CHARACTERIZATION OF BIPHASIC CALCIUM
PHOSPHATE WITH RATIO OF 70/30 BEFORE AND AFTER
IMPLANTED
INTO SHEEP’S BONE
DINI NOVIALISA
DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES BOGOR AGRICULTURAL UNIVERSITY
STATEMENT ON THESIS
I hereby declare that thesis entitled characterization of biphasic calcium
phosphate with ratio of 70/30 before and after implanted into sheep’s bone is my work under direction of the supervising committee and has not been submitted in any form to any college. The source of information is derived or quoted from the published or unpublished work by other authors mentioned in the text and listed in the reference at the end of this thesis.
I hereby assign the copyright of my papers to the Bogor Agriculture University.
ABSTRACT
DINI NOVIALISA. Characterization of Biphasic Calcium Phosphate with Ratio of 70/30 Before and After Implanted into Sheep’s Bone. Supervised by KIAGUS DAHLAN.
Nowadays, biphasic calcium phosphate is used for biomaterial to cure bone damage. This study focuses only on mixing ratio of 70% mass of hydroxyapatite and 30% mass of tricalcium phosphate with chitosan and acetic acid as the hardening. Pure HA had done by wet precipitation in sintering process at temperature of 900 oC while β-TCP is at temperature of 1000 oC. Mechanical method of BCP is proven to be a non toxic material and could absorb by the mineral body. Implanted BCP was characterized with XRD, FTIR, SEM-EDS and bone characterization with Vickers Hardness. In XRD analysis, the result is adding chitosan makes BCP less accuracy of pure HA and β-TCP. FTIR shows composition of BCP sample with HA, TCP, H2O, AKB, and N-H bending.
a paper submitted
in partial fulfillment of the requirement for bachelor degree
Faculty of Mathematic and Natural Sciences
CHARACTERIZATION OF BIPHASIC CALCIUM
PHOSPHATE WITH RATIO OF 70/30 BEFORE AND AFTER
IMPLANTED
INTO SHEEP’S BONE
DINI NOVIALISA
DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES BOGOR AGRICULTURAL UNIVERSITY
Title : Characterization of Biphasic Calcium Phosphate with Ratio of 70/30 Before and After Implanted into Sheep’s Bone
Name : Dini Novialisa
NIM : G74100046
Approved by
Dr Kiagus Dahlan Supervisor
Known by
Dr Akhiruddin Maddu Head of Physics Department
PREFACE
Alhamdulillah, blessing to God and The prophet Muhammad the author prayed that can be given to completed this research proposal entitled
“Characterization of Biphasic Calcium Phosphate With Ratio of 70/30 Before and After Implanted into Sheep’s Bone”. The proposal is structured as a condition of graduation degree program in Physics Departement of Bogor Agricultural University.
The author would like to express sincere appreciation of my parents who always support me, Dr. Kiagus Dahlan for his guidance, Setia Utami Dewi, M.Si for suggestions to help completing this research and Nur Aisyah Nuzulia, M.Si for her patience to teach theories of this research. I am also thankful to M. N. Indro M.Sc and the other lecturers for the advices and encouragement. For all of my friends at Physics students, IKPMR, Student Executive Board, I feel grateful to have you in my life.
Nonetheless, the author also welcome for any critical feedback and advice from readers in order to maintain it as a successful project. I hope this paper could be useful and become reference of the other researches.
CONTENT
TABLE LIST viii
FIGURE LIST viii
APPENDIX LIST viii
INTRODUCTION 1
Background 1
Hypotheses 2
Objective 2
Benefit 2
MATERIALS AND METHOD 3
Place and Time 3
Materials and Equipments 3
Experimental Method 3
RESULTS AND DISCUSSION 5
XRD Analysis of BCP Implant 6
FTIR Analysis of BCP Implant 8
Analysis of Post Operation Material 8
Bone Characteristics of Post Operation 12
CONCLUSIONS AND SUGGESTION 14
Conclusions 14
Suggestions 14
REFERENCES 15
TABLE LIST
1 Lattice parameter of BCP sample 7
2 Macroscopic changes in implant and bone after implanted.* 12 3 Vickers Hardness Number of Implanted Bone in Three Month 13
FIGURE LIST
1 XRD patterns of HA and β-TCP sample 5
2 XRD patterns of BCP powder 7
3 XRD patterns of BCP pellet 7
4 FTIR spectra of BCP powder and BCP pellet 8
5 FTIR spectra of implanted samples in each month 9
6 SEM image showing physical structures of 1 month after implanted 10 7 SEM image showing physical structures of 2 months after implanted 11 8 SEM image showing physical structures of 3 months after implanted 11
APPENDIX LIST
1 Flowchart of Research 17
2 Pictures of Research Properties 18
3 Lattice parameter formulation match to HA 19
4 Lattice parameter formulation match to TCP 21
5 JCPDS references 22
6 FTIR Spectra of Samples 23
7 SEM Characterization 25
1 materials as implant are stone, coral, eggshells and etc, have same characteristics of apatite materials as bone graft such as bioactive, biocompatible, and bioresorbable.2 It can be used to repair, restore, and replace damage bone tissue.3
One of the apatite materials that have same component with calcium phosphate minerals in bone are hydroxyapatite (HA), Ca10(PO4)6(OH)2.4 HA as
bone filler can induce more new bone growth and accelerate bone healing processes. Problems can arise in clinical situations due to the slow resorption rate of pure HA.5 Nowadays, to overcome this problem some research have used another calcium phosphate minerals to get better result for bone repair. Tricalcium phosphate (β-TCP), Ca3(PO4)2, has biodegradability in faster replacement of the
material with bone tissue.6 Precipitation process by mixing a solution of
phosphate resources at higher sintering temperature successfully formed β-TCP phase. It showed biodegradability or bioresorbability that is more readily than HA ceramics.5,7
One common way to enhance the degradation properties of calcium phosphate scaffolds is to combine a high soluble phase (β-TCP) with an insoluble phase (HA) to create material called BCP ceramics.8 A mixture of HA and β-TCP produces biphasic calcium phosphate (BCP) which possesses the reactivity of β -TCP and the stability of HA, providing more bioactivity, involving more new bone growth, and ensuring better resistance of the implants to strain.9 In mechanical process, it will be expected that mixing of both calcium phosphate phases can form materials that have osteoconductive and osteoinductive properties which increase bone growth from fracture condition.2 The concept is based on an optimum balance of the more stable phase of HA and more soluble β -TCP. The material is soluble and gradually dissolves in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium.10
Synthetic material is well crystallized, while bone mineral is a mixture of amorphous and crystalline phase. With HA 70:30 β-TCP ratio, different structure and component of bone will have same quality of crystalline because of biomaterial. From Yessie (2007) research about in vivo study, bone apatite crystalline has a nonlinear relation to the age, however younger rats is more crystalline than older.11 Making a material that has osteoconductive and osteoinductive should be adapted to what the bone needs. Young bone need more calcium to its growth,
with a high amount of HA will give more stability to bone’s healing. This
research suggests a new kind of biomaterial components that generate BCP with highly successful for the present of 70 HA/30 β-TCP.
2
Biomatlante ) has capability of resorbable and active properties that are well suited to human bone. Based on economic point, the material is considered expensive and still imported, so the material is not affordable by our Indonesian society.12 In this thesis, it is used chicken eggshell as starting material to have lower of the cost production and can be reachable by whole society. Utilization of eggshell has proven by wise drop methods to form a phase of HA and β-TCP.7,13 Adding chitosan with acetic acid as solvent of BCP is for forming BCP so it can be implanted into the bone. Picture of BCP pellet can be seen in Appendix 2.
Results of bone remodeling will show in X-Ray Diffraction (XRD), Fourier Transform Infra Red (FTIR) and Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy (SEM-EDS) analysis. Data of bone analysis is obtained by Hardness Vickers Test. Hardness is usually redefined as the resistance of material to indentation by another solid body under static or dynamic loading. Hardness or indentation test measures hardness by driving an indenter with a specific geometry into the polished surface of material with a known load for a specific time.14
Hypotheses
Ratio 70/30 of HA/β-TCP will determine the ability of material for accelerating the bone healing process in young bone sheep. It will drive more bone tissue to appear. With 30% β-TCP of BCP material, the absorption of material occurs after postoperative implantation. After the callus appeared that shows bone healing process, the bone will absorb the material and speed up the formation process of new bone tissue by the stability properties of 70% HA.
Objective
This research is conducted to analyze BCP material with ratio of 70/30 that was implanted into sheep’s bone. Ability to regenerate bone tissue by material to be shown in the results will determine future research for better improvement in bone healing. Characterization with X-Ray diffraction (XRD), Fourier Transform Infra Red (FTIR), Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDS), and Vickers Hardness Test will show that material have great connection to bone for the healing process.
Benefit
3
MATERIALS AND METHOD
Place and Time
This research was conducted from June to November 2013 which took place in IPB-Biophysics Materials Laboratory while sample analysis (XRD and FTIR) were performed in IPB-Analysis of Materials Laboratory. Characterization of SEM-EDS and Vickers Hardness Test was done in BATAN. Bone implant operation was done in IPB-Surgical and Radiology Laboratory, Faculty of Veterinary.
Materials and Equipments
The Equipments are erlenmeyer, beaker glass, crucibles, mortar, aluminum foil, whatman paper, Mohr pipette, magnetic stirrer, hotplate, analytical scales, furnace, digital thermometer, Furnace Nebhertherm, Furnace Vulcan, burette, infusion device, X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy and Vickers Hardness Tool.
The materials are eggshells, pro-analysis ammonium hydrogen phosphate ((NH4)2HPO4) and phosphoric acid (H3PO4), chitosan (C6H11NO4), acetic acid
(CH3COOH), aqua bidesand aquades.
Experimental Method
Preparations
Preparation is started by cleaning up the eggshells from any kind of dirt then drying it in room temperature for a while. Next process is calcinations, a thermal treatment process in presence of air applied to solid materials to bring about a thermal decomposition of calcium carbonate (CaCO3) into calcium oxide
(CaO) at 1000 oC. Calcination is be heated for about 3 hours of temperature increment and 5 hours of holding time using Furnace Nebertherm.
Synthesis of Biphasic Calcium Phosphate by Mechanical Method
Hydroxyapatite (HA) synthesis was precipitated in wise drop method for 90 minutes. A solution of 0.3 M(NH4)2HPO4 in 100 ml of distilled water was
slowly added into 0.5 M CaO in 100 ml of distilled water while it was stirred about 60 minutes and then aged for overnight. Samples then filtered by using Whatman paper and dried in furnace with temperature 110 oC with holding time for 5 hours. It followed by sintering process at temperature of 900 oC with holding time for 5 hours. Samples were crushed and get characterization by using XRD and FTIR.
Synthesis of β-Tricalcium Phosphate (β-TCP) started with precipitation by using burette for 2 hours. A solution of 1.2 M CaO in 100 ml of distilled water and solution of 0.8 M H3PO4 in 100 ml of distilled water was stirred in hot plate
4
sintering process at a temperature of 1000 oC with holding time for 5 hours. After that, sample was be mashed and then characterized by using XRD and FTIR.
HA and β-TCP samples were mixed by using magnetic stirrer. For each implant of 10 gram BCP, it has 7 gram of HA and 3 gram of β-TCP. These samples were mixed with 0.2 gram of chitosan and acetic acid to form a gel. Ensure to get the sample is in tube shaped with a diameter and length of 6-6.5 mm (see pictures in Appendix 2) and get it into incubator with 50 oC of temperature for sterilization. Flowchart of research is shown in Appendix 1.
Implantation of BCP into Tibia Sheep’s bone
BCP is implanted in 9 sheep aged 1-2 years old. All male sex of sheep divided into 3 groups of 3 animals each for 30 days, 60 days and 90 days observation the healing process in bone. Before the operation, BCP materials were sterilized by exposure to ultraviolet light. The sheep were anaesthetized by intravenous injection of xylazin 2%. Right os tibia of sheep is used for material implant and the left os tibia for control requirement. Materials were implanted with about 6 mm length or should not be entered into the bone marrow. Each material implantation was performed under the same veterinary surgeon. The edges of the bone membrane sutured with a traumatic suture and the incision was closed with silk suture. Antibiotics were injected into the sheep post surgery. All operations and postoperative procedures already had followed according to the animal welfare act and the NIH guide for care and use of laboratory animals. Animals were euthanized at 3 months, postoperatively.
XRD and FTIR Characterization
BCP before and after formed into pellet is characterized by both XRD and FTIR to know the component phase of the sample. XRD analysis is used for determining phase and lattice parameter of both samples. FTIR characterization is used to get functional group of sample and support the results of XRD data.
After 3 months BCP had implanted, material implant is characterized to know the macroscopic and microscopic changes of BCP. By using XD-610 Shimadzu XRD characterization, it had Cu as target with wavelength 1.54060 x 10-10 m and then discharge the sample of 200 mg on aluminum plate with diameter of 2 cm. XRD pattern was being recorded in the range of 2θ from 10° to 80°. Results were compared with data analysis Joint Committee on Powder Diffraction Standards (JCPDS). Lattice parameter is measured by angle references emergence of HA and β-TCP. FTIR characterization used ABB MB 3000 model FTIR Spectroscopy. Two milligram of sample compacted into a pellet with a hundred of KBr to be irradiated by infra red with wave number in a range of 4000-400 cm-1.
SEM-EDS Characterization and Vickers Hardness Test
5 was using Phenom Machine. Some spot were used to get percent concentration of each composition in the sample.
Vickers Hardness used HV-1000 with magnification lens by 40 and using 50 gram of indenter with 10 second of suppression time. Sample measurement by Vickers Test is formed in (0.5 x 0.25 x 0.02) cm3 size and giving buffer with resin and hardness after had being polishing to refine the surfaces. Hardness Vickers Number (HV) is measured with 5 different spot area, 3 spots at the surfaces of bone and the other 2 spots at the inner bone. Each spot have different HV that were representing the hardness of bone.
RESULTS AND DISCUSSION
Wise drop method to synthesis HA with 3 hours stirring from eggshell proven showing pure HA by Putri.13 Figure 1 shows XRD pattern of HA sample. XRD peak showed matching with JCPDS 09-0432 as HA reference major peaks of 2θ at 31.78 , 32.196 and 32.92 . Impurities that changed phase of calcium phosphate because of unequal increasing temperature shows one peak at 25.93 as OCP (Octa Calcium Phosphate). Although this peak has 40% of highest relative intensity, it not concluded as impurity. The presence of OCP will not affect BCP material that will be explained later.
From Hardiyanti, synthesis of β-TCP has optimum temperature by 1000 C. This temperature indicates 2 phases of HA and β-TCP, which is more
dominated by β-TCP.7 Figure 1 shows XRD pattern of β-TCP sample. XRD peak showed matching with JCPDS 09-0169 as β-TCP reference peaks of 2θ at 27.8o, 31.04o, and 34.38o. Impurities are also shown in β-TCP material that will affect in BCP implant that will be explained later.
Figure 1 XRD patterns of HA and β-TCP sample
6
XRD Analysis of BCP Implant
From the Bragg Law, the peak positions provide both of the crystal structure and the lattice parameter for each phase that is contained in the powder sample. The diffraction beam intensity provides a measure of the distribution and position of atoms within the crystal.15 Reference major peaks 2θ of HA is at 31.8 (2 1 1), 32.2 (1 1 2), and 32.9 (3 0 0) meanwhile major peak of β-TCP is at 27.77 (2 1 4), 31.03 (2 0 10) and 34.37 (2 0 0) is attached in Appendix 5 for JCPDS references.
In this research, it used mechanical method to mix samples of HA and β -TCP. This method is successfully making BCP without any significantly changes in material phases. Material implant shows BCP peak before formed into pellets (BCP powder) and BCP peak after formed into pellets (BCP pellet) which consist of HA, β-TCP and OCP. XRD pattern of BCP powder and BCP pellet is shown in Figure 2 and Figure 3.
From Figure 2, it is found 36 experimental peaks matching with 87 HA reference peaks meanwhile from 523 β-TCP reference peaks, 61 experimental peak matching is found. Relative Intensity Ratio (RIR) at 31.0778 (2 1 1) is 61% of HA, 32.968 (2 0 11) is 38.98% of β-TCP and the others is OCP. Whereas in Figure 3 showed 36 experimental peak matching with HA and 37 experimental peak matching with β-TCP. RIR of HA at 31.778 (2 0 11) and β-TCP at 32.981 (3 0 6) is 63% and 36.97% respectively. Chitosan also affected to shift of the diffraction peaks that decreased peak matching with the references. Matching peaks of samples by XRD characterization can be seen in Appendix 3 and Appendix 4.
By using ratios and measuring peak areas, the RIR method can be used to determine the concentrations. It can be concluded that higher the percentage shows higher concentration of samples. The mismatch of BCP ratio was caused by the presence of β-TCP that is also known to enhance decomposition of HA. It is postulated that β-TCP accelerates HA decomposition due to the thermal expansion coefficient mismatch between the intimately mixed phase.16 Existence of OCP was also presented at HA and β-TCP materials. However, the existence of OCP did not matter for ceramics used for implantation because synthetic OCP showed its osteoconductive characteristics that is faster in biodegrade rate than β -TCP.17
Lattice parameter of BCP sample is shown in Table 1 where it changed because of chitosan presence. Accuracy of each sample is reduced caused by the bonding of composite BCP-chitosan. One of the most important characteristics of chitosan, for tissue engineering applications, is its ability to be shaped into various structures, such as pellet to improve their process capability and mechanical properties. Chitosan presents a wide range of properties that make it appropriate for tissue engineering applications, namely, its biodegradability, biocompatibility, antibacterial activity, wound healing properties, and bioadhesive character.18 The bonding extension arise due to appear of chitosan ties. Adding more mass of chitosan will decrease accuracy of lattice parameter and peak number of HA and
7
Figure 2 XRD patterns of BCP powder
Figure 3 XRD patterns of BCP pellet
Table 1 Lattice parameter of BCP sample
8
FTIR Analysis of BCP Implant
Figure 4 FTIR spectra of BCP powder and BCP pellet
Figure 4 shows FTIR spectra of BCP powder and BCP pellet. The first indication for HA formation that assigned to the stretching mode of hydroxyl groups (OH-) is at 3572 cm-1 and 3448 cm-1. While it was at about 1041 cm-1
which arise due to factor group splitting of the stretching asymmetry vibration (ν3) fundamental vibration mode of the PO43- band. The bands at 455 cm-1 and at
563-671 cm-1 correspond to bending vibration (ν2) and bending asymmetry vibration
(ν4) of the PO43- ion, respectively. The asymmetry bands of HA in ν3 and ν4
indicate HA is not entirely amorphous as shown in XRD pattern. (See Appendix 6 for FTIR peak spectra)
OCP is presented by H2O band at 1643 cm-1 that followed by XRD peaks in
BCP pellets form. This band had growing amounts after combined with chitosan because of temperature given while it was drying. The present of chitosan and acetic acid in BCP pellet does not affect the emergence of a new bond. Besides of too little amount, this bond is disappeared by temperature incubator of chitosan melting point that affected on the lattice parameter accuracy only.
Analysis of Post Operation Material
In FTIR spectra of implant, many peaks showed interaction of implant with bone. As seen in Figure 5 that shows FTIR spectra of implanted sample in each month. The spectrum of bone exhibits all the most intense bands observed in the spectrum of hydroxyapatite (at 500-700 cm-1 and 900-1200 cm-1) and that of collagen (in the 1200-1700 cm-1 and 2800-3700 cm-1 regions), being nearly coincident with the sum of the respective profiles. Nevertheless, there are some
3-9
Figure 5 FTIR spectra of implanted samples in each month
new bands (namely at around 870 cm-1 and 1400-1450 cm-1) originated from carbonate substitutions in the crystal lattice of hydroxyapatite.19FTIR spectra of
sample is appropriate with Figueredo et al research about bone graft materials.19 Bands of BCP pellets still existed at 455-571 cm-1 and 602 cm-1 of bending
vibration (ν2) and bending asymmetry vibration (ν4) of the PO43- ion, respectively.
While band at 1041 cm-1 is ν3 PO43- stretching asymmetry as well as stretching absorptions from CH2 wagging and bending vibrations superimposed with those
from asymmetric stretching (ν3, usually as a double band) vibrations of CO3
2-groups, present as ionic substitutes in the apatite crystal.19 This band is characteristic of a type A carbonated apatite. The bands at 2854 cm-1 and 2924-2932 cm-1 also showed CH2 asymmetry stretching.
Surface remodeling refers to the resorption or deposition of bone material on the external surface of the bone.20 A special characteristic of bioactive materials is their ability to form a direct bond between the tissue and the implanted material resulting in a uniquely strong interface. Bioactivity has been associated with the formation of bone apatite on the surfaces of biomaterial.21 The addition of new bonds that appeared on the material after implanted, showed bone remodeling process and the bioactivity of BCP.
10
sample after having connection to bone could possibly cause irregularity in ratio number. Besides, there is a natural tendency for a living organism to respond a foreign object when the material was exposed to a living organism. It caused by protein adsorption, cell adhesion and calcium-phosphate homeostasis.22 In bone remodeling process, ions were the activator. Phosphate was the most needed ion to accelerate bone healing process. High absorption of phosphate is showed in 1 month of material implant. In 2 months of material implant, calcium absorbed as much as phosphate and it has same condition in 3 months of material implant.
Besides the ion activator, uneven mixing cause irregularity in ratio number of Ca/P, as seen in Figure 6, 7, 8 by SEM of physical structure images for each month after implantation. The grains size is not same in each location. To obtain the same grains size, the mechanical method can be given a stirred in hot plate rather than just mixing without any treatment.
11
Figure 7SEM image showing physical structures of 2 months after implanted
12
The result of BCP macroscopic analysis also concluded, in 3 months post-operative of BCP 70:30 with 10% chitosan mixing with the implant still not absorb completely in bone. Table 3 shows macroscopically changes of post operation, in condition, had chosen best sample out of the three. Operations still have visible threads after 60 days and implant protrusions at bone surfaces which have white color. Growth tissue at internal implant that cover implant and degradation sign of implant is happen in 60 days post operation. Some material implant also found in bone marrow that means error operation or not suitable material size to its implantation place.
Physical state of implant that was too tough caused the problems. Bone cell is difficult to penetrate the implant so then bone healing became hampered. Porosity in implants is also needed for bone engineering applications since it facilitates transport of nutrients and oxygen and enables tissue infiltration into the pores. The challenge however is to reach an optimum density which can provide the desirable mechanical properties while still maintaining a porous structure.16
Table 2 Macroscopic changes in implant and bone after implanted.* Characteristics Evaluation Time (Day Post-Operation)
30 60 90
Implant Condition Ud Ud Ud
Implant Color White White Bone Color
Growth Tissue at Periosteum - + ++
*Best condition of three sample implanted bone; undegradated (Ud) degradated (Deg) (-) none (+) little (++) enough (+++) more.
Bone Characteristics of Post Operation
Bone analysis was done by data comparison of implanted and control bone in Hardness Vickers Number (HVN or HV). Two spots are tested on the inner surface table and three spots on the outer surface table were made on each specimen. Inner and outer surfaces should have different measure in normal bone (without case of remodeling bone). The average of HV is shown in Table 3.
13 stiffer and more dense.20 HV number of bone control showed decreasing caused by unavailability of needed nutrients in bone and blood.
Table 3 Vickers Hardness Number of Implanted Bone in Three Month HV Bone Implant HV Bone Control Inner
surfaces
Outer surfaces
Inner surfaces
14
CONCLUSIONS AND SUGGESTION
Conclusions
Characterization with FTIR, SEM-EDS and Vickers Hardness Test showed a connection of material to bone for the bone healing process. Although the ability of material to regenerate bone tissue is less fast than the control, BCP material with ratio of 70/30 is proven to be a good material for implant in minor damage bone because of its biocompatibility, bioresorbability, osteoconductive and osteoinductive characteristic from biological waste synthesized. BCP material also proved to providing more bioactivity, involving more new bone growth and gradually dissolves in the body.
By using mechanical method to form BCP material, there is no phase transformation happen because it changes only caused by thermal properties. BCP analysis data showed that HA and β-TCP also brought out OCP, indicated by XRD and FTIR characterization. Impurity in BCP ceramics was caused by result of material before mixing process. In pellet formed, BCP have many peaks major that showed HA, β-TCP and OCP. The presence of OCP did not matter in material implantation because it biodegraded in faster than β-TCP. Adding more mass of chitosan and acetic acid will decrease accuracy of lattice parameter and peak number of HA and β-TCP due to ion bonding.
Implant characterization after implanted into sheep bone proved to have protein adsorption, cell adhesion and calcium-phosphate homeostasis. These types of responds are showed by the result of HV. Irregularity of HV was impacted by unavailability of needed nutrients in bone and blood.
In SEM-EDS images showed implant for 1 month has the biggest Ca/P because phosphate was firstly absorbed by bone. Damage bones need phosphate to activated bone tissue for remodeling bone. It concluded that BCP with chitosan and acetic acid showed biocompatibility properties because of showing any inflammation but too slow for healing process and bioactive. Low absorption capability of implant is showed with intact implant samples even after 3 months of implantation. It caused by too hard of implant so that was difficult for bone tissues and blood cells to penetrate. Which is means slow healing process due to implant sample.
Suggestions
15
REFERENCES
1. Warastuti Y and Abbas B. 2011. Sintesis dan Karakterisasi Pasta Injectable Bone Substitute Iradiasi Berbasis Hidroksiapatit. Jurnal Ilmiah Aplikasi Isotop dan Radiasi, 7 (2011) No. 2.
2. Laurencin CT and Yusuf K. Bone Graft Substitute Materials. Orthopedic Surgery. Expert Rev Med Devices (2006) 3(1) pp 49-57.
3. Darwis D. 2008. Biomaterial untuk Keperluan Klinis. http://nhc.batan.go.id/. [September 13th 2013].
4. Wahl DA and Czrenuszka JI. Collagen-Hydroxyapatite for Hard Tissue Repair. Europe Cells and Material, 11 (2006) pp 43-56.
5. Piatelli A, Scarano A and Manganot C. Clinical And Histologic Aspects Of Biphasic Calcium Phosphate Ceramic (BCP) Used In Connection With Implant Placement. Biomaterials 17 (1996) pp 1767-1770.
6. Velayati, CE. 2013. Sintesis Komposit Biomaterial (β-Ca3(PO4)2)–(ZrO)
Berbasis Cangkang Telur Ayam Ras Dengan Variasi Komposisi Dan Pengaruhnya Terhadap Porositas, Kekerasan, Mikrostruktur, Dan Konduktivitas Listriknya. [Skripsi]. Malang: Universitas Negeri Malang. 7. Hardiyanti. 2013. Sintesis Dan Karakterisasi Β-Tricalcium Phosphate Dari
Cangkang Telur Ayam Dengan Variasi Suhu Sintering. [Skripsi]. Bogor: Institut Pertanian Bogor.
8. Legeros RZ et al. The Effect of Fluoride on the Bone Mineralization of Young Wistar Rats. Advancement of Life Science. Amvo Publishing Company, Taiwan (2003) pp 29-33.
9. Daculsi G et al. Macroporous Calcium Phosphate Ceramics For Long Bone Surgery in Human Dogs. Clinical and Histological Study. Journal Biomedical Material 11 (1990) pp 379-396.
10. Daculsi G. Biphasic Calcium Phosphate Concept Applied To Artificial Bone, Implant Coating And Injectable Bone Substitute. Biomaterials 19 (1998) pp 1473-1478.
11.Sari YW et al. Nanostructure in Bone Apatite. Biomed 06, IFMBE Proceedings 15 (2009) pp 118-121.
12.Yolanda, Henkky. 2013. Influence of Distribution of Hidroksiapatit (HA) On The Strength Of the Composite Matrix Albumen. http://library.gunadarma.ac.id. [March 14th 2013].
13.Putri, AAM. 2011. Metode Single Drop Pada Pembuatan Hidroksiapatit Berbasis Cangkang Telur. [Skripsi]. Bogor: Institut Pertanian Bogor.
14.Langton CM and Njeh CF. 2004. Series of Medical Physics and Biomedical Engineering: Physical Measurement of Bone. London: Institute of Physics Publishing.
15.Flewitt PEJ and Wild RK. 2003. Physical Method for Materials Characterization. London: Institute of Physics Publishing.
16.Chetty, Avashnee et al. 2012. Hydroxyapatite: Synthesis, Properties, And Applications. New York: Nova Science Publishers.
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18.Rita, Ana et al. Scaffolds Based Bone Tissue Engineering: The Role of Chitosan. Tissue Engineering (2011), Part B 17 (5).
19.Figueiredo MM, Gamelas JAF and Martins AG. Characterization of Bone and Bone-Based Graft Materials Using FTIR Spectroscopy. http:www.intechopen.com[September 13th 2013].
20.Cowin SC and Hegedus DH. Bone Remodeling I: Theory of Adaptive Elasticity. Journal of Elasticity 6 (1976) No. 3.
21.Legeroz et al. Biphasic Calcium Phosphate Bioceramics: Preparations, Properties and Application. Journal of Material Science: Materials in Medicine 14 (2003) pp 201-209.
22.Shi, Donglu. 2004. Biomaterials and Tissue Engineering. Germany: Springer-Verlag Berlin Heidelberg
23.Elhaney et al.Mechanical Properties of Cranial Bone. Journal Biomehnzics 3 (1970) pp 495-511.
17
Appendix 1 Flowchart of Research
BCP Making by Mechanical Method Synthesis of
Hydroxyapatite
Synthesis of β -Tricalcium Phosphate
XRD and FTIR Characterization
XRD and FTIR Characterization
XRD and FTIR Characterization
BCP + Chitosan and Acetic Acid (Pellet form)
XRD and FTIR Characterization
Materials Implanted into Tibia Sheep’s Bone
18
Appendix 2 Pictures of Research Properties
Presipitation Pellets form of BCP
Position of Sample in Bone Polisher tool
Hardness Test Tool
19
Appendix 3 Lattice parameter formulation match to HA
Where,
∑ ∑ ∑ ∑
∑ ∑ ∑ ∑
∑ ∑ ∑ ∑
BCP Before Implanted
2
XRD patterns of BCP sample that matched with HA peak references
Inte
nsit
y (Counts
20
2
XRD patterns of BCP sample that matched with HA peak references
Inte
nsit
y (Counts
21
Appendix 4 Lattice parameter formulation match to β-TCP
BCP Before Implanted
2
XRD patterns of BCP sample that matched with β-TCP peak references
BCP After Implanted
2
XRD patterns of BCP sample that matched with β-TCP peak reference
Inte
nsit
y (Counts
)
Inte
nsit
y (Counts
22
Appendix 5 JCPDS references
a. Hydroxyapatite
23
Appendix 6 FTIR Spectra of Samples
BCP Sample before Pellet Form
24
1 Month of Implant Sample
2 Months of Implant Sample
25
Appendix 7 SEM Image and Distribution of Element Concentrations
SEM image of 1 month implant sample
Distribution of element concentrations from 1 month implant sample
Ca/P =
Element Name
Confi dence
(%)
Spot 1 Spot 2 Spot 3 Spot 4 Spot 5
Mean
C E C E C E C E C E
Calcium 100 19 0.8 35.8 1 17.2 0.8 26.7 0.8 12.9 0.8 22.32 Oxygen 100 67.2 1.2 47.8 3.3 69.6 1.1 57 1.7 76.4 0.9 63.6 Phosphorus 100 13.7 0.9 10.3 1.9 13.2 0.9 16.3 1 10.6 0.9 12.82
Carbon 100 - - 6 2.7 - - - 6
26
SEM image of 2 months implant sample
Distribution of element concentrations from 2 months implant sample
Element
Confid ence
(%)
Spot 1 Spot 2 Spot 3 Spot 4 Spot 5 Spot 6
Mean
C E C E C E C E C E C E
Ca 100 20 0.8 27.3 0.8 12.1 0.9 30.8 0.8 12.6 0.9 18.4 0.8 20.2 O 100 65.9 1.2 54.6 1.7 75.9 0.9 19.9 0.9 67.1 1.1 68.5 1.1 58.65 P 100 14.1 0.9 18.1 0.9 11.9 0.9 49.3 2.1 12.8 0.8 13 0.9 19.86
N 100 - - - 7.5 3.4 - - 7.5
Sum 100 - 100 - 99.9 - 100 - 100 - 99.9 -
Ca/P =
27
SEM image of 3 months implant sample
Distribution of element concentrations from 3 months implant sample Element
Name
Confi dence
(%)
Spot 1 Spot 2 Spot 3 Spot 4
Mean
C E C E C E C E
Calcium 100 36 0.8 18.8 0.8 32.6 0.9 15.5 0.8 25.725 Oxygen 100 41.9 2.6 67 1.3 44.6 2.6 73 1 56.625 Phosphorus 100 22.1 0.9 14.2 0.9 16.3 1.2 11.5 1 16.025
Carbon 100 - - - - 6.5 2.2 - - 6.5
Sum 100 100 100 100 -
Ca/P =
28
Appendix 8 Vickers Hardnes Number of Implanted Bone
d average
=
HV =
Hardness Vicker Number of Bone in a Month after Implanted Control Bone D-13
29
Hardness Vicker Number of Bone in 3 Months after Implanted Control Bone D-2
d1 d2 d average (d average)2 HV HV average
3.6 3.6 0.09000 0.008100 11.44691
11.009457 3.49 3.5 0.08738 0.007634 12.14504
3.98 3.95 0.09913 0.009826 9.436415 2.86 2.86 0.07150 0.005112 18.13683
15.942147 3.32 3.25 0.08213 0.006745 13.74747
Implant Bone D-5
d1 d2 d average (d average)2 HV HV average
3.09 3.09 0.07725 0.005968 15.53733
14.728935 3.07 3.07 0.07675 0.005891 15.74043
3.4 3.38 0.08475 0.007183 12.90904 2.64 2.64 0.06600 0.004356 21.28558
30
Curriculum Vitae
The writer was born at Pekanbaru November 10th 1992. She is the first of three children from Mr. Syafe’i and Mrs. Lismar Yuhaimi. She studied at TK AISIYAH II in 1998, SDN 030 Pekanbaru and graduated in 2004, SMP N 1 Pekanbaru and graduated in 2007, SMA N 1 Pekanbaru and graduated in 2010 and accepted as physics student at Bogor Agricultural University by USMI (Undangan Seleksi Masuk IPB).
