected. The indirect effect could be inducement of ions, amino acids, and proteins by a surface charge. For nucleation of an accumulated layer, Ca ions are predominantly adsorbed on the N-surface relative to the other cations in the medium, such as Na, K, and Mg [13]. In actual
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Fig. 4. SEM photographs of the adhering MC3T3-E1 cells on the overgrown bone-like layer on the ceramic HAp cultured for 4 days. (a) On the nongrown surface of ceramic Hap; (b) on the layer grown by soaking for 1 day; (c) on the layer grown by soaking for 3 days; and (d)–(f) on the layer grown by soaking for 7 days.
Fig. 5. Relation between soaking time and number of adhering cells after cultivation. (Oblique bar shows cultivation for 3 days, and black bar shows cultivation for 7 days.)
fact, bone-like crystal growth on the N-surface in SBF was accelerated by inducement of Ca2+ to the surface. It was confirmed that the bone-like layer was also grown in the culture medium. The surface of the grown layer was very smooth, and then it was not similar to the bone-like layer grown in SBF [14]. It was suggested that these differences in the state of the surface were caused by the differences in the conditions of the layer formation between SBF and the culture medium. The culture medium contained proteins and amino acids, while the SBF had no organic matter. To detect the manipulation mechanism of cell proliferation by material is very important for creating new biomaterials. The polarized ceramic HAp has a great expectation to a possibility of faculty cell manipulation by the polarization effect. For that purpose the manipulation factor of the cell growth on the polarized ceramic HAp was investigated in detail. From in vitro estimations, a remarkable relation between the cell growth and the bone-like layer growth
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in SBF was discovered. Both the bone-like layer growth in SBF and the cell proliferation were accelerated on the N-surface [9–12]. It was reported that a surface charge was effective on these layer growths, and the resulting surface of the grown layer was different between SBF and the culture medium. From these results, it was suggested that the layer grown in the culture medium was due to an acceleration factor of cell proliferation. Thus, the relationship between the bone-like layer growth and the cell proliferation was estimated. The deposition from the culture medium was fast and already observed after 1 day of soaking. The adsorption of proteins from body fluids onto the material surface occurred fast when the biomaterial is implanted in the body [15,16]. For cell cultivation, the culture medium included serum protein and the adsorption of proteins can be carried out as one of the first events when the material was soaked in the medium. Therefore, it was suggested that the initial deposit from the medium was adsorbed serum protein. After protein adsorption, a thin layer of the amorphous calcium phosphate appeared on the surface of the ceramic HAp by soaking in the medium [17]. A very important phenomenon is that the amorphous calcium phosphate layer containing serum proteins can be formed on the biomaterial, because the layer activates bone formation [18]. The quantity of the layer growth was in proportion to the soaking time, and the shape of the layer was gradually modified from a film to a stitch structure. The adhering MC3T3-E1 cells on the ceramic HAp were spindle shaped. The adhering cells on the grown layer by soaking for 1 day were similar to those on the ceramic HAp. From these results, the layer formed at 1 day was not enough to influence the cell adhesion. Part of the adhering cells on the ceramic HAp was flat shaped on the layer grown by soaking for 3 days (Fig. 4(c)). Therefore, the grown layer after 3 days soaking in aMEM with 10% FBS was effective for reinforcing the cell adherence. The difference in the surfaces that were soaked in the medium for 1 and 3 days was covering the ceramic HAp surface either imperfectly or perfectly. Therefore, it was considered that not only protein adsorption from the culture medium, but also formation of the thin layer of the amorphous calcium phosphate including serum protein had an important role in the cell adherence. It was suggested that MC3T3-E1 cells were closely contacted with the grown layer, because the surface structure of the substrate was distinctly visible through the flattened adhering cells. The layer that was grown by soaking in a-MEM with 10% FBS for 7 days was sufficient to reinforce the adherence of all the adhering cells, because all of the adhering cells had a flat-shape on the grown layer. Consequently, reinforcement of the cell adhesion strength is related to the formation of the amorphous calcium phosphate layer containing serum protein.
Fig. 6. Schematic drawing of the effect of surface charge on the cell behavior. The polarization effect is shown by red dotted line. Serum protein and amino acids were adsorbed on each ceramic HAp surface. Calcium ion was significantly guided by the polarization effect on the N-surface, and then the bone-like layer growth was accelerated while taking serum protein and amino acid within the structure (left). Moreover, adhering cells were activated on the grown bone-like layer. On the other hand, floating cells with a negative charge were significantly guided to the P-surface such that the bone-like layer growth was decelerated by the surface charge (right). However, cells were not activated after adhesion due to deceleration of the bone-like layer growth.
On the bone-like layer formed by soaking for 1 day, the number of adhering cells was greater than that of the non-soaked ceramic HAp. It was indicated that the bone-like layer formed by soaking for 1 day in the culture medium was effective for cell proliferation. The number of adhering cells gradually increased with the amount of the grown layer as shown in Fig. 5. Therefore, it was also revealed that the growth layer was one of the acceleration factors of cell proliferation. However, the polarization effect was indicated to be more effective than the grown layer for cell proliferation as mentioned above. It was suggested that the direct and indirect effects of the polarized ceramic HAp play roles in the cell growth. Fig. 6 shows the discovery of this study with respect to the polarization effect on the cells and the surrounding environment. The bone-like layer growth was accelerated on the N-surface, and then the acceleration of cell proliferation was induced. Although the cell adhesion was accelerated on the P-surface, on the other hand, the cell proliferation was reduced. This shows the importance of the bone-like layer growth on the biomaterial surface and bioactivity of ceramic HAp by polarization.
5. Conclusions The effects of polarization on cell behavior were investigated by cell cultivation on electrically polarized ceramic HAp, and then the acceleration of the cell proliferation was confirmed on the N-surface of the polarized ceramic HAp. Moreover, the relationship
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between the bone-like layer growth and the cell growth was revealed for elucidation of the polarization effect on the cell proliferation. As a result, the cell proliferation was accelerated by the bone-like formed layer. The number of adhering cells then gradually increased with the amount of the grown layer. It was discovered that the bone-like layer significantly reinforced the cell adherence. Consequently, the grown layer formed on the specimen was one of the cell activation factors. It was considered that cells on the N-surface of the polarized ceramic HAp was more proliferated than those on the ceramic HAp, because the layer growth is accelerated on the N-surface. From a comparison of the polarization effect with and without the bone-like layer, however, it was suggested that the polarization was effective for cell growth not only indirectly but also directly. Finally, we confirmed that manipulation of cells on the material surface could be possible by the polarization effect.
Acknowledgements This work was partly supported by a grant-in-aid for Scientific Research (A) #10305047 from the Ministry of Education, Science, Sports and Culture of Japan. The author (MO) thanks the Okura Memorial Foundation.
References [1] LeGeros RZ, LeGeros JP. An introduction to bioceramics. In: Hench LL, Wilson J, editors. Singapore: World Scientific, 1993. p. 139–80. [2] Klein CT, Driessen AA, de Groot K, van den Hooff A. Biodegradation behavior of various calcium phosphate materials in bone tissue. J Biomed Mater Res 1983;17:769–84. [3] Ducheyne P, Van Raemdonck W, Heughebaert JC, Heughebaert M. Structural analysis of hydroxyapatite coatings on titanium. Biomaterials 1986;7(2):97–103.
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[4] de Groot K, Geesink R, Klein CT, Serekain P. Plasma sprayed coatings of hydroxylapatite. J Biomed Mater Res 1987;21: 1375–81. [5] Kuroyama Y, Aoki H, Higashikata M, Yoshizawa K, Nakamura S, Ohgaki M, Akao M. Solubility and tissue reaction of a-TCP plasma-coated layer on titanium in subcutaneous tissue. J Jpn Soc Dent Mater Dev 1993;12:528–34. [6] Ohgaki M, Yamashita K. Preparation and biological behavior of a-TCP and HAp-coatings. Phos Res Bull 1998;8:37–42. [7] Yamashita K, Kitagaki K, Umegaki T. Thermal instability and proton conductivity of ceramic hydroxyapatite at high temperature. J Am Ceram Soc 1995;78:1191–7. [8] Nakamura S, Takeda H, Yamashita K. Proton transport polarization and depolarization of hydroxyapatite ceramics. J Appl Phys 2001;89:5386–92. [9] Yamashita K, Oikawa N, Umegaki T. Acceleration and deceleration of bone-like crystal growth on ceramic hydroxyapatite by electric poling. Chem Mater 1996;8:2697–700. [10] Ohgaki M, Nakamura S, Yamashita K. Vectorial Effects on selective cell adhesion of electrically poled hydroxyapatite ceramics. Bioceramics 1999;12:187–90. [11] Ohgaki M, Kizuki T, Katsura M, Yamashita K. Manipulation of selective cell adhesion and growth by surface charges of electrically polarized hydroxyapatite. J Biomed Mater Res 2001;56:366–73. [12] Ohgaki M, Katsura M, Kizuki T, Yamashita K. Electrovectorial effect of crystal formation and cell proliferation. Trans Mater Res Soc Japan 2000;25:169–72. [13] Shimabayashi S, Tamura C, Nakagaki M. Adsorption of hydroxyl ion on hydroxyapatite. Chem Pharm Bull 1981;29:3090–8. [14] Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res 1990;24: 721–34. [15] Ziats NP, Miller KM, Anderson JM. In vitro and in vivo interactions of cells with biomaterials. Biomaterials 1988;9:5–13. [16] Vroman L. The life of an artificial device in contact with blood: initial events and their effects on its final state. Bull NY Acad Med 1988;64:352–7. [17] Neo M, Kotani S, Nakanura T, Yamamuro T, Ohtsuki C, Kokubo T, Bando Y. A comparative study of ultrastructures of the interfaces between four kinds of surface-active ceramic and bone. J Biomed Mater Res 1992;26:1419–32. [18] Ducheyne P. Stimulation of biological function with bioactive glass. MRS Bull 1998;23:43–9.
Effect of bone-like layer growth from culture medium on adherence of osteoblast-like cells Takashi Kizukia,b,*, Masataka Ohgakia, Mihoko Katsuraa, Satoshi Nakamuraa, Kazuaki Hashimotob, Yoshitomo Todab, Shigekazu Udagawab, Kimihiro Yamashitaa a
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kandasurugadai, Chiyoda, Tokyo 1010062, Japan b Industrial Inorganic Chemistry, Graduate School of Engineering, Chiba Institute of Technology, 2-17-1, Tsudanuma, Narashino City, Chiba 2750016, Japan Received 21 December 2001; accepted 10 June 2002
Abstract The electrical polarization of ceramic HAp had an effect on the acceleration of bone restoration. Cell behavior in the bone-like growth layer was investigated. The deposits on the ceramic HAp was grown and formed layers by soaking in a-minimum essential medium supplemented with 10% fetal bovine serum (a-MEM supplemented with 10% FBS). The shapes of the adhering cells on the grown layer gradually changed from spindle to flat with growth of the layer. On the totally grown layer that was grown on the ceramic HAp by soaking in a-MEM supplemented with 10% FBS for 7 days, all the adhering cells were flat and the surface was filled with the grown cells. From these results, it was revealed that the grown layer on the ceramic HAp is one of the activation factors of cell growth. Consequently, cell growth was reinforced by acceleration of the layer growth on the negatively charged surface of the polarized ceramic HAp. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Hydroxyapatite; Polarization effect; Bone-like layer; Osteoblast-like cells; Cell behavior
1. Introduction A ceramic HAp with a high biocompatibility is used as the bioactive material in the orthopedic and dental fields. When the ceramic HAp is implanted, a bone-like apatite layer is grown between the natural bone and ceramic HAp [1,2]. This phenomenon is a peculiar reaction of the ceramic HAp, while other implantable materials are epidolied by fibroblast cells. Numerous studies using HAp coatings on metals and other ceramics to produce a high biocompatibility have achieved good recovery on a broken part of hard tissue [3–6]. From these results, it was shown that the characteristics of the material surface are very important for contact with the tissues. Recently, it was discovered that the ceramic HAp was electrically polarizable and that the polarized HAp had *Corresponding author. Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kandasurugadai, Chiyoda, Tokyo 1010062, Japan. E-mail address: [email protected] (T. Kizuki).
outstanding effects on the bone-like crystal growth in simulated body fluid (SBF) [7–9]. The bone-like crystal growth was accelerated on the negatively charged surface and decelerated on the positively charged surface of a polarized ceramic HAp. Moreover, the osteoblastlike cell (MC3T3-E1 cell) proliferation was influenced by the polarization effect [10,11]. Control of cell proliferation by the polarization effect was confirmed in not only MC3T3-E1 but also in fibroblastic cells (L-929 cell) and nerve cells (SK-N-SH cell) [11,12]. On the polarized ceramic HAp, the cell proliferation was accelerated on the negatively charged surface and decelerated on the positively charged surface as well as the bone-like layer growth. We postulated that the cell proliferation on the polarized ceramic HAp should have a relationship with the bone-like crystal growth. In this study, the cell behavior on the polarized ceramic HAp was investigated. The relation between the cell proliferation and bone-like layer was estimated by the cell cultivation test on the surface that has a different bone-like layer quantity.
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 4 3 0 - 1
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2. Materials and methods Cell behavior on the polarized ceramic HAp was investigated in Section 2.2 as an investigation of polarization effect. Cell behavior on the bone-like layer that cell growth could be manipulated was investigated in Section 2.3 as elucidation of acceleration of the cell proliferation. 2.1. Materials HAp powder was synthesized by a precipitation reaction from calcium nitrate (Ca(NO3)2) and ammonium dihydrogenphosphate ((NH4)2HPO4). In a stirred aqueous suspension of Ca(NO3)2, an aqueous solution of (NH4)2HPO4 was gradually added. While (NH4)2HPO4 was being added, the pH of the solution was kept at 10.5 by added NH4 solution. The obtained slurry was filtered, and then freeze dried. After ground to under 100 mesh, the HAp powder was calcined at 8001C for 1 h in air. The calcined HAp powder was formed into a pellet using a uni-axial press at 148 MPa. The ceramic HAp was prepared by sintering the HAp pellet at 12501C for 2 h under H2O vapor flow. The obtained ceramic HAp was characterized by X-ray diffractometry (XRD) and infrared spectroscopy (IR). The ceramic HAp was sandwiched between a pair of Pt electrode plates, and electrically polarized in a dc field of 10 kV cm 1 at 4001C for 1 h. The samples were cooled to room temperature under the electric field polarization. Polarizability of ceramic HAp was measured by thermal stimulated depolarization current (TSDC) measurement, as reported in Ref. [7]. The negatively and positively charged surfaces of the polarized specimens were respective classified as the N- and P-surface. The surface of the non-polarized ceramic HAp was denoted as the O-surface. 2.2. Cell cultivation Osteoblast-like cells (MC3T3-E1) were used in this study. MC3T3-E1 cells were cultured in an a-minimum essential medium (a-MEM) supplemented with 10% fetal bovine serum (FBS) in an incubator containing 5% CO2 at 371C. As a separate system, each 2 ml suspension of MC3T3-E1 cells (5.5 104 cells ml 1) was divided into the culture dishes with each specimen. The specimens were separately placed in the culture dish to estimate the effect of each polarized surface. Similarly, a 2 ml suspension of MC3T3-E1 cells (2.4 104 cells ml 1) was divided into a +35 mm culture dish with every specimen to estimate the reciprocal action of the polarized surfaces as a combined system. The cell behavior was investigated by proliferation estimation and scanning electron microscopy (SEM) observations. Adhering cells were fixed by the 2.5% glutalaldehyde
and 1% OsO4 solutions for 1 h. The cells were then dried by a critical point drier after dehydration with ethanol (50–100%). Morphological estimation of the adhering cells was carried out by SEM observations after being coated by Pt–Pd sputtering. 2.3. Formation of the bone-like layer and cell cultivation The ceramic HAp was soaked in 2 ml of culture medium (a-MEM supplemented with 10% FBS) in an incubator containing 5% CO2 in air at 371C to form the bone-like layer. One of the first reactions is adsorption of serum proteins to material surface, when biomaterial is implanted, as reported by many researchers. It is necessary to take account of the adsorption behavior of the serum proteins for more realistic investigation of bone-like apatite formation on the ceramic HAp in this study. Therefore, the culture medium containing serum bovine protein is employed in this study. The medium was changed every 2 or 3 days, and all of the procedures were carried out under a germ-free condition. After soaking in the culture medium for 1, 3, 5, and 7 days, the specimens were collected and washed with 0.1 m PBS (phosphate buffer solution). Formation of the bone-like layer on the ceramic HAp was observed by SEM. MC3T3-E1 cells were cultured in a-MEM supplemented with 10% FBS in an incubator containing 5% CO2 in air at 371C. Each 2 ml suspension of MC3T3-E1 cells (1.1 104 cells ml 1) was placed in a 24-well culture microplate with the ceramic HAp having the overgrown bone-like layer and non-soaked ceramic HAp as the control. The cell cultivation was carried out 2 times. After the cell cultivation for 3 and 7 days, the cell behavior was investigated by proliferation estimation and SEM observations. Cell counts were performed with every three samples by a hemocytometer, and the averaged values were adopted as the number of cells. Adhering cells were fixed by the 2.5% glutalaldehyde and 1% OsO4 solutions for 1 h, and then coated by Pt– Pd sputtering before the SEM observations.
3. Results 3.1. Polarization effect on the cell behavior The ceramic HAp used in this study was identified as pure HAp by XRD (Fig. 1) and IR (Fig. 2). The obtained ceramic HAp had an average relative density of 9671%. The sintered ceramic HAp was used in this study after electrical polarization. Polarization of the ceramic HAp was confirmed by TSDC measurement. The polarization effect was estimated by cell cultivation. It was confirmed that the polarization effect controlled the MC3T3-E1 cell proliferation, from the cell cultivation on the polarized ceramic HAp in the
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adhering cells, and the highest growth site was the grain boundary. Intensity (a.u.)
3.2. Estimation of relation between the bone-like layer and the cell proliferation
0
10
20
30
40
50
CuKα 2θ (deg.) Fig. 1. XRD pattern and IR spectra of the synthesized ceramic HAp obtained by sintering at 12501C in H2O vapor for 2 h.
adsorbed H2O
Transmittance
adsorbed H2O
PO43-
PO43-
OHstr.
OHlib. 4000
3400
2800
2200
1600
1000
400
Wave number (cm-1) Fig. 2. SEM photographs of the ceramic HAp surfaces: (a) before cell cultivation; and (b) after cell cultivation for 4 days.
Table 1 Cell growth rate of MC3T3-E1 cell on polarized and non-polarized HAp ceramics in combined system
Cultivation for 2 days Cultivation for 4 days
O-surface
N-surface
P-surface
100 100
75.7 95.8
145.7 108.5
Values are expressed as a percentage based on the cell numbers cultured on O-surface.
separate system. In the combined system, the P-surface had the highest number of adhering cells compared to the other surfaces in the middle of the cell cultivation (Table 1). In the latter part of the cell cultivation, the number of adhering cells on each surface was not different again. As a result of detailed SEM observations of the cell adhering surface, it was revealed that the overgrown layer existed together with adhering cells. It was discovered that the surface condition of the grown layer was smooth. This layer was found around the
Non-soaked ceramic HAp surface was shown in Fig. 1(a). The ceramic HAp was soaked in a-MEM supplemented with 10% FBS for investigation of the relationship between the bone-like layer and the cell growth. The SEM observations showed that the deposits were already formed on the surface of the ceramic HAp after only 1 day of soaking (Fig. 3(b)). Spherical deposits with a diameter from 100 to 200 nm were uniformly dispersing on the ceramic HAp surface. The surface of the bone-like layer grown by soaking in aMEM supplemented with FBS for 3 days is shown in Fig. 3(c). The deposited particles were gradually grown and formed a thin layer by soaking in the culture medium, and then it completely covered the surface of the specimen. The grain-boundary was not clearly observed when the layer was overgrown by soaking for 5 days (Fig. 3(d)). When soaking from 3 to 7 days, a conspicuous transformation of the surface condition was not discovered against the growth of bone-like layer. The volume of the bone-like layer grown after 7 days of soaking was the largest of the soaked specimens (Fig. 3(e)). As a peculiar case, the bone-like grown layer after soaking in the culture medium for 14 days had a stitch-shaped structure (Fig. 3(f)). It was revealed that the layer included carbon, and the Ca/P ratio of the layer was lower than that of the ceramic HAp based on EDS and EPMA analyses. As a result of the cell cultivation on the grown layer, it was confirmed that the MC3T3-E1 cells grew well on the grown layer. After cell cultivation for 3 days, the cells adhering on the ceramic HAp surface were spindle-like in shape, and then formed a gap junction (Fig. 4(a)). On the grown layer by soaking in the a-MEM supplemented with 10% FBS for 1 day, the morphology of the adhering cells was similar to those on the ceramic HAp (Fig. 4(b)). Flattened MC3T3-E1 cells were observed on the layer grown by soaking in the a-MEM supplemented with 10% FBS for 3 days (Fig. 4(c)). With an increase in the thickness of the grown layer, the shapes of the adhering cells were gradually modified from spindle to flat. Fig. 4(d)–(f) show the adhering cells on the layer grown by soaking in the a-MEM supplemented with 10% FBS for 7 days. The shapes of all the adhered MC3T3-E1 cells were flat (Fig. 4(d)) and formed a tight junction, and few spaces existed among the cells (Fig. 4(e)). Moreover, the surface structure of the ceramic HAp was distinctly visible through the adhering cells (Fig. 4(f)). After cell cultivation on the grown layer that was prepared by soaking in a-MEM supplemented
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Fig. 3. SEM photographs of the ceramic HAp surfaces with bone-like layer overgrown by soaking in culture medium. (a) Non-grown surface of ceramic Hap; (b) bone-like layer grown by soaking for 1 day; (c) bone-like layer grown by soaking for 3 days; (d) bone-like layer grown by soaking for 5 days; (e) bone-like layer grown by soaking for 7 days; and (f) bone-like layer grown by soaking for 14 days.
with 10% FBS for 7 days, the surface was not similar to the grown layer or non-soaked ceramic HAp. The grown layer was leveled by phagocytosis of the cells, and it had nanosized pores. From the adhering cell count after the cultivation for 3 days, the adhered cells were estimated more on the layer grown in the a-MEM supplemented with 10% FBS for 1 day than on the surface of the non-soaked ceramic HAp. The number of adhering cells gradually increased with the soaking time, that is, the amount of the grown layer. This tendency was remarkably confirmed after the cell cultivation for 7 days (Fig. 5). A significant acceleration of the cell proliferation appeared on the bone-like layer that was grown from 3 to 5 days.
4. Discussion It was confirmed that MC3T3-E1 cell proliferation in the separate-system was controlled on the polarized
ceramic HAp by the cell cultivation, and that this result agreed with a previous report [10–12]. On the other hand, MC3T3-E1 cells adhered more on the P-surface than on the O-, and N-surfaces in the combined system. From these results, acceleration of the cell proliferation on the N-surface in the separate system and acceleration of the cell adhesion on the P-surface in the combined system were confirmed. It was considered that the obvious cell proliferation effect on the N-surface could be confirmed for long-term cultivation, because the number of cells on the N-surface compared poorly with the P-surface at the start, but had a similar number of adhering cells in the latter part of the cell cultivation. It is conceivable that the polarization effect on cell proliferation was both directly and indirectly affected. The indirect effect could be inducement of ions, amino acids, and proteins by a surface charge. For nucleation of an accumulated layer, Ca ions are predominantly adsorbed on the N-surface relative to the other cations in the medium, such as Na, K, and Mg [13]. In actual
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Fig. 4. SEM photographs of the adhering MC3T3-E1 cells on the overgrown bone-like layer on the ceramic HAp cultured for 4 days. (a) On the nongrown surface of ceramic Hap; (b) on the layer grown by soaking for 1 day; (c) on the layer grown by soaking for 3 days; and (d)–(f) on the layer grown by soaking for 7 days.
Fig. 5. Relation between soaking time and number of adhering cells after cultivation. (Oblique bar shows cultivation for 3 days, and black bar shows cultivation for 7 days.)
fact, bone-like crystal growth on the N-surface in SBF was accelerated by inducement of Ca2+ to the surface. It was confirmed that the bone-like layer was also grown in the culture medium. The surface of the grown layer was very smooth, and then it was not similar to the bone-like layer grown in SBF [14]. It was suggested that these differences in the state of the surface were caused by the differences in the conditions of the layer formation between SBF and the culture medium. The culture medium contained proteins and amino acids, while the SBF had no organic matter. To detect the manipulation mechanism of cell proliferation by material is very important for creating new biomaterials. The polarized ceramic HAp has a great expectation to a possibility of faculty cell manipulation by the polarization effect. For that purpose the manipulation factor of the cell growth on the polarized ceramic HAp was investigated in detail. From in vitro estimations, a remarkable relation between the cell growth and the bone-like layer growth
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in SBF was discovered. Both the bone-like layer growth in SBF and the cell proliferation were accelerated on the N-surface [9–12]. It was reported that a surface charge was effective on these layer growths, and the resulting surface of the grown layer was different between SBF and the culture medium. From these results, it was suggested that the layer grown in the culture medium was due to an acceleration factor of cell proliferation. Thus, the relationship between the bone-like layer growth and the cell proliferation was estimated. The deposition from the culture medium was fast and already observed after 1 day of soaking. The adsorption of proteins from body fluids onto the material surface occurred fast when the biomaterial is implanted in the body [15,16]. For cell cultivation, the culture medium included serum protein and the adsorption of proteins can be carried out as one of the first events when the material was soaked in the medium. Therefore, it was suggested that the initial deposit from the medium was adsorbed serum protein. After protein adsorption, a thin layer of the amorphous calcium phosphate appeared on the surface of the ceramic HAp by soaking in the medium [17]. A very important phenomenon is that the amorphous calcium phosphate layer containing serum proteins can be formed on the biomaterial, because the layer activates bone formation [18]. The quantity of the layer growth was in proportion to the soaking time, and the shape of the layer was gradually modified from a film to a stitch structure. The adhering MC3T3-E1 cells on the ceramic HAp were spindle shaped. The adhering cells on the grown layer by soaking for 1 day were similar to those on the ceramic HAp. From these results, the layer formed at 1 day was not enough to influence the cell adhesion. Part of the adhering cells on the ceramic HAp was flat shaped on the layer grown by soaking for 3 days (Fig. 4(c)). Therefore, the grown layer after 3 days soaking in aMEM with 10% FBS was effective for reinforcing the cell adherence. The difference in the surfaces that were soaked in the medium for 1 and 3 days was covering the ceramic HAp surface either imperfectly or perfectly. Therefore, it was considered that not only protein adsorption from the culture medium, but also formation of the thin layer of the amorphous calcium phosphate including serum protein had an important role in the cell adherence. It was suggested that MC3T3-E1 cells were closely contacted with the grown layer, because the surface structure of the substrate was distinctly visible through the flattened adhering cells. The layer that was grown by soaking in a-MEM with 10% FBS for 7 days was sufficient to reinforce the adherence of all the adhering cells, because all of the adhering cells had a flat-shape on the grown layer. Consequently, reinforcement of the cell adhesion strength is related to the formation of the amorphous calcium phosphate layer containing serum protein.
Fig. 6. Schematic drawing of the effect of surface charge on the cell behavior. The polarization effect is shown by red dotted line. Serum protein and amino acids were adsorbed on each ceramic HAp surface. Calcium ion was significantly guided by the polarization effect on the N-surface, and then the bone-like layer growth was accelerated while taking serum protein and amino acid within the structure (left). Moreover, adhering cells were activated on the grown bone-like layer. On the other hand, floating cells with a negative charge were significantly guided to the P-surface such that the bone-like layer growth was decelerated by the surface charge (right). However, cells were not activated after adhesion due to deceleration of the bone-like layer growth.
On the bone-like layer formed by soaking for 1 day, the number of adhering cells was greater than that of the non-soaked ceramic HAp. It was indicated that the bone-like layer formed by soaking for 1 day in the culture medium was effective for cell proliferation. The number of adhering cells gradually increased with the amount of the grown layer as shown in Fig. 5. Therefore, it was also revealed that the growth layer was one of the acceleration factors of cell proliferation. However, the polarization effect was indicated to be more effective than the grown layer for cell proliferation as mentioned above. It was suggested that the direct and indirect effects of the polarized ceramic HAp play roles in the cell growth. Fig. 6 shows the discovery of this study with respect to the polarization effect on the cells and the surrounding environment. The bone-like layer growth was accelerated on the N-surface, and then the acceleration of cell proliferation was induced. Although the cell adhesion was accelerated on the P-surface, on the other hand, the cell proliferation was reduced. This shows the importance of the bone-like layer growth on the biomaterial surface and bioactivity of ceramic HAp by polarization.
5. Conclusions The effects of polarization on cell behavior were investigated by cell cultivation on electrically polarized ceramic HAp, and then the acceleration of the cell proliferation was confirmed on the N-surface of the polarized ceramic HAp. Moreover, the relationship
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between the bone-like layer growth and the cell growth was revealed for elucidation of the polarization effect on the cell proliferation. As a result, the cell proliferation was accelerated by the bone-like formed layer. The number of adhering cells then gradually increased with the amount of the grown layer. It was discovered that the bone-like layer significantly reinforced the cell adherence. Consequently, the grown layer formed on the specimen was one of the cell activation factors. It was considered that cells on the N-surface of the polarized ceramic HAp was more proliferated than those on the ceramic HAp, because the layer growth is accelerated on the N-surface. From a comparison of the polarization effect with and without the bone-like layer, however, it was suggested that the polarization was effective for cell growth not only indirectly but also directly. Finally, we confirmed that manipulation of cells on the material surface could be possible by the polarization effect.
Acknowledgements This work was partly supported by a grant-in-aid for Scientific Research (A) #10305047 from the Ministry of Education, Science, Sports and Culture of Japan. The author (MO) thanks the Okura Memorial Foundation.
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