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Development of a novel aqueous hydroxyapatite suspension for stereolithography applied to bone tissue engineering
Author’s Accepted Manuscript Development of a Novel Aqueous Hydroxyapatite Suspension for Stereolithography Applied to Bone Tissue Engineering Zhen Wang, Chuanzhen Huang, Jun Wang, Bin Zou www.elsevier.com/locate/ceri
PII: DOI: Reference:
S0272-8842(18)33169-9 https://doi.org/10.1016/j.ceramint.2018.11.063 CERI20037
To appear in: Ceramics International Received date: 30 July 2018 Revised date: 9 November 2018 Accepted date: 9 November 2018 Cite this article as: Zhen Wang, Chuanzhen Huang, Jun Wang and Bin Zou, Development of a Novel Aqueous Hydroxyapatite Suspension for Stereolithography Applied to Bone Tissue Engineering, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.11.063 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.
Development of a Novel Aqueous Hydroxyapatite Suspension for Stereolithography Applied to Bone Tissue Engineering Zhen Wanga, Chuanzhen Huanga*, Jun Wangb, Bin Zoua a.
Center for Advanced Jet Engineering Technologies (CaJET), Key Laboratory of
High-efficiency and Clean Mechanical Manufacture (Ministry of Education), National Demonstration Center for Experimental Mechanical Engineering Education (Shandong University), School of Mechanical Engineering, Shandong University, Jinan 250061, China b.
School of Mechanical and Manufacturing Engineering, The University of New South Wales (UNSW), Sydney, NSW 2052, Australia
*
Corresponding author: Tel. and Fax: +86 531 88396913. E-mail address: [email protected] (C. Z. Huang).
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Abstract: Stereolithography (SLA) has a good ability to form complex bodies, while hydroxyapatite (HA) ceramic has good mechanical and biological properties because of its similarity in composition with natural bone. Some researchers tried combining SLA technology with HA bioceramic for bone tissue engineering. However, the ceramic suspension used in SLA generally has a relatively higher viscosity and are not environmentally friendly. In this paper, a novel aqueous HA suspension for SLA applied to bone tissue engineering was developed. The effect of surfactant concentration, solid loading and particle size on the rheological property of the HA suspension was investigated to preliminarily determine the formulation of the suspension. By a viscosity
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test and a sedimentation experiment, a conclusion was drawn that the surfactant PAA-NH4 had an optimum concentration of about 0.3mg/m2. In addition, the maximum value of viscosity reached up to 7.3Pa·s, when the solid loading was 56vol%. Taking account of viscosity of suspension and the strength of sintered parts, the particle size of 1μm was relatively reasonable to be used in the HA suspension. According to the study of curing behaviour, the suspension had a critical exposure of 20.3mJ/cm2 and a penetration depth of 50.7μm. The final sintered HA parts provided a strength of 37MPa meeting the requirement of cancellous bone, and was similar to original HA powders in chemical composition implying an ideal biological performance. Therefore, this kind of HA suspension has a broad prospect in bone tissue engineering applied to the treatment of bone disease for thousands of people. Keywords: Stereolithography; Hydroxyapatite; Suspension; Bioceramic 1. Introduction Recently, number of people suffering from various orthopaedics diseases caused by traffic accidents, bone tumors, and infections is increasing gradually [1-2]. Although, bone can heal small segment defects by itself, it is still a big challenge for treating non-healable sized bone defects. Therefore, a bone transplant surgery is usually performed on patients to help restore their natural bone function, including three main strategies such as autograft, allograft and xenograft. However, each of the mentioned strategies has its pre-operation or post-operation limitations. The autograft has always been regarded as the gold standard of the bone transplantation, but the source of the
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autograft is restricted seriously, besides, it can cause a secondary damage i.e. physical and mental illness to patients. The allograft and xenograft have much broader sources, but they are usually rejected by the immune system of the receiving patients. Fortunately, with the development of science and technology, a new strategy, bone tissue engineering, was proposed to treat bone defects. Tissue engineering (TE) is a promising field for the reconstruction of bone, which is an interdisciplinary field that applies the principles of engineering and life sciences to develop biological substitutes for restoring, maintaining, or improving the tissue function [3]. Scaffolds play a very important role in bone tissue engineering so as to provide a suitable environment for cell migration, proliferation and vascularization, besides acting as a supporting structure [4-6]. Some ideal properties are required for the bone tissue engineering scaffold [7-11], for example, the scaffold should have appropriate pore size, morphology and porosity, in addition, the material for scaffold should have osteoinduction and good biocompatibility. Moreover, the controllable biodegradability is also a desired property of the bone tissue engineering scaffold which should have a suitable degradation rate corresponding to the cell growth rate. Additionally, the scaffold should have sufficient mechanical strength to support human body, and appropriate elasticity modulus to match that of bone. To satisfy the requirement of scaffold, additive manufacturing, commonly called 3D printing, is a very promising technology and is also a greatly suitable way to manufacture scaffolds, especially, stereolithography (SLA) has
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relatively high processing accuracy among the additive manufacturing technologies, which can fabricate intricate structures and complex surfaces to meet the specific needs of scaffolds. Although most of the commercial materials for SLA are light curing resins, researchers have developed some kinds of ceramic suspensions [12-14], which were formulated by mixing ceramics powders with the light curing resins to finally fabricate ceramic parts after exposure to ultraviolet (UV) light. Wu et al. developed an approach to fabricate dense zirconia-toughened alumina ceramics with excellent properties by SLA [15]. Chartier et al. fabricated bioceramic implants by SLA with a developed HA suspension [16-17]. However, these resin-based suspensions have relatively higher viscosity and are not environmentally friendly. Hydroxyapatite (HA) with the chemical formula Ca10(PO4)6(OH)2, which is the basic composition of the bone mineral component, has excellent biological and mechanical performances [18]. Therefore, in this paper a novel aqueous HA suspension was developed for SLA applied to bone tissue engineering. To be used in SLA, the ceramic suspensions should have the following properties: (1) A low viscosity to make sure the scraper of a SLA machine can spread ceramic suspensions homogeneously and levelly layer by layer; (2) A sufficient cured depth to confer an overcure at the interface so as to provide a good cohesion between two cured layers. (3) A narrow cured width to offer a high accuracy of manufacturing for complex shaped parts. 5
A higher solid loading suspension always means a lower shrinkage of the sintered ceramic parts, but that also represents a higher viscosity of the suspension and a worse reactivity of the UV reactive system. As a consequence, it is of prime important to make a balance between the solid loading and the curing reactivity. Taking this into account, the effect of some parameters such as surfactant concentration, solid loading and particle size on rheological property of ceramic suspensions was investigated to initially identify a formulation of ceramic suspensions. Subsequently, the curing behavior and mechanical property was explored to further optimize the formulation. Finally, an XRD analysis was implemented on sintered HA ceramic parts to make sure that the HA parts still remained good biological property without any change of chemical composition.
2. Materials and methods 2.1. Powder surface modification In order to reduce the viscosity of ceramic suspensions, ammonium polyacrylate (PAA-NH4, Adamas) was used as surfactant to modify the surface of HA powders. A small amount of surfactant has an obvious influence on rheological property but a minimal impact on curing behavior. In this respect, the amount of surfactant was preliminarily investigated. Three kinds of HA ceramic powders (DKN, China) with different particle sizes and specific surface areas (d50=0.3μm, 1μm, 5μm, and BET=6m2/g, 2.1m2/g, 0.6m2/g, respectively) were used as the ceramic filler in the following study. 6
Firstly, PAA-NH4 was dissolved in the deionized water at 35℃-45℃, stirring for 10 minutes. Secondly, the HA powders dispersed in the solution by an ultrasonic homogenizer (1500F, Scientz) at 1500W and 20KHz for 10 minutes. Subsequently, after being milled for 4 hours by a planetary ball milling, the suspension was freeze dried by a lyophilizer. Following, the dried HA powders modified by PAA-NH4 were passed through a 100-mesh sieve to get rid of agglomerated particles. The change of particle size, before and after surface modification, was measured by a dynamic laser light scattering (MS-2000, Malvern). Then, the wettability was investigated by a contact angle meter (JC2000D1, Shanghai Zhongchen).
2.2. HA suspension preparation Generally, UV curable ceramic suspensions, used in stereolithography, contain following components: monomer, diluent (or solvent), ceramic powder, photoinitiator and some additives such as dispersant, defoamer and light absorber. The aqueous solvent has a lower viscosity than the resin-based solvent at the same temperature and shear rate, besides, it is much more environmentally friendly to conform with the encouragement of modern society. Considering these advantages of the aqueous solvent, it was used to dissolve monomers and HA particles in this paper. Acrylamide (AM) and N-N’ methylenebisacrylamide (MBAM) were selected as the organic monomer system to be dissolved in deionized water. Subsequently, a certain amount of glycerol and modified HA were added into the solution in an ultrasonic 7
homogenizer (1500F, Scientz) at 1500W and 20KHz for 15 minutes. At last, the photocure-1173 (HongTai, China) was mixed with the suspension by a planetary ball mill for 10 hours at a revolving speed of 500rpm-1000rpm. In order to preliminarily determine a formulation of HA suspension and to achieve a better manufacturability, the rheological property of the suspensions with different surfactant concentration, solid loading and HA particle size was measured by the rheometer (Kinexus, Malvern) at a shear rate from 0.1-100s-1. In addition, a zeta potential meter (Zetaplus, Brookhaven) was used to analyse the effect of the surfactant PAA-NH4 on viscosity of the suspension, and a sedimentation experiment was conducted to test the stability of the suspension.
2.3. Stereolithography of the HA suspension The HA suspension was added into the vat of a SLA machine (SPS450B, Shaanxi Hengtong Co. Ltd, China) to fabricate green parts. The platform was moved to a position 0.1mm under the top surface of the suspension by an elevator, which was controlled by a computer. When the UV laser scanned the surface of the suspension, the photoinitiator splits primitive radicals. Subsequently, the radicals react with the monomer to produce a polymer chain, and then the polymer chain propagates continuously with more monomers to form a chain reaction. At last, it terminates when two of the reactive polymer chains combine together [19], as shown in equation (1). Therefore, HA particles were anchored by the crosslinked polymers. 8
S h R0 R M R 0 1 Rn 1 M Rn Rn Rm Pm+n
(1)
where S is the photo initiator, hγ is the photon, R0 is the primitive radical, M is the monomer, R is the reactive polymer chain, and P is the final polymer molecule. Then, the platform moved down 0.1mm to submerge the solidified layer by the new suspension, at the same time, a scraper was required to level the surface and absorb the bubble. After the suspension was solidified layer by layer, a resulting green part was obtained with HA particles and crosslinked polymers. The schematic diagram of SLA process is shown in Fig.1. The investigation on the curing behavior of the ceramic suspension is important to form a strong green part with a high dimensional precision. Especially, the HA solid loading in the suspension has a significant influence on the curing behavior. Therefore, the cured depth (Cd) and the cured width (Cw) was measured by a digital caliper, then the critical exposure (Ec) was calculated by fitting in Origin software. Subsequently, the dimensional precision of green parts was measured by a laser microscope (Keyence, Japan). Finally, an orthogonal array for experiment was designed to further optimize the formulation, which had been preliminarily identified by the exploration on rheological property and curing behaviour.
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2.4. Debinding and sintering To get a strong HA ceramic part used for bone tissue engineering and remove the toxic substances for cells, four processes of rinsing, drying, debinding and sintering were required on the green part. After the green part was rinsed with alcohol, the residual water and alcohol in the part needed to be dried. A special drying approach was used [20] based on PEG400 liquid desiccant to make sure a uniform extraction rate in all directions of the part. In order to avoid cracking of the green part and transforming of the HA composition, during debinding and sintering, the maximum temperature (1180℃) and heating rate must be controlled very well. Referred to the study of Daculsi et al. [21] and through continuous experimental trying, finally a heating route in a vacuum furnace was determined as shown in Fig.2. The organic polymers in green parts were removed before temperature rising to about 500℃, so a very low heating rate was conducted in this stage to reduce deformation and to avoid cracking. Subsequently, the temperature was heated to 1080℃ with a little higher heating rate, which actually was a pre-sintering stage from 500℃ to 1080℃. After the HA parts were cooled to room temperature, a new sintering stage was conduct to 1180℃ with a higher heating rate. Although this heating route was time-consuming, the sintered HA parts were a little bit stronger. Aiming to be applied in bone tissue engineering field, the sintered HA ceramic should require an adequate mechanical property, especially compression strength, and also a small shrinkage. Therefore, the effect of particle size on the mechanical property 10
was investigated by a compression test on a material testing machine (Instron8801, USA) at a speed of 1mm/min. Additionally, the shrinkage rate of sintered HA parts was measured by a digital caliper. Taking account to that a transformation of chemical composition of the HA can cause the biological property declining, the original HA powders and the final HA scaffolds were evaluated by the XRD (DMAX-2500PC, Rigaku, Japan) with CuKα radiation (λ=1.5418A°) in the 2θ range of 20°–60° by the step scanning mode, with a step size of 0.02° and step duration of 0.5s.
3. Results and discussion 3.1. Characterization of powder To explore the relationship between the size of the HA particle and the rheological property of the suspension, the dynamic laser light scattering (MS-2000, Malvern) was used to determine the HA particle size before and after surface modification. Fig.3 indicates that the particle size increases after surface modification, especially for small particles. Although the three particle sizes increased by 27%-110%, the ceramic particles can still be used in suspension, because there was no significant agglomeration. Before mixing the modified HA with the aqueous solution, the wettability of the original HA and the modified HA was determined to predict the trend of rheological
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property of the HA suspension. Fig.3 shows that the contact angles of the three different particles were obviously dropped. So a ceramic suspension with homogeneously dispersed particles and lower viscosity was foreseen, used in SLA to fabricate bone tissue engineering scaffolds.
3.2. Rheological property of the HA suspension In order to require an ability of self-levelling, ceramic suspensions require a viscosity lower than 3Pa·s at 30s−1shear rate [22]. In this study, a developed scraper in the SLA machine spread ceramic suspensions on a platform with a constant speed (4mm/s), so the spreading shear rate was 40s−1, calculated by equation (2):
𝛾̇ =
𝑣 𝑡
(2)
where 𝛾̇ is the shear rate, v is the spreading speed, and t is the thickness of a spread layer (0.1mm). Based on the rheological property of the HA suspension, the formulation of the novel suspension was attempted to be preliminarily determined by single-factor analysis in the following study. 3.2.1. Effect of surfactant concentration The effect of surfactant concentration on rheological property was firstly investigated to reduce the viscosity of the suspension, because a small amount of surfactant has an obvious influence. PAA-NH4 is a kind of organic polymer with a long 12
carbon chain. One end of the chain can be absorbed on the surface of HA ceramic particles, and the other end stretch out to liquid. The surface modified particles disperse more homogeneously attributing to the steric-hinerance effect of the long chain and the electrostatic repulsive force of the (–COO-) group, as shown in Fig.4. The viscosity of suspension with different particle sizes and surfactant concentration was tested. As shown in Fig.5, the viscosity of the suspension with 5μm HA particle size was the lowest among the three particle seizes. Moreover, the viscosity reduced with the concentration of PAA-NH4 increasing about from 0mg/m2 to 0.3mg/m2, and then almost plateauing from 0.3mg/m2 to 0.35mg/m2. Subsequently, the viscosity rose up with the concentration of PAA-NH4 over 0.35mg/m2. The zeta potential represents the electrostatic repulsion, which is the major factor causing this phenomenon, as shown in Fig.6. More specifically, there were more surface charges on the surface of the HA particle because of the absorption of PAA-NH4. The surface charges thus improved the surface potential of the HA particle, and increased the electrostatic repulsion. Therefore, the suspension dispersed more uniformly, which attributed to better fluidity and lower viscosity. When the concentration of PAA-NH4 was over 0.35mg/m2, the adsorption capacity of the HA particle reached a saturation point. At the same time, the concentration of macromolecules dissolved in the liquid improved, which formed bridges between particles to hinder the suspension flowing. Furthermore, a sedimentation experiment was conducted to further determine the concentration of PAA-NH4. Fig.7 illustrates that the storage time (1 day, 5 days or 10
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days) is the most influential factors in the sedimentation of HA particles. Only 80.3% of particles, without PAA-NH4 modification, remained uniformly in the suspension after 10 days. The remaining particles increased at first with the increase of the concentration of PAA-NH4, and then it dropped after the concentration of PAA-NH4 over 0.3mg/m2. Therefore, the result shows that PAA-NH4 with a concentration of about 0.3mg/m2 is suitable for the developed HA suspension.
3.2.2. Effect of solid loading According to the previous studies of T. Chartier [23] and H. M. Shaw [24], the suspension used in SLA should contains at least 50vol% ceramic particles in order to provide a dense part after debinding and sintering. However, a high solid loading suspension represents a high viscosity and a low cured depth. To preliminary determine the ceramic volume fraction in the formulation, the relationship between solid loading and viscosity was investigated. Different amounts of HA (50vol%, 52vol%, 54vol%, 56vol%) were added into the aqueous solution. Fig.8 illustrates that the viscosity of the suspension increased rapidly with the solid loading increasing. When the solid loading was 56vol%, the maximum value of viscosity reached up to 7.3Pa·s, which was too high to be used by a SLA machine. The Krieger-Dougherty [25] equation can be used to describe this relationship: =
(
)
[ ]
(3)
where, η is the viscosity of a suspension contained the φ volume percent of solid 14
particles, ηs is the viscosity of the medium and φmax is the maximum packing fraction of solid particles in the suspension. [η] is the intrinsic viscosity depended on the shape of the particles, here it was assumed that the particles are spheres, [η]=2.5. As the volume fraction of HA particles in a suspension increases, it implies that the particles become more numerous in each unit volume. In other words, the distance between particles is shortened, so the interaction force increases which causes the flow resistance to improve i.e. the viscosity increases. However, the experimental curve does not fit well with the calculated curve, which may be attributed to the assumption that the particles were spheres. In fact, the HA particles in the suspension have no regular shapes.
3.2.3. Effect of particle size Based on the study of the relationship between solid loading and viscosity, a formulation of suspension contained 52% or 54vol% HA powders was determined. Then the effect of particle size on rheological property was investigated to further reduce the viscosity of the suspension. As shown in Fig.9, all the suspensions revealed typical shear thinning behaviour that the viscosity decreased with an increase in the share rate. In addition, there were some differences among the suspensions with different particle sizes. The viscosity of the suspension with bigger particle size was much lower, especially at the low share rates, and the gap of viscosity narrowed as the share rate increased. When a suspension contains ceramic with the same volume
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fraction, in a given volume unit, the smaller the particle is, the more particles present. Maybe the increasing viscosity can be explained by the increase of the interaction forces between the closer particles. From the results, the suspension with 0.3μm HA particles was not suitable for SLA processing because of high viscosity.
3.3. Curing behaviour of the suspension An aqueous HA suspension, which contained 52% or 54vol% HA with a particle size of 1μm, was prepared to be cured by the SLA machine so as to evaluate the curing behaviour. 3.3.1. Cured depth The cured depth (Cd) can be derived from the Beer-Lambert law, as described in equation (4): 𝐸
𝐶𝑑 = 𝐷𝑝 ln ( ) 𝐸𝑐
(4)
where Dp is the depth of light penetration, E is the exposure (the radiant energy on a unit area) and Ec is the critical exposure (the minimal exposure to initiate polymerization). For a ceramic loaded suspension, Dp is a function of the volume fraction of powders, the particle size and the refractive index difference between the UV curable solution and the ceramic powder [13]. Exposure (E) can be calculated by some parameters of the SLA machine, as described in equation (5):
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𝐸=
2 𝑃0 𝜋𝜔0 𝑣𝑠
(5)
where P0 (300mW) is the laser power, ω0 (120μm) is the beam radius, and vs (500-5000mm/s) is the scanning speed of the UV laser. To obtain a good cohesion between layers, the cured depth should be at least more than the layer thickness. The main purpose of this chapter is to evaluate the cured depth, so as to make sure green parts can be formed. Fig.10 shows that the cured depth increases with the increase of exposure, but the tendency seems to be slower and slower, indicating that it is not feasible to increase the cured depth only by raising the exposure because of the limitation on laser power and scanning speed. Additionally, a higher solid loading suspension shows a lower cured depth attributing to the scattering and absorption of ceramic particles [26]. Therefore, considering that the cured depth should more than the layer thickness (100μm), the suspension with 52vol% HA ceramics was finally determined to avoid green parts laminating. Subsequently, according to equation (4), a critical exposure (Ec=20.3mJ/cm2) and a penetration depth (Dp=50.7μm) of the suspension were fitted out by Origin software.
3.3.1. Cured width The cured width (Cw) is also a very important factor in SLA processing, directly influencing the dimensional precision of green parts. Fig.11 indicates that a higher laser exposure represents a wider cured line. Additionally, the relationship between cured
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width and exposure is not linear growth, which is similar to the correlation between cured depth and exposure. The cured width increased rapidly as solid loading increased, especially at a high laser exposure. This can be explained by the scattering and absorption of the laser by the HA particles in suspension. The great amount of HA particles indeed hinder the penetration of laser, and scatter the laser to the surrounding. In this respect, a suspension with 52vol% is more reasonable to meet our requirement of fabricating porous HA scaffolds with high dimensional precision for bone tissue engineering.
3.3.3. Dimensional precision The developed HA suspension intends to be formed to porous scaffolds used for bone tissue engineering, so the dimensional precision is of prime important, especially for pore shape. There was an intricate effect of the solution (water, AM, MBAM, glycerol) on the dimensional precision, so a multiple-factor analysis was implemented. In this respect, an orthogonal experiment was designed to further optimize the formulation, preliminarily determined by the rheological property and curing behaviour. Firstly, green parts with small square holes (1mm×1mm) was fabricated from different suspensions, which contained deionized water (62wt%, 67wt%, 72wt%), AM (20wt%, 25wt%, 30wt%), MBAM (0.5wt%, 1wt%, 1.5wt%) and glycerol. Obviously, there were only three independent factors with three levels in this experimental design, and the concentration of glycerol is not an independent factor, which can be calculated by the 18
other three. Then, the dimensional precision (p) was described as equation (6), in order to make the following analysis.
p 1
d m d
(6)
where d is the designed value, m is the measured value. That is to say, a higher p represents a higher dimensional precision. According to the rule of orthogonal experiment, the design and results of the experiment was shown in Table.1 and Table.2. The effect of the three factors on the dimensional precision is quite intricate. However, from the results of the orthogonal experiment, it can be concluded that the concentration of MBAM has a more significant influence on dimensional precision at the given levels. In addition, an optimized combination (67wt% water, 25wt% AM, 1.5wt% MBAM and 6.5wt% glycerol) was proposed, with a maximum dimensional precision of 92%.
3.4. Characterization of the sintered HA Based on the results of rheological property and curing behaviour of the suspension, a preliminary formulation was determined, which has 52vol% HA ceramic with a particle size of 1μm or 5μm, and with a PAA-NH4 concentration of 0.3mg/m2. In the following, the formulation was further optimized by mechanical property and shrinkage rate of the sintered HA parts. Fig. 12 shows a green HA part and a sintered one. 19
A bigger particle size in ceramic suspensions indeed represents a lower viscosity, but it also causes a poor strength of sintered parts. Fig. 13 shows that the strength is decreasing rapidly as the particle size increases. The compression strength of HA parts sintered from 5μm particles was only 15MPa. When the particle size decreased to 1μm, the strength reached up to 37MPa meeting the requirement of cancellous bone [27]. This can be explained by that a smaller particle has a bigger specific surface, so it possesses a higher surface energy to drive sintering, subsequently to get a denser part. However, in the respect of shrinkage rate, there was no such an obvious difference as the compression strength between particle sizes of 1μm and 5μm, because the debinding and sintering processes were combined action between the elimination of polymers and the densification of ceramic particles. Considering this, although the shrinkage of the part sintered from 1μm particles was 14%, a little higher than that of 5μm particles, the HA particles with a size of 1μm in the suspension was a relatively reasonable choice. As the specific forming mechanism of SLA, the shrinkage cannot be avoided. But the shrinkage rate can be taken account to CAD files to conduct dimension compensation. Additionally, we will optimize the formulation of suspension and the parameters of processing in our further study. In order to make sure that there was no change of chemical composition, the sintered HA parts were analyzed by an XRD machine. From the result of Fig.14, few differences between the original HA powder and the sintered HA part could be detected, which implies an ideal biological performance, like good biocompatibility, 20
osteoconductivity and osteoinductivity, just as the original HA had.
4. Conclusion In this paper, a novel aqueous HA suspension was developed for SLA applied to bone tissue engineering. The effect of surfactant concentration, solid loading and particle size on the rheology property was investigated to preliminarily determine a formulation of the suspension. Subsequently, the curing behaviour was explored to further optimize the solid loading based on the cured depth and width. Taking account to mechanical property and shrinkage rate, the suspension with HA particle size of 1μm was finally confirmed. The deionized water was used as solvent to dissolve AM and MBAM, which could be cured by UV after mixing with the photoinitiator. According to the results, among the three kinds of suspension with different particle sizes of 0.3μm, 1μm and 5μm, the viscosity decreased with an increase in the particle size. By the viscosity test and sedimentation experiment, a conclusion was drawn that the surfactant PAA-NH4 had an optimum concentration of about 0.3mg/m2.The PAA-NH4 coated on the surface of the HA particles had a maximum value, and the extra PAA-NH4 would spread in the liquid to block flowing, which explained the increased viscosity. Besides, considering of viscosity and curing activity, the suspension with 52% HA solid loading was finally determined, which had a critical exposure of 20.3mJ/cm2 and a penetration depth of 50.7μm. The compression strength of the sintered HA parts reached up to 37MPa 21
meeting the requirement of cancellous bone. Although the sintered part shrank by about 14%, but that does not deter its promising application prospect in the biomedical field to benefit thousands of patients. From the XRD pattern, the final HA part was similar to the original HA powders in chemical composition, which may imply ideal biological performances. Taking full advantage of SLA in fabricating complex bodies and the good biological performance of HA bioceramic, the aqueous HA suspension for SLA applied to bone tissue engineering was developed. Although the strength and shrinkage of the sintered HA parts are not perfect, the formulation of the aqueous suspension will be further optimized in the future study. In addition, much attention will be paid to the study of the mechanical property and biological performance of HA bone tissue engineering scaffolds, fabricated by SLA with this suspension. To promote the HA scaffolds into clinical application early, it requires concerted efforts of researchers from interdisciplinary fields. Acknowledgements The work is supported by National Natural Science Foundation of China (No. 51675312) and the National Key Research and Development Program of China (No. 2016YFB1101801).
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through
a
stereolithography-based additive manufacturing, Ceramics International, 43 (2017) 968-972. [16] M. Lasgorceix, E. Champion, T. Chartier, Shaping by microstereolithography and sintering of macro–micro-porous silicon substituted hydroxyapatite, Journal of the European Ceramic Society, 36 (2016) 1091-1101. [17] J. Brie, T. Chartier, C. Chaput, C. Delage, B. Pradeau, F. Caire, M.P. Boncoeur, J.J. Moreau, A new custom made bioceramic implant for the repair of large and complex craniofacial bone defects, J Craniomaxillofac Surg, 41 (2013) 403-407. [18] J.B. Park, Biomaterials science and engineering, IEEE Transactions on Biomedical Engineering, 1 (1985) 990-990. [19] G. Odian, Principles of Polymerization, McGraw-Hill, New York, 1970. [20] E. Landi, A. Tampieri, G. Celotti, S. Sprio, Densification behaviour and mechanisms of synthetic hydroxyapatites, Journal of the European Ceramic Society, 20 (2000) 2377-2387. [21] G. Daculsi, R.Z. Legeros, G. Grimandi, A. Soueidan, E. Aguado, E. Goyenvalle, J. Legeros, Effect of sintering process of HA/TCP bioceramics on microstructure, dissolution, cell proliferation and bone ingrowth, Key Engineering Materials, 361-363 (2008) 1139-1142. [22] B.A. Horri, P. Ranganathan, C. Selomulya, H. Wang, A new empirical viscosity
25
model for ceramic suspensions, Chemical Engineering Science, 66 (2011) 2798-2806. [23] T. Chartier, M. Ferrato, J.F. Baumard, Influence of the debinding method on the mechanical properties of plastic formed ceramics, Journal of the European Ceramic Society, 15 (1995) 899-903. [24] H.M. Shaw, M.J. Edirisinghe, Removal of binder from ceramic bodies fabricated using plastic forming methods. American Ceramic Society Bulletin, 72 (1993) 94-99. [25] I.M. Krieger, T.J. Dougherty, A mechanism for non-newtonian flow in suspensions of rigid spheres, Transactions of the Society of Rheology, 3 (2000) 137-152. [26] J. Tarabeux, V. Pateloup, P. Michaud, T. Chartier, Development of a numerical simulation model for predicting the curing of ceramic systems in the stereolithography process, Journal of the European Ceramic Society, 38 (2018). [27] A.H. Burstein, D.T. Reilly, M. Martens, Aging of bone tissue: mechanical properties, Journal of Bone & Joint Surgery American Volume, 58 (1976) 82.
26
Fig.1. Schematic diagram of SLA
27
Fig.2. Debinding and sintering for the green parts
28
Fig.3. Change of the HA before and after surface modification (the concentration of PAA-NH4 is 0.3mg/m2)
29
Fig.4. PAA-NH4 absorbed on the surface of a HA particle
30
Fig.5. Viscosity versus surfactant concentration (52% HA)
31
Fig.6. Zeta potential versus surfactant concentration (52% HA with a particle size of 1μm)
32
Fig.7. Remained particles in the suspension (52vol% HA with a particle size of 1μm)
33
Fig.8. Effect of HA solid loading on viscosity (5μm particle size and 0.3mg/m2 PAA-NH4)
34
Fig.9. Viscosity of the suspension with different particle sizes (52vol% HA and 0.3mg/m2 PAA-NH4)
35
Fig.10. Cured depth versus exposure
36
Fig.11. Cured width versus exposure
37
Fig.12. HA parts fabricated by SLA (a. green part, b. sintered part)
38
Fig.13. Compression strength and shrinkage rate of the sintered HA parts.
39
Fig.14. XRD pattern of HA (with a step size of 0.02° and step duration of 0.5s)
40
Table.1 Factors and levels
Factor 1 2
A (Water wt%) 62 67
B (AM wt%) 20 25
C (MBAM wt%) 0.5 1
3
72
30
1.5
Level
(1wt% photoinitiator 1173, 52vol% HA and 0.3mg/m2 PAA-NH4)
41
Table.2 Results of the orthogonal experiment Dimensional precision (%)
Factor
Experimental number A
B
C
1
1
1
1
87
2
1
2
2
85
3
1
3
3
89
4
2
1
2
83
5
2
2
3
92
6
2
3
1
83
7
3
1
3
90
8
3
2
1
91
9
3
3
2
85
K1
261
260
261
-
K2
258
268
253
-
K3
266
257
271
-
k1
87.0
86.7
87.0
-
k2
86.0
89.3
84.3
-
k3
88.7
85.7
90.3
-
R
2.7
3.6
6.0
-
Order Optimized level
C>B>A A2
B2
Optimal combination
C3 A2B2C3
42
-
PII: DOI: Reference:
S0272-8842(18)33169-9 https://doi.org/10.1016/j.ceramint.2018.11.063 CERI20037
To appear in: Ceramics International Received date: 30 July 2018 Revised date: 9 November 2018 Accepted date: 9 November 2018 Cite this article as: Zhen Wang, Chuanzhen Huang, Jun Wang and Bin Zou, Development of a Novel Aqueous Hydroxyapatite Suspension for Stereolithography Applied to Bone Tissue Engineering, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.11.063 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.
Development of a Novel Aqueous Hydroxyapatite Suspension for Stereolithography Applied to Bone Tissue Engineering Zhen Wanga, Chuanzhen Huanga*, Jun Wangb, Bin Zoua a.
Center for Advanced Jet Engineering Technologies (CaJET), Key Laboratory of
High-efficiency and Clean Mechanical Manufacture (Ministry of Education), National Demonstration Center for Experimental Mechanical Engineering Education (Shandong University), School of Mechanical Engineering, Shandong University, Jinan 250061, China b.
School of Mechanical and Manufacturing Engineering, The University of New South Wales (UNSW), Sydney, NSW 2052, Australia
*
Corresponding author: Tel. and Fax: +86 531 88396913. E-mail address: [email protected] (C. Z. Huang).
1
Abstract: Stereolithography (SLA) has a good ability to form complex bodies, while hydroxyapatite (HA) ceramic has good mechanical and biological properties because of its similarity in composition with natural bone. Some researchers tried combining SLA technology with HA bioceramic for bone tissue engineering. However, the ceramic suspension used in SLA generally has a relatively higher viscosity and are not environmentally friendly. In this paper, a novel aqueous HA suspension for SLA applied to bone tissue engineering was developed. The effect of surfactant concentration, solid loading and particle size on the rheological property of the HA suspension was investigated to preliminarily determine the formulation of the suspension. By a viscosity
2
test and a sedimentation experiment, a conclusion was drawn that the surfactant PAA-NH4 had an optimum concentration of about 0.3mg/m2. In addition, the maximum value of viscosity reached up to 7.3Pa·s, when the solid loading was 56vol%. Taking account of viscosity of suspension and the strength of sintered parts, the particle size of 1μm was relatively reasonable to be used in the HA suspension. According to the study of curing behaviour, the suspension had a critical exposure of 20.3mJ/cm2 and a penetration depth of 50.7μm. The final sintered HA parts provided a strength of 37MPa meeting the requirement of cancellous bone, and was similar to original HA powders in chemical composition implying an ideal biological performance. Therefore, this kind of HA suspension has a broad prospect in bone tissue engineering applied to the treatment of bone disease for thousands of people. Keywords: Stereolithography; Hydroxyapatite; Suspension; Bioceramic 1. Introduction Recently, number of people suffering from various orthopaedics diseases caused by traffic accidents, bone tumors, and infections is increasing gradually [1-2]. Although, bone can heal small segment defects by itself, it is still a big challenge for treating non-healable sized bone defects. Therefore, a bone transplant surgery is usually performed on patients to help restore their natural bone function, including three main strategies such as autograft, allograft and xenograft. However, each of the mentioned strategies has its pre-operation or post-operation limitations. The autograft has always been regarded as the gold standard of the bone transplantation, but the source of the
3
autograft is restricted seriously, besides, it can cause a secondary damage i.e. physical and mental illness to patients. The allograft and xenograft have much broader sources, but they are usually rejected by the immune system of the receiving patients. Fortunately, with the development of science and technology, a new strategy, bone tissue engineering, was proposed to treat bone defects. Tissue engineering (TE) is a promising field for the reconstruction of bone, which is an interdisciplinary field that applies the principles of engineering and life sciences to develop biological substitutes for restoring, maintaining, or improving the tissue function [3]. Scaffolds play a very important role in bone tissue engineering so as to provide a suitable environment for cell migration, proliferation and vascularization, besides acting as a supporting structure [4-6]. Some ideal properties are required for the bone tissue engineering scaffold [7-11], for example, the scaffold should have appropriate pore size, morphology and porosity, in addition, the material for scaffold should have osteoinduction and good biocompatibility. Moreover, the controllable biodegradability is also a desired property of the bone tissue engineering scaffold which should have a suitable degradation rate corresponding to the cell growth rate. Additionally, the scaffold should have sufficient mechanical strength to support human body, and appropriate elasticity modulus to match that of bone. To satisfy the requirement of scaffold, additive manufacturing, commonly called 3D printing, is a very promising technology and is also a greatly suitable way to manufacture scaffolds, especially, stereolithography (SLA) has
4
relatively high processing accuracy among the additive manufacturing technologies, which can fabricate intricate structures and complex surfaces to meet the specific needs of scaffolds. Although most of the commercial materials for SLA are light curing resins, researchers have developed some kinds of ceramic suspensions [12-14], which were formulated by mixing ceramics powders with the light curing resins to finally fabricate ceramic parts after exposure to ultraviolet (UV) light. Wu et al. developed an approach to fabricate dense zirconia-toughened alumina ceramics with excellent properties by SLA [15]. Chartier et al. fabricated bioceramic implants by SLA with a developed HA suspension [16-17]. However, these resin-based suspensions have relatively higher viscosity and are not environmentally friendly. Hydroxyapatite (HA) with the chemical formula Ca10(PO4)6(OH)2, which is the basic composition of the bone mineral component, has excellent biological and mechanical performances [18]. Therefore, in this paper a novel aqueous HA suspension was developed for SLA applied to bone tissue engineering. To be used in SLA, the ceramic suspensions should have the following properties: (1) A low viscosity to make sure the scraper of a SLA machine can spread ceramic suspensions homogeneously and levelly layer by layer; (2) A sufficient cured depth to confer an overcure at the interface so as to provide a good cohesion between two cured layers. (3) A narrow cured width to offer a high accuracy of manufacturing for complex shaped parts. 5
A higher solid loading suspension always means a lower shrinkage of the sintered ceramic parts, but that also represents a higher viscosity of the suspension and a worse reactivity of the UV reactive system. As a consequence, it is of prime important to make a balance between the solid loading and the curing reactivity. Taking this into account, the effect of some parameters such as surfactant concentration, solid loading and particle size on rheological property of ceramic suspensions was investigated to initially identify a formulation of ceramic suspensions. Subsequently, the curing behavior and mechanical property was explored to further optimize the formulation. Finally, an XRD analysis was implemented on sintered HA ceramic parts to make sure that the HA parts still remained good biological property without any change of chemical composition.
2. Materials and methods 2.1. Powder surface modification In order to reduce the viscosity of ceramic suspensions, ammonium polyacrylate (PAA-NH4, Adamas) was used as surfactant to modify the surface of HA powders. A small amount of surfactant has an obvious influence on rheological property but a minimal impact on curing behavior. In this respect, the amount of surfactant was preliminarily investigated. Three kinds of HA ceramic powders (DKN, China) with different particle sizes and specific surface areas (d50=0.3μm, 1μm, 5μm, and BET=6m2/g, 2.1m2/g, 0.6m2/g, respectively) were used as the ceramic filler in the following study. 6
Firstly, PAA-NH4 was dissolved in the deionized water at 35℃-45℃, stirring for 10 minutes. Secondly, the HA powders dispersed in the solution by an ultrasonic homogenizer (1500F, Scientz) at 1500W and 20KHz for 10 minutes. Subsequently, after being milled for 4 hours by a planetary ball milling, the suspension was freeze dried by a lyophilizer. Following, the dried HA powders modified by PAA-NH4 were passed through a 100-mesh sieve to get rid of agglomerated particles. The change of particle size, before and after surface modification, was measured by a dynamic laser light scattering (MS-2000, Malvern). Then, the wettability was investigated by a contact angle meter (JC2000D1, Shanghai Zhongchen).
2.2. HA suspension preparation Generally, UV curable ceramic suspensions, used in stereolithography, contain following components: monomer, diluent (or solvent), ceramic powder, photoinitiator and some additives such as dispersant, defoamer and light absorber. The aqueous solvent has a lower viscosity than the resin-based solvent at the same temperature and shear rate, besides, it is much more environmentally friendly to conform with the encouragement of modern society. Considering these advantages of the aqueous solvent, it was used to dissolve monomers and HA particles in this paper. Acrylamide (AM) and N-N’ methylenebisacrylamide (MBAM) were selected as the organic monomer system to be dissolved in deionized water. Subsequently, a certain amount of glycerol and modified HA were added into the solution in an ultrasonic 7
homogenizer (1500F, Scientz) at 1500W and 20KHz for 15 minutes. At last, the photocure-1173 (HongTai, China) was mixed with the suspension by a planetary ball mill for 10 hours at a revolving speed of 500rpm-1000rpm. In order to preliminarily determine a formulation of HA suspension and to achieve a better manufacturability, the rheological property of the suspensions with different surfactant concentration, solid loading and HA particle size was measured by the rheometer (Kinexus, Malvern) at a shear rate from 0.1-100s-1. In addition, a zeta potential meter (Zetaplus, Brookhaven) was used to analyse the effect of the surfactant PAA-NH4 on viscosity of the suspension, and a sedimentation experiment was conducted to test the stability of the suspension.
2.3. Stereolithography of the HA suspension The HA suspension was added into the vat of a SLA machine (SPS450B, Shaanxi Hengtong Co. Ltd, China) to fabricate green parts. The platform was moved to a position 0.1mm under the top surface of the suspension by an elevator, which was controlled by a computer. When the UV laser scanned the surface of the suspension, the photoinitiator splits primitive radicals. Subsequently, the radicals react with the monomer to produce a polymer chain, and then the polymer chain propagates continuously with more monomers to form a chain reaction. At last, it terminates when two of the reactive polymer chains combine together [19], as shown in equation (1). Therefore, HA particles were anchored by the crosslinked polymers. 8
S h R0 R M R 0 1 Rn 1 M Rn Rn Rm Pm+n
(1)
where S is the photo initiator, hγ is the photon, R0 is the primitive radical, M is the monomer, R is the reactive polymer chain, and P is the final polymer molecule. Then, the platform moved down 0.1mm to submerge the solidified layer by the new suspension, at the same time, a scraper was required to level the surface and absorb the bubble. After the suspension was solidified layer by layer, a resulting green part was obtained with HA particles and crosslinked polymers. The schematic diagram of SLA process is shown in Fig.1. The investigation on the curing behavior of the ceramic suspension is important to form a strong green part with a high dimensional precision. Especially, the HA solid loading in the suspension has a significant influence on the curing behavior. Therefore, the cured depth (Cd) and the cured width (Cw) was measured by a digital caliper, then the critical exposure (Ec) was calculated by fitting in Origin software. Subsequently, the dimensional precision of green parts was measured by a laser microscope (Keyence, Japan). Finally, an orthogonal array for experiment was designed to further optimize the formulation, which had been preliminarily identified by the exploration on rheological property and curing behaviour.
9
2.4. Debinding and sintering To get a strong HA ceramic part used for bone tissue engineering and remove the toxic substances for cells, four processes of rinsing, drying, debinding and sintering were required on the green part. After the green part was rinsed with alcohol, the residual water and alcohol in the part needed to be dried. A special drying approach was used [20] based on PEG400 liquid desiccant to make sure a uniform extraction rate in all directions of the part. In order to avoid cracking of the green part and transforming of the HA composition, during debinding and sintering, the maximum temperature (1180℃) and heating rate must be controlled very well. Referred to the study of Daculsi et al. [21] and through continuous experimental trying, finally a heating route in a vacuum furnace was determined as shown in Fig.2. The organic polymers in green parts were removed before temperature rising to about 500℃, so a very low heating rate was conducted in this stage to reduce deformation and to avoid cracking. Subsequently, the temperature was heated to 1080℃ with a little higher heating rate, which actually was a pre-sintering stage from 500℃ to 1080℃. After the HA parts were cooled to room temperature, a new sintering stage was conduct to 1180℃ with a higher heating rate. Although this heating route was time-consuming, the sintered HA parts were a little bit stronger. Aiming to be applied in bone tissue engineering field, the sintered HA ceramic should require an adequate mechanical property, especially compression strength, and also a small shrinkage. Therefore, the effect of particle size on the mechanical property 10
was investigated by a compression test on a material testing machine (Instron8801, USA) at a speed of 1mm/min. Additionally, the shrinkage rate of sintered HA parts was measured by a digital caliper. Taking account to that a transformation of chemical composition of the HA can cause the biological property declining, the original HA powders and the final HA scaffolds were evaluated by the XRD (DMAX-2500PC, Rigaku, Japan) with CuKα radiation (λ=1.5418A°) in the 2θ range of 20°–60° by the step scanning mode, with a step size of 0.02° and step duration of 0.5s.
3. Results and discussion 3.1. Characterization of powder To explore the relationship between the size of the HA particle and the rheological property of the suspension, the dynamic laser light scattering (MS-2000, Malvern) was used to determine the HA particle size before and after surface modification. Fig.3 indicates that the particle size increases after surface modification, especially for small particles. Although the three particle sizes increased by 27%-110%, the ceramic particles can still be used in suspension, because there was no significant agglomeration. Before mixing the modified HA with the aqueous solution, the wettability of the original HA and the modified HA was determined to predict the trend of rheological
11
property of the HA suspension. Fig.3 shows that the contact angles of the three different particles were obviously dropped. So a ceramic suspension with homogeneously dispersed particles and lower viscosity was foreseen, used in SLA to fabricate bone tissue engineering scaffolds.
3.2. Rheological property of the HA suspension In order to require an ability of self-levelling, ceramic suspensions require a viscosity lower than 3Pa·s at 30s−1shear rate [22]. In this study, a developed scraper in the SLA machine spread ceramic suspensions on a platform with a constant speed (4mm/s), so the spreading shear rate was 40s−1, calculated by equation (2):
𝛾̇ =
𝑣 𝑡
(2)
where 𝛾̇ is the shear rate, v is the spreading speed, and t is the thickness of a spread layer (0.1mm). Based on the rheological property of the HA suspension, the formulation of the novel suspension was attempted to be preliminarily determined by single-factor analysis in the following study. 3.2.1. Effect of surfactant concentration The effect of surfactant concentration on rheological property was firstly investigated to reduce the viscosity of the suspension, because a small amount of surfactant has an obvious influence. PAA-NH4 is a kind of organic polymer with a long 12
carbon chain. One end of the chain can be absorbed on the surface of HA ceramic particles, and the other end stretch out to liquid. The surface modified particles disperse more homogeneously attributing to the steric-hinerance effect of the long chain and the electrostatic repulsive force of the (–COO-) group, as shown in Fig.4. The viscosity of suspension with different particle sizes and surfactant concentration was tested. As shown in Fig.5, the viscosity of the suspension with 5μm HA particle size was the lowest among the three particle seizes. Moreover, the viscosity reduced with the concentration of PAA-NH4 increasing about from 0mg/m2 to 0.3mg/m2, and then almost plateauing from 0.3mg/m2 to 0.35mg/m2. Subsequently, the viscosity rose up with the concentration of PAA-NH4 over 0.35mg/m2. The zeta potential represents the electrostatic repulsion, which is the major factor causing this phenomenon, as shown in Fig.6. More specifically, there were more surface charges on the surface of the HA particle because of the absorption of PAA-NH4. The surface charges thus improved the surface potential of the HA particle, and increased the electrostatic repulsion. Therefore, the suspension dispersed more uniformly, which attributed to better fluidity and lower viscosity. When the concentration of PAA-NH4 was over 0.35mg/m2, the adsorption capacity of the HA particle reached a saturation point. At the same time, the concentration of macromolecules dissolved in the liquid improved, which formed bridges between particles to hinder the suspension flowing. Furthermore, a sedimentation experiment was conducted to further determine the concentration of PAA-NH4. Fig.7 illustrates that the storage time (1 day, 5 days or 10
13
days) is the most influential factors in the sedimentation of HA particles. Only 80.3% of particles, without PAA-NH4 modification, remained uniformly in the suspension after 10 days. The remaining particles increased at first with the increase of the concentration of PAA-NH4, and then it dropped after the concentration of PAA-NH4 over 0.3mg/m2. Therefore, the result shows that PAA-NH4 with a concentration of about 0.3mg/m2 is suitable for the developed HA suspension.
3.2.2. Effect of solid loading According to the previous studies of T. Chartier [23] and H. M. Shaw [24], the suspension used in SLA should contains at least 50vol% ceramic particles in order to provide a dense part after debinding and sintering. However, a high solid loading suspension represents a high viscosity and a low cured depth. To preliminary determine the ceramic volume fraction in the formulation, the relationship between solid loading and viscosity was investigated. Different amounts of HA (50vol%, 52vol%, 54vol%, 56vol%) were added into the aqueous solution. Fig.8 illustrates that the viscosity of the suspension increased rapidly with the solid loading increasing. When the solid loading was 56vol%, the maximum value of viscosity reached up to 7.3Pa·s, which was too high to be used by a SLA machine. The Krieger-Dougherty [25] equation can be used to describe this relationship: =
(
)
[ ]
(3)
where, η is the viscosity of a suspension contained the φ volume percent of solid 14
particles, ηs is the viscosity of the medium and φmax is the maximum packing fraction of solid particles in the suspension. [η] is the intrinsic viscosity depended on the shape of the particles, here it was assumed that the particles are spheres, [η]=2.5. As the volume fraction of HA particles in a suspension increases, it implies that the particles become more numerous in each unit volume. In other words, the distance between particles is shortened, so the interaction force increases which causes the flow resistance to improve i.e. the viscosity increases. However, the experimental curve does not fit well with the calculated curve, which may be attributed to the assumption that the particles were spheres. In fact, the HA particles in the suspension have no regular shapes.
3.2.3. Effect of particle size Based on the study of the relationship between solid loading and viscosity, a formulation of suspension contained 52% or 54vol% HA powders was determined. Then the effect of particle size on rheological property was investigated to further reduce the viscosity of the suspension. As shown in Fig.9, all the suspensions revealed typical shear thinning behaviour that the viscosity decreased with an increase in the share rate. In addition, there were some differences among the suspensions with different particle sizes. The viscosity of the suspension with bigger particle size was much lower, especially at the low share rates, and the gap of viscosity narrowed as the share rate increased. When a suspension contains ceramic with the same volume
15
fraction, in a given volume unit, the smaller the particle is, the more particles present. Maybe the increasing viscosity can be explained by the increase of the interaction forces between the closer particles. From the results, the suspension with 0.3μm HA particles was not suitable for SLA processing because of high viscosity.
3.3. Curing behaviour of the suspension An aqueous HA suspension, which contained 52% or 54vol% HA with a particle size of 1μm, was prepared to be cured by the SLA machine so as to evaluate the curing behaviour. 3.3.1. Cured depth The cured depth (Cd) can be derived from the Beer-Lambert law, as described in equation (4): 𝐸
𝐶𝑑 = 𝐷𝑝 ln ( ) 𝐸𝑐
(4)
where Dp is the depth of light penetration, E is the exposure (the radiant energy on a unit area) and Ec is the critical exposure (the minimal exposure to initiate polymerization). For a ceramic loaded suspension, Dp is a function of the volume fraction of powders, the particle size and the refractive index difference between the UV curable solution and the ceramic powder [13]. Exposure (E) can be calculated by some parameters of the SLA machine, as described in equation (5):
16
𝐸=
2 𝑃0 𝜋𝜔0 𝑣𝑠
(5)
where P0 (300mW) is the laser power, ω0 (120μm) is the beam radius, and vs (500-5000mm/s) is the scanning speed of the UV laser. To obtain a good cohesion between layers, the cured depth should be at least more than the layer thickness. The main purpose of this chapter is to evaluate the cured depth, so as to make sure green parts can be formed. Fig.10 shows that the cured depth increases with the increase of exposure, but the tendency seems to be slower and slower, indicating that it is not feasible to increase the cured depth only by raising the exposure because of the limitation on laser power and scanning speed. Additionally, a higher solid loading suspension shows a lower cured depth attributing to the scattering and absorption of ceramic particles [26]. Therefore, considering that the cured depth should more than the layer thickness (100μm), the suspension with 52vol% HA ceramics was finally determined to avoid green parts laminating. Subsequently, according to equation (4), a critical exposure (Ec=20.3mJ/cm2) and a penetration depth (Dp=50.7μm) of the suspension were fitted out by Origin software.
3.3.1. Cured width The cured width (Cw) is also a very important factor in SLA processing, directly influencing the dimensional precision of green parts. Fig.11 indicates that a higher laser exposure represents a wider cured line. Additionally, the relationship between cured
17
width and exposure is not linear growth, which is similar to the correlation between cured depth and exposure. The cured width increased rapidly as solid loading increased, especially at a high laser exposure. This can be explained by the scattering and absorption of the laser by the HA particles in suspension. The great amount of HA particles indeed hinder the penetration of laser, and scatter the laser to the surrounding. In this respect, a suspension with 52vol% is more reasonable to meet our requirement of fabricating porous HA scaffolds with high dimensional precision for bone tissue engineering.
3.3.3. Dimensional precision The developed HA suspension intends to be formed to porous scaffolds used for bone tissue engineering, so the dimensional precision is of prime important, especially for pore shape. There was an intricate effect of the solution (water, AM, MBAM, glycerol) on the dimensional precision, so a multiple-factor analysis was implemented. In this respect, an orthogonal experiment was designed to further optimize the formulation, preliminarily determined by the rheological property and curing behaviour. Firstly, green parts with small square holes (1mm×1mm) was fabricated from different suspensions, which contained deionized water (62wt%, 67wt%, 72wt%), AM (20wt%, 25wt%, 30wt%), MBAM (0.5wt%, 1wt%, 1.5wt%) and glycerol. Obviously, there were only three independent factors with three levels in this experimental design, and the concentration of glycerol is not an independent factor, which can be calculated by the 18
other three. Then, the dimensional precision (p) was described as equation (6), in order to make the following analysis.
p 1
d m d
(6)
where d is the designed value, m is the measured value. That is to say, a higher p represents a higher dimensional precision. According to the rule of orthogonal experiment, the design and results of the experiment was shown in Table.1 and Table.2. The effect of the three factors on the dimensional precision is quite intricate. However, from the results of the orthogonal experiment, it can be concluded that the concentration of MBAM has a more significant influence on dimensional precision at the given levels. In addition, an optimized combination (67wt% water, 25wt% AM, 1.5wt% MBAM and 6.5wt% glycerol) was proposed, with a maximum dimensional precision of 92%.
3.4. Characterization of the sintered HA Based on the results of rheological property and curing behaviour of the suspension, a preliminary formulation was determined, which has 52vol% HA ceramic with a particle size of 1μm or 5μm, and with a PAA-NH4 concentration of 0.3mg/m2. In the following, the formulation was further optimized by mechanical property and shrinkage rate of the sintered HA parts. Fig. 12 shows a green HA part and a sintered one. 19
A bigger particle size in ceramic suspensions indeed represents a lower viscosity, but it also causes a poor strength of sintered parts. Fig. 13 shows that the strength is decreasing rapidly as the particle size increases. The compression strength of HA parts sintered from 5μm particles was only 15MPa. When the particle size decreased to 1μm, the strength reached up to 37MPa meeting the requirement of cancellous bone [27]. This can be explained by that a smaller particle has a bigger specific surface, so it possesses a higher surface energy to drive sintering, subsequently to get a denser part. However, in the respect of shrinkage rate, there was no such an obvious difference as the compression strength between particle sizes of 1μm and 5μm, because the debinding and sintering processes were combined action between the elimination of polymers and the densification of ceramic particles. Considering this, although the shrinkage of the part sintered from 1μm particles was 14%, a little higher than that of 5μm particles, the HA particles with a size of 1μm in the suspension was a relatively reasonable choice. As the specific forming mechanism of SLA, the shrinkage cannot be avoided. But the shrinkage rate can be taken account to CAD files to conduct dimension compensation. Additionally, we will optimize the formulation of suspension and the parameters of processing in our further study. In order to make sure that there was no change of chemical composition, the sintered HA parts were analyzed by an XRD machine. From the result of Fig.14, few differences between the original HA powder and the sintered HA part could be detected, which implies an ideal biological performance, like good biocompatibility, 20
osteoconductivity and osteoinductivity, just as the original HA had.
4. Conclusion In this paper, a novel aqueous HA suspension was developed for SLA applied to bone tissue engineering. The effect of surfactant concentration, solid loading and particle size on the rheology property was investigated to preliminarily determine a formulation of the suspension. Subsequently, the curing behaviour was explored to further optimize the solid loading based on the cured depth and width. Taking account to mechanical property and shrinkage rate, the suspension with HA particle size of 1μm was finally confirmed. The deionized water was used as solvent to dissolve AM and MBAM, which could be cured by UV after mixing with the photoinitiator. According to the results, among the three kinds of suspension with different particle sizes of 0.3μm, 1μm and 5μm, the viscosity decreased with an increase in the particle size. By the viscosity test and sedimentation experiment, a conclusion was drawn that the surfactant PAA-NH4 had an optimum concentration of about 0.3mg/m2.The PAA-NH4 coated on the surface of the HA particles had a maximum value, and the extra PAA-NH4 would spread in the liquid to block flowing, which explained the increased viscosity. Besides, considering of viscosity and curing activity, the suspension with 52% HA solid loading was finally determined, which had a critical exposure of 20.3mJ/cm2 and a penetration depth of 50.7μm. The compression strength of the sintered HA parts reached up to 37MPa 21
meeting the requirement of cancellous bone. Although the sintered part shrank by about 14%, but that does not deter its promising application prospect in the biomedical field to benefit thousands of patients. From the XRD pattern, the final HA part was similar to the original HA powders in chemical composition, which may imply ideal biological performances. Taking full advantage of SLA in fabricating complex bodies and the good biological performance of HA bioceramic, the aqueous HA suspension for SLA applied to bone tissue engineering was developed. Although the strength and shrinkage of the sintered HA parts are not perfect, the formulation of the aqueous suspension will be further optimized in the future study. In addition, much attention will be paid to the study of the mechanical property and biological performance of HA bone tissue engineering scaffolds, fabricated by SLA with this suspension. To promote the HA scaffolds into clinical application early, it requires concerted efforts of researchers from interdisciplinary fields. Acknowledgements The work is supported by National Natural Science Foundation of China (No. 51675312) and the National Key Research and Development Program of China (No. 2016YFB1101801).
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Fig.1. Schematic diagram of SLA
27
Fig.2. Debinding and sintering for the green parts
28
Fig.3. Change of the HA before and after surface modification (the concentration of PAA-NH4 is 0.3mg/m2)
29
Fig.4. PAA-NH4 absorbed on the surface of a HA particle
30
Fig.5. Viscosity versus surfactant concentration (52% HA)
31
Fig.6. Zeta potential versus surfactant concentration (52% HA with a particle size of 1μm)
32
Fig.7. Remained particles in the suspension (52vol% HA with a particle size of 1μm)
33
Fig.8. Effect of HA solid loading on viscosity (5μm particle size and 0.3mg/m2 PAA-NH4)
34
Fig.9. Viscosity of the suspension with different particle sizes (52vol% HA and 0.3mg/m2 PAA-NH4)
35
Fig.10. Cured depth versus exposure
36
Fig.11. Cured width versus exposure
37
Fig.12. HA parts fabricated by SLA (a. green part, b. sintered part)
38
Fig.13. Compression strength and shrinkage rate of the sintered HA parts.
39
Fig.14. XRD pattern of HA (with a step size of 0.02° and step duration of 0.5s)
40
Table.1 Factors and levels
Factor 1 2
A (Water wt%) 62 67
B (AM wt%) 20 25
C (MBAM wt%) 0.5 1
3
72
30
1.5
Level
(1wt% photoinitiator 1173, 52vol% HA and 0.3mg/m2 PAA-NH4)
41
Table.2 Results of the orthogonal experiment Dimensional precision (%)
Factor
Experimental number A
B
C
1
1
1
1
87
2
1
2
2
85
3
1
3
3
89
4
2
1
2
83
5
2
2
3
92
6
2
3
1
83
7
3
1
3
90
8
3
2
1
91
9
3
3
2
85
K1
261
260
261
-
K2
258
268
253
-
K3
266
257
271
-
k1
87.0
86.7
87.0
-
k2
86.0
89.3
84.3
-
k3
88.7
85.7
90.3
-
R
2.7
3.6
6.0
-
Order Optimized level
C>B>A A2
B2
Optimal combination
C3 A2B2C3
42
-