Sclerostin Alters Tumor Cell Characteristics of Oral Squamous Cell Carcinoma and May Be a Key Player in Local Bone Invasion.

Localized jawbone invasion is a milestone in the progression of oral squamous cell carcinoma (OSCC). The factors that promote this process are not well understood. Sclerostin is known to be involved in bone metabolism and there are preliminary reports of its involvement in bone tumors and bone metastasis. To identify a possible involvement of sclerostin in the bone invasion process of OSCC, sclerostin expression was analyzed in vitro in two different human OSCC tumor cell lines by quantitative real-time polymerase chain reaction (qRT-PCR), and the effect of recombinant human (rh)-sclerostin treatment on tumor cell capabilities was evaluated using proliferation, migration, and invasion assays. Undifferentiated human mesenchymal stem cells (hMSCs) were osteogenically differentiated and co-cultured with OSCC tumor cells to demonstrate potential interactions and migration characteristics. Sclerostin expression was evaluated in clinical cases by immunohistochemistry at the OSCC-jawbone interface in a cohort of 15 patients. Sclerostin expression was detected in both OSCC tumor cell lines in vitro and was also detected at the OSCC-jawbone interface in clinical cases. Tumor cell proliferation rate, migration and invasion ability were increased by rh-sclerostin treatment. The migration rate of tumor cells co-cultured with osteogenically differentiated hMSCs was increased. The results presented are the first data suggesting a possible involvement of sclerostin in the bone invasion process of OSCC, which deserves further investigation and may be a potential approach for drug-based tumor therapy.

Development of a system of heparin multilayers on titanium surfaces for dual growth factor release.

The aim of the present study was to establish a modular platform of poly-L-lysine-heparin (PLL-Hep) polyelectrolyte multilayer (PEM) coatings on titanium surfaces for dual growth factor delivery of recombinant human bone morphogenic protein 2 (rhBMP2) and recombinant human vascular endothelial growth factor 165 (rhVEGF165) in clinically relevant quantities. Release characteristics for both growth factors differed significantly depending on film architecture. rhBMP2 induced activation of alkaline phosphatase in C2C12 cells and proliferation of human mesenchymal stem cells (hMSCs). rhVEGF mediated induction of von Willebrand factor (vWF) in hMSCs and proliferation of human umbilical vein endothelial cells. Osteogenic and angiogenic effects were modified by variation in cross-linking and architecture of the PEMs. By creating multilayer films with distinct zones, release characteristics and proportion of both growth factor delivery could be tuned and surface-activity modified to enhance angiogenic or osteogenic function in various ways. In summary, the system provides a modular platform for growth factor delivery that allows for individual composition and accentuation of angiogenic and osteogenic surface properties.

Porous 3D Scaffolds Enhance MSC Vitality and Reduce Osteoclast Activity.

In the context of an aging population, unhealthy Western lifestyle, and the lack of an optimal surgical treatment, deep osteochondral defects pose a great challenge for the public health system. Biodegradable, biomimetic scaffolds seem to be a promising solution. In this study we investigated the biocompatibility of porous poly-((D,L)-lactide-ε-caprolactone)dimethacrylate (LCM) scaffolds in contrast to compact LCM scaffolds and blank cell culture plastic. Thus, morphology, cytotoxicity and metabolic activity of human mesenchymal stromal cells (MSC) seeded directly on the materials were analyzed after three and six days of culturing. Further, osteoclastogenesis and osteoclastic activity were assessed using reverse-transcriptase real-time PCR of osteoclast-specific genes, EIA and morphologic aspects after four, eight, and twelve days. LCM scaffolds did not display cytotoxic effects on MSC. After three days, metabolic activity of MSC was enhanced on 3D porous scaffolds (PS) compared to 2D compact scaffolds (CS). Osteoclast activity seemed to be reduced at PS compared to cell culture plastic at all time points, while no differences in osteoclastogenesis were detectable between the materials. These results indicate a good cytocompatibility of LCM scaffolds. Interestingly, porous 3D structure induced higher metabolic activity of MSC as well as reduced osteoclast activity.

Biomimetic Designer Scaffolds Made of D,L-Lactide-ɛ-Caprolactone Polymers by 2-Photon Polymerization.

IMPACT STATEMENT: In tissue engineering (TE), the establishment of cell targeting materials, which mimic the conditions of the physiological extracellular matrix (ECM), seems to be a mission impossible without advanced materials and fabrication techniques. With this in mind we established a toolbox based on (D,L)-lactide-ɛ-caprolactone methacrylate (LCM) copolymers in combination with a nano-micromaskless lithography technique, the two-photon polymerization (2-PP) to mimic the hierarchical structured and complex milieu of the natural ECM. To demonstrate the versatility of this toolbox, we choose two completely different application scenarios in bone and tumor TE to show the high potential of this concept in therapeutic and diagnostic application.

Biomimetic multilayer coatings deliver gentamicin and reduce implant-related osteomyelitis in rats.

Implant-related infections like periprosthetic joint infections (PJI) are still a challenging issue in orthopedic surgery. In this study, we present a prophylactic anti-infective approach based on a local delivery of the antibiotic gentamicin. The local delivery is achieved via a nanoscale polyelectrolyte multilayer (PEM) coating that leaves the bulk material properties of the implant unaffected while tuning the surface properties. The main components of the coating, i.e. polypeptides and sulfated glycosaminoglycans (sGAG) render this coating both biomimetic (matrix mimetic) and biodegradable. We show how adaptions in the conditions of the multilayer assembly process and the antibiotic loading process affect the amount of delivered gentamicin. The highest concentration of gentamicin could be loaded into films composed of polypeptide poly-glutamic acid when the pH of the loading solution was acidic. The concentration of gentamicin on the surface could be tailored with the number of deposited PEM layers. The resulting coatings reveal a bacteriotoxic effect on Staphylococcus cells but show no signs of cytotoxic effects on MC3T3-E1 osteoblasts. Moreover, when multilayer-coated titanium rods were implanted into contaminated medullae of rat tibiae, a reduction in the development of implant-related osteomyelitis was observed. This reduction was more pronounced for the multifunctional, matrix-mimetic heparin-based coatings that only deliver lower amounts of gentamicin.

Influence of the Microstructure and Silver Content on Degradation, Cytocompatibility, and Antibacterial Properties of Magnesium-Silver Alloys In Vitro.

Implantation is a frequent procedure in orthopedic surgery, particularly in the aging population. However, it possesses the risk of infection and biofilm formation at the surgical site. This can cause unnecessary suffering to patients and burden on the healthcare system. Pure Mg, as a promising metal for biodegradable orthopedic implants, exhibits some antibacterial effects due to the alkaline pH produced during degradation. However, this antibacterial effect may not be sufficient in a dynamic environment, for example, the human body. The aim of this study was to increase the antibacterial properties under harsh and dynamic conditions by alloying silver metal with pure Mg as much as possible. Meanwhile, the Mg-Ag alloys should not show obvious cytotoxicity to human primary osteoblasts. Therefore, we studied the influence of the microstructure and the silver content on the degradation behavior, cytocompatibility, and antibacterial properties of Mg-Ag alloys in vitro. The results indicated that a higher silver content can increase the degradation rate of Mg-Ag alloys. However, the degradation rate could be reduced by eliminating the precipitates in the Mg-Ag alloys via T4 treatment. By controlling the microstructure and increasing the silver content, Mg-Ag alloys obtained good antibacterial properties in harsh and dynamic conditions but had almost equivalent cytocompatibility to human primary osteoblasts as pure Mg.

Analysis of the influence of the macro- and microstructure of dental zirconium implants on osseointegration: a minipig study.

OBJECTIVES: It was the aim of this study to analyze the influence of implant design and surface topography on the osseointegration of dental zirconium implants. STUDY DESIGN: Six different implant designs were tested in the study. Nine or 10 test implants were inserted in the frontal skull in each of 10 miniature pigs. Biopsies were harvested after 2 and 4 months and subjected to microradiography. RESULTS: No significant differences between titanium and zirconium were found regarding the microradiographically detected bone-implant contact (BIC). Cylindric zirconium implants showed a higher BIC at the 2-month follow-up than conic zirconium implants. Among zirconium implants, those with an intermediate Ra value showed a significantly higher BIC compared with low and high Ra implants 4 months after surgery. CONCLUSIONS: Regarding osseointegration, titanium and zirconium showed equal properties. Cylindric implant design and intermediate surface roughness seemed to enhance osseointegration.

Investigations on the secondary structure of polypeptide chains in polyelectrolyte multilayers and their effect on the adhesion and spreading of osteoblasts.

Inspired by the composition of the native extracellular matrix, biomimetic polyelectrolyte multilayers were assembled from polypeptides and the glycosaminoglycan chondroitin sulfate (CS). To investigate whether peptide conformation imposes an effect on the cell biological functions of osteoblasts, the secondary structure was analyzed by in situ infra-red and circular dichroism spectroscopy. Multilayers composed of polypeptides and CS reveal a predominantly random coiled conformation and impede osteoblast spreading. On the contrary, polypeptide chains in assemblies of poly-L-lysine and poly-L-glutamic acid (PGA) primarily adopt an intermolecular ß sheet structure and reveal an increased area of spread, which consequently supports the proliferation of osteoblasts. When CS is replaced by PGA in mixed multilayers, we observe a structural rearrangement from random coils to ß sheets with a concomitant improved cell response. We conclude that polypeptide conformation in biomimetic multilayer assemblies affects osteoblast response by altering the stiffness of the multilayer.

Colloidal force spectroscopy and cell biological investigations on biomimetic polyelectrolyte multilayer coatings composed of chondroitin sulfate and heparin.

To promote osteoblast adhesion and proliferation on (bio)material surfaces, biomimetic coatings resembling the natural extracellular matrix (ECM) are desirable. The glycosamino glycans (GAGs) chondroitin sulfate (CS) and heparin (HEP) are promising candidates for a biomimetic coating since they are two of the most prevalent noncollagenous biomolecules constituting the ECM. Coatings containing CS and HEP were prepared employing the "layer by layer" technique yielding polyelectrolyte multilayers (PEMs). Physicochemical and mechanical characterization of the coatings were performed by means of streaming potential measurements and colloidal force spectroscopy. The capability of the coatings to support cell adhesion, spreading, proliferation, and maintenance of an osteoblastic phenotype was assessed with SaOS osteosarcoma cells. We demonstrate that PEMs constructed from CS as the polyanion display a low Young's modulus correlated with poorly supported cell adhesion and proliferation. When the CS was adsorbed onto a stiffer polypeptide PEM basis, the Young's modulus increased, and the cell response was significantly improved. For HEP coatings an intermediate Young's modulus and moderate cell adhesion and spreading were observed. No significant changes in stiffness or cell response were detected when HEP was adsorbed onto the polypeptide film.

Improved initial osteoblast functions on amino-functionalized titanium surfaces.

Adhesion and spreading of cells on biomaterials are integrin-mediated processes. But recent findings indicate a key role of the cell membrane associated matrix substance hyaluronan (HA) in interface interactions. Because HA is a negatively charged molecule we assume that a biomaterial surface with an opposed charge could boost the first contact of the cell to the surface. Polished cp titanium (R(a)=0.19 microm) was coated with an amino-group containing plasma polymer (Ti PPA). For this purpose, a microwave excited, pulsed, low-pressure plasma was used. Additionally, collagen was immobilized on Ti PPA with polyethylene glycol diacid (PEG-DA), catalyzed by carbodiimide (CDI). The physico-chemical surface analytical techniques like XPS, FT-IR, water contact angle and zeta-potential verified the retention of the allylamine precursor structure. Human osteoblasts were cultured in serum-free Dulbecco's modified Eagle medium (DMEM). Adhesion and cell cycle phases were calculated by flow cytometry. Spreading and actin cytoskeleton were visualized by confocal microscopy. Gene expression of osteogenic markers was detected by real-time RT-PCR. Ti PPA is significantly advantageous concerning initial adhesion and spreading during the first hours of the cell contact to the surface. The proliferation of osteoblasts is positively influenced. Gene expression of the differentiation marker bone sialoprotein was upregulated after 24h. Our results demonstrate that functionalization of titanium with positively charged amino-groups is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface.