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. Author manuscript; available in PMC: 2021 Jun 14.
Published in final edited form as: J Orthop Res. 2015 May 21;33(7):979–987. doi: 10.1002/jor.22850

Machine Oil Inhibits the Osseointegration of Orthopaedic Implants by Impairing Osteoblast Attachment and Spreading

Lindsay A Bonsignore 1,2, Victor M Goldberg 1, Edward M Greenfield 1,2,3
PMCID: PMC8201705  NIHMSID: NIHMS1692964  PMID: 25676177

Abstract

The most important factor contributing to short-term and long-term success of cementless total joint arthroplasties is osseointegration. Osseointegration leads to a direct structural and functional connection between living bone and the surface of an implant. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration. For example, specific lots of hip replacement Sulzer Inter-OP™ acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil residues. However, the effect of machine oil on implant integration is unknown. Therefore, the goal of this study was to determine if machine oil inhibits the osseointegration of orthopaedic implants. To test this hypothesis in vivo we used our murine model of osseointegration where titanium alloy implants are implanted into a unicortical pilot hole in the mid-diaphysis of the femur. We found that machine oil inhibited bone-to-implant contact and biomechanical pullout measures. Machine oil on titanium alloy discs inhibited early stages of MC3T3-E1 osteogenesis in vitro such as attachment and spreading. Inhibition of osteoblast attachment and spreading occurred in both areas with and without detectable oil. Osteoblast growth was in turn inhibited on discs with machine oil due to both a decrease in proliferation and an increase in cell death. Later stages of osteogenic differentiation and mineralization on titanium alloy discs were also inhibited. Thus, machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells. These results support routine testing by manufacturers of machine oil residues on orthopaedic implants.

Keywords: orthopaedic implants, osseointegration, osteoblasts, bacterial debris, machine oil

Total joint arthroplasty (TJA) is one of the most successful interventions in surgery. It provides patients with advanced degenerative joint disease improved function, decreased pain and the opportunity to resume a more active lifestyle. There are over 600,000 TJAs performed each year in the United States.1,2 Although, TJA provides excellent 10 to 15 year outcomes, aseptic loosening remains a major clinical problem.3,4 Currently, 35 to 45% of TJAs are performed on patients below age 65.5 Many of these patients will require a revision surgery unless improvements are made to increase implant longevity. The most important factor contributing to short-term and long-term success of cementless TJAs is osseointegration.6

First described by Branemark in the 1950 s, osseointegration leads to a direct structural and functional connection between living bone and the surface of an implant.7 Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization procedures and impair osseointegration.8,9 The FDA regulatory guidelines stipulate that medical device manufacturers identify possible residues, establish a residue limit, stay below that limit, and document and validate cleanliness as part of an ongoing process.10 However, the FDA guidelines do not specify contamination limits or appropriate analytic techniques. In that regard, from 2003 to 2010 the FDA recalled 26 medical devices due to process contamination.11 For example, specific lots of hip replacement Sulzer Inter-OP™ acetabular shells were recalled because they were associated with impaired osseointegration and early failure rates. The recalled shells were later found to be contaminated with bacterial debris and machine oil residues.12,13 This type of hip replacement acetabular shell had previously shown excellent fixation with good short-term and long-term clinical success in patients.14–16 However, the recalled lots of Sulzer Inter-OP™ implants had a 33% early loosening rate and at the time of revision had either gross implant motion or micromotion due to failure of bony ingrowth.17,18 Histopathology from patients with failed implants revealed a fibrous tissue response at the bone implant interface and an inflammatory response that was characterized by the presence of lymphocytes, histiocytes, and foreign body giant cells.18 Although these implants lacked fixation and had a severe inflammatory response, they did not have any evidence of clinical infection.17,18 Therefore, it is likely that the contaminants on these lots of recalled implants inhibited bone ingrowth and thereby caused the subsequent lack of implant fixation.

Contaminants that were found on the recalled lots of Sulzer Inter-OP™ implants included machine oil residues.13 Lathes used in the manufacturing of implants utilize machine oil to lubricate their sliding surface tracks and minimize friction, heat, and wear.19 During manufacturing of the recalled lots of Sulzer Inter-OPTM implants, the machine oil may have contaminated the cutting fluid that is sprayed onto the implant to lubricate and cool the surface during machining.12 Reuse of the cutting fluid could have resulted in the machine oil also being sprayed onto the implants. Machine oil contaminants were not removed from implants during the sterilization process possibly because the manufacturer had eliminated a nitric acid wash in their production steps.12

After machine oil residue contaminants were identified on the lots of recalled Sulzer Inter-OPTM implants, a study examining the tissue response to machine oil concluded that oil alone does not replicate the inflammatory response seen in the tissues from patients with failed sockets.20 However, in that study the machine oil was directly injected into the knee joint and the effect of oil on implant integration was not examined. Therefore, the goal of our study was to determine if machine oil is one of the types of contaminants that inhibit the osseointegration of orthopaedic implants. We therefore measured the effects of machine oil on titanium alloy surfaces in vivo using our murine osseointegration model,8,9 and in vitro using MC3T3-E1 pre-osteoblast cells.

Methods

Implants

Titanium alloy screws (0.8 mm diameter, 3.5 mm length, Ti – 6Al – 4V, Grade 5, AMS120/5BT, Antrin Miniature Specialties Inc, Fallbrook, CA) were utilized as implants. These screw shaped implants were rigorously cleaned with five alternating treatments of alkali ethanol (0.1 N NaOH and 95% ethanol at 32 °C) and 25% nitric acid, as we previously described.8,21 Mobil Vactra Oil #2 (Exxon Mobil, Irving TX), a commercial machine oil that is commonly used to lubricate small to medium size machines, was sterilized by gamma irradiation at 2 kGy (Radiation Resources Core Facility, Case Western Reserve University).20 Machine oil (5 μl) was pipetted on rigorously cleaned implants. Excess machine oil was allowed to drain off for one hour in a sterile hood.

Animals and Surgical Procedure

The experimental protocol was approved by the Case Western Reserve University School of Medicine Institutional Animal Care and Use Committee. Six-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were maintained in the Animal Resources Center of Case Western Reserve University. Mice were randomly assigned to receive rigorously cleaned implants or implants with machine oil. Implants were placed into unicortical pilot holes in the anterior diaphysis of the femur as we previously described.8,9 The mice tolerated the surgery well and showed no sign of inflammation or infection. Mice were sacrificed one week after implantation because this time point allows sufficient osseointegration so that significant effects of contaminants can be measured.8,9

Histomorphometric Analysis

Preparation of 100 μm undecalcified sections was performed in the Case Western Reserve Department of Orthopaedic’s Hard Tissue Histology Core Facility as we previously described.8 The percentage of bone-to-implant contact (BIC) and the percentage of peri-implant bone were measured by a blinded observer as previously described.8 Briefly, the percentage of peri-implant bone was determined in a region of interest encompassing the entire area between the implant threads and includes all of the bone in that region irrespective of whether or not it is in direct contact with the implant. BIC was determined as the percentage of the length of implant surface in direct contact with bone in a region of interest extending from the periosteal surface of the cortex to the tip of the last implant thread. BIC was also measured on the inner half and outer half of the implant threads. The inner half was defined as the region containing the troughs of the screw threads to the midpoint between the troughs and the peaks, while the outer half was defined as the remainder of the screw threads including the peaks.

Biomechanical Analysis

Pullout testing was performed as previously described,8,9 except a Test Resources 100 R Series Single Column Frame (Test Resources, Shakopee, MN) with a 100 R Controller was used. Ultimate force, stiffness and work to failure were determined from the resultant load versus displacement curves according to ASTM standards (F543-07).

Titanium Alloy Discs

Ti – 6Al – 4V discs (12.5 mm diameter, 4 mm height, Grade 5, ELI Bar, ASTM F136; Titanium Industries, Wood Dale, IL) were rigorously cleaned as described above.8,21 Machine oil (5 μl) was pipetted onto the disc and spread over the entire surface of the disc with a pipette tip. Discs sat for one hour in a sterile hood before cell seeding.

Cell Culture

MC3T3-E1 pre-osteoblast cells were maintained in minimum essential media (Thermo Scientific Hyclone, Logan, UT) with 10% fetal bovine serum (FBS, Hyclone), L-glutamine (CellGro), non-essential amino acids (CellGro) and penicillin-streptomycin. Cells were seeded on titanium alloy discs at 2.5 × 104 cells/cm2 in alpha minimum essential media with 10% FBS, L-glutamine and antibiotic/antimycotic (Invitrogen, Carlsbad, CA). Media was replaced after three days and every three days thereafter with differentiation media, which consisted of the media described above supplemented with 50 μg/ml ascorbate (Gibco, Grand Island, NY) and 5 mM beta-glycerophosphate (Sigma, St. Louis, MO).22

Cellular Counting and Cellular Spreading

Cells were fixed, stained with Texas Red phalloidin and DAPI, and viewed as previously described.9 Autofluorescence of oil was viewed with the 360/40 nm excitation and 470/40 nm emission filters. Ten random 200X microscopic fields were analyzed per disc. The number of cells and the percent of spread cells were determined using Image J software (NIH, Bethesda, MD). Cells were considered spread if they had cellular processes extending outward from the cell and filamentous actin within extended processes.

DNA Quantification and Alkaline Phosphatase Activity

Cells were lysed in 2% Triton X-100 and DNA was quantified using PicoGreen dsDNA Quantitation reagent (Molecular Probes) as described23 using a standard curve of calf thymus DNA (Sigma). Alkaline phosphatase activity was measured as described9 using a standard curve of ρ-nitrophenol (Sigma).

BrdU ELISAs

5-bromo-2’-deoxyuridine (BrdU) ELISAs were performed using the Cell Proliferation ELISA system (GE Healthcare Lifesciences, Piscataway, NJ) as recommended by the manufacturer. Cells were cultured for one day prior to analysis in media containing 10 μM BrDU but without ribo- and deoxyribonucleotides because they can compete with BrdU.

LDH Release

Lactate dehydrogenase (LDH) release was measured as described24 using the Cytotoxicity Kit (Roche Applied Sciences, Indianapolis, IN) and are reported as percentage of LDH release caused by lysis in 2% Triton X-100.

Mineralization

Media was replaced every three days with differentiation media further supplemented with 50 ng/ml recombinant human bone morphogenetic protein 2 (R&D Systems, Minneapolis, MN). Thirty days after plating, cells were incubated with 20 μM xylenol orange (Sigma).25 The next day, fluorescent xylenol orange staining was viewed and the percentage of surface area mineralized was calculated using ImageJ software.

Statistical analysis

Statistical analysis was performed using SigmaStat Software 3.0 (Systat Software, San Jose, CA). All data sets passed normality and equal variance testing, therefore parametric analyses were performed. The specific statistical test used and sample size for each group is listed in the figure captions. All data are shown as mean ± standard error.

Results

To test the hypothesis that machine oil inhibits osseointegration of orthopaedic implants, we compared rigorously cleaned implants and implants with machine oil in our murine model.8 Fluorescence microscopy showed that machine oil was only detectable within the troughs of the implant threads (Fig. 1A). Rigorously cleaned implants and implants with machine oil were then implanted into the femur of mice and osseointegration was analyzed after one week. Histomorphometric measurements of the rigorously cleaned implants (Fig. 2A and B) were similar to those we found previously.8,9 However, BIC was significantly inhibited on implants with machine oil by 32% (Fig. 2A). Peri-implant bone was defined as bone in between the implant threads and was not affected by machine oil (Fig. 2B). Since the machine oil was detectable mainly within the troughs of the implant threads (Fig. 1A), we determined whether BIC is primarily inhibited in that region by measuring the BIC in both the inner and outer half of the screw threads. Oil impaired BIC on the inner half of the screw threads by 38% (Fig. 2C) but did not significantly affect BIC on the outer half. Therefore, areas on implants with detectable oil correspond to increased inhibition of BIC. The impaired BIC is illustrated in the representative histological cross-sections at one week after implantation (Fig. 2D).

Figure 1. – Autofluorescence of Machine Oil on Implants and Titanium Alloy Discs.

Figure 1. –

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Fluorescent microscopic images demonstrating autofluorescence of machine oil on implants (A) or titanium alloy discs before (B) or one hour after (C) the addition of media.

Figure 2. – Machine Oil Inhibits Bone-to-Implant Contact.

Figure 2. –

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The percentage of BIC (A) and the percentage of peri-implant bone (B) at one week after implantation. The percentage of BIC on the outer half and inner half of implant threads (C). Representative histological cross-sections, which are closest to the means in C (D). White arrows denote BIC on the outer half and yellow arrows denote BIC on inner half. Statistical analysis was by student’s t-test. n = 7 mice per group.

To further examine the effects of machine oil on osseointegration, we performed biomechanical pullout testing at one week after implantation. Machine oil significantly inhibited biomechanical pullout measures of ultimate force by 27% (Fig. 3A), stiffness by 25% (Fig. 3B), and work to failure by 30% (Fig. 3C). The effects of machine oil are illustrated in the representative force versus displacement curves in Fig. 3D.

Figure 3. – Machine Oil Inhibits Biomechanical Pullout Testing Measures.

Figure 3. –

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Ultimate force (A), stiffness (B) and work to failure (C) at one week after implantation. Representative force versus displacement curves that are closest to the means in A, B and C (D). Statistical analysis was by student’s t-test. n = 11–13 mice per group.

To determine whether machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells, we examined MC3T3-E1 pre-osteoblast cells cultured on titanium alloy discs. Initially, a thin coat of machine oil covered the entire surface of the disc (Fig. 1B). However, when aqueous cell culture media was added to the discs, the hydrophobic machine oil coalesced into distinct areas (Fig. 1C). Machine oil significantly inhibited attachment of MC3T3-E1 pre-osteoblast cells by 50 to 57%, as analyzed by DNA measurements at one and two hours after plating (Fig. 4A). Fluorescent images of cells at two hours after plating revealed a heterogeneous distribution of cells on discs with machine oil, compared to the homogenous distribution of cells on rigorously cleaned discs (left panels, Fig. 4B). Areas with very few cells corresponded to areas with detectable oil (right panels, Fig. 4B). Areas of discs with detectable machine oil had 80 to 90% fewer cells than rigorously cleaned discs at one and two hours after plating (Fig. 4C). Interestingly, the number of cells was also significantly decreased in areas without detectable machine oil by 50% at one hour after plating (Fig. 4C). Osteoblast spreading was also inhibited on discs with machine oil. The percentage of spread cells was significantly impaired in areas with detectable machine oil by >85% at all time points (Fig. 5A). The percentage of spread cells was also significantly reduced in areas without detectable oil at all time points, although this effect is stronger at the earlier time points (Fig. 5A). The impaired spreading in areas with and without detectable machine oil is illustrated in the representative microscopic images of cells at twenty-four hours (Fig. 5B).

Figure 4. – Machine Oil Inhibits Osteoblast Attachment.

Figure 4. –

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DNA measurement of MC3T3-E1 pre-osteoblast cells (A). Representative images of cells stained with phalloidin and DAPI at two hours after plating (left panels) and oil autofluorescence images of the same fields (right panels) (B). Cell counts on rigorously cleaned discs or on discs with machine oil, in both areas without and with detectable oil (C). Statistical analysis was by Two-Way ANOVA, * denotes p < 0.05 compared to rigorously cleaned discs. Each data point represents the mean ± SEM of two or three independent experiments. Each experiment contained 3–4 discs per group, each assayed in triplicate.

Figure 5. – Machine Oil Inhibits Osteoblast Spreading.

Figure 5. –

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The percentage of spread cells on rigorously cleaned discs or on discs with machine oil, in both areas without and with detectable oil (A). Representative images of cells stained with phalloidin and DAPI at twenty-four hours after plating (B). Data is from same experiments as Figure 4C. Statistical analysis was by Two-Way ANOVA, * denotes p < 0.05 compared to rigorously cleaned discs. Each data point represents the mean ± SEM of two independent experiments. Each experiment contained 3–4 discs per group, each assayed in triplicate.

We also examined later stages of osteoblast cell growth, differentiation and mineralization. Osteoblast growth is inhibited in areas with and without detectable oil by >65% as measured by cellular number at one day after plating (Fig. 6A). DNA measurements at three, six, and nine days also demonstrated significantly inhibited osteoblast growth on discs with machine oil (Fig. 6B). This reduced growth is due to both decreased proliferation and increased cell death as both BrdU incorporation and LDH release were significantly altered on discs with machine oil (Fig. 6C–D). Machine oil also significantly impaired osteoblast differentiation and mineralization. Alkaline phosphatase activity was significantly inhibited by 62% at nine days after plating (Fig. 7A). This inhibition of alkaline phosphatase activity was due to a combination of the reduced number of cells and reduced alkaline phosphatase per cell. Thus, alkaline phosphatase activity per cell was significantly inhibited by oil by 44% at nine days, as assessed by normalization of alkaline phosphatase activity to DNA (Fig. 7B). Mineralization was also significantly inhibited on discs with machine oil by 98% at thirty days after plating (Fig. 7C). The impaired mineralization with machine oil is illustrated in the representative discs stained with xylenol orange shown in Figure 7D.

Figure 6. – Machine Oil Inhibits Osteoblast Growth.

Figure 6. –

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Cell counts on rigorously cleaned discs or on discs with machine oil, in both areas without and with detectable oil (A). DNA measurements (B), BrdU incorporation (C), or LDH release (D). Statistical analysis was by Two-Way ANOVA, * denotes p < 0.05 compared to rigorously cleaned discs. Each data point represents the mean ± SEM of three independent experiments. Each experiment contained 3–4 discs per group, each assayed in triplicate.

Figure 7. – Machine Oil Impairs Osteoblast Differentiation and Mineralization.

Figure 7. –

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Alkaline phosphatase activity normalized to area (A) or to DNA (B). The percentage of surface area mineralized as assessed by xylenol orange staining at thirty days after plating (C). Representative discs stained with xylenol orange that are closest to the mean in C (D). Statistical analysis was by Two-Way ANOVA, * denotes p < 0.05 compared to rigorously cleaned. Each data point represents the mean ± SEM of three independent experiments. Each experiment contained 3–4 discs per group, each assayed in triplicate.

Discussion

Osseointegration is crucial for early fixation as well as long-term success of orthopaedic implants. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration.8 For example, specific lots of hip replacement Sulzer Inter-OPTM acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil.11,12 We have previously shown that bacterial LPS adherent to the implants inhibits osseointegration.9 Therefore, the goal of this study was to determine whether machine oil inhibits the osseointegration of orthopaedic implants in our murine model. We found that machine oil inhibited biomechanical pullout measures of osseointegration and the histomorphometric measure of BIC but not peri-implant bone. These in vivo results are similar to what we have previously found with ultrapure LPS adherent to the implants.9

Machine oil on titanium alloy discs inhibited all stages of MC3T3-E1 osteogenesis in vitro including attachment, spreading, growth, differentiation, and mineralization. Thus, machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells. A mechanism that may be responsible for those effects is that the hydrophobic machine oil may impair surface adsorption of hydrophilic proteins, such as fibronectin and vitronectin, that facilitate osteoblast attachment and spreading on orthopaedic surfaces.26–30 Interestingly, osteoblast attachment, and spreading were inhibited in both areas with and without detectable machine oil. This result suggests that there may be low levels of oil contaminants in areas without detectable oil.

Inhibition of osteoblast differentiation and mineralization is likely a result of the impaired osteoblast attachment, spreading, and growth. Our results also suggest a direct inhibitory affect of machine oil on osteoblast differentiation. For example, alkaline phosphatase activity per cell is significantly inhibited by the machine oil and mineralization is almost completely inhibited. In contrast, adherent LPS inhibited the later stages of osteogenic differentiation and mineralization without affecting attachment, spreading, or growth.9 Inhibition of distinct steps may lead to synergistic impairment of osseointegration when both machine oil and LPS are present. Such synergy might account for the impaired osseointegration in the failed lots of Sulzer Inter-OPTM implants.17,18 It is also possible that the inflammation induced by the failed implants18 further contributed to their impaired osseointegration.

A limitation of this study was that only one concentration of machine oil was examined. A future study examining the effect of varying amounts of oil may be useful. For example, not all of the patients who received the recalled Sulzer Inter-OPTM implants required a revision surgery.11,17 This may be because not all of the implants were contaminated or that implant failure depended upon the quantity of the machine oil present.13 An additional potential limitation is that the observed effects might be due to irradiation of the machine oil. However, 10–20 fold higher irradiation doses were used in the previous study of machine oil20 and implants are often sterilized by radiation. Limitations of the mouse model used in this study include the trans-cortical site of implantation and the resultant absence of physiological loading. However, non-loading models are useful for examining surface effects of implants before testing in a loading model.31 More importantly for a study of surface effects, the vigorous cleaning protocol does not detectably affect the surface characteristics of the titanium alloy implants.21,32 Despite these limitations, this is the first study that we know of that has examined the effect of machine oil contaminants on osseointegration.

In conclusion, we found that machine oil on implant surfaces inhibits BIC and biomechanical measures of osseointegration. In addition, machine oil on titanium alloy discs inhibited osteoblast attachment, spreading, growth, differentiation and mineralization in vitro. These results differ from the previous study of machine oil, which concluded that intra-articular oil injections do not induce an inflammatory response but that study did not examine implant integration.20 The results of our study support routine testing by manufacturers for machine oil on orthopaedic implants.

Acknowledgements

We thank Joscelyn Tatro for assistance with surgeries, Sarah McBride for assistance with image analysis, and Teresa Pizzuto for histological preparation. We would also like to thank John Mulvihill for gamma irradiation of the oil. This work was supported by NIH T32 AR07505 (LAB), the Harry E. Figgie III MD Professorship (EMG), and the Radiation Resources Core Facility of the Case Comprehensive Cancer Center (P30CA043703).

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