Roel Kuijer

Cobalt and chromium ions reduce human osteoblast-like cell activity in vitro, reduce the OPG to RANKL ratio, and induce oxidative stress.

Metal‐on‐metal hip arthroplasty is associated with elevated levels of cobalt and chromium ions. The effects of cobalt and chromium ions on cell number, activity, expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) and oxidative stress on human osteoblast‐like cells were addressed. Saos‐2 cells were supplemented with Co2+, Cr3+, or Co2+ + Cr3+ (1:2) at 0, 1, 10, and 100 µg/L and incubated for 24, 48, 72, and 96 h. Cell activity was assessed by MTT‐assay and cell number by Crystal Violet staining. RNA levels of OPG and RANKL were evaluated using real‐time quantitative polymerase chain reaction (qPCR). Compared to controls Co2+ reduced cell numbers: at 10 µg/L by 17 ± 8% after 48 h and at 100 µg/L after 24 h by 35 ± 8%. Cr3+ decreased cell numbers at 10 µg/L after 48 and 72 h. Co2+ + Cr3+ combined at 1 µg/L lowered cell numbers after 24 and 96 h (17 ± 13, resp. 13 ± 4%). The 10 and 100 µg/L concentrations reduced cell numbers significantly after 24, 48, and 96 h. Cr3+ reduced osteoblast activity at 1, 10, and 100 µg/L at all incubation times. The strongest reduction (11 ± 1%) was seen at 100 µg/L after 96 h. The OPG/RANKL ratio was reduced after 72 h with almost all Co2+ and Cr3+ concentrations. After 96 h, glutathione, superoxide dismutase, and catalase levels were indicative for an oxidative stress response in all samples. In conclusion, cobalt and chromium ions reduce human osteoblast activity, reduce OPG/RANKL ratio and lead to oxidative stress.

Microbial biofilm growth versus tissue integration on biomaterials with different wettabilities and a polymer-brush coating.

Biomaterials-associated infections (BAI) constitute a major clinical problem and often necessitate implant replacement. In this study, the race for the surface between Staphylococcus epidermidis ATCC 35983 and U2OS osteosarcoma cells is studied on biomaterials with different wettabilities and on a polymer-brush coating. S. epidermidis was deposited on the different surfaces in a parallel plate flow chamber and then U2OS cells were seeded. Subsequently, staphylococci and U2OS cells were allowed to grow simultaneously on the surfaces for 48 h under low flow conditions. The presence of staphylococci reduced cell growth on all surfaces, but adhering cells spread equally well in the absence and presence of staphylococci. A hydrophilic polymer-brush coating discouraged bacterial and cellular adhesion and growth. Thus, whereas the biomaterials evaluated support both biofilm formation and tissue integration, polymer-brush coatings support neither. Therewith, the outcome of the race for the surface on these surfaces remains uncertain, emphasizing the need for biofunctionalized surfaces that discourage biofilm formation and support tissue growth at the same time.

In Vitro Interactions between Bacteria, Osteoblast-Like Cells and Macrophages in the Pathogenesis of Biomaterial-Associated Infections

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.

Mammalian cell growth versus biofilm formation on biomaterial surfaces in an in vitro post-operative contamination model

Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a new in vitro model for the post-operative bacterial contamination of implant surfaces and investigate the effects of contamination on the balance between mammalian cell growth and bacterial biofilm formation. U2OS osteosarcoma cells were seeded on poly(methyl methacrylate) in different densities and allowed to grow for 24 h in a parallel-plate flow chamber at a low shear rate (0.14 s(-1)), followed by contamination with Staphylococcus epidermidis ATCC 35983 at a shear rate of 11 s(-1). The U2OS cells and staphylococci were allowed to grow simultaneously for another 24 h under low-shear conditions (0.14 s(-1)). Mammalian cell growth was severely impaired when the bacteria were introduced to surfaces with a low initial cell density (2.5 × 10(4) cells cm(-2)), but in the presence of higher initial cell densities (8.2 × 10(4) cells cm(-2) and 17 × 10(4) cells cm(-2)), contaminating staphylococci did not affect cell growth. This study is believed to be the first to show that a critical coverage by mammalian cells is needed to effectively protect a biomaterial implant against contaminating bacteria.

Both Hyaluronan and Collagen Type II Keep Proteoglycan 4 (Lubricin) at the Cartilage Surface in a Condition That Provides Low Friction during Boundary Lubrication

Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superficial zone (SZ). This study aimed at better understanding the mechanism of PRG4 localization at the cartilage surface. We show direct interactions between surface bound HA and freely floating PRG4 using the quartz crystal microbalance with dissipation (QCM-D). Freely floating PRG4 was also shown to bind with surface bound collagen type II fibrils. Albumin, the most abundant protein of the synovial fluid, effectively blocked the adsorption of PRG4 with HA, through interaction with C and N terminals on PRG4, but not that of PRG4 with collagen type II fibrils. The above results indicate that collagen type II fibrils strongly contribute in keeping PRG4 in the SZ during cartilage articulation in situ. Furthermore, PRG4 molecules adsorbed very well on mimicked SZ of absorbed HA molecules with entangled collagen type II fibrils and albumin was not able to block this interaction. In this last condition PRG4 adsorption resulted in a coefficient of friction (COF) of the same order of magnitude as the COF of natural cartilage, measured with an atomic force microscope in lateral mode.

Treatment of the degenerated intervertebral disc; closure, repair and regeneration of the annulus fibrosus

Degeneration of the intervertebral disc (IVD) and disc herniation are two causes of low back pain. The aetiology of these disorders is unknown, but tissue weakening, which primarily occurs due to inherited genetic factors, ageing, nutritional compromise and loading history, is the basic factor causing disc degeneration. Symptomatic disc herniation mainly causes radicular pain. Current treatments of intervertebral disc degeneration and low back pain are based on alleviating the symptoms and comprise administration of painkillers or surgical methods such as spinal fusion. None of these methods is completely successful. Current research focuses on regeneration of the IVD and particularly on regeneration of the nucleus pulposus. Less attention has been directed to the repair or regeneration of the annulus fibrosus, although this is the key to successful nucleus pulposus, and therewith IVD, repair. This review focuses on the importance of restoring the function of the annulus fibrosus, as well as on the repair, replacement or regeneration of the annulus fibrosus in combination with restoration of the function of the nucleus pulposus, to treat low back pain. Copyright

recA mediated spontaneous deletions of the icaADBC operon of clinical Staphylococcus epidermidis isolates : a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC.

Human periosteum-derived cells from elderly patients as a source for cartilage tissue engineering?

The aim of this study was to establish the potential of human periosteum‐derived cells from elderly patients as a cell source for cartilage tissue engineering by optimizing culture conditions for both proliferation and differentiation. Periosteum was obtained from the tibiae of nine patients. Biopsies were prepared for routine histological examination. Periosteum‐derived cells were allowed to grow out from the remaining tissue, and were expanded in minimum essential medium containing D‐valine (MEM–DV). Fetal bovine serum (FBS) or substitutes, fibroblast growth factor‐2 (FGF‐2), insulin‐like growth factor‐1 (IGF‐1) and non‐essential amino acids were added to study proliferation. For differentiation of cells, serum‐free medium was used supplemented with one or more isoforms of transforming growth factor‐β (TGFβ) and/or IGF‐1. Samples were analysed for expression of collagens type I, II and X by competitive RT–PCR, immunohistochemically, and histologically using Alcian blue staining. In all samples the cambium layer could hardly be detected. Periosteum‐derived cells proliferated in serum‐containing MEM–DV. Optimal proliferation was found when this medium was supplemented with 100 ng/ml FGF‐2 and non‐essential amino acids. Chondrogenesis was detected in 59% of micromasses that were cultured with TGFβ isomers, and in 83% of the samples cultured in media to which two TGFβ isoforms were added. Periosteum from elderly humans (mean age 66, range 41–76 years) has chondrogenic potential and remains an attractive cell source for cartilage tissue engineering. By expanding cells in MEM–DV, the selection of progenitor cells might be favoured, which would result in a higher cartilage yield for tissue engineering applications. Copyright

Enzymatic Breakdown of Type II Collagen in the Human Vitreous

PURPOSE To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in the vitreous. Western blot and slot blot analyses were used to further identify vitreal type II collagen breakdown products in three age groups with average ages of 25, 45, and 65 years. Purified type II collagen was cleaved by MMPs that are known to occur naturally in the vitreous to elucidate what possible type II collagen breakdown products could thus be formed in the human vitreous. RESULTS By means of both immunohistochemistry and slot blot analysis, col2-3/4C-short was detected in the vitreous. Using Western blot analysis, a range of type II collagen breakdown products was found, mostly in younger eyes, but none of these products contained the neoepitope that characterizes the col2-3/4C-short molecule. Digestion of purified type II collagen by MMPs did not give the same breakdown products as found in the vitreous. CONCLUSIONS The presence of collagen degradation products in the human vitreous supports the hypothesis that enzymatic breakdown is most likely an active process in this extracellular matrix. Based on the size of the degradation products found by Western blot analysis, it is likely that in addition to MMPs, other proteolytic enzymes able to digest type II collagen are also active.

Control of oxygen release from peroxides using polymers

An important limitation in cell therapy for the regeneration of tissue is the initial lack of oxygen. After implantation of large 3D cell-seeded structures, cells die rather than contribute to tissue regenerating. Here we’ve tested oxygen-releasing materials to improve cell survival and growth after implantation. Calcium peroxide (CaO2) in a polymer matrix was used as source of oxygen. Two polymers were tested in order to slow down and extend the period of oxygen release, poly(D,L-lactic acid) and poly(lactic-co-glycolic acid). Compared to CaO2 particles, both releasing systems showed an initially higher and shorter oxygen release. Human mesenchymal stromal cells cultured on casted films of these oxygen-releasing composites required catalase to proliferate, indicating the production of cytotoxic hydrogen peroxide as intermediate. Poly(D,L-lactic acid) and poly(lactic-co-glycolic acid) are less suited for slowly oxygen-releasing materials. Catalase was able to reduce the cytotoxic effect of H2O2.Graphical Abstract