The biomimetic design and 3D printing of customized ...

Materials and Design 152 (2018) 30–39

Contents lists available at ScienceDirect

Materials and Design journal homepage: www.elsevier.com/locate/matdes

The biomimetic design and 3D printing of customized mechanical properties porous Ti6Al4V scaffold for load-bearing bone reconstruction Boqing Zhang a,1, Xuan Pei a,1, Changchun Zhou a,⁎, Yujiang Fan a, Qing Jiang a, Alfredo Ronca b, Ugo D'Amora b, Yu Chen c, Huiyong Li a, Yong Sun a, Xingdong Zhang a a b c

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, Naples 80125, Italy Department of Applied Mechanics, Sichuan University, Chengdu 610064, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Scaffolds showed biomimetic structure design and customized mechanical properties. • Scaffolds with 66.1%–79.5% porosity were biomimetic designed by using diamond lattice pore units array formation. • Scaffolds within a wide range of compressive strength from 36 to 140 MPa were successfully fabricated. • Animal results indicated that the damaged load-bearing bones were well reconstructed.

a r t i c l e

i n f o

Article history: Received 30 January 2018 Received in revised form 26 April 2018 Accepted 26 April 2018 Available online 27 April 2018 Keywords: Biomimetic architectures Simulation and modeling Ti6Al4V scaffolds Mechanical properties 3D printing

a b s t r a c t The Ti6Al4V alloy is one of the most commonly used in orthopedic surgery. Mechanical property of implant contributes important biological functions for load-bearing bone tissue reconstruction. There is a significant need for design and fabrication of porous scaffold with customized mechanical properties for bone tissue engineering. In this paper, bionic design and fabrication of porous implants were studied by using finite element analysis (FEA) and 3D printing techniques. Novel porous architectures were built up with diamond lattice pore structure arraying units. With finite element analysis, the structure weak points under pressure were simulated so that the mechanical properties of the implants were optimized. Porous implants with different porosities and mechanical properties were precisely fabricated by selected laser melting (SLM), one of powder bed fusion additive manufacturing techniques. The biocompatibility and repair effect were studied by in vivo experiments. Animal results indicated that the damaged load-bearing bones were well reconstructed. New generated bones embedded and fitted into the designed porous implants. The optimized design and precisely manufactured implants are conducive to bone tissue repair and reconstruction. © 2018 Elsevier Ltd. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (C. Zhou). 1 These authors contributed equally.

https://doi.org/10.1016/j.matdes.2018.04.065 0264-1275/© 2018 Elsevier Ltd. All rights reserved.

B. Zhang et al. / Materials and Design 152 (2018) 30–39

1. Introduction The ideal scaffold for bone tissue reconstruction should resemble natural bone in both structural and mechanical properties. Autologous bone graft represents the gold standard for healing bone defects but, unfortunately, it is not always available in suitable shape or quantity and even when it is available, it will inevitably damage the donor site tissues. As an ideal bone tissue scaffold, not only the materials chemistry but also the three-dimensional (3D) porous structure is considered as a crucial role for bone regeneration [1–3]. With the help of this structure containing micropores and interconnected pores, a temporary support for cell proliferation and tissue infiltration, as well as a microenvironment for transportation of nutrients and waste products can function well [4,5]. For load-bearing bone regions, the needs for suitable mechanical strength require better biomimetic design and fabrication of bone tissue implants for these applications [6–8]. Owing to its excellent mechanical properties and biocompatibility, titanium alloy has drawn a lot of attention as bone implants [9–14]. But the elastic modulus of bulk titanium is too high compared with natural bone and the unmatched elastic modulus will lead to stress shielding. Since the first porous titanium structure was manufactured and reported in the late 1960s, the interest keeps increasing. The superiorities are obvious compared with bulk titanium: density, elastic modulus and mechanical strength are adjustable to avoid stress-shielding. In the past, various methods have been developed to manufacture porous titanium body including sintering with powders, solid-state foaming by expansion of argon-filled pores [15], and sintering of titanium fibers [16]. However, too many complex parameters affect on porosity, pore shape and pore size. Therefore, it is difficult to fabricate idea implants with accuracy architectures through conventional methods [17,18]. Recently, 3D printing technology has made it become possible to fabricate biomaterials with complex architectures [19–26]. Selected laser melting (SLM), a typical additive manufacturing technique with a super ability to fabricate 3D complicated architecture with customized pore, has drawn a lot of attention [27–33]. With high power laser energy, SLM printer melt selected area of powder directly to manufacture objects layer by layer, therefore objects can be manufactured accurately [34–36]. What is more important that SLM could produce highly complicated implants with personal-customized architectures for different patients in accordance with their CT data [37]. With Finite Element Analysis (FEA), the weakness of structure under pressure can be easily simulated so the structure can be predesigned and optimized [38,39]. Some literatures have proposed that the ideal bone tissue engineering scaffold owns macro-pores of about 300–900 μm and porosity of 60–95% [40–42]. But because lack of ability to precisely manufacture porous structure, the mechanism of porosities influence biological properties still needs further exploration [43,44]. Furthermore, researchers found difficulties in design of biomimetic mechanical properties, especially elastic modulus. One key problem is that conventional manufacturing methods cannot fabricate scaffolds with precise pore shape, size, location and inter-connectivity. Therefore, researchers have a strong interest in 3D printing technology that enables accurate manufacturing of porous architectures [27,41,45–49]. Additive manufacturing techniques such as SLM can obtain precise porous titanium implants with pore size of 400–1000 μm, which exhibit excellent osteointegration performance in vivo [50]. However, few reports have clarified the true effect of porous structures in terms of mechanical properties and biological functions. In this research, an ideal biomimetic architecture that has both excellent biocompatibility and mechanical properties for load-bearing bone reconstruction was proposed. We investigated the effect of porosity design on mechanical properties and bone regeneration. The scaffolds were designed with constant pore size and pore shape but varied in support struts, which allows precisely control scaffold architectures and mechanical properties. Porous scaffolds with five strut sizes (0.2,

31

0.25, 0.3, 0.35, 0.4 μm) and constant pore size and shape were fabricated by SLM method. Scaffold structures were analyzed by using micro-focus computed tomography (micro-CT) and compressive mechanical test. Moreover, in-vivo animal experiments were conducted to evaluate the biological properties of fabricated 3D porous scaffolds. Biomimetic architectures with appropriate porosities and mechanical properties allow bone ingrowth and avoid stress shielding. This proposed porous 3D biomimetic titanium bone implants fabricated by SLM represents potential bone substitute candidates. 2. Experimental 2.1. Biomimetic mechanical property implants molding Fig. 1 shows the strategies and technique routes for manufacturing of personal customized porous titanium scaffolds. The pore units were inspired by the natural cancellous bone tissues. After simulating and optimizing by FEA, these diamond-like pores were applied as the filler pores. The outer shape of the implants was biomimetic modeling according the CT data information. The bone implant with adjustable mechanical properties was designed by computer-aided design (CAD) and pre-simulation. A porous implant was filled by arrayed basic diamondlike pore units. A diamond-like pore unit is a truss structure in which one node is tetrahedrally surrounded by four other nodes coming from the crystal structure of the diamond crystal. Five different implants composed with the similar porous unit were designed by constant pore size and varied support strut in diameter of 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm and 0.4 mm, noted as D0.20, D0.25, D0.30, D0.35, D0.40 (Fig. 2), respectively. The specimens were designed by modeling software and their details parameters were obtained by modeling information as shown in Fig. 2(e). The printing Ti6Al4V were spherical powders with particle size of 50–100 μm (Fig. 2(a)). The scaffolds were built up with array diamond-like units as shown in Fig. 2(b). Cylinders specimens (∅10 × 17 mm, as shown in Fig. 2(c) were prepared for the mechanical test and final implants were prepared as Fig. 2(d) for in vivo experiments (∅5 × 30 mm) using the five types porous units mentioned above. 2.2. Materials and fabrication process Ti6Al4V powder with particle size of 50–100 μm and density of 4.5 g/cm3 was used as printing materials. An SLM system (M2, Concept laser, German) was used in this research. Specimens were manufactured with a 70 W laser and laser scanning speed of 500 mm/s. the thickness of the powder was kept 50 μm. After manufacturing, specimens were wire-electrode cut off from base and heat-treated at 820 °C for 1.5 h immediately in a vacuum environment to eliminate the internal stress voiding cracks. 2.3. Surface bio-activation of implants The acid pickling treatment was conducted before surface bioactivation of implants. Specimens were immersed in 0.7%wt hydrofluoric acids for 10 min to clean up oxide and unmelted metal powder. Magnetic stirrer was conducted to make sure homogeneity. After acid pickling to dislodge oxide scales and contamination, a thin hydroxyapatite layer was coated at the surface of titanium to promote osteoconductivity and osteoinductivity. Briefly, the treated specimens were placed into a container, in which the air was pumped out to a negative pressure state by a vacuum pump. The electrolyte containing 2.5 mM CaCl2·6H2O, 1.5 mM NH4H2PO4, and 0.15 M NaCl was then added to the container so that the electrolyte could penetrate the inner pores of the samples completely. Specimens were carried out in an electrochemical workstation (PARSTAT 2273, Princeton Applied Research, USA). Pulsed current method and three electrodes were adopted. Specimens, platinum plate and saturated calomel electrode

32

B. Zhang et al. / Materials and Design 152 (2018) 30–39

Fig. 1. The strategies and technique routes for manufacturing of personal customized porous titanium scaffolds.

were used as the cathode, anode, and reference electrode, respectively. The electrolyte temperature was controlled at about 85 °C during the process. 2.4. Characterization of porous implants 2.4.1. Biomimetic architectures characterization The biomimetic architectures of porous implants were characterized by SEM and micro-CT system. Specimens were firstly observed with a scanning electron microscopy (SEM, JSE-5900LV, Japan). The voltage

was set to 5.0 kV and the magnification was set to ×30–×500. Then, the Microfocus X-ray computed tomography technique was used to analyze the in-site situation of acquired implants in animal experiments. A micro-CT system (SCANCO vivaCT 40, Switzerland) was used for this, specimens for the mechanical test as well as for in vivo experiment were scanned and images consisting of 2910 × 2910 × 2028 pixel per specimens with a pixel of (22 μm)2 were reconstructed using Materialise's interactive medical image control system (Materialise, Belgium). Mechanical test specimen reconstructed models were compared with CAD model and were colored by diversity. In vivo

Fig. 2. Biomimetic architectures of porous Ti6Al4V implants. (a) SEM image of the printing Ti6Al4V powders with particle size of 50–100 μm, (b) single diamond-like unit, (c) standard specimen for the mechanical test, (d) implant for in vivo experiment, (e) detail parameters and characters for the designed specimens.

B. Zhang et al. / Materials and Design 152 (2018) 30–39

experiment specimens were marked blue and bone tissue was marked yellow. Osteogenesis was calculated by regenerative bone volume and porosity of implants. Compact and cancellous bone area was selected at defects of compact and cancellous bone. 2.4.2. Porosity Porosity was calculated automatically during specimen CAD building. After manufacturing, the porosity was measured on the basis of Archimedes method and Theoretical formula calculation. The scaffold was dipped into in water and suspended from an analytical balance to obtain wet weight (Wa). Theoretical formula are calculated as follows, Buoyancy in water ð f Þ : V0 ¼

Wa−Ww ρw  g

f ¼ Wa−Ww ¼ ρw  g  V 0

ε¼

  V0 1−  100% V

healed in previous research [51–53]. After one month observation, the osteonecrosis model was found not self-healed and then the necrosis hole was expanded and sterilized specimens with D0.4 pore structure were implanted properly. After 4 and 6 months, dogs were sacrificed with the overdose of intravenously administered high concentrated muriate. All specimens and bone tissue around were maintained and scanned by a micro-CT machine mentioned above. After one month paraformaldehyde fixation, tissue slices were cut and observed with a light microscope. All surgeries were conducted by experienced orthopedics. All experiments were approved by the Animal Care and Use Committee of Sichuan University.

ð1Þ

3. Results

ð2Þ

3.1. Architectures characterization

where, V′ is the volume of outer contour of the scaffold minus open pores volume. Then, the porosity of the scaffold can be calculated as follows, ð3Þ

where Wa and Ww are the sample weights in air and water, respectively. V is the volume of outer contour of the scaffold which was calculated by the size of the scaffolds. ρw is the pure water density, which is 0.9975 g/cm3 at 20 °C. The printed porous scaffold porosity ‘ε’ was calculated with Eq. (3). All measurement was conducted at room temperature. 2.4.3. Mechanical properties simulation Single units rather than whole cylinders were used in finite element analysis to decrease calculation. The boundary condition of these units was set to simulate stress condition at the core of scaffold as followed: the bottom was fixed and force was applied at the top indirectly through a layer of the rigid cube to make sure synchronous displacement and mimic real load condition. For the same reason, displacement was limited in other directions except load-paralleled direction along samples. Simulations were conducted with Solidworks built-in simulation module with max element size of 0.05 mm. Materials of the unit was built-in Ti6Al4V (solution-anneal) with a yield strength of 827.4 MPa, the max strength of 1050 MPa and elastic modulus of 104.8 GPa. The load was applied gradually to simulate quasi static compression. When the load converged, the software automatically stopped and the load was regarded as the max load of this unit. Indirectly, stress and strain were calculated from displacement and load of rigid cube. 2.4.4. Mechanical properties test The compressive test was conducted with a crosshead speed of 1 mm/min (Precision Universal Tester Autograph AG-X, Japan). According to ISO 13314:2011 (E), porous cylinders (diameter: 10 mm, length: 17 mm, with 1 mm compact cylinder on both ends to void stress concentration, three cylinders of each type) were used in this test. The stiffness was calculated from the slope of the curve in the linear elastic region. 2.5. In vivo experiments Beagle dogs were used as the animal model in this research. Osteonecrosis model was built one month before implantation according to previous research [51–53]. Briefly, an electronic drill with ∅5 mm diameter was used to made 20 mm deep hole from outboard shank into caput femoris. Then, liquid nitrogen was poured into the hole and made around bone tissue necrosis by low temperature. This animal osteonecrosis model was proved that it is hard to be self-

33

SEM images of specimens were conducted before and after acid pickling of implants as shown in Fig. 3. Before acid pickling, the surface appearances were similar between implants of different pore sizes. The main body of specimens was sintered compactly and some titanium powders were observed attached on strut caused remarkable irregularities of the surface (Fig. 3(c)). In terms of pore structure, pore size was controlled at about 660 μm and pores were arrayed regularly and interconnected. The pore shape was similar to the diamond-like structures and a little bit accumulation powders were found at the edge of pores. After acid pickling, most of the residual powders were removed and the inner surface of the implant became smoother. With removal of the residual powders accumulation, the whole architectures of the implants achieved to a precisely duplicate as the designed diamond-like porous structures. 3.2. Porosity results The designed parameters and characters of the specimens were shown as Fig. 2(e). The measured porosity results were shown in Fig. 3(d), from D0.2 to D0.4, the designed porosity of specimens decreased linearly from 79.5% to 66.1% with max pore size constant at 650 ± 20 μm, and specific surface area decreased from 13.47/mm to 8.29/mm. Generally, porosities differences were less 10% higher than the designed porosity due to adhesion of residual titanium powders. After acid treatment, porosities increased and showed a letter bit higher than the designed porosities. 3.3. Micro-CT results To detect the accuracy of SLM process and the discrepancy between specimens and CAD model, Micro-CT measurement was conducted to analyze the implants. The discrepancy between specimens and CAD model was shown in Fig. 4. The deviation was marked with color and which generally lower than 48.5 μm. Accumulation was observed perpendicular to the printing direction. The discrepancy was quantified from percentage differences between designed porosities and measured porosities. From D0.2 to D0.4, diversities were 7.9%, 7.2%, 5.3% and 0.7%, respectively. 3.4. Mechanical properties results As shown in Fig. 5(a) and (d), the ultimate compressive strengths of D0.20, D0.25, D0.30, D0.35, D0.40 were 36.45 MPa, 56.63 MPa, 85.81 MPa, 109.20 MPa and 140.26 MPa, respectively. The FEAsimulated ultimate compressive strengths of D0.2, D0.25, D0.3, D0.35, and D0.4 were 35.30 MPa, 53.61 MPa, 81.94 MPa, 109.44 MPa and 144.91 MPa, respectively. The results showed a good linear increase trend. The maximum compressive strength implant was 4.1 times the minimum one. It indicated that there is a lot of design space to adjust the mechanical properties. The elastic moduli of D0.20, D0.25, D0.30,

34

B. Zhang et al. / Materials and Design 152 (2018) 30–39

Fig. 3. SEM images of specimens with (a)–(c) before acid pickling (e) and (f) after acid pickling. (d) Porosities of different specimens.

D0.35, D0.40 were 1.22 GPa, 2.00 GPa, 3.02 GPa, 3.79 GPa and 5.15 GPa, respectively. The FEA-simulated elastic moduli of D0.20, D0.25, D0.3, D0.35 and D0.40 were 1.21 GPa, 2.00 GPa, 3.02 GPa, 3.79 GPa and

5.15 GPa, respectively. The elastic moduli increased with the support struts increased. It also showed a linear increase trend as the compressive strength. The maximum elastic moduli of the D0.40 implant were

Fig. 4. (a) Discrepancy between specimens and CAD model. Green color represents better match with the design model, red and blue color represents plus and minus errors. The unit is mm. (b) and (d) The accumulation at the bottom of pores; (c) Quantified errors for different porosities implants.

B. Zhang et al. / Materials and Design 152 (2018) 30–39

35

Fig. 5. Mechanical properties of different specimens. (a) Stress-strain curves with load speed of 1 mm/min, (b)–(c) actual and simulated compressive strength and elastic modulus; (d) compressive strength as well as elastic modulus; (e), (f) FEA results of D0.20. The red color indicated more deformation of the implant, and the red arrow point shows the weakest area in the scaffold.

4.26 times bigger than the minimum one (D0.2). As shown in Fig. 5(b), the simulated compressive strength showed a good fit to that of the experimental test. Generally, the simulated compressive strength showed a little smaller than the tested data. For the elastic modulus as shown in Fig. 5(c), the simulated elastic modulus of different implants showed the consistent increase trend with increasing of the struts. However, the simulated elastic modulus was higher than that of the experimental test results. Fig. 5(d) showed compressive strength as well as elastic modulus. Fig. 5(e) showed the simulated the compressive strength and elastic modulus results of the D0.20 implant. The results indicated that with varying the porosity of the specimens, the mechanical properties can be modulated in a wide range, which is beneficial to biomimetic design of bone tissues scaffolds. Fig. 5(f) showed an example of the FEA results of D0.20. The red color in Fig. 5(f) indicated more deformation of the implant, and the red arrow point shows the weakest area in the scaffold.

3.5. In-vivo experimental results Biocompatibility and reconstruction effect were studied on beagle dogs. The load-bearing implant sites and X-ray results after six implantations were shown in Fig. 6. It was observed that no infection of the surgical sites, implants dislocation, or adverse reactions such as inflammation or foreign body reaction around the implantation sites. Beagles limp on its right legs in the first two weeks, but they can walk freely after one month implantation. After 4 months, the implants had little effect on their movement. After 6 months, new bone tissues gradually grow into the porous implants. The damaged legs were completely repaired, and it was hard to detect the initial injury from the beagle's gait walking. Bone reconstruction effect was analyzed by micro-CT and histological slices. As shown in Fig. 7, new bone volume in cubes (5 mm × 5 mm × 5 mm) at the cancellous bone area as well as the compact bone area

Fig. 6. Porous specimens are implanted into beagle's right hind femoral head. (a) Front X-ray result, (b) side X-ray result after six implantations. The implants are in the red circles as arrow pointed.

36

B. Zhang et al. / Materials and Design 152 (2018) 30–39

Fig. 7. (a)–(c) The reconstructed models after implantation. (d) The regenerative bone volume for cancellous and compact bones.

were calculated and considered as osteogenesis. Reconstructed data showed that 4 months after implantation, both cancellous and compact bone tissues were observed in the porous implants. Bone tissue grew into the pores and more than 80% volume at the compact bone area

and 35% at the cancellous bone area were filled with new bone tissues. After 6 month implantation, bone mineral density further increased, more than 70% volume of cancellous bone and 86% volume of compact bones were observed in the implants.

Fig. 8. Morphology images of the cancellous bone area. (a) The slice morphology showed that the specimen of D0.35 after implantation for 4 months, (b) for 6 months, magnification: 20×. (c) The slice morphology showed that the specimen of D0.35 after implantation for 4 months, (d) for 6 months, magnification: 4×, respectively.

B. Zhang et al. / Materials and Design 152 (2018) 30–39

37

Fig. 9. Morphology images of the compact bone area. (a) The slice morphology showed that the specimen of D0.35 after implantation for 4 months, (b) for 6 months, magnification: 20×. (c) The slice morphology showed that the specimen of D0.35 after implantation for 4 months, (d) for 6 months, magnification: 4×, respectively.

Both the cancellous area and compact area were detected through histological studies. As shown in Figs. 8 and 9, hard tissue slice morphology showed that after implantation for 4 months, obvious bone tissue structures, as well as Haversian canals can be observed around implants in both cancellous and compact area. New bone tissues at compact bone area seemed more compact than the tissues at the cancellous area. New generated cancellous bones seemed sparse and filled with porous adipose tissues. No obvious boundaries between implants and bone tissues were observed due to the 3D interconnected biomimetic porous architecture design. Two more months later, more complete bone tissues were observed. Many bone marrow tissues were observed among cancellous bones. 4. Discussion SLM technology makes it possible to precisely control mechanical properties, as well as architectures make it benefit to modulate the biological effects for bone regeneration. To achieve that goal, a series of implants with constant pore size and varied mechanical properties were manufactured by additive manufacturing technique. SLM can accurately fabricate the outer shape and inter porous microstructures of bone implants. In this research, we designed and prepared porous structure with diamond-like pores. These novel porous structures have many advantages compared with other microstructures. Firstly, because of the truss structure system in the scaffolds, it shows more stable under multi-directional stress and isotropy mechanical strength. Materials only bearing compression stress instead of shear tress and force are contributed averagely among tetrahedral support strut under pressure. Secondly, it is capable to design the porosity and support struts to mimic the mechanical properties of natural bone tissues. Furthermore, interconnected 3D pores are benefit for bone ingrowth. Body fluid, nutrition, and waste for cells can be transported among those interconnected

pores. In order to verify the printing accuracy, a comprehensive study between design CAD models and printing specimens were compared via micro-CT data. Results indicated that the manufacture process have a remarkable influence on pore structure. Therefore, it is need to carefully control the processing parameters during the scaffolds fabrication so as to obtain true duplication [54]. Based on the statistical analysis, it is found that the dimensional accuracy and mechanical properties are sensitive to printing parameters such as laser power, laser scanning speed and powder layer thickness. Furthermore, printing of different scaffolds with varied strut diameters, the evolution of the molten pool morphology, surface roughness and dimensional accuracy would be different. Our studies indicated that the average width and depth of the molten pool, the lower surface roughness and dimensional deviation decrease with the increase of scanning speed and hatch spacing. However, the upper surface roughness was found to be almost constant under different processing parameters due to it is affected by the printing powders morphology [55]. It was also found that accumulation appeared at bottom of pores along printing direction. This is probably due to the spherical powder falling into the laser melted bath and aggregated during the SLM molding process. For the same reason, powders were observed attach at top of the strut and few powders were found below as shown in Figs. 3(a), (b) and (c) and 4(b) and (d). These residual powder and accumulation may lead to error of porosity, pore structure. However, the discrepancy of specimens was less than 8% as shown in Fig. 4. These accumulation and residual powders were partially bound to the main body and it can be removed by subsequent acid pickling processes. The discrepancy can be considered during design molding to achieve desired dimensions. During acid pickling, the souring of acid solution corroded surface of specimens. However, looseness and roughness of unmelted metal powder and accumulation make it faster to corrode and drop off than the main body. After 10 min acid pickling, most of particles and

38

B. Zhang et al. / Materials and Design 152 (2018) 30–39

accumulation were removed with porosities decreased about 10% and of more accurate with designed porosities. Yet, the drop of particles gave surface necessary roughness for cell adhesion. Mechanical properties are very important for load-bearing bone tissues reconstruction [56]. Implants with too high stiffness would bear most stress under pressure but bone tissue cannot be stimulated by stress. Literatures have proved that stress promotes bone tissue growth and help bone reconstruction [57]. Conversely, too low stiffness would cause too much loads on bones and lead to bone fracture. Therefore, implants with suitable stiffness need to be biomimetically designed and fabricated for load-bearing bones. Under stress and torque loading, biomimetic strength was required either to avoid implants sliding, loosening and fracture after implantation. Moreover, adequate strength may decrease the chance that implants fracture during and after surgery. In this research, the compressive test was conducted to evaluate the strength of specimens, and stiffness was calculated from the stressstrain curves. Ti6Al4V is brittle materials and the specimens fractured at 45 degrees to the loading direction, which indicated that the designed architectures of the implants are isotropic in the mechanical properties. Ultimate strength varied from 36.45 MPa to 140.26 MPa and elastic modulus varied from 1.21 GPa to 5.15 GPa with the increase of strut size. Besides, elongation indicating plasticity increased either. FEA data showed the same trend with experimental test data with ultimate strength accurately predicted, therefore FEA had a significance to predict mechanical properties of implants. Besides, stress cloud map showed the weak spot of the structure in the struts. The change of strut size would lead to the change of compressive strength and elastic modulus. For biomimetic mechanical properties design consideration, our study proposed specimens with higher compressive strengths and variable stiffness within cancellous bone(compressive strength: 10–20 MPa; elastic modulus: 0.05–0.5GPa) and compact bone (compressive strength: 70–150 MPa; elastic modulus: 7–30 GPa). At early implantation stage, high strength decreased the risk of failure, and proper stiffness could attract and stimulate osteoblasts grow into the scaffolds and promote osteointegration. With the bone tissue ingrowth and maturation, the newly formed composite structures will rebuild the mechanical properties of damaged bone sites. As previously reported [40] that porosities have an important effect on bone formation and reconstruction, porosities beyond 60% were regarded as suitable for bone reconstruction. As shown in Fig. 5(e), the implants compressive strength increased about 100 MPa accompanied with decreasing about 10% porosities when adjusting struts from D0.2 to D0.4. These results indicated that the mechanical properties of the implants can be flexibly adjusted in a wide range without changing the porous structures too much. In vivo animal model was used to study the biological performance of biomimetic architectures porous Ti6Al4V scaffolds. The primary goal is to access if the implants manufactured by SLM can successfully reconstruct the bone tissues for load-bearing sites. D0.4 pore structure due to its high compressive strength and proper elastic modulus was selected. According to slice morphology and micro-CT results, after 4 months implantation, new bone tissues were observed grew inward implants in both cancellous bone and compact bone areas. Haversian canal appeared and boundaries between implants and bone tissue were observed. At compact bone area, the pores of implants were full of new bone tissue, and bone tissues at cancellous bone area were sparse. It is a very interesting phenomenon that our biomimetic architectures porous Ti6Al4V scaffolds can promote both the cancellous and compact bones reconstruction. Two more months later, the boundary between cancellous bone and compact bones became more obvious which was just like the natural bone tissues structures. Bone marrow was observed after 6 months implantation which is very important to hematopoietic function. Compact bone, which bears most stress of human body, has a high density, stiffness and strength. The porous Ti6Al4V implants with comparable lower stiffness would take a concentrated stress, which stimulates compact bones regeneration. Therefore,

compact bone tissues were observed at the compact bone area. At the same time, osteointegration also happened at the cancellous bone area. However, implants possess higher stiffness compared with cancellous bones afford most stress which caused less stress stimulation for the cancellous area. Therefore, after 4 months implantation, fewer bone tissues were observed in the implants at the cancellous bone area. However, much more compact bones tissues were observed at the compact bone area. This phenomenon strongly proved that mechanics stimulation plays an important role in bone regeneration.

5. Conclusions This research provides an effective method to build orthopedic implants with personalized shape and adjustable mechanical properties. Biomimetic architectures porous Ti6Al4V scaffolds were custom-made with suitable mechanical properties for load-bearing bone tissue reconstruction. The diamond-lattice porous structure was proposed and manufactured by using SLM methods for femoral-head repair implants. The struts diameters, pore size and porosity of the implants can be easily revised to achieve bionic design of bone tissue implants. These novel porous structures show isotropic mechanical properties which can be predesigned to match the mechanical properties of natural bones. Biomimetic architectures design and accurate manufacture of porous Ti6Al4V implants provide a versatility way for reconstruction of individualized load-bearing bone tissues.

Acknowledgments This work was supported by the National Key Research and Development Program of China (2016YFC1102000), Sichuan Province Major Scientific & Technological Achievements Transformation Demonstration Project (2016CZYD0004), China, Science & Technology Department of Sichuan Province Key Projects (2017SZ0001), China; Science & Technology Support Program of Sichuan Province (2016GZ0196), China. References [1] C. Zhou, K. Wang, X. Pei, Z. Dong, Y. Hong, X. Zhang, Combination of fused deposition modeling and gas foaming technique to fabricated hierarchical macro/microporous polymer scaffolds, Mater. Des. 109 (2016) 415–424. [2] X. Pei, L. Ma, B. Zhang, J. Sun, Y. Sun, Y. Fan, Z. Gou, C. Zhou, X. Zhang, Creating hierarchical porosity hydroxyapatite scaffolds with osteoinduction by threedimensional printing and microwave sintering, Biofabrication 9 (4) (2017), 045008. [3] H. Yoshikawa, N. Tamai, T. Murase, A. Myoui, Interconnected porous hydroxyapatite ceramics for bone tissue engineering, J. R. Soc. Interface 6 (2009) S341–S348. [4] I. Henriksson, P. Gatenholm, D.A. Hagg, Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds, Biofabrication 9 (1) (2017). [5] S.Y. Chen, J.C. Huang, C.T. Pan, C.H. Lin, T.L. Yang, Y.S. Huang, C.H. Ou, L.Y. Chen, D.Y. Lin, H.K. Lin, T.H. Li, J.S.C. Jang, C.C. Yang, Microstructure and mechanical properties of open-cell porous Ti-6Al-4V fabricated by selective laser melting, J. Alloys Compd. 713 (2017) 248–254. [6] C. Zhou, X. Ye, Y. Fan, F. Qing, H. Chen, X. Zhang, Synthesis and characterization of CaP/Col composite scaffolds for load-bearing bone tissue engineering, Compos. Part B 62 (2014) 242–248. [7] A. Liu, M. Sun, X. Yang, C. Ma, Y. Liu, X. Yang, S. Yan, Z. Gou, Three-dimensional printing akermanite porous scaffolds for load-bearing bone defect repair: an investigation of osteogenic capability and mechanical evolution, J. Biomater. Appl. 31 (5) (2016) 650–660. [8] M. Niinomi, Y. Liu, M. Nakai, H. Liu, H. Li, Biomedical titanium alloys with Young's moduli close to that of cortical bone, Regen Biomater. 3 (3) (2016) 173–185. [9] A.C.A. Yánez, O. Martel, H. Afonso, D. Monopoli, Gyroid porous titanium structures: a versatile solution to be used as scaffolds in bone defect reconstruction, Mater. Des. 140 (2018) 21–29. [10] S. Bahl, A.S. Krishnamurthy, S. Suwas, K. Chatterjee, Controlled nanoscale precipitation to enhance the mechanical and biological performances of a metastable beta TiNb-Sn alloy for orthopedic applications, Mater. Des. 126 (2017) 226–237. [11] A. Cuadrado, A. Yanez, O. Martel, S. Deviaene, D. Monopoli, Influence of load orientation and of types of loads on the mechanical properties of porous Ti6Al4V biomaterials, Mater. Des. 135 (2017) 309–318. [12] B. Van Hooreweder, Y. Apers, K. Lietaert, J.P. Kruth, Improving the fatigue performance of porous metallic biomaterials produced by Selective Laser Melting, Acta Biomater. 47 (2017) 193–202.

B. Zhang et al. / Materials and Design 152 (2018) 30–39 [13] A. Kumar, K.C. Nune, R.D.K. Misra, Design and biological functionality of a novel hybrid Ti-6Al-4V/hydrogel system for reconstruction of bone defects, J. Tissue Eng. Regen. Med. 12 (2018) 1133–1144. [14] S. Li, X. Li, W. Hou, K.C. Nune, R.D.K. Misra, V.L. Correa-Rodriguez, Z. Guo, Y. Hao, R. Yang, L.E. Murr, Fabrication of open-cellular (porous) titanium alloy implants: osseointegration, vascularization and preliminary human trials, Sci. China Mater. 61 (2018) 525–536. [15] N. Davis, J. Teisen, C. Schuh, D. Dunand, Solid-state foaming of titanium by superplastic expansion of argon-filled pores, J. Mater. Res. 16 (05) (2011) 1508–1519. [16] J.W. Vehof, P.H. Spauwen, J.A. Jansen, Bone formation in calcium-phosphate-coated titanium mesh, Biomaterials 21 (19) (2000) 2003–2009. [17] C. Wang, Y. Liu, Y. Fan, X. Li, The use of bioactive peptides to modify materials for bone tissue repair, Regen. Biomater. 4 (3) (2017) 191–206. [18] P. Tian, F. Peng, D. Wang, X. Liu, Corrosion behavior and cytocompatibility of fluoride-incorporated plasma electrolytic oxidation coating on biodegradable AZ31 alloy, Regen. Biomater. 4 (1) (2017) 1–10. [19] T. Ahlfeld, G. Cidonio, D. Kilian, S. Duin, A.R. Akkineni, J.I. Dawson, S. Yang, A. Lode, R.O.C. Oreffo, M. Gelinsky, Development of a clay based bioink for 3D cell printing for skeletal application, Biofabrication 9 (3) (2017), 034103. [20] N. Sears, P. Dhavalikar, M. Whitely, E. Cosgriff-Hernandez, Fabrication of biomimetic bone grafts with multi-material 3D printing, Biofabrication 9 (2) (2017). [21] H. Shao, X. Ke, A. Liu, M. Sun, Y. He, X. Yang, J. Fu, Y. Liu, L. Zhang, G. Yang, S. Xu, Z. Gou, Bone regeneration in 3D printing bioactive ceramic scaffolds with improved tissue/material interface pore architecture in thin-wall bone defect, Biofabrication 9 (2) (2017), 025003. [22] J. Wolff, M. Herberstein, Three-dimensional printing spiders: back-and-forth glue application yields silk anchorages with high pull-off resistance under varying loading situations, J. R. Soc. Interface 14 (127) (2017). [23] K.C. Nune, R.D.K. Misra, S.M. Gaytan, L.E. Murr, Biological response of nextgeneration of 3D Ti-6Al-4V biomedical devices using additive manufacturing of cellular and functional mesh structures, J. Biomater. Tissue Eng. 4 (10) (2014) 755–771. [24] K.C. Nune, A. Kumar, R.D.K. Misra, S.J. Li, Y.L. Hao, R. Yang, Functional response of osteoblasts in functionally gradient titanium alloy mesh arrays processed by 3D additive manufacturing, Colloids Surf. B: Biointerfaces 150 (2017) 78–88. [25] Y. Chen, T. Li, Z. Jia, F. Scarpa, C.-W. Yao, L. Wang, 3D printed hierarchical honeycombs with shape integrity under large compressive deformations, Mater. Des. 137 (2018) 226–234. [26] Y. Chen, T. Li, F. Scarpa, L. Wang, Lattice metamaterials with mechanically tunable Poisson's ratio for vibration control, Phys. Rev. Appl. 7 (2) (2017). [27] S. Arabnejad, R. Burnett Johnston, J. Pura, B. Singh, M. Tanzer, D. Pasini, Highstrength porous biomaterials for bone replacement: a strategy to assess the interplay between cell morphology, mechanical properties, bone ingrowth and manufacturing constraints, Acta Biomater. 30 (2016) 345–356. [28] W. Chen, Y. Xie, G. Imbalzano, J. Shen, S. Xu, S. Lee, P. Lee, Lattice Ti structures with low rigidity but compatible mechanical strength: design of implant materials for trabecular bone, Int. J. Precis. Eng. Manuf. 17 (6) (2016) 793–799. [29] L. Dobrzański, A. Dobrzańska-Danikiewicz, P. Malara, T. Gaweł, L. Dobrzański, A. Achtelik-Franczak, Fabrication of scaffolds from Ti6Al4V powders using the computer aided laser method, Arch. Metall. Mater. 60 (2) (2015). [30] N. Otawa, T. Sumida, H. Kitagaki, K. Sasaki, S. Fujibayashi, M. Takemoto, T. Nakamura, T. Yamada, Y. Mori, T. Matsushita, Custom-made titanium devices as membranes for bone augmentation in implant treatment: modeling accuracy of titanium products constructed with selective laser melting, J. Craniomaxillofac. Surg. 43 (7) (2015) 1289–1295. [31] S. Zhang, Q. Wei, L. Cheng, S. Li, Y. Shi, Effects of scan line spacing on pore characteristics and mechanical properties of porous Ti6Al4V implants fabricated by selective laser melting, Mater. Des. 63 (2014) 185–193. [32] X. Pei, B. Zhang, Y. Fan, X. Zhu, Y. Sun, Q. Wang, X. Zhang, C. Zhou, Bionic mechanical design of titanium bone tissue implants and 3D printing manufacture, Mater. Lett. 208 (2017) 133–137. [33] K.C. Nune, R.D. Misra, S.M. Gaytan, L.E. Murr, Interplay between cellular activity and three-dimensional scaffold-cell constructs with different foam structure processed by electron beam melting, J. Biomed. Mater. Res. A 103 (5) (2015) 1677–1692. [34] Z. Zheng, L. Wang, M. Jia, L. Cheng, B. Yan, Microstructure and mechanical properties of stainless steel/calcium silicate composites manufactured by selective laser melting, Mater Sci Eng C Mater Biol Appl 71 (2017) 1099–1105. [35] S.M. Ahmadi, R. Hedayati, R.K.A.K. Jain, Y. Li, S. Leeflang, A.A. Zadpoor, Effects of laser processing parameters on the mechanical properties, topology, and microstructure of additively manufactured porous metallic biomaterials: a vector-based approach, Mater. Des. 134 (2017) 234–243. [36] V. Crupi, E. Kara, G. Epasto, E. Guglielmino, H. Aykul, Static behavior of lattice structures produced via direct metal laser sintering technology, Mater. Des. 135 (2017) 246–256.

39

[37] H. Liu, W. Li, C. Liu, J. Tan, H. Wang, B. Hai, H. Cai, H. Leng, Z. Liu, C. Song, Incorporating simvastatin/poloxamer 407 hydrogel into 3D-printed porous Ti6Al4V scaffolds for the promotion of angiogenesis, osseointegration and bone ingrowth, Biofabrication 8 (4) (2016), 045012. [38] K. Kang, Y. Koh, J. Son, J. Yeom, J. Park, H. Kim, Biomechanical evaluation of pedicle screw fixation system in spinal adjacent levels using polyetheretherketone, carbonfiber-reinforced polyetheretherketone, and traditional titanium as rod materials, Compos. Part B 130 (2017) 248–256. [39] F. Naddeo, A. Naddeo, N. Cappetti, Novel “load adaptive algorithm based” procedure for 3D printing of lattice-based components showing parametric curved microbeams, Compos. Part B 115 (2017) 51–59. [40] C.C. Zhou, C.Y. Deng, X.N. Chen, X.F. Zhao, Y. Chen, Y.J. Fan, X.D. Zhang, Mechanical and biological properties of the micro-/nano-grain functionally graded hydroxyapatite bioceramics for bone tissue engineering, J. Mech. Behav. Biomed. Mater. 48 (2015) 1–11. [41] Z. Wang, C. Wang, C. Li, Y. Qin, L. Zhong, B. Chen, Z. Li, H. Liu, F. Chang, J. Wang, Analysis of factors influencing bone ingrowth into three-dimensional printed porous metal scaffolds: a review, J. Alloys Compd. 717 (2017) 271–285. [42] J. Cattalini, A. Hoppe, F. Pishbin, J. Roether, A. Boccaccini, S. Lucangioli, V. Mourino, Novel nanocomposite biomaterials with controlled copper/calcium release capability for bone tissue engineering multifunctional scaffolds, J. R. Soc. Interface 12 (110) (2015), 20150509. [43] G.S. Selders, A.E. Fetz, M.Z. Radic, G.L. Bowlin, An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration, Regen. Biomater. 4 (1) (2017) 55–68. [44] Y. Chen, L. Wang, Harnessing structural hierarchy to design stiff and lightweight phononic crystals, Extreme Mech. Lett. 9 (2016) 91–96. [45] G. Armencea, C. Berce, H. Rotaru, S. Bran, D. Leordean, C. Coada, M. Todea, C.A. Jula, D. Gheban, G. Baciut, M. Baciut, R.S. Campian, Micro-CT and histological analysis of Ti6Al7Nb custom made implants with hydroxyapatite and SiO2-TiO2 coatings in a rabbit model, Clujul Med. 88 (3) (2015) 408–414. [46] H. Cho, Y. Cho, H. Han, Finite element analysis for mechanical response of Ti foams with regular structure obtained by selective laser melting, Acta Mater. 97 (2015) 199–206. [47] L. Mullen, R. Stamp, W. Brooks, E. Jones, C. Sutcliffe, Selective Laser Melting: a regular unit cell approach for the manufacture of porous, titanium, bone in-growth constructs, suitable for orthopedic applications, J Biomed Mater Res B Appl Biomater 89 ( (2) (2009) 325–334. [48] S. Van Bael, Y. Chai, S. Truscello, M. Moesen, G. Kerckhofs, H. Van Oosterwyck, J. Kruth, J. Schrooten, The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds, Acta Biomater. 8 (7) (2012) 2824–2834. [49] P. Mengucci, A. Gatto, E. Bassoli, L. Denti, F. Fiori, E. Girardin, P. Bastianoni, B. Rutkowski, A. Czyrska-Filemonowicz, G. Barucca, Effects of build orientation and element partitioning on microstructure and mechanical properties of biomedical Ti6Al-4V alloy produced by laser sintering, J. Mech. Behav. Biomed. Mater. 71 (2017) 1–9. [50] Z. Bagheri, D. Melancon, L. Liu, R. Johnston, D. Pasini, Compensation strategy to reduce geometry and mechanics mismatches in porous biomaterials built with Selective Laser Melting, J. Mech. Behav. Biomed. Mater. 70 (2017) 17–27. [51] W.X. Jiang, P.F. Wang, Y.L. Wan, D.S. Xin, M. Fan, A simple method for establishing an ostrich model of femoral head osteonecrosis and collapse, J. Orthop. Surg. Res. 10 (2015). [52] F.W.G. Costa, G.A.D. Brito, R.M.A. Pessoa, E.C. Studart-Soares, Histomorphometric assessment of bone necrosis produced by two cryosurgery protocols using liquid nitrogen: an experimental study on rat femurs, J. Appl. Oral Sci. 19 (6) (2011) 604–609. [53] H.T. Jin, B.J. Xia, N.Z. Yu, B.J. He, Y. Shen, L.W. Xiao, P.J. Tong, The effects of autologous bone marrow mesenchymal stem cell arterial perfusion on vascular repair and angiogenesis in osteonecrosis of the femoral head in dogs, Int. Orthop. 36 (12) (2012) 2589–2596. [54] S.L. Sing, F.E. Wiria, W.Y. Yeong, Selective laser melting of lattice structures: a statistical approach to manufacturability and mechanical behavior, Robot. Comput. Integr. Manuf. 49( (2018) 170–180. [55] X. Han, H. Zhu, X. Nie, G. Wang, X. Zeng, Investigation on selective laser melting AlSi10Mg cellular lattice strut: molten pool morphology, surface roughness and dimensional accuracy, Materials 11 (3) (2018) 392. [56] Y. Zhang, R. Luo, J. Tan, J.X. Wang, X. Lu, S.X. Qu, J. Weng, B. Feng, Osteoblast behaviors on titania nanotube and mesopore layers, Regen. Biomater. 4 (2) (2017) 81–87. [57] F. Naddeo, N. Cappetti, A. Naddeo, Novel “load adaptive algorithm based” procedure for 3D printing of cancellous bone-inspired structures, Compos. Part B 115 (2017) 60–69.