Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Development of a surface to increase retinal pigment epithelial cell (ARPE-19) proliferation under reduced serum conditions.

Age related macular degeneration of the eye is brought about by damage to the retinal pigment epithelium (RPE) and is a major cause of adult blindness. One potential treatment method is transplantation of RPE cells grown in vitro. Maintaining RPE cell viability and physiological function in vitro is a challenge, and this must also be achieved using materials that can be subsequently used to deliver an intact cell sheet into the eye. In this paper, plasma polymerisation has been used to develop a chemically modified surface for maintaining RPE cells in vitro. Multiwell plates modified with a plasma copolymer of allylamine and octadiene maintained RPE cell growth at a level similar to that of TCPS. However, the addition of bound glycosaminoglycans (GAGs) to the plasma polymerised surface significantly enhanced RPE proliferation. Simply adding GAG to the culture media had no positive effect. It is shown that a combination of plasma polymer and GAG is a promising method for developing suitable surfaces for cell growth and delivery, that can be applied to any substrate material.

Development of a surface to enhance the effectiveness of fibroblast growth factor 2 (FGF-2).

Growth factors (GFs) play an important role in biological processes such as cell proliferation, differentiation and angiogenesis. GFs are known to bind to glycosaminoglycans (GAGs) in the extracellular matrix, aiding projection from degradation and pooling the GFs for quick response to biological stimuli in vivo. GFs are typically expensive and have a relatively short half-life in culture media, requiring regular replenishment. Here the cooperative binding of GF to a plasma polymerised surface decorated with heparin, and the subsequent culture of primary human dermal fibroblasts (HDFs) is investigated. A simple one-step technique suitable for coating a wide range of different substrates was utilised. Substrates such as culture-ware, scaffolds, bandages and devices for implantation could be coated. The modified surface was compared to standard culture techniques of addition of GF to the media. Results demonstrate that surface bound heparin and FGF-2 have a greater effect on cell proliferation especially at reduced serum concentrations. With performance equivalent to supplementing the media achieved at as little as 1% total FGF-2 added. The protective cooperative effect of FGF-2-GAG bound to modified surface at the interface could lead to reduced costs by reduction of FGF-2 required. Furthermore, for applications such as chronic non-healing wounds, bandages can be produced modified by plasma and decorated with GAGs that could utilise and protect important GFs. This would effectively re-introduce important biomolecules which are protected by GAG binding into a harsh environment.

The influence of substrate stiffness gradients on primary human dermal fibroblasts.

Materials mechanical properties are known to be an important regulator of cellular processes such as proliferation, differentiation and migration, and have seen increasing attention in recent years. At present, there are only few approaches where the mechanical properties of thin films can be controllably varied across an entire surface. In this work, we present a technique for controlled generation of gradients of surface elastic moduli involving a weak polyelectrolyte multilayer (PEM) system of approximately 100 nm thickness and time dependent immersion in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Uniform surface chemistry across the gradient and wettability was provided by the addition of a 10 nm thick plasma polymer layer deposited from vapour of either allylamine or acrylic acid. We used the resultant stiffness gradients (0.5-110 MPa in hydrated state) to investigate the adhesion, morphology and proliferation on human dermal fibroblasts (HDFs). We show that substrate mechanical properties strongly influence HDF cell fate. We also found that in the experimental range of surface properties used in this study, the surface stiffness was a stronger driving force to cells fate compared to chemistry and wettability.

Fatherhood status and risk of prostate cancer: nationwide, population-based case-control study.

Previous studies have shown a decreased risk of prostate cancer for childless men; however, the cause of the association remains to be elucidated. The aim of our study was to assess the risk of prostate cancer by fatherhood status, also considering potential confounding factors. In a case-control study in Prostate Cancer data Base Sweden 2.0, a nationwide, population-based cohort, data on number of children, marital status, education, comorbidity and tumor characteristics obtained through nationwide healthcare registers and demographic databases for 117,328 prostate cancer cases and 562,644 controls, matched on birth year and county of residence, were analyzed. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) for prostate cancer overall and by risk category, adjusting for marital status and education. Childless men had a decreased risk of prostate cancer compared to fathers, OR = 0.83 (95% CI = 0.82-0.84), and risk was lower for low-risk prostate cancer, OR = 0.74 (95% CI = 0.72-0.77), than for metastatic prostate cancer, OR = 0.93 (95% CI = 0.90-0.97). Adjustment for marital status and education attenuated the association in the low-risk category, adjusted OR = 0.87 (95% CI = 0.84-0.91), whereas OR for metastatic cancer remained virtually unchanged, adjusted OR = 0.92 (95% CI = 0.88-0.96). Our data indicate that the association between fatherhood status and prostate cancer to a large part is due to socioeconomic factors influencing healthcare-seeking behavior including testing of prostate-specific antigen levels.

Glycosaminoglycan (GAG) binding surfaces for characterizing GAG-protein interactions.

Glycosaminoglycans play an important role in tissue organisation through interactions with a diverse range of proteins, growth factors and other chemokines. In this report, we demonstrate the GAG-binding 'fingerprint' of two important GAG-binding proteins - osteoprotogerin and TIMP-3. The technique uses a straightforward method for attaching GAGs to assay surfaces in a non-covalent manner using plasma polymerization that leaves the adsorbed GAG able to participate in subsequent ligand binding. We show that OPG and TIMP-3 bind preferentially to different GAGs in a simple ELISA and that this binding does not correlate directly with simple GAG properties such as degree of sulfation. The methods outlined in this report can be easily applied to tissue engineering scaffolds in order to exploit the potential of surface-bound GAGs in influencing the structure of engineered tissues.

Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions.

The interactions of glycosaminoglycans (GAGs) with proteins underlie a wide range of important biological processes. However, the study of such binding reactions has been hampered by the lack of a simple frontline analysis technique. Previously, we have reported that cold plasma polymerization can be used to coat microtiter plate surfaces with allyl amine to which GAGs (e.g., heparin) can be noncovalently immobilized retaining their ability to interact with proteins. Here, we have assessed the capabilities of surface coats derived from different ratios of allyl amine and octadiene (100:0 to 0:100) to support the binding of diverse GAGs (e.g., chondroitin-4-sulfate, dermatan sulfate, heparin preparations, and hyaluronan) in a functionally active state. The Link module from TSG-6 was used as a probe to determine the level of functional binding because of its broad (and unique) specificity for both sulfated and nonsulfated GAGs. All of the GAGs tested could bind this domain following their immobilization, although there were clear differences in their protein-binding activities depending on the surface chemistry to which they were adsorbed. On the basis of these experiments, 100% allyl amine was chosen for the generation of a microtiter plate-based "sugar array"; X-ray photoelectron spectroscopy revealed that similar relative amounts of chondroitin-4-sulfate, dermatan sulfate, and heparin (including two selectively de-sulfated derivatives) were immobilized onto this surface. Analysis of four unrelated proteins (i.e., TSG-6, complement factor H, fibrillin-1, and versican) illustrated the utility of this array to determine the GAG-binding profile and specificity for a particular target protein.