Antti Soininen

Poly(dimethylsiloxane) electrospray devices fabricated with diamond-like carbon–poly(dimethylsiloxane) coated SU-8 masters

This study presents coupling of a poly(dimethylsiloxane) (PDMS) micro-chip with electrospray ionization-mass spectrometry (ESI-MS). Stable electrospray is generated directly from a PDMS micro-channel without pressure assistance. Hydrophobic PDMS aids the formation of a small Taylor cone in the ESI process and facilitates straightforward and low-cost batch production of the ESI-MS chips. PDMS chips were replicated with masters fabricated from SU-8 negative photoresist. A novel coating, an amorphous diamond-like carbon-poly(dimethylsiloxane) hybrid, deposited on the masters by the filtered pulsed plasma arc discharge technique, improved significantly the lifetime of the masters in PDMS replications. PDMS chip fabrication conditions were observed to affect the amount of background peaks in the MS spectra. With an optimized fabrication process (PDMS curing agent/silicone elastomer base ratio of 1/8 (w/w), curing at 70 degree C for 48 h) low background spectra were recorded for the analytes. The performance of PDMS devices was examined in the ESI-MS analysis of some pharmaceutical compounds and amino acids.

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.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

Background and purpose Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. Methods We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. Results In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166× progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). Interpretation Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

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.

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.

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.

Cell adhesion and osteogenic differentiation on three-dimensional pillar surfaces

We hypothesized that when compared with conventional two-dimensional (2D) cultures, substrates containing 3D micropillars would allow cells to grow at levels, activating their cytoskeleton to promote osteogenesis. Fibroblasts, osteoblast-like cells, and mesenchymal stem cells (MSCs) were studied. Planar substrates were compared with 200-nm-, 5-μm-, and 20-μm-high pillars of Ormocomp®, Si, diamond-like carbon, or TiO(2). Scanning electron microscopy and staining of actin cytoskeleton showed 7.5-h adhesion to pillar edges and 5-day stretching between adhesion contacts > 100-μm distances of fibroblast and MSC in 3D networks, whereas SaOS-2 cells adhered flatly and individually on horizontal and vertical surfaces. ERK and ROCK immunostaining at 14 and 21 days confirmed activation of the cytoskeleton. In contrast to expectations, success to induce osteogenesis was dominated by the cytocompatibility of the substrate over the 3D structure. This was shown using early alkaline phosphatase, intermediate osteopontin, and late mineralization markers, together with bone nodule formation, which were seen in planar substrates and low-profile TiO(2) pillars, but were poor in the 20-μm landscape. The lack of intercellular contacts seems to halt the osteogenesis-promoting effects of cytoskeletal organization and tension described earlier.

Do changing toll-like receptor profiles in different layers and grades of osteoarthritis cartilage reflect disease severity?

Objective. Cartilage degeneration in osteoarthritis (OA) leads to release of potential danger signals. The aim of our study was to profile OA cartilage for the Toll-like receptor (TLR) danger signal receptors. Methods. Osteochondral cylinders from total knee replacements were graded using OA Research Society International score and stained for proteoglycans, collagenase-cleaved type II collagen, and TLR 1–10, which were analyzed histomorphometrically. Results. Grade 1 OA lesions contained 22%–55% TLR 1–9-positive cells in the surface zone, depending on the TLR type. In Grade 2 TLR, immunoreactivity was 60%–100% (p < 0.01) and it was even higher in Grades 3 and 4 (p < 0.01 vs Grade 1). TLR-positive cells in Grade 1 middle zone were low, 0–19.9%, but were 5.1%–32.7% in Grade 2 (p < 0.01) and 34%–83% in Grades 3–4 samples (p < 0.001). TLR values in Grade 5 were low (14.3%–28.7%; p < 0.001). In Grades 3–4 OA, cartilage matrix stained strongly for TLR. In Grade 1, COL2-3/4M was restricted to chondrocytes, but was increasingly seen in matrix upon progress of OA to Grade 4, and then declined. Conclusion. Cells in the gliding surface zone are fully equipped with TLR in mild OA. Their proportion increases and extends to the middle or even the deep zone, reflecting OA progression. COL2A-3/4M staining suggests Endo180-mediated intake for intralysosomal degradation by cathepsins in Grade 1, but in higher grades this chondrocyte-mediated clearance fails and the matrix demonstrates extensive collagenase-induced damage. Detached and/or partially degraded matrix components can then act as endogenous danger signals (damage-associated molecular patterns or DAMP) and stimulate increasingly TLR-equipped chondrocytes to inflammation. At the peak inflammatory response, soluble TLR may exert negative feedback, explaining in part the low TLR levels in Grade 5 OA.

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.

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.