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Gel casting of hydroxyapatite with naphthalene as pore former
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Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash, Ritwik Sarkar, Santanu Bhattacharyya
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S0272-8842(14)01791-X http://dx.doi.org/10.1016/j.ceramint.2014.11.053 CERI9498
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Cite this article as: Smruti Rekha Dash, Ritwik Sarkar, Santanu Bhattacharyya, Gel casting of hydroxyapatite with naphthalene as pore former, Ceramics International, http: //dx.doi.org/10.1016/j.ceramint.2014.11.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash, Ritwik Sarkar and Santanu Bhattacharyya Department of Ceramic Engineering National Institute of Technology, Rourkela, Odisha-769008, India
Corresponding author and First author: Smruti Rekha Dash, Ph.D. Research Scholar Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail of the corresponding author: [email protected] Telephone: Mob: +91 9778184420
Second author: Ritwik Sarkar, Associate Professor, Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail: [email protected] Telephone: +91 661-2462203
Third author: Santanu Bhattacharyya, Professor, Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail: [email protected] Telephone: +91 661-2462205
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Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash*, Ritwik Sarkar, Santanu Bhattacharyya Department of Ceramic Engineering, National Institute of Technology, Rourkela 769008, Odisha, India
Abstract The present study discusses the processing and characterization of HA scaffolds which have been processed through a combination of gel casting and fugitive addition. The additional use of Napthalene (NA) as fugitive in the gel casting process provides an additional parameter for tailoring the porosity over and above that obtained via gel casting route. The motive behind the use of combination process was to prepare a porous hydroxyapatite (HA) scaffold with wide porosity range along with sufficient mechanical strength. In house prepared HA powder was used as the staring powder and Methyl acrylamide - N, N’-Methylene-bis-acrylamide (MAM-MBM) system was used for gel casting. Varying amount of Naphthalene (10-40vol% data) and HA solid loading (27-54 vol %) were used for scaffold preparation. The maximum solid loading that could be casted was 48 vol%. The sintered porous Hydroxyapatite-Naphthalene (HA-NA) scaffolds had been characterized for phase stability, microstructure, pore size and pore morphology. The highest compressive strength was 7.51MPa recorded for 22% porosity samples, and it was 0.75 MPa for 81% porosity samples. Scaffolds prepared with lower solid loading had good pore interconnectivity. In vitro bioactivity study of the scaffolds showed dissolution of scaffold up to 3 weeks in SBF solution followed by deposition of calcium phosphate as apatite layer. MTT assay results showed acceptable limit of cell growth and proliferation. Keywords: Hydroxyapatite; Gel casting; Naphthalene, compressive strength, in vitro aging, MTT assay * Corresponding author. Tel: +91-661-2462205. E-mail address: [email protected] (S. R. Dash).
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1. Introduction Porous Hydroxyapatite (HA) and other calcium phosphate based materials are finding wider applicability as scaffold material in the area of bio-implants and tissue engineering [1]. For effective and successful use of HA based porous scaffold, the scaffold material should have open pores with certain degree of interconnectivity for better bone bonding and osteo-integration. A large number of methods have been tried by different research groups for the preparation and characterization of HA based scaffold like polymeric sponge method [2], addition of fugitives [3-6], starch consolidation method [7], foaming [8,9], protein consolidation method [10], freeze casting [11-13], multipass extrusion [14], gel casting [15] etc. Polymeric sponge method or replication method is one of the oldest scaffold fabrications which are safe and easy to reproduce. First proposed by Schwarzwalder and coworkers [16] this method can produce a wide variety of scaffold microstructure depending on the pore architecture of polymer, solid loading and number of coating [17]. However, the basic microstructure of the scaffolds prepared by this method is strongly dependent on polymer microstructure and pore size and microstructure with similar pore architecture is difficult to reproduce. In the natural fugitive method, additives are mixed with the powder or dispersed in the slurry and shaped in green condition. The fugitive burn off during sintering leaving behind mostly open pores, although some claim that degree of interconnectivity of struts is poor. Starch consolidation is another method which has widespread acceptability for porous scaffold processing. Lyckfeldt et al [18] used potato starch granules in ceramic slurry and the slurry was consolidated by heating at 600C. During sintering, the starch granules burn off leaving behind pores. This route could produce ceramics with 30-70% porosity. Dhara et al [19] have used ovalbumin as foaming agent and have produced porous samples by 3
gelcasting. The degree of porosity and interconnectivity depended on water: albumin ratio, solid loading, sintering temperature etc. Research has also been conducted on the use of Bovine Serum Albumin (BSA) [20] for producing alumina based foams. The use of BSA could produce porous scaffold with 8-20% porosity along with good interconnectivity. Freeze Casting method [21] have also been used to prepare macroporous HA. Kohet al [22] have used unidirectional freeze casting method to prepare HA scaffold. Ghazanfari et.al [23] have studied the effect of nano-silica addition on the properties and microstructure of freeze casted HA-nSiO2 composite. Macroporous HA has also been prepared by multipass extrusion method [24]. The fabricated scaffold had unidirectional micro channel at the exterior part and a central canal in the interior. This type of microstructure was expected to provide better bone bonding and osteointegration. Gel casting [25-29] is another popular processing route which has been widely used for the processing of both porous and dense ceramics. The first report of gel casting was made by Omatete and Janney [30, 31].It described the gel casting process and the advantages like near net shape forming, machinability and ease production that gels casting provided over other processes [32]. The basic process involves in-situ polymerization and gelation of organic additives which are used along with dispersed slurry. The gelation of monomers lead to rapid setting of cast bodes. The organic monomers and polymers when removed on heating create a network of porous bodies with interconnectivity and the pores are mostly spherical [33]. The microstructure and porosity is dependent on the nature of additives, solid loading and diapersability of powder in the slurry [34, 35]. Although MAM and MBAM based monomers have been traditionally used, due to health concern, other more ecofriendly monomers like agarose, food quality gel are also being used. Roshanbinfar et al [36] have used gel casting route with agarose gel to prepare porous HA scaffold. Casting was done by shake gelcasting
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method at 60 Hz frequency. Li at al [37] have used a combination of gel casting and rapid prototyping to prepare -TCP porous scaffold. Anil Asif at al [38] have prepared HA scaffold using food grade gel and gelling medium to prepare porous HA scaffold. Therefore, from the above discussion it is evident that gel casting is a very successful method for preparing porous scaffold. However, in all the gel casting method, the porosity and pore shape size is dependent on the type and amount of monomer. Therefore, in this study, an attempt has been made to modify the microstructure and the pore architecture by a combination of fugitive and gel casting. The additional use of Napthalene (NA) as fugitive has provided an additional parameter for controlling the porosity and pore connectivity. Therefore, the present work differs from the conventional gel casting or freeze casting method studied earlier on two counts. Firstly, in this study, combined use of monomer and fugitive has resulted in high degree of porosity along with variation in pore size which more than that achieved by gel casting alone. Secondly, most of the reports on gel casting have been made in the solid loading range of 40-70%. However, in the present study, the effect of low solid loading (25-54 volume %) has been investigated. The casted samples were sintered at 1250 oC and characterized for pore shape and size, strength, microstructure, invitro aging and cytotoxicity test.
2. Experimental Procedure All the samples in this study were prepared by Gel Casting of Hydroxyapatite (HA). The HA powder (Ca/P = 1.67) was synthesized in house from wet chemical route using diammonium hydrogen phosphate ((NH4)2HPO4) (Merck, India, GR grade 99.9% purity) and calcium nitrate (Ca(NO3)2·4H2O) (Merck, India, GR grade 99.9% purity). The mixing of the precursors and the precipitation of the hydroxides was carried out at room 5
temperature which varied from 25 OC to 30 OC depending on the season. The mixed precursors were homogenized by stirring for 2 hours at 900 rpm in a magnetic stirrer. 1:1 Ammonia solution was drop wise added from a burette (dropping speed 1ml/min) to effect the precipitation.
The added ammonia was in excess of the
stoichiometric amount in order to maintain the solution pH between 10.5- 11 and the stirring was continued for another 2 hours. Subsequently, the as formed precipitates were aged for 24 hours washed with deionized water to bring down the pH near 7-8. The washed precipitates were oven dried for 24 hours. The dried amorphous precipitates were ground to fine powder and calcined at 850 oC at a heating rate of 3o/min for crystallization of HA. The particle size of HA was between 0.5 μm and 5 μm and BET specific surface area was 14.9 m2/gm. Gel Casting slurries were prepared from calcined HA, Methyl Acrylamide (MAM) (C2H3CONH2), Merck, India (GR grade) and Methylene-Bis-Acrylamide (MBAM) [(C2H3CONH)2 CH2],Merck, India (GR grade), Ammonium Persulphate (NH4)2S2O8 (APS),Merck, India (GR grade)and N, N, N, N Tetra Methylethylene Diamine (TEMED),Merck, India (GR grade 99.9% purity). While MAM was the chain former, MBAM acted as branching former. On the other hand, APS and TEMED performed the initiator and catalyzer reactions respectively. For preparing stable dispersed HA slurry, 1.5% Darvan C (Vanderbilt, USA) was used as deflocculant. The different components required for slurry preparation were mixed in sequence and the mixed slurry was stirred for 6 hrs for dispersing and stabilizing the slurry. The rheology of the slurries were measured as a function of solid loading and the maximum solid loading (54 vol %) corresponding to the acceptable limit of pourability was taken as the maximum permissible solid loading for successful gel casting. For creating higher degree of porosity in HA over and above that created by Gel Casting itself, Naphthalene (NA) particles (-300+200μm) were added to stable HA slurry and the mixture of HA + NA
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were stirred for 30-45minutesfor homogeneous mixing of NA particles in the slurry. Table-1 shows the slurry composition and NA content and Fig 1 gives detailed process flow diagram for scaffold preparation. The HA + NA mixed slurry was transferred to a petroleum jelly-coated Teflon mold (46mm
i.d. x 5mm height). Post mould filling, the
mould was gently tapped to remove entrapped air as well as for even distribution of slurry in the mould The casts were dried in water bath (40 OC) for 2 days following which the casts were demoulded and oven dried at 800C for 24 hrs. The samples were carefully heated till 650oC (heating rate 2o/min) and a holding time of 2 hours was provided at that temperature for facilitating easy removal of monomers, initiators, accelerators, other organics as well as NA. Finally the samples were heated to 12500C (holding time 2 hours) at a heating rate of 3o/min for final densification of samples. 3. Characterization The thermal decomposition behavior of as dried gel was studied by DSC-TG (Netzsch STA/409 C) till 1000 oC in an argon atmosphere. The heating rate was 10OC/ minute and -Al2O3was used as the reference material. The rheological behavior of HA slurry was measured using Rheometer (Rheolab QC, Anton Paar USA). Rheological tests were performed at 25 °C in the shear rate in the range 0.1-250 s-1, The phases in sintered samples were determined through XRD(Panalytical, Netherland) using Cu K radiation ( = 1.542 nm) fitted with Ni filter. The samples were scanned in the 2 range 25° to 60° in continuous mode with a step sizeof 0.02 degree/sec. The apparent porosity of the samples was measured through Archimedes principle using water as liquid medium. The linear shrinkage of samples was determined by measuring the diameter of samples before and after sintering. The microstructure of the scaffold was observed in a Field Emission Scanning Electron Microscopy (FESEM) (Nova Nano 450, FEI USA). The samples were sputter coated with gold for 3 minutes in Argon atmosphere (Quorum Technology, 7
Q150R ES). EDAX (Energy Dispersive X-ray analysis) (Bruker USA, Model 127 eV) analysis was carried out on SBF aged samples for identifying the apatite deposits. Some samples were also tested through Raman Spectroscopy (LabRAM HR (UV) spectrometer) for confirmation of apatite layer. Thelasersourcewas514nmforminimizingthefluorescence effect of the sample. The porosity and pore size distribution was determined by Mercury
Intrusion Porosimeter (Quantachrome/PM-33-13). The compressive strength of the porous materials was determined on cylindrical sample (15 mm
x 6 mm) in Universal
Testing Machine (H10KS Tinius Olsen, UK) following ASTM standard test method for compressive strength measurement (ASTM C773-88 (2011) [39] . The samples were tested with a load cell was 10 KN, cross head speed 0.5 mm/minute and Compressive Strength (CCS) was obtained using the formula: CCS=P/A Where, P = Load in KN and A = Cross sectional area of the cylindrical sample. The permeability of the samples was determined from liquid flow measurement using Darcy’s equation [40] Q= (KpPA)/L Where,Q=volume flow rate of liquid through the sample, Kp= specific permeability coefficient, P= pressure drop across the sample, L=flow length or thickness of the test sample, A=area of cross-section of the sample, = fluid viscosity. The permeability coefficient Kp depends on the nature of the fluid porosity and pore size as well as pore connectivity combination of porous material. Higher Kp value indicate ease of flow through the material which implies that pore interconnectivity is of high degree. In vitro bioactivity of sintered porous body was tested in simulated body fluid (SBF) by immersing specimen in SBF at 37 ± 1°C for different time periods ranging from 3 days to 8
7 weeks in a BOD incubator. The SBF was prepared in the laboratory as per Tas’s specification [41]. The SBF solution was replaced with fresh SBF solution at regular intervals to compensate for the decrease in ion concentration of SBF solution as a result of apatite layer formation on the specimens. The aged samples after its removal from SBF solution, were thoroughly washed with deionized water, and dried at room temperature. Formation and growth of apatite layer was studied by observing FESEM image of SBF aged surfaces. Selected areas of aged samples were also analyzed by EDAX and Raman spectroscopy for compositional analysis of the deposits. The phosphorus and calcium ion concentration in SBF solution before and after immersion for different time periods were studied in an Atomic Absorption Spectrophotometer (Perkin Elmer AS 800). The cell viability and cell proliferation activity was studied through MTT assay using L929 mouse osteoblast [42-44]. The formazon crystals formed were dissolved by adding dimethyl sulfoxide and the absorbance was measured at 540nm using spectrophotometer (Systronics double beam spectrophotometer 2203).
4. Results& Discussion Fig.2 (a) shows DSC-TG diagram of as dried gel. The DSC curve has a number of stages. At 100°C, it has an endothermic peak associated with weight loss of about 5%on account of removal of adsorbed water from gel. The exothermic peak at 230 OC is due to the oxidative decomposition of polyacrylamides which starts around 2200C. Between 210-2300C, the endothermic peak appears due to an imidic reaction which releases ammonia gas. This reaction completes by 3750C. Following this, decomposition of amide groups and breakdown of polymer structure takes place up to 6000C [45, 46]. By 6000C, the weight loss of gel was nearly complete. At still higher temperature (between 650 °C and 850 °C), a broad peak can be observed without any significant weight loss. The occurrence of the broad peaks can be correlated with the crystallization of HA from its 9
amorphous state first in its disordered structure and subsequently its progressive transformation to ordered HA. This fact was verified by a series of XRD plots for powder calcined in the temperature range of 7500C to 9500C (Fig 2(b) and 2 (c)). It is seen that at the first crystallization temperature (750oC), the d-spacing of HA is more than of JCPDS value (Table- 2) and with increase in temperature the d-spacing shifts to ideal value for HA (i.e. closer to ideal HA diffraction peak). It is also seen that at 8500C, the d-spacing matches with the JCPDS value (File No 84-1998). Further, the exothermic peak at 9500C corresponds to another crystallization peak of HA but the d- spacing at this temperature differs from JCPDS value (84-1998). On the basis of the above findings, the calcination temperature for HA was fixed at 850 °C. Fig. 3 (a) and 3 (b) show the effect solid loading on the shear stress-shear rate behavior and viscosity-shear rate respectively of HA slurries. The shear stress-shear rate plots have been obtained in both upturn (increasing shear rate) and downturn (decreasing shear rate) mode. It was observed that slurries with solid loading less than 30 vol% were very fluid and showed negative shear stress value. Only slurries of 30 vol% and higher solid loading showed characteristics shear thinning behavior and shear stress increased with shear rate rapidly during at low shear rate and more gradually at higher shear rate with typical hysteresis between shear stress and shear rate. Considering the fact that the slurries would show power
law behavior, the shear thinning constant (n) was calculated from the slope of ln() vs ln( J ) plot. It was observed that all the slurries showed shear thinning behavior with n value of 0.25 to 0.40. Fig. 3 (c) shows the representative shear stress-shear rate graph for 54 vol% solid loading. The difference in shear stress in the upturn and downturn mode at zero shear shear rate is gel or an index of structural buildup. Fig. 3 (d) shows the variation of gel for all the composition between 30 and 57vol% which shows that the index of structural buildup increases with solid loading. This implies that at higher solid loading, the samples will have a lower casting time and the cast may have low porosity and it may be difficult to cast a sluury with very high gel. Fig 3. (e) shows the variation of steady state viscosity (measured at 200 s-1) with solid loading for all the compositions. It is seen that the steady state viscosity is 7 Pa.s for 20 vol% solid loading and it is 12 Pa.s for 27 vol%. The low viscosity of these two slurries resulted in poor cast properties and samples cracked after drying. However, the viscosity is 22 Pa.s for 32 vol% and it could produce crack free samples. The viscosity increased further with increase in solid loading and at 48 vol% solid loading the maximum in viscosity (220 Pa.s) corresponding to “pourable” and castable slurry was observed. Beyond that, the slurries were not pourable. Therefore, the maximum solid loading in this investigation was restricted to 48 vol%. 10
Figure 4and Figure 5 show the XRD pattern of sintered HA and sintered HA-NA respectively. It is observed that there is no change in peak position of HA till 1250oC and also naphthalene has no effect on phase (Fig. 5)implying that HA phase is stable till 1250oC.The d-value of the three characteristics peaks of HA (d=2.812Ao, 2.776Ao, 2.718Ao) matches with the standard d value of HA (JCPDS 84-1998). But at 13500C the hydroxyapatite decomposes to give -TCP and Calcium oxide (CaO) as shown in Figure 4. Fig. 6 (a) and 6 (b) shows the effect of addition of NA on porosity and strength of the gel casted scaffolds, respectively. It was found that with the increase in the amount of NA, the porosity increased and simultaneously the bulk density and compressive strength decreased. Without NA addition, the porosity was 22 ±3 % and corresponding strength was 7.51±0.1MPa. As already explained, NA was added to create higher porosity sample. At 40wt. %NA addition, the porosity was nearly 75 % and strength reduced to about 1MPa which is close to the literature value [47-49] for this level of porosity. At still higher NA content (50% & 60%NA), the sample strength could not be measured because high porosity resulted in mechanically weak and fragile samples. Therefore, the strength limiting factor restricted the maximum percentage of NA to 40%. Compressive strength values for samples were in accordance with the literature as well as close to the reported compressive strength of human cancellous bone ranges between (2-12 MPa). [50-52]. A plot of compressive strength ( ) against porosity (P) shows that strength ( ) exponentially decreases with increase in volume fraction porosity and follows the relation.
V
V 0 exp(bP )
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Where, b is constant and 0 is the compressive strength for zero porosity samples. A plot of ln ( ) vs volume fraction porosity (P) [Fig. 6(c)] yields a straight line with a negative slope. The slope gives the value of ‘b’ and for this study it was found to be 0.04.The negative slope indicates that as porosity increases, loading carrying capacity of the scaffolds decreases exponentially due to lesser interconnection among solid materials. In all the gel cast sintered samples, porosity and pore size depended on naphthalene content. Fig. 7(a-e) shows the pore size distribution of porous HA ceramics. Fig. 7 (a) represents gel casted sample without addition of naphthalene which shows pore within a narrow size and mostly consisting of micro pores (<50μm). On the other hand, Fig. 7 (be) show a higher population of macro pores (100±20 μm to 200±20μm) and less micro pores (<50μm). At higher naphthalene content, not only the porosity increases, but the agglomeration of naphthalene also causes an increase in the pore size which could not be eliminated in the pore removal process during sintering It is also seen that at higher naphthalene addition, not only the pore size but also the pore population changes to higher value. Thus by using the combination of NA and gel casting wide variety of porous structure could be produced. Fig. 8 shows the microstructure of a sintered sample (30NA). It is seen from the microstructure that the sintered scaffold contains macro pores of 20-100μm. The encircled area in the image (Fig. 8) shows that the pore on the strut walls are connected which indicate that the scaffold has interconnectivity. The permeability of scaffold materials with different porosity level and pore size is shown in Fig.9. A comparison of 30NA sample with that of 40NA show that porosity and permeability has 1:1 correspondence, i.e. 10% increase in the porosity resulted in an equal increase in permeability value, assuming interconnected pores. The highest permeability of 12
30.2 x 10 -11m2 is obtained for 40NA samples. However, in the present investigation, even the minimum permeability is much higher than the reported value of 3.9x 10-11 m2 [46]. Hence the combination of gel casting with NA addition as additional pore forming agent results in a porous scaffold with significant interconnectivity and high permeability.
4.1 In vitro bioactivity and the formation of apatite layer: (a) Effect of aging days on apatite layer formation: Figure10 (a-g) shows the effect of aging time on the apatite layer of 30 NA after immerse in SBF of 3, 5, 7, 14, 17, 21 and 28 days respectively. The images show that after 3 days of aging a few white granular spots are seen which correspond to the apatite particle formation (Fig 10a &.10b). In order to confirm that the deposited particles correspond to apatite, EDAX was carried out on the spots as shown in Figure 11(a). Although HA and apatite are composed of similar elements, the presence of carbon on the spots prove that the deposited layers are of apatite. Some other aged samples (40 NA) were also characterized by Raman spectroscopy. The Raman spectra are shown in Figure 11(b). The spectrum shows characteristic peak at 960 cm-1 assigned to symmetric stretching mode (1) of the tetrahedral (PO4)3- group of (P O) bond and the presence of this peak confirms the deposition of apatite layer and the peak position matches with the reported value for apatite [53]. With increase in aging period (7 days and 14 days) the area covered by the apatite layer increases (Fig. 10c and 10.d). Further, the shape of the deposited apatite layer changes with aging time (Fig.10 e-g). At lower aging time, it has particle like morphology, at intermediate time it has flower morphology and at the highest aging time it has petal like appearance. It is also observed from the above Figures that, the surface coverage also increases with increase in ageing time. Thus, not only the 13
apatite morphology changes with aging time but also the area covered by the apatite layer changes with aging time.
(b) Effect of porosity on apatite layer formation: Fig. 12 shows the FESEM image of HA scaffolds after soaking in SBF for 21days. The microstructure shows apatite formation on the HA surface. The apatite could be recognized as white precipitates (encircle area) on sample surface. Fig.12a represents the microstructure of only gel casted scaffold without any naphthalene. It is seen that there are a few spots which correspond to apatite deposition. Fig.12 (b-e) represents the microstructure with progressively higher amount of naphthalene and consequently higher porosity. From the micrographs, it could be observed that higher porosity sample (40NA) had more apatite layer than those corresponding to lower porosity (10NA). The higher deposition rate of apatite in higher porosity samples can be related to faster dissolution of ion and its migration through interconnected porous network of higher surface area [5356]. Beyond 21 days no significant change in surface area coverage could be observed and all samples were almost fully covered with apatite deposits.
4.2 Dissolution Behaviour of Hydroxyaptite in SBF The dissolution behaviour of HA was measured by estimating Ca+2 concentration in SBF solution.The variation of Ca2+ concentration in SBF solution with dissolution time containing the investigated porous HA is shown in Fig.13. It is seen that Ca2+ concentration in SBFincreases slowly in the first week of dissolution and after 1 week the concentration increases rapidly till 3 weeks (in some case upto 4 weeks) time. The increase of Ca2+ in the SBF solution confirms the dissolution of HA at pH 7.4 [47]. The
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degree of dissolution of HA in SBF is depend on impregnation by SBF and higher porosity sample, higher volume of impregnation is achiened.Similar observations were also noted by Ereiba et al [54]. Rapid response of the porous bioactive surfaces could be correlated with the dissolution of initially formed phosphate and its reprecipitation carbonated apatite from the supersaturated solution [55]. Thus it could be concluded that the increases in Ca+2 concentrations in SBF till 21 days of ageing correspond to its dissolution in SBF and the decrease in Ca+2concentration after 21 days till 2 days correspond to precipitation of apatite from solution. On the other hand, the weight loss increased till 21-28 days and then gradually decreased due to the deposition of CaP compound from SBF on the scaffold surface (Fig. 14). Therefore, the dissolution is dominant till 28 days and apatite growth process is dominant after that period. Fig.15. shows the effect of ageing time on the pH change of SBF solution. The pH of SBF increases during the initial period of ageing i.e till 21days which can be correlated with dissolution of HA in SBF. The dissolution leads to increase the Ca+2 concentrations. Due to the dissolution of Ca+2, the pH increase. Thereafter, the pH decreases as the released Ca+2 reacts with HPO42- to produce CaP layer as per the reaction given below [56]. .
5Ca2 3HPO42 - + OH- o Ca5 (PO4 )3 OH + H 2O
It is anticipated that Ca2+ and HPO4
2-
(1)
ions in the solution form apatite nuclei which
deposit either on the surface of hydroxyapatite or in the SBF solution. Those nucleated on the surface of hydroxyapatite further grew into an apatite coating layer, while those
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nucleated in the solution form into apatite precipitates [57]. During the growth of the apatite coating or precipitates, the pH of the solution decreased due to the consumption of OH- in the solution according to reaction (1).
4.3 Cytotoxicity Behaviour The cytotoxicity/cell viability of the hydroxyapatite scaffolds was assessed by the MTT cytotoxicity assay as shown in Fig. 16.The cell viability and cell proliferation activity was studied through MTT 3(4, 5-dimethylthiazol-2yl)-2-5diphenyltetrazolium bromide) (SIGMA, USA) assay using L929 mouse osteoblast after 72 hours incubation period at 37 oC in 5% CO2. The sterile scaffold along with control was placed in UV sterilized 96 well plates (Nunc, Germany) in 200 μl DMEM (Dulbecco’s modified Eagle medium) at a concentration of 10 4 MG 63 cells per well. The dehydrogenase enzymes of the active cell reduced the yellow tetrazolium MTT into intracellular purple formazan. The concentration of formazon produced was proportional to the number of viable cells present in the media [42, 44, 58]. The formazon crystals were dissolved by adding dimethyl sulfoxide and the absorbance was measured at 540nm. The resultsshow that the cells were able
to grow and proliferate favoourably. The results also indicate the porosity provides favourable sites for cell proliferartion. However, in the present study ahigher porosity sample (40NA) exhibited only marginally higher cell viability than less porous sample (20 NA) (Fig. 17). Thus, from the above observation, it can be concluded that both low and porosity scaffold prepared in this study permit proper cell proliferation and are expected to show acceptable cytocompatibility along with cell viability properties.
5. Conclusions The present work dealt with the processing and characterization of porous HA scaffold. The porous hydroxyapatite scaffold was prepared by incorporation of volatile material (Naphthalene) as a pore forming agent in gel casted samples. By modifying the solid loading, naphthalene content, the porosity and compressive strength could be altered over a wide range. Porous HA scaffold with 60% porosity had open and interconnected pores. Compressive strength of 4.1 MPa was recorded for 50% porous scaffold (30NA sample) and compressive strength of 2.3 MPa was observed for 60% porosity (40NA sample). The processed scaffolds had wide pore size ranging from 10–150μm. These pores were also found to be interconnected in the macro porous structure. Invitro aging confirmed the deposition of apatite layer on HA surface and the deposition rate varied 16
with sample porosity. MTT results indicate that the processed HA scaffold had appreciable cytocompatibility and therefore may have a potential for use as scaffold material.
Acknowledgements This research was financially supported by National Institute of Technology,
Rourkela,
India.
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[15] S. H. Sadraie, K. Roshanbinfar, G. Kaka, J. Hoseini, Mechanical and Structural Study of Hydroxyapatite(HA)Scaffolds Produced by Gel Casting and Sponge Replication Methods,Journal of Biomaterials and Tissue Engineering 4 (2014) 1–5. [16] K Schwarzwalder, AV Somers, Methods of making porous ceramic articles Polymeric sponge coating, (1963) US Pat 3090094. [17] P Sepulveda, JGP Binner, SO Rogero, OZ Higa, JC Bressiani, Production of porous hydroxyapatite by the gel casting of foams and cytotoxic evaluation. J Biomed Mater Res 50 (2000), 27–34. [18] Lyckfeldt O, Ferreira JMF, Processing of porous ceramics by starch consolidation, J Eur Ceram Soc 18 (1998) 131–40. [19] S. Dhara, P.Bhargava, Simple Direct Casting Route to Ceramic Foams, J. Am. Ceram. Soc 86 (2003) 1645–1650. [20] A.R. Studart, U.T. Gonzenbach, E. Tervoort, L.J.Gauckler Processing Routes to Macroporous Ceramics: A Review, J. Am. Ceram. Soc., 89 (2006) 1771–1789. [21] S. Deville, E. Saiz, A.P. Tomsia, Freeze casting of hydroxyapatite scaffolds for bone tissue engineering, Biomaterials 27 (2006) 5480-5489. [22] Y.H. Koh, E.J. Lee, B.H. Yoon, J.H. Song, H.E. Kim, Effect of polystyrene addition on freeze casting of ceramic/camphene slurry for ultra-high porosity ceramics with aligned pore channels, J. Am. Ceram. Soc. 89 (2006) 3646-3653. [23] S.M Ghazanfari, A Zamanian, Effect of nanosilica addition on the physicomechanical properties,pore morphology, and phase transformation of freeze cast hydroxyapatite scaffolds, J Mater Sci 49 (2014) 5492–5504. [24] D. Jang, R. Franco, S. Sarkar, B. Lee, Fabrication of Porous Hydroxyapatite Scaffolds as
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[34] P. Sepulveda, J.G.P. Binner, Processing of celluar ceramics by foaming and in situ polymerization of monomers, J. Eur. Ceram. Soc. 19 (1999) 2059–2066. [35] L.A. Cyster, D.M. Grant, S.M. Howdle, F.R.A.J. Rose, D.J. Irvine, D. Freeman, C.A. Scotchford, K.M. Shakesheff, The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering, Biomaterials 26 (2005) 697–702. [36] K. Roshanbinfar, M Ansari, Investigating of Mechanical and Biological Properties of Porous Hydroxyapatite Scaffolds Produced by Novel Shake Gel Casting Method, J. Biomater. Tissue Eng. 3 (2013) 1-5. [37] S. H. Li, J. R. Wijn, P. Layrolle, K. Groot; Novel method to manufacture porous hydroxyapatite by Dual- Phase mixing, J. Am. Ceram. Soc, 86 (2003) 65-72 [38] A. Asif, R. Nazir, T. Riaz , N. Ashraf , S Zahid , R. Shahid, A. Rehman • A. Chaudhry • I Rehman, Influence of processing parameters and solid concentration on microstructural properties of gel-casted porous hydroxyapatite, J Porous Mater 21 (2014) 31–37. [39] ASTM C773 – 88 (2011), Standard Test Method for Compressive (Crushing) Strength of Fired Whiteware Materials, ASTM International, West Conshohocken, PA. [40] James S. Reed, Principles of Ceramics Processing, 2nd edition, John Wiley & Sons, Inc., New York, 1995. [41] A. Cuneyt. Tas, Synthesis of biomimetic Ca-hydroxyapatite powders at 37 0C in synthetic body fluids, Biomaterials 21 (2000) 1429-1438. [42] L.L. Hench, J. Wilson, An Introduction to Bioceramics, World Scientific, Singapore, (1993) 139-180. [43] P.W Hui, P.C Leung, A Sher, Fluid conductance of cancellous bone graft as a predictor for graft-host interface healing. J. Biomechanics 29 (1996) 123-132.
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[44] W. Suchanck, M. Yoshimura, Processing and properties of hydroxyapatite based biomaterials for use as hard tissue replacement implants, J. Mater. Res. 13 (1998) 94– 117. [45] E. Sachlos, J.T. Czernuszka, Making tissue engineering scaffolds work, review on the application of solid freeform fabrication technology to the production of tissue engineering scaffolds, J. Eur. Cell Mater. 5 (2003) 29–40. [46] B.S. Chang, C.K. Lee, K.S. Hong, H.J. Youn, H.S. Ryu, S.S. Chung, et al., Osteo conduction at porous hydroxyapatite with various pore configurations, Biomaterials 21 (2000) 1291–129. [47] H. Yoshikawa, A. Myoui, Bone tissue engineering with porous hydroxyapatite ceramics, J. Artif. Organs 8 (2005) 131–136.. [48] T. Abdurrahim, I. Sopyan, Recent Progress on the Development of Porous Bioactive Calcium Phosphate for Biomedical Applications, Bone. (2008) 213–229. [49] I. Sopyan, N.S. Sulaiman, D. Gustiono, N. Herdianto, Porous hydroxyapatite gelatin composites with functions of bone substitutes and drug releasing agents: a preliminary study, BioMEMS and Nanotechnology II, SPIE, Int. Soc. Opt. Eng. 6036 (2006) doi:10.1117/12.639846. [50] L. Bakhtiari, H. Reza, S. Mohamad, M. Ali, Investigation of biphasic calcium phosphate / gelatin nanocomposite scaffolds as bone tissue engineering, Ceram. Int. 36 (2010) 2421–2426. [51] G. Negroiu, R. M. Piticescu, G. C. Chitanu, I. N. Mihailescu, L. Zdrentu, M. Miroiu, Biocompatibility evaluation of a novel hydroxyapatite polymer coating for medical implants ( in vitro tests ), J. Mater. 19 (2008) 1537–1544. [52] F. Balas, S. Sa, In vitro structural changes in porous HA / -TCP scaffolds in simulated body fluid, Acta Biomater. 5 (2009) 2738–2751.
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[53] B. Wopenka, J. D. Pasteris, A mineralogical perspective on the apatite in bone, Mater. Sci. Eng.-C, 25 (2005) 131–143. [54] K.M.T. Ereiba, G. Mostafa, G. Gamal, H. Said, In vitro study of iron doped hydroxyapatite, J. Biophys. Chem. 04 (2013) 122–130. [55] H. Kim, T. Himeno, T. Kokubo, T. Nakamura, Process and kinetics of bonelike apatite formation on sintered hydroxyapatite in a simulated body fluid, Electronics. 26 (2005) 4366 - 4373. [56] K. Hyakuna, T.Yamamuro, Y.Kotoura, M. Oka, T. Nakamura, T. Kitsugi,
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Fig.1. Flowchart of processing of Gel casted porous HA scaffolds using Naphthalene. Fig. 2(a) DSC-TG of as dried gel. Fig. 2(b) XRD plots of precipitated HA powder calcined in the temperature range of 750 0C to 950 0C. Fig. 2(c) XRD plots (triplet) for powder calcined in the temperature range of 750 0C to 950 0
C showing the peak shifting of HA.
Fig. 3(a) Shear stress versus shear rate plot as a function of HA solid loading. Fig. 3(b) Viscosity-shear rate plot as a function of HA solid loading. Fig.3(c) Shear stress – shear rate plot for 54 vol% solid loading. Fig. 3(d) Variation of gel with solid loading. Fig. 3(e) Variation of steady state viscosity as a function of HA solid loading. Fig.4 XRD pattern of sintered HA at different temperature (1000-1300 OC). Fig. 5 XRD pattern of sintered HA and HA-NA scaffold. Fig.6 (a) Variation of porosity as a function of naphthalene content. Fig. 6(b) Variation of compressive strength as a function of porosity. Fig.6 (c) Semi-logarithmic plot of compressive strength and porosity. Fig 7. (a-e) Pore size distribution of sintered HA scaffolds prepared by gel casting (a) 0 NA, (c) 10 NA, (c) 20 NA, (d) 30 NA, (e) 40NA. Fig. 8. Interconnection of pores on the strut wall. Fig.9. Permeability and porosity of Porous scaffolds as a function of Naphthalene content. Fig. 10 (a-g). Effect of aging time on the apatite layer of 30 NA after immerse in SBF of 3, 5, 7, 14, 17, 21 and 28 days respectively. Fig.11 (a). EDAX image of 30 NA after 17 days immersion in SBF. Fig. 11(b) Raman spectroscopy study of immersed scaffold (40NA 14 days) 24
Fig.12. a, b, c, d, e represent FESEM of 0NA, 10NA, 20NA, 30NA, 40NAin 21days. Fig.13.Dissolution Behavior of Hydroxyapatite in SBF. Fig.14. Dissolution of porous HA as function of Immersion Time Fig. 15. Effect of Immersion time on pH of porous HA scaffolds Fig.16. Fluorescence microscopy images of mouse osteoblasts cultured in HA scaffolds after 24h incubation in cell culture medium. (a) Control, (b) 20NA, (c) 40NA. Fig.17. Cell viability index of HA scaffolds. Proliferation is shown relative to control.
Tables Table 1 Slurry composition for scaffold preparation. Table 2 d-spacing of HA at different calcined temperature (750-950 oC)
25
Figure1
Methyl Acrylamide (MAM)
DI water
Methylene Bisacrylamide (MBAM)
Mixing Mixed solution (Water: monomer: cross-linker= 45:4.8:0.2 in mass ratio) Mixing HA powder added to the solution Addition of Naphthalene powder Initiator (APS): Catalyst (TEMED) (1:1) Casting into Teflon mould
Gelation
Demoulding
Slow controlled drying
Removal of binders and organics at 650 0C o
Sintering at 1250 C for 2 h
Fig. 1.
Figure 2(a)
Fig. 2(a).
Figure 2(b)
Fig. 2(b).
Figure 2(c)
Fig. 2 (c).
Figure 3(a)
Fig. 3(a).
Figure 3(b)
Fig. 3 (b)
Figure 3(c)
Fig. 3(c)
Figure 3(d)
Fig. 3 (d).
Figure 3(e)
Fig. 3(e).
Figure 4
Fig. 4.
Figure 5
Fig. 5.
Figure 6(a)
Fig. 6 (a).
Figure 6(b)
Fig. 6 (b)
Figure 6(c)
Fig. 6 (c).
Figure 7(a-e)
a
b
c d
e
Fig 7. (a-e)
Figure 8
Fig. 8.
Figure 9
Fig. 9.
Figure 10 (a-g)
a
b
Particle Shape
C
d
Flower shape
e
f
Petal like apatite
g
Fig. 10 (a-g)
Figure 11 (a)
Fig.11 (a).
Figure 11(b)
Fig. 11(b).
Figure 12
a
b
. c
d
e
Fig.12 (a-e).
Figure 13
Fig. 13.
Figure 14
Fig. 14.
Figure 15
Fig. 15.
Figure 16
Fig. 16(a-c).
Figure 17
Fig. 17.
Table 1 Slurry composition for scaffold preparation Sample Name
HA
Naphthalene
(Vol.%)
(Vol. %)
0NA
54.0
-
10NA
48.0
10
20NA
43.0
20
30NA
37.0
30
40NA
32.0
40
50NA
27.0
50
Table 2 d-spacing of HA at different calcined temperature (750-950 oC).
d-value (oA)
Calcination temperature
750
2.819, 2.784, 2.726
800
2.818, 2.783, 2.724
850
2.812, 2.775, 2.718
900
2.812, 2.774, 2.716
950
2.809, 2.774, 2.716
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Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash, Ritwik Sarkar, Santanu Bhattacharyya
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PII: DOI: Reference:
S0272-8842(14)01791-X http://dx.doi.org/10.1016/j.ceramint.2014.11.053 CERI9498
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Ceramics International
Received date: Revised date: Accepted date:
15 February 2014 4 November 2014 7 November 2014
Cite this article as: Smruti Rekha Dash, Ritwik Sarkar, Santanu Bhattacharyya, Gel casting of hydroxyapatite with naphthalene as pore former, Ceramics International, http: //dx.doi.org/10.1016/j.ceramint.2014.11.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash, Ritwik Sarkar and Santanu Bhattacharyya Department of Ceramic Engineering National Institute of Technology, Rourkela, Odisha-769008, India
Corresponding author and First author: Smruti Rekha Dash, Ph.D. Research Scholar Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail of the corresponding author: [email protected] Telephone: Mob: +91 9778184420
Second author: Ritwik Sarkar, Associate Professor, Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail: [email protected] Telephone: +91 661-2462203
Third author: Santanu Bhattacharyya, Professor, Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha-769 008, India. E Mail: [email protected] Telephone: +91 661-2462205
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Gel casting of hydroxyapatite with naphthalene as pore former Smruti Rekha Dash*, Ritwik Sarkar, Santanu Bhattacharyya Department of Ceramic Engineering, National Institute of Technology, Rourkela 769008, Odisha, India
Abstract The present study discusses the processing and characterization of HA scaffolds which have been processed through a combination of gel casting and fugitive addition. The additional use of Napthalene (NA) as fugitive in the gel casting process provides an additional parameter for tailoring the porosity over and above that obtained via gel casting route. The motive behind the use of combination process was to prepare a porous hydroxyapatite (HA) scaffold with wide porosity range along with sufficient mechanical strength. In house prepared HA powder was used as the staring powder and Methyl acrylamide - N, N’-Methylene-bis-acrylamide (MAM-MBM) system was used for gel casting. Varying amount of Naphthalene (10-40vol% data) and HA solid loading (27-54 vol %) were used for scaffold preparation. The maximum solid loading that could be casted was 48 vol%. The sintered porous Hydroxyapatite-Naphthalene (HA-NA) scaffolds had been characterized for phase stability, microstructure, pore size and pore morphology. The highest compressive strength was 7.51MPa recorded for 22% porosity samples, and it was 0.75 MPa for 81% porosity samples. Scaffolds prepared with lower solid loading had good pore interconnectivity. In vitro bioactivity study of the scaffolds showed dissolution of scaffold up to 3 weeks in SBF solution followed by deposition of calcium phosphate as apatite layer. MTT assay results showed acceptable limit of cell growth and proliferation. Keywords: Hydroxyapatite; Gel casting; Naphthalene, compressive strength, in vitro aging, MTT assay * Corresponding author. Tel: +91-661-2462205. E-mail address: [email protected] (S. R. Dash).
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1. Introduction Porous Hydroxyapatite (HA) and other calcium phosphate based materials are finding wider applicability as scaffold material in the area of bio-implants and tissue engineering [1]. For effective and successful use of HA based porous scaffold, the scaffold material should have open pores with certain degree of interconnectivity for better bone bonding and osteo-integration. A large number of methods have been tried by different research groups for the preparation and characterization of HA based scaffold like polymeric sponge method [2], addition of fugitives [3-6], starch consolidation method [7], foaming [8,9], protein consolidation method [10], freeze casting [11-13], multipass extrusion [14], gel casting [15] etc. Polymeric sponge method or replication method is one of the oldest scaffold fabrications which are safe and easy to reproduce. First proposed by Schwarzwalder and coworkers [16] this method can produce a wide variety of scaffold microstructure depending on the pore architecture of polymer, solid loading and number of coating [17]. However, the basic microstructure of the scaffolds prepared by this method is strongly dependent on polymer microstructure and pore size and microstructure with similar pore architecture is difficult to reproduce. In the natural fugitive method, additives are mixed with the powder or dispersed in the slurry and shaped in green condition. The fugitive burn off during sintering leaving behind mostly open pores, although some claim that degree of interconnectivity of struts is poor. Starch consolidation is another method which has widespread acceptability for porous scaffold processing. Lyckfeldt et al [18] used potato starch granules in ceramic slurry and the slurry was consolidated by heating at 600C. During sintering, the starch granules burn off leaving behind pores. This route could produce ceramics with 30-70% porosity. Dhara et al [19] have used ovalbumin as foaming agent and have produced porous samples by 3
gelcasting. The degree of porosity and interconnectivity depended on water: albumin ratio, solid loading, sintering temperature etc. Research has also been conducted on the use of Bovine Serum Albumin (BSA) [20] for producing alumina based foams. The use of BSA could produce porous scaffold with 8-20% porosity along with good interconnectivity. Freeze Casting method [21] have also been used to prepare macroporous HA. Kohet al [22] have used unidirectional freeze casting method to prepare HA scaffold. Ghazanfari et.al [23] have studied the effect of nano-silica addition on the properties and microstructure of freeze casted HA-nSiO2 composite. Macroporous HA has also been prepared by multipass extrusion method [24]. The fabricated scaffold had unidirectional micro channel at the exterior part and a central canal in the interior. This type of microstructure was expected to provide better bone bonding and osteointegration. Gel casting [25-29] is another popular processing route which has been widely used for the processing of both porous and dense ceramics. The first report of gel casting was made by Omatete and Janney [30, 31].It described the gel casting process and the advantages like near net shape forming, machinability and ease production that gels casting provided over other processes [32]. The basic process involves in-situ polymerization and gelation of organic additives which are used along with dispersed slurry. The gelation of monomers lead to rapid setting of cast bodes. The organic monomers and polymers when removed on heating create a network of porous bodies with interconnectivity and the pores are mostly spherical [33]. The microstructure and porosity is dependent on the nature of additives, solid loading and diapersability of powder in the slurry [34, 35]. Although MAM and MBAM based monomers have been traditionally used, due to health concern, other more ecofriendly monomers like agarose, food quality gel are also being used. Roshanbinfar et al [36] have used gel casting route with agarose gel to prepare porous HA scaffold. Casting was done by shake gelcasting
4
method at 60 Hz frequency. Li at al [37] have used a combination of gel casting and rapid prototyping to prepare -TCP porous scaffold. Anil Asif at al [38] have prepared HA scaffold using food grade gel and gelling medium to prepare porous HA scaffold. Therefore, from the above discussion it is evident that gel casting is a very successful method for preparing porous scaffold. However, in all the gel casting method, the porosity and pore shape size is dependent on the type and amount of monomer. Therefore, in this study, an attempt has been made to modify the microstructure and the pore architecture by a combination of fugitive and gel casting. The additional use of Napthalene (NA) as fugitive has provided an additional parameter for controlling the porosity and pore connectivity. Therefore, the present work differs from the conventional gel casting or freeze casting method studied earlier on two counts. Firstly, in this study, combined use of monomer and fugitive has resulted in high degree of porosity along with variation in pore size which more than that achieved by gel casting alone. Secondly, most of the reports on gel casting have been made in the solid loading range of 40-70%. However, in the present study, the effect of low solid loading (25-54 volume %) has been investigated. The casted samples were sintered at 1250 oC and characterized for pore shape and size, strength, microstructure, invitro aging and cytotoxicity test.
2. Experimental Procedure All the samples in this study were prepared by Gel Casting of Hydroxyapatite (HA). The HA powder (Ca/P = 1.67) was synthesized in house from wet chemical route using diammonium hydrogen phosphate ((NH4)2HPO4) (Merck, India, GR grade 99.9% purity) and calcium nitrate (Ca(NO3)2·4H2O) (Merck, India, GR grade 99.9% purity). The mixing of the precursors and the precipitation of the hydroxides was carried out at room 5
temperature which varied from 25 OC to 30 OC depending on the season. The mixed precursors were homogenized by stirring for 2 hours at 900 rpm in a magnetic stirrer. 1:1 Ammonia solution was drop wise added from a burette (dropping speed 1ml/min) to effect the precipitation.
The added ammonia was in excess of the
stoichiometric amount in order to maintain the solution pH between 10.5- 11 and the stirring was continued for another 2 hours. Subsequently, the as formed precipitates were aged for 24 hours washed with deionized water to bring down the pH near 7-8. The washed precipitates were oven dried for 24 hours. The dried amorphous precipitates were ground to fine powder and calcined at 850 oC at a heating rate of 3o/min for crystallization of HA. The particle size of HA was between 0.5 μm and 5 μm and BET specific surface area was 14.9 m2/gm. Gel Casting slurries were prepared from calcined HA, Methyl Acrylamide (MAM) (C2H3CONH2), Merck, India (GR grade) and Methylene-Bis-Acrylamide (MBAM) [(C2H3CONH)2 CH2],Merck, India (GR grade), Ammonium Persulphate (NH4)2S2O8 (APS),Merck, India (GR grade)and N, N, N, N Tetra Methylethylene Diamine (TEMED),Merck, India (GR grade 99.9% purity). While MAM was the chain former, MBAM acted as branching former. On the other hand, APS and TEMED performed the initiator and catalyzer reactions respectively. For preparing stable dispersed HA slurry, 1.5% Darvan C (Vanderbilt, USA) was used as deflocculant. The different components required for slurry preparation were mixed in sequence and the mixed slurry was stirred for 6 hrs for dispersing and stabilizing the slurry. The rheology of the slurries were measured as a function of solid loading and the maximum solid loading (54 vol %) corresponding to the acceptable limit of pourability was taken as the maximum permissible solid loading for successful gel casting. For creating higher degree of porosity in HA over and above that created by Gel Casting itself, Naphthalene (NA) particles (-300+200μm) were added to stable HA slurry and the mixture of HA + NA
6
were stirred for 30-45minutesfor homogeneous mixing of NA particles in the slurry. Table-1 shows the slurry composition and NA content and Fig 1 gives detailed process flow diagram for scaffold preparation. The HA + NA mixed slurry was transferred to a petroleum jelly-coated Teflon mold (46mm
i.d. x 5mm height). Post mould filling, the
mould was gently tapped to remove entrapped air as well as for even distribution of slurry in the mould The casts were dried in water bath (40 OC) for 2 days following which the casts were demoulded and oven dried at 800C for 24 hrs. The samples were carefully heated till 650oC (heating rate 2o/min) and a holding time of 2 hours was provided at that temperature for facilitating easy removal of monomers, initiators, accelerators, other organics as well as NA. Finally the samples were heated to 12500C (holding time 2 hours) at a heating rate of 3o/min for final densification of samples. 3. Characterization The thermal decomposition behavior of as dried gel was studied by DSC-TG (Netzsch STA/409 C) till 1000 oC in an argon atmosphere. The heating rate was 10OC/ minute and -Al2O3was used as the reference material. The rheological behavior of HA slurry was measured using Rheometer (Rheolab QC, Anton Paar USA). Rheological tests were performed at 25 °C in the shear rate in the range 0.1-250 s-1, The phases in sintered samples were determined through XRD(Panalytical, Netherland) using Cu K radiation ( = 1.542 nm) fitted with Ni filter. The samples were scanned in the 2 range 25° to 60° in continuous mode with a step sizeof 0.02 degree/sec. The apparent porosity of the samples was measured through Archimedes principle using water as liquid medium. The linear shrinkage of samples was determined by measuring the diameter of samples before and after sintering. The microstructure of the scaffold was observed in a Field Emission Scanning Electron Microscopy (FESEM) (Nova Nano 450, FEI USA). The samples were sputter coated with gold for 3 minutes in Argon atmosphere (Quorum Technology, 7
Q150R ES). EDAX (Energy Dispersive X-ray analysis) (Bruker USA, Model 127 eV) analysis was carried out on SBF aged samples for identifying the apatite deposits. Some samples were also tested through Raman Spectroscopy (LabRAM HR (UV) spectrometer) for confirmation of apatite layer. Thelasersourcewas514nmforminimizingthefluorescence effect of the sample. The porosity and pore size distribution was determined by Mercury
Intrusion Porosimeter (Quantachrome/PM-33-13). The compressive strength of the porous materials was determined on cylindrical sample (15 mm
x 6 mm) in Universal
Testing Machine (H10KS Tinius Olsen, UK) following ASTM standard test method for compressive strength measurement (ASTM C773-88 (2011) [39] . The samples were tested with a load cell was 10 KN, cross head speed 0.5 mm/minute and Compressive Strength (CCS) was obtained using the formula: CCS=P/A Where, P = Load in KN and A = Cross sectional area of the cylindrical sample. The permeability of the samples was determined from liquid flow measurement using Darcy’s equation [40] Q= (KpPA)/L Where,Q=volume flow rate of liquid through the sample, Kp= specific permeability coefficient, P= pressure drop across the sample, L=flow length or thickness of the test sample, A=area of cross-section of the sample, = fluid viscosity. The permeability coefficient Kp depends on the nature of the fluid porosity and pore size as well as pore connectivity combination of porous material. Higher Kp value indicate ease of flow through the material which implies that pore interconnectivity is of high degree. In vitro bioactivity of sintered porous body was tested in simulated body fluid (SBF) by immersing specimen in SBF at 37 ± 1°C for different time periods ranging from 3 days to 8
7 weeks in a BOD incubator. The SBF was prepared in the laboratory as per Tas’s specification [41]. The SBF solution was replaced with fresh SBF solution at regular intervals to compensate for the decrease in ion concentration of SBF solution as a result of apatite layer formation on the specimens. The aged samples after its removal from SBF solution, were thoroughly washed with deionized water, and dried at room temperature. Formation and growth of apatite layer was studied by observing FESEM image of SBF aged surfaces. Selected areas of aged samples were also analyzed by EDAX and Raman spectroscopy for compositional analysis of the deposits. The phosphorus and calcium ion concentration in SBF solution before and after immersion for different time periods were studied in an Atomic Absorption Spectrophotometer (Perkin Elmer AS 800). The cell viability and cell proliferation activity was studied through MTT assay using L929 mouse osteoblast [42-44]. The formazon crystals formed were dissolved by adding dimethyl sulfoxide and the absorbance was measured at 540nm using spectrophotometer (Systronics double beam spectrophotometer 2203).
4. Results& Discussion Fig.2 (a) shows DSC-TG diagram of as dried gel. The DSC curve has a number of stages. At 100°C, it has an endothermic peak associated with weight loss of about 5%on account of removal of adsorbed water from gel. The exothermic peak at 230 OC is due to the oxidative decomposition of polyacrylamides which starts around 2200C. Between 210-2300C, the endothermic peak appears due to an imidic reaction which releases ammonia gas. This reaction completes by 3750C. Following this, decomposition of amide groups and breakdown of polymer structure takes place up to 6000C [45, 46]. By 6000C, the weight loss of gel was nearly complete. At still higher temperature (between 650 °C and 850 °C), a broad peak can be observed without any significant weight loss. The occurrence of the broad peaks can be correlated with the crystallization of HA from its 9
amorphous state first in its disordered structure and subsequently its progressive transformation to ordered HA. This fact was verified by a series of XRD plots for powder calcined in the temperature range of 7500C to 9500C (Fig 2(b) and 2 (c)). It is seen that at the first crystallization temperature (750oC), the d-spacing of HA is more than of JCPDS value (Table- 2) and with increase in temperature the d-spacing shifts to ideal value for HA (i.e. closer to ideal HA diffraction peak). It is also seen that at 8500C, the d-spacing matches with the JCPDS value (File No 84-1998). Further, the exothermic peak at 9500C corresponds to another crystallization peak of HA but the d- spacing at this temperature differs from JCPDS value (84-1998). On the basis of the above findings, the calcination temperature for HA was fixed at 850 °C. Fig. 3 (a) and 3 (b) show the effect solid loading on the shear stress-shear rate behavior and viscosity-shear rate respectively of HA slurries. The shear stress-shear rate plots have been obtained in both upturn (increasing shear rate) and downturn (decreasing shear rate) mode. It was observed that slurries with solid loading less than 30 vol% were very fluid and showed negative shear stress value. Only slurries of 30 vol% and higher solid loading showed characteristics shear thinning behavior and shear stress increased with shear rate rapidly during at low shear rate and more gradually at higher shear rate with typical hysteresis between shear stress and shear rate. Considering the fact that the slurries would show power
law behavior, the shear thinning constant (n) was calculated from the slope of ln() vs ln( J ) plot. It was observed that all the slurries showed shear thinning behavior with n value of 0.25 to 0.40. Fig. 3 (c) shows the representative shear stress-shear rate graph for 54 vol% solid loading. The difference in shear stress in the upturn and downturn mode at zero shear shear rate is gel or an index of structural buildup. Fig. 3 (d) shows the variation of gel for all the composition between 30 and 57vol% which shows that the index of structural buildup increases with solid loading. This implies that at higher solid loading, the samples will have a lower casting time and the cast may have low porosity and it may be difficult to cast a sluury with very high gel. Fig 3. (e) shows the variation of steady state viscosity (measured at 200 s-1) with solid loading for all the compositions. It is seen that the steady state viscosity is 7 Pa.s for 20 vol% solid loading and it is 12 Pa.s for 27 vol%. The low viscosity of these two slurries resulted in poor cast properties and samples cracked after drying. However, the viscosity is 22 Pa.s for 32 vol% and it could produce crack free samples. The viscosity increased further with increase in solid loading and at 48 vol% solid loading the maximum in viscosity (220 Pa.s) corresponding to “pourable” and castable slurry was observed. Beyond that, the slurries were not pourable. Therefore, the maximum solid loading in this investigation was restricted to 48 vol%. 10
Figure 4and Figure 5 show the XRD pattern of sintered HA and sintered HA-NA respectively. It is observed that there is no change in peak position of HA till 1250oC and also naphthalene has no effect on phase (Fig. 5)implying that HA phase is stable till 1250oC.The d-value of the three characteristics peaks of HA (d=2.812Ao, 2.776Ao, 2.718Ao) matches with the standard d value of HA (JCPDS 84-1998). But at 13500C the hydroxyapatite decomposes to give -TCP and Calcium oxide (CaO) as shown in Figure 4. Fig. 6 (a) and 6 (b) shows the effect of addition of NA on porosity and strength of the gel casted scaffolds, respectively. It was found that with the increase in the amount of NA, the porosity increased and simultaneously the bulk density and compressive strength decreased. Without NA addition, the porosity was 22 ±3 % and corresponding strength was 7.51±0.1MPa. As already explained, NA was added to create higher porosity sample. At 40wt. %NA addition, the porosity was nearly 75 % and strength reduced to about 1MPa which is close to the literature value [47-49] for this level of porosity. At still higher NA content (50% & 60%NA), the sample strength could not be measured because high porosity resulted in mechanically weak and fragile samples. Therefore, the strength limiting factor restricted the maximum percentage of NA to 40%. Compressive strength values for samples were in accordance with the literature as well as close to the reported compressive strength of human cancellous bone ranges between (2-12 MPa). [50-52]. A plot of compressive strength ( ) against porosity (P) shows that strength ( ) exponentially decreases with increase in volume fraction porosity and follows the relation.
V
V 0 exp(bP )
11
Where, b is constant and 0 is the compressive strength for zero porosity samples. A plot of ln ( ) vs volume fraction porosity (P) [Fig. 6(c)] yields a straight line with a negative slope. The slope gives the value of ‘b’ and for this study it was found to be 0.04.The negative slope indicates that as porosity increases, loading carrying capacity of the scaffolds decreases exponentially due to lesser interconnection among solid materials. In all the gel cast sintered samples, porosity and pore size depended on naphthalene content. Fig. 7(a-e) shows the pore size distribution of porous HA ceramics. Fig. 7 (a) represents gel casted sample without addition of naphthalene which shows pore within a narrow size and mostly consisting of micro pores (<50μm). On the other hand, Fig. 7 (be) show a higher population of macro pores (100±20 μm to 200±20μm) and less micro pores (<50μm). At higher naphthalene content, not only the porosity increases, but the agglomeration of naphthalene also causes an increase in the pore size which could not be eliminated in the pore removal process during sintering It is also seen that at higher naphthalene addition, not only the pore size but also the pore population changes to higher value. Thus by using the combination of NA and gel casting wide variety of porous structure could be produced. Fig. 8 shows the microstructure of a sintered sample (30NA). It is seen from the microstructure that the sintered scaffold contains macro pores of 20-100μm. The encircled area in the image (Fig. 8) shows that the pore on the strut walls are connected which indicate that the scaffold has interconnectivity. The permeability of scaffold materials with different porosity level and pore size is shown in Fig.9. A comparison of 30NA sample with that of 40NA show that porosity and permeability has 1:1 correspondence, i.e. 10% increase in the porosity resulted in an equal increase in permeability value, assuming interconnected pores. The highest permeability of 12
30.2 x 10 -11m2 is obtained for 40NA samples. However, in the present investigation, even the minimum permeability is much higher than the reported value of 3.9x 10-11 m2 [46]. Hence the combination of gel casting with NA addition as additional pore forming agent results in a porous scaffold with significant interconnectivity and high permeability.
4.1 In vitro bioactivity and the formation of apatite layer: (a) Effect of aging days on apatite layer formation: Figure10 (a-g) shows the effect of aging time on the apatite layer of 30 NA after immerse in SBF of 3, 5, 7, 14, 17, 21 and 28 days respectively. The images show that after 3 days of aging a few white granular spots are seen which correspond to the apatite particle formation (Fig 10a &.10b). In order to confirm that the deposited particles correspond to apatite, EDAX was carried out on the spots as shown in Figure 11(a). Although HA and apatite are composed of similar elements, the presence of carbon on the spots prove that the deposited layers are of apatite. Some other aged samples (40 NA) were also characterized by Raman spectroscopy. The Raman spectra are shown in Figure 11(b). The spectrum shows characteristic peak at 960 cm-1 assigned to symmetric stretching mode (1) of the tetrahedral (PO4)3- group of (P O) bond and the presence of this peak confirms the deposition of apatite layer and the peak position matches with the reported value for apatite [53]. With increase in aging period (7 days and 14 days) the area covered by the apatite layer increases (Fig. 10c and 10.d). Further, the shape of the deposited apatite layer changes with aging time (Fig.10 e-g). At lower aging time, it has particle like morphology, at intermediate time it has flower morphology and at the highest aging time it has petal like appearance. It is also observed from the above Figures that, the surface coverage also increases with increase in ageing time. Thus, not only the 13
apatite morphology changes with aging time but also the area covered by the apatite layer changes with aging time.
(b) Effect of porosity on apatite layer formation: Fig. 12 shows the FESEM image of HA scaffolds after soaking in SBF for 21days. The microstructure shows apatite formation on the HA surface. The apatite could be recognized as white precipitates (encircle area) on sample surface. Fig.12a represents the microstructure of only gel casted scaffold without any naphthalene. It is seen that there are a few spots which correspond to apatite deposition. Fig.12 (b-e) represents the microstructure with progressively higher amount of naphthalene and consequently higher porosity. From the micrographs, it could be observed that higher porosity sample (40NA) had more apatite layer than those corresponding to lower porosity (10NA). The higher deposition rate of apatite in higher porosity samples can be related to faster dissolution of ion and its migration through interconnected porous network of higher surface area [5356]. Beyond 21 days no significant change in surface area coverage could be observed and all samples were almost fully covered with apatite deposits.
4.2 Dissolution Behaviour of Hydroxyaptite in SBF The dissolution behaviour of HA was measured by estimating Ca+2 concentration in SBF solution.The variation of Ca2+ concentration in SBF solution with dissolution time containing the investigated porous HA is shown in Fig.13. It is seen that Ca2+ concentration in SBFincreases slowly in the first week of dissolution and after 1 week the concentration increases rapidly till 3 weeks (in some case upto 4 weeks) time. The increase of Ca2+ in the SBF solution confirms the dissolution of HA at pH 7.4 [47]. The
14
degree of dissolution of HA in SBF is depend on impregnation by SBF and higher porosity sample, higher volume of impregnation is achiened.Similar observations were also noted by Ereiba et al [54]. Rapid response of the porous bioactive surfaces could be correlated with the dissolution of initially formed phosphate and its reprecipitation carbonated apatite from the supersaturated solution [55]. Thus it could be concluded that the increases in Ca+2 concentrations in SBF till 21 days of ageing correspond to its dissolution in SBF and the decrease in Ca+2concentration after 21 days till 2 days correspond to precipitation of apatite from solution. On the other hand, the weight loss increased till 21-28 days and then gradually decreased due to the deposition of CaP compound from SBF on the scaffold surface (Fig. 14). Therefore, the dissolution is dominant till 28 days and apatite growth process is dominant after that period. Fig.15. shows the effect of ageing time on the pH change of SBF solution. The pH of SBF increases during the initial period of ageing i.e till 21days which can be correlated with dissolution of HA in SBF. The dissolution leads to increase the Ca+2 concentrations. Due to the dissolution of Ca+2, the pH increase. Thereafter, the pH decreases as the released Ca+2 reacts with HPO42- to produce CaP layer as per the reaction given below [56]. .
5Ca2 3HPO42 - + OH- o Ca5 (PO4 )3 OH + H 2O
It is anticipated that Ca2+ and HPO4
2-
(1)
ions in the solution form apatite nuclei which
deposit either on the surface of hydroxyapatite or in the SBF solution. Those nucleated on the surface of hydroxyapatite further grew into an apatite coating layer, while those
15
nucleated in the solution form into apatite precipitates [57]. During the growth of the apatite coating or precipitates, the pH of the solution decreased due to the consumption of OH- in the solution according to reaction (1).
4.3 Cytotoxicity Behaviour The cytotoxicity/cell viability of the hydroxyapatite scaffolds was assessed by the MTT cytotoxicity assay as shown in Fig. 16.The cell viability and cell proliferation activity was studied through MTT 3(4, 5-dimethylthiazol-2yl)-2-5diphenyltetrazolium bromide) (SIGMA, USA) assay using L929 mouse osteoblast after 72 hours incubation period at 37 oC in 5% CO2. The sterile scaffold along with control was placed in UV sterilized 96 well plates (Nunc, Germany) in 200 μl DMEM (Dulbecco’s modified Eagle medium) at a concentration of 10 4 MG 63 cells per well. The dehydrogenase enzymes of the active cell reduced the yellow tetrazolium MTT into intracellular purple formazan. The concentration of formazon produced was proportional to the number of viable cells present in the media [42, 44, 58]. The formazon crystals were dissolved by adding dimethyl sulfoxide and the absorbance was measured at 540nm. The resultsshow that the cells were able
to grow and proliferate favoourably. The results also indicate the porosity provides favourable sites for cell proliferartion. However, in the present study ahigher porosity sample (40NA) exhibited only marginally higher cell viability than less porous sample (20 NA) (Fig. 17). Thus, from the above observation, it can be concluded that both low and porosity scaffold prepared in this study permit proper cell proliferation and are expected to show acceptable cytocompatibility along with cell viability properties.
5. Conclusions The present work dealt with the processing and characterization of porous HA scaffold. The porous hydroxyapatite scaffold was prepared by incorporation of volatile material (Naphthalene) as a pore forming agent in gel casted samples. By modifying the solid loading, naphthalene content, the porosity and compressive strength could be altered over a wide range. Porous HA scaffold with 60% porosity had open and interconnected pores. Compressive strength of 4.1 MPa was recorded for 50% porous scaffold (30NA sample) and compressive strength of 2.3 MPa was observed for 60% porosity (40NA sample). The processed scaffolds had wide pore size ranging from 10–150μm. These pores were also found to be interconnected in the macro porous structure. Invitro aging confirmed the deposition of apatite layer on HA surface and the deposition rate varied 16
with sample porosity. MTT results indicate that the processed HA scaffold had appreciable cytocompatibility and therefore may have a potential for use as scaffold material.
Acknowledgements This research was financially supported by National Institute of Technology,
Rourkela,
India.
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Fig.1. Flowchart of processing of Gel casted porous HA scaffolds using Naphthalene. Fig. 2(a) DSC-TG of as dried gel. Fig. 2(b) XRD plots of precipitated HA powder calcined in the temperature range of 750 0C to 950 0C. Fig. 2(c) XRD plots (triplet) for powder calcined in the temperature range of 750 0C to 950 0
C showing the peak shifting of HA.
Fig. 3(a) Shear stress versus shear rate plot as a function of HA solid loading. Fig. 3(b) Viscosity-shear rate plot as a function of HA solid loading. Fig.3(c) Shear stress – shear rate plot for 54 vol% solid loading. Fig. 3(d) Variation of gel with solid loading. Fig. 3(e) Variation of steady state viscosity as a function of HA solid loading. Fig.4 XRD pattern of sintered HA at different temperature (1000-1300 OC). Fig. 5 XRD pattern of sintered HA and HA-NA scaffold. Fig.6 (a) Variation of porosity as a function of naphthalene content. Fig. 6(b) Variation of compressive strength as a function of porosity. Fig.6 (c) Semi-logarithmic plot of compressive strength and porosity. Fig 7. (a-e) Pore size distribution of sintered HA scaffolds prepared by gel casting (a) 0 NA, (c) 10 NA, (c) 20 NA, (d) 30 NA, (e) 40NA. Fig. 8. Interconnection of pores on the strut wall. Fig.9. Permeability and porosity of Porous scaffolds as a function of Naphthalene content. Fig. 10 (a-g). Effect of aging time on the apatite layer of 30 NA after immerse in SBF of 3, 5, 7, 14, 17, 21 and 28 days respectively. Fig.11 (a). EDAX image of 30 NA after 17 days immersion in SBF. Fig. 11(b) Raman spectroscopy study of immersed scaffold (40NA 14 days) 24
Fig.12. a, b, c, d, e represent FESEM of 0NA, 10NA, 20NA, 30NA, 40NAin 21days. Fig.13.Dissolution Behavior of Hydroxyapatite in SBF. Fig.14. Dissolution of porous HA as function of Immersion Time Fig. 15. Effect of Immersion time on pH of porous HA scaffolds Fig.16. Fluorescence microscopy images of mouse osteoblasts cultured in HA scaffolds after 24h incubation in cell culture medium. (a) Control, (b) 20NA, (c) 40NA. Fig.17. Cell viability index of HA scaffolds. Proliferation is shown relative to control.
Tables Table 1 Slurry composition for scaffold preparation. Table 2 d-spacing of HA at different calcined temperature (750-950 oC)
25
Figure1
Methyl Acrylamide (MAM)
DI water
Methylene Bisacrylamide (MBAM)
Mixing Mixed solution (Water: monomer: cross-linker= 45:4.8:0.2 in mass ratio) Mixing HA powder added to the solution Addition of Naphthalene powder Initiator (APS): Catalyst (TEMED) (1:1) Casting into Teflon mould
Gelation
Demoulding
Slow controlled drying
Removal of binders and organics at 650 0C o
Sintering at 1250 C for 2 h
Fig. 1.
Figure 2(a)
Fig. 2(a).
Figure 2(b)
Fig. 2(b).
Figure 2(c)
Fig. 2 (c).
Figure 3(a)
Fig. 3(a).
Figure 3(b)
Fig. 3 (b)
Figure 3(c)
Fig. 3(c)
Figure 3(d)
Fig. 3 (d).
Figure 3(e)
Fig. 3(e).
Figure 4
Fig. 4.
Figure 5
Fig. 5.
Figure 6(a)
Fig. 6 (a).
Figure 6(b)
Fig. 6 (b)
Figure 6(c)
Fig. 6 (c).
Figure 7(a-e)
a
b
c d
e
Fig 7. (a-e)
Figure 8
Fig. 8.
Figure 9
Fig. 9.
Figure 10 (a-g)
a
b
Particle Shape
C
d
Flower shape
e
f
Petal like apatite
g
Fig. 10 (a-g)
Figure 11 (a)
Fig.11 (a).
Figure 11(b)
Fig. 11(b).
Figure 12
a
b
. c
d
e
Fig.12 (a-e).
Figure 13
Fig. 13.
Figure 14
Fig. 14.
Figure 15
Fig. 15.
Figure 16
Fig. 16(a-c).
Figure 17
Fig. 17.
Table 1 Slurry composition for scaffold preparation Sample Name
HA
Naphthalene
(Vol.%)
(Vol. %)
0NA
54.0
-
10NA
48.0
10
20NA
43.0
20
30NA
37.0
30
40NA
32.0
40
50NA
27.0
50
Table 2 d-spacing of HA at different calcined temperature (750-950 oC).
d-value (oA)
Calcination temperature
750
2.819, 2.784, 2.726
800
2.818, 2.783, 2.724
850
2.812, 2.775, 2.718
900
2.812, 2.774, 2.716
950
2.809, 2.774, 2.716
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