Theo G. van Kooten

Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats

Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibroblast population in relation to fibrotic changes after IRI in rats was investigated. A model of unilateral renal IRI (45 min of ischemia) was used in F344 rats that were reconstituted with R26-human placental alkaline phosphatase transgenic BM to quantify BMDC contribution to the renal interstitial myofibroblast population over time. After IRI, transient increases in collagen III transcription and interstitial protein deposition were observed, peaking on days 7 and 28, respectively. Interstitial infiltrates of BMDC and myofibroblasts reached a maximum on day 7 and gradually decreased afterward. Over time, an average of 32% of all interstitial alpha-smooth muscle actin-positive myofibroblasts coexpressed R26-human placental alkaline phosphatase and, therefore, were derived from the BM. BMD myofibroblasts produced procollagen I protein and therefore were functional. The postischemic kidney environment was profibrotic, as demonstrated by increased transcription of TGF-beta and decreased transcription of bone morphogenic protein-7. TGF-beta protein was present predominantly in interstitial myofibroblasts but not in BMD myofibroblasts. In conclusion, functional BMD myofibroblasts infiltrate in the postischemic renal interstitium and are involved in extracellular matrix production.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Biodegradable nanocomposite hydrogel structures with enhanced mechanical properties prepared by photo-crosslinking solutions of poly(trimethylene carbonate)–poly(ethylene glycol)–poly(trimethylene carbonate) macromonomers and nanoclay particles☆

Soft hydrogels with elasticity modulus values lower than 100kPa that are tough and biodegradable are of great interest in medicine and in tissue engineering applications. We have developed a series of soft hydrogel structures from different methacrylate-functionalized triblock copolymers of poly(ethylene glycol) (PEG) with poly(trimethylene carbonate) (PTMC) by photo-crosslinking aqueous solutions of the macromonomers in 2.5 and 5wt.% colloidal dispersions of clay nanoparticles (Laponite XLG). The length of the PTMC blocks of the macromonomers and the clay content determined the physicomechanical properties of the obtained hydrogels. While an increase in the PTMC block length in the macromonomers from 0.2 to 5kg/mol resulted in a decrease in the gel content, the addition of 5wt.% Laponite nanoclay to the crosslinking solution lead to very high gel contents of the hydrogels of more than 95%. The effect of PTMC block length on the mechanical properties of the hydrogels was not as pronounced, and soft gels with a compressive modulus of less than 15kPa and toughness values of 25kJm(-3) were obtained. However, the addition of 5wt.% Laponite nanoclay to the formulations considerably increased the compressive modulus and resilience of the hydrogels; swollen nanocomposite networks with compressive modulus and toughness values of up to 67kPa and 200kJm(-3), respectively, could then be obtained. The prepared hydrogels were shown to be enzymatically degradable by cholesterol esterase and by the action of macrophages. With an increase in PTMC block length in the hydrogels, the rates of mass loss increased, while the incorporated Laponite nanoclay suppressed degradation. Nanocomposite hydrogel structures with a designed gyroid pore network architecture were prepared by stereolithography. Furthermore, in the swollen state the porous gyroid structures were mechanically stable and the pore network remained fully open and interconnected.

Macrophage-mediated erosion of gamma irradiated poly(trimethylene carbonate) films

A macrophage culture model was used to investigate the erosion of gamma irradiated poly(trimethylene carbonate) (PTMC) films. When the PTMC films were incubated in the culture medium, but physically separated from the cells by a membrane, no erosion occurred. In contrast, when the J774A macrophages were directly cultured on PTMC films, they adhered to the films and were found to have eroded the polymer surface. Macrophages adhered to gamma irradiated poly(epsilon-caprolactone) (PCL) controls as well, but to a lesser extent than to the PTMC films. In this case, no signs of erosion were observed. Human skin fibroblasts cultured on PTMC and PCL films as controls also adhered to the films but did not erode the surfaces. The effect of enzymes and reactive oxygen species that can be secreted by macrophages on the erosion process was assessed using aqueous solutions of cholesterol esterase, lipoprotein lipase, esterase, potassium superoxide, and hydrogen peroxide. The PTMC films eroded in aqueous enzyme solutions as well as in aqueous superoxide solutions. Cholesterol esterase and superoxide anion radicals seem to be most involved in the macrophage-mediated erosion of PTMC. This macrophage culture model is useful in assessing the influence of macrophages on the in vivo biodegradability of polymers and in elucidating the biodegradation mechanisms involved.

An annulus fibrosus closure device based on a biodegradable shape-memory polymer network

Injuries to the intervertebral disc caused by degeneration or trauma often lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). This can compress nerves and cause lower back pain. In this study, the characteristics of poly(D,L-lactide-co-trimethylene carbonate) networks with shape-memory properties have been evaluated in order to prepare biodegradable AF closure devices that can be implanted minimally invasively. Four different macromers with (D,L-lactide) to trimethylene carbonate (DLLA:TMC) molar ratios of 80:20, 70:30, 60:40 and 40:60 with terminal methacrylate groups and molecular weights of approximately 30 kg mol(-1) were used to prepare the networks by photo-crosslinking. The mechanical properties of the samples and their shape-memory properties were determined at temperatures of 0 °C and 40 °C by tensile tests- and cyclic, thermo-mechanical measurements. At 40 °C all networks showed rubber-like behavior and were flexible with elastic modulus values of 1.7-2.5 MPa, which is in the range of the modulus values of human annulus fibrosus tissue. The shape-memory characteristics of the networks were excellent with values of the shape-fixity and the shape-recovery ratio higher than 98 and 95%, respectively. The switching temperatures were between 10 and 39 °C. In vitro culture and qualitative immunocytochemistry of human annulus fibrosus cells on shape-memory films with DLLA:TMC molar ratios of 60:40 showed very good ability of the networks to support the adhesion and growth of human AF cells. When the polymer network films were coated by adsorption of fibronectin, cell attachment, cell spreading, and extracellular matrix production was further improved. Annulus fibrosus closure devices were prepared from these AF cell-compatible materials by photo-polymerizing the reactive precursors in a mold. Insertion of the multifunctional implant in the disc of a cadaveric canine spine showed that these shape-memory devices could be implanted through a small slit and to some extent deploy self-sufficiently within the disc cavity.

Resorbable elastomeric networks prepared by photocrosslinking of high-molecular-weight poly(trimethylene carbonate) with photoinitiators and poly(trimethylene carbonate) macromers as crosslinking aids

Resorbable and elastomeric poly(trimethylene carbonate) (PTMC) networks were efficiently prepared by photoinitiated crosslinking of linear high-molecular-weight PTMC. To crosslink PTMC films, low-molecular-weight PTMC macromers with methacrylate end groups were synthesized and used as crosslinking aids. By exposing PTMC films containing only photoinitiator (Irgacure(®) 2959) or both photoinitiator and PTMC macromers to ultraviolet light, PTMC networks with high gel contents (87-95%) could be obtained. The crosslink density could be readily varied by adjusting the irradiation time or the amount of crosslinking aid used. The formed networks were flexible, with low elastic modulus values ranging from 7.1 to 7.5MPa, and also showed excellent resistance to creep in cyclic tests. In vitro experiments showed that the photocrosslinked PTMC networks could be eroded by macrophages, and upon incubation in aqueous cholesterol esterase enzyme- or potassium dioxide solutions. The rate of surface erosion of photocrosslinked PTMC networks was significantly lower than that observed for films prepared from linear PTMC. These resorbable PTMC elastomeric networks are compatible with cells and may find application in tissue engineering and controlled release.

Prevention of posterior capsular opacification

Posterior capsular opacification (PCO) is a common complication of cataract surgery. The development of PCO is due to a combination of the processes of proliferation, migration, and transdifferentiation of residual lens epithelial cells (LECs) on the lens capsule. In the past decades, various forms of PCO prevention have been examined, including adjustments of techniques and intraocular lens materials, pharmacological treatments, and prevention by interfering with biological processes in LECs. The only method so far that seems effective is the implantation of an intraocular lens with sharp edged optics to mechanically prevent PCO formation. In this review, current knowledge of the prevention of PCO will be described. We illustrate the biological pathways underlying PCO formation and the various approaches to interfere with the biological processes to prevent PCO. In this type of prevention, the use of nanotechnological advances can play a role.

Biomaterial-stem cell interactions and their impact on stem cell response

In this review, current research in the field of biomaterial properties for directing stem cells are discussed and placed in a critical perspective. Regenerative medicine, in which stem cells play a crucial role, has become an interdisciplinary field between cell biology and materials science. New insights are generated, different approaches to determine material features and stem cell properties are implemented, but also many misconceptions exist. According to the current state-of-the-art and combined with basic principles from two different disciplines the topic is critically addressed. We take into account what seem to be the most important material properties and their influence towards stem cells but also the various stem cells available with respect to their origin, tissue source and culturing conditions.

Physicochemical and biological evaluation of poly(ethylene glycol) methacrylate grafted onto poly(dimethyl siloxane) surfaces for prosthetic devices

Poly(dimethyl siloxane) (PDMS) was surface-polymerized with poly(ethylene glycol)methacrylate (PEGMA) by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous media at room temperature. Modification of the PDMS surface followed a three-step procedure: (i) PDMS surface hydroxylation by UV/ozone exposure, immediately followed by (ii) covalent attachment of the initiator, 1-trichlorosilyl-2-(chloromethylphenyl)ethane, onto the hydroxylated PDMS, via chemical vapor deposition; finally (iii) PDMS surface-polymerization of PEGMA by ATRP. Modified PDMS was characterized by water contact angle measurement, SEM, FTIR-ATR, and XPS. Results showed that modified surfaces had a hydrophilic character, given the water contact angles around 60°; FTIR-ATR and XPS analysis confirmed the presence of polymerized PEGMA on the surface of PDMS and the adhesion of Staphylococcus aureus GB 2/1 and Streptococcus salivarius GB 24/9 onto the modified surfaces was inhibited 94% and 81%, respectively. Finally, the modified PDMS showed no evidence of cytotoxic effects in in vitro assays using human skin fibroblasts.

Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.