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Pore size and pore volume effects on alumina and TCP ceramic scaffolds
Materials Science and Engineering C 23 (2003) 479 – 486 www.elsevier.com/locate/msec
Pore size and pore volume effects on alumina and TCP ceramic scaffolds Susmita Bose a, Jens Darsell a, Martha Kintner b, Howard Hosick b, Amit Bandyopadhyay a,* a
School of Mechanical and Materials Engineering, Washington State University, P.O. Box 642920, Pullman, WA 99164 2920, USA School of Biological Sciences and Molecular Biosciences, Washington State University, P.O. Box 644236, Pullman, WA 99164 4236, USA
b
Accepted 18 July 2002
Abstract Controlled porosity alumina and h-tricalcium phosphate ceramic scaffolds with pore sizes in the range of 300 – 500 Am and pore volumes in the range of 25 – 45% were processed using the indirect fused deposition process. Samples having different pore sizes with constant volume fraction porosity and different volume fractions porosity with a constant pore size were fabricated to understand the influence of porosity parameters on mechanical and biological properties. In vitro cell proliferation studies were carried out with OPC1 human osteoblast cell line for 28 days with different scaffolds. Variation in pore size did not show any conclusive differences, but samples with higher volume fraction porosity showed some evidence of increased cell growth. Volume fraction porosity also showed a stronger influence on the mechanical properties under uniaxial compression loading. Compression strength dropped significantly for samples with higher volume fraction porosity, but changed marginally when only the pore size was varied. D 2002 Elsevier Science B.V. All rights reserved.
1. Introduction Rapid prototyping (RP) or solid freeform fabrication (SFF) has evolved over the past decade as a potential manufacturing approach to making functional structures, directly from CAD files, having controlled shape as well as internal architecture. This is an approach in which threedimensional objects are fabricated on a fixtureless platform from a CAD file without any part-specific tools or dies, using polymeric, ceramic or metallic materials [1]. RP is a layered manufacturing approach in which a CAD file is sliced in the Z-direction in a virtual environment and then each slice is built one on top of the other. The first layer is built on a fixtureless platform and then the platform indexes down. The next layer is built on top of the previous layer. This process continues until the fabrication of the part is complete. Several commercial RP techniques are available including stereolithography (SLA), selective laser sintering (SLS) and fused deposition modeling (FDM). Most of these processes are used to build parts using polymers, but some are capable of fabricating metals and ceramics as well. Porous ceramics have been studied as scaffold materials for small-scale bone defects as well as spinal fusion appli*
Corresponding author. Tel.: +1-509-335-8654; fax: +1-509-335-4662. E-mail address: [email protected] (A. Bandyopadhyay).
cations. Different research groups have used numerous processing routes to fabricate porous ceramic structures. White et al. [2,3] have reported replamineform process to fabricate porous ceramic implants that duplicate the microstructure of corals. Hulbert et al. [4,5] reported the fabrication of porous alumina ceramics using a pore former or foaming agent that evolves gases during sintering at elevated temperatures leaving pores behind. Klein et al. [6] made porous hydroxyapatite (HAp) ceramic blocks using HAp slurry mixed with foaming agent followed by sintering at elevated temperature. All of these porous structures had randomly arranged pores with a wide variety of sizes and volume fraction porosity. In recent years, the RP techniques have also been used to fabricate porous structures for biomedical applications. The SLS [7], 3-D printing [8,9], stereolithography [10,11] and FDM [12 – 14] processes have been used to fabricate porous structures for scaffold-related applications. RP is used in two different ways, the direct and the indirect, to produce these porous structures. In the direct route, the desired part is directly formed from the CAD file to the final form. In the indirect route, first, a negative of the desired structure is made with polymer, which acts as a mold, and then the actual part is cast from the polymer mold. The direct route is the most desirable one, though it may require the development of feedstock materials and hardware modifications. The indirect approach can be used
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-4931(02)00129-7
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with any commercial hardware, as all the machines are capable of making polymer prototypes. Moreover, this route uses a traditional lost-mold ceramic processing approach in conjunction with slurry or gel casting. In the present work, fused deposition modeling (FDMk), one of the commercially available RP processes, was used to make porous alumina and tricalcium phosphate (TCP) ceramic structures using the indirect route. These porous structures have three-dimensionally interconnected porosity. The main advantage of this process is the simultaneous design of both the micro- and the macrostructure of the part using the CAD program. During mold making, different porosity parameters of the polymeric molds can be tailored by optimizing the diameter of the extruded polymeric filament (called ‘‘road-width’’), the distance between two polymer roads (called ‘‘road-gap’’), and the thickness of each layer (called ‘‘slice thickness’’) during the layered manufacturing process. Moreover, by changing the deposition angles of polymeric roads for each layer, the pores can be oriented layer by layer. The present work on mechanical and biological responses was carried out with porous ceramic structures having constant pore sizes but different pore volumes, and constant pore volume with different pore sizes, using alumina and h-tricalcium phosphate (h-TCP) ceramics. Controlled porosity cylindrical structures were
tested under uniaxial compressive loading to understand the influence of porosity parameters on the mechanical properties. In vitro assessment with the OPC1 human osteoblast cell line was conducted to evaluate the influence of porosity parameters on cell proliferation.
2. Processing of porous ceramics Processing of porous ceramics has three steps including (a) mold microstructure design, (b) ceramic slurry development and (c) binder burnout and sintering. Fig. 1 shows the schematic of the process flow chart for porous ceramic structures. The molds were made using a Stratasys FDM 1650 machine with commercially available ICW06 thermoplastic polymers. Polymeric molds were made with different road-gap and road-width to have different pore size and volume fraction porosity in the final structures. Slice thickness was varied between 0.25 and 0.35 mm, road-gaps were varied between 0.5 and 1 mm and road-widths were varied between 0.4 and 0.625 mm. Mold diameter was kept constant at 1.725 cm. Water-based ceramic slurries were made with high-purity 10D (surface area 10 m2/gm) alumina powder doped with 500 ppm MgO (Baikowski International, NC) and food-grade TCP powders (Monsanto, CA) for alumina and
Fig. 1. The schematic of the process flow chart for porous ceramic structures. (a) Polymer filament. (b) Fused deposition modeling process. (c) Polymer mold. (d) Slurry-infiltrated mold. (e) Porous ceramic structure with a porosity gradient from center to the outside.
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TCP scaffolds, respectively. 1-Butanol was used as an antifoaming agent for both powders. Darvan-821A (RT Vanderbilt, CT) and D-3021 (Rohm and Haas, PA) were used as dispersant for alumina and TCP slurries, respectively. Binder B-1001 (Rohm and Haas) was used as binder for both the slurries. Ceramic powder, antifoaming agent and dispersant were added to water and then ball milled for 5 –16 h in a polyethylene bottle. The required amount of binder was added to the mixture just before the infiltration. A 1.5 wt.% sample of Darvan 821A was found optimum for alumina powders, and 3.5 wt.% D-3021 was found optimum for TCP powders. Slurry compositions were optimized using a Brookfield viscometer. For same solids loading, TCP slurry was always more viscous than alumina and TCP slurry required longer ball-milling time as well. The slurry was infiltrated into the porous polymeric molds and the green structures were dried for 2 days. The structures were then subjected to a binder burnout and sintering cycle. During the binder burnout heating cycle, the mold polymer leaves the part, creating the pores. Issues related to the development of binder removal and sintering cycles have already been discussed in Refs. [15,16]. By changing the distance between the polymer roads and their width, different types of porosity can be created. For in vitro testing, controlled porosity samples made of alumina and TCP of 2-mm thickness and 1.2 cm diameter were processed. For the first set of samples, the volume fraction porosity was changed from 29% to 35% to 44% keeping the pore size constant at 300 Am. For the second set of samples, pore size was varied from 300 to 380 to 480 Am, keeping the volume fraction porosity constant at 44%. For uniaxial compression testing, cylindrical samples of 2 cm long and 1.2 cm diameter were processed having the same porosity parameters as in the case of in vitro samples.
3. Results 3.1. Physical structure evaluation Porous structures were characterized for their physical, mechanical and biological properties to understand the
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influence of porosity parameters. Shrinkage was measured as a part of physical property measurement to understand the sintering behavior of these ceramics. Nonuniform shrinkage causes cracking and warping in the final product. Moreover, controlled shrinkage information can be utilized in the design stage to make necessary design modifications. Shrinkage during sintering is dependent on the solids loading of the ceramic slurry and final sinter density. Shrinkage also varies as a function of volume fraction porosity in these samples in which higher volume fraction porosity showed a lower amount of shrinkage [15]. For the porous alumina samples, linear shrinkage varied from 23% to 25%, while shrinkage of TCP varied between 17% and 19%. The shrinkage variations were primarily due to the variations in pore volume, as the solids loading remained constant. Sintered density for porous alumina samples was between 3.78 and 3.80 g/cm3 (>95% theoretical density). For TCP, the density varied between 2.90 and 2.95 g/cm3 (f90% theoretical density). Fig. 2 shows an optical micrograph of 2D porous samples with different volume fraction porosity where pore size was kept constant at 300 Am. Using this approach, structures with porosity gradient, i.e., varying porosity from one end to the other end can also be processed. 3.2. Mechanical property evaluation Strength degradation is a serious concern in porous ceramics. As the total volume fraction porosity increases, the failure strength decreases. Cylindrical porous samples of 12 mm diameter and 20 mm long (L/D=1.6) were used for this study. Uniaxial compression tests were performed using an Instron 1331 servo hydraulic machine under stroke control mode at a stroke rate 0.5 mm/min. At least 20 samples of each porosity level were tested. Fig. 3 shows the influence of pore volume and pore size on the mechanical properties of 3-D honeycomb alumina samples. Fig. 4 shows the same for the TCP ceramics. It is clear from both Figs. 3 and 4, that with increasing volume fraction porosity, compressive strength decreases. Compressive strengths of alumina ceramics are about two orders of magnitude higher
Fig. 2. An optical micrograph of 2-D porous samples having different volume fraction porosity where pore size is kept constant at 300 Am.
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Fig. 3. The influence of pore volume and pore size on the mechanical properties of 3-D honeycomb alumina samples.
than those of the TCP ceramics. Similar influences of volume fraction porosity on compressive strength have also been reported with other materials having similar porosity [17]. As expected, failure planes in all of these samples were parallel to the loading axis. The influence of pore size on the strength degradation is not clear from these figures. In fact, for a constant pore volume, pore size should not have any effect on the strength degradation behavior. The relative variation due to pore size effect may be due to the height differences of the pores for the change in slice thickness in the polymer molds, which produces some minor influence
of shape. Any clear trend due the pore size alone could not be delineated from these experimental data. 3.3. In vitro analysis In vitro tests were performed on porous scaffolds of alumina and TCP ceramics using OPC1 cells. The OPC1 cell line is a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone
Fig. 4. The influence of pore volume and pore size on the mechanical properties of 3-D honeycomb TCP ceramics.
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phenotype from passage 10 to at least passage 30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ ml).Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell system to evaluate bone development and cell/biomaterial interactions, and may be useful to screen for putative bone-differentiating factors [18].The OPC1 cells were cultured in a standard medium made of McCoy’s 5A (with l-glutamine, without phenol red and sodium bicarbonate) (Sigma, St. Louis, MO, USA), supplemented with 10% fetal bovine serum, 2.2 g/l sodium bicarbonate, 0.1 g/l penicillin and 0.1 g/l streptomycin. The selection of a nutrient medium is strongly influenced by type of cell, type of culture and degree of necessary chemical definition. Cells’ splitting was done 3 days before the cell culture. During the splitting process, the same medium was used supplemented with 10% bovine calf serum. Cultures were incubated at 37 jC in a humidified 5% CO2 atmosphere. A total of 100,000 cells per matrix were seeded onto 15 matrices in a 10-cm tissue culture dish. Sufficient medium was present (15 ml) to completely cover the matrices. Three matrices were removed from random regions of the culture vessel on days 2, 7, 14, 21 and 28 after seeding. These were then evaluated by MTT assay. This
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assay quantitates the ability of mitochondrial dehydrogenases to metabolize 3-[4,5-2-yl]-2,5-diphenyl-tetrazolium bromide to an insoluble formazan [19]. The amount of formazan is directly proportional to the total number of living cells. This enzyme assay produces a colored product, which is quantitated in a microplate reader at a wavelength of 570 nm. During the MTT assay, after removal of the growth media, a 1-mg/ml solution of MTT in phenol redfree DME was added to the scaffold in a 1-cm culture vessel (0.5 ml) or to the cells on the bottom of a 1-cm culture vessel (0.3 ml).The reaction was allowed to proceed for 1 h at 37 jC, at which time micrographs were taken on a WILD stereomicroscope to evaluate coverage of the scaffold by the cells. The cells were returned to 37 jC for 3 h more, and then the MTT solution was removed. Formazan crystals were solubilized in isopropanol and the optical density at 570 nm was determined on a Cambridge Technology Series 700 microplate reader. Fig. 5a and b shows the effect of different volume fraction porosity on OPC1 cell growth for alumina and TCP scaffolds in which the pore size was kept constant at 300 Am. Both alumina and TCP ceramics showed similar behavior for volume fraction porosity samples. The initial part of the cell growth for up to 14 days on these scaffolds
Fig. 5. (a) and (b) shows the effect of volume fraction porosity on OPC1 cell growth for alumina and TCP scaffolds. A570 in Y-axis shows numbers that was the read out from a microplate reader at 570 nm wavelength. Higher number indicates the presence of increasing number of living cells.
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Fig. 6. (a) and (b) shows the effects of pore size on the (a) alumina and (b) TCP scaffolds having a constant pore volume of 44%.
Fig. 7. (a) and (b) shows the SEM images of alumina and TCP matrices showing the cell – matrix interaction.
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was relatively fast for 29 and 35 vol.% samples followed by slower growth for the next 14 days. For the 44 vol.% samples of both alumina and TCP, there was a consistent growth rate throughout the 28 days of the test period. For the 29 and 35 vol.% alumina samples, most of the cell growth took place during the first 14 days. A relatively slower growth was observed for the third and fourth week of the study. In general, for both the cases, 44 vol.% samples showed highest cell growth up to 28 days, though the cell growth behavior was quite similar up to the first 14 days. From all the batches, samples were taken out for stereomicroscopy to see the cell attachment on the scaffolds. It was found that within the first 14 days, both 29 and 35 vol.% samples, cells had grown to cover almost all the surfaces. For the case of 44 vol.% porosity samples, having higher volume fraction porosity, and higher surface area per unit volume, cells took longer time to fully cover the scaffold surface. Having the same initial cell density for all the scaffolds, the higher cell growth behavior can be explained due to the higher surface area for the 44 vol.% scaffolds. Fig. 6a and b shows the effects of pore size on the alumina and TCP scaffolds having a constant pore volume of 44%. Both 29 and 44 vol.% samples were studied and pore sizes were varied between 300 and 480 Am. All the samples showed similar cell growth behavior and there was no conclusive evidence for pore size effect on these scaffolds in this size range. As the cell size is significantly smaller than the pore size of these scaffolds, no specific changes were expected due to the variation of pore sizes. Moreover, samples having constant pore volume but different pore sizes had similar total surface area. Scanning electron microscopy was conducted with some of these alumina and TCP scaffolds. Fig. 7a and b shows SEM images of alumina and TCP matrices, illustrating the cell – matrix interaction. It was observed that cells tend to attach more intimately with TCP matrix than alumina. Also it was noted that cells grew on both the top and the bottom surfaces of TCP scaffolds during cell growth experiments, but for the alumina scaffolds, it was primarily the top surfaces where most of the cell growth occurred.
4. Summary Numerous researchers are working on porous ceramic scaffolds primarily for bone-graft applications. In most of these cases, the porosities are random in nature. The primary aim of this work was to understand and delineate the influence of different porosity parameters on the cell growth behavior on bio-inert alumina and bio-resorbable TCP ceramics. Pore size and pore volumes were identified as two parameters to evaluate. Samples with constant pore volume with varying pore size, and constant pore size with varying pore volume were fabricated. Pore size was varied between 300 and 480 Am and pore volume was varied between 29 and 44 vol.%. As the pore volume was varied,
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the total surface area per unit volume was also varied, though the surface area was almost constant for samples with different pore sizes. It was found that materials chemistry had minimal influence on cell growth behavior. Between pore size and pore volume, it was pore volume that had some influence on cell growth, where higher volume fraction porosity resulted in an increase in cell proliferation. Pore size did not show any specific influence on cell growth behavior for this size range of porosity. It is believed that the higher cell proliferation for samples with high volume fraction porosity are primarily due to the effect of higher surface area/unit volume. When mechanical properties of these structures were compared, it was found that pore volume also had a stronger influence on the mechanical properties of these ceramics, in which compressive strength decreased significantly with an increase in total pore volume.
Acknowledgements Authors would like to acknowledge the financial support from the National Science Foundation through CAREER grant DMI 9874971. Authors would also like to thank Travis Sonnett and Dr. Randy Kintner for their experimental help.
References [1] H.L. Marcus, D.L. Bourell, Advanced Materials & Processes 9 (1993) 28 – 35. [2] E.W. White, J.N. Webber, D.M. Roy, E.L. Owen, R.T. Chiroff, R.A. White, Journal of Biomedical Materials Research Symposium 6 (1975) 23 – 27. [3] R.T. Chiroff, E.W. White, J.N. Webber, D.M. Roy, Journal of Biomedical Materials Research Symposium 6 (1975) 29 – 45. [4] S.F. Hulbert, S.J. Morrison, J.J. Klawitter, Tissue reaction to three ceramics of porous and non-porous structures, Journal of Biomedical Materials Research Symposium 6 (1975) 347 – 374. [5] S.F. Hulbert, J.R. Matthews, J.J. Klawitter, B.W. Sauer, R.B. Leonard, Effect of stress on tissue ingrowth into porous aluminum oxide, Journal of Biomedical Materials Research Symposium 5 (1974) 85 – 97. [6] C.P.A.T. Klein, P. Patka, A comparison between HA and Whitlockite macroporous ceramics implanted in dog femurs, in: T. Yamamura, L.L. Hench, J. Wilson (Eds.), Handbook of Bioactive Ceramics: II. Calcium Phosphate and Hydroxylapatite Ceramics, CRC Press, Boca Raton, FL, 1990, pp. 53 – 60. [7] G. Lee, J.W. Barlow, W.C. Fox, T.B. Aufdermorte, Biocompatibility of SLS formed calcium phosphate implants, Proceedings of the Solid Freeform Fabrications 1996, The University of Texas, Austin, TX, 1996, pp. 15 – 21. [8] US patent 5,518,680. [9] US patent 5,490,962. [10] T.M. Chu, J.W. Halloran, Hydroxyapatite scaffolds with controlled porosity, Bioceramics: Materials and Applications Symposium, 100th Annual Meeting Abstract Book, American Ceramic Society, Westerville, OH, 1998, p. 9. [11] R. Garg, R.K. Prud’homme, I.A. Aksay, Orthopaedic implants by ceramic stereolithography, Bioceramics: Materials and Applications Symposium, 100th Annual Meeting Abstract Book, American Ceramic Society, Westerville, OH, 1998, p. 5.
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[12] S. Bose, S. Sugiura, A. Bandyopadhyay, Processing of controlled porosity ceramic structures via fused deposition process, Scripta Materialia 41 (9) (1999) 1009 – 1014. [13] P. Teung, R.K. Panda, S.C. Danforth, A. Safari, Fabrication of porous hydroxyapatite ceramics by solid freeform fabrication technique, Bioceramics: Materials and Applications Symposium, 100th Annual Meeting Abstract Book, American Ceramic Society, Westerville, OH, 1998, p. 5. [14] S. Bose, M. Avila, A. Bandyopadhyay, Processing of Bioceramic Implants via Fused Deposit on Process, Solid Freeform Fabrication Symposium Proceedings, vol. 9, UT Austin, Austin, TX, 1998, pp. 629 – 636. [15] R. Atisivan, S. Bose, A. Bandyopadhyay, Porous mullite preforms via fused deposition, Journal of the American Ceramic Society 84 (1) (2001) 221 – 223.
[16] A. Bandyopadhyay, S.C. Danforth, A. Safari, Effects of processing history on thermal debinding, Journal of Materials Science 35 (16) (2000) 3983 – 3988. [17] A. Hattiangadi, A. Bandyopadhyay, Strength degradation of porous ceramics under uniaxial compressive loading, Journal of the American Ceramic Society 83 (11) (2000) 2730 – 2736. [18] S.R. Winn, G. Randolph, H. Uludag, S.C. Wong, G.A. Hair, J.O. Hollinger, Establishing an immortalized human osteoprecusor cell line (OPC 1), Journal of Bone and Mineral Research 14 (10) (1999) 1721 – 1733. [19] P.W. Sylvester, H.P. Birkenfeld, H.L. Hosick, K.P. Briski, Fatty acid modulation of epidermal growth-factor induced mouse mammary cell proliferation in vitro, Experimental Cell Research 214 (1994) 145.
Pore size and pore volume effects on alumina and TCP ceramic scaffolds Susmita Bose a, Jens Darsell a, Martha Kintner b, Howard Hosick b, Amit Bandyopadhyay a,* a
School of Mechanical and Materials Engineering, Washington State University, P.O. Box 642920, Pullman, WA 99164 2920, USA School of Biological Sciences and Molecular Biosciences, Washington State University, P.O. Box 644236, Pullman, WA 99164 4236, USA
b
Accepted 18 July 2002
Abstract Controlled porosity alumina and h-tricalcium phosphate ceramic scaffolds with pore sizes in the range of 300 – 500 Am and pore volumes in the range of 25 – 45% were processed using the indirect fused deposition process. Samples having different pore sizes with constant volume fraction porosity and different volume fractions porosity with a constant pore size were fabricated to understand the influence of porosity parameters on mechanical and biological properties. In vitro cell proliferation studies were carried out with OPC1 human osteoblast cell line for 28 days with different scaffolds. Variation in pore size did not show any conclusive differences, but samples with higher volume fraction porosity showed some evidence of increased cell growth. Volume fraction porosity also showed a stronger influence on the mechanical properties under uniaxial compression loading. Compression strength dropped significantly for samples with higher volume fraction porosity, but changed marginally when only the pore size was varied. D 2002 Elsevier Science B.V. All rights reserved.
1. Introduction Rapid prototyping (RP) or solid freeform fabrication (SFF) has evolved over the past decade as a potential manufacturing approach to making functional structures, directly from CAD files, having controlled shape as well as internal architecture. This is an approach in which threedimensional objects are fabricated on a fixtureless platform from a CAD file without any part-specific tools or dies, using polymeric, ceramic or metallic materials [1]. RP is a layered manufacturing approach in which a CAD file is sliced in the Z-direction in a virtual environment and then each slice is built one on top of the other. The first layer is built on a fixtureless platform and then the platform indexes down. The next layer is built on top of the previous layer. This process continues until the fabrication of the part is complete. Several commercial RP techniques are available including stereolithography (SLA), selective laser sintering (SLS) and fused deposition modeling (FDM). Most of these processes are used to build parts using polymers, but some are capable of fabricating metals and ceramics as well. Porous ceramics have been studied as scaffold materials for small-scale bone defects as well as spinal fusion appli*
Corresponding author. Tel.: +1-509-335-8654; fax: +1-509-335-4662. E-mail address: [email protected] (A. Bandyopadhyay).
cations. Different research groups have used numerous processing routes to fabricate porous ceramic structures. White et al. [2,3] have reported replamineform process to fabricate porous ceramic implants that duplicate the microstructure of corals. Hulbert et al. [4,5] reported the fabrication of porous alumina ceramics using a pore former or foaming agent that evolves gases during sintering at elevated temperatures leaving pores behind. Klein et al. [6] made porous hydroxyapatite (HAp) ceramic blocks using HAp slurry mixed with foaming agent followed by sintering at elevated temperature. All of these porous structures had randomly arranged pores with a wide variety of sizes and volume fraction porosity. In recent years, the RP techniques have also been used to fabricate porous structures for biomedical applications. The SLS [7], 3-D printing [8,9], stereolithography [10,11] and FDM [12 – 14] processes have been used to fabricate porous structures for scaffold-related applications. RP is used in two different ways, the direct and the indirect, to produce these porous structures. In the direct route, the desired part is directly formed from the CAD file to the final form. In the indirect route, first, a negative of the desired structure is made with polymer, which acts as a mold, and then the actual part is cast from the polymer mold. The direct route is the most desirable one, though it may require the development of feedstock materials and hardware modifications. The indirect approach can be used
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-4931(02)00129-7
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with any commercial hardware, as all the machines are capable of making polymer prototypes. Moreover, this route uses a traditional lost-mold ceramic processing approach in conjunction with slurry or gel casting. In the present work, fused deposition modeling (FDMk), one of the commercially available RP processes, was used to make porous alumina and tricalcium phosphate (TCP) ceramic structures using the indirect route. These porous structures have three-dimensionally interconnected porosity. The main advantage of this process is the simultaneous design of both the micro- and the macrostructure of the part using the CAD program. During mold making, different porosity parameters of the polymeric molds can be tailored by optimizing the diameter of the extruded polymeric filament (called ‘‘road-width’’), the distance between two polymer roads (called ‘‘road-gap’’), and the thickness of each layer (called ‘‘slice thickness’’) during the layered manufacturing process. Moreover, by changing the deposition angles of polymeric roads for each layer, the pores can be oriented layer by layer. The present work on mechanical and biological responses was carried out with porous ceramic structures having constant pore sizes but different pore volumes, and constant pore volume with different pore sizes, using alumina and h-tricalcium phosphate (h-TCP) ceramics. Controlled porosity cylindrical structures were
tested under uniaxial compressive loading to understand the influence of porosity parameters on the mechanical properties. In vitro assessment with the OPC1 human osteoblast cell line was conducted to evaluate the influence of porosity parameters on cell proliferation.
2. Processing of porous ceramics Processing of porous ceramics has three steps including (a) mold microstructure design, (b) ceramic slurry development and (c) binder burnout and sintering. Fig. 1 shows the schematic of the process flow chart for porous ceramic structures. The molds were made using a Stratasys FDM 1650 machine with commercially available ICW06 thermoplastic polymers. Polymeric molds were made with different road-gap and road-width to have different pore size and volume fraction porosity in the final structures. Slice thickness was varied between 0.25 and 0.35 mm, road-gaps were varied between 0.5 and 1 mm and road-widths were varied between 0.4 and 0.625 mm. Mold diameter was kept constant at 1.725 cm. Water-based ceramic slurries were made with high-purity 10D (surface area 10 m2/gm) alumina powder doped with 500 ppm MgO (Baikowski International, NC) and food-grade TCP powders (Monsanto, CA) for alumina and
Fig. 1. The schematic of the process flow chart for porous ceramic structures. (a) Polymer filament. (b) Fused deposition modeling process. (c) Polymer mold. (d) Slurry-infiltrated mold. (e) Porous ceramic structure with a porosity gradient from center to the outside.
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TCP scaffolds, respectively. 1-Butanol was used as an antifoaming agent for both powders. Darvan-821A (RT Vanderbilt, CT) and D-3021 (Rohm and Haas, PA) were used as dispersant for alumina and TCP slurries, respectively. Binder B-1001 (Rohm and Haas) was used as binder for both the slurries. Ceramic powder, antifoaming agent and dispersant were added to water and then ball milled for 5 –16 h in a polyethylene bottle. The required amount of binder was added to the mixture just before the infiltration. A 1.5 wt.% sample of Darvan 821A was found optimum for alumina powders, and 3.5 wt.% D-3021 was found optimum for TCP powders. Slurry compositions were optimized using a Brookfield viscometer. For same solids loading, TCP slurry was always more viscous than alumina and TCP slurry required longer ball-milling time as well. The slurry was infiltrated into the porous polymeric molds and the green structures were dried for 2 days. The structures were then subjected to a binder burnout and sintering cycle. During the binder burnout heating cycle, the mold polymer leaves the part, creating the pores. Issues related to the development of binder removal and sintering cycles have already been discussed in Refs. [15,16]. By changing the distance between the polymer roads and their width, different types of porosity can be created. For in vitro testing, controlled porosity samples made of alumina and TCP of 2-mm thickness and 1.2 cm diameter were processed. For the first set of samples, the volume fraction porosity was changed from 29% to 35% to 44% keeping the pore size constant at 300 Am. For the second set of samples, pore size was varied from 300 to 380 to 480 Am, keeping the volume fraction porosity constant at 44%. For uniaxial compression testing, cylindrical samples of 2 cm long and 1.2 cm diameter were processed having the same porosity parameters as in the case of in vitro samples.
3. Results 3.1. Physical structure evaluation Porous structures were characterized for their physical, mechanical and biological properties to understand the
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influence of porosity parameters. Shrinkage was measured as a part of physical property measurement to understand the sintering behavior of these ceramics. Nonuniform shrinkage causes cracking and warping in the final product. Moreover, controlled shrinkage information can be utilized in the design stage to make necessary design modifications. Shrinkage during sintering is dependent on the solids loading of the ceramic slurry and final sinter density. Shrinkage also varies as a function of volume fraction porosity in these samples in which higher volume fraction porosity showed a lower amount of shrinkage [15]. For the porous alumina samples, linear shrinkage varied from 23% to 25%, while shrinkage of TCP varied between 17% and 19%. The shrinkage variations were primarily due to the variations in pore volume, as the solids loading remained constant. Sintered density for porous alumina samples was between 3.78 and 3.80 g/cm3 (>95% theoretical density). For TCP, the density varied between 2.90 and 2.95 g/cm3 (f90% theoretical density). Fig. 2 shows an optical micrograph of 2D porous samples with different volume fraction porosity where pore size was kept constant at 300 Am. Using this approach, structures with porosity gradient, i.e., varying porosity from one end to the other end can also be processed. 3.2. Mechanical property evaluation Strength degradation is a serious concern in porous ceramics. As the total volume fraction porosity increases, the failure strength decreases. Cylindrical porous samples of 12 mm diameter and 20 mm long (L/D=1.6) were used for this study. Uniaxial compression tests were performed using an Instron 1331 servo hydraulic machine under stroke control mode at a stroke rate 0.5 mm/min. At least 20 samples of each porosity level were tested. Fig. 3 shows the influence of pore volume and pore size on the mechanical properties of 3-D honeycomb alumina samples. Fig. 4 shows the same for the TCP ceramics. It is clear from both Figs. 3 and 4, that with increasing volume fraction porosity, compressive strength decreases. Compressive strengths of alumina ceramics are about two orders of magnitude higher
Fig. 2. An optical micrograph of 2-D porous samples having different volume fraction porosity where pore size is kept constant at 300 Am.
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Fig. 3. The influence of pore volume and pore size on the mechanical properties of 3-D honeycomb alumina samples.
than those of the TCP ceramics. Similar influences of volume fraction porosity on compressive strength have also been reported with other materials having similar porosity [17]. As expected, failure planes in all of these samples were parallel to the loading axis. The influence of pore size on the strength degradation is not clear from these figures. In fact, for a constant pore volume, pore size should not have any effect on the strength degradation behavior. The relative variation due to pore size effect may be due to the height differences of the pores for the change in slice thickness in the polymer molds, which produces some minor influence
of shape. Any clear trend due the pore size alone could not be delineated from these experimental data. 3.3. In vitro analysis In vitro tests were performed on porous scaffolds of alumina and TCP ceramics using OPC1 cells. The OPC1 cell line is a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone
Fig. 4. The influence of pore volume and pore size on the mechanical properties of 3-D honeycomb TCP ceramics.
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phenotype from passage 10 to at least passage 30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ ml).Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell system to evaluate bone development and cell/biomaterial interactions, and may be useful to screen for putative bone-differentiating factors [18].The OPC1 cells were cultured in a standard medium made of McCoy’s 5A (with l-glutamine, without phenol red and sodium bicarbonate) (Sigma, St. Louis, MO, USA), supplemented with 10% fetal bovine serum, 2.2 g/l sodium bicarbonate, 0.1 g/l penicillin and 0.1 g/l streptomycin. The selection of a nutrient medium is strongly influenced by type of cell, type of culture and degree of necessary chemical definition. Cells’ splitting was done 3 days before the cell culture. During the splitting process, the same medium was used supplemented with 10% bovine calf serum. Cultures were incubated at 37 jC in a humidified 5% CO2 atmosphere. A total of 100,000 cells per matrix were seeded onto 15 matrices in a 10-cm tissue culture dish. Sufficient medium was present (15 ml) to completely cover the matrices. Three matrices were removed from random regions of the culture vessel on days 2, 7, 14, 21 and 28 after seeding. These were then evaluated by MTT assay. This
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assay quantitates the ability of mitochondrial dehydrogenases to metabolize 3-[4,5-2-yl]-2,5-diphenyl-tetrazolium bromide to an insoluble formazan [19]. The amount of formazan is directly proportional to the total number of living cells. This enzyme assay produces a colored product, which is quantitated in a microplate reader at a wavelength of 570 nm. During the MTT assay, after removal of the growth media, a 1-mg/ml solution of MTT in phenol redfree DME was added to the scaffold in a 1-cm culture vessel (0.5 ml) or to the cells on the bottom of a 1-cm culture vessel (0.3 ml).The reaction was allowed to proceed for 1 h at 37 jC, at which time micrographs were taken on a WILD stereomicroscope to evaluate coverage of the scaffold by the cells. The cells were returned to 37 jC for 3 h more, and then the MTT solution was removed. Formazan crystals were solubilized in isopropanol and the optical density at 570 nm was determined on a Cambridge Technology Series 700 microplate reader. Fig. 5a and b shows the effect of different volume fraction porosity on OPC1 cell growth for alumina and TCP scaffolds in which the pore size was kept constant at 300 Am. Both alumina and TCP ceramics showed similar behavior for volume fraction porosity samples. The initial part of the cell growth for up to 14 days on these scaffolds
Fig. 5. (a) and (b) shows the effect of volume fraction porosity on OPC1 cell growth for alumina and TCP scaffolds. A570 in Y-axis shows numbers that was the read out from a microplate reader at 570 nm wavelength. Higher number indicates the presence of increasing number of living cells.
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Fig. 6. (a) and (b) shows the effects of pore size on the (a) alumina and (b) TCP scaffolds having a constant pore volume of 44%.
Fig. 7. (a) and (b) shows the SEM images of alumina and TCP matrices showing the cell – matrix interaction.
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was relatively fast for 29 and 35 vol.% samples followed by slower growth for the next 14 days. For the 44 vol.% samples of both alumina and TCP, there was a consistent growth rate throughout the 28 days of the test period. For the 29 and 35 vol.% alumina samples, most of the cell growth took place during the first 14 days. A relatively slower growth was observed for the third and fourth week of the study. In general, for both the cases, 44 vol.% samples showed highest cell growth up to 28 days, though the cell growth behavior was quite similar up to the first 14 days. From all the batches, samples were taken out for stereomicroscopy to see the cell attachment on the scaffolds. It was found that within the first 14 days, both 29 and 35 vol.% samples, cells had grown to cover almost all the surfaces. For the case of 44 vol.% porosity samples, having higher volume fraction porosity, and higher surface area per unit volume, cells took longer time to fully cover the scaffold surface. Having the same initial cell density for all the scaffolds, the higher cell growth behavior can be explained due to the higher surface area for the 44 vol.% scaffolds. Fig. 6a and b shows the effects of pore size on the alumina and TCP scaffolds having a constant pore volume of 44%. Both 29 and 44 vol.% samples were studied and pore sizes were varied between 300 and 480 Am. All the samples showed similar cell growth behavior and there was no conclusive evidence for pore size effect on these scaffolds in this size range. As the cell size is significantly smaller than the pore size of these scaffolds, no specific changes were expected due to the variation of pore sizes. Moreover, samples having constant pore volume but different pore sizes had similar total surface area. Scanning electron microscopy was conducted with some of these alumina and TCP scaffolds. Fig. 7a and b shows SEM images of alumina and TCP matrices, illustrating the cell – matrix interaction. It was observed that cells tend to attach more intimately with TCP matrix than alumina. Also it was noted that cells grew on both the top and the bottom surfaces of TCP scaffolds during cell growth experiments, but for the alumina scaffolds, it was primarily the top surfaces where most of the cell growth occurred.
4. Summary Numerous researchers are working on porous ceramic scaffolds primarily for bone-graft applications. In most of these cases, the porosities are random in nature. The primary aim of this work was to understand and delineate the influence of different porosity parameters on the cell growth behavior on bio-inert alumina and bio-resorbable TCP ceramics. Pore size and pore volumes were identified as two parameters to evaluate. Samples with constant pore volume with varying pore size, and constant pore size with varying pore volume were fabricated. Pore size was varied between 300 and 480 Am and pore volume was varied between 29 and 44 vol.%. As the pore volume was varied,
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the total surface area per unit volume was also varied, though the surface area was almost constant for samples with different pore sizes. It was found that materials chemistry had minimal influence on cell growth behavior. Between pore size and pore volume, it was pore volume that had some influence on cell growth, where higher volume fraction porosity resulted in an increase in cell proliferation. Pore size did not show any specific influence on cell growth behavior for this size range of porosity. It is believed that the higher cell proliferation for samples with high volume fraction porosity are primarily due to the effect of higher surface area/unit volume. When mechanical properties of these structures were compared, it was found that pore volume also had a stronger influence on the mechanical properties of these ceramics, in which compressive strength decreased significantly with an increase in total pore volume.
Acknowledgements Authors would like to acknowledge the financial support from the National Science Foundation through CAREER grant DMI 9874971. Authors would also like to thank Travis Sonnett and Dr. Randy Kintner for their experimental help.
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