In vitro and in vivo study of 3D-printed porous Tantalum scaffolds for

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In vitro and in vivo study of 3D-printed porous Tantalum scaffolds for repairing bone defects Yu Guo, Kai Xie, Wenbo Jiang, Lei Wang, Guoyuan Li, Shuang Zhao, Wen Wu, and Yongqiang Hao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01094 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018

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ACS Biomaterials Science & Engineering

In vitro and in vivo study of 3D-printed porous Tantalum scaffolds for repairing bone defects Yu Guo1#, Kai Xie1#, Wenbo Jiang3#, Lei Wang1*, Guoyuan Li2, Shuang Zhao1, Wen Wu1, Yongqiang Hao1,3*

1Shanghai

Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery,

Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 200011, Shanghai, China 2Department

of Orthopaedic Surgery, The First Affiliated Hospital of USTC, Department of

Orthopaedic Surgery, Division of Life Sciences and Medicine, University of Science and Technology of China, 230001, Hefei, China 3Clinical

and Translational Research Center for 3D Printing Technology, Shanghai Ninth

People’s Hospital, Shanghai Jiao Tong University School of Medicine, 200011, Shanghai, China # These authors contributed to this work equally. * To whom correspondence should be addressed: E-mail: [email protected] (YQ Hao); [email protected] (L Wang)

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Abstract Porous tantalum (Ta) scaffold is a novel implant material widely used in orthopedics including joint surgery, spinal surgery, bone tumor surgery, and trauma surgery. However, porous Ta scaffolds manufactured using the traditional method have many disadvantages. In this study, we used selective laser melting (SLM) technology to manufacture porous Ta scaffolds, and the pore size was controlled to 400 μm. The compressive strength and elastic modulus of the porous scaffolds were evaluated in vitro. To evaluate the osteogenesis and osseointegration of Ta scaffolds manufactured by SLM technology, cytocompatibility in vitro and osseointegration ability in vivo were evaluated. This porous Ta scaffold group showed superior cell adhesion and proliferation results of human bone mesenchymal stem cells (hBMSCs) compared with the control porous Ti6Al4V group. Moreover, the alkaline phosphatase (ALP) activity at day 7 and the semi-quantitative analysis of Alizarin red staining at day 21 demonstrated that osteogenic differentiation of hBMSCs was enhanced in the Ta group. The porous Ta scaffold was implanted into a cylindrical bone defect with a height and diameter of 1 and 0.5 cm, respectively, in the lateral femoral condyle of New Zealand rabbits. Radiographic analysis showed that the new bone formation in Ta scaffolds was higher than that in Ti6Al4V scaffolds. Histological images indicated that compared with porous Ti6Al4V scaffolds, Ta scaffolds increased bone ingrowth and osseointegration. The porous Ta scaffold manufactured by SLM not only has a regular pore shape and connectivity, but also has controllable elastic modulus and compressive strength. Moreover, the osteogenesis and osseointegration results in vitro and in vivo were improved compared to those of the porous Ti6Al4V scaffold manufactured using the same technology. These findings demonstrate that

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the porous Ta scaffold manufactured by SLM is potentially useful for orthopedic clinical application.

Keywords: porous Ta, selective laser melting, orthopedic implant, osteogenesis, osseointegration

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1 Introduction In orthopedic clinical activities, autologous or allogeneic bone grafting techniques are widely used in the treatment of bone defects.1 However, these technologies have their inevitable shortcomings, such as donor shortages, potential disease transmission, and rejection reactions.2 Therefore, artificial bone materials have attracted great attention. Among these artificial bone materials, the titanium (Ti6Al4V) alloy is widely used because of its good biocompatibility, strong corrosion resistance, and suitable mechanical strength. However, the Ti6Al4V alloy has many disadvantages, such as lack of bioactivity and mismatched mechanical properties, which limit its application. At present, many studies have made the Ti6Al4V material biologically active by surface modification.3-4 Tantalum (Ta) is a bioactive metal with good biocompatibility and corrosion resistance.

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However, due to the large modulus of

elasticity and weight, the clinical application of Ta has been greatly limited. Therefore, many studies have tried to find various methods to make Ta metal materials with suitable mechanical properties. For instance, many studies have used Ta in Ti-Ta-Nb-Zr alloys to obtain Ta metals with a low modulus of elasticity and weight, 8-9 and have generated a porous structure to reduce its weight and modulus of elasticity.10-13 Previous studies have shown that designing implants with a porous structure allows them to obtain the appropriate mechanical properties and biological activities14. Nevertheless, porous Ta manufactured by the traditional method has many shortcomings, such as irregular pore size and undesirable mechanical properties, which limit its use as a bone substitute. Bone tissue regeneration is a complex process involving multiple factors. This process is related to the biological activity, morphology, and surface area of the implants.15 Compared

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with dense material, porous metal material has the advantages of a lower elastic modulus, larger surface area, and higher pore connectivity; thus, they are suitable for bone ingrowth and osseointegration. Moreover, the porous structure facilitates the growth of surrounding tissue into the material, while nutrients can also enter the interior of the scaffolds, thereby promoting osseointegration and bone repair.11 Scaffolds can be manufactured by advanced additive manufacturing technology such as selective laser melting (SLM) technology.16-18 The currently most widely used metal implants in orthopedic surgery are made of Ti6Al4V. Nevertheless, Ti6Al4V implants may fail because of insufficient integration into surrounding bone. As a novel implant, porous Ta has good biocompatibility, excellent biological activity, good corrosion resistance, and suitable biomechanical properties.19-21 Currently, Ta-related products have been used in clinical practice.22 The implants used clinically need to have good biocompatibility and mechanical strength, as well as biomechanical properties that match the human bones, such as the elastic modulus. Mismatching the elastic modulus can seriously affect the regeneration of bone tissue.23 However, human bones are subject to various factors such as age, nutritional status, and hormones, which makes it very difficult to create an implant that matches the bone, especially when cavitary bone defects are present. There are two types of bones in the human body: cancellous bone and cortical bone. Their modulus of elasticity ranges from 0.5 to 20 GPa. This is far from the elastic modulus of the most widely used Ti6Al4V alloy (about 114 GPa).24 This mismatch in the elastic modulus is considered to be the main cause of stress shielding,25-26 which can affect bone tissue regeneration and even lead to secondary fractures. The porous structures exhibit their unique advantages here. First, the porous structure can effectively

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reduce the elastic modulus of the inner plant and prevent the occurrence of stress occlusion effects.27 Furthermore, the porous structure allows the surrounding bone tissue and vascular tissue to grow into the scaffold, facilitating early stabilization, which is beneficial in the repair of bone tissue and conducive to the early rehabilitation. It has been reported that porous Ta has good biocompatibility and suitable mechanical properties making it suitable for clinical applications.28 In summary, a method for producing porous Ta scaffolds with suitable mechanical properties with controlled pore size and porosity is required. High-power lasers in Laser Engineered Net Shaping is one of the traditional fabrication procedures29-30; however, the implants manufactured by this method cannot achieve matching of the elastic modulus, and the pore size and porosity is not controlled. SLM technology allows for the generation of scaffolds with suitable porosity, pore size, and mechanical properties.31-36 With the advent of 3D printing technology, we can design implants of various shapes through computer software, and then manufacture them by electron beam or laser-forming technology.31 In this study, SLM technology was first employed to manufacture porous Ta scaffolds. We then evaluated the interaction between human mesenchymal stem cells (hBMSCs) and the porous Ta scaffolds in comparison to control Ti6Al4V scaffolds. The osseointegration and osteogenesis properties of these scaffolds were also assessed.

2 Experimental Section 2.1 Materials and methods 2.1.1 Preparation of porous scaffolds

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Porous Ti6Al4V and Ta scaffolds were manufactured using an SLM system (Arcam A1, Arcam AB, Mölndal, Sweden) through 3D printing technology (porosity, 73.8±1.6% and 68.3±1.1%; pore size, 316±71 μm and 334±86 μm). Briefly, the 3D model was generated and transmitted to the 3D printer with computed aided design (CAD) software in the form of an STL format file. Medical-grade Ta powder was manufactured by hydrogenation treatment, crushing ball milling treatment, screening treatment, shaping treatment, and dehydrogenation treatment. The particle size of the powder ranged from 200 µm to 400 µm. The laser melts the metal powder and then the metal is coagulated. A new layer of powder then spreads to the next layer, and so on until the whole structure formed. The entire manufacturing process is in a vacuum environment (∼10−4 to 10−5 mbar). The porous scaffolds used for in vivo (Ø5 mm × L10 mm) and in vitro (Ø10 mm × H2 mm) experiments underwent ultrasonic washing to remove metal powder after printing was completed. 2.1.2 Mechanical evaluations Samples were prepared according to national standards, diameter of 10 mm and height of 20 mm, were used to evaluate the compressive strength and elastic modulus of porous Ti6Al4V and Ta scaffolds (featuring the pore sizes and porosity mentioned in the preceding paragraph). The mechanical strength test used the testing machine (Instron-5569; Instron, USA). The elastic modulus of a porous material is determined by a stress-strain curve. Three samples were selected for each group for mechanical testing (Table 1). 2.1.3 Sample characterization The microstructure of the scaffold surface was observed by scanning electron microscopy (SEM, S-4800; Hitachi, Japan). Elements energy dispersive spectroscopic (EDS) detection was

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conducted to detect the elements contained in the scaffolds. Prior to the SEM scan, all samples were coated with platinum (Pt) to increase conductivity. 2.1.4 Cell culture hBMSCs were isolated using the method previously reported. All experiments were strictly in accordance with the policies of the Ninth People’s Hospital of Shanghai Jiao Tong University. The donor (male, 46 y) was healthy without any disease that may affect the results of this study. Before bone marrow donation, we obtained written informed consent using guidelines approved by the Ethical Committee on the Use of Human Subjects at the Shanghai Jiao Tong University. The hBMSCs were cultured in α-MEM (Hyclone, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Tauranga, New Zealand) at 37 °C in a moist atmosphere with 5% CO2. The α-MEM medium was changed every 2 days. After 96 hours of culture, the unattached cells were cleared, and the dishes were washed twice with phosphate buffer solution (PBS). The 2-4th generation stem cells were trypsinized and used in subsequent experiments. 2.1.5 Cell proliferation and morphology The sample used in the in vitro experiment was a cylinder; 2 mm in height and 10 mm in diameter. hBMSCs were seeded at 1×104 cells/ml on the surface of scaffolds placed in 24-well plates. The cells were incubated in α-MEM with 10% FBS at 37 °C in 5% CO2. Cell proliferation was detected 1, 3, 5, and 7 days after cell-to-material co-culture. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technology, Japan). The reagent was added to the well at a ratio of 1:10 and then incubated for 2 hours. The optical

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density (OD) was tested using an ELX800 absorbance microplate reader (Bio-Tek, USA) at 405 nm. hBMSCs (1×104/ml) were seeded on porous Ti6Al4V scaffolds and Ta scaffolds and incubated for 7 days, α-MEM medium, which was changed every 2 days. After removing the α-MEM medium, each sample was washed with PBS three times, fixed in 2.5% glutaraldehyde solution overnight at 4 °C, and then dehydrated in a gradient alcohol solution (50%, 60%, 70%, 80%, 90%, and 100%) for 15 min. After dehydration, each sample was dried at 37 °C. The cell morphology was visualized using a scanning electron microscope (SEM, S-4800; Hitachi, Japan). Each sample needs to be coated with a layer of Pt on the surface to increase conductivity. hBMSCs (1×104/ml) were seeded on porous Ti6Al4V scaffolds and Ta scaffolds and cultured as described above. After removing the α-MEM medium on day 7, each sample was washed with PBS three times and then fixed in 4% paraformaldehyde solution for 30 min at room temperature. Thereafter, each sample was stained with phalloidin (Cytoskeleton, PHDG1-A) for 30 min at room temperature for cytoskeleton staining, and nuclei were counterstained with 4′,6 diamidino-2-phenylindole (Sigma, D9542-10MG, DAPI) for 15 min. Images were taked by an Olympus confocal laser scanning microscope. The cell overall area to the nucleus area (CN ratio) was measured using ImageJ software. Each experiment was repeated three times, and three samples were used in each experiment per group. 2.1.6 In vitro osteogenic differentiation assays hBMSCs (1×104/ml) were seeded on porous Ti6Al4V scaffolds and Ta scaffolds and cultured as described above. The ALP activity was detected after the stem cells were co-

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cultured with the scaffold for 7 days using the ALP Activity Assay Kit (P0321; Beyotime, China), following the protocol. First, the medium was carefully removed, and the dishes were washed three times with PBS. Next, the scaffold material was transferred to a new 24-well cell culture plate and the 0.2% Triton X-100 solution was added. Finally, ALP activity was detected at a wavelength of 405 nm. After 21 days of incubation, the scaffold was transferred to a new 24-well cell culture plate, washed three times with PBS, and then fixed with 4% paraformaldehyde for 30 min at room temperature. After the alizarin red staining, scaffolds were washed with PBS solution until the PBS solution was clarified. Thereafter, the mineralized nodules that had been stained were dissolved with 10% cetylpyridinium chloride (C9002-25G; Sigma, USA) for semiquantitative detection by determining absorbance at 562 nm. All experiments were repeated three times, with three samples per group in each experiment. 2.1.7 Quantitative real-time PCR (RT-PCR) hBMSCs were seeded on the samples at a density of 5×104/well in 24-well culture plates. After the cells were completely adhered, the α-MEM complete medium was replaced with the osteogenic induction solution (ascorbic acid, dexamethasone, and β glycerol phosphate added to complete medium). The medium was changed every 2 days. Osteogenesis-related genes were detected after 7 and 14 days of co-culture with the scaffold. The expression of osteopontin (OPN), osteocalcin (OCN), ALP, collagen type-1 (Col-1), and runt-related transcription factor2 (Runx2) was examined to detect the osteogenic differentiation of stem cells. Total RNA of stem cells co-cultured with the scaffold was extracted using the TRIzol method at each observation point and then reverse transcription into cDNA was performed by using

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PrimeScript RT Master Mix (Takara). RT-PCR reactions were performed using SYBR Premix Ex Taq II (Takara) on a CFX96 PCR System (Bio-Rad). The housekeeping gene GAPDH was used as a control. The primers used are provided in Table 2. 2.1.8 Animals and surgical procedures For in vivo experiments, we used samples with a diameter of 5 mm and a height of 10 mm. The experiment was carried out in accordance with the Chinese animal experimentation laws and was approved by the Ethical Committee of Shanghai Jiao Tong University (Reference number: SYXK (Hu) 2012-0007 and SCXK (Hu) 2009-0018). Thirty New Zealand rabbits (aged 18 weeks) with an average weight of 2.5−3.5 kg were included. Two different scaffolds were implanted into the right hind legs of each rabbit. The rabbits were anesthetized with phenobarbital sodium via intravenous injection. The surgical areas were shaved and sterilized. We made an incision about 3 cm long to expose the condyles of the femur. A cylindrical defect with a diameter of 5 mm and depth of 10 mm was drilled in the right hind leg femur of the rabbit, and different scaffolds were inserted into the defects. The incision was sutured layer by layer and disinfected again. Each rabbit was intravenously injected with 40000 U penicillin for 3 days to prevent infection. Each rabbit was also subcutaneously injected with Alizarin red and calcein 7 days and 3 days before sacrifice. 2.1.9 Medical imageology evaluation At 4, 8, and 12 weeks after surgery, the rabbits were sacrificed via intravenous injection of air under anesthesia. The rabbit femoral condyle specimen was taken out and immediately fixed with 4% paraformaldehyde solution for 3 days. Thereafter, the samples were washed overnight in water. The samples were scanned with Micron X-ray 3D Imaging System (Y. Cheetah,

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Germany) in order to detect bone regeneration. The scanning resolution was approximately 17 μm. 2.1.10 Histological analysis After micro-CT analysis, the samples were embedded in methyl methacrylate (MMA). The embedded specimens were made into hard tissue sections using the cutting-grinding system (Buehler 11-1280-250, USA) system. Each specimen was selected from three regions and sectioned (distal, middle, and proximal, respectively) so that we could observe the growth and integration of bone tissue in different parts of the scaffolds. The tissue sections were stained with Stevenel’s blue and van Geison’s picro-fuchsin. The images were captured by a Nikon SMZ 1500 stereoscopic zoom microscope (Nikon Instruments Inc., Melville, NY). Specimens embedded in MMA were also visualized with SEM (S4800; Hitachi, Japan; imaging mode of backscattered electron; accelerating voltage of 10.0 kV; working distance of 13.5 mm). 2.1.11. Statistical analysis Data are presented as mean ± standard deviation. Data were analyzed using analysis of variance and then pairwise multiple comparisons were performed using Fisher's least significant difference method. Statistical analysis was performed using SPSS 19.0. P < 0.05 considered the difference to be significant.

2.2 Results 2.2.1 Implant characterization The surface characteristics of Ti6Al4V and Ta scaffolds detected using SEM were presented with a 3D structure (Figure 1). The images showed homogeneous porous structures

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and pore size (Table 1). 2.2.2 Mechanical evaluations The compressive strength was 78.54 MPa in the Ta group and 71.04 MPa in the Ti6Al4V group. The average elastic modulus was 2.34 GPa in the Ta group and 2.27 GPa in the Ti6Al4V group. The specific data are provided in Table 1. From the test results of the mechanical properties, the elastic modulus of the two stents manufactured by 3D printing technology is smaller than that of human cortical bone, which can effectively avoid the stress shielding effect. 2.2.3 In vitro cell proliferation and morphology Figure 2a shows the SEM results of stem cell adhesion on the scaffolds of porous Ta and Ti6Al4V groups after 7 days of culture. The cells were completely spread out with cellular pseudopod extensions in the Ta group. The cell numbers in the Ta group were higher than those in the Ti6Al4V group. The results of the laser confocal microscope were also consistent with the SEM results. Cells on Ta group were spread better than those on Ti6Al4V group (Figure 2b), and the overall cell area to the nucleus area (CN ratio) was higher in the Ta scaffolds than in the Ti6Al4V scaffolds (Figure 2c). The cell proliferation experiment results show an increase in proliferation with increasing incubation time in both the porous Ta and Ti6Al4V groups (Figure 2d). The scaffolds had very good biocompatibility, although there was no significant difference between the Ta and the control groups on days 1 and 3. However, proliferation in the Ta scaffold group was significantly higher than that in the Ti6Al4V scaffold group at days 5 and 7 (Figure 2e). 2.2.4 Osteogenic differentiation evaluations

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ALP activity was tested at day 7 in two groups, which was higher in the Ta group than in the Ti6Al4V group (Figure 3a). Alizarin red staining showed more mineralized nodules on Ta scaffolds (Figure 3b). On the 21st day, alizarin red staining and semi-quantitative analysis showed that calcium nodules were formed on the scaffolds of each group, but the calcium nodules were significantly more on the Ta group than on the Ti6Al4V alloy group (Figure 3c). 2.2.5 Quantitative RT-PCR analysis of gene expression Osteogenic differentiation was evaluated on days 7 and 14 by quantitative RT-PCR analysis of the expression of the osteogenic markers Runx2, ALP, OCN, OPN, and Col-1. At both time points, Runx2, ALP, and Col-1 gene expression levels were higher in the Ta group than in the Ti6Al4V group (Figure 4a, b, e). In contrast, the levels of OCN and OPN gene expression did not differ between the groups on day 7 but were significantly higher in the Ta group on day 14 (Figure 4c, d). These results showed that the new porous Ta scaffolds promoted osteogenic differentiation of hBMSCs. 2.2.6 X-rays, micro-CT, and histological evaluation All experimental animals recovered well after surgery, and no loosening or dislocation of the implants was found by X-ray examination. Rejection reaction was also not observed in the animals. Porous implants were stable and integrated with the host bone at each of the time points. X-ray results indicated that new bone was well integrated with the scaffolds and that there was no postoperative dislocation of the scaffolds (Figure 5a). At 4 weeks, the new bone was mainly confined around the scaffolds; at 8 weeks, in addition to the new bone around the stent, new bone tissue also appeared inside the scaffolds; at 12 weeks, the bone tissue and the stent

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formed a good integration. Micro-CT analysis was also carried out to characterize the bone regeneration in scaffolds (Figure 5b). Histological analysis was performed to evaluate the osteogenesis and osseointegration of Ti6Al4V and Ta scaffolds. At 4 weeks, both groups had new bone formation, and there was a significant gap between the new bone and the scaffolds, indicating that the effect of osseointegration was not satisfactory. At the 8th and 12th weeks, the amount of new bone increased significantly, and the gap between the bone tissue and the scaffolds was significantly reduced (Figure 6a). The new bone area and thickness of the Ta group was larger than that of the Ti6Al4V group at 12 weeks. In the Ti6Al4V group, the gaps between the new bone tissue and the scaffolds seen at week 4 was still observed by week 12. Therefore, osseointegration in the Ta group was significantly better than that in the control group. Furthermore, after 4 weeks, many bone lining cells were observed around the scaffolds in the Ta group, indicating that there was already active bone regeneration at this point. The volume of regenerated bone increased with increasing operation time (Figure 6b). Notably, the quantitative histomorphology results showed that new bone mass in the Ta scaffold group was higher than that of the control group at each time point. SEM examination of bone regeneration and osseointegration at different positions at 4, 8, and 12 weeks also indicated that bone regeneration was superior in the Ta group compared to that in the Ti6Al4V group (Figure 6c). 2.2.7 Analysis of bone formation and resorption. New bone and bone reconstruction in the tissue was observed with fluorescence calcein/alizarin red double labeling (Figure 7a). The ratio of calcein fluorescence to alizarin 15



red fluorescence represents bone regeneration and reconstruction; during the stage of callus formation, a higher ratio indicates a higher rate of bone formation. In this study, the calceinto-alizarin-red area ratio was elevated (Figure 7b), which suggested accelerated bone formation in both groups; however, the stimulatory effect was clearly stronger in the Ta group, which indicates that the new porous scaffolds more effectively induced new bone formation. At 12 weeks, the ratios were still elevated in all cases, which implied that the bone-remodeling process was ongoing; the ratio remained higher with the Ta scaffolds than with the Ti6Al4V scaffolds.

2.3 Discussion The orthopedic implants used clinically are required provide a bridge for bone-defect or -fracture repair, which can be in the form of an oral implant37 or maxillofacial treatment,38-39 or can be used to restore lost function in orthopedic patients.40 These implants must possess the appropriate mechanical strength and biocompatibility, as well as having the ability to concurrently promote, or at least not impede, bone regeneration and osseointegration. Our experimental results demonstrated that porous Ta scaffolds fabricated using SLM have suitable mechanical properties, biocompatibility, and biological activity. This indicates that porous Ta scaffolds can be tailored to exhibit the enhanced biocompatibility and mechanical-adapted properties necessary for clinical application. Previous studies have shown that Ta metal is a suitable bone substitute for clinical applications.41 However, the excessively high modulus of elasticity and weight of the Ta metal itself hinders its widespread clinical application. Therefore, manufacturing Ta metals into

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implants that meet clinical application standards is a problem yet to be solved. In our study, we use 3D printing technology to produce Ta metal scaffolds with a controlled pore size, suitable porosity, proper mechanical properties, and free geometry. Moreover, our results show that the porous Ta scaffolds not only retain the original biological activity of the Ta metal, but also have biomechanical adaptability, making it more suitable for bone defect repair. Due to the good biocompatibility and the osseointegration and osteogenesis properties, Ta is a promising metal for use as a substitute for bone defect repair. Our previous study showed that the porous structure can reduce the weight and modulus of elasticity of the material.42 At the same time, the surrounding tissue and nutrients can enter the space inside the scaffold, which is beneficial to promote bone tissue regeneration. In a previous study, we have shown that the porous Ti6Al4V scaffold with a porosity of approximately 400 μm had the best result in terms of both biocompatibility and osseointegration.42 Ti6Al4V alloys and Ta scaffolds manufactured by SLM technology have a suitable elastic modulus that can match that of human bones. The elastic modulus of human bones have differing results depending on the test method.43 The elastic modulus of human cortical bone can reach 20 GPa, while the elastic modulus of cancellous bone is only about 3 GPa.24,44 The elastic modulus of the two porous samples that we designed was 2.34 GPa in the Ta group and 2.27 GPa in the Ti6Al4V group, which was close to the values for human cancellous bone. In order to avoid the stress shielding effect, the elastic modulus can be used as an important indicator for the manufacture of implants. The elastic modulus of the Ti6Al4V scaffolds produced in this experiment was found to be consistent with that reported in previous studies.45 It has been reported that the elastic modulus of the femur of the rabbit's hind leg is about 18.96

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GPa, which is close to the elastic modulus of human bone. Therefore, this study used a rabbit femoral condyle defect model.45 The experimental results of this study also indicate that the use of this animal model as a model for simulating human bone defects is reasonable. The ALP activity in the Ta group was significantly higher than that in the control group, which can be interpreted in many ways. First, the porous Ta scaffold induced hBMSC osteogenic differentiation. Second, the cell morphology also changes due to the presence of a porous stereoscopic spatial structure, as evidenced by SEM results and confocal staining results. The ability of the cells to differentiate into osteoblasts may be enhanced by these morphological changes.46 The stem cells touch at multiple points, which may also promote osteogenic differentiation. The semi-quantitative results of alizarin red staining performed on the 21st day also indicated that the porous Ta group resulted in increased osteogenic differentiation in hBMSCs than the porous Ti6Al4V group. However, an accurate explanation of this result requires further study. To examine the mechanism by which the porous Ta scaffolds promote the osteogenic differentiation of cells, we examined the expression of osteogenic related genes (Runx2, ALP, OCN, OPN, and Col-1) in both groups using quantitative RT-PCR. Runx2, ALP, and Col-1 were significantly higher in the Ta group compared to the control group. OCN and OPN did not differ significantly between the two groups on day 7; however, on day 14, OCN and OPN expression was significantly higher in the experimental group compared to the control group. This upregulation of these genes indicates that the porous Ta scaffolds promote the osteogenic differentiation of hBMSCs. Furthermore, this study showed that the surface properties of Ta scaffolds, such as topological structure and roughness, may play an important role in cellular

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osteogenic differentiation; however, this result needs further study. This study demonstrates that Ta scaffolds have a superior ability to promote bone regeneration and osseointegration compared to porous Ti6Al4V scaffolds in vivo. This result is mainly due to the advanced additive manufacturing technology that gives the Ta scaffolds good pore connectivity, regular pore size, and proper mechanical properties. Of course, the biological activity of Ta metal itself should also be taken into consideration. The dynamic process of bone tissue regeneration on the stent surface and inside the stent was observed in in vivo experiments at 4, 8, and 12 weeks. Initially, a poorly integrated fibrous callus appeared on the surface of the material; in the mid-term, a partially integrated cartilage callus appeared; finally, bone tissue formed inside the stent. In this model, the whole scaffold was surrounded by bone, which may explain why both the ends and the middle had similar bone ingrowth. In vivo experiments demonstrated that the 3D printed porous Ta scaffolds significantly promoted bone tissue regeneration and osseointegration compared to Ti6A14V scaffolds. Our results demonstrate that the SLM-assisted porous Ta scaffold is a novel material with the potential to replace porous Ti6Al4V scaffolds in clinical applications. However, the manufacturing method of the porous Ta scaffolds is complicated, and the manufacturing cost is high. Future studies should aim to improve the manufacturing process of porous Ta. In addition, the biological functions of porous Ta scaffolds need to be further investigated and the mechanism should be explored in more depth. 3 Conclusions Porous Ta scaffolds were successfully fabricated using SLM technology. We evaluated the mechanical properties, biocompatibility, and the osseointegration and osteogenesis

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properties of porous Ta scaffolds. In vitro studies indicated that the SLM-Ta scaffolds promoted stem cell proliferation, adhesion, and osteogenic differentiation compared with porous Ti6Al4V scaffolds. In addition, micro-CT showed that the integration of new bone tissue into the scaffold increased over time. Histological analysis revealed that the amount of new bone mass and osseointegration in the Ta scaffold group was higher than that in the control group. Furthermore, improved osseointegration was observed around the Ta scaffolds in vivo. Therefore, porous Ta has high potential for orthopedic clinical application as a novel bone substitute.

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Author Information Corresponding Author: Yongqiang Hao *E-mail: [email protected] Lei Wang *E-mail: [email protected]

Author Contributions Yu Guo, Kai Xie, and Wenbo Jiang carried out the experiments. Guoyuan Li, Shuang Zhao, and Wen Wu conducted the analysis and interpreted the data. Yongqiang Hao and Lei Wang revised the manuscript for intellectual content. All authors read and approved the final manuscript.

Notes The authors declare no competing financial interest.

Acknowledgements This work was jointly supported by the National Key Research and Development Program of China, no. 2016YFC1100600 (subproject 2016YFC1100604), the Technology Commission of Shanghai (12DZ1940203), the Technology Support Project of the Science and Technology Commission of Shanghai (15411951200), the New Cutting-Edge Technology Project of ShenKang Hospital Development Center of Shanghai (16CR3025A), and Doctoral

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Innovation Fund Projects from Shanghai Jiao Tong University School of Medicine (DLY201506).

Supporting Information Available: Additional computer images for illustrating materials designed for in vivo and in vitro experiments. The images of porous scaffolds used for in vivo experiments. SEM of the adhesion experiment of Ti6Al4V disc and Tantalum disc with same roughness.

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Figure Legends Figure 1. (a) Porous Ta and porous Ti6Al4V scaffolds used for in vitro experiments (diameter × height: 10 × 2 mm). (b) SEM micrographs of blank samples of the scaffolds. (c) EDS analysis of the porous Ta and porous Ti6Al4V scaffolds.

Figure 2. (a) SEM micrographs showing hBMSCs adhered on porous Ta and Ti6Al4V scaffolds. Morphological analysis (b) and DAPI and phalloidin fluorescence labeling (c) of hBMSCs adhered on porous scaffolds. We measured three different fields per sample and three separate samples for each group (*P < 0.05, versus Ti6Al4V group). (d) Cell adhesion on the tested scaffolds. High OD values represent high cell numbers, high cell viability, and high cell adhesion. (e) Cell growth on porous Ta and Ti6Al4V scaffolds. A higher OD value indicates that a higher number of cells grew or remained “alive” in the Ta group (*P < 0.05, versus Ti6Al4V group).

Figure 3. (a) ALP activity on day 7 and (c) semiquantitative analysis of calcium nodules on day 21 of culture on porous scaffolds. A high OD value indicates increased formation of calcium nodules on Ta scaffolds (*P < 0.05, versus Ti6Al4V group). (b) Alizarin red staining of scaffolds.

Figure 4. Expression of osteogenic related genes: Runx2 (a), ALP (b), OCN (c), OPN (d), and Col-1 (e); n = 3/group; *P < 0.05, versus Ti6Al4V group.

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Figure 5. (a) Radiographic follow-up images (anteroposterior-lateral). (b) Micro-CT images of femur specimens.

Figure 6. Hard tissue section stained by van Gieson staining (a) and histomorphometric analysis of Ti6Al4V and Ta scaffolds (b) at 4, 8, and 12 weeks after surgery. The red-stained tissue represents bone tissue; at 4 weeks, the amount of new bone tissue in the scaffolds is thin and irregular. Osteoblasts seam with bone-lining cells, indicating active bone formation. *P < 0.05, versus Ti6Al4V group. (c) SEM micrographs of bone apposition and bone microstructure on porous scaffolds at different positions at 4, 6, and 12 weeks. White: implant; grey: new bone.

Figure 7. (a) Bone-remodeling assessment through calcein/Alizarin red double-labeling in rabbit femoral condyle at 4, 8, and 12 weeks after implantation with Ti6Al4V or Ta scaffolds. (b) Semi quantitative analysis of bone-remodeling. *P < 0.05.

Table 1. Mechanical strength, porosity, and pore size of porous Ti6Al4V and Ta scaffolds

Table 2. Primers used for quantitative RT-PCR analysis

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For Table of Contents Use Only In vitro and in vivo study of 3D-printed porous Tantalum scaffolds for repairing bone defects Yu Guo1#, Kai Xie1#, Wenbo Jiang3#, Lei Wang1* Guoyuan Li2, Shuang Zhao1, Wen Wu1, ,Yongqiang Hao1,3*

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In vitro and in vivo study of 3D-printed porous Tantalum scaffolds for

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