Cinnamon Oil and Chitosan Coating on Orthopaedic Implant Surface for Prevention of Staphylococcus Epidermidis Biofilm Formation.

S. Epidermidis is among the most frequently isolated microorganisms found in -infection related to implanted devices and the formation of biofilm will be more resistant compared to the planktonic form. This study was carried out to determine the effect of coating on stainless steel orthopaedic implants surfaces with cinnamon oil and chitosan as bioadhesive to prevent biofilms formation of S. Epidermidis.The rod shaped stainless steel 316 L orthopaedic implant with 5 mm diameters was coated 2 times using a mixture of cinnamon oil and chitosan 3% and 2% respectively with serial concentration of cinnamon from 0.125% to 2%. The coated implants were then put into tubes that contained bacterial suspension and incubated. Subsequently, the implants were washed with PBS solution followed by MTT soulution and isopropanol acid solution. that related to biofilm formation. The results were expressed in numbers which represents the absorbance level at ELISA readings on 575 nm (A575) wavelength.The stainless steel implant coated with chitosan and cinnamon oil 2% and 1% has lower absorbance level compared with the absorbance level of S.Epidermidis biofilm only. This study showed that mixture of cinnamon oil and chitosan coated on the surface of stainless steel orthopaedic implant has an effect against S.Epidermidis biofilm formation with the minimum cinnamon oil concentration of 1%.


INTRODUCTION
Staphylococcus Epidermidis (S. Epidermidis) as one of Coagulase-Negative Staphylococci (CoNS) is among the most frequently isolated microorganisms found related to complications following surgical implantation of prosthetic devices 1

. National Nosocomial Infection Surveillance
System reported that CoNS are among the five most commonly reported pathogens in hospitals. Epidemiologically, it is also associated with prosthetic joints and other implanted devices in the community 2 . The incidence of infection following hip surgery using prosthetic device was around 0.06%-11.8% and following open fracture which underwent ORIF, the incidence of infection was around 13.9% 3,4 . Bacteria that attach to implanted devices secrete extracellular polymeric substances, forming the matrix of microbial biofilms. It is a thin layer where the colonisation of bacteria will be 10-1000 fold more resistant to antibiotics and external environment compared with the bacteria in planktonic form 5 . Furthermore, ineffective attempts of polymorphonuclear neutrophils penetration into the biofilm may result in periprosthetic osteolysis specifically for orthopaedic implants due to the release of cytotoxic and proteolytic substances 6 . Therefore, because of such ability of Staphylococcus epidermidis to attach, colonise and form biofilm on the implanted biomaterials surface, it is important to develop and implement therapeutic strategies and effective infection control measures, particularly for the orthopaedic implanted devices 2 . One of the strategies is to coat the orthopaedic implant using Cinnamon oil that has an antimcrobial effect. Cinnamon oil is made from Cinnamonum burminii which is found in Indonesia that also called Indonesian cassia or Cinnamon stick. It is known to have antibacterial effect against several bacteria, such as Streptococcus mutans, Lactobacillus plantarum, and S. Epidermidis 7,8,9 . Chitosan is a natural bio-degradable and non-toxic biopolysaccharide derived from chittin that is widely used in medicine especially in wound management, drug transporter, and implant coatings. It has biocompatibility, bioadhesive, bacteriostatic, antithrombogenic and osteoconductive properties 10,11 . Nowadays, the bioadhesive properties are promising due to their ability to effectively bind to metal substances, to agglutinate a wide variety of mammalian cells, relative safety to human tissue, and the formulation of film related to drug delivery systems 12,13,14 . The application of chitosan as bioadhesive can provide a long stay, adequate drug penetration, high efficienct and acceptability 15 . Chitosan also has a wide spectrum antimicrobial activity, however the outcome depends on various factors 16,17 .
Previous study have shown that cinnamon oil coated onto Kwire has an antimicrobial effect in planktonic level of S. Epidermidis 7 . Therefore, this study was expected to be linked and become the advanced research extension of that study to determine the effect of coating stainless steel orthopaedic implants surfaces with cinnamon oil and chitosan as bioadhesive to prevent biofilms formation of S. Epidermidis and to determine the minimum effective concentration of cinnamon oil in chitosan.

Bacterial isolates
Staphylococcus Epidermidis was used as bacterial isolates in this study. It was obtained from body fluid -collected from Sardjito General Hospital,Yogyakarta, Indonesia, and identified in the Microbiology Department, Universitas Gadjah Mada, Yogyakarta, Indonesia. The bacteria were cultured in Trypticase Soy Broth (TSB) media, incubated for 18 to 24 hours at 37 0 C, and then suspended into tube filled with 10 cc NaCL 0.9% until similar turbidity with McFarland standard solution was achieved 18 . One hundred microliters of the suspension was taken, added with 9.9 ml TSB media, mixed until homogenous to get bacterial concentration of 10 6 CFU/ml.

Antimicrobials
Cinnamon oil was made from cinnamon stick (Cinnamomum Burmanii) obtained from local market in Indonesia with authentification by botanical experts. One kilogram of Cinnamon stick and four litres of water were boiled and connected with modified Clavenger device. The device gathered the oil by steam distillation. Sodium Sulphate Exicatus was added to remove watermarks.
This was an in vitro basic science study using rod shaped stainless steel 316 L orthopaedic implant with 5 mm diameters and 10 mm length. It was coarsened using silica sand with grid blasting method to achieve uniformity of coarseness which was confirmed by surface profilometry. The coating was done twice using a mixture of cinnamon oil and chitosan 3% and 2% respectively with serial concentration of cinnamon from 0.125% to 2%. The coated implants were then put into tubes that contained bacterial suspension and incubated in 37 0 Celcius within 24 hours. Subsequently, the implants were washed by phosphatebuffered saline (PBS) solution followed by MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and afterwards isopropanol acid solution was added to dissolve the MTT solution that had reacted with biofilm.
The results were expressed in numbers which represented the absorbance level at ELISA readings on 575 nm (A575) wavelength. Higher number indicated turbidity resulting from the greater formation of biofilm of the bacteria. The experiment was done three times to seek the average value. For comparison, a tube containing S. Epidermidis biofilm was useed as the standard for biofilm formation, whereas a tube containing TSB only was used as standard for the absence of biofilm formation.

RESULTS
This study showed that stainless steel implant coated with chitosan and cinnamon oil 2% and 1% has lower number, whereas cinnamon oil 0.5%, 0.25%, and 0.125% has higher number compared with the absorbance level of S.Epidermidis biofilm only. It can be concluded that chitosan and cinnamon oil 2% and 1% have less biofilm formation of S. Epidermidis compared with tube filled with full S. Epidermidis biofilm.

DISCUSSION
Coagulase-negative staphylocci constitute the majority of the commensal bacterial microflora and widely distributed throughout the surface of the human body, and therefore it becomes one of the most common species responsible for infection 2 . Biofilm acts as a basic survival mechanism against environmental influences including host immune responses and antimicrobial agents. Moreover, microorganisms within biofilms are less susceptible to antimicrobial treatment compared with their planktonic  19 . Therefore, it is important to prevent formation of biofilm on orthopaedic implanted devices to avoid one of the catastrophic complications in orthopaedic surgery ie. infection 6,12,20 . The use of materials and coatings that release antimicrobials effect can be applied to prevent colonization and consequent biofilm formation. It is considered as one of the practical strategies due to release of antimicrobials effect in lethal concentration for any local planktonic cell before they form biofilm 6 .
Cinnamon is recognised for the presence of cinnalmaldehyde which is highly electronegative and proanthocyanidins that can interfere with biological processes. Both of the components significantly contribute to the antibacterial properties. Due to its safe antibacterial properties, it can be applied as natural food preservatives in many practices 21 . Cinnamon oil has the most potent bactericidal effect against different important pathogens compared to 20 other essential oils used in traditional medicine 21,22 . It has an effective antimicrobial agent including against S. Epidermidis due to biofilm formation inhibition, existing biofilms detachment, and strong bactericidal effect 23 . The effectivity of cinnamon oil in preventing S. Epidermidis biofilm formation has similar effect in terms of its antimicrobial properties compared with antibacterial combination in the form of rifampicin with clindamycin or gentamycin and rifampicin with levofloxacin 20 . In the future, it is expected that cinnamon oil can be used as one of the option in the effort to prevent biofilm formation especially in prosthetic devices.
Previous study about effectiveness of cinnamon oil cream based showed that cinnamon oil has antimicrobial effect against S. Epidermidis biofilm formation when it coated in the K-wire 7 . Compared with that study, this study used chitosan for its adhesive properties. Chitosan can act as good adhesive media due to its abilities to bind with metal. A study conducted by Greene (2008) showed chitosan had the capacity to be sufficiently bonded to stainless steel medical devices. It can also be added with therapeutic antimicrobial agents. The potentiality of chitosan as a coating for orthopaedic devices is proved by biocompatibility, zone of inhibition, and elution tests, whereas functional simulated bone testing suggests the coating strength is sufficient to be used in these applications 12 . Besides that, chitosan can act as a wound dressing material due to its hydrogel-forming properties that have been considered to be advantageous. There are four factors that affect the antimicrobial activity of chitosan. Those factors are microbial factors related to microorganism species and cell age, intrinsic factors of chitosan, physical state, and the environmental factors involving pH and temperature 24 . It also has an antimicrobial activity againsts Gram-positive and Gram-negative bacteria with lower toxicity towards mammalian cells 16,17 . However, the outcome of its antimicrobial properties is severely affected by various intrinsic and extrinsic factors including the physical states of chitosan. Various physical forms of chitosan and its derivates are used in different areas, whereas those in the forms of beads, films, fibers, and hydrogels are commonly used in biomedical applications 24 .
There are several factors that affect the formation of biofilm onto the surface of biomaterial, including the surface roughness 25 . The biofilm formation and its attachment increases as the increase of high surface free energy and/or wettability due to roughness of the surfaces 25,26 . Stainless steel that was used in this study has high surface free energies and it is usually more hydrophillic. Moreover, rough surfaces tend to show greater bacterial attachment and colonisation due to its hydrophilicity 25 . Therefore, the higher level of absorbance showed in the concentration of cinnamon oil 0.5%, 0.25%, and 0.125% might be due to low to none antimicrobial effect of cinnamon oil and the coating of the stainless steel increase the surface roughness.
Although the findings in this study demonstrate the ability of cinnamon oil and chitosan in preventing biofilm formation of S. Epidermidis, further research regarding the durability of cinnamon oil and chitosan at the implant surface and the effect of cinnamon oil and chitosan in the body tissue should be carried out before it can be applied in the clinical settings.

CONCLUSION
This study showed that a mixture of cinnamon oil and chitosan as bioadhesive coated on the surface of stainless steel orthopaedic implant has an effect against S.Epidermidis biofilm formation with the minimum cinnamon oil concentration of 1%.

ACKNOWLEDGMENT
The authors fully acknowedge Titik Nuryastuti MD, PhD whose help, stimulating suggestions, knowledge, and experience helped us at all times of the study.