ADLN PERPUSTAKAAN UNIVERSITAS AIRLANGGA DAFTAR PUSTAKA

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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. Skripsi FMIPA IPB. Dewi, S.U., 2009. Pembuatan Komposit Kalsium Fosfat – Kitosan dengan Metode Sonikasi. Tesis Sekolah Pascasarjana Institut Pertanian Bogor. Ekawati, D., 2008. Pengaruh Temperatur Sinter Terhadap Karakteristik Aluminium Grafit dengan Wetting Agent Tembaga. Skripsi Fakultas Teknik Universitas Indonesia, Earl, J.S. 2006. Hydrothermal synthesis of hydroxyapatite, Journal of Physics: Conference Series 26 (2006) 268–271. Ficai, A., Andronescu, Ecaterina. Voicu, Georget F., Denisa. 2011. Advances in Composite Materials for Medicine and Nanotechnology. Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science: Romania Gunawarman, M.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. 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. Istifarah. 2012. Sintesis Hidroksiapatit dari Tulang Sotong (Sepia Officinalis L.) dengan Metode Hidrotermal untuk Tujuan Bone Repair, Laporan PKMP 2012. Ivankovic, H., Orlic, S., Kranzelic, D., Tkalcec, E. 2010. Highly Porous Hydroxyapatite Ceramics for Engineering Applications, Advances in Science and Technology Vol. 63 (2010) pp 408-413, Switzerland. Kaewsichana, L., 2011. Effects of sintering temperatures on micro-morphology, mechanical properties, and bioactivity of bone scaffolds containing calcium silicate. ScienceAsia 37 (2011): 240–246.

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Kahtan, Khalaf A.K. 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. Kutz, Myer. 2003. Standard Handbook of Biomedical Engineering and Design. McGrawHill: New York Lesson CR., Lesson TS, 1989, Human Structure, Toronto-Philadelphia, BC Decker 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. Muralithran, G. Ramesh, S. 1999. The effects of sintering temperature on the properties of hydroxyapatite. Ceramics International 26 (2000) 221-230. Paljar, K., Orlic, S., Tkalcec, E., Ivankovic, H., 2009, Preparation of Silicon Doped Hydroxyapatite. Croatia : Faculty of Chemical Engineering and Technology, University of Zagreb. Park, J. & Lakes R.S., 2007, Biomaterials, An Introduction, Third Edition, Springer Science + Business Media, LLC, New York, USA. Pratiwi, K.D., 2011. Dinamika Sel Darah Putih Pada Domba Lokal yang Diimplantasi Material Tulang Hidroksiapatit-Trikalsium Fosfat (HA-TKF) dan HidroksiapatitKitosan (HA-Kitosan). Fakultas Kedokteran Hewan, Institut Pertanian Bogor, Jakarta. Prokopiev & Sevostianov. 2006. Dependence of the mechanical properties of sintered hydroxyapatite on the sintering temperature. Materials Science and Engineering A 431 (2006) 218–227. Rini, D.K. 2010. Pembuatan Biphasic Calcium Phosphate (BCP) dengan Metode Hidrotermal.Institut Pertanian Bogor. Rismawati, D.R., 2008, Sintesis Hidroksiapatit Menggunakan Bahan Dasar Batu Gamping. Skripsi FMIPA Unair Surabaya.

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Setyowati, F.F., 2008. Pengaruh Suhu Sintering terhadap Resistivitas Bahan Ferroelektrik Ba0,6Sr0,4TiO3 pada Berbagai Suhu Pengukuran. Skripsi tidak diterbitkan. Malang: Universitas Negeri Malang. Sloane, Ethel. 1996. Anatomi dan Fisiologi Untuk Pemula. Penerbit Buku Kedokteran: EGC. Jakata W. Suchanek. M. Yoshimura.1998. Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J Mater Res, 13 (1998), 94–117. Wijayanti, F., 2010, Variasi Komposisi Cobalt - Chromium Pada Komposit Co-Cr-HAP Sebagai Bahan Implan, Skripsi FSAINTEK Unair.

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Artikel Jurnal TELAAH Fisika LIPI

Artikel untuk jurnal Telaah (Siswanto-Unair) (2) Saya Ke [email protected] 8 Okt Kepada Yth.BapakPrihandoko. Assalamu'alaikumwr.wb. Bersamainisayakirimartikeluntukditerbitkan di jurnaltelaahfisika LIPI.Semogadapatditerima.Mohonbantuandankerjasamanya.Dua kali sayakirimkealamat [email protected] dan [email protected] failure. Sayadapat email bapakdari Pak AgusFisika LIPI. Terimakasihataskerjasamanya. Wassalam Aminatun (anggotapeneliti) DepartemenFisika- FakultasSainsdanTeknologi UniversitasAirlangga Surabaya 2. bambangprihandoko KeSaya 8 Okt Koreksi awal sebelum masuk reviewer. Nanti kirimkan lagi   

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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: [email protected] 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.

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c

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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: [email protected]

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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References [1]

Lesson CR., Lesson TS, Human Structure, Toronto-Philadelphia, BC Decker Inc., 1989

[2]

Park, J. dan Lakes R.S., Biomaterials, An Introduction, Third Edition, Springer Science + Business Media, LLC, New York, USA. 2007

[3]

Hui, P., Meena, S.L., Singh, G., Agarawal, R.D., Prakash, S., Synthesis of Hydroxyapatite Bio-Ceramic Powder by Hydrothermal Method, Journal of Minerals & Materials Characterization & Engineering, 2010; Vol.9, No.8: 683692.

[4]

Rismawati, DR, Hydroxyapatite Synthesis Using Basic Materials Limestone. Skripsi Faculty of Mathematical and Sciences, Airlangga University, Surabaya, 2008

[5]

Gunawarman,

Malik,

A.,

Mulyadi

S.,

Riana,

Hayani,

A.

Physical and Mechanical Characteristics of Bovine Bone Weight Life Variations as Reference of Design Material Implant,

Proceeding of Annual National

Seminar Mechanical Engineering, ITS, Surabaya, 2010 [6]

Istifarah, Aminatun, Widyanti P., Synthesis of Hydroxyapatite from Cuttlefish (Sepia Officinalis L.) Bone

by Hydrothermal Method for

Bone Repair,

Proceedings of the National Seminar on Applied Physics III, Unair, Surabaya, 2012. [7]

Muralithran, G. Ramesh, S.,The Effects of Sintering Temperature on the Properties of Hydroxyapatite. Ceramics International, 2000; 221-230.

[8]

Prokopiev. Sevostianov, Dependence of the Mechanical Properties of Sintered Hydroxyapatite on the Sintering Temperature. Materials Science and Engineering A 431 2006; 218–227.

[9]

Monmaturapoj, Naruporn. Yatongchai, Chokchai, Effect of Sintering on Microstructure and Properties of Hydroxyapatite Produced by Different Synthesizing Methods. Journal of Metals, Materials and Minerals, 2010;Vol.20. No.2.

[10]

Earl, J.S.

Hydrothermal Synthesis of Hydroxyapatite, Journal of Physics:

Conference Series 2006; 26: 268–271.

LAPORAN PENELITIAN


Drs. SISWANTO


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[11]

Paljar, K., Orlic, S., Tkalcec, E., Ivankovic, H., Preparation of Silicon Doped Hydroxyapatite. Croatia : Faculty of Chemical Engineering and Technology, University of Zagreb, 2009.

[12]

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, 2009; vol.28, no.10.

LAPORAN PENELITIAN


Drs. SISWANTO

ADLN PERPUSTAKAAN UNIVERSITAS AIRLANGGA DAFTAR PUSTAKA

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