The comparison of properties of Ti-6Aluminum-4Vanadium porous scaffolds fabricated through low-power selective laser Melting and electron beam melting

Jun Hu; Yiwei Wang; Minjie Fan; Qingqiang Yao; Pengfei Zheng

doi:10.4103/digm.digm_46_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 8 | Issue : 1 | Page : 12

The comparison of properties of Ti-6Aluminum-4Vanadium porous scaffolds fabricated through low-power selective laser Melting and electron beam melting

Jun Hu¹, Yiwei Wang², Minjie Fan², Qingqiang Yao³, Pengfei Zheng⁴
¹ Department of Trauma Center, Northern Jiangsu People's Hospital, Yangzhou, Jiangsu, China
² Department of Orthopaedic Surgery, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu, China
³ Department of Orthopaedic Surgery, Nanjing First Hospital; Key Lab of Biomaterial and Additive Manufacturing Research, Institute of Digital Medicine, Nanjing Medical University, Nanjing, Jiangsu, China
⁴ Department of Orthopaedic Surgery, Children's Hospital of Nanjing Medical University; Department of Orthopaedic Surgery, Nanjing First Hospital; Key Lab of Biomaterial and Additive Manufacturing Research, Institute of Digital Medicine, Nanjing Medical University, Nanjing, Jiangsu, China

Date of Submission	01-Dec-2021
Date of Decision	03-Feb-2022
Date of Acceptance	25-Feb-2022
Date of Web Publication	07-Jun-2022

Correspondence Address:
Pengfei Zheng
Department of Orthopaedic surgery, Children's Hospital of Nanjing Medical University, 8^th South Jiangdong Road, Jianye Distrcit, Nanjing 210008
China
Qingqiang Yao
Department of Orthopaedic Surgery, Nanjing First Hospital, 68^th Changle Road, Qinhuai District, Nanjing 210006
China

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/digm.digm_46_21

Abstract

Background and Purpose: Three-dimensional printing (3DP) selective laser melting (SLM) and electron beam melting (EBM) technique can construct porous Ti-6Aluminum-4Vanadium (Ti-6Al-4V) scaffolds with special microstructural and biomechanical properties. However, it is still needed to be tested for bone tissue engineering. Materials and Methods: To investigate the microstructure and surface modification of a porous titanium scaffold, 3DP-SLM technique was used, and the mechanical and biological performance of the scaffolds was compared with that fabricated by EBM technique. Ti-6Al-4V scaffolds were computer-designed and fabricated using low-power SLM (L-SLM). The microstructure morphologies of L-SLM Ti-6Al-4V (L-SLM-Ti) scaffolds were determined and compared with EBM-fabricated Ti-6Al-4V (EBM-Ti) scaffolds. Each scaffold was immersed with marrow clot for 1 h until fully combined with bone mesenchymal stem cells in clots. The biomechanical and cellular response of these two kinds of Ti-6Al-4V scaffolds were compared. Results: The L-SLM-Ti scaffolds showed a microstructure closer to the designed parameters than that of the EBM-Ti scaffolds. The L-SLM-Ti scaffold fibers had a rougher surface than the EBM-Ti scaffolds. Meanwhile, L-SLM-Ti scaffolds had a lower elasticity modulus and lower bearing force than EBM-Ti scaffold. Cell proliferation and the relative expression levels of OPN, COL1, and RUNX2 in L-SLM-Ti scaffolds was apparently higher than in the EBM-Ti scaffolds, with no significant difference found between the percentage of live cells found in L-SLM-Ti and EBM-Ti scaffolds. Conclusion: 3DP-Ti-6Al-4V scaffolds fabricated by L-SLM and designed with rougher surfaces and larger pore sizes may have more reasonable biomechanical properties and increased biological performance than traditional EBM-Ti scaffolds. These L-SLM-Ti scaffolds might be suitable candidates for bone defect repair.

Keywords: Bone defect, Electron beam melting, Porous scaffold, Selective laser melting, Titanium alloy

How to cite this article:
Hu J, Wang Y, Fan M, Yao Q, Zheng P. The comparison of properties of Ti-6Aluminum-4Vanadium porous scaffolds fabricated through low-power selective laser Melting and electron beam melting. Digit Med 2022;8:12

How to cite this URL:
Hu J, Wang Y, Fan M, Yao Q, Zheng P. The comparison of properties of Ti-6Aluminum-4Vanadium porous scaffolds fabricated through low-power selective laser Melting and electron beam melting. Digit Med [serial online] 2022 [cited 2023 Jun 9];8:12. Available from: http://www.digitmedicine.com/text.asp?2022/8/1/12/346893

Introduction

Bone defects are among the most common diseases in orthopedic surgery and can lead to pain, deformity, and dysfunction.^[1] At present, severe bone defects that exceed the ability of bone self-regeneration need bone transplantation.^[2] However, bone transplantation can have several side effects, such as additional invasive surgical procedures, infection, disease transmission, or rejection reaction.^[3]

With the development of tissue engineering, bone tissue engineering (BTE) has become a potential technology for repairing bone defects by transporting cells or biological factors to the target using bioengineering scaffolds.^[4] Previous studies have shown that ideal bone scaffolds should possess the following characteristics: (i) biocompatibility; (ii) a suitable surface for cell attachment, proliferation, and differentiation; (iii) a highly porous structure with an interconnected pore network for cell ingrowth and transport of nutrients and metabolic waste; and (iv) mechanical properties that meet the requirements of surrounding tissues, to reduce or eliminate stress shielding, and those of anatomic loading, to avoid mechanical failure.^[5] Although various types of scaffold, such as biopolymers, bioglass, and bioceramics, have been processed and applied to the manufacture of BTE scaffolds, there are still no suitable scaffolds available for bone engineering.^{[4],[6],[7],[8]}

Ti-6Aluminum-4Vanadium (Ti-6Al-4V) is a kind of bone defect filling material which has been widely used in clinic.^[9],[10] Three-dimensional (3D) printing additive manufacturing (AM) has emerged as a promising fabrication technology for metal parts. Previous studies have shown that 3D printed (3DP) porous Ti-6Al-4V scaffolds have the advantages of excellent connectivity, high surface area, and appropriate mechanical strength and elastic modulus, making them promising for repairing bone defects.^[11] 3DP porous Ti-6Al-4V scaffolds with fully interconnected pores, adjustable pore sizes, and appropriate mechanical properties can now be fabricated using several different AM techniques, with selective laser melting (SLM)^[9],[12] and electron beam melting (EBM) the most commonly used.^[10],[13]

Due to the power of sintering, speed of scanning, and speed of scaffold molding of the fabrication techniques, SLM and EBM systems can construct porous Ti-6Al-4V scaffolds with diverse microstructural features. SLM can fabricate Ti-6Al-4V scaffolds with precise size control and smooth surfaces, while EBM can build scaffolds with rougher surfaces. Therefore, EBM Ti-6Al-4V (EBM-Ti) scaffolds may need a larger pore size to guarantee good connectivity properties.^{[14],[15],[16],[17]} As scaffolds with rough surfaces are considered to have more surface for cell attachment and bone in growth, porous Ti-6Al-4V scaffolds constructed using EBM techniques were considered more suitable for BTE. However, unlike SLM Ti-6Al-4V (SLM-Ti) scaffolds, EBM-Ti scaffolds may be stronger than physical bone, which would hinder bone regeneration due to the stress-shielding effect.^[18],[19] As a result, 3DP-Ti-6Al-4V scaffolds with suitable microstructure and biomechanical properties still need to be tested for bone engineering.

In this study, we prepared a porous Ti-6Al-4V scaffold with a relatively larger pore size and rough fiber surface using a low-power SLM (L-SLM) technique. We predicted that the larger pore sizes and slim fibers produced by L-SLM would give porous Ti-6Al-4V scaffolds with biomechanical properties close to those of physical bone, which may reduce the obstruction of bone in growth caused by the stress-shielding effect. Meanwhile, the rougher surface would promote cell adhesion to the surface of the scaffold fibers. As a result, this L-SLM Ti-6Al-4V (L-SLM-Ti) scaffold may have an increased capacity for bone regeneration using the modified microstructure and biomechanical properties, compared with traditional EBM-Ti scaffolds.

Materials and Methods

Sample preparation

Computer aided design

Use the computer-aided design (CAD) software, M3D Medgraphic, to design the porous Ti-6Al-4V scaffolds (Boholo, Shanghai, China). The scaffold is designed to have a large pore size structure: diameter 8 mm, thickness 4 mm, 100% pore interconnection, 64% porosity, 200 μm wire diameter, 0/90° laying pattern, 800 μm pore diameter [Figure 1]a and [Figure 1]b.

Figure 1: Design, construction, macrostructure and chemical element distribution low power-selective laser melting-Ti-6Aluminum-4Vanadium (L-SLM Ti-6Al-4V) scaffold and electron beam melting (EBM)-fabricated Ti-6Al-4V scaffold. (a and b) Structural parameters of scaffolds designed with large pore sizes. (c and d) Macrostructure of scaffolds fabricated via Selective laser melting and electron beam melting. (e and f) Energy dispersive spectroscopy shows different elements distribution between L-SLM Ti scaffold and EBM-fabricated Ti-6Al-4V scaffold.

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Construction of low power-selective laser melting-Ti-6Aluminum-4Vanadium scaffolds

Use a pulsed fiber laser SLM system (Concept Laser M2, Germany) following the equipment specification to print Ti-6Al-4V ELI (Grade 23) powder into the L-SLM-Ti scaffolds. The diameters of Ti-6Al-4V particles used in this study ranged from 5 μm to 46 μm, with a mean diameter of 25 μm and standard deviation (SD) of ±14 μm. A rake system pave Ti-6Al-4V powder to about 50 mm thickness layer by layer on the working table inside an airtight space in which the air pressure is controlled at −2 × 10⁻³ mBar, using high purity helium as a protective gas. A laser beam gun (acceleration voltage, 80 kV) melted the Ti-6Al-4V powder at the set locations, according to the CAD file prepared in advance. The configuration of the laser beam was based on the fringe exposure during the scanning process with a low laser power of 95W and a move velocity of 710 mm/s. After one layer was paved, the working table went down, and the rake system continued to pave a new layer of Ti-6Al-4V powder. Repeat the above process until the Ti-6Al-4V scaffold is fully paved. All of the SLM printers were set to the same. Every finished product was blew and rinsed by high-speed phosphate-buffered saline (PBS) for three times.

Construction of electron beam melting-Ti-6Aluminum-4Vanadium scaffolds

The EBM-Ti scaffolds were printed using an EBM system (Arcam A1, Arcam AB, Mölndal, Sweden) following the manufacturer's protocol. Ti-6Al-4V ELI (Grade 23) powder with a diameter of 25 ± 14 μm (ranging from 5 μm to 46 μm) was used. Stereolithography data converted from the CAD data were then transferred to the EBM machine. Ti-6Al-4V powder was preheated to 65°C. Electron beam scanning was used to generate a cross section layer by fusing the powder together. Subsequently, a homogeneous powder layer was applied on the process platform in a vacuum chamber at constant high temperature (approx. 700°C). The electron beam scanned the powder layer line-by-line and melted the loose powder particles at programmed locations, forming a compact layer with the desired shape. The process platform was then lowered by the thickness of one layer, and a new powder layer was applied, after which the process was repeated. This process was performed under vacuum (~10⁻⁴–10⁻⁵ mBar). All samples were ultrasonically cleaned sequentially in acetone, ethanol, and deionized water for ~15 min each.

Cell seeding

Before the experiment, ethylene oxide was used for scaffold sterilization. The marrow clots (MC)-enriched scaffold was prepared following our previous protocol.^[20] In brief, New Zealand white rabbits (5–6 months old) were used for bone marrow extraction and bone mesenchymal stem cell (BMSC) isolation. Bone marrow was aspirated from the posterior superior iliac crest, and mononuclear cells were isolated by density gradient centrifugation. Next, cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Austria) supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Buffer, and 1% antibiotics (penicillin 8000 U/mL and streptomycin 8000 U/mL) in a 37°C, 5% CO₂ incubator. Osteoblastic, chondrogenic, and adipogenic differentiation were tested to confirm the identity of BMSCs. Each scaffold was immersed and mixed with MC for 1 h until fully combined. These scaffolds were cultured in low-glucose DMEM for 4 days, and change the DMEM every day before the scaffolds were all clear. The scaffolds were then cultured using osteogenesis induction medium (Gibco, Thermo Fisher Scientific Inc., US) containing high-glucose DMEM, 10% FBS, 1% penicillin-streptomycin, 10 mmol/L β-glycerophosphate (β-GP), 50 μM ascorbic acid, and 100nM dexamethasone (Dex) for 28 days in vitro. Scaffolds were culture in vitro and sampled on day 1 and after 1, 2, 4 weeks. At each time point, test five samples for each group (n = 5).

Macrostructure and microstructure morphology observations

Macrostructure morphology

The macrostructure morphologies of the L-SLM-Ti and EBM-Ti scaffolds were observed by pictures.

Microstructure morphology

The microstructure morphologies were observed using scanning electron microscopy (SEM) following our previously reported protocol.^[21] In short, the sample was incubated overnight in 10% formalin and then dehydrated with a series of graded ethanol solutions. Next, the samples were dried at room temperature overnight, plated with gold, and observed using an SEM scanner (JEOL, Tokyo, Japan) at an accelerating voltage of 5 keV. Porosity was calculated using the usual liquid displacement method in ethanol. For the pore morphologies, the changes of pore contents were observed and identified by inverted microscope. The surface roughness of the scaffold was measured using a surface measuring system (SV-3200, Mitutoyo, Japan).^[22] The scaffold was placed on the positioning table with the upper part set in the upward direction. The crank shaft was placed in a downward orientation to measure the roughness of the top surface of the scaffold. The roughness was calculated as the arithmetic mean roughness (Ra), which represented the average absolute distance in the direction of the normal vector to the section curve of the surface based on the center line average within the sampling length.

Mechanical testing

Mechanical testing of specimens was conducted using an Instron 4502 uniaxial testing system (Instron Ltd., High Wycombe, UK). Secure the scaffolds securely with a pliers to ensure that the fixed and test surface is smooth. Next, each tested scaffold (uncultured sample) underwent compression for one time of 1 mm/min until reaching a 2 mm displacement. The compressive strengths of the five samples in each group were recorded at 20% strain (0.8 mm displacement), calculating the mean ± SD.

Measurements of the cellular response

The measurements of the cellular response were performed using a Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA) as previously described.^[20] Briefly, samples were washed with PBS and incubated in dilute dye solution for 15 min at room temperature. These samples were fixed with 4% glutaraldehyde for 1 h and photographed using confocal laser scanning microscopy (CLSM, Eclipse E600W, Nikon, Tokyo, Japan). The percentage of live cells was calculated using Zen 2011 software (Carl Zeiss, Oberkochen, Germany).

Cell proliferation was measured as follows. Scaffolds with BMSCs were seeded into a 24-well plate (Corning, USA) and incubated for 24 h. Old media was then removed and 1 mL of leaching liquor of each group was added into the 24-well plate and incubated for 1, 7, 14, and 28 days. The normal medium was used as a control. At each time point, 1 ml of CCK-8 solution reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well in the 24-well plate after incubating at 37°C for 3 h. The cell viability was measured using an ultraviolet spectrophotometer (GE Healthcare, Stockholm, Sweden) at 490 nm.

Cell differentiations were measured by real-time polymerase chain reaction (PCR). The relative expression levels of OCN, OPN, COL1, RUNX2 were used as the differentiation markers of BMSCs. After BMSCs were stimulated for 7 and 14 days, cells were harvested for RNA isolation. Then total RNA was reversed by reverse transcriptional kit according to the below transcription reaction procedure: 25°C for 10 min; 37°C for 120 min; 85°C for 5 min. Finally, the real-time PCR amplification scheme was carried out according to the manufacturer's instructions of ROCH. The sequences of primers are: OCN-F: 5'-CACTCCTCGCCCTATTGGC-3'; OCN-R: 5'-CCCTCCTGCTTGGACACAAAG-3';

OPN-F: 5'-CTCCATTGACTCGAACGACTC-3';

OPN-R: 5'-CAGGTCTGCGAAACTTCTTAGAT-3';

COL1-F: 5'-GAGGGCCAAGACGAAGACATC-3';

COL1-R: 5'-CAGATCACGTCATCGCACAAC-3';

RUNX2-F: 5'-TGGTTACTGTCATGGCGGGTA-3';

RUNX2-R: 5'-TCTCAGATCGTTGAACCTTGCTA-3'.

Statistical analysis

Results were presented as means ± SD. The difference between the groups was analyzed by one-way Analysis of variance with least significant difference. A P < 0.05 was considered to be significantly different.

Results

Microstructures of low power-selective laser melting Ti-6Aluminum-4Vanadium and electron beam melting-Ti-6Aluminum-4Vanadium scaffolds

[Table 1] shows the differences between the parameters of the obtained scaffold microstructures, as determined by SEM, and the CAD-designed parameters. The L-SLM-Ti scaffold more closely matched the designed values than the EBM-Ti scaffold, demonstrating a smaller pore size difference (33 ± 20 μm vs. 81 ± 37 μm, P < 0.05) and fiber diameter difference (28 ± 10 μm vs. 87 ± 38 μm, P < 0.05).

Table 1: Structural data for three-dimensional printing Ti-6Aluminum.4Vanadium scaffolds by scanning electron microscopy

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Surface roughness of three-dimensional printing scaffolds produced by low power-selective laser melting and electron beam melting processes

[Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d combined with SEM images of L-SLM scaffold we described before shows that fibers of both the L-SLM-Ti and EBM-Ti scaffolds had particle-covered rough surfaces.^[20] There were no metal particle dissociation and pore obstruction observed in the samples. Due to the lower laser power, the L-SLM-Ti scaffold had a larger amount of un-molten and semi-molten powder particles, with metal particle diameters in the micrometer range. The Ra values of the L-SLM-Ti and EBM-Ti scaffolds were 12.6 ± 4.2 μm and 5.7 ± 2.3 μm, respectively. The L-SLM-Ti scaffold had a rougher surface than the EBM-Ti scaffold.

Figure 2: Scanning electron microscopy images of low power-selective laser melting-Ti-6Aluminum-4Vanadium (L-SLM Ti-6Al-4V) scaffold and electron beam melting (EBM)-fabricated Ti-6Al-4V scaffold. (a) ×30 scanning electron microscopy images of the L-SLM Ti scaffold. (b, c and d) ×30, ×300, and ×1000 scanning electron microscopy images of the EBM-fabricated Ti-6Al-4V scaffold. Both scaffolds had a particle-covered rough surface. The L-SLM Ti scaffold had a larger number of unmolten and semi-molten powder particles because of the lower laser power. SLM: Power-selective laser melting, EBM: Electron beam melting. SLM: Power-selective laser melting, EBM: Electron beam melting.

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Biomechanical test

[Figure 3] shows that the average maximum bearing force of the L-SLM-Ti scaffold was 1.89 ± 0.15 kN, with an average elastic modulus of 10.88 ± 0.34 GPa, while the EBM-Ti scaffold had an average maximum bearing force of 3.48 ± 0.21 kN and an average elastic modulus of 12.81 ± 0.39 GPa. The EBM-Ti scaffold needed a larger pressure than the L-SLM-Ti scaffold at each point of displacement [5%, 10%, 15%, and 20%; [Figure 3]b]. Therefore, the scaffold produced by SLM, which had a slimmer fiber and larger pore size, had a lower elasticity modulus (P < 0.05) and lower bearing force (P < 0.05) than that produced by the EBM process.

Figure 3: Biomechanical test of low power-selective laser melting-Ti-6Aluminum-4Vanadium (L-SLM Ti-6Al-4V) scaffold and electron beam melting (EBM)-fabricated Ti-6Al-4V two types scaffold. (a) Maximal bearing force and elastic modulus comparison of L-SLM Ti scaffold and EBM-fabricated Ti-6Al-4V scaffold. (b) EBM-fabricated Ti-6Al-4V scaffold needed a larger pressure than L-SLM Ti scaffold at each point of 5%, 10%, 15%, and 20% displacement. SLM: Power-selective laser melting, EBM: Electron beam melting. *P < 0.05, **P < 0.01, ***P < 0.001.

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Effect of marrow clots enrichment on cell adhesion, proliferation and differentiation

[Figure 4]a, [Figure 4]b, [Figure 4]c shows the viability and proliferation measurement of cells grown on L-SLM-Ti scaffold after 4 weeks of culture in vitro from confocal laser scanning microscopy (CLSM, Eclipse E600W, Nikon, Tokyo, Japan) images of the Live/Dead assay. [Figure 4]d, [Figure 4]e, [Figure 4]f shows the equivalent results for the EBM-Ti scaffold. The assay of both the L-SLM-Ti and EBM-Ti scaffolds showed that high percentage of green-stained live cells and low percentage of red-stained dead cells was observed after 4 weeks of culture in vitro. However, the percentage of live cells was not significantly different between the two scaffolds [P<0.05, [Figure 4]g].

Figure 4: Cellular response measurements of marrow clots-Ti-6Al-4V-6Al-4V scaffold. (a–f) Confocal laser scanning microscopy showing cell growth performance on the low power-selective laser melting-Ti-6Aluminum-4Vanadium (L-SLM Ti-6Al-4V) scaffolds and the electron beam melting (EBM)-fabricated Ti-6Al-4V scaffolds at 4 weeks. The living cell emits a green fluorescent light and the dead cell emits a red fluorescent light. (g) Percentage of live cells between the L-SLM Ti and EBM-fabricated Ti-6Al-4V scaffolds. (h) Cell proliferation evaluated by CCK-8 assay. *Statistically significant differences between groups, SLM: Power-selective laser melting, EBM: Electron beam melting. P < 0.05, **P < 0.01, ***P < 0.001.

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Cell proliferation was evaluated using the CCK-8 assay, which found that both the L-SLM-Ti and EBM-Ti scaffolds were good for cell proliferation with low cytotoxicity. Cell proliferation was significantly larger in the L-SLM-Ti scaffold than in the EBM-Ti scaffold at 7, 14, and 28 days [P < 0.05, [Figure 4]h]. Cell differentiations were evaluated by analyzing the relative expression levels of OCN, OPN, COL1, and RUNX2. We found that both L-SLM-Ti and EBM-Ti scaffolds could stimulate cell differentiation. The relative expression levels of OPN, COL1, and RUNX2 were higher in the L-SLM-Ti scaffold than in the EBM-Ti scaffold at 14 days [P < 0.05, [Figure 5]]. Therefore, these results indicated that the L-SLM-Ti scaffold with a rougher surface was more conducive to cell proliferation.

Figure 5: Cell differentiations measurements of marrow clots-Ti-6Al-4V-6Al-4V scaffold. (a) No significant different expression levels of OCN between L-SLM Ti-6Al-4V and electron beam melting (EBM)-fabricated Ti-6Al-4V scaffolds at 7 or 14 days, (b and c) The relative expression levels of OPN and COL1 were higher in the low power-selective laser melting-Ti-6Aluminum-4Vanadium (L-SLM Ti-6Al-4V) scaffold than in the EBM-fabricated Ti-6Al-4V scaffold at both 7 and 14 days (P < 0.05), (d) The relative expression levels of RUNX2 were higher in the L-SLM Ti scaffold than in the EBM-fabricated Ti-6Al-4V scaffold only at 14 days (P < 0.05). **P < 0.01.

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Discussion

The purpose of our current study was to compare the biomechanical and biological properties of low-power SLM-manufactured Ti-6Al-4V porous scaffolds with EBM-Ti scaffold, and test their characteristics to determine whether the L-SLM-Ti scaffold met the standards for BTE.

The results showed that L-SLM-Ti scaffolds had a rougher surface due to the larger amount of unmolten and semi-molten metal particles on the surface of the fibers compared with the EBM-Ti scaffolds. This increased fiber surface might improve the cell adhesion and extracellular matrix accumulation of the Ti scaffold.

However, these metal particles can theoretically separate from the scaffold and block pores to reduce the level of nutrient exchange. Moreover, dissociated metal particles might have a negative effect on the bio-compatibility and bio-safety of the scaffold.^[23] The L-SLM-Ti scaffold was designed and manufactured using layer-by-layer addition of slim Ti-6Al-4V fibers (200 μm) to afford a relatively large pore size (800 μm). SEM images showed that the size of the Ti-6Al-4V particles ranged from 5 to 46 μm. In theory, the obtained scaffolds had a pore size larger than the metal particles, which might allow these dissociative metal particles to be washed off easily, eliminating their potential side effects. The results showed no significant difference in live cell rate between the L-SLM-Ti and EBM-Ti scaffolds, demonstrating that the L-SLM-Ti scaffold, which had a larger pore size, might be suitable for bone engineering.

Chemical composition measurements showed that a large amount of carbon (C) and oxygen (O) were present on the surface of the 3DP-Ti scaffolds.^[20] This indicated that metal oxidization might occur on the surface of fibers and form a thin layer of oxides on the surface, usually resulting in improved bioactivity, which can prevent continuous oxidization and enhance the corrosion resistance.^[24] This favors a decrease in the risk of rejection and enhanced biocompatibility.^[25],[26]

Previous studies have shown that the mechanical properties of 3DP scaffolds were determined by the shape, structure, and manufacturing method.^[27],[28] Biomechanical measurements showed that the L-SLM-Ti scaffold had a lower strength (1.89 ± 0.15 kN, 3.48 ± 0.21 kN, P < 0.05) and elastic modulus (10.88 ± 0.34 GPa, 12.81 ± 0.39 GPa, P < 0.05) than the EBM-Ti scaffolds. Previous studies showed that the different percentage of martensitic α/β occurred by different sintering temperature, and the microstructure difference between EBM and SLM scaffolds may be the main reason for the biomechanical differences.^[18] We hypothesize that the outcomes of this study might corroborate the aforementioned findings. As a result, compared with the EBM-Ti scaffold, the L-SLM-Ti scaffold with the current architecture might have an increased bone regeneration capacity due to the reduced stress-shielding effect. Meanwhile, CCK-8 measurements showed that cell proliferation inside the L-SLM-Ti scaffold was higher than in the 3DP-EBM scaffolds after 4 weeks of culture in vitro. Cell differentiation results at 14 days also showed the better effects of L-SLM-Ti scaffold than 3DP-EBM scaffold. These results demonstrated that the L-SLM-Ti scaffold with large pore size might have a superior capacity for bone regeneration due to the reasonable biomechanical properties and improved biological performance.

To our knowledge, this study represents the first report of a 3DP-Ti-6Al-4V porous scaffold with a rougher surface and larger pore size through a low-power SLM process possessing a good elastic modulus and biological properties. However, this study has several limitations, as follows: (i) a lack of comparison with different structure designs, such as different porosities, pore sizes, and basic structures (cubic, diagonal, pyramidal); (ii) only in vitro experiments were performed, but different effects may occur in dynamic body fluid or in vivo; and (iii) biomechanical measurements were only performed under compression force, with further work required for comprehensive biomechanical measurements.

Conclusion

As a novel microstructure and surface modification technique for porous Ti-6Al-4V scaffolds, the design of 3D microstructures with large pore sizes and their fabrication using L-SLM might be an effective method for building up 3DP-Ti scaffolds with reasonable biomechanical and biological performances. The L-SLM-Ti scaffold might be a suitable candidate for bone defect repair.

Acknowledgments

We thank Dr. Xu Yan and Dr. Zhou Jin for assistance in performing the language modification and data analysis.

Financial support and sponsorship

This study was supported by the Jiangsu Provincial Key Research and Development Program (CN) (BE2019608, BE2015613, BE2016763) and the National Natural Science Foundation of China (81601612).

Conflicts of interest

Qingqiang Yao is an Editor-in-Chief of the journal. The article was subject to the journal's standard procedures, with peer review handled independently of this editor and his research groups.

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