Advanced Materials Research Vol. 747 (2013) pp 123-126 © (2013) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.747.123
Preparation of Porous Hydroxyapatite as Synthetic Scaffold Using Powder Deposition and Sintering and Cytotoxicity Evaluation Eko Pujiyanto1,a, Alva Edy Tontowi2,b, Muhammad Waziz Wildan2,c and Widowati Siswomihardjo3,d 1
Industrial Engineering Department, Sebelas Maret University, Indonesia
2
Mechanical and Industrial Engineering Department, Gadjah Mada University, Indonesia 3
Dentistry Faculty, Gadjah Mada University, Indonesia a
[email protected],
[email protected], c
[email protected],
[email protected]
Keywords: Synthetic scaffold, porous hydroxyapatite, powder deposition and cytotoxicity.
Abstract. This study prepared porous hydroxyapatite (porous HA) as synthetic scaffold and find out chemical properties, porosity, compressive strength and cytotoxicity properties. Porous HA was prepared by powder deposition and sintering from HA-PMMA mixed powder. Porous HA characterizations were conducted by XRD, XRF, SEM-EDX and mercury porosimetry analysis. In vitro cytotoxicity testing of porous HA was conducted by MTT method using vero cells. Porous HA has porosity on the interval 62.79 to 69.67% and compressive strength on the interval 1.53 to 3.71 MPa. Optimal porous HA has porosity is 62.79% with compressive strength is 3.71 MPa. Mercury porosimetry analysis showed that optimal porous HA has interconnective porosity up to 88.25% with pore size on the interval 0.05-355 µm and median pore is 52.64 µm. There was no significantly difference in the death percentage of vero cells caused HA powder and optimal porous HA (p= 0.158) but concentration of optimal porous HA were significantly effect on the percentage of vero cells death (p=0.003). Introduction Bone destruction would disrupt the function of the body, thus requiring a bone grafting. Until now, most of the materials used in bone grafting are autograft. In addition to having many advantages, autograft have some drawbacks. In this case, synthetic bone graft probably use in bone grafting because of limitations of autograft [1]. Based on the function, autograft serves as a scaffold. Synthetic scaffold should have properties similar to the micro and macro autograrft [2]. Some physical property of synthetic scaffold are porosity between 50-95 % with pore inter-connective, pore size of 100-150 µm to support osteoconduction process [3, 10]. Pore size smaller than 50 µm is still possible to osteoconduction process [5]. The required compressive strength synthetic scaffold differ to porous human bones is 2-12 MPa [2]. In addition to meet physical and mechanical properties, a synthetic scaffold should be biocompatible with the human body. The first step is to test the biocompatibility properties of synthetic scaffold is in vitro cytotoxicity test. Materials that are widely used to make synthetic scaffold is hydroxyapatite (HA). HA can be synthesized by hydrothermal method from gypsum. This method produces HA better than conventional methods. In addition to a much faster process, this method produces smaller crystal size and greater surface area [6]. Local gypsum (Tasikmalaya, Indonesia) was synthesized into HA by microwave-hydrothermal method [7]. HA powder that synthesized from local gypsum will be used to make porous HA as synthetic scaffold. One of the good synthetic scaffold criteria is should be made that match the defects in bone of individual patients. This problem can be solved by powder deposition process and sintering. Finally, the need for synthetic scaffold that biocompatible with the human body, suitable physical-mechanical properties and customized that are quickly needs to be developed.
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Materials and Methods Powder Deposition and Sintering HA powder was used in the experiment that synthesized by Pujiyanto, et.al [7]. HA powder was mixed for 6 hours with 30, 40, 50, 60 and 70 weight percent PMMA powder. HA-PMMA mixtures were placed in the hopper nozzle in deposition machine tool. Powder deposition process was done by deposition machine tool [8]. Geometric deposition followed ASTM C-773 was a cylindrical shape with height of 12.7 mm and diameter of 6.35 mm. Powder deposition process carried out by feeding speed 2 mm/min. Specimens were sintered at a temperature of 140oC for 30 minutes into green bodies. The next step, green bodies were sintering at temperature 1150oC, 1250oC, 1350oC and 1450oC and holding time of 2 hours with heating rate 8oC/minute as result porous HA. Characterizations Porous HA Phase analysis of porous HA was performed on a X-ray Diffraction (Simadzu, Japan). Element composition of porous HA was performed on EDX machine (JEOL JSM-6360LA, Japan). Porosity properties of porous HA was measured by Archimedes methods and mercury porosimetry analysis. While the characterization of microstructure using SEM (JEOL JSM-6360LA, Japan). The compressive strength test was carried out according to ASTM C 773–88 standard. Porous HA was tested in compression on Universal Testing Machine (Falter, England) with maximum 200 N load cells. Cytotoxicity Evaluation Cytotoxicity in vitro testing properties of HA powder and porous HA were performed by MTT method using vero cells. Testing procedures of in vitro cytotoxicity properties in accordance document CCRC-02-010-00 (Faculty of Pharmacy, Gadjah Mada University) on procedure method of MTT cytotoxicity test [9]. HA powder and porous HA were placed in wells that contained 100 µL vero cells. All wells divided into 4 samples groups, the first group as medium control, the second group as cell control, the third as sample control and the last one as treatment. The treated groups were divided into 8 sub groups with the concentration 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/ml. Each sub group consisted of 3 wells as replication. After 24 hours, viable cells was determined by the result of OD value using ELISA reader and finally the cell death percentage was determined. Results and Discussion Powder Deposition and Sintering Figure 1 showed result of the first layer of powder deposition using deposition machine with a thickness of 1 mm. The results of this first layer showed good shapes. Problems begin to arise when it would conduct a second deposition layer. Powder deposition process for second layer cause geometric shapes shifting. To overcome this problem and to maintain powder deposition process, powder deposition process was done layer by layer using molding. Powder deposition process was done manually on molding. HA powder was mixed 40, 50, 60 and 70 weight percent PMMA powder have been success into green bodies. Sintering successfully occurred only in HA+40wt% PMMA green bodies. Figure 2 showed photo of sintering unsuccessfully and figure 3 showed photo of sintering successfully.
Fig. 1. The first layer of powder deposition
Fig. 2. Specimen HA+70 wt% PMMA sintering at 1450oC
Fig. 3. Specimen HA+40 wt% PMMA sintering at 1450oC
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Characterizations Porous HA Table 1 showed summary of porosity and compressive strength for 4 specimens with 5 replications. Figure 4 showed that if sintering temperature increased than porosity decreased from 69.67% to 62.79% and compressive strength increased from 1.526 MPa to 3.706 MPa. Optimal porous HA occored on porosity is 62.79% and compressive strength is 3.706 MPa. Based on these results and considering characteristics of the scaffold [2, 3], these results include in the range of scaffold characteristics. Figure 5 showed SEM micrograph of HA-4 specimen.
4 3
80
Porosity 69.67
3.71 67.72
2.78
2
0 1050
70 65
1.88
1
75
1.53
64.19 62.79 Compressive strength
1150 1250 1350 1450 Sintering temperature (oC)
60
Porosity (%)
Compressive strength (MPa)
Table 1. Summary of porosity and compressive strength porous HA Sintering temperature Holding time Porosity Compressive strength Specimen o ( C) (hour) (%) (MPa) HA-1 1150 2 69.666 1.526 HA-2 1250 2 67.716 1.878 HA-3 1350 2 64.189 2.781 HA-4 1450 2 62.793 3.706
55 50 1550
Fig. 4. Effect of sintering temperature on porosity and compressive strength
Fig. 5. SEM micrograph of HA-4 specimen
The results of EDX analysis of optimal porous HA showed Ca is 42.43% and P is 15%. The molar ratio of Ca/P of optimal porous HA is 1.62 closed to theoretical value of molar ratio of Ca/P of HA is 1.67 or closed to molar ratio of Ca/P of TCP is 1.5 [10]. The results of the analysis of 10 highest peaks of XRD patterns of optimal porous HA closed to JCPDS number 09-169 (β-TCP), 09348 (α-TCP) and 25-1137 (TTCP). This means that the phase compositions of optimal porous HA are α-TCP, β-TCP and TTCP. Mercury porosimetry analysis in optimal porous HA showed interconnective porosity is 65.63%, closed porosity is 8.74% and total porosity is 74.37%. Percentage interconnective porosity to total porosity of optimal porous HA is 88.25%. Ideally, the percentage interconnective porosity of synthetic scaffold is 100%. Mercury porosimetry analysis in optimal porous HA showed pore size 0.05-355 µm, median diameter pore is 52.644 µm and average pore diameter is 16.846 µm. The results of chemical, porosity and compressive strength properties meet the criteria for the scaffold, so that porous HA potentially developed into synthetic scaffold. Cytotoxicity Evaluation Cytotoxicity in vitro testing data of HA powder and optimal porous HA are showed in Table 2. The results of the t test between HA powder and optimal porous HA is p = 0.158 ( α = 0.05). These results indicate that there was no significantly difference in the death percentage of vero cells caused by the optimal porous HA and HA powder. It can be concluded that there was no significantly difference in cytotoxicity properties of optimal porous HA and HA powder. Analysis of variance test for optimal porous HA is p = 0.003 (α = 0.05) that means concentrations optimal porous HA were significantly effect on the death percentage of vero cells.
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Table 2. The death percentage of vero cells by HA powder and optimal porous HA
Num. 1 2 3 4 5 6 7 8
Concentration (µg/ml) 1000 500 250 125 62.5 31.25 15.63 7.81
Death percentage (%) HA powder Optimal porous HA 19.94 29.45 19.07 44.03 22.66 17.64 6.26 23.35 1.50 9.95 -7.33 10.58 -8.88 -1.94 -11.01 -2.18
Conclusion Conclusions of this study are sintering process to make green body of HA performed at temperature of 140oC for 30 minutes with composition 60 wt% HA and 40 wt% PMMA. Optimal porous HA are occurred at sintering temperature at 1450oC and holding time 2 hours and heating rate 8oC/minute. Porosity optimal porous HA is 62.79% and compressive strength optimal porous HA is 3.706 MPa. Based on mercury porosimetry analysis, porous HA has interconnective porosity up to 88.25% with pore size on the interval 0.05-355 µm with a pore median is 52.64 µm. These results meet the criteria for the scaffold, so that porous HA potentially developed into synthetic scaffold. HA powder and optimal porous HA was no significantly difference in vitro cytotoxicity properties. This means that sintering process at temperatures at 1450oC with holding time 2 hours and heating rate 8oC/minute did not significantly effect on cytotoxicity properties of HA powder and optimal porous HA. References [1] S. N. Parikh, Bone Graft Substitutes: Past, Present and Future, Journal Postgraduate Medicine. 48 (2002) 142-148. [2] I. Sopyan, M. Mel, S. Ramesh, K. A. Khalid, Porous Hydroxyapatite for Artificial Bone Application, Science and Technology of Advanced Materials. 8 (2007) 116-123. [3] S. F. Hulbert, S. J. Morisson, J. J. Klawitter, Tissue Reaction to Three Ceramics of Porous and Non-Porous Structures, Journal Biomedical Material Research. 6 (1972) 347-354. [4] M. Doblare, J.M Garcıa., M.J. Gomez, Modelling Bone Tissue Fracture and Healing: A Review, Engineering Fracture Mechanics. 71 (2004) 1809–1840. [5] B. S. Chang, C. K. Lee, K. S. Hong, H. J. Youn, H. S. Ryu, S. S. Chung, K. W. Park, Osteoconduction at Porous Hydroxyapatite with Various Pore Configurations, Biomaterials. 21 (2000) 1291-1298. [6] H. Katsuki,S. Furuta, Microwave Versus Conventional Hydrothermal Synthesis of Hydroxyapatite Crystals from Gypsum, Journal American Ceramic Society. 87 (1999) 22572259. [7] E. Pujiyanto, W. Siswomihardjo , I. D. Ana , A. E. Tontowi and M.W. Wildan, Cytotoxicity of Hydroxyapatite Synthesized from Local Gypsum, Proceedings BME Days. (2006) 92-95. [8] E. Pujiyanto, W. Siswomihardjo , I. D. Ana , A. E. Tontowi and M.W. Wildan, Porous Hydroxyapatite–Zirconia Composites Prepared by Powder Deposition and Pressureless Sintering, Advanced Materials Research 445 (2012) 463-468. [9] S. Junedi, Document Number CCRC-02-010-00, Faculty of Pharmacy, Gadjah Mada University,Yogyakarta, 2011. [10] D. E. C. Corbridge, Phosphorus: an Outline of Its Chemistry, Biochemistry and Technology, Elsevier, Amsterdam, 1990.
