**2. Active and passive implant coatings**

#### **2.1. Osteoconductive coatings**

The definition of osteoconduction means that a material or coating "guides" the bone healing or formation. In case of coatings this means that the bone formation is "guided" to grow or attach to the coating surface (a passive coating) [18]. An orthopaedic implant with such a surface treatment or coating provides an ideal substrate for bone aposition which results in improved implant fixation and a possible prolonged lifespan, with a decreased risk of implant loosening and possibly infection [10, 12].

#### *2.1.1. Apatite coatings*

The initial idea of hydroxyapatite (HA) coatings originated from the use of calciumphosphates as a material to stimulate osteogenesis, like tricalciumphosphate (TCP). During the last decades, studies have shown that HA and TCP are suitable for the production of ceramic scaffolds to serve as a bone substitute. Studies on the stability of sintered TCP (Ca3(PO4)2) and HA (Ca10(PO4)6(OH)2) have shown that TCP ceramics dissolve over 10 times faster in acidic and alkaline environments compared to HA [19]. Explaining the rationale behind the current use of TCP for resorbable bone scaffolds and the use of HA for implant coatings.

Since the proposed use of HA as a coating, in the late 1980's by Geesink*et al.*[14-16], several implant designs have been used in the clinic, e.g. partially coated or fully coated hip implants. Fully coated implants achieved complete bone remodeling around the implant, with very good fixation properties. The major disadvantage of these fully coated implants was that in case of revision surgery (either for implant infection or component failure) removal of the implant resulted in massive bone trauma due to the fixation of the implant to the bone. By redefining the coating location to the taper only, a good fixation could be achieved with limited problems at the time of a revision surgery [15]. However this design allowed the formation of stress shielding due to the pressure of the stem against the cortical wall. Due to this strain an implant can get loose, resulting in bone loss or a cortical wall fracture. Still HA coatings sintered to an implant surface has proven itself to be the most successful implant coating made, with 20 years of clinical experience [8, 20].

#### *2.1.2. Hydroxyapatite application methods for metallic surfaces*

There are several ways to coat a metallic alloy, like titanium or stainless steel, with HA. The techniques to coat such a metallic implant include; dip coating [21], sputter coating [22], pulsed laser deposition [23], hot pressing and hot isostatic pressing [24], electrophoretic deposition [25], electrostatic spraying [26, 27], thermal spraying [28], and sol–gel [29]. Some of these techniques are still experimental, thermal spraying, in particular. Plasma spraying is the most accepted method for the production of HA coatings [30]. Plasma spraying requires high temperatures which may damage the HA crystallinity and create unwanted or amorphous phases, with HA ablation from the coated surface as a possible result [28]. Every technique has its advantages and disadvantages. For example, the thickness, the bonding strength and the properties of the HA-composition may be influenced by the application technique. Techniques such as thermal spraying and sputter coating are used for surfaces or substrates (e.g. porous titanium implants) which are difficult to coat. Techniques such as electrophoretic deposition and sol-gel may coat more complex substrates such as porous alloys, still the production of crack free coatings remains challenging (Table 2).

#### **2.2. Osteoinductive coatings**

the accompanying bone fractures, soft tissue infection, and inflammation result in fixation

In order to decrease the amount of implant infections and prevent the implants from loosening, coatings can be applied to the surface of the implant. These coatings may vary from (antibiotic) releasing to non-releasing coatings. In general non-releasing coatings (like hydroxyapatite) are applied by thermal-processes, while releasing coatings (like RGD or antibiotic-containing coatings) are mostly applied to the surface by dip or spray coating, due to their limited thermal

Since the principle of the "race for the surface" dictates that early tissue integration may also reduce the infection risk, a coating promoting tissue integration may also be regarded as a passive method to reduce the amount of infections. In order to promote this tissue integration, one of the biggest leaps forward in the improvement of implant fixation and "the race for the surface" in favour of eukaryotic cells might be the use of hydroxyapatite (HA) coatings on the surface of a metallic implant [12, 14-16]. Although in the beginning it was believed that uncemented prostheses, including the HA-coated implants, would have a higher infection percentage compared to implants fixated with an antibiotic-releasing bone-cement, long-term studies showed a comparable infection percentage and a longer survival in favour of the uncemented prosthesis [5, 17]. These HA-based coatings (and their derivatives) are still one of the most frequently used implant coatings in the field of orthopaedic surgery and trauma, resulting in improved implant ingrowth and a longer lifespan of the prosthesis [8]. A combined situation of a coating with both antimicrobial and osteoconductive properties, is yet to be

The definition of osteoconduction means that a material or coating "guides" the bone healing or formation. In case of coatings this means that the bone formation is "guided" to grow or attach to the coating surface (a passive coating) [18]. An orthopaedic implant with such a surface treatment or coating provides an ideal substrate for bone aposition which results in improved implant fixation and a possible prolonged lifespan, with a decreased risk of implant

The initial idea of hydroxyapatite (HA) coatings originated from the use of calciumphosphates as a material to stimulate osteogenesis, like tricalciumphosphate (TCP). During the last decades, studies have shown that HA and TCP are suitable for the production of ceramic scaffolds to serve as a bone substitute. Studies on the stability of sintered TCP (Ca3(PO4)2) and

issues and an increased infection risk during revision surgery [13].

**1.3. Implant coatings**

48 Modern Surface Engineering Treatments

stability.

found.

**2. Active and passive implant coatings**

loosening and possibly infection [10, 12].

**2.1. Osteoconductive coatings**

*2.1.1. Apatite coatings*

Although biomimetic HA coatings improve the osteoconductivity of metal implants, they do not influence the osteoinductivity. In general osteoinductive coatings are described as coatings which induce bone formation of undifferentiated cells in the surrounding tissue to ultimately promote osteointegration of bone to the coating (active coatings). In order to promote the differentiation of immature progenitor cells to an osteoblastic lineage, attempts to integrate functional biological agents such as growthfactors into biomimetic coatings have been realized [33, 34]. Several of these coatings have been studied extensively, the most important coatings are described below.


tant role in cell adhesion, cell proliferation and differentiation. RGD peptides coated to a surface can initiate these processes in their direct vicinity. The major advantage of using peptide coatings instead of protein coatings is that peptides are smaller and more stable compared to proteins. This allows more peptides to be coated to a surface, which results in a more dense coating. Studies have shown that the flanking amino acids in a RGD containing peptide are of great importance for their efficiency. *In vitro* studies show promising results, where RGD enhances (human) cell adhesion, proliferation and differentiation in the osteogenic lineage [35]. An *in vivo* study of an HA/HA-RGD coating with antibiotic release showed that the HA-RGD coating performed at least equally well as the HA-only coating [33]. Still these

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Bone morphogenic proteins (BMP's), which belong to the transforming growth factor-β (TGFβ) superfamily, are generally accepted osteoinductive additives for per-operative use to enhance bone remodeling. Due to the lack of a local delivery system, capable of a sustained release, relatively high dosages of BMP's (e.g. BMP-2) are being used in the clinic. The use of BMP's has a locally higher incidence of tumorigenesis as a major disadvantage. Other osteo‐ genic BMP's, such as BMP-4 [36, 37] and BMP-7 [38], are also potent inducers of bone regen‐ eration. Knippenberg et al showed that BMP7, in contrast to BMP2 may be a more chondrogenic growth factor in contrast to BMP2 which was described as a more osteogenic growhtfactor [39]. BMP-2 works in 2 concentration dependent directions, at low dosages it promotes bone remodeling, while at high dosage it promotes bone resorption. Therefore a lowdose releasing BMP-2 coated implant may be the most optimal [40]. While many techniques to incorporate BMP's result in a burst release, they can also be incorporated into the crystal latticework of coatings to establish a gradual release system [34, 40]. As such the incorporated proteins can be released gradually and steadily at a low pharmacological level; not rapidly as in a single high-dose burst [41]. In conclusion, incorporation of osteoinductive coatings may seem attractive but, release rate, potential carcinogenesis, inactivation of the compound (e.g.

due to changes in temperature, pH), and bonding to the implant remain of concern.

Since vascularization of infected tissue is often compromised and a bacterial biofilm is formed, which results in poor penetration of antibiotics, systemic antibiotics are not sufficient to treat a bone infection properly [10-12]. To achieve a local dose high enough to treat the infection, this would involve a systemic overdose of the antibiotic (possibly resulting in kidney and liver failure and damage to the function of the inner ear). The best solution to this problem is to have a local delivery system, this suggests an approach for the treatment of orthopaedic infections. Still in many cases the prosthesis can be rescued by infection treatment *in situ*, without a surgical procedure [13, 42, 43]. The use of local antibiotics by antibiotic-loaded bone cement, either as beads or spacers often placed after implant removal in the remaining infected cavity. The general treatment procedure requires at least two surgical procedures, one to

coatings remain experimental by application.

*2.2.2. BMP coatings*

**3. Antimicrobial coatings**

**Table 2.** Different coating techniques to apply hydroxyapatite (HA) on an implant.

#### *2.2.1. RGD coatings*

Extracellular matrix proteins contain a short functional domain of three aminoacids, arginine (R), glycine (G) and asparagine (D), the so-called RGD-domain. This domain plays an impor‐ tant role in cell adhesion, cell proliferation and differentiation. RGD peptides coated to a surface can initiate these processes in their direct vicinity. The major advantage of using peptide coatings instead of protein coatings is that peptides are smaller and more stable compared to proteins. This allows more peptides to be coated to a surface, which results in a more dense coating. Studies have shown that the flanking amino acids in a RGD containing peptide are of great importance for their efficiency. *In vitro* studies show promising results, where RGD enhances (human) cell adhesion, proliferation and differentiation in the osteogenic lineage [35]. An *in vivo* study of an HA/HA-RGD coating with antibiotic release showed that the HA-RGD coating performed at least equally well as the HA-only coating [33]. Still these coatings remain experimental by application.

### *2.2.2. BMP coatings*

Bone morphogenic proteins (BMP's), which belong to the transforming growth factor-β (TGFβ) superfamily, are generally accepted osteoinductive additives for per-operative use to enhance bone remodeling. Due to the lack of a local delivery system, capable of a sustained release, relatively high dosages of BMP's (e.g. BMP-2) are being used in the clinic. The use of BMP's has a locally higher incidence of tumorigenesis as a major disadvantage. Other osteo‐ genic BMP's, such as BMP-4 [36, 37] and BMP-7 [38], are also potent inducers of bone regen‐ eration. Knippenberg et al showed that BMP7, in contrast to BMP2 may be a more chondrogenic growth factor in contrast to BMP2 which was described as a more osteogenic growhtfactor [39]. BMP-2 works in 2 concentration dependent directions, at low dosages it promotes bone remodeling, while at high dosage it promotes bone resorption. Therefore a lowdose releasing BMP-2 coated implant may be the most optimal [40]. While many techniques to incorporate BMP's result in a burst release, they can also be incorporated into the crystal latticework of coatings to establish a gradual release system [34, 40]. As such the incorporated proteins can be released gradually and steadily at a low pharmacological level; not rapidly as in a single high-dose burst [41]. In conclusion, incorporation of osteoinductive coatings may seem attractive but, release rate, potential carcinogenesis, inactivation of the compound (e.g. due to changes in temperature, pH), and bonding to the implant remain of concern.

### **3. Antimicrobial coatings**

*2.2.1. RGD coatings*

Extracellular matrix proteins contain a short functional domain of three aminoacids, arginine (R), glycine (G) and asparagine (D), the so-called RGD-domain. This domain plays an impor‐

**Technique Advantage Disadvantage Thickness Ref**

• Processing in controlled

• Only coats visible area • Expensive and time consuming • Unable to coat complex 3D

• Risk for amorphous coating

• See sputter coating • See sputter coating 1 - 10 µm [23, 25]

• Only coats visible area

• Cracks in coating

temperature

porous substrates

• Expensive

• High sintering temperatures

Coating decomposition due to high

• Rapid cooling may result in amorphous coating

• High sintering temperatures • Thermal expansion results in amorphous coating • Fragile due to thickness

• Differences in coating elastisity

• Interaction with or changes due to the encapsulation material

• Fragility

• High deposition rates • Only coats visible area

• Dense coatings • Unable to coat complex 3D

**Table 2.** Different coating techniques to apply hydroxyapatite (HA) on an implant.

porous substrates

< 1 µm [29]

0.02 - 2 µm [22]

1 - 10 µm [26, 27, 31]

0.1 - 200 µm [25]

30 – 200 µm [25, 28]

0.05 - 2 mm [21, 25]

0.1 – 10 mm [24, 25, 32]

atmosphere

Sol-Gel • Coats 3D complex porous substrates

50 Modern Surface Engineering Treatments

• Low processing temperatures

• Uniform coating thickness on flat

Uniform coating thickness • High deposition rates • Coatscomplex 3D poroussubstrates

Coatings applied quickly • Coatscomplex 3D poroussubstrates

• Relatively cheap • Very thin coatings

Sputter coating • Uniform coating thickness on flat substrates

> substrates • Relatively cheap

Pulsed Laser Deposition

Electrostatic Spray Deposition

Electrophoretic Deposition

Thermal spraying, Plasma spraying

Hot Pressing, Hot Isostatic Pressing and Sintering

Dip Coating • Inexpensive

Since vascularization of infected tissue is often compromised and a bacterial biofilm is formed, which results in poor penetration of antibiotics, systemic antibiotics are not sufficient to treat a bone infection properly [10-12]. To achieve a local dose high enough to treat the infection, this would involve a systemic overdose of the antibiotic (possibly resulting in kidney and liver failure and damage to the function of the inner ear). The best solution to this problem is to have a local delivery system, this suggests an approach for the treatment of orthopaedic infections. Still in many cases the prosthesis can be rescued by infection treatment *in situ*, without a surgical procedure [13, 42, 43]. The use of local antibiotics by antibiotic-loaded bone cement, either as beads or spacers often placed after implant removal in the remaining infected cavity. The general treatment procedure requires at least two surgical procedures, one to 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 a primary hip.

of silver is considered to be antimicrobial and its mode of function is multifactorial. Ionic silver not only reacts easily with amines and microbial DNA to prevent bacterial replication, but also with sulfhydryl groups of metabolic enzymes of the bacterial electron transport chain, resulting in their inactivation [52, 53]. This also forms its treat to large scale clinical applications, since it can also inhibit eukaryotic metabolic function in a patient. Therefore a local release of silver ions is preferable. In contrast to lead and mercury silver does not appear to have cumulative toxic effects in the body, suggesting its potential as a coating component. The use of silver in releasing coatings currently spans from central venous catheters to urinary tract catheters and coated orthopaedic implants, with limited *in vivo* antimicrobial effectiveness as a main problem. While some studies show that a silver coated surface can minimize the infection risks by lowering the bacterial load [54-57], to date, pre-clinical studies and random‐ ized controlled trials of silver coated catheters, implants and external fixation pins were not

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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

This is defined as all testing modalities performed in controlled laboratory conditions, so

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

**• 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].

able to prove its antimicrobial efficacy [52, 58-61].

outside of a living organism or its natural setting (Table 3).

**4. Coating evaluation**

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

*4.1.1. Cytotoxicity tests*

performing an MTT-assay.

categories: *in vitro* and *in vivo* testing.

#### **3.1. Antibiotic releasing coatings**

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 interest in the research and development of such coatings.

#### **3.2. Silver-based coatings**

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 of silver is considered to be antimicrobial and its mode of function is multifactorial. Ionic silver not only reacts easily with amines and microbial DNA to prevent bacterial replication, but also with sulfhydryl groups of metabolic enzymes of the bacterial electron transport chain, resulting in their inactivation [52, 53]. This also forms its treat to large scale clinical applications, since it can also inhibit eukaryotic metabolic function in a patient. Therefore a local release of silver ions is preferable. In contrast to lead and mercury silver does not appear to have cumulative toxic effects in the body, suggesting its potential as a coating component. The use of silver in releasing coatings currently spans from central venous catheters to urinary tract catheters and coated orthopaedic implants, with limited *in vivo* antimicrobial effectiveness as a main problem. While some studies show that a silver coated surface can minimize the infection risks by lowering the bacterial load [54-57], to date, pre-clinical studies and random‐ ized controlled trials of silver coated catheters, implants and external fixation pins were not able to prove its antimicrobial efficacy [52, 58-61].
