Rad Rapid Prototyping

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Case studyFabrication of artificialbioactive bone usingrapid prototyping

Zhongzhong Chen, Dichen Li,

Bingheng Lu, Yiping Tang,

Minglin Sun and Zhen Wang

The authors

Zhongzhong Chen,Dichen Li,Bingheng Lu,Yipng Tang, andMinglin Sunare 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 MedicalUniversity, Xian, Peoples Republic of China.

Keywords

Rapid prototypes, Bones

Abstract

A new technique based on rapid prototyping (RP) is proposed to

fabricate the mould of artificial bone composed of a nontoxicsoluble 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. Byinjecting self-setting calcium phosphate cement (CPC) with bone

morphogenetic protein (BMP, a kind of bone growth factors) intothe cavities of the mould, the CPC solidified and the microporescan be formed after the internal 3D scaffolds is dissolved, finally

the artificial bioactive bone can be produced. This approach isbetter than the traditional fabrication process, which the latter

method cannot fabricate an artificial bone with inter-connectivemicropores so as to realize the osteo-induction for lack of

bioactivity. Through animal experiments, it shows that thesimulated inter-structure could provide artificial bone withproper voids for the growth of the bone tissue and the quick

activation, and hence effectively speed up the bone growth bymeans of activating osteo-conduction and osteo-induction. So,

the new method of fabricating artificial bone with biologicalbehaviors is justified.

Electronic access

The Emerald Research Register for this journal is

available atwww.emeraldinsight.com/researchregister

The current issue and full text archive of this journal isavailable atwww.emeraldinsight.com/1355-2546.htm

Introduction

In bone graft operations, the preferred treatment is

the use of autologous bone with a view to attain

biocompatibility and bio-affinity. 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, posesevere 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 artificial boneis, 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 solidification (AJS)

system is developed, which can built up the mould

of artificial bone with customized geometric

contour and internal porous architectures.

Mould fabrication of artificial boneBiomimetic 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

Rapid Prototyping Journal

Volume 10 Number 5 2004 pp. 327333

q Emerald Group Publishing Limited ISSN 1355-2546

DOI 10.1108/13552540410562368

Received: 10 April 2003Reviewed: 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).

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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.

(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 influence 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 artificial bone after

degradation.

Fabrication material

Considering the working principle of layered

fabrication, the fabrication material should have

proper plasticity, ductility and viscosity, so that the3D scaffolds can be built up and would not distort

after solidification. 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 refined 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 filaments can be extruded through the nozzle

Figure 1(a) Solid CAD model of the artificial bone(b) Geometrical modelling of the internal microtubule structurein one cross-section

Figure 2 Internal microtubule structure CAD modeling of theartificial bone

Fabrication of artificial bioactive bone using rapid prototyping

Zhongzhong Chenet al.


Volume 10 Number 5 2004 327333

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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 filament

is deposited layer by layer in the areas defined by

CAD model to build a 3D part (Figure 3).

In the fabrication process, after feeding DS intotwo 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 fine filament with the platform

moving inX-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 fine filament,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 filament is constant. Then the diameter

of the filament can be controlled accurately by

regulating the moving speed of the platform.

The faster the moving speed of the platform,

the thinner the filament 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 avoidmelting 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

filaments.

Fabrication process of the 3D scaffolds

After extruding from the jet, DS can be shaped

into fine filament by extrusion, stretching and

solidification (Figure 5). It can be concluded that

the shape and size of the filament are mainlydetermined in the Rs1 region. In the fabrication

process, the platform repeats accelerating and

decelerating motions. Assuming that the platform

moves at an acceleration ofa from the static state,

until Vreaches V 0, the platform tends to keep a

uniform motion from ts1 to ts2, and then it begins to

move at a deceleration of2aand finally reaches a

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

Figure 4Mould of the artificial bone fabricated on AJS system





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static state (Figure 6a). According to the motion

state, it can be deduced that the shape of the

filament is thick at both end-s and thin in the

middle (Figure 6b).According to the feature of the filament 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 filament at each point of Haversian

canals. In the same way, Volkmans canals can be

formed by extruding the filament 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

Haversian canals so as to increase the discharge of

DS and hence the filament 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, whichare made deliberately a little thicker than the actual

size, considering that the surface of filament

scaffolds will be dissolved slightly by succeeding

filling process.

Porosity

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 artificial bone. It can be adjustedby changing the heightH, which is the proper

height of deposit for the fabrication of the internal

3D scaffolds (Figure 9). It has been confirmed by

experiments that whenH is less than 1 mm, the

interference or destruction between two adjacent

Figure 5Schematic diagram of the filament fabrication

Figure 6Motion state of the platform and the shape of thefabricated filament

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

Figure 7Schematic diagram of the longitudinal spatial

orientation of Haversian canals and Volkmans canals

Figure 9Schematic diagram of the height H





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filament scaffolds will occur. Furthermore,

the thermal influencing area of the nozzle would

melt the previous filament scaffolds. On the other

hand, whenHis 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 ifHis 2 mm,which can ensure the simulation accuracy and give

enough space for heat dissipation.

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

fluidity, 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 filament 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 solidification of CPC, the artificial bone

is produced (Figure 10).

Porous architecture and property

The porous morphology of the artificial bone can

be examined by a microscope and a scanning

electron microscopy (SEM). In order to prevent

micropores from being damaged by a cuttingscalpel, the artificial 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 artificial

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) increasegradually outwards (Figure 12). Therefore, it

shows that the micropores can be formed with the

DS scaffolds dissolving gradually when CPC

solidifies.

The porosity of the artificial bone is

63.2 per cent, evaluated by the toluene infiltration

displacement method. The compressive strength is

18Mpa, 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 resultsanalysis

The implantation experiments are conducted in

order to evaluate the biological characteristics and

osteogenesis of the artificial bone. Twelve mature

canines are prepared for implantation

experiments. Segmental bony defects are inflicted

at the middle shaft of each canine radius and filled

with the artificial bioactive bone respectively

(Figure 13). Operative sites are examined by X-ray

views every four weeks after surgery in order to

Figure 10Filling process

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





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evaluate the healing. The artificial 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 completelyrepaired (Figure 15).

The experimental results prove that the

artificial bone has a good biocompatibility and

biodegradability, and the validity of using DS to

fabricate the interior microstructure of artificial

bone is confirmed. 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.

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

Figure 13 Implantation of the artificial bone Figure 14The microstructure observed under microscope eightweeks after implantation (X100) I remaining implant II Cartilagetissue





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Conclusions

The fabrication technique of bioactive artificial

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 artificial bone can be obtained by

regulating the processing parameters.

Furthermore, acting as a carrier scaffold into

which the BMP is induced, the artificial bone with

interior 3D micropores can provide a porous

network for the circulation of the tissue fluid and

hence can speed up osteogenesis and realize bone

transformation.

Note

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

Further reading

James, W. (1993), Fundamental Fluid Mechanics for ThePracticing Engineer, Marcel Dekker, New York, NY.

Lu, B.H. (1998), Rapid Prototyping and Rapid Tooling, ShaanxiScience & Technology Press, Xian.Richard, O.C. (1998), Growth and differentiation of human

bone marrow osteoprogenitors on novel calciumphosphate cements,Biomaterials, Vol. 19 No. 20,pp. 1845-54.

Robert, C. (1999), Guided tissue fabrication from periosteumusing preformed biodegradable polymer scaffolds,Biomaterials, Vol. 20 No. 21, pp. 2007-18.

Song, J. (2000), Bio-medical Materials, Tianjin University Press,Tianjin.

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





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