What is biofilm?

Peri-implant infections are a particular type of infection produced by a biomaterial in the body. The microorganisms in these infections take advantage of the weak interface between the antibodies and the surface of the implant [11]-[13].

Because planktonic bacteria are associated with orthopedic implants, these infections create multicellular communities on the implant's surface, which are challenging to eradicate. Bacterial adhesion, in general terms, has five stages [5],[14]-[16]:

• Stage one: Planktonic bacteria adhere reversibly to the surface implant. This adhesion depends on characteristics such as the surface material, hydrodynamics, electrostatic interactions, surface roughness, etc. [1].

• Stage two: The adhered bacteria send information to the nearby bacteria and secrete proteins with adhesive components, which makes the adhesion irreversible in a few hours [1].

• Stage three: The bacterial community colonizes the surface and creates a biofilm matrix that will encapsulate the bacteria. This results in a complex structure which will cover the colony [1].

• Stage four: The bacterial colony starts to secrete extracellular polymeric substances (EPS) and develops a complex microorganism known as a mature biofilm. This biofilm consists mainly of bacteria, polysaccharides, proteins, and DNA. In this last stage, some ducts are created to spare more bacteria, spreading the infection to different body parts [1].

Preventing bacterial adhesion and anchorage to the implant's surface is crucial for preventing further infections [17]. Biofilms protect bacteria from the immune system and antibiotics, and, once they have created a biofilm, treating the infection with antibiotics becomes harder [1]. It has been found that the dose of antibiotics needed to treat some bacteria in biofilms is 1 000 times higher than that needed in the first adhesion stage [14], [18]. Many authors such as [19] and [20] have stated that modifying the implant surface, either chemically or physically, may lead to antibacterial surfaces.

Regarding the physical modification of the surface, micro-and nano-topographies are essential in creating antibacterial surfaces. However, the exact values are still unknown [21]-[23].

Surface roughness parameters used to measure bacterial adhesion

Surface roughness is the way in which the topography of a surface can be described in terms of how high the peaks are and how deep the valleys are on a surface after manufacturing. Surface roughness is just one of the many characteristics that influence bacterial adhesion [24]-[28].

Surface roughness can be measured with different parameters: profile roughness parameters (Ra, Rq...) calculated in a profile (line), and area roughness parameters (Sa, Sq...) calculated in a completed area. Profile parameters, especially Ra, are the most commonly reported, but they cannot describe the surface thoroughly, providing a reduced portion of surface information [26]-[28].

Area roughness parameters better describe the surface as a whole. Some parameters can provide specific information about the surface, and their use depends on the application [29]-[32]. The surface roughness parameters described below are reported in the ISO 25178 - 2 standard and are used to measure surface roughness for antibacterial adhesion [33].

• Sa - Arithmetic mean deviation: This parameter is the arithmetic mean of an absolute value corresponding to the height of the sampling area. This parameter does not adequately describe the surface topography because different surfaces with different peaks and valleys can have the same Sa.

• Sq - Squared mean height: This parameter is similar to Sa and is related to the surface energy.

• Ssk - Asymmetry: This parameter describes the distribution of peaks of the topography. The height difference is distributed uniformly if a surface has a Ssk = 0.

• Sku - Kurtosis: This parameter describes the spikiness of the surfaces if the peaks are uniformly distributed.

• Sdr - Surface area ratio: This is one of the most important parameters to describe an antibacterial surface. A surface with a Sdr = 0 is ideally 'smooth'. This parameter can also identify the differences between two surfaces with the same Sa.

The measurement of these parameters is affected by the equipment used. Some studies describe how contact and no-contact equipment affect measurements, so this criterion should be considered in future research [32], [34].

How do nano- and micro-roughness surfaces affect adhesion?

Surface roughness has been widely studied with the aim of preventing bacterial adhesion to metal surfaces. Many authors have described the roughness values found to be the most appropriate in this regard. However, they disagree on each type of topography [13], [35].

Some authors have stated that some surface feature patterns potentially determine the bacteria's early location and adhesion. In this vein, nanoscale topographies with an organized pattern reduce bacterial adhesion by 40-95% [36],[37].

In a study on bacterial adhesion to titanium nitride, [38] found that surfaces with a roughness lower than 0,066 urn had better anti-adhesion properties. Other studies such as [39] argue that the adhesion of bacteria is minimal if the surface has a roughness value or Ra of 160 nm. This study evaluated stainless steel surfaces with Ra between 30 and 890 nm. A different study [41] evaluated five types of surfaces with Ra between 172,5 and 45,2 nm, finding that bacterial adhesion was restrained on the nanoscale surface.

While investigating different polishing surfaces, [41] found that bacteria had less adhesion in metal surfaces with higher porosity. The authors found increased bacterial adhesion in surfaces with a Ra smaller than 0,2 um. In another study, no relation between superficial roughness and bacterial adhesion could be found [42].

In contrast, the authors of [43] argue that there is a negative correlation between bacterial adhesion and metal surfaces with a roughness of less than 6 nm. They found that bacteria prefer a smoother surface under static culture conditions if the roughness is smaller than 0,23-6,13 nm. When the roughness is more significant than 6-30 nm, bacteria prefer surfaces with higher roughness values. The authors also found that a higher roughness does not necessarily benefit adhesion; it will increase the production of EPS to create a biofilm. It will help protect the bacteria because deep valleys trap them and provide cover from shear force [16].

On the other hand, some authors such as [44], [45], and [46], among others, have stated that nano-topography has an antibacterial effect, as peaks with sizes similar to those of the bacteria exert strain forces on the bacterial membrane, causing it to break. This phenomenon is known as the cicada wings effect.

[43] suggests that increasing the surface roughness more significantly than 1,24 urn will increase bacterial adhesion. [47] support this idea, stating that surfaces with a roughness higher than 0,4 um increase the risk of peri-implant diseases. They support the conclusions of [48], i.e., metal surfaces of 130-360 nm are desired in avoiding bacterial adhesion. A similar idea is shared by [49]: surfaces with flaws such as groves, gaps, or cracks provide a favorable environment for bacteria [39]. These authors suggest that bacteria adhere to surfaces that correspond to their size since this maximizes their contact area [50]-[52].

[20] suggest that the adhesion of the bacterium Staphylococcus aureus decreases by using surfaces with a roughness value under 1,51 nm and a unidirectional surface texture. Some related results were obtained [53]. In this study, the adhesion of Escherichia coli and Staphylococcus aureus was reduced by 55,6 and 40,5%, respectively, by using a titanium surface smaller than 6 nm (surface peak density). The study by [15] with the same bacteria found similar results using pattern surfaces of 1 um. A similar result with the same value of roughness was found by [54].

As is the case of [55], some authors agree that, despite the vast amount of works, there is not enough information about the adequate topography. However, in general, the best approximation would be a nanoscale surface roughness with a structure similar structure to that of bone tissue [56].

In [57], by analyzing the way in which bacterial adhesion is evaluated, the authors found that using parameters such as Ra may not be the most accurate approach. Ra is an arithmetic mean or the absolute value of the vertical deviation from the mean line of the profile [31], [58], [59]. In other words, Ra is described as the arithmetic mean deviation of an assessed profile, which is the average of the peaks and valleys of the surface with regard to a centerline [35]. According to this, there could be different surfaces with the same Ra and significant differences in peaks and valleys [60]. Therefore, other superficial roughness parameters should be considered in future research, such as Rq, Rsk, or even area roughness parameters such as Sq, Sq, Ssk, and Sku [61].

Other works [61], [66] have used Sq, Ssk, and Sku to measure surface roughness, finding, for instance, that Staphylococcus epidermis creates biofilms on surfaces with high Ssk values and that smoother surfaces have the best antibacterial properties. Despite not providing an exact value for the surface parameters to create antibacterial surfaces, the findings are consistent with the idea that a surface with low peaks and valleys hinders the adhesion of bacteria.

Moreover, bacteria will avoid surfaces with a roughness smaller than their size, as this increases their contact area, which can damage the bacterial membrane [46], [66].