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In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres
Acta Biomaterialia 6 (2010) 2525–2531
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres q L.H. Li a,b, K.P. Kommareddy a, C. Pilz a, C.R. Zhou b, P. Fratzl a, I. Manjubala a,* a b
Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany Department of Materials Sciences and Engineering, Jinan University, Guangzhou 510630, China
a r t i c l e
i n f o
Article history: Received 24 December 2008 Received in revised form 16 February 2009 Accepted 24 March 2009 Available online 31 March 2009 Keywords: PLLA Chitosan Hydroxyapatite Scaffolds Cell culture
a b s t r a c t Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-L-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells, guide their growth and regenerate tissue in three dimensions. As bone is a highly complex material with different levels of hierarchy in structure that is composed of organic–inorganic composite as well as proteoglycans and other minor components, the scaffold designed for bone should be able to match such hierarchy and composition. The ideal scaffold has been defined by many researchers [1,2] and several methods have been used to achieve the ideal scaffold, including particle leaching, solution casting, sheet melting and gas formation [3–5]. However, the architecture and structure of the scaffolds were not controllable and reproducible by these methods. A combination of computer-aided design (CAD) and the rapid prototyping (RP) technique allows one to control the macro-
q Research presented at the E-MRS 2008 Symposium on New Scaffolds for Tissue Engineering: Materials and Processing Methods, organized by Dr. W. Swieszkowski and Prof. D.W. Hutmacher. * Corresponding author. Tel.: +49 331 567 9408; fax: +49 331 567 9402. E-mail address: [email protected] (I. Manjubala).
and micro-architecture of the scaffolds and reproduce them successfully [6–8]. Potentially suitable biomaterials for use in bone tissue engineering include: (i) ceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), which are similar to the inorganic component of bone and possess good osteoconductivity or osteoinductivity; (ii) natural polymers, such as chitosan, collagen and hyaluronic acid, which possess good biocompatibility but have poor mechanical strength; and (iii) synthetic polymers and copolymers based on poly(methylmethacrylate), poly-L-lactic acid (PLLA), polycaprolactone, etc., which are biodegradable and have high mechanical strength. Scaffolds composed of only natural/synthetic polymer or ceramic are too flexible and too brittle, respectively. Scaffolds based on these materials have been reported utilizing various RP techniques [9–12]. Since natural bone is a reinforced organic/inorganic composite (collagen/HA composite), a polymerbased HA composite is a promising material for use as bone implants [13–16]. Chitosan composites in combination with either HA or PLLA have already been investigated as bulk material or in scaffold form [17–21]. PLLA is more stable than chitosan, but the biocompatibility of chitosan is much better than that of PLLA, and a combination of these two, as reported earlier, would serve well as a stable
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.03.028
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polymer network for scaffolding purposes. Therefore, the aim of our research work was to develop a three-component organic– inorganic composite scaffold, composed of PLLA, chitosan and HA, with a defined internal architecture and biocompatible properties suitable for osteoconduction and osteoinduction. These scaffolds include HA microspheres, which can be useful as drug delivery vehicles, have been proved to be a good substrate on which pre-osteoblastic cells can proliferate and can form a threedimensional (3-D) network of extracellular matrix.
2. Materials and methods 2.1. Materials Chitosan (mol. wt. 150,000, 85% deacetylated, flakes) was obtained from Fluka Biochemical Ltd. and purified. Hydroxyapatite microspheres (average diameter 10 lm) were purchased from Plasma Biotal, UK. PLLA (inherent viscosity 3.0 dl g 1) was purchased from Boehringer Ingelheim Pharma GmbH, Germany; acetic acid and sodium hydroxide were from Merck GmbH, and absolute ethanol (analytical grade) was from Fischer Organics, Germany.
2.2. Scaffold preparation Molds of a woodpile structure with the required parameters were designed with commercial CAD software. The CAD design was transferred to a 3-D wax printer (Solidscape, Model Maker II) that prints two different wax materials, build wax and support wax. Layers of parallel struts were built, each layer turned 90° with respect to the previous one, laying side-by-side but separated from each other, like a woodpile. The width and thickness of one strut was 500 lm, resulting in a final strut spacing of 500 lm. After the molds were printed, the support wax was removed in Dewasol solution (Solidscape Inc. USA) at 55 °C with constant stirring and the structure was air-dried. A highly viscous chitosan solution was obtained by dissolving chitosan flakes in 2 wt.% acetic acid solution. The chitosan solution and HA powder were mixed together at different ratios (60:40 and 50:50) to produce a homogeneous slurry. For three-component scaffolds, a freshly prepared 2 wt.% PLLA chloroform solution was mixed with an equal amount of the chitosan–HA slurry. The composite slurry was then poured into the molds under vacuum. The slurry-filled molds were kept in a freezer ( 80 °C) for 3 h, freezedried for 24 h ( 60 °C) and then immersed in sodium hydroxide/ ethanol solution under constant stirring and finally in ethanol to remove the wax molds. The obtained scaffolds were dried at 55 °C and stored in vacuum.
2.3. Physicochemical characterization The microstructure of the scaffolds was observed with an environmental scanning electron microscope (Quanta 600 FEG, FEI Europe) at low vacuum and low voltage (15.0 kV). The phase composition of the composite was analyzed with a powder X-ray diffractometer (D8 X-ray diffractometer, Brucker AXS Ltd., UK) using a Cu Ka radiation source. Data were collected from 20 to 60° (2h values), with a step size of 0.02° and a counting time of 2 s per step. To analyze the mechanical property of these scaffolds, a compression test was performed at room temperature using a Zwick universal testing machine (Zwick Z010, Zwick GmbH, Germany) with a constant strain rate of 2 mm min 1. The compressive modulus (E) was determined from the stress–strain curve by linear regression of the slope in the initial elastic region.
2.4. Cell seeding experiments The biocompatibility of the PLLA composite scaffold was investigated with MC3T3-E1 pre-osteoblastic cells according to the protocol reported previously [17]. Chitosan composite scaffolds, chitosan/HA 50:50 and chitosan/HA 60:40 were used for comparison. The cell culture medium used was modified Eagle’s minimum essential medium with 4.5 g l 1 glucose, 10% fetal calf serum, 10 lg ml 1 ascorbic acid and 30 lg ml 1 gentamicin. About 5 105 cells were suspended in culture medium, dispersed over the scaffolds and cultured for about 5 weeks, with the medium changed twice per week. More than 10 scaffolds from each of the composites were used in the experiment and during every medium change the scaffolds were observed with a phase-contrast microscope (Nikon Eclipse TS100, Germany) for cell adhesion and proliferation. 2.5. Alkaline phosphatase enzyme activity Cell differentiation was analyzed with alkaline phosphatase (ALP) enzyme activity after 2, 3, 4 and 5 weeks. The cell-seeded scaffolds were washed with phosphate-buffered saline (PBS), crushed and air-dried under a laminar airflow for 10 min and frozen at 20 °C for 1 h, then lysed with 0.5% Triton X-100 solution for 30 min at room temperature. Next, 20 ll of the lysed product was added to 1 ml of ALP enzyme working reagent (ALP Test Kit Rolf Greiner Biochemica GmbH, Germany) and the mixture was incubated at 37 °C. Absorbance of these samples was measured at 405 nm at 37 °C with a ultraviolet–visible spectrophotometer (UV–vis system, CN 9451, Agilent Technology, Germany). Two samples were measured for every time period and the results are expressed in units of U l 1. 2.6. Histological analysis The scaffolds seeded with cells were rinsed with PBS twice and fixed with 4% paraformaldehyde at 4 °C. The fixed scaffolds were dehydrated in graded series of ethanol and embedded in polymethyl methacrylate resin and sectioned as 100 lm blocks and 5 lm slices for staining. Thick sections were stained with 20% Giemsa stain to observe the cell distribution and the thin sections were stained with Gömori stain to observe the extracellular matrix formation. 2.7. Cell morphology analysis by environmental scanning electron microscopy (ESEM) Scaffolds cultured with cells were washed twice in PBS and kept at 4 °C for 20 min. The cells were fixed with 2% glutaraldehyde and 4% paraformaldehyde at room temperature overnight. The fixed scaffolds were dehydrated in a graded series of ethanol and critical point dried. The cryofractured samples were coated with a thin layer of palladium using a sputter coater (BALTEC, SCD 050) for 30 s under vacuum. Cell morphology on the surface and cross-sections was observed with an environmental scanning electron microscope under low vacuum conditions. 3. Results 3.1. Scaffold characterization The composite scaffolds produced by the RP technique are shown in Fig. 1, along with the blue wax molds. The width of a single strut as well as the distance between two struts was 500 lm. The morphology of cryofractured sections of the scaffolds as
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ber of irregularities at the pore corners due to post-treatment of the scaffolds with sodium hydroxide/ethanol solution. A very interesting phenomenon is that the polymers in the composite form parallel layers along the vertical direction, the direction of freeze-drying (Fig. 2c). The distribution of HA microspheres is uniform and dense, with few micropores between adjacent microspheres. A thin film of polymer entangles the neighboring particles tightly, as seen from Fig. 2d. The X-ray diffraction (XRD) patterns of the raw HA powder and PLLA/chitosan/HA composite scaffold are shown in Fig. 3a. The raw HA powder is highly crystalline in nature, while the crystallinity of HA decreases in the composite, as observed by the broadening of the peaks, but there is no shift in the peak position. These results shows that HA has good compatibility with PLLA and chitosan, and the preparation did not affect the phase composition of the HA. In the XRD spectra no peak due to chitosan was seen due to its low concentration. Compression tests were performed to examine the mechanical properties of the PLLA/chitosan/HA composite scaffolds and to compare them to the chitosan/HA 50:50 and chitosan/HA 60:40 composites. The stress–strain curves resemble the behavior of cellular structures (Fig. 3b). The compressive modulus of the chitosan/HA 50:50, chitosan/HA 60:40 and PLLA composites are 1.07, 1.31 and 1.46 MPa, respectively. 3.2. Cell proliferation and differentiation Fig. 1. (a) Blue wax molds prepared by the RP technique after removing the support wax. The diameter of the molds was 10 mm. (b) PLLA–chitosan–HA composite scaffolds produced by freeze-drying (diameter of the scaffolds are 9 mm).
observed by ESEM is shown in Fig. 2. The overview of a scaffold cross-section (Fig. 2a) shows uniform pore channels through the scaffolds, while the focused pore channel in Fig. 2b shows a num-
The ingrowth of the cells into the pore channels and the tissue formation were observed with ongoing culture time using phasecontrast light microscopy. Fig. 4 shows the growth of MC3T3-El cells on all the scaffolds of chitosan/HA 50:50, chitosan/HA 60:40 and PLLA composites after culture periods of 2 and 5 weeks. After 2 weeks, the cells on each scaffold have already begun to grow around the corner of the pores, and multilayer of cells are visible.
Fig. 2. Environmental scanning electron micrographs of the scaffolds. (a) Overview of a scaffold cross-section showing the square pore channels. (b) A single pore channel clearly showing the pore walls. (c) The layered structure of the polymer film seen in vertical cross-section. (d) HA microspheres connected by polymer film; a few micropores are visible.
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Fig. 4. Phase-contrast light micrographs of cells cultured on the scaffolds. (a and b) Chitosan/HA 50:50, (c and d) chitosan/HA 60:40 and (e and f) PLLA composites after culture for 2 and 5 weeks. The pore edges are marked with rectangles and the cell layers are shown with arrows.
Fig. 3. (a) XRD patterns of PLLA composite powder and pure HA powder. The patterns of HA are indexed according to JCPDS 9-432 and there seems to be no difference between the two patterns except for a decrease in crystallinity. (b) Representative stress–strain curves of compression tests measured for all composite scaffolds showing the plateau region of stress for increasing strain resembling cellular structure.
After 5 weeks, the 2-D images show that the multilayers of cells did not increase much because the cells also proliferated along the height of the pore channel. There is an apparently thicker tissue layer in the chitosan/HA 50:50 scaffold and PLLA composite scaffold than in chitosan/HA 60:40 scaffold. ALP enzyme activity was used as a biochemical marker for determining the differentiation of the pre-osteoblast phenotype to the mature osteoblast phenotype. Fig. 5 shows the ALP enzyme activity of the cells on three different scaffolds with culture time. There is an increasing tendency of ALP enzyme activity initially (up to 3–4 weeks) in all the scaffolds, indicating the differentiation of MC3T3-E1 pre-osteoblastic cells into mature osteoblasts. It is interesting to note that the ALP values of the PLLA composite scaffolds were close to those of other two samples until 3 weeks, was increased at 4 weeks but decreased at 5 weeks.
Fig. 5. ALP enzyme activity of MC3T3-E1 pre-osteoblast cells cultured on composite scaffolds at different time periods.
by arrows) both in pore channels and in micropores connecting the HA microspheres.
3.3. Histological analysis
3.4. Cell morphology observed by ESEM
The sections of the cell-seeded scaffolds after 5 weeks of culture stained with Giemsa and Gömori stains are shown in Fig. 6. It was observed that the cells proliferated on the struts and also inside the pore channel in a circular form, forming a round channel, as seen in Fig. 6b. A thick cell layer can be clearly seen around the corner of the pores. Fig. 6(c) and (d) shows the extracellular matrix (stained brown) on the scaffold consisting of a fibrous network (indicated
The morphology and distribution of the MC3T3-E1 cells attached on the scaffolds observed by ESEM are shown in Fig. 7. The uniform distribution of the cells over the HA microspheres in one of the struts after 3 weeks of culture is seen in Fig. 7a. Most of the cells were polygonal in shape, fully spread and formed multilayers (Fig. 7b). The cells were proliferating not only in the pore channel and on the outer surface, but also inside the micropores
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Fig. 6. Optical micrographs of stained scaffold sections after 5 weeks of culture. Giemsa staining shows the cells (a) surrounding the struts and (b) inside the pore channel, where the circular growth of the cells can be seen clearly; (c and d) Gömori staining revealing the extracellular matrix produced by the cells distributed on both macro- and micropores (marked with arrows).
Fig. 7. ESEM images of the cultured cells on the scaffolds surface. (a) One strut of the scaffold covered with cells showing distribution of microspheres and cells on it (3 weeks of culture); (b) multilayer cell growth (3 weeks of culture); (c) cells inside the micropore spreading their fine filopodia to attach to the surface; and (d) a single pore channel of the scaffold being filled with a circular cellular channel in three dimensions (5 weeks of culture).
of the struts. Fig. 7c focuses deeper inside one of the micropores. It shows layers of cells on the outside of the pore and a cell inside the pore with many filopodia. The fine filopodia generated by the cells not only anchor onto the surface of the materials but also interconnect cells with each other. Fig. 7d presents an overview of one of the pore channels filled with cells 5 weeks after culture, showing that the cells grow in a circular channel towards the interior of the square pore channel. The square pore was rounded
by the cells and the diameter decreased from 500 lm to about 200 lm.
4. Discussion It is challenging to design a scaffold similar to trabecular bone architecture whilst considering the properties of the fluid in-flow
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structure. In this research, we used an indirect RP technique incorporating a wax printer to construct 3-D porous scaffolds with a defined pore size and interconnected pore channels, and a subsequent freeze drying technique to induce microporosity. Due to the interconnected pores, the composite scaffolds were successfully obtained by vacuum-assisted infusion of the homogeneous solution. The diameter of the pore channel in the scaffolds is about 500 lm, which facilitates cell immigration and colonization, and fluid and nutrient exchange. The coarse surface of HA, the gaps between particles and the fine polymer films generated a tough surface and micropores in the struts. There were parallel polymer layers (see Fig. 4b), formed by PLLA and chitosan due to continuous freeze-drying process, along the direction of airflow. Between the layers, the polymers formed a complex network and entangled the HA microspheres tightly. This structure can be linked to the structure of mature cortical bone [22,23], where collagen fibrils are assembled into a lamellar structure, which has been described in analogy to plywood. However, the results of the compressive test show that these scaffolds can be used as non-load-bearing bone alternatives. The stress–strain curves for the scaffolds resemble typical curves of the compression of foams or cellular solids [24], where the strength or modulus is related to the relative density and architecture of the material. These curves suggest that the scaffold has an initial elastic region (marked as ‘‘a”), followed by a constant plateau (Fig. 3b) where the stress shielding effect of the scaffold can be seen, then followed by a large increase in stress (marked as ‘‘b”), suggesting that the scaffold can serve as an energy-absorbing material, such as a protective layer. The high strength of the composite scaffold can be explained by the contribution of PLLA, which has good mechanical strength. ESEM observation of the composites (Fig. 4) shows that the strong PLLA film and chitosan fibres form an interpenetrating network, which holds the HA particles tightly. Comparing this with the data for human bone, the compressive strength of the composite scaffold is still far from that of cortical bone (strength of 130–180 MPa, modulus of 12–18 103 MPa) and cancellous bone (strength of 4–12 MPa, modulus of 0.1– 0.5 103 MPa) [25–27], but is closer to cartilage (strength of 4– 59 MPa and modulus of 1.9–14.4 MPa) [28] or the initial soft callus material that forms during bone fracture, which has modulus of less than 1 GPa. Since the mechanical property was measured in the dry state and behaved similar to cellular structures, the strength should definitely be increased in wet conditions [24]. Therefore these materials can be used as a substitute for cartilage or initial fracture healing callus, which can remodel and develop into bone tissue. It is good to have scaffolds with such mechanical properties and structure that can facilitate the proliferation and growth of cells without any tension or stress shielding, while the bulk dense material, having a large elastic modulus, produces tension during cell growth and the cells are squeezed in a limited space. Well-interconnected structures and pore channels are important and basic issues in tissue engineering as they allow the migration of cells into the scaffold and permit good diffusion of nutrients, eventually distributing substances throughout the structure [29,30]. The ability of the scaffolds to enhance tissue regeneration was observed with MC3T3-E1 pre-osteoblastic cells. The surface properties of the materials are very important for the adhesion of proteins, which requires toughness and hydrophilicity. HA and chitosan are hydrophilic materials, and the composite scaffold have a porous morphology that is suitable for cell proliferation as well as nutrition supply. The cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores (Fig. 6c). PLLA composites have the equivalent tissue formation as the other two scaffolds after 5 weeks of culture (see Fig. 7). Fur-
thermore, the cells grew not only around the corner of the macropores of the scaffold but also in the micropores of the strut. The presence of PLLA in the composite scaffolds increased the initial cell proliferation and differentiation process up to 4 weeks, as revealed by the tissue growth and ALP enzyme activity. In the later stages, at 5 weeks, a decrease in ALP was observed for PLLA composite scaffolds which might be due to a partial degradation of the polymer. Furthermore, the micropores between the HA microspheres would provide a good environment for the slow release of growth factors and drugs, should the microspheres be loaded with them.
5. Conclusions In conclusion, PLLA composite scaffolds consisting of chitosan and HA microspheres were shown to have a good interconnected porous nature, with few micropores. The mechanical properties of the composite scaffolds resemble those of cellular foams, where the strength can be increased in fluid environments. The proliferation of MC3T3-E1 osteoblastic cells showed 3-D tissue growth in the porous scaffolds, and an increase in ALP production is an indication of the differentiation of pre-osteoblastic cells into a mature osteoblastic phenotype. Histological analysis showed that the cells grow not only in the macropores, but also in micropores of the struts. Such 3-D porous scaffolds could be employed as a potential biomaterial for bone reconstruction as non-load-bearing implants. Acknowledgements L.H. Li thank DAAD for the grant of exchange fellowship. The generous gift of the cells from Dr. Franz Varga, Ludwig Boltzmann Institute of Osteology, Vienna is kindly acknowledged. The authors thank Mrs. Rona Pitschke for critical point drying of the samples. References [1] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. [2] Yang SF, Leong KF, Chua CK. The design of scaffolds for use in tissue engineering. Part 1. Traditional approaches. Tissue Eng 2002;8:679–89. [3] Li LH, Zhang R, Li QQ, Zhou CR. Use of SCF-CO2 technique to fabricate scaffolds. Tissue Eng 2006;12:1064. [4] Mathieu LM, Mueller TL, Bourban P, Pioletti DP, Müller R, Manson JE. Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering. Biomaterials 2006;27:905–16. [5] Kim SS, Park MS, Jeon O, Choi CY, Kim BS. Poly(lactide-co-glycolide)/ hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 2006;27:1399–409. [6] Yeong WY, Chua CK, Leong KF, Chandrasekaran M. Rapid prototyping in tissue engineering: challenges and potential. Trends Biotechnol 2004;22:643–52. [7] Wilson CE, Bruijn JD, Blitterswijk CA, Verbout AJ, Dhert WJA. Design and fabrication of standardized hydroxyapatite scaffolds with a defined macroarchitecture by rapid prototyping for bone-tissue-engineering research. J Biomed Mater Res Part A 2003;68A:123–32. [8] Leong KF, Cheah CM, Chua CK. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials 2003;24(13):2363–78. [9] Wiria FE, Leong KF, Chua CK, Liu Y. Poly-e-caprolactone/hydroxyapatite for tissue engineering scaffold fabrication via selective laser sintering. Acta Biomater 2007;3:1–12. [10] Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modelling. J Biomed Mater Res 2001;55(2):203–16. [11] Sachlos E, Reis N, Ainsley C, Derby B, Czernuszka JT. Novel collagen scaffolds with predefined internal morphology made by solid free form fabrication. Biomaterials 2003;24(8):1487–97. [12] Tan KH, Chua CK, Leong KF, Cheah CM, Gui WS, Tan WS. Selective laser sintering of biocompatible polymers for applications in tissue engineering. Bio-Med Mater Eng 2005;15(1,2):113–24.
L.H. Li et al. / Acta Biomaterialia 6 (2010) 2525–2531 [13] Devin JE, Attawia MA, Laurencin CT. Three-dimensional degradable porous polymer–ceramic matrices for use in bone repair. J Biomater Sci Polymer 1996;8:661–9. [14] Marra KG, Szem JW, Kumta PN, DiMilla PA, Weiss LE. In vitro analysis of biodegradable polymer blend/hydroxyapatite composites for bone tissue engineering. J Biomed Mater Res 1999;47:324–35. [15] Laurencin CT, Lu HH. Polymer–ceramic composites for bone-tissue engineering. Bone Eng 2000:462–72. [16] Maquet V, Boccaccini AR, Pravata L, Notingher I, Jérôme R. Porous poly (ahydroxyacid)/bioglass, composite scaffolds for bone tissue engineering. I. Preparation and in vitro characterisation. Biomaterials 2004;25:4185–94. [17] Manjubala I, Woesz A, Pilz C, Rumpler M, Fratzl-Zelman N, Roschger P, Stampfl J, Fratzl P. Biomimetic mineral–organic composite scaffolds with controlled internal architecture. J Mater Sci 2005;6:1111–9. [18] Li LH, Ding S, Zhou CR. Preparation and biological evaluation of PLA/chitosan composite materials. J Biomed Eng 2003;20:398–400. [19] Rumpler M, Woesz A, Varga F, Manjubala I, Klaushofer K, Fratzl P. Threedimensional growth behaviour of osteoblasts on biomimetic hydroxylapatite scaffolds. J Biomed Mater Res 2007;81A:40–50. [20] Manjubala I, Scheler S, Bossert J, Jandt KD. Mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique. Acta Biomater 2007;2:75–84.
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Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres q L.H. Li a,b, K.P. Kommareddy a, C. Pilz a, C.R. Zhou b, P. Fratzl a, I. Manjubala a,* a b
Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany Department of Materials Sciences and Engineering, Jinan University, Guangzhou 510630, China
a r t i c l e
i n f o
Article history: Received 24 December 2008 Received in revised form 16 February 2009 Accepted 24 March 2009 Available online 31 March 2009 Keywords: PLLA Chitosan Hydroxyapatite Scaffolds Cell culture
a b s t r a c t Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-L-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells, guide their growth and regenerate tissue in three dimensions. As bone is a highly complex material with different levels of hierarchy in structure that is composed of organic–inorganic composite as well as proteoglycans and other minor components, the scaffold designed for bone should be able to match such hierarchy and composition. The ideal scaffold has been defined by many researchers [1,2] and several methods have been used to achieve the ideal scaffold, including particle leaching, solution casting, sheet melting and gas formation [3–5]. However, the architecture and structure of the scaffolds were not controllable and reproducible by these methods. A combination of computer-aided design (CAD) and the rapid prototyping (RP) technique allows one to control the macro-
q Research presented at the E-MRS 2008 Symposium on New Scaffolds for Tissue Engineering: Materials and Processing Methods, organized by Dr. W. Swieszkowski and Prof. D.W. Hutmacher. * Corresponding author. Tel.: +49 331 567 9408; fax: +49 331 567 9402. E-mail address: [email protected] (I. Manjubala).
and micro-architecture of the scaffolds and reproduce them successfully [6–8]. Potentially suitable biomaterials for use in bone tissue engineering include: (i) ceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), which are similar to the inorganic component of bone and possess good osteoconductivity or osteoinductivity; (ii) natural polymers, such as chitosan, collagen and hyaluronic acid, which possess good biocompatibility but have poor mechanical strength; and (iii) synthetic polymers and copolymers based on poly(methylmethacrylate), poly-L-lactic acid (PLLA), polycaprolactone, etc., which are biodegradable and have high mechanical strength. Scaffolds composed of only natural/synthetic polymer or ceramic are too flexible and too brittle, respectively. Scaffolds based on these materials have been reported utilizing various RP techniques [9–12]. Since natural bone is a reinforced organic/inorganic composite (collagen/HA composite), a polymerbased HA composite is a promising material for use as bone implants [13–16]. Chitosan composites in combination with either HA or PLLA have already been investigated as bulk material or in scaffold form [17–21]. PLLA is more stable than chitosan, but the biocompatibility of chitosan is much better than that of PLLA, and a combination of these two, as reported earlier, would serve well as a stable
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polymer network for scaffolding purposes. Therefore, the aim of our research work was to develop a three-component organic– inorganic composite scaffold, composed of PLLA, chitosan and HA, with a defined internal architecture and biocompatible properties suitable for osteoconduction and osteoinduction. These scaffolds include HA microspheres, which can be useful as drug delivery vehicles, have been proved to be a good substrate on which pre-osteoblastic cells can proliferate and can form a threedimensional (3-D) network of extracellular matrix.
2. Materials and methods 2.1. Materials Chitosan (mol. wt. 150,000, 85% deacetylated, flakes) was obtained from Fluka Biochemical Ltd. and purified. Hydroxyapatite microspheres (average diameter 10 lm) were purchased from Plasma Biotal, UK. PLLA (inherent viscosity 3.0 dl g 1) was purchased from Boehringer Ingelheim Pharma GmbH, Germany; acetic acid and sodium hydroxide were from Merck GmbH, and absolute ethanol (analytical grade) was from Fischer Organics, Germany.
2.2. Scaffold preparation Molds of a woodpile structure with the required parameters were designed with commercial CAD software. The CAD design was transferred to a 3-D wax printer (Solidscape, Model Maker II) that prints two different wax materials, build wax and support wax. Layers of parallel struts were built, each layer turned 90° with respect to the previous one, laying side-by-side but separated from each other, like a woodpile. The width and thickness of one strut was 500 lm, resulting in a final strut spacing of 500 lm. After the molds were printed, the support wax was removed in Dewasol solution (Solidscape Inc. USA) at 55 °C with constant stirring and the structure was air-dried. A highly viscous chitosan solution was obtained by dissolving chitosan flakes in 2 wt.% acetic acid solution. The chitosan solution and HA powder were mixed together at different ratios (60:40 and 50:50) to produce a homogeneous slurry. For three-component scaffolds, a freshly prepared 2 wt.% PLLA chloroform solution was mixed with an equal amount of the chitosan–HA slurry. The composite slurry was then poured into the molds under vacuum. The slurry-filled molds were kept in a freezer ( 80 °C) for 3 h, freezedried for 24 h ( 60 °C) and then immersed in sodium hydroxide/ ethanol solution under constant stirring and finally in ethanol to remove the wax molds. The obtained scaffolds were dried at 55 °C and stored in vacuum.
2.3. Physicochemical characterization The microstructure of the scaffolds was observed with an environmental scanning electron microscope (Quanta 600 FEG, FEI Europe) at low vacuum and low voltage (15.0 kV). The phase composition of the composite was analyzed with a powder X-ray diffractometer (D8 X-ray diffractometer, Brucker AXS Ltd., UK) using a Cu Ka radiation source. Data were collected from 20 to 60° (2h values), with a step size of 0.02° and a counting time of 2 s per step. To analyze the mechanical property of these scaffolds, a compression test was performed at room temperature using a Zwick universal testing machine (Zwick Z010, Zwick GmbH, Germany) with a constant strain rate of 2 mm min 1. The compressive modulus (E) was determined from the stress–strain curve by linear regression of the slope in the initial elastic region.
2.4. Cell seeding experiments The biocompatibility of the PLLA composite scaffold was investigated with MC3T3-E1 pre-osteoblastic cells according to the protocol reported previously [17]. Chitosan composite scaffolds, chitosan/HA 50:50 and chitosan/HA 60:40 were used for comparison. The cell culture medium used was modified Eagle’s minimum essential medium with 4.5 g l 1 glucose, 10% fetal calf serum, 10 lg ml 1 ascorbic acid and 30 lg ml 1 gentamicin. About 5 105 cells were suspended in culture medium, dispersed over the scaffolds and cultured for about 5 weeks, with the medium changed twice per week. More than 10 scaffolds from each of the composites were used in the experiment and during every medium change the scaffolds were observed with a phase-contrast microscope (Nikon Eclipse TS100, Germany) for cell adhesion and proliferation. 2.5. Alkaline phosphatase enzyme activity Cell differentiation was analyzed with alkaline phosphatase (ALP) enzyme activity after 2, 3, 4 and 5 weeks. The cell-seeded scaffolds were washed with phosphate-buffered saline (PBS), crushed and air-dried under a laminar airflow for 10 min and frozen at 20 °C for 1 h, then lysed with 0.5% Triton X-100 solution for 30 min at room temperature. Next, 20 ll of the lysed product was added to 1 ml of ALP enzyme working reagent (ALP Test Kit Rolf Greiner Biochemica GmbH, Germany) and the mixture was incubated at 37 °C. Absorbance of these samples was measured at 405 nm at 37 °C with a ultraviolet–visible spectrophotometer (UV–vis system, CN 9451, Agilent Technology, Germany). Two samples were measured for every time period and the results are expressed in units of U l 1. 2.6. Histological analysis The scaffolds seeded with cells were rinsed with PBS twice and fixed with 4% paraformaldehyde at 4 °C. The fixed scaffolds were dehydrated in graded series of ethanol and embedded in polymethyl methacrylate resin and sectioned as 100 lm blocks and 5 lm slices for staining. Thick sections were stained with 20% Giemsa stain to observe the cell distribution and the thin sections were stained with Gömori stain to observe the extracellular matrix formation. 2.7. Cell morphology analysis by environmental scanning electron microscopy (ESEM) Scaffolds cultured with cells were washed twice in PBS and kept at 4 °C for 20 min. The cells were fixed with 2% glutaraldehyde and 4% paraformaldehyde at room temperature overnight. The fixed scaffolds were dehydrated in a graded series of ethanol and critical point dried. The cryofractured samples were coated with a thin layer of palladium using a sputter coater (BALTEC, SCD 050) for 30 s under vacuum. Cell morphology on the surface and cross-sections was observed with an environmental scanning electron microscope under low vacuum conditions. 3. Results 3.1. Scaffold characterization The composite scaffolds produced by the RP technique are shown in Fig. 1, along with the blue wax molds. The width of a single strut as well as the distance between two struts was 500 lm. The morphology of cryofractured sections of the scaffolds as
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ber of irregularities at the pore corners due to post-treatment of the scaffolds with sodium hydroxide/ethanol solution. A very interesting phenomenon is that the polymers in the composite form parallel layers along the vertical direction, the direction of freeze-drying (Fig. 2c). The distribution of HA microspheres is uniform and dense, with few micropores between adjacent microspheres. A thin film of polymer entangles the neighboring particles tightly, as seen from Fig. 2d. The X-ray diffraction (XRD) patterns of the raw HA powder and PLLA/chitosan/HA composite scaffold are shown in Fig. 3a. The raw HA powder is highly crystalline in nature, while the crystallinity of HA decreases in the composite, as observed by the broadening of the peaks, but there is no shift in the peak position. These results shows that HA has good compatibility with PLLA and chitosan, and the preparation did not affect the phase composition of the HA. In the XRD spectra no peak due to chitosan was seen due to its low concentration. Compression tests were performed to examine the mechanical properties of the PLLA/chitosan/HA composite scaffolds and to compare them to the chitosan/HA 50:50 and chitosan/HA 60:40 composites. The stress–strain curves resemble the behavior of cellular structures (Fig. 3b). The compressive modulus of the chitosan/HA 50:50, chitosan/HA 60:40 and PLLA composites are 1.07, 1.31 and 1.46 MPa, respectively. 3.2. Cell proliferation and differentiation Fig. 1. (a) Blue wax molds prepared by the RP technique after removing the support wax. The diameter of the molds was 10 mm. (b) PLLA–chitosan–HA composite scaffolds produced by freeze-drying (diameter of the scaffolds are 9 mm).
observed by ESEM is shown in Fig. 2. The overview of a scaffold cross-section (Fig. 2a) shows uniform pore channels through the scaffolds, while the focused pore channel in Fig. 2b shows a num-
The ingrowth of the cells into the pore channels and the tissue formation were observed with ongoing culture time using phasecontrast light microscopy. Fig. 4 shows the growth of MC3T3-El cells on all the scaffolds of chitosan/HA 50:50, chitosan/HA 60:40 and PLLA composites after culture periods of 2 and 5 weeks. After 2 weeks, the cells on each scaffold have already begun to grow around the corner of the pores, and multilayer of cells are visible.
Fig. 2. Environmental scanning electron micrographs of the scaffolds. (a) Overview of a scaffold cross-section showing the square pore channels. (b) A single pore channel clearly showing the pore walls. (c) The layered structure of the polymer film seen in vertical cross-section. (d) HA microspheres connected by polymer film; a few micropores are visible.
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Fig. 4. Phase-contrast light micrographs of cells cultured on the scaffolds. (a and b) Chitosan/HA 50:50, (c and d) chitosan/HA 60:40 and (e and f) PLLA composites after culture for 2 and 5 weeks. The pore edges are marked with rectangles and the cell layers are shown with arrows.
Fig. 3. (a) XRD patterns of PLLA composite powder and pure HA powder. The patterns of HA are indexed according to JCPDS 9-432 and there seems to be no difference between the two patterns except for a decrease in crystallinity. (b) Representative stress–strain curves of compression tests measured for all composite scaffolds showing the plateau region of stress for increasing strain resembling cellular structure.
After 5 weeks, the 2-D images show that the multilayers of cells did not increase much because the cells also proliferated along the height of the pore channel. There is an apparently thicker tissue layer in the chitosan/HA 50:50 scaffold and PLLA composite scaffold than in chitosan/HA 60:40 scaffold. ALP enzyme activity was used as a biochemical marker for determining the differentiation of the pre-osteoblast phenotype to the mature osteoblast phenotype. Fig. 5 shows the ALP enzyme activity of the cells on three different scaffolds with culture time. There is an increasing tendency of ALP enzyme activity initially (up to 3–4 weeks) in all the scaffolds, indicating the differentiation of MC3T3-E1 pre-osteoblastic cells into mature osteoblasts. It is interesting to note that the ALP values of the PLLA composite scaffolds were close to those of other two samples until 3 weeks, was increased at 4 weeks but decreased at 5 weeks.
Fig. 5. ALP enzyme activity of MC3T3-E1 pre-osteoblast cells cultured on composite scaffolds at different time periods.
by arrows) both in pore channels and in micropores connecting the HA microspheres.
3.3. Histological analysis
3.4. Cell morphology observed by ESEM
The sections of the cell-seeded scaffolds after 5 weeks of culture stained with Giemsa and Gömori stains are shown in Fig. 6. It was observed that the cells proliferated on the struts and also inside the pore channel in a circular form, forming a round channel, as seen in Fig. 6b. A thick cell layer can be clearly seen around the corner of the pores. Fig. 6(c) and (d) shows the extracellular matrix (stained brown) on the scaffold consisting of a fibrous network (indicated
The morphology and distribution of the MC3T3-E1 cells attached on the scaffolds observed by ESEM are shown in Fig. 7. The uniform distribution of the cells over the HA microspheres in one of the struts after 3 weeks of culture is seen in Fig. 7a. Most of the cells were polygonal in shape, fully spread and formed multilayers (Fig. 7b). The cells were proliferating not only in the pore channel and on the outer surface, but also inside the micropores
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Fig. 6. Optical micrographs of stained scaffold sections after 5 weeks of culture. Giemsa staining shows the cells (a) surrounding the struts and (b) inside the pore channel, where the circular growth of the cells can be seen clearly; (c and d) Gömori staining revealing the extracellular matrix produced by the cells distributed on both macro- and micropores (marked with arrows).
Fig. 7. ESEM images of the cultured cells on the scaffolds surface. (a) One strut of the scaffold covered with cells showing distribution of microspheres and cells on it (3 weeks of culture); (b) multilayer cell growth (3 weeks of culture); (c) cells inside the micropore spreading their fine filopodia to attach to the surface; and (d) a single pore channel of the scaffold being filled with a circular cellular channel in three dimensions (5 weeks of culture).
of the struts. Fig. 7c focuses deeper inside one of the micropores. It shows layers of cells on the outside of the pore and a cell inside the pore with many filopodia. The fine filopodia generated by the cells not only anchor onto the surface of the materials but also interconnect cells with each other. Fig. 7d presents an overview of one of the pore channels filled with cells 5 weeks after culture, showing that the cells grow in a circular channel towards the interior of the square pore channel. The square pore was rounded
by the cells and the diameter decreased from 500 lm to about 200 lm.
4. Discussion It is challenging to design a scaffold similar to trabecular bone architecture whilst considering the properties of the fluid in-flow
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structure. In this research, we used an indirect RP technique incorporating a wax printer to construct 3-D porous scaffolds with a defined pore size and interconnected pore channels, and a subsequent freeze drying technique to induce microporosity. Due to the interconnected pores, the composite scaffolds were successfully obtained by vacuum-assisted infusion of the homogeneous solution. The diameter of the pore channel in the scaffolds is about 500 lm, which facilitates cell immigration and colonization, and fluid and nutrient exchange. The coarse surface of HA, the gaps between particles and the fine polymer films generated a tough surface and micropores in the struts. There were parallel polymer layers (see Fig. 4b), formed by PLLA and chitosan due to continuous freeze-drying process, along the direction of airflow. Between the layers, the polymers formed a complex network and entangled the HA microspheres tightly. This structure can be linked to the structure of mature cortical bone [22,23], where collagen fibrils are assembled into a lamellar structure, which has been described in analogy to plywood. However, the results of the compressive test show that these scaffolds can be used as non-load-bearing bone alternatives. The stress–strain curves for the scaffolds resemble typical curves of the compression of foams or cellular solids [24], where the strength or modulus is related to the relative density and architecture of the material. These curves suggest that the scaffold has an initial elastic region (marked as ‘‘a”), followed by a constant plateau (Fig. 3b) where the stress shielding effect of the scaffold can be seen, then followed by a large increase in stress (marked as ‘‘b”), suggesting that the scaffold can serve as an energy-absorbing material, such as a protective layer. The high strength of the composite scaffold can be explained by the contribution of PLLA, which has good mechanical strength. ESEM observation of the composites (Fig. 4) shows that the strong PLLA film and chitosan fibres form an interpenetrating network, which holds the HA particles tightly. Comparing this with the data for human bone, the compressive strength of the composite scaffold is still far from that of cortical bone (strength of 130–180 MPa, modulus of 12–18 103 MPa) and cancellous bone (strength of 4–12 MPa, modulus of 0.1– 0.5 103 MPa) [25–27], but is closer to cartilage (strength of 4– 59 MPa and modulus of 1.9–14.4 MPa) [28] or the initial soft callus material that forms during bone fracture, which has modulus of less than 1 GPa. Since the mechanical property was measured in the dry state and behaved similar to cellular structures, the strength should definitely be increased in wet conditions [24]. Therefore these materials can be used as a substitute for cartilage or initial fracture healing callus, which can remodel and develop into bone tissue. It is good to have scaffolds with such mechanical properties and structure that can facilitate the proliferation and growth of cells without any tension or stress shielding, while the bulk dense material, having a large elastic modulus, produces tension during cell growth and the cells are squeezed in a limited space. Well-interconnected structures and pore channels are important and basic issues in tissue engineering as they allow the migration of cells into the scaffold and permit good diffusion of nutrients, eventually distributing substances throughout the structure [29,30]. The ability of the scaffolds to enhance tissue regeneration was observed with MC3T3-E1 pre-osteoblastic cells. The surface properties of the materials are very important for the adhesion of proteins, which requires toughness and hydrophilicity. HA and chitosan are hydrophilic materials, and the composite scaffold have a porous morphology that is suitable for cell proliferation as well as nutrition supply. The cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores (Fig. 6c). PLLA composites have the equivalent tissue formation as the other two scaffolds after 5 weeks of culture (see Fig. 7). Fur-
thermore, the cells grew not only around the corner of the macropores of the scaffold but also in the micropores of the strut. The presence of PLLA in the composite scaffolds increased the initial cell proliferation and differentiation process up to 4 weeks, as revealed by the tissue growth and ALP enzyme activity. In the later stages, at 5 weeks, a decrease in ALP was observed for PLLA composite scaffolds which might be due to a partial degradation of the polymer. Furthermore, the micropores between the HA microspheres would provide a good environment for the slow release of growth factors and drugs, should the microspheres be loaded with them.
5. Conclusions In conclusion, PLLA composite scaffolds consisting of chitosan and HA microspheres were shown to have a good interconnected porous nature, with few micropores. The mechanical properties of the composite scaffolds resemble those of cellular foams, where the strength can be increased in fluid environments. The proliferation of MC3T3-E1 osteoblastic cells showed 3-D tissue growth in the porous scaffolds, and an increase in ALP production is an indication of the differentiation of pre-osteoblastic cells into a mature osteoblastic phenotype. Histological analysis showed that the cells grow not only in the macropores, but also in micropores of the struts. Such 3-D porous scaffolds could be employed as a potential biomaterial for bone reconstruction as non-load-bearing implants. Acknowledgements L.H. Li thank DAAD for the grant of exchange fellowship. The generous gift of the cells from Dr. Franz Varga, Ludwig Boltzmann Institute of Osteology, Vienna is kindly acknowledged. The authors thank Mrs. Rona Pitschke for critical point drying of the samples. References [1] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. [2] Yang SF, Leong KF, Chua CK. The design of scaffolds for use in tissue engineering. Part 1. Traditional approaches. Tissue Eng 2002;8:679–89. [3] Li LH, Zhang R, Li QQ, Zhou CR. Use of SCF-CO2 technique to fabricate scaffolds. Tissue Eng 2006;12:1064. [4] Mathieu LM, Mueller TL, Bourban P, Pioletti DP, Müller R, Manson JE. Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering. Biomaterials 2006;27:905–16. [5] Kim SS, Park MS, Jeon O, Choi CY, Kim BS. Poly(lactide-co-glycolide)/ hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 2006;27:1399–409. [6] Yeong WY, Chua CK, Leong KF, Chandrasekaran M. Rapid prototyping in tissue engineering: challenges and potential. Trends Biotechnol 2004;22:643–52. [7] Wilson CE, Bruijn JD, Blitterswijk CA, Verbout AJ, Dhert WJA. Design and fabrication of standardized hydroxyapatite scaffolds with a defined macroarchitecture by rapid prototyping for bone-tissue-engineering research. J Biomed Mater Res Part A 2003;68A:123–32. [8] Leong KF, Cheah CM, Chua CK. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials 2003;24(13):2363–78. [9] Wiria FE, Leong KF, Chua CK, Liu Y. Poly-e-caprolactone/hydroxyapatite for tissue engineering scaffold fabrication via selective laser sintering. Acta Biomater 2007;3:1–12. [10] Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modelling. J Biomed Mater Res 2001;55(2):203–16. [11] Sachlos E, Reis N, Ainsley C, Derby B, Czernuszka JT. Novel collagen scaffolds with predefined internal morphology made by solid free form fabrication. Biomaterials 2003;24(8):1487–97. [12] Tan KH, Chua CK, Leong KF, Cheah CM, Gui WS, Tan WS. Selective laser sintering of biocompatible polymers for applications in tissue engineering. Bio-Med Mater Eng 2005;15(1,2):113–24.
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