**4. Coating evaluation**

remove the infected implant and the surrounding affected tissue, combined with the placement of a spacer or antibiotic-loaded beads to fill up to void [44-46]. The second operation is required to remove the spacer or beads after a couple of weeks or months. Once the infection is regarded as treated sufficiently, a new implant or prosthesis is implanted. If the treatment was not successful, new beads can be placed, which will require a third operation for the removal of the beads [44-46]. Due to the high costs and the tremendous burden for the patient a one-step procedure would be preferable. An antimicrobial coating directly on the surface of the newly placed implant, in case of revision surgery after infection, could prevent the infection form reoccurring, but such a coating may also work as a prophylactic in the case of the placement of

Already in clinical use in other medical specialties (e.g. in sutures and central venous and urinary tract cathethers), antibiotic releasing coatings remain mainly experimental in the field of orthopaedic and trauma surgery. For orthopaedic applications gentamicin, vancomycin, rifampicin, and tobramycin are the most frequently used local antiobiotics in case of an implant infection. There are several published *in vitro* and *in vivo* studies based on the use of these antibiotic drugs for an orthopaedic implant coating. Poly-L-lactide (PLLA) coatings with rifampicin on a fracture fixation plate, placed on the tibia of rabbits, showed good results on both antimicrobial properties and acceptance of the host-tissue within 28 days after surgery [47]. Also the direct application of minocycline and rifampicin on titanium, placed in the distal femur of a rabbit, lead to good results on prevention of device colonization and infection prevention within a week after surgery [48]. A combined osteoconductive/antimicrobial coating (HA/tobramycin) on titanium, evaluated in the proximal tibia of a rabbit indicated the potential of a combined coating for infection prevention as well as implant incorporation [49]. A recent study on a combined osteoconductive/osteoinductive/antimicrobial coating (HA/RGD/gentamicin) on stainless steel showed promising results on bone integration and antibiotic release characteristics [33]. Furthermore antibiotic releasing coatings on biodegrad‐ able substances could replace antibiotic containing PMMA-beads, in this case no implant coating would be necessary. A study on gentamicin coated poly(trimethylene carbonate) (PMTC), a biodegradable polymer, showed good results on antibiotic release, biofilm inhibi‐ tion and biodegradability, suggesting to be a good substitute for PMMA-beads [50]. A recent report on a prospective study of the first antibiotic releasing tibial nail has shown promising clinical results with no deep surgical wound infections within the first six months after implantation [51]. The major disadvantage for these coatings which they will face in the near future is the increasing number of antibiotic-resistant bacterial strains. This is the main reason why antimicrobial coatings, based on disinfectants or non-traditional antibiotics, are of great

Silver is (amongst copper, lead and mercury) a potent antimicrobial heavy metal which has been related to medicine for many centuries. Instead of its metallic state, only the ionic state

a primary hip.

**3.1. Antibiotic releasing coatings**

52 Modern Surface Engineering Treatments

interest in the research and development of such coatings.

**3.2. Silver-based coatings**

Newly developed coatings need evaluation before implementation in the clinic to prevent possible adverse reactions to the coating. This evaluation includes mechanical testing and cytotoxicity and biocompatibility tests. In general these tests can be subdivided in two categories: *in vitro* and *in vivo* testing.

### **4.1.** *In vitro* **evaluation**

This is defined as all testing modalities performed in controlled laboratory conditions, so outside of a living organism or its natural setting (Table 3).

### *4.1.1. Cytotoxicity tests*

Cytotoxicity tests can be subdivided in cell viability, cell adhesion and cell spreading assays and are usually performed with fibroblastic cell lines (e.g. A529 [62], MC3T3-E1 [62-65], L929 [66], MG-63 [67, 68]). Cell viability assays evaluate the toxicity of a compound present in the vicinity of the cells either in solution or in a solid state. During these tests the material to be tested is incubated in cell culture medium. The resulting pre-conditioned culture medium is then used for cell-culture to evaluate the viability of the cells after exposure to the extracted medium from the material to be tested. Depending on the material, also direct cell culture on the material surface can be performed. The viability of the cells can e.g. be assessed by performing an MTT-assay.

**• The MTT-assay** is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte‐ trazolium bromide (MTT, or another tetrazolium salt) to formazan by the enzyme succinate dehydrogenase in the mitochondria of living cells. The formed purple product can be measured spectrophotometrically and provides a direct measurement of the cell viability based on mitochondrial activity, hence energy metabolism [55, 64-70].


**Ref.** 

**Detects** 

**Detection method** 

**Analytical method** 

**Prokaryotic cultures**

Table 4. **Table 3:** *In vitro* analytical methods – part 2

adhesion on the material surface [64, 70, 71].

JIS Z 2801

ASTM E-2810

**Table 3.** *In vitro* analytical methods – part 2

Agar diffusion

[70, 73]

Bacterial growth inhibition

Bacterial growth

Bacterial growth

Bacterial growth

• Colony formation

Bacterial growth inhibition

[70]

[65]

Zone of inhibition, antibiotic potential of test compound

• Distance antibiotic containing object to colony defines potency of

antibiotic compound and its release system

Bacterial growth inhibition

Bacterial growth

MIC-MBC-assay

**Other**

• OD 600 measurements (MIC)

• Quantitative bacterial culture (MBC)

• Elevation in OD 600 indicates bacterial growth

• Colony formation

[54, 55, 65, 69]

[62, 75]

Tissue specific staining

Light microscopy

Immunocytochemistry

 **Cell adhesion assays** evaluate the potential of an implant surface to allow cell adhesion by culturing cells directly on the surface of the material to be tested. After allowing the cells to adhere to the surface, non-adhering cells are washed of the implant surface after which the adhering cells are double-stained with fluorescein diacetate (FDA) and ethidium bromide. In this live/death staining, FDA will stain the cytoplasm of intact cells green, while ethidium bromide will stain the DNA of dead cells red. The cell adhesion can be assessed by fluorescence microscopy. The ratio between the FDA-positive and ethidium bromide-positive cells provides insight into the live-dead percentage and thus biocompatibility of the culture surface. If the cells are only incubated with FDA, cell lysis allows quantification by fluorescence spectrophotometry. The level of fluorescent signal is an indication of cell

 **Cell spreading assays** evaluate the potential of a surface to allow cell adhesion and proliferation including matrix formation in the case of e.g. osteocytes. There are multiple ways to assess this surface property. One of the methods described is the use of cell staining directly on the surface after cell culture on the material surface by e.g. crystal violet staining or by an actin staining based on phalloidin. The crystal violet staining is a DNA staining in which cells are fixed on the cultured surface, then incubated with crystal violet to stain the cellular DNA. After washing the stained cells the dye is released by the incubation in a weak acid. The released dye can be measured on a spectrophotometer and providing a quantitative measure for the amount of cells present on the surface. A phalloidin-based staining allows staining of the actin cytoskeleton and cellular organization on the surface of a material. This is a direct cell staining which is visualized by fluorescence microscopy. In most cases the phalloidin

Optical imaging

Fluorescence or bioluminescence

• Fluorescence → emission after excitation

• Bioluminescence → auto-emission

[76]

Presence of light emitting cells (e.g. cell growth or biofilm)

• Fluorescence → GFP

• Bioluminescence → luciferase

DNA/RNA expression

Fluorescence

PCR

• SYBR-green related dyes

Chemiluminescence

Western blot

SEM/TEM

• Scanning electron microscopy

• Transmission electron

microscopy

Electron microscopy

• Sputtering with gold or carbon for

visualization

Protein expression

[62, 63]

Modern Orthopaedic Implant Coatings — Their Pro's, Con's and Evaluation Methods

[63]

[62-64, 66, 67,

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55

Evaluation of bacterial biofilm or extracellular matrix formation

• SEM → Cell to surface interactions

• TEM → Cell to cell interactions

73, 74]

**Table 3.** *In vitro* analytical methods – part 1

Table 3. **Table 3:** *In vitro* analytical methods –


 **Cell adhesion assays** evaluate the potential of an implant surface to allow cell adhesion by culturing cells directly on the surface of the material to be tested. After allowing the cells to adhere to the surface, non-adhering cells are washed of the implant surface after which the adhering cells are double-stained with fluorescein diacetate (FDA) and ethidium bromide. In this live/death staining, FDA will stain the cytoplasm of intact cells green, while ethidium bromide will stain the DNA of dead cells red. The cell adhesion can be assessed by fluorescence microscopy. The ratio between the FDA-positive and ethidium bromide-positive cells provides insight into the live-dead percentage and thus biocompatibility of the culture surface. If the cells are only incubated with FDA, cell lysis allows quantification by fluorescence spectrophotometry. The level of fluorescent signal is an indication of cell

 **Cell spreading assays** evaluate the potential of a surface to allow cell adhesion and proliferation including matrix formation in the case of e.g. osteocytes. There are multiple ways to assess this surface property. One of the methods described is the use of cell staining directly on the surface after cell culture on the material surface by e.g. crystal violet staining or by an actin staining based on phalloidin. The crystal violet staining is a DNA staining in which cells are fixed on the cultured surface, then incubated with crystal violet to stain the cellular DNA. After washing the stained cells the dye is released by the incubation in a weak acid. The released dye can be measured on a spectrophotometer and providing a quantitative measure for the amount of cells present on the surface. A phalloidin-based staining allows staining of the actin cytoskeleton and cellular organization on the surface of a material. This is a direct cell staining which is visualized by fluorescence microscopy. In most cases the phalloidin

**Table 3.** *In vitro* analytical methods – part 2

Table 4. **Table 3:** *In vitro* analytical methods – part 2

adhesion on the material surface [64, 70, 71].

**Ref.** 

**Detects** 

**Detection method** 

**Analytical method** 

Table 3. **Table 3:** *In vitro* analytical methods –

**Eukaryotic cultures**

[55, 64-70]

54 Modern Surface Engineering Treatments

Cell viability by metabolic activity

Spectrophotometric

Tetrazolium based assay

• MTT

• XTT

**Table 3.** *In vitro* analytical methods – part 1

• MTS

metabolically active cells

Cell adhesion

Fluorescence

FDA based assay

• Fluorescein diacetate

• DAPI

• Fluorescein diacetate

• DAPI

→ nuclei of every cell (blue)

→ cytoplasm of healthy cells (green)

• Reduction of a tetrazolium salt (yellow) to formazan (purple) by

[64, 70, 71]

[62]

Cell viability by DNA content

Spectrophotometric

Crystal violet

Spectrophotometric

SRB

• Sulforhodamine B

compared to control situation

Cell density based on protein content

• protein staining, released dye indicates amount of cells present

compared to control situation

Actin staining

Fluorescence

Phalloidinbased assay

• Rhodamine

• DAPI

• Rhodamine

• DAPI

Osteogenic cells

Fluorescence

Alizarin Red S

ALP

• Alkaline phosphatase

Spectrophotometric

• Enzymatic assay

→ staining of calcium deposition (red)

ALP activity is a marker for osteogenic potential of a cell

• Enzymatic turnover of p-nitrophenyl phosphate to p-nitrophenol

→ nuclei (blue)

→ actin cytoskeleton (red)

• DNA staining, released dye indicates level of cell viability

[72]

[64, 68, 69, 73]

[62]

[62, 63, 69, 74]

[65, 70]

Cell viability

Fluorescence

Live/dead staining

• Fluorescein diacetate

• Ethidium bromide

• Fluorescein diacetate

• Ethidium bromide

→ cytoplasm of healthy cells (green)

→ nuclei of death cells (red)

**• Cell adhesion assays** evaluate the potential of an implant surface to allow cell adhesion by culturing cells directly on the surface of the material to be tested. After allowing the cells to adhere to the surface, non-adhering cells are washed of the implant surface after which the adhering cells are double-stained with fluorescein diacetate (FDA) and ethidium bromide. In this live/death staining, FDA will stain the cytoplasm of intact cells green, while ethidium bromide will stain the DNA of dead cells red. The cell adhesion can be assessed by fluores‐ cence microscopy. The ratio between the FDA-positive and ethidium bromide-positive cells provides insight into the live-dead percentage and thus biocompatibility of the culture surface. If the cells are only incubated with FDA, cell lysis allows quantification by fluores‐ cence spectrophotometry. The level of fluorescent signal is an indication of cell adhesion on the material surface [64, 70, 71].

a standardized amount of bacteria is exposed to the preconditioned buffer/medium. After 24 hours of incubation the optical density can be measured at 600 nm, the lowest concen‐ tration which shows no increased optical density compared to the uncultured control condition defines the MIC, while the lowest concentration which shows no bacterial growth after incubation of the MIC-cultures on agar plates for another 24 hours defines the MBC

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57

**• In vitro biofilm formation** on a surface can be confirmed by incubating the surface in a bacterial suspension, rinsing the surface with an isotonic buffer (PBS) and use sonication to release the bacteria from the surface for quantitative culture. Or fix the bacteria on the surface with 2.5% glutaraldehyde/PBS for evaluation with SEM. This method can easily be trans‐

**• International standards** provide guidelines of how to assess coating stability and function, e.g. ISO 10993-5 provides guidelines for *in vitro* medical device evaluation. The Japanese Industrial Standard Z 2801 (JIS) describes a test for contact killing by the incubation of bacteria on a potential antimicrobial surface. Culturing of this surface provides insight on the antimicrobial properties of the evaluated coating [70, 73]. The American Standard E-2810 (American Society for Testing and Materials, ASTM)) describes a test for contact killing by the application of a bacteria loaded agar onto the coated surface, after incubation the number

This is defined as all testing modalities performed in a controlled group of living organ‐ isms, often including clinically relevant parameters and a broad range of imaging techni‐

The first models concerning orthopaedic conditions date back to the late 19th century, primarily focusing on osteomyelitis [77]. Rodet described 2 basic methods to establish an osteomyelitis in a rabbit, the first one by inflicting a fracture and subsequent intravenous injection of the bacterium, resulting in an osteomyelitic leasion in the area of the fracture. The second method was performed by merely injecting bacteria intravenously, which resulted in a systemic

The most well-known model for osteomyelitis is the model by Norden *et al.*; this model describes the direct percutaneous injection, directly into the tibial intramedullary cavity of a rabbit, of both a scleroting agent (sodium morrhuate, bile salts from codfish) and *S. aureus*[78]. Andriole *et al.* however, established one of the first osteomyelitis models with a foreign object. Their model was based on a tibial fracture and subsequent tibial stabilization by a stainless steel pin, contaminated with *S. aureus* [79]. Together with the model by Norden, the model of Andriole mainly form the basis for current animal models used for the evaluation of implant coatings. In general, rabbits are still the most frequently used animal species for these exper‐ imental studies, but there have also been successful models in mice, rats, dogs and sheep. During the years, models increased in complexity and included multifactorial parameters.

infection with periosteal leasions leading to local osteomyelitis [77].

[55].

ferred to the *in vivo* situation.

of viable bacteria is determined [70].

**4.2.** *In vivo* **evaluation**

ques (Table 4).


#### *4.1.2. Antimicrobial coating tests*

In the case of antimicrobial coatings the effect of the coating on bacteria can be assessed with a wide variety of assays, with the most well-known being the agar diffusion test where the release of an antimicrobial compound into the agar leads to an inhibition zone around the releasing material.

**• Bacterial viability** can be assessed by a minimal inhibitory concentration (MIC)/ minimal bactericidal concentration (MBC) assay. In this assay the releasing material is allowed to release its effective compound into a buffer or culture medium over a certain time span. The acquired pre-conditioned buffer/medium is then used in a bacterial culture setting in which a standardized amount of bacteria is exposed to the preconditioned buffer/medium. After 24 hours of incubation the optical density can be measured at 600 nm, the lowest concen‐ tration which shows no increased optical density compared to the uncultured control condition defines the MIC, while the lowest concentration which shows no bacterial growth after incubation of the MIC-cultures on agar plates for another 24 hours defines the MBC [55].


#### **4.2.** *In vivo* **evaluation**

**• Cell adhesion assays** evaluate the potential of an implant surface to allow cell adhesion by culturing cells directly on the surface of the material to be tested. After allowing the cells to adhere to the surface, non-adhering cells are washed of the implant surface after which the adhering cells are double-stained with fluorescein diacetate (FDA) and ethidium bromide. In this live/death staining, FDA will stain the cytoplasm of intact cells green, while ethidium bromide will stain the DNA of dead cells red. The cell adhesion can be assessed by fluores‐ cence microscopy. The ratio between the FDA-positive and ethidium bromide-positive cells provides insight into the live-dead percentage and thus biocompatibility of the culture surface. If the cells are only incubated with FDA, cell lysis allows quantification by fluores‐ cence spectrophotometry. The level of fluorescent signal is an indication of cell adhesion on

**• Cell spreading assays** evaluate the potential of a surface to allow cell adhesion and proliferation including matrix formation in the case of e.g. osteocytes. There are multiple ways to assess this surface property. One of the methods described is the use of cell staining directly on the surface after cell culture on the material surface by e.g. crystal violet staining or by an actin staining based on phalloidin. The crystal violet staining is a DNA staining in which cells are fixed on the cultured surface, then incubated with crystal violet to stain the cellular DNA. After washing the stained cells the dye is released by the incubation in a weak acid. The released dye can be measured on a spectrophotometer and providing a quantita‐ tive measure for the amount of cells present on the surface. A phalloidin-based staining allows staining of the actin cytoskeleton and cellular organization on the surface of a material. This is a direct cell staining which is visualized by fluorescence microscopy. In most cases the phalloidin based stainings are counterstained with DAPI to stain the cells nuclei, which allows visualization of the individual cells and their cytoskeleton [62, 64, 68,

**• Assays to assess the osteogenic potential**, quantify the osteogenic potential of a coating. This can be determined by using cultured cells on the coating surface for an alkaline phosphatase assay (ALP). The ALP assay determines the ALP activity within the tissue, which is related to osteogenesis and bone deposition on the coating surface. Another method to assess the osteogenic potential of a coated surface is an alizarin red s staining, which stains

In the case of antimicrobial coatings the effect of the coating on bacteria can be assessed with a wide variety of assays, with the most well-known being the agar diffusion test where the release of an antimicrobial compound into the agar leads to an inhibition zone around the

**• Bacterial viability** can be assessed by a minimal inhibitory concentration (MIC)/ minimal bactericidal concentration (MBC) assay. In this assay the releasing material is allowed to release its effective compound into a buffer or culture medium over a certain time span. The acquired pre-conditioned buffer/medium is then used in a bacterial culture setting in which

the material surface [64, 70, 71].

56 Modern Surface Engineering Treatments

calcified tissue [62, 63, 69, 74].

*4.1.2. Antimicrobial coating tests*

releasing material.

69, 73].

This is defined as all testing modalities performed in a controlled group of living organ‐ isms, often including clinically relevant parameters and a broad range of imaging techni‐ ques (Table 4).

The first models concerning orthopaedic conditions date back to the late 19th century, primarily focusing on osteomyelitis [77]. Rodet described 2 basic methods to establish an osteomyelitis in a rabbit, the first one by inflicting a fracture and subsequent intravenous injection of the bacterium, resulting in an osteomyelitic leasion in the area of the fracture. The second method was performed by merely injecting bacteria intravenously, which resulted in a systemic infection with periosteal leasions leading to local osteomyelitis [77].

The most well-known model for osteomyelitis is the model by Norden *et al.*; this model describes the direct percutaneous injection, directly into the tibial intramedullary cavity of a rabbit, of both a scleroting agent (sodium morrhuate, bile salts from codfish) and *S. aureus*[78]. Andriole *et al.* however, established one of the first osteomyelitis models with a foreign object. Their model was based on a tibial fracture and subsequent tibial stabilization by a stainless steel pin, contaminated with *S. aureus* [79]. Together with the model by Norden, the model of Andriole mainly form the basis for current animal models used for the evaluation of implant coatings. In general, rabbits are still the most frequently used animal species for these exper‐ imental studies, but there have also been successful models in mice, rats, dogs and sheep. During the years, models increased in complexity and included multifactorial parameters.

The bone bonding properties of apatite-coated implants were first evaluated in dogs by Geesink *et al*. [16]. After the development of these apatite-coated implants, Vogely *et al.* described a rabbit proximal tibial model for the evaluation of hydroxyapatite coated titanium implants in an implant site infection [80]. Darouiche et al. were one of the first who described a rabbit lateral femoral condyle model for the evalauation of antimicrobial coatings on titanium [48]. Poultsides *et al*. described a haematogeneous implant contamination model by MRSA [81]. Moojen et al. evaluated a combined coating with both osteoconductive (periapatite) and antimicrobial (tobramycin) properties in a proximal tibial implant infection model in rabbits [49]. Also, Moskowitz *et al*. developed antibiotic multilayer implant coatings with an antibiotic release of over 4 weeks in a 2 stage rabbit distal femoral condyle infection model. The first surgical stage contained the initial infection with the insertion of a pre-colonized peg, the second surgical stage was the removal of the peg and implantation of the antibiotic coated implant [65]. Alt *et al*. was one of the first to describe a coating which combined osteoconductive (hydroxyapatite), osteoinductive (RGD) and antibiotic (gentamicin) properties in an experi‐ mental rabbit implant infection model [33].

easy to obtain and have a relatively low burden for the patient. An X-ray only provides detailed information about the mineralized tissue (or the lack thereof) and the disease

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59

**• CT (computed tomography)** is a 3D-imaging technique which uses X-rays to construct a 3D image of the mineralized tissue in a patient. It generally provides more in-depth data about the density of the mineralized tissue and bone remodeling compared to X-rays, however imaging of metallic implants can result in scattering of the X-rays resulting in a blur around

**• DEXA (dual energy X-ray absorptiometry)** is often incorrectly stated as a bone scan. The use of 2 different energy levels of the X-ray beam allows accurate determination of the bone mineral density. DEXA is the most common imaging technique to diagnose osteoporosis

**• MRI (magnetic resonance imaging)** does, in contrast to other imaging techniques, not rely on ionizing radiation but on the magnetic spin of protons. Due to the high water content (and thus protons) of soft tissue, MRI is one of the main imaging techniques to assess the musculoskeletal tissues, like cartilage and tendons. MRI only allows indirect imaging of bony structures due to the limited water content of the bone. The main drawbacks for MRI are the duration of the imaging acquisition and the inability for it to be used in combination

**• PET (positron emission tomography)** is based on the detection of the annihilation event of a positron with an electron (beta-decay). Every annihilation-event results in 2 gammaphotons in an opposite direction from the point of decay. The detections of the photons on the detectorring of the scanner results in a 3D image [97]. 18F is one of the most frequently used isotopes (connected to a carrier molecule) to serve as a PET-tracer in orthopaedic research. 18F-fluorodeoxyglucose (FDG) is used for the detection of infection and inflam‐ mation (figure 2) and 18F-fluoride as a tracer for bone remodeling [85, 94, 98]. With signal specificity as its advantage, PET does not provide anatomical information, merely the location of the tracer uptake. This is the main reason why PET and CT are often combined

**Figure 2.** FDG PET of an uninfected implant versus an infected implant in the proximal part of a rabbit tibia, six weeks after surgery. The increased tracer uptake around the infected implant (black area) depicts the local osteomyeliticlea‐

related changes accompanied with it [15, 83, 84].

with metallic implants [76, 84].

in the clinic.

sion.

the implant, rendering data-analysis difficult [76, 85, 86].

and is seldomly used in *in vivo* coating assessment studies [86].

#### *4.2.1. Clinical parameters*

Body weight and temperature provide general information about the animal's physical condition, with weight loss and fever in case of an infection. Leucocyte differentiation provides a detailed overview of the percentages of lymphocytes, neutrophillic granulocytes, monocytes, basophilic granulocytes and eosinophilic granulocytes in the total leucocyte population. An elevated number of leucocytes or a shift in differentiation indicates a bacterial infection. The ESR is based on the fibrinogen balance in the blood. In case of an inflammation or infection the fibrinogen levels increase, resulting in agglutination of erythrocytes with sedimentation as a result. CRP is an acute phase protein whose levels rapidly increase in case of inflammation or infection. ESR and CRP both only indicate the presence of inflammation or infection, never the cause or the location [82].

#### *4.2.2. Imaging modalities*


easy to obtain and have a relatively low burden for the patient. An X-ray only provides detailed information about the mineralized tissue (or the lack thereof) and the disease related changes accompanied with it [15, 83, 84].

The bone bonding properties of apatite-coated implants were first evaluated in dogs by Geesink *et al*. [16]. After the development of these apatite-coated implants, Vogely *et al.* described a rabbit proximal tibial model for the evaluation of hydroxyapatite coated titanium implants in an implant site infection [80]. Darouiche et al. were one of the first who described a rabbit lateral femoral condyle model for the evalauation of antimicrobial coatings on titanium [48]. Poultsides *et al*. described a haematogeneous implant contamination model by MRSA [81]. Moojen et al. evaluated a combined coating with both osteoconductive (periapatite) and antimicrobial (tobramycin) properties in a proximal tibial implant infection model in rabbits [49]. Also, Moskowitz *et al*. developed antibiotic multilayer implant coatings with an antibiotic release of over 4 weeks in a 2 stage rabbit distal femoral condyle infection model. The first surgical stage contained the initial infection with the insertion of a pre-colonized peg, the second surgical stage was the removal of the peg and implantation of the antibiotic coated implant [65]. Alt *et al*. was one of the first to describe a coating which combined osteoconductive (hydroxyapatite), osteoinductive (RGD) and antibiotic (gentamicin) properties in an experi‐

Body weight and temperature provide general information about the animal's physical condition, with weight loss and fever in case of an infection. Leucocyte differentiation provides a detailed overview of the percentages of lymphocytes, neutrophillic granulocytes, monocytes, basophilic granulocytes and eosinophilic granulocytes in the total leucocyte population. An elevated number of leucocytes or a shift in differentiation indicates a bacterial infection. The ESR is based on the fibrinogen balance in the blood. In case of an inflammation or infection the fibrinogen levels increase, resulting in agglutination of erythrocytes with sedimentation as a result. CRP is an acute phase protein whose levels rapidly increase in case of inflammation or infection. ESR and CRP both only indicate the presence of inflammation or infection, never

**• Optical imaging (based on fluorescence and bioluminescence)** is based on the detection of light emitted from the body. The use of fluorescently labeled antibodies results in a very specific signal, although resulting in a very local detection, it also requires a large amount of antibodies when used in humans. This renders large scale use in humans not yet profitable [76]. Also bioluminescence can be used to visualize infection. The main disadvantage of bioluminescence is the requirement of the luciferase gene in the cell to be detected, meaning the use of genetically modified organisms in case of detection by either autologous cells or bacteria. E.g. a bacterium expressing luciferase can be used to monitor an implant infection initiated with that bacterium [76]. Both methods are currently available in laboratory animal

**• X-ray** is by far the oldest imaging technique and the most frequently used imaging technique to assess fractures, implant fixation, location and loosening, but also for the differential diagnosis of bone diseases like osteomyelitis. The use of X-rays is cost effective, they are

mental rabbit implant infection model [33].

*4.2.1. Clinical parameters*

58 Modern Surface Engineering Treatments

the cause or the location [82].

*4.2.2. Imaging modalities*

setting.


**Figure 2.** FDG PET of an uninfected implant versus an infected implant in the proximal part of a rabbit tibia, six weeks after surgery. The increased tracer uptake around the infected implant (black area) depicts the local osteomyeliticlea‐ sion.


**Ref.** 

**Detects** 

**Detection method** 

**Analytical method** 

Table 6. **Table 4:** *In vivo* analytical methods – part 2

**Imaging modalities**

Optical imaging

**Table 4.** *In vivo* analytical methods – part 2

[76]

Presence of light emitting cells

Fluorescence or bioluminescence

• Fluorescence emission after excitation

• Bioluminescence auto-emission

Electromagnetic radiation

X-ray

CT

• Computed tomography

DEXA

• Dual energy X-ray absorptiometry

Electromagnetic radiation

• X-ray (3D)

• Fluorescence → GFP

• Bioluminescence → luciferase

[33, 49, 76, 78-

Bone, bone pathology and metal objects

Bone, bone pathology and metal objects

81, 84, 87-94]

[76, 84, 86, 88,

91, 94]

[86]

Bone density

Electromagnetic radiation

• Dual energy X-ray of same object

of bone density

Soft tissue

Nuclear magnetic resonance

MRI

• Magnetic resonance imaging

• Proton magnetic spin resonance (3D)

• Detection of tissues with a high water content

Active bone remodeling

γ-radiation

Bone scintigraphy

• Single photon emission (2D)

• 99mTc –MDP → increased osteogenesis

• 67Ga-citrate → leucocyte activation (e.g. infection)

• Difference in signal between both X-rays allows calculation

[76, 84, 88]

[84, 88, 95]

Modern Orthopaedic Implant Coatings — Their Pro's, Con's and Evaluation Methods

[76, 88]

Active bone remodeling

γ-radiation

SPECT

• Single photon emission computed

tomography

• Single photon emission (3D)

• 99mTc –MDP → increased osteogenesis

• 67Ga-citrate → leucocyte activation (e.g. infection)

Active bone remodeling and infection

γ-radiation

PET

• Positron emission tomography

• Positron mediated dual photon emission

(3D)

• 18F-Fluorodeoxyglucose → inflammation and infection

• 18F-Sodiumfluoride → active bone remodeling

[76, 84, 88, 91-

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94]

61

**Table 4.** *In vivo* analytical methods – part 1

Table 5. **Table 4:** *In vivo* analytical methods – part 1


**Table 4.** *In vivo* analytical methods – part 2

Table 6. **Table 4:** *In vivo* analytical methods – part 2

**Ref.** 

**Detects** 

**Detection method** 

**Analytical method** 

Table 5. **Table 4:** *In vivo* analytical methods – part 1

**Clinical parameters**

[49, 78, 79, 81,

60 Modern Surface Engineering Treatments

General physical condition

Weighing scale

Body weight

**Table 4.** *In vivo* analytical methods – part 1

• Weight loss after surgical intervention, returns to preoperative values within first weeks after intervention.

• Persistent weight loss indicates animal discomfort,

infection or another systemic event related to device or

intervention.

General physical condition

Thermal probe

Temperature

days after surgery.

• Post-operative thermal elevation, returns to normal within

• Persistent elevation indicates infection or another systemic

event related to device or intervention.

87]

[49, 81]

[49, 78, 80, 81,

Infection by increase of erythrocyte sedimentation

Anticoagulated blood

ESR

• Erythrocyte sedimentation rate

• Capillary tube

• Elevation in first post-operative week due to surgical

intervention.

• Remains elevated in case of infection.

Infection by increase of CRP levels

Serum/plasma

CRP

∙ C-reactive protein

• ELISA

• Elevation in first post-operative week due to surgical

intervention.

• Remains elevated in case of infection.

Infection by shift in leucocyte distribution

Anticoagulated blood

Leucocyte count and

Leucocyte differentiation

• Cell count

• ↑ Neutrophils and monocytes

• ↑ Lymphocytes

• ↑ Basophils and eosinophils

and/or allergic reactions

→ inflammatory processes

→ viral infection or tumor

88]

[84]

[49, 65, 78, 80,

81, 84, 88]

→ bacterial infection


**• A bone scan** (bone scintigraphy) is based on the direct detection of gamma radiation originating from the injected tracer molecule (often 99mTc, 67Ga or 111In) connected to a specific ligand which allows tissue specific binding and thus imaging. A bone scan provides twodimensional images of the patient, which are sufficient in the clinic for the diagnosis [84].

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**• SPECT** (single photon emission computed tomography) on the other hand allows acquisi‐ tion of three-dimensional images, providing more insight in size and localization of certain pathology. In general, bone scan/SPECT-tracers have a longer half-life than PET tracers making them more cost-effective to produce. Just like PET, SPECT provides limited

**Figure 3.** The use of calcium binding fluorophores, depicted in 50 micron PMMA sections, of a rabbit tibial intrame‐ dullary nail model, to address normal bone remodeling and bone remodeling in case of an implant infection. Calcein green was injected at 2 weeks, xylenol orange at 4 weeks and calcein blue at 6 weeks. In the case of normal bone remodeling, calcium deposition is detected around the implant, combined with bone remodeling of the cortical wall. In case of an implant infection the most calcium deposition is located in the outer cortical wall depicting the periosteal

**• Calcium binding fluorophores** (like calcein green, blue and xylenol orange) are being used for the *in vivo* labeling of the calcium deposition at the time of injection. The use of different fluorophores, emitting at different wavelengths, allow post-mortem visualization of the calcium deposition during the experimental follow-up [96]. This provides the opportunity to determine implant ingrowth and bone remodeling in a normal healthy situation and periosteal elevation and calcification during the progression of an osteomyelitis (Figure 3).

elevation and calcification during the 6 week follow-up.

*4.2.3. Ex vivo analysis*

anatomical information and is therefore often combined with CT in the clinic [76].

 **PET (positron emission tomography)** is based on the detection of the annihilation event of a positron with an electron (beta-decay). Every annihilation-event results in 2 gamma-photons in an opposite direction from the point of decay. The detections of the photons on the detectorring of the scanner results in a 3D image [97]. 18F is one of the most frequently used isotopes (connected to a carrier molecule) to serve as a PET-tracer in orthopaedic research. 18F-fluorodeoxyglucose (FDG) is used for the detection of infection and inflammation (figure 2) and 18F-fluoride as a tracer for bone remodeling [85, 94, 98]. With signal specificity as its advantage, PET does not provide anatomical information, merely the location of the tracer uptake. This is the main

**Table 4.** *In vivo* analytical methods – part 3

Table 7. **Table 4:** *In vivo* analytical methods – part 3

reason why PET and CT are often combined in the clinic.


**Figure 3.** The use of calcium binding fluorophores, depicted in 50 micron PMMA sections, of a rabbit tibial intrame‐ dullary nail model, to address normal bone remodeling and bone remodeling in case of an implant infection. Calcein green was injected at 2 weeks, xylenol orange at 4 weeks and calcein blue at 6 weeks. In the case of normal bone remodeling, calcium deposition is detected around the implant, combined with bone remodeling of the cortical wall. In case of an implant infection the most calcium deposition is located in the outer cortical wall depicting the periosteal elevation and calcification during the 6 week follow-up.

#### *4.2.3. Ex vivo analysis*

**Ref.** 

**Detects** 

**Detection method** 

**Analytical method** 

Table 7. **Table 4:** *In vivo* analytical methods – part 3

**Other –** *Ex vivo*

reason why PET and CT are often combined in the clinic.

[96]

62 Modern Surface Engineering Treatments

Calcium deposition during bone remodeling (color)

• Calcein blue

Fluorescence (excitation/emission)

Calcium binding fluorophores

**Table 4.** *In vivo* analytical methods – part 3

• Calcein blue

• Calcein green

• Tetracycline

• Xylenol orange

• Alizarin red S

• Calcein blue (370 / 435 nm)

• Calcein green (470 / 530 nm)

• Tetracycline (390 / 570 nm)

• Xylenol orange (470 / 610 nm)

• Alizarin red S (550 / 620 nm)

• Calcein green

• Tetracycline

• Xylenol orange

• Alizarin red S

→ red

→ orange/red

→ yellow

→ green

→ blue

[33, 49, 75, 79-

Tissue specific staining (including bacteria)

Light microscopy

Histology

• Paraffin

• PMMA

 **PET (positron emission tomography)** is based on the detection of the annihilation event of a positron with an electron (beta-decay). Every annihilation-event results in 2 gamma-photons in an opposite direction from the point of decay. The detections of the photons on the detectorring of the scanner results in a 3D image [97]. 18F is one of the most frequently used isotopes (connected to a carrier molecule) to serve as a PET-tracer in orthopaedic research. 18F-fluorodeoxyglucose (FDG) is used for the detection of infection and inflammation (figure 2) and 18F-fluoride as a tracer for bone remodeling [85, 94, 98]. With signal specificity as its advantage, PET does not provide anatomical information, merely the location of the tracer uptake. This is the main

• Haematoxylin/eosin staining (general)

• Masson Goldnertrichrome staining (general)

• Gram staining (bacteria)

• Wear particles (e.g. polyethylene by polarized light)

• Immunostaining (antibody specific)

81, 84, 87, 90-

94]

[65, 80]

Surface treatment, bacterial biofilm, wear particles

Electron microscopy

SEM

• Scanning electron microscopy

• Surface assessment of non-metallic substrates by gold or

carbon sputtering

• Metallic substrates can be assessed directly

[48, 49, 65, 78-

Bacterial growth under specific circumstances

(Selective) culture media

Bacterial/bone culture

• Tissue swaps

• Bone homogenates

• Tryptic soy agar/broth

• Tellurite glycine agar (selective for

coagulase negative Staphylococci)

• Quantification by colony count or OD 600 measurements

81, 84, 87, 89-

94]

**• Calcium binding fluorophores** (like calcein green, blue and xylenol orange) are being used for the *in vivo* labeling of the calcium deposition at the time of injection. The use of different fluorophores, emitting at different wavelengths, allow post-mortem visualization of the calcium deposition during the experimental follow-up [96]. This provides the opportunity to determine implant ingrowth and bone remodeling in a normal healthy situation and periosteal elevation and calcification during the progression of an osteomyelitis (Figure 3). **• Histology** is a commonly used method to assess the tissue in the implant area on e.g. tissue morphology or bacterial presence. Tissue sections with metallic implants generally require embedding in methylmethacrylate, instead of paraffin, with the inability to allow immu‐ nostainings as a major drawback. Still it provides the unique opportunity to assess the tissueimplant interface [33, 49, 57, 80].

**References**

Med J. (1918). Jan 5;, 1(2975), 12-3.

tive Study]. (2009). Dec;, 80(6), 639-45.

and Control; (2011).

(2012).

1588-95.

[1] Adams, J. E. A Simple Method of Mechanical Fixation for Fracture of Long Bones. Br

Modern Orthopaedic Implant Coatings — Their Pro's, Con's and Evaluation Methods

http://dx.doi.org/10.5772/55976

65

[2] Devas, M. B. Arthroplasty of the hip: a review of 110 cup and replacement arthro‐ plasties. The Journal of bone and joint surgery British (1954). Nov;36-B(4):561-6., 1954

[3] Judet, J, & Judet, R. The use of an artificial femoral head for arthroplasty of the hip joint. The Journal of bone and joint surgery British (1950). May;32-B(2):166-73., 1950

[4] Surveillance Report: Annual epidemiological reportreporting on 2009 surveillance data and 2010 epidemic intelligence data. http://ecdc.europa.eu/en/publications/ publications/0910\_sur\_annual\_epidemiological\_report\_on\_communicable\_diseas‐ es\_in\_europe.pdfAccessed December 2012): European Centre for Disease prevention

[5] Dale, H, Hallan, G, Espehaug, B, Havelin, L. I, & Engesaeter, L. B. Increasing risk of revision due to deep infection after hip arthroplasty. Acta orthopaedica. [Compara‐

[6] Healthcare-associated infections- Fact sheethttp://www.who.int/gpsc/country\_work/ gpsc\_ccisc\_fact\_sheet\_en.pdfAccessed December 2012): World Health organisation;

[7] Surveillance of healthcare-associated infections in Europehttp://www.ecdc.euro‐ pa.eu/en/publications/Publications/120215\_SUR\_HAI\_2007.pdfAccessed December

[8] Capello, W. N, Antonio, D, Jaffe, J. A, Geesink, W. L, Manley, R. G, Feinberg, M. T, & Hydroxyapatite-coated, J. R. femoral components: 15-year minimum followup. Clini‐

[9] Tannast, M, Najibi, S, & Matta, J. M. Two to twenty-year survivorship of the hip in 810 patients with operatively treated acetabular fractures. The Journal of bone and joint surgery American volume. [Comparative Study, Evaluation Studies, Research

[10] Gristina, A. G. Biomaterial-centered infection: microbial adhesion versus tissue inte‐ gration. Science. [Research Support, U.S. Gov't, P.H.S.]. (1987). Sep 25;, 237(4822),

[11] Busscher, H. J, Van Der Mei, H. C, Subbiahdoss, G, & Jutte, P. C. van den Dungen JJ, Zaat SA, et al. Biomaterial-associated infection: locating the finish line in the race for

[12] Arciola, C. R, Campoccia, D, Speziale, P, Montanaro, L, & Costerton, J. W. Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms

2012): European Centre of Disease prevention and Control; (2007).

cal orthopaedics and related research. (2006). Dec;, 453, 75-80.

Support, Non-U.S. Gov't]. (2012). Sep 5;, 94(17), 1559-67.

the surface. Sci Transl Med. (2012). Sep 26;4(153):153rv10.

**• Electronmicroscopy** allows analysis of the implant surface (with or without coating) after distraction from the surrounding tissue. This can include analysis of the bone matriximplant interface but also analysis of a formed biofilm [31].
