Advancements in three-dimensional titanium alloy mesh scaffolds fabricated by electron beam melting for biomedical devices: mechanical and biological aspects

Science China Materials, Dec 2017

We elucidate here the process-structure-property relationships in three-dimensional (3D) implantable titanium alloy biomaterials processed by electron beam melting (EBM) that is based on the principle of additive manufacturing. The conventional methods for processing of biomedical devices including freeze casting and sintering are limited because of the difficulties in adaptation at the host site and difference in the micro/macrostructure, mechanical, and physical properties with the host tissue. In this regard, EBM has a unique advantage of processing patient-specific complex designs, which can be either obtained from the computed tomography (CT) scan of the defect site or through a computeraided design (CAD) program. This review introduces and summarizes the evolution and underlying reasons that have motivated 3D printing of scaffolds for tissue regeneration. The overview comprises of two parts for obtaining ultimate functionalities. The first part focuses on obtaining the ultimate functionalities in terms of mechanical properties of 3D titanium alloy scaffolds fabricated by EBM with different characteristics based on design, unit cell, processing parameters, scan speed, porosity, and heat treatment. The second part focuses on the advancement of enhancing biological responses of these 3D scaffolds and the influence of surface modification on cell-material interactions. The overview concludes with a discussion on the clinical trials of these 3D porous scaffolds illustrating their potential in meeting the current needs of the biomedical industry.

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Advancements in three-dimensional titanium alloy mesh scaffolds fabricated by electron beam melting for biomedical devices: mechanical and biological aspects

Advancements in three-dimensional titanium alloy mesh scaffolds fabricated by electron beam melting for biomedical devices: mechanical and biological aspects Krishna Chaitanya Nune 1 Shujun Li 0 R. Devesh Kumar Misra 1 0 Institute of Metals Research, Chinese Academy of Sciences , Shenyang 11016 , China 1 Biomaterials Science and Engineering Laboratory Metallurgical, Materials and Biomedical Engineering Department, University of Texas at El Paso , TX 79968 , USA We elucidate here the process-structure-property relationships in three-dimensional (3D) implantable titanium alloy biomaterials processed by electron beam melting (EBM) that is based on the principle of additive manufacturing. The conventional methods for processing of biomedical devices including freeze casting and sintering are limited because of the difficulties in adaptation at the host site and difference in the micro/macrostructure, mechanical, and physical properties with the host tissue. In this regard, EBM has a unique advantage of processing patient-specific complex designs, which can be either obtained from the computed tomography (CT) scan of the defect site or through a computeraided design (CAD) program. This review introduces and summarizes the evolution and underlying reasons that have motivated 3D printing of scaffolds for tissue regeneration. The overview comprises of two parts for obtaining ultimate functionalities. The first part focuses on obtaining the ultimate functionalities in terms of mechanical properties of 3D titanium alloy scaffolds fabricated by EBM with different characteristics based on design, unit cell, processing parameters, scan speed, porosity, and heat treatment. The second part focuses on the advancement of enhancing biological responses of these 3D scaffolds and the influence of surface modification on cell-material interactions. The overview concludes with a discussion on the clinical trials of these 3D porous scaffolds illustrating their potential in meeting the current needs of the biomedical industry. Electron beam melting; 3D printing; tissue engineering; mechanical properties; biocompatibility INTRODUCTION Evolution of additive manufacturing Additive manufacturing is a rapid prototype (RP) technique involving layer-by-layer processing of three-dimensional (3D) complex structures as directed by the computer-aided design (CAD) [1,2]. Additive manufacturing can be utilized to fabricate complex structures of relevance to aerospace and patient-specific implants. For instance, biomedical devices can be fabricated using the CAD program or computed tomography (CT) scan of a damaged site that provides a 3D visualization of the damaged or defect region. The CT scan data is subsequently transformed into a 3D design using a commercial software (MIMICS), which generates a STL (STereoLithography) file. Next this file is fed as an input file to the 3D printing system, to print the scaffold or component. The fabrication of porous materials has been explored since 1940s, when pores were introduced in aluminum through the addition of mercury to the melt [3]. However, the idea of introducing porous materials in the biomedical industry started only in 1970s with the aim to enhance osseointegration and enable bone ingrowth to potentially replace conventional solid/dense implants with porous coatings [4]. In this regard, several engineering materials such as metals, ceramics, polymers, and composites were rendered porous through the adaptation of advanced manufacturing approaches [5– 16]. In load-bearing orthopedic applications, irrespective April 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 of the manufacturing technique, brittle behavior of ceramics and poor strength of polymers limit their applications for bone implants. Thus, titanium, cobaltchromium, stainless steel, tantalum, and magnesium alloys are considered worthy of research. Titanium and its alloys have been widely used for several decades as monolithic and solid implants. They have inherent advantages of specific strength, low stiffness, good corrosion and fatigue resistance in physiological medium and have been extensively used for dental, orthopedic, and maxillofacial applications [17,18]. Titanium alloy has superior biological and mechanical properties, in comparison to other metals, because of the favorable microstructure and stable passive film. 3D printing of porous structures of titanium alloys have the ability to reduce modulus mismatch in the context of biomedical devices [3,19–21]. Thermo-mechanically processed solid titanium alloys have higher elastic modulus (~90–115 GPa) compared to the surrounding tissue (cortical hard bone: ~15–25 GPa; cancellous or trabecular soft bone with open cellular structure: ~0.1–4.5 GPa) [22– 24]. This leads to stress-shielding, because of which the implant takes the maximum load [25]. An important consequence of biomechanical mismatch is the possibility of reduced rate of bone healing [25]. Hence, a viable approach to alleviate biomechanical mismatch is to fabricate open cellular porous structures that are characterized by significantly reduced modulus and reduced density. Another approach is alloy design. In the last decade, with the advancement in manufacturing techniques, porous metal structures have been fabricated by additive manufacturing (AM) (layer-by-layer fabrication) from precursor powders using electron beam melting (EBM) and selective laser melting (SLM) [26–28]. The following sections summarize the advancements in design, structure, process, and properties of porous titanium alloy components fabricated by EBM for orthopedic applications. Design considerations In additive manufacturing, the properties of the fabricated component are governed by the unit cell, while the design is dictated by the resolution offered by the additive manufacturing process/equipment [1]. For ARCAM A1 the resolution is ±0.4 mm based on the information in the user’s manual. Irrespective of the above, compressive strength, endurance and elastic modulus can be optimized to ensure the long term success of the implant [1,29–32]. Vascularization is another aspect that merits consideration during design, because vascularization promotes diffusion of nutrients that is necessary for cell proliferation and differentiation. Biomedical devices of different alloys including titanium alloys, stainless steel and cobalt-chromium alloys have been actively considered for 3D printing [1]. The aspects that are of significance in the design and processing of optimal architecture for bone tissue engineering applications include: (a) high porosity-high strength combination with elastic modulus similar to bone, (b) interconnected porous structure with clinically relevant geometry that allows oxygen and exchange of nutrients, and facilitates efficient cell seeding and production of extracellular matrix (ECM), and (c) appropriate chemistry and topography for cell attachment, proliferation, and differentiation [33,34]. With regard to regeneration of an injured tissue, mimicking cellular and physiological environment, involving neighboring cells, extracellular matrix, flow of physiological fluid, mechanical, chemical and topographical cues is a primary requirement [1,35,36]. Considering that the response of cells depends on the available environment, the ECM needs to be considered as part of the environment to ensure cell-material interaction and to provide a route for sensing chemical and physical cues [1,37–39]. Hence, tissue engineered scaffolds are expected to play a determining role in ECM modeling and governing cell-material interaction during the regeneration process. In this regard, scaffolds are required to be non-toxic and biocompatible [1,40,41]. They are also expected to provide structural support to cells during tissue regeneration/remodeling [1,42,43]. The scaffold with desired physical and chemical properties dictates tissue engineering. For example, the elastic modulus of ECM dictates differentiation of cells into different lineage. High stiffness is important for bone forming cells [1,44,45]. In the case of cardiac muscle cells, the geometry of scaffold is an important issue in aligning cells, which may enable directional contraction of tissue from the perspective of efficient blood transfer [1,46]. In regard to bone tissue regeneration, pore size and percentage of porosity impact osteogenesis [18]. In the case of bio-inert materials, porosity and pore size generally do not affect osteogenesis, but with regard to bioactive materials, porosity and pore size significantly influence osteogenesis [47–49]. It is pertinent to keep in mind that porosity is not the cause for hypoxia, which is considered responsible for encouraging osteochondral ossification [1,50]. During design, degree of porosity and pore size are important considerations from the per456 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d.Sp.r.in.ge.r-.V.er.lag. G.m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 spective of long term structural support. Vascularization availability of micro-vessels is required for the transport of oxygen and nutrients, otherwise cells may die [1,51]. Thus, ensuring desired growth of new blood vessels and adequate space for growth is a necessity [1,51]. An important area of application of additive manufacturing relates to the design of scaffolds to perform a particular function. For example, electroactive scaffolds with directional porosity enabled alignment of nerve cells and acted as a guiding cue for growth [52]. Electrical stimulation also promoted nerve growth factor (NGF) [53]. Scaffolds play a determining role in transmitting the applied external stimuli to the cells and enabling the biological functions to be controlled [1,54,55]. Repair of the annulus fibrous rupture was accomplished by loading the scaffolds with mesenchymal stem cells, eliminating herniation of nucleus pulposus tissue and stabilization of disk height [1,56]. In summary, tissue-specific scaffolds are preferred because the complex structure can be mimicked and the damaged tissue can be restored. Moreover, 3D scaffold with interconnected porous structure provides space for vascularization, and serves as a template for the regeneration of tissue. Also, the porous structure can be designed to suit a particular tissue-specific environment, e.g., directionality for nerve regeneration. The aforementioned requirements can be conveniently met by designing 3D scaffolds, which is not feasible using conventional approaches. 3D ADDITIVE MANUFACTURING METHODS FOR BONE TISSUE ENGINEERING Prior to the development of 3D additive manufacturing methods, the methods adopted to fabricate 3D porous scaffolds include freeze drying, solvent casting with particulate leaching, fiber bonding, membrane lamination, and gas foaming [16,57–59]. In these methods, it is difficult to control the pore size, shape, and pore interconnectivity [60]. These challenges can now be met via rapid prototyping or 3D printing, as described in the following section below. Patient specific scaffolds or devices with tailored dimensions and porosity can be conveniently processed by the additive manufacturing method. Electron beam melting An EBM system (Fig. 1) to process 3D porous scaffolds has an electron column, where a focused electron beam is produced and scans a uniformly raked layer of powder, which is gravity fed from the feed stock [1,61]. The ARCAM A1 system, using a DC current of ~10 mA and anode potential of 60 kV drives a tungsten filament in a 3 kW electron gun. A combination of electromagnetic lenses focuses the electron beam (beam spotsize (FWHM) 0.2–1.0 mm). The system operates in vacuum of < 104 Torr. The resolution is ±0.025 mm [ARCAM user’s manual]. The selective melting of powder is based on CAD design. The powder bed is initially heated to ~750°C, for instance, in the case of titanium alloy powder [1,27,62–64]. In general, powders are heated to 0.5–0.6Tm (Tm: melting temperature of powder). Packing, powder size, beam energy, powder conductivity and heat of fusion are important contributors. The selective melting of powder that is added in the build direction produces a complex 3D structure. Also, presented in Fig. 1 is an SLM system, also referred as selective laser sintering (SLS) (Fig. 1), which is a conventional particle-bonding system, using a laser beam (e.g., CO2 laser) in lieu of electron beam [61]. Illustrations of biomedical devices produced via additive manufacturing are presented in Fig. 2 [62]. Near net-shape components can be produced by additive manufacturing with little or no machining. Titanium alloy components processed by EBM were characterized by a columnar structure and ~50–60 nm thick lamellar plates with indentation hardness of ~2.5 GPa, which was similar to the hardness of ~2.3 GPa obtained for SLM processed component. Co-Cr alloy (Co-26Cr-6Mo-0.2C) was processed by EBM to fabricate femoral (knee) prototypes as a reticulated mesh structure (density of 1.5 g cm−3) [1,64] characterized by array of orthogonal Cr23C6 carbides. The microstructure of the Co-Cr alloy was ~2 µm in the build plane, perpendicular to the build direction, and the carbide columns were present in the vertical plane, parallel to the build direction. The resulting structure had indentation hardness of 4.4 GPa, yield strength of ~0.51 GPa and tensile strength of ~1.45 GPa [1,65]. The fabricated knee exhibited a hardness of 5.9 GPa, while the prototype knee in the annealed condition had a lower hardness of 4.7 GPa [1,65]. The reticulated mesh strut structure had a high density of carbide arrays with indentation hardness of 6.2 GPa. The success achieved in obtaining a complex monolith and a reticulated mesh structure of Co-Cr alloy by EBM, that exhibited high strength, led to the conclusion that complex monoliths with multifunctional characteristics can be processed by EBM. The properties of titanium alloy fabricated by EBM and SLM are presented in Table 1 [65]. REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials Part I: Mechanical properties It is important to obtain desired mechanical properties during fabrication of 3D scaffolds. This is essential for structural integrity, till such time new tissue is regenerated [66]. The mechanical properties depend on the specific anatomical location of application, i.e., soft or hard tissue [67,68]. The compressive strength of human bone is in the range of ~2–12 MPa for cancellous bone and ~100–230 MPa for cortical bone. The elastic modulus of cortical bone, cancellous bone and titanium alloy scaffold are ~15–25 GPa, ~0.1–4.5 GPa and ~110 GPa, respectively [69]. Scaffolds of strength less than the bone are additionally supported by plates, screws, or nails. The mismatch of elastic moduli between the scaffold and bone with a higher elastic modulus than host tissue leads to stress shielding and consequent bone resorption. In a recent study focused on understanding the mechanical property-porosity relationship (pore size ~350–1000 µm) led to reduced elastic modulus of the porous implant, and minimized stress shielding, in vivo [1,70]. However, the 458 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d.Sp.r.in.ge.r-.V.er.lag. G.m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 scaffolds can rupture and give away if their strength is less than the host bone for load-bearing situations. It is desired that the scaffolds exhibit mechanical properties (elastic modulus and strength) similar to bone [1,71]. Ideally, it is preferred that a scaffold which substitutes for the bone, is modeled as a structure with multiple layers so that the mechanical properties vary across the structure. This can be made possible by having a gradient or graded structure with different porosity across the scaffold, which provides a means to vary the stiffness [72]. It is important to keep in mind that the mechanical strength depends on apparent density of the scaffold, where the thickness zone of inter-pore walls and pore size to beam thickness ratio dictates the mechanical strength of 3D printed porous scaffolds [73,74]. In general, the mechanical properties of 3D printed scaffolds depend on unit cell, pore architecture, pore volume, and percentage porosity. To ensure long term endurance and the ability to withstand impact, it is necessary that the fabricated materials have a good combination of strength and energy absorption capability, as discussed below. It is recognized that scaffolds with high interconnected porosity, though, preferred for tissue regeneration, but they may lack adequate mechanical strength and energy absorption capability. The mutually opposing requirement of high porosity and mechanical strength in conjunction with high energy absorption is a challenge. Role of unit cell design Recently, the effect of cell shape on mechanical properties and deformation mechanism was studied in EBM fabricated Ti-6Al-4V reticulated mesh structure with different unit cell elements (cubic, G7, rhombic dodecahedron) [75]. These different unit cells provide a means to effectively balance the strength, elastic modulus and degradation behavior in cellular solids. It was observed that in the case of cellular structures with different unit cell, the underlying fatigue mechanism involved interaction of cyclic ratcheting and fatigue crack growth on the struts. This was closely related to coupling of buckling and bending deformation of the strut. On increasing the buckling deformation on the struts via design of cell shape, the cyclic ratcheting rate of the meshes during cyclic deformation was decreased and the compressive fatigue strength was increased. While increasing the bending deformation of the struts, the contribution of fatigue crack growth in the struts led to significant fatigue damage of mesh [75]. Moreover, the presence of rough surface and pores contained in the struts significantly deteriorated the compressive fatigue strength of the struts. It was also observed that by altering the influence of bending deformation, the stress-strain behavior transformed from brittle to ductile. It was demonstrated that Ti-6Al-4V cellular materials with high strength, low modulus, and desirable mechanical deformation behavior can be processed by EBM, involving tuning of cell design (Fig. 3) [75]. Table 2 summaries the properties of titanium alloy fabricated with different unit cell design by EBM [75]. Role of porosity A comparative study of Ti-6Al-4V and Ti2448 alloy on the effect of porosity on mechanical properties indicated that both the compressive strength and yield strength decreased with increased porosity for both the alloys [76]. It was observed that Ti-6Al-4V alloy with identical porosity (75%) built using the same unit cell shape exhibited higher compressive strength and Young’s modulus of REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials ~60 MPa and 3.0 ± 0.6 GPa, respectively in comparison to the Ti2448 alloy sample of ~38 MPa and ~1.44 ± 0.2 GPa. They concluded that β-type Ti2448 porous structure had at least twice strength-to-modulus ratio than Ti-6Al-4V porous components with identical porosity, and was highly attractive for biomedical applications. Role of scan speed The effect of scan speed in EBM on the surface and mechanical properties of the scaffolds was also studied [77]. Higher scan speeds, led to a rough strut surface with 460 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d .Sp.r.in.ge.r-.V.er.lag. G. m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 pores/defects (~40 µm) because of partial sintering of particles. It was observed that lower electron beam scan speed (150 mm s−1) led to more input energy, which produced strong struts with less number of pores/defects and smooth surface. This suggested that the surface properties can be tailored by adjusting the scan speed of the electron beam. Other researchers observed that the orientation during the building process had an influence on surface roughness because of change in energy flow [78,79]. Role of complex design architectures (Graded/Gradient) The natural bone has a functionally graded structure. The outer dense region referred as cortical bone has elastic modulus of ~15–25 GPa, while the inner porous region referred as cancellous bone has an elastic modulus of ~0.1–4.5 GPa. There is a bone marrow cavity in the center [1,80]. Thus, to obtain a replica of natural bone, 3D scaffolds with graded porosity were studied, since porosity is related to the elastic modulus according to Gibson-Ashby equation: E=E0(ρ/ρ0)2 [81]. In this regard, titanium alloy rod with graded density, characterized by a lower density inner foam (density ~0.6 g cm−3; stiffness ~0.3 GPa), surrounded by a foam with relatively higher density (~1.1 g cm−3; stiffness ~2.2 GPa) was fabricated [28]. Graded/gradient porous structures are envisioned to provide optimum combination of elastic modulus, strength and toughness. Recent studies focused on bone ingrowth suggested that the cellular structures should have high degree of porosity. However, highly porous structures are expected to have low mechanical strength. It is absolutely essential that cellular structures are characterized by adequate strength and ductility combination in conjunction with the ability to absorb high energy [1]. In this regard, a complex structure mimicking bone involving graded/gradient Ti-6Al-4V mesh structure using EBM was fabricated [82]. It was observed that graded and uniform mesh structures had an elastic regime up to the peak stress, followed by a plateau region, where the stress fluctuated (Fig. 4). Furthermore, it was noted that both low and high density regions of the graded mesh structure first experienced uniform deformation, followed by fracture in the low density region, when crush band was formed. Finite element analysis (FEA) was used to indicate stress concentration regions in the mesh and a discontinuous stress transfer was noted in graded materials at the nodes, but not in the gradient material (Fig. 5) [82]. This study outlined the advantage of gradient mesh design over graded and uniform mesh design, which avoids the weakness of brittle behavior and is characterized by high strength-high ductility combination and high energy absorption. Role of post-fabrication heat treatment Several post-fabrication treatments such as stress relieving, surface heat treatment, and hot isostatic pressing (HIP) have been explored for porous scaffolds [83,84]. The process of annealing or aging implemented after fabrication led to a different microstructure, where the mechanical properties are sensitive to the microstructure [85]. It was observed that the annealed sample consisted of single β phase and exhibited large plasticity, low Young’s modulus and good fatigue properties. On aging, precipitation of α phase occurred that increased the hardness, strength and Young’s modulus and at low stress, cyclic ratcheting was the primary factor that led to fatigue failure [85]. Role of post-fabrication surface modification Recently, several surface modification methods have been explored on porous scaffolds to promote cellular interApril 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials actions with the host tissue, beyond what is obtained by 3D printing. However, to obtain uniform surface modification on 3D cellular structure is a challenge. In this regard, the traditional surface treatment methods such as grinding, grit blasting, ion implantation, thermal spraying, etc. cannot be employed because they do not help in obtaining a homogeneous surface modification throughout the entire structure [86–90]. Hence, the recent focus was on chemical and/or electrochemical methods because acid-based solution can penetrate through the porous structures through the interconnected pores, producing a homogeneous surface throughout the entire structure [86,91–93]. Micro-arc oxidation was observed not to influence the mechanical properties such as maximum stress, yield stress, plateau stress, and energy absorption, but had a significant effect on the elastic modulus. Mechanical properties were significantly affected and decreased in scaffolds with higher relative densities [93]. A combination of chemical etching and electrochemical polishing of porous structures as a post-fabrication surface modification step led to an increase in internal porosity and a decrease in effective strut thickness resulting in overall decreased mechanical properties [86]. In another study, acid-alkali surface treatment induced substantial mass loss and, hence, significant loss of both static and fatigue properties [94]. Thus, large surface area of 3D printed porous scaffolds, was responsible for a significant impact on mechanical properties induced by surface modification. Irrespective of the above, an improvement in fatigue strength was noticed on chemically etching with same etchant compared to the as-fabricated samples of Ti-6Al4V alloy, but not in the case of CoCrMo alloy [83,84]. The differences were attributed to low notch (surface roughness and micro-pitting after etching) sensitivity of CoCrMo alloy with more ductility than Ti-6Al-4V alloy. Given that the notch is a stress raiser, the fatigue properties were improved in a Ti-6Al-4V alloy after etching because of reduced surface roughness and removal of partially melted powder particles. In contrast, in CoCrMo alloy, while the particles were removed, micro-pits were formed on the surface, leading to insignificant difference in surface roughness. Thus, selecting an appropriate etchant and exposure time is important to enhance mechanical properties. Part II: Biocompatibility and biological response —influence of surface modification A practical approach to alleviate the undesired biomechanical mismatch is to use a 3D interconnected 462 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d.Sp.r.in.ge.r-.V.er.lag. G.m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 SCIENCE CHINA Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REVIEWS porous foam or mesh structure, which will have a significantly low modulus compared to a solid and dense implant. Recently, the interplay between 3D printed foam and mesh structure and osteoblast functions were studied and compared with a solid Ti-6Al-4V alloy [1,95,96]. As expected, the seeding efficiency was low for both mesh and foam structures, but there was significant cell spreading and cell bridging of osteoblasts (MC3T3-E1). Fig. 6 illustrates morphology of osteoblasts on mesh and foam structures [1,95,96]. The alkaline phosphate (ALP) assay results suggested that the enzyme activity increased with time for foam and mesh structures, irrespective of pore size. The response of different cells to pore size varies [97]. For example, pore size of ~5–15 μm was considered suitable for fibroblast, ~70–120 μm for chondrocytes, ~100– 400 μm for osteoblast ingrowth [19,98–100]. Moreover, interconnected porous structures with gradient distribuApril 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials tion of pores can repair and reconstruct more than one tissue, even though the microenvironment varies for different tissues [1]. For instance, functionally graded porous titanium alloy fabricated to meet the requirements of tissue engineering and to reduce stress shielding and high energy absorption indicated that the structure facilitated flow of nutrients and cell proliferation across the entire structure [101,102]. It was concluded that the 464 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d .Sp.r.in.ge.r-.V.er.lag. G. m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 SCIENCE CHINA Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REVIEWS various stages of cell maturation can co-exist in different regions of the gradient mesh structure. Surface modification of 3D printed structures To enhance the cell-material interaction and osseointegration potential beyond what is possible via 3D architecture, surface modification is being explored. A number of studies have been conducted on Ti-6Al-4V cellular structures fabricated by EBM to study biocompatibility of surface modified structures. One such study focused on determining the suitability of different Ti-6Al-4V surfaces produced by EBM as a matrix for attachment, proliferation, and differentiation of osteoblasts [103]. It was noted that osteoblast proliferation and differentiation can be changed by modifying the surface characteristics of Ti6Al-4V mesh. Titanium alloy was observed to provide a highly favorable surface for osteoblast adhesion and osteogenic differentiation. In another study, it was shown that the oxide layer adsorbed fibronectin promoted osteoblast attachment and osteointegration through binding of adsorbed fibronectin to surface-expressed osteoblast integrins [104]. Studies also reported the influence of surface nanotopography on the enhanced expression of osteogenic markers [105–107]. More recently, mesh structures were modified via micro-arc oxidation and anodization approach (Figs 7 and 8) [96,97]. The combination of macro-, micro-, and nanoporous architecture obtained in the surface modified Ti-6Al-4V mesh structures mimicked natural bone-like ECM topography that promotes bone formation at nanoscale (regulating protein conformation and signal transduction) and microscale (impacting initial cell attachment and proliferation) [83,108–117]. The unique combination of microporous/nanoporous bioactive titania and interconnected porous architecture of microarc/ anodized titanium alloy mesh structures provided a multimodal surface roughness from nano-to-micro-tomacroscale regime and increased the bioactivity of the asfabricated mesh structure [91,92]. This provided a favorable osteogenic microenvironment for tissue ingrowth and is envisioned to facilitate stronger primary and secondary fixation of the implant device. The interconnected porous architecture also enables circulation of physiological fluid, oxygen, nutrients, and cells and mimics the flow conditions of natural bone. Additionally, in the context of long term structural integrity, the transport of cells through the porous mesh structure helps in mechanical interlocking because the confluent sheet connects the pores and integrates with the structure [91,92]. The coating of 3D titanium implants with tantalum and chitosan-hydroxyapatite composite improved diabetesinduced impaired osteogenesis of these implants [118,119]. While evidence of clinical studies indicated high failure rate for titanium implants in diabetic patients, where reactive oxygen species (ROS) were produced at the implant/bone interface [120–122]. DiabetesApril 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials induced accumulation of ROS also influenced the viability of cells on the titanium surface [123]. In this regard, tantalum coating on titanium indicated superior integration of tissue in diabetes mellitus impaired osteogenesis in sheep by suppressing the ROS-mediated p38 MAPK pathway. In a similar way, studies performed by chitosan-hydroxyapatite composite coating on 3D mesh structures improved diabetes-induced impaired osteointegration via the reactivation of Wnt/β-catenin pathway [118]. Contrary to the aforementioned benefits, coatings were believed to loosen the biomaterial/coating interface because of inherent brittleness and poor adhesion [124–126]. In another study, the Ti-6Al-4V mesh structures fabricated by EBM technique were biologically surface modified using BMP-2 (bone morphogenetic protein-2) and decellularized extracellular matrix. The bioactivity of the surface was improved and the modified surface was expected to enhance the fixation of the implant with the surrounding bone and improve the long-term stability [127,128]. Biological surface modification: incorporation of therapeutic drugs into 3D printed architectures The incorporation of osteoinductive growth factors such as bone morphogenetic proteins (BMP) into 3D printed mesh structures or surface treatment of 3D mesh structures with BMP can be considered as viable approaches to increase osteoinductivity and facilitate bone tissue regeneration in the scaffold. Incorporation of other drugs such as dexamethasone, hydrogels, simvastatin, antimicrobial agents, and VEGF has also been explored [127– 130]. The potential role of topography of the material in determining the molecular assembly of BMP-2 and subsequent osteoblast functions indicated a significant difference in organization and assembly of protein [127]. It was proposed that 300 µm pore size mesh structure with interconnected porous architecture was superior to mesh structures with 600 µm and 900 µm pore size respectively, in terms of cell proliferation and mineralization. Also, a favorable effect of pore size on protein adsorption was noted, which led to the formation of small and dense dendrites of protein on mesh structures, that provided a favorable osteogenic microenvironment. It was envisaged that the unique microstructure of mesh along with preadsorbed protein (BMP-2) provided the conditions favorable for bone tissue formation in terms of cell adhesion, proliferation, expression level of proteins, and mineralization. On incorporating BMP-2, the osteoinductive potential of the material was increased, influencing cell signaling cascade to differentiate osteoblast cells into a more matured osteogenic phenotype. In another study, 3D printed scaffolds containing proteins were observed to successfully deliver BMP-2 that promoted bone formation in relation to scaffold without BMP-2 [1,131]. More recently, the advances in printing technology have provided an opportunity to load drugs 466 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d.Sp.r.in.ge.r-.V.er.lag. G.m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 during post-fabrication surface treatment [132–135]. It is pertinent to indicate that vascularization directly impacts in vivo bone formation. In reality, scaffolds with interconnected porous structure and with pore size of ~300–400 µm are favorable for the ingrowth of blood capillaries [1,136,137]. Thus, the long-term success of a 3D scaffold under in vivo conditions is largely governed by the effectiveness of the scaffold to support angiogenesis [138–140]. Angiogenesis refers to new blood capillaries that are formed from pre-existing blood vessels at the host site. Vascularization occurs during wound healing [1,141] and is a slow process, requiring several days or weeks for it to completely take place in porous scaffolds with interconnected pores [140,141]. When there is insufficient vascular network, the cells experience a condition of hypoxia/necrosis because of restricted supply of oxygen/nutrients [142]. The amount of oxygen that is supplied to the cells, is governed by the diffusion length, which is ~150–200 µm from the blood vessel [141–146]. A primary limitation associated with 3D scaffold vascularization is adequate supply of blood in the center of scaffolds, when the blood capillaries are absent [140]. For instance, rapid growth of blood vessels occurs in scaffolds with pore size greater than ~250 µm [1,147]. Additive manufacturing that allows fabrication of 3D scaffolds with predefined architecture, is most likely to enable cell alignment, spatial organization of cells, tune porosity with respect to mechanical properties and vascularization [1,52,148–150]. On the other hand, conventional methods such as freeze casting, gas foaming, and particle leaching that produce a porous architecture with random distribution of pores results in a torturous pathway for new blood capillaries [1,151]. This slowdowns the growth of blood capillaries and transport of nutrients and oxygen [1,141]. In the case of large scaffolds, pre-treatment is generally preferred for rapid growth of blood capillaries. To accomplish this, growth factors such as vascular endothelial growth factor (VEGF) play a role in angiogenesis and faster healing [1,152,153]. Furthermore, vascularization provides osteoprogenitor cells at the defect site, promoting new bone formation [154,155]. To ensure that vascularization occurs at the center of the 3D scaffold, pre-seeding of scaffolds with endothelial cells was proposed [156,157]. As a consequence, a link is established between the blood vessels of host tissues and endothelial cells which promotes vascularization. Furthermore, coating of endothelial cells and VEGF encourages the formation of blood vessels and promotes new bone formation [1,158]. The development of vascular and circulation networks is important for homeostasis and the regeneration ability of the bone. Based on the aforementioned findings, the future of bone tissue engineering will depend on developing tissue specific porous 3D scaffolds and incorporate the recent findings to ensure tissue regeneration [1]. IN VIVO STUDIES AND CLINICAL TRIALS OF EBM FABRICATED 3D SCAFFOLDS 3D printed titanium alloy mesh structure scaffolds have the potential to restore anatomically complex patient specific areas such as spinal fusion, dental, temporomandibular joint and craniomaxillofacial surgery [159,160]. Spinal fusion mesh/cage devices were introduced in 1988 to attain spine interbody (cervical disk) fusion and are now widely practiced. Interbody fusion cage made of poly-ether-ether-ketone (PEEK) was used for spinal disorder treatment. But, it does not integrate well with surrounding bone tissue and forms fibrous tissue capsule. To overcome this disadvantage, 3D porous Ti mesh/cage using EBM, was developed [160]. Research is in progress to 3D print titanium alloys to produce customized titanium prosthesis in limb salvage surgery to replace bones lost due to sarcoma [161]. Studies have demonstrated that porous Ti implants into the frontal skull of domestic pigs exhibited enhanced bone formation within the implant [162]. The 3D mesh/cage constructs provide necessary support for cells to proliferate and differentiate. They are believed to be a good choice for bone regeneration and hard tissue replacement [58,163]. The control of pore size, pore geometry and porosity distribution, cell behavior in vitro and in vivo environments were significantly improved and good biocompatibility was obtained [18,164–166]. It was shown that pore size in the range of 300–900 µm using EBM-fabricated mesh provided favorable condition for cell differentiation at an early stage, and bone cells adhered to the struts and ligaments of mesh by forming numerous cytoplasmic extensions [95]. Within 21 days, bone cells colonized the entire architecture bridging the pores. For these architectures, the stiffness for Ti-6Al-4V implants was in the range of ~2–20 GPa, appropriate for elimination of stress shielding. A recent in vivo study on porous Ti mesh/cage fabricated by EBM and implanted at cervical disc levels (C3/ C4 or C4/C5), in 12 mature female Small Tail Han sheep (1–2 years old, 35–50 kg) indicated that rapid bone ingrowth is a possibility, together with superior osseointegration and mechanical stability. This demonstrated their April 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 REVIEWS . . . . . . . . . . . . . . . . . . . . . . . . . . SCIENCE CHINA Materials potential for clinical application in comparison to PEEK [160]. After three months, post-implantation, micro CT (computer generated) imaging and histological staining showed the growth of neighboring bone tissue into porous Ti mesh, partially fusing the two vertebral bodies. The bone tissue bonded closely with Ti mesh/cage, with majority of bone tissue around the struts in the interior and peripheral regions of the mesh (Fig. 9) [160]. A comparative study showed that the culture of human osteoblast-like cells on Ti-6Al-4V led to enhanced osteogenic differentiation in comparison to PEEK implant [167]. The 3D-printed titanium mesh implants have also been shown to be excellent candidates for the repair of bifrontal skull defects and maxillary and orbital floor reconstruction [168,169]. The 3D architecture provides improved stability after fixation of mandibular fracture, compared to the plates and screws because of their 3D configuration instead of greater plate thickness or screw length [170]. This is supposed to facilitate good blood supply to the bone tissue. More recently, clinical trials of 468 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e.C.hi.na. P.r.es.s a.n.d .Sp.r.in.ge.r-.V.er.lag. G. m.b.H. G.e.rm.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 bimaxillary surgery and mandibular reconstruction using EBM fabricated titanium alloy on a woman has also shown highly encouraging results [171]. In summary, the EBM is potentially a valuable tool to fabricate patient specific prosthesis with complex shapes/architectures and internal features, that can precisely match the bone tissueimplant interface and contribute to the improvement in the quality of life of the patient. CONCLUSIONS The process-structure-property relationships in three-dimensional implantable titanium alloy biomaterials processed by EBM is elucidate here. EBM has the benefit of processing patient-specific complex designs, which can be either obtained from the CT scan of the defect site or through a CAD program. We have summarized the evolution and underlying reasons that have motivated 3D printing of scaffolds for tissue regeneration. There are two parts of review for obtaining ultimate functionalities. The first part focuses on obtaining the ultimate functionalities in terms of mechanical properties of 3D titanium alloy scaffolds fabricated by EBM with different characteristics based on design, unit cell, processing parameters, scan speed, porosity, and heat treatment. The second part focuses on the advancement of enhancing biological response of these 3D scaffolds and the influence of surface modification on cell-material interactions. In the end a discussion on the clinical trials of these 3D porous scaffolds illustrates their potential in meeting the current needs of the biomedical industry. 1 2 3 4 5 6 Kumar A, Nune KC, Murr LE, et al. Biocompatibility and mechanical behaviour of three-dimensional scaffolds for biomedical devices: process–structure–property paradigm. Int Mater Rev, 2016, 61: 20–45 Schubert C, van Langeveld MC, Donoso LA. Innovations in 3D printing: a 3D overview from optics to organs. Br J Ophthalmol, 2014, 98: 159–161 Simancik F. Introduction: the strange world of cellular metals. In: Degischer HP, Kriszt B (Eds). Handbook of cellular metals. Weinheim: Wiley-VCH Verlag, 2002, 1–4 Weber JN, White EW. Carbon-metal graded composites for permanent osseous attachment of non-porous metals. Mater Res Bull, 1972, 7: 1005–1016 Klawitter JJ, Weinstein AM. The status of porous materials to obtain direct skeletal attachment by tissue ingrowth. Acta Orthop Belg 1974, 40: 755–765 White EW, Weber JN, Roy DM, et al. Replamineform porous biomaterials for hard tissue implant applications. J Biomed Mater Res, 1975, 9: 23–27 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 April 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e.C. h.in.a. P.r.e.ss. a.n.d.S.p.ri.ng.e.r-.V. e.rl.ag. .G.m.b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 63 64 470 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e .C.hi.na. P.r.es.s a.n.d .Sp.r.in.ge.r-.V.er.lag. G. m.b.H. G.e.r m.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 98 99 100 April 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e. C. h.in.a. P.r.e.ss. a.n.d. S.p.ri.ng.e.r-.V. e.rl.ag. .G.m. b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 132 133 134 472 . . . . . . . . . . . . . . . . . . . . . . . .©. .Sc.ie.nc.e .C.hi.na. P.r.es.s a.n.d .Sp.r.in.ge.r-.V.er.lag. G. m.b.H. G.e.r m.a.n.y .20.17. . . . . . . . . . . . . April 2018 | Vol. 61 No. 4 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 171 Acknowledgements Nune KC and Misra RDK acknowledge support from the Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso. All the authors acknowledge different reference sources that enabled the authors to prepare this overview article. The authors have done their best in citing all the relevant articles, however, inadvertently there may be situations where an April 2018 | Vol. 61 No. 4 . . . . . . . . . . . . . . .©. .S.ci.en.c.e. C. h.i n.a. P.r.e.ss. a.n.d. S.p.ri.ng.e.r-.V. e.rl.ag. .G.m. b.H. .G.er.m. a.n.y.2.01.7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 article has not been appropriately cited. All figures adapted from different references and reference number is indicated in the figure caption. Li S greatly acknowledges support of the Key Research Program of Frontier Science, CAS (QYZDJ-SSW-JSC031-02) Author contributions Nune KC and Misra RDK wrote the paper and Li S contributed to the discussion. Krishna Chaitanya Nune is a senior research scientist in the Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, USA. His research interests are in biomaterials. R. Devesh Kumar Misra is a professor and Chair of the Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, USA. The research interests are in biomaterials, nanostructured materials, deformation and fracture, and additive manufacturing. Krishna Chaitanya Nune1, 李述军2, R. Devesh Kumar Misra1*


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Krishna Chaitanya Nune, Shujun Li, R. Devesh Kumar Misra. Advancements in three-dimensional titanium alloy mesh scaffolds fabricated by electron beam melting for biomedical devices: mechanical and biological aspects, Science China Materials, 2017, 455-474, DOI: 10.1007/s40843-017-9134-x