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nanosized hydroxyapatite composite structures by stereolithography
Acta Biomaterialia 9 (2013) 5989–5996
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Preparation of designed poly(D,L-lactide)/nanosized hydroxyapatite composite structures by stereolithography A. Ronca a,⇑, L. Ambrosio a, D.W. Grijpma b,c a
Institute of Composite and Biomedical Materials, National Research Council of Italy, Piazzale Tecchio 80, 80125 Naples, Italy MIRA Institute for Biomedical Technology and Technical Medicine, Department of Polymer Chemistry and Biomaterials, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands c W.J. Kolff Institute, Department of Biomedical Engineering, University Medical Center Groningen and University of Groningen, P.O. Box 196, 9700 AD Groningen, The Netherlands b
a r t i c l e
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Article history: Received 10 September 2012 Received in revised form 12 November 2012 Accepted 4 December 2012 Available online 8 December 2012 Keywords: Nano-composites Biodegradable networks CAD designed scaffold Stereolithography Bone tissue engineering
a b s t r a c t The preparation of scaffolds to facilitate the replacement of damaged tissues and organs by means of tissue engineering has been much investigated. The key properties of the biomaterials used to prepare such scaffolds include biodegradability, biocompatibility and a well-defined three-dimensional 3-Dpore network structure. In this study a poly(D,L-lactide)/nanosized hydroxyapatite (PDLLA/nano-Hap) composite resin was prepared and used to fabricate composite films and computer designed porous scaffolds by micro-stereolithography, mixing varying quantities of nano-Hap powder and a liquid photoinitiator into a photo-crosslinkable PDLLA-diacrylate resin. The influence of nano-Hap on the rheological and photochemical properties of the resins was investigated, the materials being characterized with respect to their mechanical, thermal and morphological properties after post-preparation curing. In the cured composites stiffness was observed to increase with increasing concentration of nanoparticles. A computer designed construct with a pore network based on the Schwarz architecture was fabricated by stereolithography using PDLLA/nano-Hap composite resins. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In bone tissue engineering the scaffold plays a fundamental role as a support for cell adhesion and proliferation and for tissue regeneration. An effective scaffold must exhibit the following properties and attributes: high porosity and a requisite pore size for the host cells; suitable surface properties to allow cell adhesion, differentiation and proliferation; adequate mechanical stability to maintain the predesigned structure; no cytotoxicity and osteoconductivity [1]. The internal pore architecture, including pore size, pore shape and pore connection pattern, are important parameters in controlling the extent of bone formation [2], influencing the path of bone regeneration, and determining the mechanical properties of the implants [3]. However, scaffolds prepared by conventional techniques often result in inhomogeneous structures with irregular pore sizes and pore size distributions, poor pore interconnectivity and inferior mechanical properties [4]. Rapid prototyping allows the preparation of scaffolds with optimal properties with regard to pore structure, geometry and connectivity, mechanical characteristics, cell seeding efficiency, and transport of nutrients and metabolites [5]. Stereolithography is an additive fabrication process that uses a liquid light-curable ⇑ Corresponding author. Tel.: +39 3283724007; fax: +39 0812425932. E-mail addresses: [email protected], [email protected] (A. Ronca).
photopolymer and a laser to create three-dimensional (3-D) structures, a technique introduced in the late 1980s [6]. Although several alternative approaches have been developed since then, stereolithography remains one of the most powerful and versatile solid freeform techniques. It allows 3-D (micro) fabrication of solid structures from models created using computer-aided design (CAD) programs [7]. The working principle of stereolithography is based on the spatially controlled solidification of a liquid photopolymerizable resin. Using a computer-controlled laser beam or digital light projection and a computer-driven building platform a 3-D object is constructed in a layer by layer fashion [8]. To do this the design of the object to be built is numerically analyzed to define the different layers that are to be manufactured [9]. The sequential polymerization of layers with different illumination patterns leads to the creation of complex 3-D objects, described by Griffith and Halloran [10]. The number of available stereolithography resins suitable for use in biomedical applications is limited [11]. General purpose photocurable materials are predominantly acrylate-based resins [12], and among these poly(ethylene glycol) dimethacrylate has been used to fabricate non-degradable cell-containing hydrogels in predesigned shapes [13]. For bone tissue engineering strong and rigid biodegradable materials are required. Poly(lactide) is such a material, with a long track record of successful application in clinical use and which has been used for the preparation of
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.12.004
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tissue engineering scaffolds. Low molecular weight poly(D,L-lactide) (PDLLA) oligomers functionalized with methacrylate groups can be crosslinked to form rigid polymer networks [14]. However, when the crosslink density of these networks is too high they are quite brittle, which limits their usefulness [15]. Composites of polymers and ceramics are being developed to enhance the mechanical properties and improve tissue interactions [16]. The development of composites which combine biodegradable polymers and bioactive ceramics or glasses for use in tissue engineering has been extensively discussed [17,18]. Hydroxyapatite (Ca10(PO4)6(OH)2) (Hap) has attracted much interest due to its chemical similarity to the calcium phosphate mineral present in biological hard tissues [19]. Hap has been used for a variety of biomedical applications, for example as a matrix for controlled drug release and as a carrier material in bone tissue engineering [20]. Orthopaedic research suggests that osteoconductivity would be optimal when synthetic Hap resembles the bone mineral with regard to composition, size and morphology [21]. In natural bone the nanometer scale of the inorganic apatite component is considered to be important for the biological and mechanical properties of the tissue [22]. Well-dispersed nanosized hydroxyapatite (nano-Hap) with an ultrafine structure has the potential to improve the performance of composites, because these particles have a high surface area to volume ratio and their surfaces have minimal defects [21]. Several nano-Hap/polymer composite tissue engineering scaffolds have been developed for use as substitute bone [23]. It remains a challenge to extend the stereolithography technique to allow the processing of nanosized composite ceramic and polymer resins to fabricate scaffolds for bone tissue regeneration. In this paper we report on the use of nano-Hap powders, dispersed in a photo-curable PDLLA-based resin, to prepare biodegradable nanocomposite structures by stereolithography. Varying amounts of nano-Hap were used, and the effects of nano-Hap content on the photo-crosslinking kinetics and network properties were investigated. In this way porous structures designed for possible use in bone tissue engineering were prepared at high resolution. 2. Materials and methods 2.1. Materials D,L-lactide was obtained from Purac Biochem (The Netherlands). 1,6-Hexanediol, stannous octoate (SnOct), methacrylic anhydride (MAAH), N-methyl-2-pyrollidone (NMP) and tocopherol were purchased from Sigma–Aldrich (The Netherlands) and used without further purification. Triethylamine (TEA) (Fluka, Switzerland) and technical grade isopropanol and acetone (Biosolve, The Netherlands) were used as received. Orasol Orange G was kindly donated by Ciba Specialty Chemicals (Switzerland). Lucirin TPO-L (ethyl2,4,6-trimethyl benzoyl phenyl phosphinate) was kindly provided by BASF (Germany). Analytical grade dichloromethane (Biosolve) was distilled from calcium hydride (Acros Organics, Belgium). Nano-sized hydroxyapatite (particle size <200 nm) was obtained from Sigma–Aldrich.
acid, the by-product of the functionalization reaction, was scavenged using TEA. An excess of 20 mol.% MAAH and TEA per hydroxyl group was used. The solution was filtered and precipitated into cold isopropanol. The PDLLA dimethacrylate macromers were isolated, washed with water and freeze-dried. 2.3. Formulation of composite resins Nano-Hap powder (particle size <200 nm) was used to prepare a series of composite PDLLA/nano-Hap resins for application in stereolithography. Homogeneous nanosized composite resins were prepared by mixing 15 g of PDLLA macromer and different amounts of nano-Hap (up to 20 wt.% with respect to macromer) in NMP. The dispersions were kept at 60 °C overnight, and sonicated for 15 min before use in order to ensure homogeneity. Dispersion viscosity was assessed at 25 °C using a Brookfield DV-E rotating spindle viscometer equipped with a small sample adapter. The shear rate was varied by adjusting the rotation speed of the spindle (Brookfield s21 spindle) between 0.6 and 4 r.p.m. 2.4. Stereolithography To fabricate PDLLA/nano-Hap composite porous structures by stereolithography 4 wt.% Lucirin-TPO photoinitiator, 0.2 wt.% tocopherol inhibitor (to prevent premature polymerization) and 0.15 wt.% Orange Orasol G dye (to set the requisite light penetration depth) were added to the PDLLA macromer solutions in NMP. Table 1 gives an overview of the different composite resins prepared. The weight percentages of the various ingredients cited in Table 1 were calculated with respect to PDLLA macromer content, 15 g of PDLLA macromer being used in each resin preparation. A commercial stereolithography apparatus (SLA) (Perfactory Mini Multilens, Envisiontec) was employed to build films and designed porous structures. In stereolithography a CAD file is used to describe the size and geometry of the structures to be built. This file is converted into STL format and virtually sliced into the layers used in the layer by layer fabrication process [7]. By convention the slices are in the X,Y-plane while the specimen is built in the Z-direction [24]. The building process involves subsequent blue light projection (wavelength 400–550 nm with a peak at 440 nm, intensity 17 mW cm2) of 1280 1024 pixels, each 32 32 lm in size. In our set-up distinct voxels in subsequent layers with thicknesses of 25 lm were cured by irradiating for times varying between 14 and 23 (s) (depending on the specific resin used). Non-reacted macromer, diluent and photoinitiator were extracted from the built structures using a 3:1 mixture of isopropanol and acetone. The extracted films and porous structures were then dried at 90 °C under a nitrogen flow for 2 days. 2.5. Characterization of PDLLA/nano-Hap composite network films To assess and characterize their physical properties, nanocomposite PDLLA macromer resins containing 4.0% Lucirin TPO were
Table 1 Overview of the formulations of the nanocomposite resins used in stereolithography.
2.2. Polymer synthesis Linear hydroxyl-telechelic poly(D,L-lactide) oligomers were synthesized on a 100 g scale by ring opening polymerization of D,L-lactide for 40 h at 130 °C under an argon atmosphere, using 0.0015 wt.% stannous octoate as a catalyst and hexanediol as bifunctional initiator. The oligomers were functionalized by reacting the terminal hydroxyl groups with methacrylic anhydride in dry dichloromethane under an argon atmosphere. Methacrylic
Composite resin
PDLLA macromer (g)
Nano-Hap (wt.%)
NMP (wt.%)
Viscosity g (Pa s)
PDLLA0 PDLLA5 PDLLA10 PDLLA20
15 15 15 15
0 5 10 20
45 45 50 50
6.36 7.89 5.27 7.05
Resins containing 4 wt.% Lucirin-TPO, 0.2 wt.% tocopherol and 0.15 wt.% Orange Orasol G were prepared using 15 g PDLLA macromer and different amounts of nanoHap and NMP diluent. All wt.% are with respect to the PDLLA macromer.
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also photo-crosslinked in the form of sheets in a UV photo-crosslinking cabinet (Ultralum crosslinking cabinet, wavelength 365 nm, intensity 3–4 mW cm2). Nanocomposite PDLLA network films were prepared by photo-crosslinking the resins in silicone rubber moulds in an inert atmosphere for 20 min. To prevent oxygen inhibition the resins in the moulds were covered with a thin fluorinated ethylene–propylene (FEP) sheet. The crosslinked networks were then extracted with a 3:1 mixture of isopropanol and acetone and subsequently dried in oven at 90 °C for 2 days under a nitrogen flow. Thermogravimetrical analysis (TGA) of the obtained nanocomposite films was performed using a TA Instruments Q 5000 machine. Specimens were heated under nitrogen from room temperature to 700 °C at a heating rate of 10 °C min1. The mechanical properties of the extracted and dried nanocomposite PDLLA networks were determined using a Zwick Z020 universal tensile tester according to the ISO 178 norm. The flexural properties were evaluated in a three-point bending test using specimens measuring 40 25 1 mm (after extraction and drying). The span width was 20 mm and the test rate 0.5 mm min1.
2.6. Scanning electron microscopy (SEM) and microcomputer tomography (l-CT) analysis of PDLLA/nano-Hap films and porous structures built by SLA SEM was performed in order to evaluate the presence and distribution of nano-Hap in the nanocomposite films, and to obtain qualitative information regarding the morphology and pore characteristics of the built PDLLA/nano-Hap structures. The specimens were gold sputtered and observed with a Leica Cambridge (Stereoscan S440) scanning electron microscope. Quantitative analysis by microcomputed tomography (lCT) was performed using a lCT apparatus (Skyscan 1072 with a resolution of 10 lm). Scanning was performed at an X-ray tube voltage of 52 kV, a current of 179 lA and a rotation angle of 180°.
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3. Results and discussion 3.1. Synthesis of poly(D,L-lactide) dimethacrylate macromers The monomer conversion and the molecular weight of the synthesized lactide oligomers can be determined by 1H NMR. A typical spectrum is depicted in Fig. 1. The degree of polymerization of the oligomers were determined from the ratios of the peak areas corresponding to the –CH2O– protons [c] at 4.1 p.p.m. from the hexanediol initiator residues and the –CHCOO– protons [a] in the repeating unit of the oligomers at 5.35 p.p.m. The number average molecular weight of the oligomers can be calculated from the degrees of polymerization. An average molecular weight of 3.0 kg mol1 was determined for the synthesized PDLLA oligomers. After functionalization with methacrylate groups and purification the degree of functionalization was determined from the peak areas corresponding to methacrylate protons [g] and [g0 ] at 5.65 and 6.2 p.p.m. and –CH2O– protons [c] at 4.1 p.p.m., which correspond to the hexanediol initiator residues. The determined degree of functionalization exceeded 98%.
3.2. Nanocomposite resin formulations For use in stereolithography a sufficiently low viscosity of the composite resin and homogeneously dispersed ceramic particles are essential. This is not only a requirement to obtain a homogeneous continuous ceramic phase in the built photo-crosslinked construct [25], but is also necessary to allow adequate spreading of the resin during manufacture. In pilot experiments the macromers were diluted with varying amounts of NMP (45–50 wt.% with respect to the PDLLA macromer), and also different amounts of nano-Hap particles (0–20 wt.% with respect to the PDLLA macromer) were added. The composite resins were heated slightly and sonicated to avoid settling and aggregation of the nano-Hap particles, after which their viscosities were determined at room temperature. These preliminary experiments indicated that both
Fig. 1. Characteristic 1H NMR spectrum of a poly(D,L-lactide) oligomer functionalized with methacrylate end groups.
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Table 2 Viscosities of the composite PDLLA resins as a function of nano-Hap content and NMP diluent concentration. Nano-Hap content
0 wt.% 5 wt.% 10 wt.% 20 wt.%
Viscosity g (Pa s) 45 wt.% NMP
50 wt.% NMP
6.36 7.89 10.37 14.61
4.22 4.69 5.27 7.05
All wt.% are with respect to the PDLLA macromer.
the amount of diluent and the amount of dispersed ceramic had significant effects on the viscosity of the resin. Furthermore, within the investigated shear rate range the viscosities of the different resins were essentially constant. In the absence of a ceramic component the viscosity decreased from 6.36 to 4.22 Pa s when the diluent concentration was increased from 45 to 50 wt.% NMP. With increasing content of the ceramic nano-Hap phasean increase in resin viscosity was observed. With the addition of 20 wt.% nanoHap the viscosity of a resin diluted with 45 wt.% NMP increased to 14.61 Pa s and that of a resin diluted with 50 wt.% NMP increased to 7.05 Pa s. The viscosities of the composite resins as a function of nano-Hap weight content are reported in Table 2. Ideally, minimal amounts of a non-reactive diluent should be used, as an excess of the latter would lead to fragile structures during the stereolithography building process. However, processing of the resin becomes more difficult at higher viscosities. The four resin compositions that had relatively low viscosities when diluent was added were selected for use in the preparation of composite films in the photo-crosslinking cabinet and the building of designed structures by stereolithography. These resins are presented in Table 1.
3.3. Physical properties of the PDLLA and nano-Hap composite network films To be able to prepare polymer–ceramic composite structures the ceramic phase must be homogeneously dispersed in the resin before and after photocuring. The powder consists of spherical particles smaller than 100 nm in diameter, with relatively few spherical aggregates. Fig. 2 shows SEM images of the nanocomposite PDLLA network films prepared from resins containing 0, 10 and 20 wt.% nano-Hap particles. It is clear from the images that the presence of nano-Hap in the resins strongly influences the surface morphology of networks. The SEM images shows that while the surface of the PDLLA network film is smooth and without defects, the surface of composite PDLLA network films containing increasing amounts of nano-Hap become more irregular and covered with particles. This implies that during photo-crosslinking not all nanoHap particles remain fully embedded within the polymer matrix. Whilst the distribution of the nano-Hap particles within the PDLLA network matrix is predominantly homogeneous, it can be seen that some aggregates with sizes of 5–10 lm have also formed. This is likely to be due to the high surface area of the nanoparticles and the consequent strong interaction between the particles. It is likely that more vigorous dispersion and the use of a surface active agent could prevent this aggregation from occurring. TGA of the composite networks was performed at temperatures ranging from 30 °C to 700 °C under a nitrogen atmosphere. Fig. 3 shows that thermal decomposition of the samples as a result of thermal degradation of the organic PDLLA matrix takes place at temperatures of approximately 250–450 °C. At temperatures higher than 450 °C no further mass loss is observed. As the ceramic phase is quite stable these residual masses can be used to determine the true composition of the composites. The amounts of residue after heating to high temperature were 0.9 ± 0.2, 4.7 ± 0.4, 9.9 ± 1.3 and 14.2 ± 1.1 wt.%, respectively, for the PDLLA0, PDLLA5, PDLLA10 and PDLLA20
Fig. 2. SEM micrographs of photo-crosslinked nanocomposite PDLLA network films prepared from resins containing 0, 10 and 20 wt.% nano-Hap particles, respectively abbreviated as PDLL0, PDLLA10, PDLLA20. (A–C) Magnification 2400, scale bar 50 lm; (D) magnification 80,000, scale bar 1 lm.
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Fig. 3. Thermogravimetric analysis of PDLLA and nano-Hap composite network films.
Fig. 4. Flexural stress–strain curves of the PDLLA/nano-Hap composite networks as a function of the amount of nano-Hap.
nanocomposite networks. The determined amount of 0.9 wt.% residual material after heating PDLLA0 is likely due to error in the TGA analysis method. The low value determined for PDLLA20 implies that at high nano-Hap contents some nano-Hap is lost during crosslinking and has not been incorporated into the photocrosslinked composite film. The concept of combining reinforcing bioceramic particles with biodegradable polymer matrices by preparing composite materials has been applied to obtain materials which are both rigid and biologically active [26]. The incorporation of Hap into PDLLA has been shown to increase the bone bonding ability of the composite [27]. The mechanical properties of linear and crosslinked PDLLA are well documented in the scientific literature [12,28]. Since the glass transition temperature of these PDLLA networks is approximately 55 °C [12], these nanocomposite network materials are rigid at room temperature. In tensile testing and in bending experiments the elasticity modulus of PDLLA was shown to be approximately 3 GPa [29]. To quantify the effect of the nano-Hap component on the mechanical properties of the photo-crosslinked PDLLA nanocomposites three-point flexural testing experiments were conducted. These PDLLA/nano-Hap composites show the characteristics of quasi-brittle materials, all specimens fracturing at a strain close to the maximum in the flexural stress–strain curve, as can be seen
Table 3 Flexural mechanical properties of PDLLA and nano-Hap composite networks determined in 3-point bending experiments.
PDLLA0 PDLLA5 PDLLA10 PDLLA20a
EF (GPa)
rmax (MPa)
emax (%)
3.1 ± 0.4 4.1 ± 0.3 4.8 ± 0.3 5.1 ± 0.6
66.0 ± 9.5 45.3 ± 9.4 42.0 ± 6.0 50.6 ± 6.5
3.5 ± 1.5 1.3 ± 0.3 1.5 ± 0.3 1.2 ± 0.2
a Note that this composition was shown to contain 15 wt.% nano-Hap (see Fig. 3 and corresponding text).
in Fig. 4. The results presented in Table 3 show that the addition of 20 wt.% nano-Hap to the PDLLA photo-crosslinkable resin increases the flexural modulus from 3.1 to 5.1 GPa. The maximum flexural strength and strain at break decrease from 66.0 MPa and 3.5%, respectively, to 50.6 MPa and 1.2%. 3.4. Photocuring behaviour of the composite resins in the stereolithography apparatus (SLA) The PDLLA/nano-Hap composite resins (also containing 4 wt.% Lucirin-TPO, 0.2 wt.% tocopherol and 0.15 wt.% Orange Orasol G) were first characterized with respect to their photocuring
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A. Ronca et al. / Acta Biomaterialia 9 (2013) 5989–5996 Table 5 Comparison of the characteristic properties of the pore network of the CAD design of the Schwarz primitive structure and the corresponding structure built by SLA.
CAD design Built by SLA
Fig. 5. Stereolithography working curves of the composite PDLLA resins containing different amounts of nano-Hap. See Table 4 for the photo-curing characteristics of the composite resins.
Table 4 Characteristic photo-curing parameters of the composite PDLLA resins in the stereolithography apparatus.
PDLLA0 PDLLA5 PDLLA10 PDLLA20
Dp (lm)
Ec (mJ cm2)
tc (s)
Eba (mJ cm2)
tba (s)
96 75 66 49
201.6 209.6 206.7 205.8
11.8 12.3 12.0 12.1
241.4 290.7 309.4 391.0
14.2 17.1 18.2 23.0
Ec and tc are the critical energy dose and corresponding exposure times. Eb and tb are the energy dose and corresponding exposure times used to build the designed structures at layer thicknesses of 25 lm. Details of the resin formulations are given in Tables 1 and 2. a The energy dose and time of exposure (Eb and Tb) used in building structures applying specific layer thicknesses are 20% larger than that calculated from the working curves (see text).
behaviour in the stereolithography apparatus. Upon exposure to light a photopolymer obeys the Beer–Lambert law of absorption and, consequently, the thickness of the solidified resin layer (cure depth Cd in lm) is controlled by the light irradiation dose E (in mJ cm2). The theoretical expression for the cured depth (Cd) is derived from the Beer–Lambert law and can be written as [7,12]:
E C d ¼ Dp ln Ec where Dp (lm) is the depth of penetration of the light and Ec (mJ cm2) is the critical exposure, i.e. the minimal exposure to effect polymerization of the macromer. The penetration of light into the composite resin is directly related to the extinction coefficient in
Porosity (%)
Specific surface area (mm1)
Pore size (lm)
63.68 64.51 ± 1.72
8.43 11.82 ± 0.45
619 ± 10 656 ± 30
the Beer–Lambert equation, and affects the Dp, a high value of the extinction coefficient of the resin leading to a small Dp. Precise control of Dp enables minimal overcure and affords accurate control of the building process. For a given resin the cure depth is determined by the illumination dose (the energy) to which the resin is exposed. This dose can be varied by adjusting the power of the light source or the illumination time. In our experiments the crosslinking energy dose was altered by varying the illumination times. Fig. 5 shows these so-called ‘‘working curves’’, the relationships between the thickness of the photocured resin layer and the illumination dose, for the different composite resins. When plotted logarithmically according to the equation above such a graph yields a straight line with slope equal to Dp and an intercept equal to Ec. It can be seen from the data in Fig. 5 that with increasing nano-Hap filler content of the composite resin the light penetration depth Dp (the slope of the working curve) decreases. The cure depth Cd which can be reached at a certain illumination dose E also decreases with filler content. Therefore, to obtain a photocured composite layer of a specific thickness the illumination dose must be increased when resins with higher amounts of nano-Hap are used. It should be noted that the cure depth appears to level off at high illumination doses, a behaviour which is most clear for resins containing filler. The penetration depths (Dp) of the blue SLA light emitted into the composite resins, and the critical illumination dosages (Ec) and corresponding illumination times (tc) to initiate network formation were evaluated as described previously, and are presented in Table 4. The data in Table 4 clearly indicate that while the critical energy for network formation of the different resins remains constant, the light penetration depth Dp decreases significantly with increasing nano-Hap content. When building at high resolutions this is quite favourable, as the resolution in the vertical direction is improved. Using building layer thicknesses (or platform step heights) of 25 lm layers of resin were sequentially photo-crosslinked by exposure to the different light pixel patterns. It was found in a previous work [12] that optimal energy doses and illumination times were approximately 20% higher than the corresponding light penetration depths determined from the working curves. Table 4 also indicates the applied energy doses and illumination times used in preparing the designed porous structures. 3.5. Design and preparation of PDLLA and nano-Hap composite porous structures with Schwarz pore network architecture As the pore network initially provides the spatial template for cell adhesion and proliferation and the deposition of extracellular
Fig. 6. Design and structure built by stereolithography of a porous structure with a Schwarz primitive pore network architecture using a PDLLA and nano-Hap composite resin containing 5 wt.% nano-Hap (Scale bars represent 1 mm).
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Fig. 7. SEM images of porous structures with Schwarz primitive pore network architecture prepared by stereolithography from PDLLA and nano-Hap composite resins containing 5 wt.% nano-Hap. Scale bars 200 lm.
matrix the scaffold morphology is an important factor determining tissue formation in tissue engineering [30]. As the nano-Hap component of the PDLLA/nano-Hap composite can induce bone formation, preparing designed porous structures from these composite resins by stereolithography will allow the preparation of sophisticated biologically active scaffolds for use in bone tissue engineering. As an example of such a complex porous structure we chose to fabricate structures with Schwarz primitive pore network architectures. The pore surface of this open pore network architecture is a triply periodic minimal surface (TPMS). TMPS surfaces are periodic in three independent directions that extend infinitely and, in the absence of self-intersection, partition the space into two labyrinths. Minimal surfaces are of ubiquitous geometry in nature, in the sense that subcellular organelles of different biological forms assume this shape [31]. The use of porous composite structures with primitive architectures, such as the Schwarz primitive structure, in bone tissue engineering is a practical application of TPMS surfaces. Kim et al. characterized the mechanical properties of a Schwarz primitive structure by simulating compressive loading [32], their simulation revealing optimal stress distributions compared with less regular structures. This triply periodic primitive structure is uniquely defined by its unit cell, and can be described by the trigonometric function:
S : cosx þ cosy þ cosz ¼ 0:4 From this CAD files that describe the surface of the pore network architecture were prepared and converted to STL, thus building files for their manufacture by stereolithography. It should be noted that addition of an offset value to this implicit function allows the design of porous structures with a Schwarz primitive architecture and varying porosities. Fig. 6A and B illustrates the design of an advanced porous structure with the Schwarz primitive pore network architecture. By illuminating the resin with different pixel patterns in sequential layers during the stereolithographic building process this porous structure with a Schwarz pore network architecture was built using the PDLLA/nano-Hap composite resin containing 5 wt.% nanohydroxyapatite (PDLLA5 resin). Fig. 6C shows a photographic image of the prepared structure after extraction and post-preparation curing. Note that in designing the structure shrinkage due to extraction of the non-reactive diluent is taken into account. Fig. 6D shows a reconstructed l-CT image of the prepared structure after extraction and drying. From the l-CT data structural parameters such as porosity, pore size and specific surface area can be determined. Table 5 compares the characteristic properties of the structure design and the determined values of the built structures. It can be seen from the results that the match is very good. The high porosity and large pore size, which both closely match the design values, will enable efficient seeding and proliferation of cells within the structure. Although the specific surface area, determined to be 11.82 mm1, is somewhat lower than that of scaffolds prepared using conventional pore-forming techniques [33], the open nature of the pore network will result in good accessibility of the pores and thus facilitate the
transport of nutrients and metabolites, both during in vitro culture and after implantation in the body. SEM images of the structures built from the PDLLA5 composite resin are shown in Fig. 7. These show that the overall Schwarz geometry and its open interconnected pore network structure is not influenced by the presence of the ceramic phase. At higher magnifications the layer by layer nature of the stereolithography process becomes visible. It can also be seen that nano-Hap particles are present on the surface of the porous structure. This is important with regard to the bone-forming properties of the scaffold, as these particles will be in direct contact with the seeded cells. Sections through the struts of the structure show that particles are also present in the bulk of the polymeric matrix. Although the particles appear to be well distributed on the surface of the structure and within the matrix, it is apparent that some nanoHap aggregation has occurred. 4. Conclusions In this study we have prepared composite resins based on PDLLA macromers and nano-Hap particles for application in stereolithography. The particles were homogenously dispersed in the resin and did not settle. With increasing ceramic component the resins became more viscous, and NMP was added as a non-reactive diluent to decrease the viscosity and allow processing by stereolithography. Photo-crosslinked composite films were prepared from these resins and characterized with respect to their physical properties. The mechanical properties showed that strong rigid materials were obtained and that with increasing nano-Hap content the elasticity modulus of the composite PDLLA/nano-Hap network materials increased. The photocuring characteristics of the different composite resins were evaluated, after which designed porous structures with a Schwarz pore network architecture were fabricated by stereolithography using a resin containing 5 wt.% nano-Hap. The prepared structures precisely matched the design. Although some aggregation of particles occurred, it was shown that the ceramic component remained well dispersed in the polymeric matrix. SEM images showed exposed ceramic particles on the pore surface, allowing interaction between the bone-forming nano-Hap and cells. References [1] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. [2] Ripamonti U, Ma S, Reddi A. The critical role of geometry of porous hydroxyapatite delivery system of bone by osteogenin, a bone morphogenic protein. Matrix 1992;12:202–12. [3] Huec JL, Schaeverbeke T, Clement D, Faber J, Rebeller AL. Influence of porosity on the mechanical resistance of hydroxyapatite ceramics under compressive stress. Biomaterials 1995;16:113–8. [4] Gloria A, Russo T, De Santis R, Ambrosio L. 3D fiber deposition technique to make multifunctional and tailor-made scaffolds for tissue engineering applications. J Appl Biomater Bioceramics 2009;3:141–52. [5] Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005;4:518–24.
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[6] Kim K, Yeatts A, Dean D, Fisher JP. Stereolithographic bone scaffold design parameters osteogenic differentiation and signal expression. Tissue Eng B 2010;16:523–39. [7] Melchels FPW, Feijen J, Grijpma DW. A review on stereolithography and its applications in biomedical engineering. Biomaterials 2010;31:6121–30. [8] Lu Y, Mapili G, Suhali G, Chen S, Roy K. A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds. J Biomed Mater Res 2006;77A:396–405. [9] Provin C, Monneret S. Complex ceramic–polymer composite microparts made by microstereolithography. Trans Elect Pack Man 2002;25:59–63. [10] Griffith ML, Halloran JW, Freeform fabrication of ceramics via stereolithography. J Am Ceram Soc 1996;79(10):2601–8. [11] Dhariwal B, Hunt E, Boland. Rapid prototyping of tissue-engineering constructs, using photopolymerizable hydrogels and stereolithography. Tissue Eng 2004;10:1316–22. [12] Melchels FPW, Feijen J, Grijpma DW. A poly(D,L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography. Biomaterials 2009;30:3801–9. [13] Arcaute K, Mann BK, Wicker RB. Stereolithography of three-dimensional bioactive poly(ethylene glycol) constructs with encapsulated cells. Ann Biomed Eng 2006;34:1429–41. [14] Storey RF, Warren SC, Allison CJ, Wiggins SJ, Puckett AD. Synthesis of bioabsorbable network from methacrylate-endcapped polyesters. Polymer 1993;34:4365–72. [15] Gurr M, Hoffmann D, Ehm M, Thomann Y, Kubler R, Mulhaupt R. Acrylic nanocomposite resins for use in stereolithography and structural light modulation based rapid prototyping and rapid manufacturing technologies. Adv Funct Mater 2008;18(16):2390–7. [16] Kim HW, Knowles JC, Kim HE. Hydroxyapatite/poly(epsilon caprolactone) composite coatings on hydroxyapatite porous bone scaffold for drug delivery. Biomaterials 2004;25:1279–87. [17] Guarino V, Taddei P, Di Foggia M, Fagnano C, Ciapetti G, Ambrosio L. Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering. Tissue Eng A 2009;15:3662–70. [18] Webster TJ, Ahn ES. Nanostructured biomaterials for tissue engineering bone. Adv Biochem Eng Biotechnol 2007;103:275–308. [19] Currey J. Biomaterial sacrificial bonds heal bone. Nature 2001;414:773–6. [20] Ginebra MP, Traykova T, Planell JA. Calcium phosphate cements as bone drug delivery systems: a review. J Control Release 2006;113:102–10.
[21] Du C, Cui FZ, Zhu XD, de Groot K. Three dimensional nano-Hap–collagen matrix loading with osteogenic cells in organ culture. J Biomed Mater Res 1999;44:407–15. [22] Rho JY, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys 1998;20:92–102. [23] Clarke KI, Graves SE, Wong ATC, Triffitt JT, Francis MJO, Czernuszka JT. Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate. J Mater Sci Mater Med 1993;4:107–10. [24] Pham DT, Gault RS. A comparison of rapid prototyping technologies. Int J Mach Tools Manuf 1998;38:1257–87. [25] Doreau F, Chaput C, Chartier T. Stereolithography for manufacturing ceramic parts. Adv Eng Mater 2000;2:493–6. [26] Bleach NC, Nazhata SN, Tanner KE, Kellomaki M, Tormala P. Effect of filler content on mechanical and dynamic mechanical properties of particulate biphasic calcium phosphate–polylactide composites. Biomaterials 2002;23:1579–85. [27] Verheyen CCPM, de Wijn JR, van Blitterswijk CA, de Groot K, Rozing PM. Hydroxylapatite/poly(L-lactide) composites: an animal study on push-out strengths and interface histology. J Biomed Mater Res 1993;27:433–44. [28] Lin PL, Fang HW, Tseng T, Lee WH. Effects of hydroxyapatite dosage on mechanical and biological behaviors of polylactic acid composite materials. Mater Lett 2007;61:3009–13. [29] Sarasua JR, Arraiza AL, Balerdi P, Maiza I. Crystallinity and mechanical properties of optically pure polylactides and their blends. Polym Eng Sci 2005;45(5):745–53. [30] Melchels FPW, Bertoldi K, Gabbrielli R, Velders AH, Feijen J, Grijpma DW. Mathematically defined tissue engineering scaffold architectures prepared by stereolithography. Biomaterials 2010;31:6909–16. [31] Rajagopalan S, Robb RA. Schwarz meets Schwann: design and fabrication of biomorphic tissue engineering scaffolds. Med Image Anal 2006;10:693–712. [32] Shin J, Kim S, Jeong D, Lee HG, Lee D, Lim JY, et al. Finite element analysis of Schwarz P surface pore geometries for tissue-engineered scaffolds. Math Probl Eng 2012. http://dx.doi.org/10.1155/2012/694194. [33] Guarino V, Lewandowska M, Bil M, Polak B, Ambrosio L. Morphology and degradation properties of PCL/HYAFF11 composite scaffolds with multi-scale degradation rate. Compos Sci Technol 2010;70:1826–37.
Contents lists available at SciVerse ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Preparation of designed poly(D,L-lactide)/nanosized hydroxyapatite composite structures by stereolithography A. Ronca a,⇑, L. Ambrosio a, D.W. Grijpma b,c a
Institute of Composite and Biomedical Materials, National Research Council of Italy, Piazzale Tecchio 80, 80125 Naples, Italy MIRA Institute for Biomedical Technology and Technical Medicine, Department of Polymer Chemistry and Biomaterials, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands c W.J. Kolff Institute, Department of Biomedical Engineering, University Medical Center Groningen and University of Groningen, P.O. Box 196, 9700 AD Groningen, The Netherlands b
a r t i c l e
i n f o
Article history: Received 10 September 2012 Received in revised form 12 November 2012 Accepted 4 December 2012 Available online 8 December 2012 Keywords: Nano-composites Biodegradable networks CAD designed scaffold Stereolithography Bone tissue engineering
a b s t r a c t The preparation of scaffolds to facilitate the replacement of damaged tissues and organs by means of tissue engineering has been much investigated. The key properties of the biomaterials used to prepare such scaffolds include biodegradability, biocompatibility and a well-defined three-dimensional 3-Dpore network structure. In this study a poly(D,L-lactide)/nanosized hydroxyapatite (PDLLA/nano-Hap) composite resin was prepared and used to fabricate composite films and computer designed porous scaffolds by micro-stereolithography, mixing varying quantities of nano-Hap powder and a liquid photoinitiator into a photo-crosslinkable PDLLA-diacrylate resin. The influence of nano-Hap on the rheological and photochemical properties of the resins was investigated, the materials being characterized with respect to their mechanical, thermal and morphological properties after post-preparation curing. In the cured composites stiffness was observed to increase with increasing concentration of nanoparticles. A computer designed construct with a pore network based on the Schwarz architecture was fabricated by stereolithography using PDLLA/nano-Hap composite resins. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In bone tissue engineering the scaffold plays a fundamental role as a support for cell adhesion and proliferation and for tissue regeneration. An effective scaffold must exhibit the following properties and attributes: high porosity and a requisite pore size for the host cells; suitable surface properties to allow cell adhesion, differentiation and proliferation; adequate mechanical stability to maintain the predesigned structure; no cytotoxicity and osteoconductivity [1]. The internal pore architecture, including pore size, pore shape and pore connection pattern, are important parameters in controlling the extent of bone formation [2], influencing the path of bone regeneration, and determining the mechanical properties of the implants [3]. However, scaffolds prepared by conventional techniques often result in inhomogeneous structures with irregular pore sizes and pore size distributions, poor pore interconnectivity and inferior mechanical properties [4]. Rapid prototyping allows the preparation of scaffolds with optimal properties with regard to pore structure, geometry and connectivity, mechanical characteristics, cell seeding efficiency, and transport of nutrients and metabolites [5]. Stereolithography is an additive fabrication process that uses a liquid light-curable ⇑ Corresponding author. Tel.: +39 3283724007; fax: +39 0812425932. E-mail addresses: [email protected], [email protected] (A. Ronca).
photopolymer and a laser to create three-dimensional (3-D) structures, a technique introduced in the late 1980s [6]. Although several alternative approaches have been developed since then, stereolithography remains one of the most powerful and versatile solid freeform techniques. It allows 3-D (micro) fabrication of solid structures from models created using computer-aided design (CAD) programs [7]. The working principle of stereolithography is based on the spatially controlled solidification of a liquid photopolymerizable resin. Using a computer-controlled laser beam or digital light projection and a computer-driven building platform a 3-D object is constructed in a layer by layer fashion [8]. To do this the design of the object to be built is numerically analyzed to define the different layers that are to be manufactured [9]. The sequential polymerization of layers with different illumination patterns leads to the creation of complex 3-D objects, described by Griffith and Halloran [10]. The number of available stereolithography resins suitable for use in biomedical applications is limited [11]. General purpose photocurable materials are predominantly acrylate-based resins [12], and among these poly(ethylene glycol) dimethacrylate has been used to fabricate non-degradable cell-containing hydrogels in predesigned shapes [13]. For bone tissue engineering strong and rigid biodegradable materials are required. Poly(lactide) is such a material, with a long track record of successful application in clinical use and which has been used for the preparation of
1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.12.004
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tissue engineering scaffolds. Low molecular weight poly(D,L-lactide) (PDLLA) oligomers functionalized with methacrylate groups can be crosslinked to form rigid polymer networks [14]. However, when the crosslink density of these networks is too high they are quite brittle, which limits their usefulness [15]. Composites of polymers and ceramics are being developed to enhance the mechanical properties and improve tissue interactions [16]. The development of composites which combine biodegradable polymers and bioactive ceramics or glasses for use in tissue engineering has been extensively discussed [17,18]. Hydroxyapatite (Ca10(PO4)6(OH)2) (Hap) has attracted much interest due to its chemical similarity to the calcium phosphate mineral present in biological hard tissues [19]. Hap has been used for a variety of biomedical applications, for example as a matrix for controlled drug release and as a carrier material in bone tissue engineering [20]. Orthopaedic research suggests that osteoconductivity would be optimal when synthetic Hap resembles the bone mineral with regard to composition, size and morphology [21]. In natural bone the nanometer scale of the inorganic apatite component is considered to be important for the biological and mechanical properties of the tissue [22]. Well-dispersed nanosized hydroxyapatite (nano-Hap) with an ultrafine structure has the potential to improve the performance of composites, because these particles have a high surface area to volume ratio and their surfaces have minimal defects [21]. Several nano-Hap/polymer composite tissue engineering scaffolds have been developed for use as substitute bone [23]. It remains a challenge to extend the stereolithography technique to allow the processing of nanosized composite ceramic and polymer resins to fabricate scaffolds for bone tissue regeneration. In this paper we report on the use of nano-Hap powders, dispersed in a photo-curable PDLLA-based resin, to prepare biodegradable nanocomposite structures by stereolithography. Varying amounts of nano-Hap were used, and the effects of nano-Hap content on the photo-crosslinking kinetics and network properties were investigated. In this way porous structures designed for possible use in bone tissue engineering were prepared at high resolution. 2. Materials and methods 2.1. Materials D,L-lactide was obtained from Purac Biochem (The Netherlands). 1,6-Hexanediol, stannous octoate (SnOct), methacrylic anhydride (MAAH), N-methyl-2-pyrollidone (NMP) and tocopherol were purchased from Sigma–Aldrich (The Netherlands) and used without further purification. Triethylamine (TEA) (Fluka, Switzerland) and technical grade isopropanol and acetone (Biosolve, The Netherlands) were used as received. Orasol Orange G was kindly donated by Ciba Specialty Chemicals (Switzerland). Lucirin TPO-L (ethyl2,4,6-trimethyl benzoyl phenyl phosphinate) was kindly provided by BASF (Germany). Analytical grade dichloromethane (Biosolve) was distilled from calcium hydride (Acros Organics, Belgium). Nano-sized hydroxyapatite (particle size <200 nm) was obtained from Sigma–Aldrich.
acid, the by-product of the functionalization reaction, was scavenged using TEA. An excess of 20 mol.% MAAH and TEA per hydroxyl group was used. The solution was filtered and precipitated into cold isopropanol. The PDLLA dimethacrylate macromers were isolated, washed with water and freeze-dried. 2.3. Formulation of composite resins Nano-Hap powder (particle size <200 nm) was used to prepare a series of composite PDLLA/nano-Hap resins for application in stereolithography. Homogeneous nanosized composite resins were prepared by mixing 15 g of PDLLA macromer and different amounts of nano-Hap (up to 20 wt.% with respect to macromer) in NMP. The dispersions were kept at 60 °C overnight, and sonicated for 15 min before use in order to ensure homogeneity. Dispersion viscosity was assessed at 25 °C using a Brookfield DV-E rotating spindle viscometer equipped with a small sample adapter. The shear rate was varied by adjusting the rotation speed of the spindle (Brookfield s21 spindle) between 0.6 and 4 r.p.m. 2.4. Stereolithography To fabricate PDLLA/nano-Hap composite porous structures by stereolithography 4 wt.% Lucirin-TPO photoinitiator, 0.2 wt.% tocopherol inhibitor (to prevent premature polymerization) and 0.15 wt.% Orange Orasol G dye (to set the requisite light penetration depth) were added to the PDLLA macromer solutions in NMP. Table 1 gives an overview of the different composite resins prepared. The weight percentages of the various ingredients cited in Table 1 were calculated with respect to PDLLA macromer content, 15 g of PDLLA macromer being used in each resin preparation. A commercial stereolithography apparatus (SLA) (Perfactory Mini Multilens, Envisiontec) was employed to build films and designed porous structures. In stereolithography a CAD file is used to describe the size and geometry of the structures to be built. This file is converted into STL format and virtually sliced into the layers used in the layer by layer fabrication process [7]. By convention the slices are in the X,Y-plane while the specimen is built in the Z-direction [24]. The building process involves subsequent blue light projection (wavelength 400–550 nm with a peak at 440 nm, intensity 17 mW cm2) of 1280 1024 pixels, each 32 32 lm in size. In our set-up distinct voxels in subsequent layers with thicknesses of 25 lm were cured by irradiating for times varying between 14 and 23 (s) (depending on the specific resin used). Non-reacted macromer, diluent and photoinitiator were extracted from the built structures using a 3:1 mixture of isopropanol and acetone. The extracted films and porous structures were then dried at 90 °C under a nitrogen flow for 2 days. 2.5. Characterization of PDLLA/nano-Hap composite network films To assess and characterize their physical properties, nanocomposite PDLLA macromer resins containing 4.0% Lucirin TPO were
Table 1 Overview of the formulations of the nanocomposite resins used in stereolithography.
2.2. Polymer synthesis Linear hydroxyl-telechelic poly(D,L-lactide) oligomers were synthesized on a 100 g scale by ring opening polymerization of D,L-lactide for 40 h at 130 °C under an argon atmosphere, using 0.0015 wt.% stannous octoate as a catalyst and hexanediol as bifunctional initiator. The oligomers were functionalized by reacting the terminal hydroxyl groups with methacrylic anhydride in dry dichloromethane under an argon atmosphere. Methacrylic
Composite resin
PDLLA macromer (g)
Nano-Hap (wt.%)
NMP (wt.%)
Viscosity g (Pa s)
PDLLA0 PDLLA5 PDLLA10 PDLLA20
15 15 15 15
0 5 10 20
45 45 50 50
6.36 7.89 5.27 7.05
Resins containing 4 wt.% Lucirin-TPO, 0.2 wt.% tocopherol and 0.15 wt.% Orange Orasol G were prepared using 15 g PDLLA macromer and different amounts of nanoHap and NMP diluent. All wt.% are with respect to the PDLLA macromer.
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also photo-crosslinked in the form of sheets in a UV photo-crosslinking cabinet (Ultralum crosslinking cabinet, wavelength 365 nm, intensity 3–4 mW cm2). Nanocomposite PDLLA network films were prepared by photo-crosslinking the resins in silicone rubber moulds in an inert atmosphere for 20 min. To prevent oxygen inhibition the resins in the moulds were covered with a thin fluorinated ethylene–propylene (FEP) sheet. The crosslinked networks were then extracted with a 3:1 mixture of isopropanol and acetone and subsequently dried in oven at 90 °C for 2 days under a nitrogen flow. Thermogravimetrical analysis (TGA) of the obtained nanocomposite films was performed using a TA Instruments Q 5000 machine. Specimens were heated under nitrogen from room temperature to 700 °C at a heating rate of 10 °C min1. The mechanical properties of the extracted and dried nanocomposite PDLLA networks were determined using a Zwick Z020 universal tensile tester according to the ISO 178 norm. The flexural properties were evaluated in a three-point bending test using specimens measuring 40 25 1 mm (after extraction and drying). The span width was 20 mm and the test rate 0.5 mm min1.
2.6. Scanning electron microscopy (SEM) and microcomputer tomography (l-CT) analysis of PDLLA/nano-Hap films and porous structures built by SLA SEM was performed in order to evaluate the presence and distribution of nano-Hap in the nanocomposite films, and to obtain qualitative information regarding the morphology and pore characteristics of the built PDLLA/nano-Hap structures. The specimens were gold sputtered and observed with a Leica Cambridge (Stereoscan S440) scanning electron microscope. Quantitative analysis by microcomputed tomography (lCT) was performed using a lCT apparatus (Skyscan 1072 with a resolution of 10 lm). Scanning was performed at an X-ray tube voltage of 52 kV, a current of 179 lA and a rotation angle of 180°.
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3. Results and discussion 3.1. Synthesis of poly(D,L-lactide) dimethacrylate macromers The monomer conversion and the molecular weight of the synthesized lactide oligomers can be determined by 1H NMR. A typical spectrum is depicted in Fig. 1. The degree of polymerization of the oligomers were determined from the ratios of the peak areas corresponding to the –CH2O– protons [c] at 4.1 p.p.m. from the hexanediol initiator residues and the –CHCOO– protons [a] in the repeating unit of the oligomers at 5.35 p.p.m. The number average molecular weight of the oligomers can be calculated from the degrees of polymerization. An average molecular weight of 3.0 kg mol1 was determined for the synthesized PDLLA oligomers. After functionalization with methacrylate groups and purification the degree of functionalization was determined from the peak areas corresponding to methacrylate protons [g] and [g0 ] at 5.65 and 6.2 p.p.m. and –CH2O– protons [c] at 4.1 p.p.m., which correspond to the hexanediol initiator residues. The determined degree of functionalization exceeded 98%.
3.2. Nanocomposite resin formulations For use in stereolithography a sufficiently low viscosity of the composite resin and homogeneously dispersed ceramic particles are essential. This is not only a requirement to obtain a homogeneous continuous ceramic phase in the built photo-crosslinked construct [25], but is also necessary to allow adequate spreading of the resin during manufacture. In pilot experiments the macromers were diluted with varying amounts of NMP (45–50 wt.% with respect to the PDLLA macromer), and also different amounts of nano-Hap particles (0–20 wt.% with respect to the PDLLA macromer) were added. The composite resins were heated slightly and sonicated to avoid settling and aggregation of the nano-Hap particles, after which their viscosities were determined at room temperature. These preliminary experiments indicated that both
Fig. 1. Characteristic 1H NMR spectrum of a poly(D,L-lactide) oligomer functionalized with methacrylate end groups.
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Table 2 Viscosities of the composite PDLLA resins as a function of nano-Hap content and NMP diluent concentration. Nano-Hap content
0 wt.% 5 wt.% 10 wt.% 20 wt.%
Viscosity g (Pa s) 45 wt.% NMP
50 wt.% NMP
6.36 7.89 10.37 14.61
4.22 4.69 5.27 7.05
All wt.% are with respect to the PDLLA macromer.
the amount of diluent and the amount of dispersed ceramic had significant effects on the viscosity of the resin. Furthermore, within the investigated shear rate range the viscosities of the different resins were essentially constant. In the absence of a ceramic component the viscosity decreased from 6.36 to 4.22 Pa s when the diluent concentration was increased from 45 to 50 wt.% NMP. With increasing content of the ceramic nano-Hap phasean increase in resin viscosity was observed. With the addition of 20 wt.% nanoHap the viscosity of a resin diluted with 45 wt.% NMP increased to 14.61 Pa s and that of a resin diluted with 50 wt.% NMP increased to 7.05 Pa s. The viscosities of the composite resins as a function of nano-Hap weight content are reported in Table 2. Ideally, minimal amounts of a non-reactive diluent should be used, as an excess of the latter would lead to fragile structures during the stereolithography building process. However, processing of the resin becomes more difficult at higher viscosities. The four resin compositions that had relatively low viscosities when diluent was added were selected for use in the preparation of composite films in the photo-crosslinking cabinet and the building of designed structures by stereolithography. These resins are presented in Table 1.
3.3. Physical properties of the PDLLA and nano-Hap composite network films To be able to prepare polymer–ceramic composite structures the ceramic phase must be homogeneously dispersed in the resin before and after photocuring. The powder consists of spherical particles smaller than 100 nm in diameter, with relatively few spherical aggregates. Fig. 2 shows SEM images of the nanocomposite PDLLA network films prepared from resins containing 0, 10 and 20 wt.% nano-Hap particles. It is clear from the images that the presence of nano-Hap in the resins strongly influences the surface morphology of networks. The SEM images shows that while the surface of the PDLLA network film is smooth and without defects, the surface of composite PDLLA network films containing increasing amounts of nano-Hap become more irregular and covered with particles. This implies that during photo-crosslinking not all nanoHap particles remain fully embedded within the polymer matrix. Whilst the distribution of the nano-Hap particles within the PDLLA network matrix is predominantly homogeneous, it can be seen that some aggregates with sizes of 5–10 lm have also formed. This is likely to be due to the high surface area of the nanoparticles and the consequent strong interaction between the particles. It is likely that more vigorous dispersion and the use of a surface active agent could prevent this aggregation from occurring. TGA of the composite networks was performed at temperatures ranging from 30 °C to 700 °C under a nitrogen atmosphere. Fig. 3 shows that thermal decomposition of the samples as a result of thermal degradation of the organic PDLLA matrix takes place at temperatures of approximately 250–450 °C. At temperatures higher than 450 °C no further mass loss is observed. As the ceramic phase is quite stable these residual masses can be used to determine the true composition of the composites. The amounts of residue after heating to high temperature were 0.9 ± 0.2, 4.7 ± 0.4, 9.9 ± 1.3 and 14.2 ± 1.1 wt.%, respectively, for the PDLLA0, PDLLA5, PDLLA10 and PDLLA20
Fig. 2. SEM micrographs of photo-crosslinked nanocomposite PDLLA network films prepared from resins containing 0, 10 and 20 wt.% nano-Hap particles, respectively abbreviated as PDLL0, PDLLA10, PDLLA20. (A–C) Magnification 2400, scale bar 50 lm; (D) magnification 80,000, scale bar 1 lm.
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Fig. 3. Thermogravimetric analysis of PDLLA and nano-Hap composite network films.
Fig. 4. Flexural stress–strain curves of the PDLLA/nano-Hap composite networks as a function of the amount of nano-Hap.
nanocomposite networks. The determined amount of 0.9 wt.% residual material after heating PDLLA0 is likely due to error in the TGA analysis method. The low value determined for PDLLA20 implies that at high nano-Hap contents some nano-Hap is lost during crosslinking and has not been incorporated into the photocrosslinked composite film. The concept of combining reinforcing bioceramic particles with biodegradable polymer matrices by preparing composite materials has been applied to obtain materials which are both rigid and biologically active [26]. The incorporation of Hap into PDLLA has been shown to increase the bone bonding ability of the composite [27]. The mechanical properties of linear and crosslinked PDLLA are well documented in the scientific literature [12,28]. Since the glass transition temperature of these PDLLA networks is approximately 55 °C [12], these nanocomposite network materials are rigid at room temperature. In tensile testing and in bending experiments the elasticity modulus of PDLLA was shown to be approximately 3 GPa [29]. To quantify the effect of the nano-Hap component on the mechanical properties of the photo-crosslinked PDLLA nanocomposites three-point flexural testing experiments were conducted. These PDLLA/nano-Hap composites show the characteristics of quasi-brittle materials, all specimens fracturing at a strain close to the maximum in the flexural stress–strain curve, as can be seen
Table 3 Flexural mechanical properties of PDLLA and nano-Hap composite networks determined in 3-point bending experiments.
PDLLA0 PDLLA5 PDLLA10 PDLLA20a
EF (GPa)
rmax (MPa)
emax (%)
3.1 ± 0.4 4.1 ± 0.3 4.8 ± 0.3 5.1 ± 0.6
66.0 ± 9.5 45.3 ± 9.4 42.0 ± 6.0 50.6 ± 6.5
3.5 ± 1.5 1.3 ± 0.3 1.5 ± 0.3 1.2 ± 0.2
a Note that this composition was shown to contain 15 wt.% nano-Hap (see Fig. 3 and corresponding text).
in Fig. 4. The results presented in Table 3 show that the addition of 20 wt.% nano-Hap to the PDLLA photo-crosslinkable resin increases the flexural modulus from 3.1 to 5.1 GPa. The maximum flexural strength and strain at break decrease from 66.0 MPa and 3.5%, respectively, to 50.6 MPa and 1.2%. 3.4. Photocuring behaviour of the composite resins in the stereolithography apparatus (SLA) The PDLLA/nano-Hap composite resins (also containing 4 wt.% Lucirin-TPO, 0.2 wt.% tocopherol and 0.15 wt.% Orange Orasol G) were first characterized with respect to their photocuring
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A. Ronca et al. / Acta Biomaterialia 9 (2013) 5989–5996 Table 5 Comparison of the characteristic properties of the pore network of the CAD design of the Schwarz primitive structure and the corresponding structure built by SLA.
CAD design Built by SLA
Fig. 5. Stereolithography working curves of the composite PDLLA resins containing different amounts of nano-Hap. See Table 4 for the photo-curing characteristics of the composite resins.
Table 4 Characteristic photo-curing parameters of the composite PDLLA resins in the stereolithography apparatus.
PDLLA0 PDLLA5 PDLLA10 PDLLA20
Dp (lm)
Ec (mJ cm2)
tc (s)
Eba (mJ cm2)
tba (s)
96 75 66 49
201.6 209.6 206.7 205.8
11.8 12.3 12.0 12.1
241.4 290.7 309.4 391.0
14.2 17.1 18.2 23.0
Ec and tc are the critical energy dose and corresponding exposure times. Eb and tb are the energy dose and corresponding exposure times used to build the designed structures at layer thicknesses of 25 lm. Details of the resin formulations are given in Tables 1 and 2. a The energy dose and time of exposure (Eb and Tb) used in building structures applying specific layer thicknesses are 20% larger than that calculated from the working curves (see text).
behaviour in the stereolithography apparatus. Upon exposure to light a photopolymer obeys the Beer–Lambert law of absorption and, consequently, the thickness of the solidified resin layer (cure depth Cd in lm) is controlled by the light irradiation dose E (in mJ cm2). The theoretical expression for the cured depth (Cd) is derived from the Beer–Lambert law and can be written as [7,12]:
E C d ¼ Dp ln Ec where Dp (lm) is the depth of penetration of the light and Ec (mJ cm2) is the critical exposure, i.e. the minimal exposure to effect polymerization of the macromer. The penetration of light into the composite resin is directly related to the extinction coefficient in
Porosity (%)
Specific surface area (mm1)
Pore size (lm)
63.68 64.51 ± 1.72
8.43 11.82 ± 0.45
619 ± 10 656 ± 30
the Beer–Lambert equation, and affects the Dp, a high value of the extinction coefficient of the resin leading to a small Dp. Precise control of Dp enables minimal overcure and affords accurate control of the building process. For a given resin the cure depth is determined by the illumination dose (the energy) to which the resin is exposed. This dose can be varied by adjusting the power of the light source or the illumination time. In our experiments the crosslinking energy dose was altered by varying the illumination times. Fig. 5 shows these so-called ‘‘working curves’’, the relationships between the thickness of the photocured resin layer and the illumination dose, for the different composite resins. When plotted logarithmically according to the equation above such a graph yields a straight line with slope equal to Dp and an intercept equal to Ec. It can be seen from the data in Fig. 5 that with increasing nano-Hap filler content of the composite resin the light penetration depth Dp (the slope of the working curve) decreases. The cure depth Cd which can be reached at a certain illumination dose E also decreases with filler content. Therefore, to obtain a photocured composite layer of a specific thickness the illumination dose must be increased when resins with higher amounts of nano-Hap are used. It should be noted that the cure depth appears to level off at high illumination doses, a behaviour which is most clear for resins containing filler. The penetration depths (Dp) of the blue SLA light emitted into the composite resins, and the critical illumination dosages (Ec) and corresponding illumination times (tc) to initiate network formation were evaluated as described previously, and are presented in Table 4. The data in Table 4 clearly indicate that while the critical energy for network formation of the different resins remains constant, the light penetration depth Dp decreases significantly with increasing nano-Hap content. When building at high resolutions this is quite favourable, as the resolution in the vertical direction is improved. Using building layer thicknesses (or platform step heights) of 25 lm layers of resin were sequentially photo-crosslinked by exposure to the different light pixel patterns. It was found in a previous work [12] that optimal energy doses and illumination times were approximately 20% higher than the corresponding light penetration depths determined from the working curves. Table 4 also indicates the applied energy doses and illumination times used in preparing the designed porous structures. 3.5. Design and preparation of PDLLA and nano-Hap composite porous structures with Schwarz pore network architecture As the pore network initially provides the spatial template for cell adhesion and proliferation and the deposition of extracellular
Fig. 6. Design and structure built by stereolithography of a porous structure with a Schwarz primitive pore network architecture using a PDLLA and nano-Hap composite resin containing 5 wt.% nano-Hap (Scale bars represent 1 mm).
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Fig. 7. SEM images of porous structures with Schwarz primitive pore network architecture prepared by stereolithography from PDLLA and nano-Hap composite resins containing 5 wt.% nano-Hap. Scale bars 200 lm.
matrix the scaffold morphology is an important factor determining tissue formation in tissue engineering [30]. As the nano-Hap component of the PDLLA/nano-Hap composite can induce bone formation, preparing designed porous structures from these composite resins by stereolithography will allow the preparation of sophisticated biologically active scaffolds for use in bone tissue engineering. As an example of such a complex porous structure we chose to fabricate structures with Schwarz primitive pore network architectures. The pore surface of this open pore network architecture is a triply periodic minimal surface (TPMS). TMPS surfaces are periodic in three independent directions that extend infinitely and, in the absence of self-intersection, partition the space into two labyrinths. Minimal surfaces are of ubiquitous geometry in nature, in the sense that subcellular organelles of different biological forms assume this shape [31]. The use of porous composite structures with primitive architectures, such as the Schwarz primitive structure, in bone tissue engineering is a practical application of TPMS surfaces. Kim et al. characterized the mechanical properties of a Schwarz primitive structure by simulating compressive loading [32], their simulation revealing optimal stress distributions compared with less regular structures. This triply periodic primitive structure is uniquely defined by its unit cell, and can be described by the trigonometric function:
S : cosx þ cosy þ cosz ¼ 0:4 From this CAD files that describe the surface of the pore network architecture were prepared and converted to STL, thus building files for their manufacture by stereolithography. It should be noted that addition of an offset value to this implicit function allows the design of porous structures with a Schwarz primitive architecture and varying porosities. Fig. 6A and B illustrates the design of an advanced porous structure with the Schwarz primitive pore network architecture. By illuminating the resin with different pixel patterns in sequential layers during the stereolithographic building process this porous structure with a Schwarz pore network architecture was built using the PDLLA/nano-Hap composite resin containing 5 wt.% nanohydroxyapatite (PDLLA5 resin). Fig. 6C shows a photographic image of the prepared structure after extraction and post-preparation curing. Note that in designing the structure shrinkage due to extraction of the non-reactive diluent is taken into account. Fig. 6D shows a reconstructed l-CT image of the prepared structure after extraction and drying. From the l-CT data structural parameters such as porosity, pore size and specific surface area can be determined. Table 5 compares the characteristic properties of the structure design and the determined values of the built structures. It can be seen from the results that the match is very good. The high porosity and large pore size, which both closely match the design values, will enable efficient seeding and proliferation of cells within the structure. Although the specific surface area, determined to be 11.82 mm1, is somewhat lower than that of scaffolds prepared using conventional pore-forming techniques [33], the open nature of the pore network will result in good accessibility of the pores and thus facilitate the
transport of nutrients and metabolites, both during in vitro culture and after implantation in the body. SEM images of the structures built from the PDLLA5 composite resin are shown in Fig. 7. These show that the overall Schwarz geometry and its open interconnected pore network structure is not influenced by the presence of the ceramic phase. At higher magnifications the layer by layer nature of the stereolithography process becomes visible. It can also be seen that nano-Hap particles are present on the surface of the porous structure. This is important with regard to the bone-forming properties of the scaffold, as these particles will be in direct contact with the seeded cells. Sections through the struts of the structure show that particles are also present in the bulk of the polymeric matrix. Although the particles appear to be well distributed on the surface of the structure and within the matrix, it is apparent that some nanoHap aggregation has occurred. 4. Conclusions In this study we have prepared composite resins based on PDLLA macromers and nano-Hap particles for application in stereolithography. The particles were homogenously dispersed in the resin and did not settle. With increasing ceramic component the resins became more viscous, and NMP was added as a non-reactive diluent to decrease the viscosity and allow processing by stereolithography. Photo-crosslinked composite films were prepared from these resins and characterized with respect to their physical properties. The mechanical properties showed that strong rigid materials were obtained and that with increasing nano-Hap content the elasticity modulus of the composite PDLLA/nano-Hap network materials increased. The photocuring characteristics of the different composite resins were evaluated, after which designed porous structures with a Schwarz pore network architecture were fabricated by stereolithography using a resin containing 5 wt.% nano-Hap. The prepared structures precisely matched the design. Although some aggregation of particles occurred, it was shown that the ceramic component remained well dispersed in the polymeric matrix. SEM images showed exposed ceramic particles on the pore surface, allowing interaction between the bone-forming nano-Hap and cells. References [1] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. [2] Ripamonti U, Ma S, Reddi A. The critical role of geometry of porous hydroxyapatite delivery system of bone by osteogenin, a bone morphogenic protein. Matrix 1992;12:202–12. [3] Huec JL, Schaeverbeke T, Clement D, Faber J, Rebeller AL. Influence of porosity on the mechanical resistance of hydroxyapatite ceramics under compressive stress. Biomaterials 1995;16:113–8. [4] Gloria A, Russo T, De Santis R, Ambrosio L. 3D fiber deposition technique to make multifunctional and tailor-made scaffolds for tissue engineering applications. J Appl Biomater Bioceramics 2009;3:141–52. [5] Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005;4:518–24.
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