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. 2019 Aug 29;477(12):2772–2782. doi: 10.1097/CORR.0000000000000954

Partially Melted Ti6Al4V Particles Increase Bacterial Adhesion and Inhibit Osteogenic Activity on 3D-printed Implants: An In Vitro Study

Kai Xie 1,2, Yu Guo 1,2, Shuang Zhao 1,2, Lei Wang 1,2, Junxiang Wu 1,2, Jia Tan 1,2, Yangzi Yang 1,2, Wen Wu 1,2, Wenbo Jiang 1,2, Yongqiang Hao 1,2,✉
PMCID: PMC6907305  PMID: 31764350

Abstract

Background

A porous Ti6Al4V implant that is manufactured using selective laser melting (SLM) has broad potential applications in the field of orthopaedic implants. The pore structure of the SLM porous Ti6Al4V implant allows for cell migration and osteogenic differentiation, which is favorable for bone ingrowth and osseointegration. However, it is unclear whether the pore structure and partially melted Ti6Al4V particles on a SLM porous Ti6Al4V implant will increase bacterial adhesion and, perhaps, the risk of implant-related infection.

Questions/purposes

(1) Is there more bacterial adhesion and colonization on SLM porous Ti6Al4V implants than on polished orthopaedic implants? (2) Do partially melted Ti6Al4V particles on SLM porous Ti6Al4V implants reduce human bone mesenchymal stem cells (hBMSCs) adhesion, viability, and activity?

Methods

To determine bacterial adhesion and biofilm formation, we incubated five different Ti6Al4V discs (polished, grit-blasted, plasma-sprayed, particle SLM porous, and nonparticle SLM porous discs) with methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Bacterial coverage on the surface of the five different Ti6Al4V discs were evaluated based on scanning electron microscopy (SEM) images quantitatively. In addition, a spread-plate method was used to quantitatively evaluate the bacterial adhesion on those implants. The biofilm formation was stained with crystal violet and semi-quantitatively determined with a microplate reader. The morphology and adhesion of hBMSCs on the five Ti6Al4V discs were observed with SEM. The cell viability was quantitatively evaluated with a Cell Counting Kit-8 assay. In addition, the osteogenic activity was determined in vitro with a quantitatively alkaline phosphatase activity assay and alizarin-red staining. For semiquantitative analysis, the alizarin-red stained mineralized nodules were dissolved and determined with a microplate reader.

Results

The polished discs had the lowest MRSA adhesion (8.3% ± 2.6%) compared with grit-blasted (19.1% ± 3.9%; p = 0.006), plasma-sprayed (38.5% ± 5.3%; p < 0.001), particle (23.1% ± 2.8%; p < 0.001), and nonparticle discs (15.7% ± 2.5%; p = 0.003). Additionally, when comparing the two SLM discs, we found that particle discs had higher bacterial coverage than nonparticle discs (23.1% ± 2.8% versus 15.7% ± 2.5%; p = 0.020). An E. coli analysis showed similar results, with the higher adhesion to particle SLM discs than to nonparticle discs (20.7% ± 4.2% versus 14.4% ± 3.6%; p = 0.011). In addition, on particle SLM porous discs, bacterial colonies were localized around the partially melted Ti6Al4V particles, based on SEM images. After a 7-day incubation period, the cell viability in the particle group (optical density value 0.72 ± 0.05) was lower than that in the nonparticle groups (optical density value: 0.87 ± 0.08; p = 0.003). Alkaline phosphatase activity, as a marker of osteogenic differentiation, was lower in the particle group than in the nonparticle group (1.32 ± 0.12 U/mL versus 1.58 ± 0.09 U/mL; p = 0.012).

Conclusion

Higher bacterial adhesion was observed on SLM porous discs than on polished discs. The partially melted Ti6Al4V particles on SLM porous discs not only enhanced bacterial adhesion but also inhibited the osteogenic activity of hBMSCs. Postprocessing treatment is necessary to remove partially melted Ti6Al4V particles on an SLM implant before further use. Additional studies are needed to determine whether an SLM porous Ti6Al4V implant increases the risk of implant-related infection in vivo.

Clinical Relevance

As implants with porous Ti6Al4V made using SLM are being designed, our preliminary findings suggest that postprocessing treatment is needed to remove partially melted Ti6Al4V particles before further use. In addition, the depth of the porous structure of the SLM implant should not exceed the maximum depth of bone ingrowth because the host immune defense cannot prevent bacterial adhesion without integration.

Introduction

Massive bone defects caused by trauma, infection, and tumor remain a challenge in orthopaedics. The development of metal additive manufacturing such as selective laser melting (SLM) offers a novel approach that may be helpful in the treatment of critical-sized bone defects by allowing for the fabrication of custom devices that permit osseointegration [33]. These custom-designed Ti6Al4V implants can be made in complex shapes, and they possess an interconnected pore structure [27], which allows for cell migration and osteogenic differentiation, which is favorable for bone growth and bone-implant osseointegration [12]. However, there is concern about whether the cell-friendly pore structure, which is susceptible to bacterial adhesion, increases the risk of implant-related infection.

Bacterial adhesion is considered the first step of implant-related infection. The initial interaction between bacteria and biomaterial provides opportunities for bacterial biofilm formation on the implant, which is crucial for the pathogenesis of implant-related infection [16]. Bacterial adhesion depends on the surface design of the implant; elements of implant surface—such as surface topography, roughness, hydrophobicity and charge—can affect bacterial adhesion and colonization [5, 25]. Microporosity on the implant’s surface provides niches that can be easily inhabited by pathogens [2]. Moreover, the large contact area provided by interconnected pore structure may increase the incidence of bacterial adhesion. In addition, a large number of partially melted Ti6Al4V particles inevitably bond to the strut during the SLM process [7, 37]. Partially melted Ti6Al4V particles can change the surface topography of the SLM porous implant by bonding to the strut and change the surface average amplitude of peaks and valleys. However, in most in vivo and in vitro experiments, researchers did not perform postprocessing treatment to remove partially melted Ti6Al4V particles after the SLM process [1, 15, 23, 38]; it is unclear whether changes in surface topography by partially melted Ti6Al4V particles influence the behavior of bacteria and the host cell, to the best of our knowledge.

Therefore, we asked: (1) Is there more bacterial adhesion and colonization on SLM porous Ti6Al4V implants than on polished orthopaedic implants? (2) Do partially melted Ti6Al4V particles on SLM porous Ti6Al4V implants reduce human bone mesenchymal stem cell (hBMSC) adhesion, viability, and activity?

Materials and Methods

Experimental Overview

This experiment consisted of two parts (Fig. 1). First, we compared the bacterial adhesion and biofilm formation on SLM porous Ti6Al4V implants with those on polished, grit-blasted, and plasma-sprayed implants. Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were cultured from five orthopaedic implants. Bacterial adhesion was determined by using scanning electron microscopy (SEM) and spread-plate analysis. The biofilm formation on five orthopaedic implants was evaluated with crystal violet staining.

Fig. 1.

Fig. 1

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This figure shows experimental design of the current study; MRSA = methicillin-resistant Staphylococcus aureus; E. coli = Escherichia coli; hBMSCs = human bone mesenchymal stem cells; SEM = scanning electron microscopy; ALP = alkaline phosphatase.

In the second part, we determined the effect of partially melted Ti6Al4V particles on the behavior of hBMSCs. The adhesion and morphology of hBMSCs on the five Ti6Al4V discs were determined with SEM. The cell viability was evaluated by using a Cell Counting Kit-8 assay. An alkaline phosphatase activity assay and alizarin-red staining were used to determine the osteogenic activity of hBMSCs on Ti6Al4V discs.

Material Preparation and Surface Characterization

We purchased the orthopaedic Ti6Al4V discs (10 mm in diameter and 2 mm high) with mirror-polished (polished group), grit-blasted (grit-blasted group), and plasma-sprayed (plasma-sprayed group) surfaces from Suzhou Kangli Orthopaedics Instrument Co, Ltd (Jiangsu, China). Polished surfaces have been used in internal fixation instruments and prostheses for a long time. Grit-blasting and plasma-spraying are considered as effective ways to create a rough surface on orthopaedic implants [33]. Therefore, we chose these three implants as control groups in the current study. We purchased SLM Ti6Al4V discs from Hunan Farsoon High-Technology Co, Ltd (Wuhan, China). These discs (10 mm in diameter and 2 mm high) were designed with Materialise Magics version 15.0 (Materialise, Leuven, Belgium) with a diamond unit cell (Fig. 2A-C). SLM was performed using an SLM machine (FS271M; Hunan Farsoon High-Technology Co, Ltd) with commercial Ti6Al4V powders (3089; AP&C Advanced Powders & Coatings Inc, Montreal, Quebec, Canada) (Fig. 2D). To further determine the effect of partially melted Ti6Al4V particles of SLM porous implants on bacterial and cell behavior, we divided the SLM porous Ti6Al4V discs into two groups: the particle group and nonparticle group. For the particle group, we washed porous Ti6Al4V discs with an ultrasonic cleaner to remove unsintered powder after the SLM process. For the nonparticle group, the SLM porous Ti6Al4V discs were sandblasted to remove partially melted Ti6Al4V particles on the surface of the implants. After postprocessing treatment, the nonparticle SLM porous Ti6Al4V discs were washed with an ultrasonic cleaner three times. The implant surfaces of five different Ti6Al4V discs were characterized using SEM (Hitachi S-4800, CamScan, Tokyo, Japan) at an accelerating voltage of 5-kV. For the particle and nonparticle groups, the pore size of the fabricated SLM discs was further measured with ImageJ 1.52a (National Institutes of Health; Bethesda, MD, USA, n = 3). The porosity of particle and nonparticle groups was determined by micro-CT (μCT 100, SCANCO Medical AG, Bassersdorf, Switzerland, n = 3). In addition, the surface average roughness parameter (Ra) was determined using two different methods (n = 3). For the polished, particle and nonparticle groups, roughness parameters were measured in a 5 × 5 μm scanning size by using atomic force microscopy (AFM, NanoMan VS, Veeco, Plainview, NY, USA), equipped with OTESPA-R3 probes (Bruker, Billerica, MA, USA). Due to the relative high surface roughness, the surface average roughness of grit-blasted and plasma-sprayed discs was determined according to national geometrical product specifications GB/T3505-09 with TIME 3220 (Beijing TIME High Technology Ltd, Beijing, China). Moreover, the contact angles of five different Ti6Al4V discs were evaluated with a contact angle detector (JY-82B, Chengde Dingsheng Testing Machine Co Ltd, Hebei, China) according to manufacturer’s instruction at room temperature (n = 3).

Fig. 2 A-F.

Fig. 2 A-F

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This figure shows (A) the diamond unit cell, (B) the lateral view and (C) the top view of the computer-aided design model of a selective laser melting porous Ti6Al4V implant, (D) the characterization of Ti6Al4V powders, (E) characterization of the surface morphology of the five Ti6Al4V implants, and (F) representative images of contact angles on five different Ti6Al4V implant.

Surface Characterization

On SEM imaging, the plasma-sprayed Ti6Al4V discs had the roughest surface. In discs in the particle group, multiple partially melted Ti6Al4V particles were observed on the strut surface. The particle size and surface morphology of the partially melted Ti6Al4V particles were very similar to those of commercial Ti6Al4V powders. In the nonparticle group, after postprocessing treatment, few partially melted Ti6Al4V particles were observed on the SLM disc surface (Fig. 2E). The mean pore size of particle and nonparticle SLM discs were 553.6 ± 24.6 μm and 569.7 ± 24.8 μm, respectively. The mean porosity of particle and nonparticle SLM discs was 70.7 ± 0.3% and 72.8 ± 0.8%, respectively. Therefore, the postprocessing treatment did not statistically increase the pore size (p = 0.055) and porosity (p = 0.062) of the SLM discs.

The mean Ra of polished, grit-blasted, plasma-sprayed, particle, and nonparticle samples were 0.048 ± 0.003 μm, 1.215 ± 0.177 μm, 6.587 ± 0.352 μm, 0.145 ± 0.008 μm, and 0.039 ± 0.006 μm, respectively. The mean contact angles of polished, grit-blasted, plasma-sprayed, particle, and nonparticle samples were 104.8 ± 1.2°, 88.7 ± 2.6°, 114.8 ± 2.1°, 111.7 ± 8.5°, and 115.5 ± 6.2°, respectively (Fig. 2F). The postprocessing treatment did not change the hydrophobicity (p = 0.680) of the SLM discs.

Bacterial Adhesion

MRSA (ATCC43300) and E. coli (ATCC25922) were cultured in tryptic soy broth at 37 °C for 18 hours, and suspensions were diluted to 1 × 104 colony forming units (CFUs)/mL. A sterile Ti6Al4V disc was placed in one well of a 24-well plate (n = 5). One-milliliter suspensions were seeded on each Ti6Al4V disc in the 24-well plate. After the discs were incubated for 24 hours at 37 °C, the nonadherent bacteria on the implant surface were carefully removed by washing them three times with phosphate-buffered saline. Ti6Al4V discs were moved to a new 24-well plate and fixed in 2.5% glutaraldehyde overnight at 4 °C. For SEM observation, Ti6Al4V discs were dehydrated through a series of graded ethanol solutions (25%, 50%, 75%, 95%, and 100%), dried, and coated with gold. Bacterial adhesion and colonization on different surfaces of the Ti6Al4V discs were observed using SEM (Hitachi S-4800, CamScan). The percent area of bacterial coverage was determined based on SEM images using Image J (NIH) according to the method of a previous report [6].

Bacterial colonization of MRSA and E. coli on Ti6Al4V discs was further observed using a spread-plate analysis (n = 5) [34]. A sterile Ti6Al4V disc was placed in one well of a 24-well plate. One milliliter of bacterial suspension (1 × 104 CFU/mL) was cultured in the 24-well plate with tryptic soy broth at 37 °C for 24 hours. Discs were washed three times using phosphate-buffered saline to remove nonadherent bacteria and moved to a new test tube. Adherent bacteria were dislodged by ultrasonication for 5 minutes. After ultrasonication, the bacterial suspension was serially diluted and incubated on tryptone soy agar. After a 24-hour incubation period at 37 °C, the bacterial colonies were counted.

We evaluated bacterial biofilm formation on the implant surface using crystal violet staining as previously described (n = 5) [14, 17]. Bacterial suspensions (1 × 104 CFU/mL) were cultured on the disc surface in 24-well plates with tryptic soy broth at 37 °C. After 24 hours of incubation, the discs were washed three times using phosphate-buffered saline to remove nonadherent bacteria and moved to a 24-well plate. Bacterial biofilm formation on the disc surface was stained with 0.1% crystal violet at room temperature for 15 minutes. The stained bacterial biofilm was then solubilized using 95% ethanol at room temperature for 30 minutes, and the optical density of solutions was measured with a microplate reader at 570 nm (Infinite 200, Tecan Trading AG, Männedorf, Switzerland).

Cell Culture

The current study was approved by our institutional ethics committee. We obtained hBMSCs from a 28-year-old patient who underwent internal fixation for a fracture at our trauma center. The donor was healthy without systemic diseases that may have influenced the behavior of hBMSCs. Informed consent was obtained from the donor before cell isolation. We obtained hBMSCs as described by Peng et al. [19]. After isolation, hBMSCs were cultured in an alpha-modified Eagle medium (Hyclone, Tauranga, New Zealand) that was supplemented by 10% fetal bovine serum (Gibco Laboratories, Gaithersburg, MD, USA) and 1% streptomycin-penicillin (Hyclone). The culture was incubated at 37 °C in a moist atmosphere with 5% CO2. We used hBMSCs at passage 2 to passage 4 for follow-up experiments.

Cell Viability and Morphology

The viability of hBMSCs on five different surfaces of the Ti6Al4V discs was evaluated with a Cell Counting Kit-8 assay (Dojindo Molecular Technology, Japan) as previously described (n = 5) [30]. A sterile disc was placed in a 24-well plate with a 1-mL cell suspension at a density of 5 × 104 cells/mL. After 1, 3, and 7 days of incubation at 37 °C in a moist atmosphere with 5% CO2, the Cell Counting Kit-8 solution was added to the culture medium. According to the manufacturer’s instructions, we measured adsorption at 450 mm using an Infinite 200 absorbance microplate reader after a 2-hour incubation period.

The morphology of hBMSCs on the five Ti6Al4V discs was observed using SEM (n = 5). A sterile disc was placed in a 24-well plate with a 1-mL cell suspension at a density of 1 × 105 CFUs/mL−1. The culture was incubated at 37 °C in a moist atmosphere with 5% CO2 for 1 day. Before SEM observation, we washed the disc three times using phosphate-buffered saline, fixed in 2.5% glutaraldehyde overnight at 4 °C, dehydrated through a series of graded ethanol solutions (25%, 50%, 75%, 95%, and 100%), dried, and coated with gold. Finally, the cell morphology was observed using SEM (Hitachi S-4800, CamScan).

Osteogenic activity

An alkaline phosphatase activity assay and alizarin-red staining were used to determine the osteogenic activity of hBMSCs on the Ti6Al4V discs (n = 5). A 1-mL cell suspension (5 × 104 CFUs/mL) was seeded on each Ti6Al4V disc. After 3 days of incubation, the culture medium was replaced with a medium to differentiate human mesenchymal stem cell osteogenesis (Cyagen Biosciences, Guangzhou, China). An assay was performed to determine alkaline phosphatase activity; discs were washed three times using phosphate-buffered saline and then moved to a new 24-well plate after 7 days of incubation. An alkaline phosphatase activity test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the alkaline phosphatase activity of hBMSCs. Calcium nodule formation was determined using alizarin-red staining, as described by Li et al. [13]. After 21 days of incubation, we fixed the discs in 4% paraformaldehyde overnight. Samples were then stained with an alizarin-red staining solution (Cyagen Biosciences) for 1 hour. We washed the stained samples with phosphate-buffered saline several times to remove nonspecific staining. Finally, the alizarin red on the implant surface was dissolved with 10% cetylpyridinium chloride (Sigma-Aldrich, St. Louis, MO, USA), and the absorbance of the sample solution was measured at 562 nm using a microplate reader (Infinite 200, Tecan Trading AG, Switzerland).

Study Outcomes

Our primary study outcomes were that SLM porous discs had higher bacterial adhesion than polished discs, and that partially melted Ti6Al4V particles increased bacterial adhesion and biofilm formation on SLM porous discs.

Our secondary study outcome was that partially melted Ti6Al4V particles reduced cell adhesion and osteogenic activity of hBMSCs.

Statistical Analysis

Data acquired from at least three independent experiments are expressed as the means ± SD. A t-test and one-way ANOVA were used to determine variance using SPSS version 23.0 (IBM Corp, Armonk, NY, USA). A p value less than 0.05 was considered statistically significant.

Results

Bacterial Adhesion and Colonization

The polished discs had the lowest MRSA adhesion (8.3% ± 2.6%) compared with grit-blasted (19.1% ± 3.9%; p = 0.006), plasma-sprayed (38.5% ± 5.3%; p < 0.001), particle (23.1% ± 2.8%; p < 0.001), and nonparticle (15.7% ± 2.5%; p = 0.003) discs. Additionally, when comparing the two SLM discs, we found that the particle discs had more adhesion than the nonparticle discs (23.1% ± 2.8% versus 15.7% ± 2.5%; p = 0.020) (Fig. 3A-B). MRSA colonies were randomly distributed on the nonparticle Ti6Al4V discs. In addition, MRSA colonization on particle SLM discs was larger than that on nonparticle Ti6Al4V discs. Notably, in contrast to random bacterial distribution on the surface of nonparticle Ti6Al4V discs, on particle Ti6Al4V discs, most MRSA colonies were located near the partially melted Ti6Al4V particles. This phenomenon was also observed in the adhesion of E. coli (Fig. 3C-D). After a 24-hour incubation period, less bacterial coverage was observed on nonparticle Ti6Al4V discs than on particle Ti6Al4V discs (20.7% ± 4.2% versus 14.4% ± 3.6%; p = 0.011). In the particle group, most E. coli colonies were located around the partially melted Ti6Al4V particles.

Fig. 3 A-D.

Fig. 3 A-D

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This image shows (A) scanning electron microscopy (SEM) observation of methicillin-resistant Staphylococcus aureus (MRSA) colonization on five Ti6Al4V implants, (B) bacterial coverage by MRSA, (C) SEM observation of Escherichia coli colonization on the five Ti6Al4V implants, and (D) bacterial coverage by E. coli. ap < 0.05 compared with the particle group; bp < 0.05 compared with the polished group.

In the spread-plate analysis of adherent bacteria dislodged from the surface of Ti6Al4V discs (Fig. 4A), both MRSA and E. coli showed greater bacterial colonization in the particle group than in the nonparticle group (Figs. 4B-C). In addition, bacterial biofilm formation was higher in the particle group than in the nonparticle group (Fig. 4D-E).

Fig. 4 A-E.

Fig. 4 A-E

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This figure shows (A) representative images of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli grown on five different Ti6Al4V implants, (B) the number of colony forming units (CFUs) of MRSA growing on the Ti6Al4V implants, (C) the number of CFUs of E. coli growing on the Ti6Al4V implants, (D) MRSA biofilm absorption of crystal violet on the Ti6Al4V implants, and (E) E. coli biofilm absorption of crystal violet on the Ti6Al4V implants. ap < 0.05 compared with the particle group; bp < 0.05 compared with the polished group; OD = optical density.

Cell Viability and Osteogenic Activity

Partially melted Ti6Al4V particles reduced the viability and osteogenic activity of hBMSCs. Cells were well-attached to the surfaces of polished, grit-blasted, and nonparticle Ti6Al4V discs, with spread and flattened morphology; however, fewer cells were observed on the surfaces of the particle discs than on nonparticle discs (Fig. 5A). Notably, in the particle group, hBMSCs were mainly attached to gaps between partially melted Ti6Al4V particles, which were associated with the spreading shape of hBMSCs and may have negatively affected osteogenic activity. After 7 days of incubation, the cell viability in the particle group (optical density value: 0.72 ± 0.05) was lower than that in the nonparticle groups (optical density value: 0.87 ± 0.08; p = 0.003) (Fig. 5B). Alizarin-red staining showed less activity in the particle group than in the nonparticle group (Fig. 5C-D). Alkaline phosphatase activity, as a marker of osteogenic differentiation, was lower in the particle group (1.32 ± 0.12 U/mL) than in the nonparticle group (1.58 ± 0.09 U/mL; p = 0.012) (Fig. 5E).

Fig. 5 A-E.

Fig. 5 A-E

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This figure shows (A) adhesion and morphology of hBMSCs after a 1-day incubation period, (B) viability of hBMSCs on the five different Ti6Al4V implants, (C) alizarin-red staining of calcium nodules after a 21-day culturing period, (D) absorption of the alizarin-red stain, and (E) the alkaline phosphatase activity of hBMSCs on the Ti6Al4V implants after 7 days of incubation. ap < 0.05 compared with the particle group; bp < 0.05 compared with the polished group; OD = optical density.

Discussion

Osteogenic properties and bacterial adhesion are among the major concerns in orthopaedic implant design [34]. Bacterial adhesion was considered the first step of implant-related infection, which could be affected by the chemical and physical properties of implant materials. Due to their inherent surface properties, ceramic materials are considered low-adhesive materials compared with commonly used metal implants [3, 31]. Generally, bacteria are more likely to adhere to materials with positively charged, hydrophilic, and rough surfaces [5]. In addition, specific topographic patterns and porous structures could provide favorable interface for both cell and bacterial adhesion [25]. Recently, 3-D printed Ti6Al4V implants have received extensive attention in in the field of hard tissue repair because of their predefined external shape and interconnected pore structure [35]. Several animal and clinical research studies have confirmed that additive, manufactured, porous implants with alterable, interconnected porosity have good bone ingrowth and bone-implant osseointegration [9, 12, 29]. However, it is unknown whether the pore structure and partially melted Ti6Al4V particles of SLM porous Ti6Al4V implants affect bacterial and cell behavior. The results of our study suggest that bacterial adhesion was higher on SLM porous Ti6Al4V discs than on polished Ti6Al4V discs. Notably, partially melted Ti6Al4V particles on SLM porous Ti6Al4V discs not only enhance bacterial adhesion but also inhibit adhesion and osteogenic activity of hBMSCs.

The study has some limitations. First, this study is an in vitro study. Implant-related infections are complicated processes that include pathogen and host tissue, which is difficult to simulate in vitro. Therefore, the results of in vitro bacterial adhesion may not be consistent with in vivo results. Further in vivo studies are needed to confirm whether an SLM porous Ti6Al4V implant increases the risk of implant-related infection in vivo. Second, we could not quantitatively calculate the number of partially melted Ti6Al4V particles we removed through postprocessing treatment, and residual partially melted Ti6Al4V particles on nonparticle SLM samples could affect results. In the current study, we used sandblasting to remove partially melted Ti6Al4V particles. However, other techniques, such as chemical etching, are also suitable for the postprocessing treatment [20]. Third, we evaluated bacterial adhesion of MRSA and E. coli in our study; these results may not be generalizable to the behavior of other microorganisms.

Bacterial Adhesion and Colonization

Our study demonstrated that bacterial adhesion SLM Ti6Al4V discs was higher than that of polished Ti6Al4V discs, and partially melted Ti6Al4V particles bonded on the SLM porous Ti6Al4V discs increase bacterial adhesion and colonization. A previous study suggested that bacterial adhesion highly depends on the dimension of the implant surface topography [32]. A surface pattern with a dimension similar to bacterial size leads to high bacterial adhesion because of a maximized bacteria-implant contact area [10], which is far below the pore size of the SLM porous Ti6Al4V implant. Therefore, bacterial colonies were randomly distributed rather than forming biofilm on the nonparticle Ti6Al4V discs. For nonparticle group, the main reason of increasing bacterial adhesion is the large contact area caused by the interconnected pore structure. For the particle group, the gap between the partially melted Ti6Al4V particles and the SLM implants provide an ideal niche for bacterial adhesion and colonization. In addition, partially melted Ti6Al4V particles on the surface of SLM implants change the average amplitude of peaks and valleys, which could increase the surface roughness. Therefore, biofilm formation was observed around the partially melted Ti6Al4V particles on the particle discs. However, as we mentioned above, the results of in vitro bacterial adhesion may not be consistent with in vivo results. Therefore, further in vivo studies are needed to determine whether SLM porous Ti6Al4V implants increase the risk of implant-related infection.

Human Cell Viability, Osteogenic Activity

Nonparticle SLM porous discs showed higher osteogenic activity than polished, grit-blasted, and plasma-sprayed samples, while partially melted Ti6Al4V particles reduced cell adhesion and the osteogenic activity of hBMSCs. The competition between host-tissue integration and bacterial biofilm formation determines the fate of orthopaedic implants [8]. Host tissue could compete with bacteria on implant surfaces to reduce bacterial adhesion through rapid and efficient bone-implant osseointegration. Individual risk factors, such as smoking, obesity, and inflammatory disease, have a negative effect on bone-implant osseointegration and could also increase the incidence of implant-related infection [4, 28, 36]. The combined results of the current and previous studies showed that a porous 3-D printed Ti6Al4V implant can enhance the osteogenic activity of mesenchymal stem cells, promote the maturation of interfacial bone tissue [22], and enhance bone-implant osseointegration [18, 24], which could help host tissue to win the race to the surface and reduce the incidence of implant-related infection. However, since the host immune defense cannot prevent bacterial adhesion before integration [21, 26], to reduce the potential risk of implant-related infection, the depth of the porous structure should not exceed the maximum depth of bone ingrowth. For particle samples, a large number of partially melted Ti6Al4V particles reduced the adhesion of hBMSCs. Partially melted Ti6Al4V particles also interfered with the potential of hBMSCs to differentiate osteogenesis, and fewer calcium nodules were observed on the surface of particle SLM Ti6Al4V discs than on nonparticle discs. Therefore, we speculate that partially melted Ti6Al4V particles negatively affect bone-implant osseointegration. On the other hand, partially melted Ti6Al4V particles on the implant could induce an inflammatory response in periprosthetic tissue, cause osteoclast-mediated bone resorption, and eventually lead to aseptic loosening of implants [11]. In view of these observations, removing partially melted Ti6Al4V particles on SLM porous implants with postprocessing treatments is necessary.

In conclusion, higher bacterial coverage was observed on SLM porous Ti6Al4V discs than on polished Ti6Al4V discs. In addition, partially melted Ti6Al4V particles on SLM porous Ti6Al4V discs not only enhanced bacterial adhesion but also inhibited the osteogenic activity of hBMSCs. Therefore, to reduce the risk of implant-related infection, the depth of the porous structure on an orthopaedic implant should not exceed the maximum depth of bone ingrowth because the host immune defense cannot prevent bacterial adhesion without integration. Postprocessing treatment is needed to remove partially melted Ti6Al4V particles on an SLM implant before further use. More well-designed in vivo studies are needed to determine whether an SLM porous Ti6Al4V implant without partially melted Ti6Al4V particles increases the risk of implant-related infection.

Acknowledgments

The first three authors contributed equally to this manuscript. We thank the funding support from the National Key R&D Program of China (2016YFC1100600), Shanghai Shen Kang Hospital Development Center (16CR3025A), Multicenter Clinical Research Project of Shanghai Jiao Tong University School of Medicine (DLY201506), and MDT Project of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (2017-1-003).

Footnotes

Each author certifies that he or she has no commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.

All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request.

Each author certifies that his or her institution approved the human protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.

This work was performed at Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

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