Cytotoxicity of Hydroxyapatite Synthesized from Local Gypsum Eko Pujiyanto1 , Widowati Siswomihardjo2 , Ika Dewi Ana2 , Alva E. Tontowi3 and M. Waziz Wildan3 1) Graduate Student, Mechanical and Industrial Engineering Department, Gadjah Mada University 1)Industrial Engineering Department, Sebelas Maret University 2) Dentistry Faculty, Gadjah MadaUniversity 3) Mechanical and Industrial Engineering Department, Gadjah Mada University E-mail :
[email protected]
Abstract Hydroxyapatite (HA) based biomaterials are being increasingly used as bone substitutes in dentistry and in reconstructive and orthopedic applications because of their good biocompatibility, osteoconductivity and/or bone-bonding properties. In this study, cytotoxicity of the hydroxyapatite synthesized from local gypsum (HA-KP) and HA-200 (Waco,Japan) were analyzed utilizing cell culture techniques. Specimens were extracted in media and then fibroblast gingiva cells were cultured in the extracts for 24 hours. Cytotoxicity was evaluated by calculation of cell death percentage. The result of ANOVA analysis (p=0.05) indicated there was a significant difference in cell death percentage between HA-KP and HA-200. Mean of cell death percentage HA KP ( 56,54 % ) was less than HA- 200 (66,74 %). This result indicated HA-KP was safer than HA-200 and HAKP indicated the possibility to be used as bone substitutes.
1. Introduction Hydroxyapatite [Ca10(PO4)6(OH)2] based biomaterials are being increasingly used as bone substitutes in dentistry and in reconstructive and orthopedic applications because of their good biocompatibility, osteoconductivity and/or bone-bonding properties [1]. Pure HA has chemical composition, biological and crystallographic closely similar with bone and teeth [2]. The present status of the pure HA as a biomaterial has already been well established [3]. Multiple techniques have been used for preparation of HA powders, as reviewed in several works. Two main ways for preparation of HAp powders are wet methods and solid state reactions. In the case of HAp fabrication, the wet methods can be divided into three groups: precipitation, hydrolysis of other calcium phosphates and hydrothermal technique [3].
HA powder have been synthesized from variety mineral, including coral, gypsum (CaSO4·2H2O) and calcite [4]. HA powder have been synthesized from local gypsum by microwave-hydrothermal method using the following chemical reaction [5,6]. 10CaSO4·2H2O + 6(NH4)2HPO4 → Ca10(PO4)6(OH)2 + 6(NH4)2SO4 + 4H2SO4+18H2O HA–KP is not know yet about the toxicity properties, whereas HA-KP must be safe as bone substitutes. This paper reported the cytotoxicity of the the hydroxyapatite synthesized from local gypsum (HA-KP) and HA-200 (Waco,Japan) as comparasion.
2. Material and Methods Synthesis of HA-KP HA-KP synthesized from local gypsum were obtained from Kulon Progo Yogyakarta, Indonesia. Gypsum powder that was obtained by pulverizing the gypsum rock. Gypsum powder (20gr) and 800 mL of 1 M diammonium hydrogen phosphate [(NH4)2HPO4] were mixed well and treated at 1000C for 20 minutes in a pyrex glass using a microwave digestion system. The system operate at frequency of 2.45 GHz. After the hydrothermal reaction, reacted sample were washed with distilled water to remove residual ion and dried. The conversion of gypsum to HA-KP was estimated from ratio of X-ray intensities of gypsum peak (d=7.261) and the HA peak (d=2.787 ) via powder X-ray defractrometry (XRD). Cytotoxicity testing of HA-KP and HA-200 HA-KP dan HA-200 were placed in wells that contained 100 µL gingival fibroblast cell suspension with density of 2x104 cells/100 µL culture cell medium. All wells divided into 3 samples groups, the first group as positive control, the second group as HA-KP and the last one as HA-200. The treated group were divided into 3 sub groups in accordance with the concentration of HA that was used in this study 5.000 µg/ml, 156,25µg/ml and
4,88µg/ml. Each sub group consisted of 3 wells as replication. After 24 hours, viable cell was determined by the result of OD value using ELISA Reader and finally the cell death percentage was determined.
Cytotoxicity testing of HA-KP and HA-200 A representative gingival fibroblast cells in this work was shown in figure 2. After 24 hours, representative viable cell was shown in figure 3.
3. Results and Discussion Synthesis of HA-KP Figure 1 shows XRD patterns of local gypsum and HA-KP. In te n s ita s
3500
HA d=2.787
3000
HA d=3.440
2500 2000 1500
Figure 2: gingival fibroblast cells before treated
1000 500
30000
15
20
Gypsum d=7.261
25
30
35
40
45
50
25000 20000
Gypsum d=4.291
15000 10000
55
60
1000C for 20 min
0 I n te n s ity s 35000 0 5 10 2 te ta
Gypsum d=3.068
5000
A
0 0
2 th e ta
10
20
30
40
50
60
70
Figure 1 : XRD paterns of local gypsum and HA-KP Table 1 shows distance (d) value of three highest peaks of HA-KP and HA-200. Table 1 : Three highest peak of HA-KP and HA-200. HA-200 HA-KP d Rel. Intent d Rel. Intent 2.81894 100 2.78711 100 3.44289 38 3.44015 37 1.84286 34 1.83563 21 From figure 1 and table 1 showed that local gypsum has been synthesized and distance value between HA-KP and HA-200 is similar.
B Figure 2: Viable cell after treated with concentration of HA-KP 5.000 µg/ml (A) and 4,88µg/ml (B)
OD value were determined using ELISA plate reader as shown in table 2. OD value indicated absorbed viable cells. Table 2. OD value OD value
Conc- HA (µg/ml)
R-1
R-2
R-3
5000.00
0.627
0.627
0.588
156.25
0.525
0.525
0.491
4.88
0.504
0.504
0.509
5000.00
0.595
0.585
0.689
156.25
0.684
0.732
0.759
4.88
0.825 1.695
0.792 1.691
0.741 1.525
HA 200
HA KP
Control
-
The higher OD value the more viable cells. Cell death percentage was determined using this formula Cell death percentage =
OD control − OD Sample x 100% OD control
The higher cell death percentage the less viable cells. Table 3 showed gingival fibroblast cells death percentage. Table 3. Cell death percentage Conc- HA
HA 200
HA KP
cell death percentage
R2 = 0,62 > 0.5 indicated regression equation that become model for describing relationship between type HA with cells death percentage. The value of P =0 less than ( p = 0.05 ) indicated type HA have significance relationship with cells death percentage. The result of ANOVA is shown in table 4 Table 4. ANOVA Sourc e
DF
HA Error Total
1
Seq SS 467.98
Adj SS 467.98
Adj MS 467.98
16 17
288.87 756.84
288.87
18.05
F
P
25.9 2
0.00
The F value (F = 25,92) and P=0 less than ( p = 0.05 ) indicated type HA have significance to cells death percentage. Mean of cell death percentage HA KP ( 56,54 % ) was less than HA- 200 (66,74 %). This result indicated HA-KP was safer than HA-200 and HA-KP can be used as bone substitutes in the future.
4. Conclusion In this study, local gypsum could be synthesized to HA at 1000C in 20 minutes using microwave. This HA (HAKP) have XRD patterns similarity with HA-200. Cytotoxicity testing HA-KP and HA-200 indicated type HA showed significant different in cells death percentage. Mean of cell death percentage HA KP ( 56,54 % ) was less than HA- 200 (66,74 %). This result indicated HAKP was safer than HA-200 and HA-KP indicated the possibility to be used as bone substitutes.
(µg/ml)
R-1
R-2
R-3
5000.00
61.70
61.70
64.08
156.25
67.93
67.93
70.01
4.88
69.21
69.21
68.91
5. Acknowledgements
5000.00
63.65
64.26
57.91
156.25
58.22
55.28
53.63
4.88
49.60
51.62
54.73
The authors are grateful to RUT-XII (Minister Research and Technology, Republics of Indonesia ) for financially supporting the research.
That value were used for determine significances type HA and concentration HA to viability gingival fibroblast cells using linear regression. Significances of mean cells death percentage be caused of type HA and concentration HA were determined using analysis of variance. Regression analysis was done using MINITAB software version 11.12 with significances 95% (p=0.05). Regression equation was shown below Cells death percentage = 76.9 - 10.2 type HA ( R2 = 0.62, P = 0)
6. References [1] C. G. Simon, Jr , J. M. Antonucci , D. W. Liu and D. Skrtic, “In vitro Cytotoxicity of Amorphous Calcium Phosphate Composites”, Journal of Bioactive and Compatible Polymers, SAGE Publications, Vol. 20, No. 3, 2005, pp 279-295 [2] M.K. Herliansyah, M. Hamdi, A.I. Ektessabi and M.W. Wildan, “Fabrication of hydroxyapatite bone graft for implant application:a literature study”, Proceedings of the First International Conference on Manufacturing and Material Processing, Kuala Lumpur, 2006, pp 559-564 [3] Suchaneck, W. and Yoshimura, M., Processing and “Properties of Hydroxyapatite Based Biomaterials for Use
as Hard Tissue Replacement Implants”, Journal Material Research, Vol. 13, No. 1, 1998, pp 94-117. [4] Ana, I.D., Fabrikasi Limbah dan Sedimentasi Alam Gipsum Menjadi Monolit Hidroksiapatit Berporus Interkonektif, Kajian Awal Rekayasa Subtitusi Tulang, Proposal Penelitian Inovatif UGM,2004 [5] Katsuki,H., Furuta,S., and Komarneni, S., “Microwave Versus Conventional Hydrothermal Synthesis of Hydroxyapatite Crystals from Gypsum”, Journal American Ceramics Society, Vol 87 , No. 8, 1999, pp. 2257-2259
[6] E. Pujiyanto, A.E. Tontowi, M.W. Wildan, W. Siswomihardjo, “Sintesis Hidroksiapatit Dari Gipsum Tasikmalaya Sebagai Bahan Baku Produk Tulang Buatan ( Kajian Awal Pengembangan Produk Tulang Buatan )”, Seminar on Aplication and Research in Industrial Technology, Jurusan Teknik Mesin dan Industri , UGM , Yogyakarta, 2006, pp IV 119-126
