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DAFTAR PUSTAKA
Arifanto. 2006. Pengaruh Atmosfer dan Suhu Sintering Terhadap Komposisi Pelet
Hidroksiapatit yang Dibuat Dari Sintesa Kimia Dengan Media Air dan SBF.
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Kahtan, Khalaf A.K. 2009. Effect of Sintering Temperature on Some Physical And
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Monmaturapoj, Naruporn. Yatongchai, Chokchai. 2010. Effect of Sintering on
Microstructure and Properties of Hydroxyapatite Produced by Different
Synthesizing Methods. Journal of Metals, Materials and Minerals, Vol.20 No.2.
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Hydroxyapatite. Croatia : Faculty of Chemical Engineering and Technology,
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Jakarta.
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Setyowati, F.F., 2008. Pengaruh Suhu Sintering terhadap Resistivitas Bahan Ferroelektrik
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Universitas Negeri Malang.
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Sebagai Bahan Implan, Skripsi FSAINTEK Unair.
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SINTESIS DAN KARAKTERISASI HIDROKSIAPATIT DARI
TULANG SOTONG (Sepia sp.) UNTUK APLIKASI BONE REPAIR
Siswanto1), Aminatun1), Suryani D.A1), Yohana M.P2), Haryati3)
1)
Program Studi S1 Fisika, Departemen Fisika, Fakultas Sains dan Teknologi
Universitas Airlangga, Surabaya
2) Program Studi S1 Teknobiomedik, Departemen Fisika, Fakultas Sains dan Teknologi
Universitas Airlangga, Surabaya
3) Program Studi Teknik Material dan Metalurgi, Fakultas Teknologi Industri, Institut
Teknologi Sepuluh Nopember Surabaya
e-mail: siswanto.fst@yahoo.co.id
Diterima: ………………
Revisi: …………………...
Disetujui:…………………
Abstrak. Penelitian ini dilakukan untuk mengetahui pengaruh variasi waktu proses hidrotermal dan pengaruh suhu
sintering terhadap karakteristik hidroksiapatit (HA) yang dihasilkan dari tulang sotong (Sepia sp.). Sintesis dilakukan
o
dengan reaksi hidrotermal antara 1M CaCO 3 dari lamellae tulang sotong dan 0,6M NH4H2PO4 pada suhu 200 C
dengan variasi waktu 10, 12, 15, 20, 27 dan 30 jam. Kemudian dilakukan sintering suhu 900°C selama 1 jam. Uji XRD
dan kuat tekan serta sitotoksisitas dengan MTT assay dilakukan untuk menentukan karakteristik HA. Hasil penelitian
menunjukkan bahwa telah berhasil dibuat hidroksiapatit dengan sumber kalsium berasal dari tulang sotong dengan
metode hidrotermal. Berdasarkan uji XRD , seluruh sampel menunjukkan 100% hidroksiapatit namun kristalinitasnya
0
masih rendah. Dengan ditambah proses sintering pada suhu 900 C selama 1 jam terjadi kenaikan kristalinitas secara
0
significant. Sampel hidroksiapatit kualitas terbaik diperoleh dari hasil proses hidrotermal pada suhu 200 C selama 12
0
jam dilanjutkan dengan proses sintering pada suhu 900 C selama 1 jam ditinjau dari kristalinitas, kuat tekan dan
sitotoksisitasnya. Sampel hidroksiapatit yang dihasilkan dari penelitian ini layak diaplikasikan sebagai bone repair,
khususnya untuk tulang concelous karena kuat tekan tertinggi (11.799 ±0.00057) MPa berada pada range tulang
concelous.
Kata kunci : Hidroksiapatit, Tulang sotong, Hidrotermal, Sintering.
Abstract: This study was conducted to determine the effect of the time variation of hydrothermal processes and the
influence of sintering temperature on the characteristics of hydroxyapatite ( HA ) were produced from bone cuttlefish
( Sepia sp . ). Synthesis performed by the reaction between CaCO3 1M from cuttlefish bone lamellae and 0.6 M
o
NH4H2PO4 at 200 C temperature with time variation 10 , 12 , 15 , 20 , 27 and 30 hours . Then do the sintering
temperature of 900 ° C for 1 h. XRD and compressive strength test and cytotoxicity by MTT assay was performed to
determine the characteristics of HA . The results showed that it has successfully made hydroxyapatite with a source of
calcium from cuttlefish bone with hydrothermal method . Based on the XRD test , all samples showed 100 %
0
hydroxyapatite but still low crystallinity . With added sintering process at 900 C for 1 hour an increase of crystallinity
significantly . Hydroxyapatite samples obtained from the best quality hydrothermal process at a temperature of 200
0
0
C for 12 hours followed by a sintering process at 900 C for 1 hour in terms of crystallinity , compressive strength and
cytotoxicity . Hydroxyapatite samples from this study worthy applied as bone repair , especially for bone concelous as
the highest compressive strength ( 11,799 ± 0.00057 ) MPa in the range concelous bone.
Keywords: hydroxyapatite, cuttlefish, cuttlefish, sintering
1.
PENDAHULUAN
Hidroksiapatit (HA) merupakan komponen terbesar (70%) dari total fase mineral tulang. HA
banyak digunakan sebagai bahan implan tulang karena sifatnya yang bioaktif dan osteokonduktif yang
dapat mendukung proses remineralisasi tulang. Di samping itu, HA juga memiliki rasio kalsium dan fosfor
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sebanyak 1,67 yang mirip dengan tulang alami. Dengan demikian, bahan ini menjadi kandidat yang ideal
untuk aplikasi klinis [1].
Hidroksiapatit (HA) dengan rumus kimia [Ca10(PO4)6(OH)2] merupakan salah satu bahan yang
paling efektif digunakan dalam bidang orthopedic sebagai bahan bone repair untuk memperbaiki bagian
tulang yang rusak karena kecelakaan atau penyakit. Kelebihan material ini disamping tahan korosi, juga
bersifat bioaktif yang dapat membentuk pertautan pada antar muka material tersebut dengan jaringan tubuh
dan antara dua tulang. HA merupakan bahan yang biokompatibel, tidak bereaksi dengan bagian-bagian
tubuh yang lain serta dapat menyatu dengan tulang. Namun, HA rapuh dan memiliki modulus elastisitas 25 kali lipat dari modulus elastisitas tulang, sehingga membatasi penggunaan HA hanya digunakan sebagai
implan yang tidak memerlukan pembebanan (non-load bearing implants) [2,3]. Hidroksiapatit (HA) sangat
banyak dibutuhkan dalam penanganan masalah tulang. Fungsinya sangat beragam, bisa sebagai bone filler,
scalfold berpori maupun sebagai material pelapis prosthesis.
Beberapa riset tentang sintesis HA telah dilakukan seperti sintesis HA dari batu koral [4], dan
dari tulang sapi (bovine) [5]. HA dari tulang sapi memiliki koneksi pori-pori yang sama seperti tulang
manusia sehingga sangat cocok untuk dijadikan tulang buatan. Hidroksiapatit dari koral, masih
menghasilkan mineral lain. Sumber HA yang telah disebutkan juga memiliki kelemahan. Sintesis HA dari
batu koral akan dapat merusak habitat bawah laut apabila pengambilannya dilakukan secara terus
menerus. Sedangkan untuk sintesis HA dari tulang sapi, hingga saat ini masih menghadapi berbagai
perdebatan terkait dengan kode etik. Di samping itu, kekurangan lainnya adalah tidak seimbangnya
kebutuhan dan persediaan donor serta munculnya permasalahan dalam hal kontrol infeksi.
Untuk mengatasi permasalahan tersebut maka dalam penelitian ini dilakukan sintesis HA dengan
menggunakan tulang sotong sebagai sumber kalsium. Dipilihnya tulang sotong (cuttlefish bone) karena
komponen utamanya adalah kalsium karbonat (CaCO3) (85%) yang bisa digunakan untuk bahan dasar
mensintesis HA[6]. Tulang sotong biasanya dimanfaatkan sebagai pakan burung kenari maupun kura-kura
dan keberadaannya di negara kita melimpah. Pada penelitian ini akan dilakukan sintesis HA dari tulang
sotong dengan metode hidrotermal untuk dikembangkan sebagai biomaterial bone repair. Pada
penelitian ini dikaji terkait dengan parameter proses sintesis yang berpengaruh terhadap kualitas
hidroksiapatit yang dihasilkan, yaitu variasi waktu proses hidrotermal serta proses sintering.
2. METODOLOGI
Bahan Penelitian
Bahan yang digunakan dalam pembuatan sampel pada penelitian ini yaitu tulang sotong, amonium
dihidrogen fosfat (NH4H2PO4), methanol dan aquades.
Bahan yang digunakan untuk MTT assay yaitu medium kultur Dulbecco’s Modification of Eagle’s
Medium (DMEM), Mili Q water, sodium bikarbonat, penicillin streptomycin, fungizone, L-glutamine,
hepes buffer solution, Fetal Bofine Serum (FBS), cell line BHK 21, Phosphate Buffer Saline (PBS), trypan
blue, larutan MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), acidified isopropanol.
Alat Penelitian
Alat yang digunakan untuk pembuatan sampel pada penelitian ini yaitu High Energy Milling HEME3D, neraca analitik, hot plate, gelas kimia, pipet, magnetic stirrer, autoklaf, oven elektrik, centrifuge,
ultrasonic chamber.
Alat yang digunakan untuk karakterisasi sampel yaitu difraktometer sinar-X untuk uji struktur
kristal, kristalinitas, ukuran kristal, rasio Ca/P dan ukuran butir dengan SEM-EDX, Autograph untuk uji
kekuatan tekan. Sedangkan alat yang digunakan untuk MTT Assay yaitu tube 50 ml, centrifuge, cawan
petri, 96-microwell plate, micropippet, sterille syiringe filter, syiringe 50 ml, ependorf tube,
hemocytometer, inkubator, microplate reader, pipet pasteur, scrapper.
Prosedur Penelitian
1. Ekstrak CaCO3 dari Tulang Sotong (Sepia sp.)
Untuk mendapatkan CaCO3, bagian lamela tulang sotong (Sepia sp.) dijadikan bubuk dengan
HEM-E3D, kemudian dipanasi pada 350°C selama 3 jam untuk menghilangkan komponen organik.
Kemudian dilakukan karakterisasi XRD untuk memastikan kandungan CaCO 3 [6]
2.
Sintesis Hidroksiapatit dengan Metode Hidrotermal
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Senyawa hidroksiapatit (HA) diperoleh dengan mereaksikan prekursor kalsium (Ca) dan
prekursor fosfat (P) dengan Ca : P = 10 : 6. Prekursor Ca diperoleh dari CaCO 3 dari tulang sotong sebanyak
100 gram. Prekursor P diperoleh dari senyawa NH4H2PO4 0,6 M. Reaksi yang akan terjadi adalah sebagai
berikut.
10 CaCO3 + 6 NH4H2PO4 + 2H2O  Ca10(PO4)6(OH)2 + 3 (NH4)2CO3 + 7 H2CO3
Berikut langkah-langkah sintesis dengan metode hidrotermal [7].
1. CaCO3 1M dan larutan NH4H2PO4 0,6M dicampur kemudian distirrer selama 30 menit.
2. Campuran larutan dipindahkan ke autoklaf.
3. Autoklaf dimasukkan ke dalam oven elektrik untuk dipanaskan hingga suhu 200 0C selama 10 jam.
Sampel dikeluarkan dari oven setelah mencapai suhu kamar.
4. Sampel yang telah kering, dicuci dengan aquades. Pencucian dilakukan berulang kali hingga
menunjukkan pH netral (7). Hal tersebut dilakukan untuk menghilangkan hasil sampingan yang
bersifat asam.
5. Pencucian yang terakhir dilakukan dengan menggunakan metanol untuk membatasi aglomerasi
partikel hidroksiapatit selama pengeringan.
6. Sampel dikeringkan dalam oven elektrik pada suhu 50oC selama 4 jam.
7. Langkah 1-6 diulangi untuk lama pemanasan yang bervariasi (12, 15, 20, 27 dan 30 jam)
3.
Proses Sintering
Setelah proses sintesis di atas selesai kemudian dilanjutkan dengan proses sintering pada suhu 900
0
C selama 1 jam [8,9]
Karakterisasi Sampel
1. Uji XRD dan Kuat Tekan (Compressive Strength)
Sampel dikarakterisasi XRD untuk mengetahui struktur kristal dan kristalinitas HA. Hasil uji XRD
tersaji dalam bentuk grafik spektrum dan tabel. Pola difraksi berupa spektrum hasil uji XRD memberikan
informasi mengenai sudut terjadinya difraksi pada atom bahan (2) pada sumbu horizontal dan besar
intensitas yang dihasilkan pada sumbu vertikal. Uji kuat tekan menggunakan Autograph. Nilai kuat tekan
dihitung dengan menggunakan persamaan berikut.

F
A
(1)
dengan F = Gaya maksimal yang dapat diterima sampel (kN)
A = Luas permukaan sampel (mm2)
σ = Kekuatan tekan (kN/mm2 atau MPa)
2.
Uji Viabilitas Sel Fibroblas
Sediaan medium kultur sel fibroblast (cell line BHK 21) dibuat dengan mencampurkan 100 ml
DMEM phenol red, 113.250 μl Mili Q water, 6.750 μl sodium bikarbonat, 100 μl penicillin streptomycin,
100 μl fungizone, 4.500 μl L.glutamine dan 2.000 μl hepes buffer. Kemudian medium kultur disimpan
dalam lemari pendingin. Sebelum digunakan, 45 ml sediaan medium kultur dimasukkan ke dalam tube 50
ml, ditambahkan 5 ml FBS, kemudian dihomogenkan. Campuran medium tersebut kemudian dipindahkan
ke dalam syringe 50 ml, dan dilakukan penyaringan dengan menggunakan sterille syringe filter ke dalam
tube 50 ml.
Sel fibroblas dari penyimpanan nitrogen cair diambil sebanyak 100 μl dengan menggunakan
micropipet, lalu dimasukkan ke dalam tabung sentrifugasi dan ditambahkan PBS hingga 10 ml, kemudian
disentrifugasi dengan kecepatan 2000 rpm, suhu 24°C, selama 10 menit. Setelah supernatan dibuang, 10
ml PBS kembali dimasukkan, kemudian disentrifugasi kembali dengan kecepatan, suhu dan waktu yang
sama, sehingga tampak pellet sel di dasar tube. Hal tersebut dilakukan agar sel menjadi bersih dari bahan
pengawet (DMSO) yang diberikan selama sel disimpan dalam nitrogen cair. Pellet yang terbentuk
dilarutkan dengan medium kultur lengkap dan dihomogenkan dengan micropippet, kemudian
dipindahkan ke dalam cawan petri dan ditambahkan medium kultur lengkap hingga volumenya mencapai
7 ml. Cawan petri tersebut dimasukkan ke dalam inkubator pada suhu 37°C dan 5% CO2 selama 48 jam.
Setelah sel bertambah banyak, maka sel fibroblas tersebut siap untuk dipanen (harvesting).
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Kultur sel fibroblas diambil dari dalam inkubator, kemudian medium sel dibuang dengan
menggunakan pipet pasteur, lalu ditambahkan PBS untuk melakukan pencucian. Pencucian dilakukan
sebanyak tiga kali. Sel dikumpulkan dengan scrapper, lalu dipindahkan ke dalam tabung sentrifugasi.
Suspensi disentrifugasi (5 menit dalam 2000 rpm) hingga terbentuk pellet sel. Setelah supernatan
dibuang, sel dicairkan dengan menggunakan medium kultur, kemudian sel diambil dan dimasukkan
medium sel sebanyak 10 μl ke dalam sebuah ependorf tube, lalu ditambahkan 80 μl PBS dan 10μl trypan
blue. Penghitungan sel dilakukan dengan menggunakan hemocytometer. Setelah dilakukan penghitungan
sel, sel diresuspensi hingga mencapai 106 sel/ml dan volume sel tiap well sebanyak 50 μl, kemudian
diinkubasi selama 24 jam.
Setelah larutan sampel dipersiapkan, sel yang telah diinkubasi selama 24 jam dikeluarkan dari
inkubator untuk diberikan perlakuan. Sampel HA dan komposit HA-kitosan dimasukkan ke dalam kolom
96-microwells plate. Disediakan pula kolom yanng tidak diberi sampel sebagai kontrol. Lalu diinkubasi
selama 4 jam.
Setelah diinkubasi 4 jam, larutan MTT dimasukkan ke tiap-tiap well sebanyak 15 μl. Kemudian
diinkubasi kembali selama 3 jam, diberikan acidified isopropanol sebanyak 150 μl pada tiap well. Lalu
plate ditempatkan pada shaker dengan kecepatan 50 rpm selama 1 jam. Pembacaan dilakukan dengan
menggunakan microplate reader dengan panjang gelombang 490 nm. Selanjutnya persentase kelompok
perlakuan dibandingkan dengan persentase kelompok kontrol diperoleh nilai OD. Nilai viabilitas sel
dihitung dengan menggunakan persamaan berikut [6]
viabilitas sel (% dari kontrol) 
nilai absorbansi kelompok perlakuan
nilai absorbansi kelompok kontrol
(2)
3. HASIL DAN PEMBAHASAN
1. Hasil uji XRD
Hidroksiapatit yang disintesis melalui proses hidrotermal 10 jam, 12 jam, 15 jam, 20 jam, 27 jam
dan 30 jam pada suhu 200oC dikarakterisasi XRD pada sudut 2Ɵ = 5o-60o. Hasil uji XRD seluruh sampel
tersebut ditunjukkan pada Gambar 1a hingga 1f. ICDD yang digunakan sebagai referensi adalah ICDD 01074-0565. Hasil analisis yang didapatkan menunjukkan bahwa kandungan dari sampel yang diuji tersebut
adalah 100% hidroksiapatit (Ca10(PO4)6(OH)2). Seluruh spektrum XRD yang terbentuk pada sampel
hidroksiapatit tersebut bersesuaian dengan ICDD acuan.
a
c
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f
e
Gambar 1. Spektrum XRD Hidroksiapatit pada Variasi waktu Hidrotermal
(a) 10 jam (b) 12 jam (c) 15 jam (d) 20 jam (e) 27 jam (f) 30 jam
Hidroksiapatit dengan sumber kalsium dari tulang sotong telah berhasil disintesis melalui proses
hidrotermal pada variasi waktu 10 jam, 12 jam, 15 jam, 20 jam, 27 jam dan 30 jam. Sebagaimana
ditunjukkan pada Gambar 1 seluruh variasi waktu tersebut di atas menghasilnya 100% hidroksiapatit. Hal
ini ditunjukkan dengan munculnya puncak-puncak difraksi yang sama dengan data ICDD acuan 01-0740565. Akan tetapi nampak bahwa hidroksiapatit yang terbentuk masih bersifat amorf ditunjukkan dengan
puncak difraksi yang masih rendah. Untuk meningkatkan kristalinitas hidroksiapatit maka dilakukan proses
sintering pada suhu 9000C selama 1 jam. Terjadi kenaikan puncak maksimum akibat proses sintering
tersebut. Adapun Puncak maksimum sampel dengan variasi waktu hidrotermal sebelum dan sesudah
sintering pada suhu 9000C selama 1 jam ditunjukkan pada Tabel 1. Hasil uji XRD seluruh sampel setelah
perlakuan sintering ditunjukkan pada Gambar 2.
a
c
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e
f
Gambar 2. Spektrum XRD Hidroksiapatit pada Variasi waktu Hidrotermal setelah proses
sintering 9000C selama 1 jam
(a) 10 jam (b) 12 jam (c) 15 jam (d) 20 jam (e) 27 jam (f) 30 jam
Tabel 1 Data Puncak Difraksi Maksimum Hidroksiapatit dengan Variasi Waktu Hidrotermal
Sampel
hidrotermal
(jam)
Puncak Difraksi Maksimum
Sebelum sintering
2Ɵ
Intensitas
(tinggi puncak)
Sesudah sintering
2Ɵ
Intensitas
(tinggi
puncak)
10
31,7857
312
31.7428
956.13
12
31,7042
472
31.7407
1163.02
15
31,7389
521
31.7267
602.64
20
31,7568
286
31.7252
471.14
27
31,6815
233
31.7699
581.23
30
31,7585
75
31.7392
908.12
Berdasarkan Tabel 1, nampak terjadi kenaikan intensitas puncak difraksi karena pengaruh sintering.
Sebelum proses sintering, seluruh sampel 100% terbentuk HA akan tetapi intensitas puncak difraksi masih
rendah. Dengan melakukan proses sintering pada suhu rekristalisasinya maka intensitas HA semakin tinggi.
Hal ini menunjukkan bahwa proses sintering mempengaruhi intensitas puncak difraksi yang dapat
dikatakan mempengaruhi kristalinitas hidroksiapatit yang dihasilkan. Selama proses sintering, atom-atom
bervibrasi menyusun diri membentuk susunan yang teratur, sehingga kristalinitasnya meningkat [10,11].
Terjadi peningkatan intensitas yang significant untuk proses hidrotermal 10, 12 dan 30 jam. Kristalinitas
tertinggi terjadi pada proses hidrotermal 12 jam. Dengan demikian sintesis hidroksiapatit dari tulang sotong
terbaik diperoleh pada suhu hidrotermal 2000C selama 12 jam yang disinterring pada suhu 9000C selama 1
jam.
Uji Kuat Tekan
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Kuat Tekan (MPa)
Nilai kuat tekan dari sampel HA setelah disinterring pada suhu 900 0C selama 1 jam disajikan
pada Gambar 3.
14
12
10
8
6
4
2
0
11.799
10.55
9.629
7.786
0
10.126
7.052
10
20
30
waktu hidrotermal (jam)
40
Gambar 3. Grafik Kuat tekan setelah proses sintering
Nilai kuat tekan HA setelah proses sintering pada suhu 900 0C selama 1 jam sebagaimana
ditunjukkan pada Gambar 3 bervariasi nilainya. Nilai ini seiring dengan nilai kristalinitas HA (Tabel 2).
Semakin tinggi kristalinitas semakin rapat susunan atom-atomnya sehingga nilai kuat tekan semakin
meningkat. Nilai kuat tekan tertinggi (11.799 ±0.00057) MPa terjadi pada proses hidrotermal 12 jam yang
memiliki nilai kristalinitas tertinggi pula. Selama proses sintering terjadi pertumbuhan butir dan
densifikasi pada tahapan Intermediate Stage. Pada tahap ini terjadi pertumbuhan butir dan struktur pori
menjadi halus. Geometri batas butir dan pori yang terjadi pada tahap ini bergantung pada laju proses
sinter. Sementara itu, pemadatan (densification) yang terjadi pada tahap ini diikuti oleh difusi volume dan
difusi batas butir [12].
Uji Sitotoksisitas
Hasil uji MTT assay dibaca dengan Elisa reader berupa nilai absorbansi (OD). Viabilitas sel
dihitung dengan Persamaan 2. Adapun viabilitas sel dari masing-masing sampel yang didapatkan tersaji
pada Gambar 4.
Viabilitas Sel (%)
100
80
60
40
20
0
10
12
15
20
27
Waktu Hidrotermal (jam)
30
Gambar 4. Uji Viabilitas Sel setelah proses sintering
Gambar 4 menunjukkan bahwa hidroksiapatit yang disintesis dari tulang sotong dengan variasi waktu
hidrotermal dan disintering pada suhu 9000C selama 1 jam tidak bersifat toksik terhadap sel fibroblast (cell
lines). Hal ini terlihat dari persentase viabilitas sel berada pada range 72,18%-90,89% masih di atas 60%
(batas toleransi toksisitas). Berdasarkan Gambar 4 di atas, terlihat bahwa viabilitas sel tertinggi adalah pada
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sampel proses hidrotermal 12 jam dengan persentase viabilitas sel 90,89%. Sementara itu, viabilitas sel
sampel hidrotermal 27 jam merupakan yang terendah dengan persentase sel hidup sebesar 72.18%.
4. KESIMPULAN
Berdasarkan hasil penelitian dapat disimpulkan sebagai berikut.
Telah berhasil dibuat hidroksiapatit dengan sumber kalsium berasal dari tulang sotong, melalui metode
hidrotermal pada suhu 2000C dengan waktu yang bervariasi yaitu 10, 12, 15, 20, 27 dan 30jam. Uji XRD
menunjukkan seluruh sampel mengandung 100% hidroksiapatit namun kristalinitasnya masih rendah.
Dengan ditambah proses sintering pada suhu 900 0C selama 1 jam terjadi kenaikan kristalinitas secara
significant. Sampel hidroksiapatit kualitas terbaik diperoleh dari hasil proses hidrotermal pada suhu 200
0
C selama 12 jam dilanjutkan dengan proses sintering pada suhu 900 0C selama 1 jam ditinjau dari
kristalinitas, kuat tekan dan sitotosisitasnya. Sampel hidroksiapatit yang dihasilkan dari penelitian ini layak
diaplikasikan sebagai bone repair, khususnya untuk tulang concelous karena kuat tekan tertinggi (11.799
±0.00057) MPa berada pada range tulang concelous.
DAFTAR PUSTAKA
[1] Hui, P., Meena, S.L., Singh, G., Agarawal, R.D., Prakash, S., 2010, Synthesis of
Hydroxyapatite
Bio-Ceramic Powder by Hydrothermal Method, Journal of Minerals & Materials Characterization
& Engineering, Vol. 9, No.8, pp.683-692, India.
[2] Park, J. dan Lakes R.S., 2007, Biomaterials, An Introduction, Third Edition, Springer Science + Business
Media, LLC, New York, USA.
[3] Garcia-Sauz F.J, Mayor, M.B., Arias, J.L., Pou, J., Leon, B. 1997. Hidroxyapatite Coatings: a Comparative
Study between Plasma Spray and Pulsed Laser Deposition Techniques, Journal of Material
Science. Material in Medicine, 861-865
[4] Rismawati, Dyah Retno, 2008, Sintesis Hidroksiapatit Menggunakan Bahan Dasar Batu Gamping.
Skripsi FMIPA Unair Surabaya.
[5] Gunawarman, Malik, A., Mulyadi S., Riana, Hayani, A. 2010. Karakteristik Fisik dan Mekanik Tulang
Sapi Variasi Berat Hidup sebagai Referensi Desain Material Implan. Seminar Nasional Tahunan
Teknik Mesin (SNMTTM) ke-9.
[6] Istifarah, Aminatun, Widyanti P., 2012. Sintesis Hidroksiapatit dari Tulang Sotong (Sepia Officinalis
L.) dengan Metode Hidrotermal untuk Tujuan Bone Repair, Prosiding Seminar nasional Fisika
Terapan III, Unair, Surabaya.
[7] Earl, J.S. 2006. Hydrothermal synthesis of hydroxyapatite, Journal of Physics: Conference Series 26
(2006) 268–271.
[8] Kaewsichana, Lupong. 2011. Effects of sintering temperatures on micro-morphology, mechanical
properties, and bioactivity of bone scaffolds containing calcium silicate. ScienceAsia 37 (2011):
240–246.
[9] Kahtan, khalaf Al-Khazraji. 2009. Effect of Sintering Temperature on Some Physical And Mechanical
Properties of Fabricated Hydroxyapatite Used For Hard Tissue Healing. Eng. & Tech. Journal,
Vol.28, No.10.
[10] Monmaturapoj, Naruporn. Yatongchai, Chokchai. 2010. Effect of Sintering on Microstructure and
Properties of Hydroxyapatite Produced by Different Synthesizing Methods. Journal of Metals,
Materials and Minerals, Vol.20 No.2.
[11] Muralithran, G. Ramesh, S. 1999. The effects of sintering temperature on the properties of
hydroxyapatite. Ceramics International 26 (2000) 221-230.
[12] Prokopiev. Sevostianov. 2006. Dependence of the mechanical properties of sintered hydroxyapatite
on the sintering temperature. Materials Science and Engineering A 431 (2006) 218–227.
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THE EFFECT OF SINTERING PROCESS ON THE
CHARACTERISTICS OF HYDROXYAPATITE FROM
CUTTLEFISH BONE (Sepia Sp.)
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2)
Aminatun1*), Siswanto1), Suryani.D.H1), Y.M.Penga2), Istifarah2), R. Apsari1)
1)
Study Program of Physics, Department of Physics, Faculty of Science and
Technology ,
Airlangga University, Surabaya-Indonesia
Study Program of Technobiomedic, Department of Physics, Faculty of Science and
Technology , Airlangga University, Surabaya-Indonesia
*)
e-mail: ami_sofijan@yahoo.co.id
Abstract
This study aimed to determine the effect of the sintering process on the characteristics of
hydroxyapatite which was produced from cuttlefish bone (sepia sp.). Hydroxyapatite was
obtained by a hydrothermal reaction between 1M CaCO3 of cuttlefish bone lamellae and
0.6M NH4H2PO4 at 200oC in 12 hours. Followed by a sintering process with variations
in the temperatures : 600°C, 800°C, and 900°C for 1 hour. Furthermore, the best results
were taken from variation in time of 2 and 3 hours. XRD, SEM-EDX, and compressive
strength test as well as MTT assay were performed to determine the characteristics of
HA. The result of this study showed that an increase in crystallinity, crystal size,
morphology, and compressive strength occurs by increasing the sintering temperature. In
addition, an increase in temperature does not cause toxic effects on the HA as shown by
cell viability which is more than 60%. Meanwhile, the increase in sintering time showed
no significant changes to HA crystallinity, yet the mechanical properties of HA could still
be improved. Based on the analysis, the optimal HA sintering temperature and time
which are 900oC and 1 hour respectively, yielded the highest crystallinity with the high
maximum diffraction peak of 1163.02 and compressive strength of (11.79900 ± 0.00057)
MPa which is suitable for the application of cancellous bone.
Keywords : Hydroxyapatite, Cuttlefish bone, Hydrothermal, Sintering
1. Introduction
Bone is a powerful tissue which forms the framework of the human body. Bone
has four main functions namely mechanical, protective, metabolic and hematopoietic
function. Protective function acts as a protector of vital organs in the body and the bone
marrow. Metabolic function acts as a backup and site of metabolism of many essential
minerals such as calcium and phosphate. Hematopoietic function acts as a venue for the
process of formation and development of blood cells [1].
Damages and bone disorders can affect the activities and functions of other
organs. To overcome various damages that occurs in the bone, some treatment with
therapy or an implant to replace or support the function of the actual bone was
performed. There are several sources in medical biomaterials for implantation such as
autograft, allograft and xenograft. Sources of biomaterials for bone implantation has a
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weakness that synthetic hydroxyapatite as an alternative materials to implant from nature
must be developed [2].
Hydroxyapatite (HA) is the largest component (60%) of the total mineral phase of
bone. HA has osteoconductive and bioactive properties that can support the process of
bone remineralization [3]. Research on HA synthesis of natural products have been made
such as HA synthetic of coral [4] and from bovine bone [5]. HA sources that have been
mentioned above have weaknesses namely the imbalance of requirements and donors
supply as well as the emergence of problems in terms of infection control. Therefore, the
researchers tried to find the source of HA which was expected to be more available with
characteristics that were not inferior to other sources, and one of which is by using
cuttlebone.
Study on Hydroxyapatite synthetic with CaCO3 source of cuttlebone has been
done by Istifarah [6] using a hydrothermal method. The study yielded HA with quite well
characteristics. However, the crystallinity of the resulting HA still needs to be increased,
as in bone repair applications,
HA with very good crystallinity to produce good
mechanical properties is necessary. Crystallinity of a material depends on several factors
such as the temperature and time of sintering.
Several studies have been conducted to determine the effect of the sintering
process on the characteristics of HA. Muralithran and Ramesh [7] proved that sintering
temperature affects the phase stability, densification properties, microstructure and
hardness (hardness) of hydroxyapatite. Prokopiev and Sevostianov [8] found that changes
in the mechanical properties of the sintered HA correlated with grain size, the bonds
between the grains, the density of the specimen and pore shape. Monmaturapoj and
Yatongchai [9] proved that the microstructure and mechanical properties increased with
increasing sintering temperature, whereas the effect of sintering time is still very small.
Based on the preliminary studies a synthesis of HA from cuttlebone with
variations in temperature and sintering time was conducted to determine the effect of
these parameters on the characteristics of hydroxyapatite which include crystallinity,
morphology, compressive strength and toxicity of the material. sintering temperature and
time were then optimized to obtain HA with the appropriate characteristics for its
application in bone repair.
2. Material and Methods
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2.1 Materials and Equipments
Materials used in the manufacture of the sample in this study was cuttlefish bone
(Sepia sp.), ethanol, ammonium dihydrogen phosphate (NH4H2PO4) and methanol.
The tools used for the manufacture of the sample in this study were High Energy
Milling HEM-E3D, electric oven, magnetic stirrer, autoclave, pH meter and furnace.
2.2 Research Procedure
The study was carried out experimentally with the following stages.
2.2.1 Extraction of CaCO3 from cuttlebone
Aragonite (CaCO3) was obtained from the lamellae of cuttlefish bone powder
which was made using HEM-E3D or mortar, then heated in a furnace at 350 ° C for
3 hours. To determine the content of CaCO3, the results were characterized by
XRD.
2.2.2 Preparation Materials
CaCO3 solution (Mr = 100) 1M was obtained by adding 100 grams of CaCO3 to 1
liter of distilled water. The solution of NH4H2PO4 (Mr = 115) 0.6 M was prepared
by dissolving 69 grams to 1 liter of distilled water.
2.2.3. Synthesis of Hydroxyapatite by Hydrothermal Method
In this synthesis process, a reaction that was expected to occur is as follows [10].
10 CaCO3 + 6 NH4H2PO4 + 2H2O  Ca10(PO4)6(OH)2 + 3 (NH4)2CO3 + 7 H2CO3
Hydrothermal synthesis method was carried out by mixing 1M CaCO 3 and 0.6 M
NH4H2PO4 solution with a magnetic stirrer for 30 minutes. Solution mixture was
transferred to the reactor and put in an Electric Oven to be heated until the temperature of
200oC with the duration of 12 hours. The results obtained, were cooled at room
temperature and then washed with distilled water using a magnetic stirrer. Washing
performed repeatedly until the results of the reaction were separated from distilled water,
was indicated by the return of the neutral pH (pH = 7). This assisted to eliminate the
acidic byproducts. The last washing was performed with methanol to limit the
agglomeration of HA during drying. Next, the sample was filtered with a filter paper and
dried in an electric oven at a temperature of 50°C until dry. Hydroxyapatite samples that
have been formed is named sample 1, which further was characterized by XRD to ensure
the formation of HA.
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2.2.4. Stages of Sintering
Hydroxyapatite resulting from previous stages was further sintered using a
furnace. Products of sintering conducted with variations in the temperature of 600°C,
800°C, and 900°C for 1hour, are respectively named sample 2, 3, and 4. Next, the best
sintering temperature based on the characterization chosen as the sintering temperature
for the second sintering with time variation of 2 and 3 hours, are respectively named
sample 5 and 6. Sintering results of time variation were then characterized.
2.2.5 Characterization
The entire sample was characterized by the crystal structure and crystalinity with XRD,
morphology and the ratio Ca / P with SEM-EDX, compressive strength and cytotoxicity
by MTT assay using fibroblast cell cultures.
3. Results and Discussion
3.1 Test Results of XRD
The XRD test results of the cuttlebone lamellae powder that have been heattreated at 350 ° C for 3 h showed 100% content of calcium carbonate (aragonite, CaCO 3)
(Figure 1).XRD spectrum of the sample showed suitability with ICDD 01-71-4891. This
is in line with the research results of Paljar et al.[11] which indicates that the heat
treatment on the cuttlefish bone lamellae does not change the content of aragonite into
calcite, not as part of dorsal.Aragonit is more easily transformed into HA than calcite.
Furthermore, in this study aragonite from the cuttlefish bone lamellae to synthesize HA
was used.
Figure 1. XRD spectrum of cuttlefish bone lamella powders
Hydroxyapatite was characterized and analyzed by XRD reference ICDD 01-0740565. The analysis showed that the content of the tested samples was 100%
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hydroxyapatite (Ca10 (PO4)6 (OH) 2). The entire XRD spectrum formed on the HA
samples correspond to ICDD reference.
1. The content of Hydroxyapatite with Sintering Temperature Variation
Figure 2.XRD spectrum of Hydroxyapatite with Sintering Temperature Variation
(a) Without sinter, (b) 600oC, (c) 800oC, (d) 900oC
Based on the test results of XRD (Fig. 2) shows that the HA samples sintered at a
temperature of 900oC (Figure 2d) has the highest intensity of the diffraction peak, which
is 1163.02. Therefore, the sample can be said to have a higher crystallinity compared to
the HA samples before sintering and sintered at a temperature of 600 oC and 800
o
C.Crystallinity of a material was figured out by looking at the parameters peak height
and FWHM bandwidth, but bcause HA samples produced a single crystal phase and no
other phases formed, the height of peaks can represent the HA crystallinity.
2. The content of Hydroxyapatite with Duration Sintering Variation
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Figure 3. XRD spectrum of Hydroxyapatite at 900oC with durationVariation: (a) 2 hours,
(b) 3 hours
Figure 3 above shows that crystallinity decreased when viewed from maximum
diffraction peak height. The HA samples sintered at 900oC for 3 hours (Figure 3b) has a
higher crystallinity compared with the HA samples sintered at 900oC for 2 hour (Figure
3a). Based on the XRD data obtained, it can also note the sample size of HA crystals. To
determine the size of the crystal, Scherrer equation was used.
t
k
0.9

BCos BCos
(1)
Where t is the crystal size (Å), λ is the wavelength of x-ray diffraction (equivalent to
1,54 Å), B is the width of the diffraction peak FWHM (radians), and (θ) is the diffraction
angle. According to the results of this study, the crystal size in the sample is as follows.
Figure 4. Comparison of crystal size with Sinter Temperature and duration variation
Figure 4 shows an increase in crystal size due to an increase in sintering
temperature. This is in contrast with the measured crystal size of the sample with
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sintering time variations. On the graph, the crystal size decreased while the sintering was
increased.
In this study, a refinement using the program Powder Cell of Window (PCW) was
also carried out. One of the information obtained from this refinement is the crystal
lattice parameters, as shown in table 1.
Table 1. Lattice parameters of HA Refinement Results
Parameter
Rp
Rwp
Rexp
a=b
c
ICSD
9,424
6,879
1
10,12
15,25
2,170
9,4167
6,897
2
13,75
15,67
2,13
9,4170
6.877
Sample
3
4
13,66
14,58
19,46
18,76
2,08
2.04
9,4185 9,4195
6,8807 6,8801
5
15,65
20,83
2,02
9,4193
6,8819
6
15,89
21,32
1.83
9.4187
6.8809
The data presented in the table shows that there is a slight increase and decrease in
the lattice parameters a and c. However, the overall sample 4 (HA sintered at a
temperature of 900oC for 1 hour) has a lattice parameter value closer to the value of the
lattice parameters of HA reference (a = 9.424 Å and c = 6.879 Å).
3.2 SEM EDX Test
SEM characterization on samples with variations in temperature and length of
time of each sinter using 500x magnification, 10000x, and 20.000x is shown in Figure 5.
Figure 5.Morphology of hydroxyapatite with sintering temperature variations.
The SEM picture above
showed no differences in morphological structure.
However, there has been little changes in the form of granules.
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Figure 6. Morphology of hydroxyapatite with sintering duration variation.
Pore diameter size of the sample was measured using a line scale in the SEM images.
The pore diameter is presented in Table 2.
Table 2. The Pore Diameter Size
Sample
Pore Diameter Size
(nm)
2
152,1-418,7
3
102,0-123,6
4
70,46-102,0
5
65,16-88,63
6
32,58-52,30
Sintering temperature increases led to a more solid material and pore shrinkage. The
longer time given in the sintering process, the smaller the size of the pores became.
Pore size of a material is closely related to the material strength. The larger pore
size showed reduced density of the material, meaning that the material is not able to
withstand the load given to it. In other words, a material which has a large pore size tends
to be brittle.
Pore size of HA samples (Table 2) shows a decrease along with the increase
sintering temperature and time. This occured because during the sintering process,
bonding between grains occurred and caused the pore size to become smaller.
3.3. Compressive Strength Test
Compressive strength test was carried out using autograph. The magnitudes obtained
were then calculated using the Equation:
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
F
A
(2)
F is the maximum force that can be accepted by the samples (kN), A is the sample
surface area (mm2) and σ is compressive strength (kN/mm2 or MPa).
Compressive strength of HA sample with variations in temperature and time sintering is
presented in the graph in Figure 7.
15
11.6348
Compressive
Strength
(MPa)
11.799
10
6.99222
9.34183
5
0
0
300
600
Sintering Temperature (o C)
900
Figure 7. Graph of Hydroxyapatite with Sintering Temperature Variation
Compressive strength of HA increased as the sintering temperature also increased
as a result of the events on the grain growth and densification in Intermediate Stage. At
this stage, grain growth occured and pore structure became smoother. Grain boundaries
and pore geometry that occured at this stage depended on the rate of sintering process.
Meanwhile, compaction (densification) that occured at this stage was followed by
volume diffusion and grain boundary diffusion.The higher the sintering temperature
was,the higher the density of the sample became. It made the sample able to withstand
the load and pressure given [12]
The highest compressive strength is owned by the sample that was heated for 3
hours, which equals to (11.92357 ± 0.00057) MPa. An increase in the compressive
strength was caused by a strong bond between the grains during the sintering process.
This event could possibly occur due to a material movement mechanism between grain
(diffusion process) and the source of energy to activate the movement. Longer time
given in the sintering process, caused more particles to bond so that the material became
stronger. Meanwhile, long sintering time affects the mechanical properties of the sample
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where the value of compressive strength samples increased by increasing the sintering
time.
12
11.8
Compressive
Strength
(MPa)
11.79900
11.88960
11.92357
11.6
11.4
11.2
11
0
1
Sintering Time (hour)
2
3
Figure 8. Graph of Hydroxyapatite with Sintering Time Variation
3.4. Cytotoxicity test by MTT Assay
MTT assay test results were read by Elisa reader absorbance value (OD). Cell
viability was calculated with the equation of in vitro technologies. The cell viability of
each sample obtained is presented in Figure 9.
Figure 9. Cell Viability of Sample with Sintering Temperature and Time Variation
Hydroxyapatite that was synthesized from cuttlefish bones was not toxic.This is indicated
by the value of live cell viability of which was above 60%.
4. Discussion
This research has been conducted on the synthesis of cuttlefish bone (Sepia sp.)
Hydrothermally.Then, sintered HA was obtained by variation of sintering temperature
and time.Variations in temperature and time of sintering aimed to produce HA with
optimal characteristics in medical applications.
The characterizations, including XRD, SEM-EDX, compressive strength, and
MTT Assay test, obtained good results. XRD test showed an increase in crystallinity with
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increasing temperature. Sample 4, HA sintered at temperature of 900 oC has a crystallinity
with the highest diffraction peak, i.e the intensity of 1163.02. This diffraction peak height
increase led to an increase in the size of the crystal. Crystal size was calculated using
Scherre requation according to the XRD analysis data. The obtained results prove that
when the sintering temperature increases, the grains in a material will bond together and
form a larger grain structure. Meanwhile, the arising effect from the increase in sintering
time could not be as certained due to fluctuating changes in the results.
Morphological structure of the sample showed a less significant change in both
the sintering temperature and time variations. The HA samples synthesized was not
homogeneous and has irregular grain and pore size. In addition, a decrease in pore size
occurred after increasing the temperature and the sintering time. Overall, the pore size of
the six samples is less than 10 microns. This proves the occurrence of densification,
where the grains bind strongly and form a very solid structure, thereby reduce the pore
size of the material.
The test results were then analyzed with SEM EDX to determine the composition
of HA. Results were taken from one point so it could not be used as a benchmark of the
ratio of Ca / P. Overall, there is an exception when it is a homogeneous sample.
However, the EDX analysis showed that all samples have a ratio of Ca / P that were not
exactly 1.67, but still close to that value. The composition of Ca / P is less appropriate
because the starting material used as a source of CaO was cuttlebone that still contained
CaCO3, so as after the reaction between CaO and (NH4) H2PO4, there were CaCO3
which did not participate in reaction and affect the amount of Ca in the sample.
Compressive strength test results showed that increasing sintering temperature
and time plays a role in increasing sintering temperature and that times play a role in
increasing the compressive strength of material. The compressive strength of samples
obtained in the range among the compressive strength forcancellous bone applications,
and the highest is samples 6 (HA sintered at a temperature of 900oC for 3 hours) equals
to (11.92357±0.00057) MPa. Compressive strength is associated with a decrease in pore
size due to densification events. This is supported by the SEM test results showing a
decrease in pore size with increases in temperature and sintering time. The higher the
density of a material, the higher the material's ability to withstand a given load will be.
Thus, the compressive strength value became higher.
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based on the results of MTT assay test, the highest cell viability was obtained on
sample 4 with the percentage of 90.89%. Meanwhile, the lowest cell viability was seen in
sample 3 with the percentage of 65.56%. The test results do not directly specify that a
substance is toxic. To detect the toxic effects of the test material (e.g. the rate of growth,
proliferation and differentiation of cells in the material) can depend on the contact and
the spread of the cells on the surface of the material. If the cells do not interact with the
material perfectly then there will be no cell growth, as well as for HA. Hydroxyapatite is
effective to stimulate the growth of bone cells in case of interaction with cells.
5.1. Conclusion
1. Sintering temperature variation affects the crystallinity, crystal size, lattice parameter
and morphology of hydroxyapatite. Crystallinity and crystal size increases with an
increase of the sintering temperature. However, in this study the influence of sintering
temperature on morphological changes in the structure of the HA of cuttlebone can
only be seen from the size of the pore, where the pore size decreased with an increase
of the sintering temperature. Meanwhile, long sintering time has an influence that is
not linear to the lattice parameters, the crystallinity and crystal size of HA.
2. Sintering temperature and time affect the compressive strength of hydroxyapatite from
cuttlebone. Higher temperature and longer sintering time, will decrease the pore size
of the sample, so that compressive strength of the sample increases.
3. This study did not find the effect of sintering temperature and time on cytotoxicity.
However, it is known that HA from the cuttlebone is not toxic because the cell
viability is more than 60%.
4. Sintering temperature and time of HA are optimal at temperatures of 900 oC for 1 hour.
This is evidenced by the high crystallinity with the height of maximum diffraction
peak of 1163.02 and compressive strength which is suitable for the application on
cancellous bone equals to (11.79900±0.00057) MPa.
5.2. Suggestion
To determine the effect of sintering time to the characteristics of hydroxyapatite
more significantly, it is necessary to vary the time with a longer interval.
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