Prevention of Biomaterial Infection by Pre-Operative Incubation with Human Cells.

BACKGROUND: Cells of tissues and biofilm forming bacteria compete for the living space on the surface of an implant. We hypothesized the incubation of the implant (titanium, polydimethylsiloxane, and polystyrene surface) with human cells before implantation as a strategy to prevent bacterial adhesion and biofilm formation. METHODS: After 24 hours of incubation with human osteogenic sarcoma SaOS-2 cells (1 × 105 cells/mL), the materials were incubated for 4.5 hours or two days with Staphylococcus aureus in serial 1:10 dilutions of 108 colony-forming units/mL. The bacterial adherence and biofilm biomass on materials pre-incubated with SaOS-2 cells were compared with our previous results on materials incubated only with bacteria or in simultaneous co-culture of SaOS-2 cells and S. aureus. Fluorescent microscopy and crystal violet stain were used. The number of viable SaOS-2 and bacterial cells present was tested using colorimetric methods (MTT, LDH) and drop plate method, respectively. RESULTS: The pre-treatment with human cells was associated with a reduction of bacterial colonization of the biomaterial at 4.5 hours and 48 hours compared with the non-pre-treated materials. The presence of bacteria decreased the number of viable human cells on all materials. ( Supplementary Fig. 1 ; see online supplementary materials at www.liebertpub.com/sur ). CONCLUSIONS: These results suggest that the pre-operative incubation of prostheses with host cells could prevent infection of biomaterials.

Competitive colonization of prosthetic surfaces by staphylococcus aureus and human cells.

Implantation of a biomaterial provides an adhesion substratum both to host cell integration and to contaminating bacteria. We studied simultaneous competitive adhesion of Staphylococcus aureus in serial 1:10 dilutions of 108 colony forming units (CFU)/mL and human osteogenic sarcoma (SaOS-2) or primary osteoblast (hOB) cells, both 1x105 cells/mL, to the surfaces of titanium, polydimethylsiloxane and polystyrene. The bacterial adherence and human cell proliferation, cytotoxicity and production of reactive oxygen species (ROS) were studied using fluorometric (fluorescent microscopy and flow cytometry) and colorimetric methods (MTT, LDH and crystal violet). The bacterial cell viability was also evaluated using the drop plate method. The presence of bacteria resulted in reduced adherence of human cells to the surface of the biomaterials, increased production of ROS, and into increased apoptosis. On the other hand, the presence of either type of human cells was associated with a reduction of bacterial colonization of the biomaterial with Staphylococcus aureus. These results suggest that increasing colonization of the biomaterial surface in vitro by one negatively affects colonization by the other. Host cell integration to an implant surface reduces bacterial contamination, which opens novel opportunities for the design of infection-resistant biomaterials in current implantology and future regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 62-72, 2017.

Osteogenic differentiation on DLC-PDMS-h surface.

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion-growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use.

Formation and retention of staphylococcal biofilms on DLC and its hybrids compared to metals used as biomaterials.

Staphylococcus epidermidis and Staphylococcus aureus cause most of the implant-related infections. Antibiotic treatment often fails and cure requires surgical intervention. It was hypothesized that biomaterial coatings resistant to biofilms offer a preventive option. Physical vapour deposited diamond-like carbon (DLC) and its polytetrafluoroethylene (DLC-PTFE-h) and polydimethylsiloxane (DLC-PDMS-h) hybrids were compared to titanium (Ti), tantalum (Ta) and chromium (Cr) thin films on silicon wafers for their resistance against formation and/or retention of biofilms produced by S. epidermidis and S. aureus in vitro. Sample surfaces were characterized for surface topography, contact angle and zeta-potential, because such properties might affect the biofilm. Biofilm was stained using calcofluor white and analysed in fluorescence microscopy using morphometry. Sixteen hour incubation was selected in pilot tests; at this checkpoint Ti, Ta, Cr and DLC-PDMS-h were almost fully covered by biofilm, but DLC and DLC-PTFE-h were only partially biofilm coated by S. epidermidis (88±26%, p<0.001 and 56±39%, p<0.001, respectively) or S. aureus (81±24%, p<0.001 and 51±26%, p<0.001, respectively). DLC and its PTFE hybrid offer a potential biofilm hostile surface coating for implants and medical devices. This ability to resist biofilm formation and attachment could not be explained by only one factor, but it seems to be related to a combination of various properties, with electrokinetic streaming potential and protein coating being particularly important for its outcome.

In vitro adhesion of staphylococci to diamond-like carbon polymer hybrids under dynamic flow conditions.

This study compares the ability of selected materials to inhibit adhesion of two bacterial strains commonly implicated in implant-related infections. These two strains are Staphylococcus aureus (S-15981) and Staphylococcus epidermidis (ATCC 35984). In experiments we tested six different materials, three conventional implant metals: titanium, tantalum and chromium, and three diamond-like carbon (DLC) coatings: DLC, DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) and DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) coatings. DLC coating represents extremely hard material whereas DLC hybrids represent novel nanocomposite coatings. The two DLC polymer hybrid films were chosen for testing due to their hardness, corrosion resistance and extremely good non-stick (hydrophobic and oleophobic) properties. Bacterial adhesion assay tests were performed under dynamic flow conditions by using parallel plate flow chambers (PPFC). The results show that adhesion of S. aureus to DLC-PTFE-h and to tantalum was significantly (P < 0.05) lower than to DLC-PDMS-h (0.671 ± 0.001 × 10(7)/cm(2) and 0.751 ± 0.002 × 10(7)/cm(2) vs. 1.055 ± 0.002 × 10(7)/cm(2), respectively). No significant differences were detected between other tested materials. Hence DLC-PTFE-h coating showed as low susceptibility to S. aureus adhesion as all the tested conventional implant metals. The adherence of S. epidermidis to biomaterials was not significantly (P < 0.05) different between the materials tested. This suggests that DLC-PTFE-h films could be used as a biomaterial coating without increasing the risk of implant-related infections.

Interactions of human bone cells with diamond-like carbon polymer hybrid coatings.

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin-TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.

Bacterial adhesion to diamond-like carbon as compared to stainless steel.

Recent studies suggest that diamond-like carbon (DLC) coatings are suitable candidates for application on biomedical devices and implants, due to their high hardness, low friction, high wear and corrosion resistance, chemical inertness, smoothness, and tissue and blood compatibility. However, most studies have neglected the potential susceptibility of DLC coatings to bacterial adhesion, which is the first step in the development of implant-related infections. This study compares adhesion of seven bacterial strains, commonly implicated in implant-related infections, to tetrahedral amorphous carbon, with their adhesion to AISI 316L surgical steel. The results show that bacterial adhesion to DLC was similar to the adhesion to commonly used stainless steel. This suggests that DLC coating can be advantageously used on implants made of AISI 316L or other materials without increasing the risk to implant-related infections.

Load-bearing biomedical applications of diamond-like carbon coatings - current status.

The current status of diamond-like carbon (DLC) coatings for biomedical applications is reviewed with emphasis on load-bearing coatings. Although diamond-like carbon coating materials have been studied for decades, no indisputably successful commercial biomedical applications for high load situations exist today. High internal stress, leading to insufficient adhesion of thick coatings, is the evident reason behind this delay of the break-through of DLC coatings for applications. Excellent adhesion of thick DLC coatings is of utmost importance for load-bearing applications. According to this review superior candidate material for articulating implants is thick and adherent DLC on both sliding surfaces. With the filtered pulsed arc discharge method, all the necessary requirements for the deposition of thick and adherent DLC are fulfilled, provided that the substrate material is selected properly.

Adhesion of staphylococcal and Caco-2 cells on diamond-like carbon polymer hybrid coating.

Staphylococci cause the majority of the nosocomial implant-related infections initiated by adhesion of planktonic bacteria to the implant surface. It was hypothesized that plasma accelerating filtered pulsed arc discharge method enables combination of the advantageous properties of diamond with the antisoiling properties of polymers. Diamond-like carbon polytetrafluoroethylene hybrid (DLC-PTFE-h) coating was produced. The adhesion of S. aureus ATCC 25923 (10(8) colony-forming units/mL) to surfaces diminished from 2.32%, 2.35%, and 2.57% of high quality DLC, titanium, and oxidized silicon, respectively, to 1.93% of DLC-PTFE-h. For S. epidermidis ATCC 35984 the corresponding figures were 3.90%, 3.32%, 3.47%, and 2.57%. Differences in bacterial adhesion between recombinant DLC-PTFE-h and other materials were statistically significant (p < 0.05). In contrast, human Caco-2 cells adhered as well to DLC-PTFE-h as to DLC, titanium, or silicon, which were all in the MTT test found to be cytocompatible. DLC-PTFE-h coating can be used to modify the surface properties of any surgical implants and is an unfavorable substrate for staphylococcal cells, but compatible with human Caco-2 cells. DLC-PTFE-h coating may help in the combat against Staphylococcus-related implant infections which usually require both antibiotics and surgical removal of the implant for cure.

Toll-like receptors in the interface membrane around loosening total hip replacement implants.

Toll-like receptors (TLRs) have been known to act as sensors of innate immunity and respond to ligands of microbial and endogenous components. Tissues and cells typical for interface membrane of foreign body reaction were analyzed to evaluate potential role of TLRs in the pathogenesis of the so called "aseptic loosening of total hip replacement." Fourteen cases of interface membrane around aseptic loose total hip replacement implants were stained by single and double immunohistochemical methods to examine cellular localization of toll-like receptor (TLR)-4 and TLR-9. Osteoarthritic synovium was used as control tissues. Cultured macrophages were used to study TLR-4 and TLR-9 mRNA levels by quantitative reverse transcriptase-polymerase chain reaction. The effect of titanium particle stimulation on macrophages was also examined in the culture. Extensive immunolocalization of TLR-4 and TLR-9 positive cells was observed in the synovial membrane-like interface membrane of foreign body granulomas compared with control synovial membranes. TLR and CD68 double staining demonstrated that the TLR positive cells in aseptic loosening were mostly monocyte/macrophages and foreign body giant cells. TLR-4 and TLR-9 mRNA expression was also found in macrophage-colony stimulating factor treated rat macrophages, but this expression decreased (p < 0.05 or less) upon stimulation with titanium particles although matrix metalloproteinase (MMP)-9 mRNA levels used as macrophage activation marker were increased (p = 0.01). The interface membrane around loosening total hip replacement implants is apparently well equipped with TLRs and, thus, probably very sensitive to various structural components of microbes and to endogenous TLR ligands. This seems to be due to recruitment of monocyte/macrophages as particles per se seemed to down-regulate some of the key TLRs. This suppression after particle phagocytosis might prevent excessive and harmful host responses, and injury to innocent bystander cells/tissues.

Innervation of the joint and role of neuropeptides.

Rheumatoid arthritis is considered to represent a disease of the synovial membrane, osteoarthritis of the hyaline articular cartilage, and osteoporosis of the bone. It can be questioned to what extent this is true and to what extent these diseases could be considered to be due to extra-articular, extra-skeletal pathology related to the neuroendocrine system. Pain is the main symptom in arthritis. This is related to prostaglandin-mediated sensitization of the primary afferent nociceptive nerves. Accordingly, nonsteroidal anti-inflammatory drugs are used in symptomatic treatment, occasionally together with opioids and tricyclic antidepressants. The midline symmetry and involvement of the richly innervated, small peripheral joints in rheumatoid arthritis have raised speculation about the role of neurogenic inflammation and neuropeptides in its pathogenesis. In contrast to the free nerve endings, the role of the proprioceptive sensors is to provide information of our actual motor performance (the afferent copy of our movements) compared to the efferent motor program, which is activated by our will to move. These include proprioceptors in the skin (e.g., Meissner corpuscles), muscles (annulospiral and flower-spray endings of the muscle spindles), Golgi tendon organs, and Ruffini end organs and Pacinian corpuscles in the superficial and deep layers of the joint capsule. Elderly people may have slow reflexes, lax joints, joint incongruity, and loss of muscle power; obesity, alcohol and medicinal use, and joint pain can be combined with poor/nonexisting capacity for repair and remodeling of the musculoskeletal tissues. Impaired biomechanics contributes to increased joint tenderness, accumulation of minor trauma (secondary osteoarthritis), and falls (osteoporotic fractures). More attention needs to be paid to aging of proprioception, not only to the terminal disease target.

Tantalum as a buffer layer in diamond-like carbon coated artificial hip joints.

The acid resistance of tantalum coated and uncoated human hip joint prostheses was studied with commercial CrCoMo acetabular cups. The samples were exposed to 10% HCl solution and the quantities of dissolved Cr, Co, and Mo were measured with proton-induced X-ray emission (PIXE). The absolute quantities were obtained with the use of Cr and Se solution standards. Tantalum coatings (thicknesses 4-6 microm) were prepared in vacuum with magnetron sputtering. Tantalum coating decreased the corrosion rate by a factor of 10(6). As a spinoff from recent wear tests on artificial hip joints it was shown that tantalum has excellent mechanical properties as an intermediate layer of diamond-like carbon (DLC) coatings. When tantalum was tested together with DLC on three metal-on-metal hip joint pairs in a hip simulator, no observable defects occurred during 15 million walking cycles with a periodic 50-300-kg load (Paul curve).