Case study Fabrication of articial bioactive bone using rapid prototyping

Zhongzhong Chen, Dichen Li, Bingheng Lu, Yiping Tang, Minglin Sun and Zhen Wang
The authors
Zhongzhong Chen, Dichen Li, Bingheng Lu, Yipng Tang, and Minglin Sun are all based at Institute of Advanced Manufacturing Technology, Xian Jiaotong University, Xian, Peoples Republic of China. Zhen Wang is a professor from the Fourth Military Medical University, Xian, Peoples Republic of China.

Introduction
In bone graft operations, the preferred treatment is the use of autologous bone with a view to attain biocompatibility and bio-afnity. However, the supply of suitable bone is limited and a risk of infection is bound to occur. In bone tissue engineering, large segmental bone defects, pose severe challenge to reconstructive surgery, because the bone substitute materials should have appropriate porosity and structural features. The traditional ways of fabricating porous scaffolds include polymer foaming technique, particulate-leaching, solid-liquid phase separation, textile technique and extrusion process, etc. But with these methods, the biomimetic scaffolds similar to morphological characteristics to the internal microtubule structure of the natural bone could not be ensured, which is essential to vascularization and tissue regeneration. Therefore, the key problem in the fabrication of articial bone is, how to construct the simulated interior microstructure with proper connectivity so as to obtain a good biological behavior and provide a porous scaffold for vascularization. Based on the building principle of fused deposition modeling (FDM) in RP, a new forming technique of air-pressure jet solidication (AJS) system is developed, which can built up the mould of articial bone with customized geometric contour and internal porous architectures.

Keywords
Rapid prototypes, Bones

Abstract
A new technique based on rapid prototyping (RP) is proposed to fabricate the mould of articial bone composed of a nontoxic soluble material. The mould has both an external structure that exactly coincides with the replaced natural bone and an internal 3D scaffolds simulating the bone microtubule structure. By injecting self-setting calcium phosphate cement (CPC) with bone morphogenetic protein (BMP, a kind of bone growth factors) into the cavities of the mould, the CPC solidied and the micropores can be formed after the internal 3D scaffolds is dissolved, nally the articial bioactive bone can be produced. This approach is better than the traditional fabrication process, which the latter method cannot fabricate an articial bone with inter-connective micropores so as to realize the osteo-induction for lack of bioactivity. Through animal experiments, it shows that the simulated inter-structure could provide articial bone with proper voids for the growth of the bone tissue and the quick activation, and hence effectively speed up the bone growth by means of activating osteo-conduction and osteo-induction. So, the new method of fabricating articial bone with biological behaviors is justied.

Mould fabrication of articial bone

Biomimetic modeling The biomimetic modeling includes the external contour CAD modeling and the internal microtubule structure CAD modeling. The corresponding slicing data can be output from the two CAD modelings, respectively. (1) CAD modeling of the external contour Based on computed tomography (CT) scanning data, the bony structural geometry model can be
Received: 10 April 2003 Reviewed: 6 May 2004 Accepted: 12 July 2004 All animal experiments described in this paper were carried out by the Animal Experiment Center in the Fourth Military Medical University (Xian, 710033, China). All operations and treatments were carried out by medical personnel, and animal boarding management was carried out by medical personnel and animal health technicians. The authors gratefully acknowledge the support of the Natural Science Foundation of China (No. 50235020).

Electronic access
The Emerald Research Register for this journal is available at www.emeraldinsight.com/researchregister The current issue and full text archive of this journal is available at www.emeraldinsight.com/1355-2546.htm
Rapid Prototyping Journal Volume 10 Number 5 2004 pp. 327333 q Emerald Group Publishing Limited ISSN 1355-2546 DOI 10.1108/13552540410562368

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Fabrication of articial bioactive bone using rapid prototyping

Rapid Prototyping Journal Volume 10 Number 5 2004 327333

Zhongzhong Chen et al.

reconstructed by the 3D modeling software. Then the data with STL format (the industry standard of RP) can be output and loaded in AJS system to fabricate the external contour of the mould. Figure 1(a) shows the solid model of a reconstructed segmental canine radius.

Figure 2 Internal microtubule structure CAD modeling of the articial bone

(2) CAD modeling of the internal microtubule structure Based on the histological observation and analysis, the osteo-structure analogous CAD model can be obtained by observing the density, shapes, apertures and distribution of Volkmanns canal and Haversian canal with electronic microscope. The corresponding mathematical model can be built up through abstracting the structural features from micrographs and histological analysis (Figure 1b, 1 Medullary cavity of bone, 2 External contour of bone, 3 Haversian system (Osteon), 4 Volkmans canals). An REV-engineering software is developed to treat these features and generate the processing data to fabricate the internal 3D scaffolds (Figure 2). In addition, the fabrication of the medullary cavity is ignored in the process, because the main work in this research is to study the inuence of porous architecture on osteogenesis, rather than in emphasizing the exact likeness with the geometric contour of the natural bone. Furthermore, in view of bone tissue engineering, the inner cavity could be formed in the center of the articial bone after degradation.
Figure 1 (a) Solid CAD model of the articial bone (b) Geometrical modelling of the internal microtubule structure in one cross-section

Fabrication material
Considering the working principle of layered fabrication, the fabrication material should have proper plasticity, ductility and viscosity, so that the 3D scaffolds can be built up and would not distort after solidication. In addition, in order to ensure that the 3D scaffolds do not collapse when injecting CPC[1] and BMP, the material is required to have proper compressive strength and water-resistance. Therefore, according to these requirements, a novel soluble material denatured sucrose (DS) is developed. Besides, and it can also serve as a stabilizer of protein to maintain the activation of BMP.

AJS system
The new AJS system is an integration of hardware, software, NC control and proper building materials. Like many other RP techniques, the AJS system builds a part, layer by layer directly from 3D CAD data. The rened DS is fed into two controllable jets and melted into a semi-molten state by heating systems. Each jet has a small nozzle on the tip, the diameter of the nozzle is 0.2 mm. The jet connects with an air compressor through a high pressure-resistant pipe on its top. Fine laments can be extruded through the nozzle

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by applying compressed air. Under the control of a computer, the on-off operation of the compressed air can be controlled by electromagnetic valves, and a 3D working platform moves according to the slicing data of the part. Therefore the DS lament is deposited layer by layer in the areas dened by CAD model to build a 3D part (Figure 3). In the fabrication process, after feeding DS into two jets, jet I and jet II are heated to 908C and 1208C, respectively, and their temperatures are kept unchanged during the whole forming process. Jet I extrudes ne lament with the platform moving in X-Y directions according to the slicing data of the external contour. After one layer is built, the platform moves 0.2 mm downwards, and continues to fabricate the next layer, then the external contour of the mould can be built up. Until the required height is enough for building the internal 3D scaffolds, jet I is cut off, the platform moves horizontally to the position under jet II, and then jet II then begins to extrude the ne lament, with the platform moving according to the processed data of the internal microtubule structure, so the internal 3D scaffolds can be fabricated. The above mentioned processes are repeated until the mould is built up (Figure 4). It is indicated that when the air pressure and the temperature of jet are given, the discharge of

extruded lament is constant. Then the diameter of the lament can be controlled accurately by regulating the moving speed of the platform. The faster the moving speed of the platform, the thinner the lament will be. Therefore, in the forming process of the mould, the AJS system using one jet with lower temperature to fabricate the external contour of the mould in order to avoid melting the previous built layers; and at the same time, using another jet with higher temperature and faster moving speed of platform to fabricate the internal 3D scaffolds with thinner laments.

Fabrication process of the 3D scaffolds

After extruding from the jet, DS can be shaped into ne lament by extrusion, stretching and solidication (Figure 5). It can be concluded that the shape and size of the lament are mainly determined in the Rs1 region. In the fabrication process, the platform repeats accelerating and decelerating motions. Assuming that the platform moves at an acceleration of a from the static state, until V reaches V 0, the platform tends to keep a uniform motion from ts1 to ts2, and then it begins to move at a deceleration of 2 a and nally reaches a

Figure 3 Two-jets structure and schematic diagram of the AJS system

Figure 4 Mould of the articial bone fabricated on AJS system

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Figure 5 Schematic diagram of the lament fabrication

Haversian canals so as to increase the discharge of DS and hence the lament can be extruded downwards further. In this way, the integrated fabrication of Haversian and Volkmans canals can be realized. Figure 8 shows a longitudinal section of the 3D scaffolds fabricated. The average diameters of Haversian canals and Volkmans canals are 350 mm and 200 mm respectively, which are made deliberately a little thicker than the actual size, considering that the surface of lament scaffolds will be dissolved slightly by succeeding lling process.

Porosity
static state (Figure 6a). According to the motion state, it can be deduced that the shape of the lament is thick at both end-s and thin in the middle (Figure 6b). According to the feature of the lament and the spatial orientations of Haversian canals and Volkmans canals (Figure 7), a forming process can be designed to fabricate Haversian canals by extruding the lament at each point of Haversian canals. In the same way, Volkmans canals can be formed by extruding the lament from one Haversian canals to the next one. Furthermore, in order to ensure the interconnection of Haversian canals between upper and lower cross-sections, the platform should pause temporarily at the point of
Figure 6 Motion state of the platform and the shape of the fabricated lament

The compressive strength of the cured CPC, the degradation rate and osteogenesis are all closely associated with porosity, and in turn, porosity depends on the volume ratio of the 3D scaffolds to the mould of the articial bone. It can be adjusted by changing the height H, which is the proper height of deposit for the fabrication of the internal 3D scaffolds (Figure 9). It has been conrmed by experiments that when H is less than 1 mm, the interference or destruction between two adjacent

Figure 8 Microstructure of the 3D scaffolds (X100) (1) Volkmans canals (2) Haversian canals

Figure 7 Schematic diagram of the longitudinal spatial orientation of Haversian canals and Volkmans canals Figure 9 Schematic diagram of the height H

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lament scaffolds will occur. Furthermore, the thermal inuencing area of the nozzle would melt the previous lament scaffolds. On the other hand, when H is more than 4 mm, the internal architecture will be too loose to simulate the natural bone, could not obtain the necessary porosity. According to the experimental analysis, suitable porosity can be obtained if H is 2 mm, which can ensure the simulation accuracy and give enough space for heat dissipation.

Figure 11 Micropores in the longitudinal section of the articial bone (X50) (1),(3) Volkmans canals (2),(4) Haversian canals

Filling process
In accordance with a certain proportion, the CPC powder, setting solution and BMP are mixed thoroughly into a slurry with good plasticity and uidity, then injecting the mixture into the cavities of the mould through a syringe. Misplacement and force action should be avoided during the curing of CPC so as not to damage the lament scaffolds or reduce the strength of cured CPC. The slurry can obtain a green strength in 20 , 30 min and reach a high strength after 4 h. At this time, the external contour of the mould can be stripped. After the complete solidication of CPC, the articial bone is produced (Figure 10).

Porous architecture and property

The porous morphology of the articial bone can be examined by a microscope and a scanning electron microscopy (SEM). In order to prevent micropores from being damaged by a cutting scalpel, the articial bone is broken off with hands. Microscopically, the porous architecture can be clearly observed in both horizontal and vertical directions (Figure 11). There is no remarkable difference in morphology between the 3D scaffolds and the porous architecture formed in the articial
Figure 10 Filling process

bone, but the diameters of Haversian canal and Volkmans canals are a little smaller, e.g. 300 mm and 150 mm respectively, which can meet the histological standard of carrier scaffold. The porous morphology of the scaffolds is examined by SEM at 20 Kv. From the energy spectrum analysis, it can be seen that there is no chemical element inside the micropore. However, the carbon content (the main element of DS) decreases and the contents of calcium and phosphorus (the main elements of CPC) increase gradually outwards (Figure 12). Therefore, it shows that the micropores can be formed with the DS scaffolds dissolving gradually when CPC solidies. The porosity of the articial bone is 63.2 per cent, evaluated by the toluene inltration displacement method. The compressive strength is 18 Mpa, which is tested with an Instron system. All these indexes can satisfy the histological standard of carrier scaffolds in bone tissue engineering.

Animal experiments and the results analysis

The implantation experiments are conducted in order to evaluate the biological characteristics and osteogenesis of the articial bone. Twelve mature canines are prepared for implantation experiments. Segmental bony defects are inicted at the middle shaft of each canine radius and lled with the articial bioactive bone respectively (Figure 13). Operative sites are examined by X-ray views every four weeks after surgery in order to

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Figure 12 SEM images of the micropore and the energy spectrum analysis

Figure 13 Implantation of the articial bone

Figure 14 The microstructure observed under microscope eight weeks after implantation (X100) I remaining implant II Cartilage tissue

evaluate the healing. The articial bones are harvested in 4, 8, 12, 24 weeks respectively after surgery and specimens are examined by histomorphological detection and SEM to evaluate the effectiveness of defect repair. Four weeks after surgery, the implant is observed having connected tightly with callus at the two margins of the bone defects. A few chondral cells are found under the microscope. Eight weeks after surgery, the microstructure in the cross-section of the remaining implant is obviously embedded by new bone. The diameter of micropore is approximately 150 , 250 mm (Figure 14). 12 weeks after implantation, large amounts of bridged callus are formed in the region of transplantation. The implant is encysted

in the callus and combines tightly with new bone. The bone defect is connected with new bone and vessels have grown into the center of the implant, which shows that the bone defects are completely repaired (Figure 15). The experimental results prove that the articial bone has a good biocompatibility and biodegradability, and the validity of using DS to fabricate the interior microstructure of articial bone is conrmed. The interior 3D porous architecture can ensure new bone growing inward rapidly and adhering tightly with the implant, which has an obvious effect on new bone regeneration and can speed up the bone defect repair.

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Figure 15 Radiographic images of the implantation experiment: (a) just after surgery and (b) at 12 weeks

regulating the processing parameters. Furthermore, acting as a carrier scaffold into which the BMP is induced, the articial bone with interior 3D micropores can provide a porous network for the circulation of the tissue uid and hence can speed up osteogenesis and realize bone transformation.

Note
1 The injectable CPC is a kind of hydroxyapatite with good biocompatibility and osteo-conduction. The production is warranted by the State Drug Administration (SDA) of China, Product No: Q/DACL-001-2000.

Further reading
James, W. (1993), Fundamental Fluid Mechanics for The Practicing Engineer, Marcel Dekker, New York, NY. Lu, B.H. (1998), Rapid Prototyping and Rapid Tooling, Shaanxi Science & Technology Press, Xian. Richard, O.C. (1998), Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements, Biomaterials, Vol. 19 No. 20, pp. 1845-54. Robert, C. (1999), Guided tissue fabrication from periosteum using preformed biodegradable polymer scaffolds, Biomaterials, Vol. 20 No. 21, pp. 2007-18. Song, J. (2000), Bio-medical Materials, Tianjin University Press, Tianjin.

Conclusions
The fabrication technique of bioactive articial bone proposed in this research is the combination of RP and bioactive factor composition. Through the analysis of the new system developed and its forming process, proper porosity and micropores size of the articial bone can be obtained by

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