T.G. van Kooten

Animal models for tracheal research.

Tracheal research covers two main areas of interest: tracheal reconstruction and tracheal fixation. Tracheal reconstructions are aimed at rearranging or replacing parts of the tracheal tissue using implantation and transplantation techniques. The indications for tracheal reconstruction are numerous: obstructing tracheal tumors, trauma, post-intubation tissue reactions, etc. Although in the past years much progress has been made, none of the new developed techniques have resulted in clinical application at large scale. Tissue engineering is believed to be the technique to provide a solution for reconstruction of tracheal defects. Although developing functional tracheal tissue from different cultured cell types is still a challenge. Tracheal fixation research is relatively new in the field and concentrates on solving fixation-related problems for laryngectomized patients. In prosthetic voice rehabilitation tracheo-esophageal silicon rubber speech valves and tracheostoma valves are used. This is often accompanied by many complications. The animal models used for tracheal research vary widely and in most publications proper scientific arguments for animal selection are never mentioned. It showed that the choice on animal models is a multi-factorial process in which non-scientific arguments tend to play a key role. The aim of this study is to provide biomaterials scientists with information about tracheal research and the animal models used.

Effect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

In vitro degradation of a biodegradable polyurethane foam, based on 1,4‐butanediisocyanate: A three‐year study at physiological and elevated temperature

Biodegradable polyesterurethanes (PUs) may be used as scaffold materials for tissue regeneration applications, because of their excellent mechanical properties. In this study, the degradation of highly porous PU foams was evaluated in vitro. The PU had amorphous soft segments of DL-lactide/epsilon-caprolactone and uniform hard segments, synthesized from 1,4-butanediisocyanate and butanediol. The foams were degraded for 3 years in a Sörensen buffer solution (pH 7.4) at 37 and 60 degrees C. Dimensions of the foams, intrinsic viscosity, mass loss, thermal properties, and composition of the remaining material were evaluated. Copolyester (CP) foams of DL-lactide/epsilon-caprolactone served as controls. The PU foams kept their dimensions for 20 weeks at 37 degrees C, whereas CP foams collapsed after 3 weeks. PU mass loss reached a maximum of 80% at both 37 and 60 degrees C. CP mass loss reached 99.9% at 60 degrees , and 92% at 37 degrees C after 3 years. The degradation processes at 37 and 60 degrees C are initially the same, but eventually degradation products with different thermal properties are being formed. (1)H NMR studies showed that the hard urethane segments of the PU do not degrade in vitro at pH 7.4. It was concluded that the PU material has favorable characteristics for a scaffold material. Compared to long-term in vivo results of the same PU these in vitro results are not representative for the in vivo situation and therefore total resorption has to be investigated in long-term in vivo studies.

An In Vivo Study of Composite Microgels Based on Hyaluronic Acid and Gelatin for the Reconstruction of Surgically Injured Rat Vocal Folds

PURPOSE The objective of this study was to investigate local injection with a hierarchically microstructured hyaluronic acid-gelatin (HA-Ge) hydrogel for the treatment of acute vocal fold injury using a rat model. METHOD Vocal fold stripping was performed unilaterally in 108 Sprague-Dawley rats. A volume of 25 μl saline (placebo controls), HA-bulk, or HA-Ge hydrogel was injected into the lamina propria (LP) 5 days after surgery. The vocal folds were harvested at 3, 14, and 28 days after injection and analyzed using hematoxylin and eosin staining and immunohistochemistry staining for macrophages, myofibroblasts, elastin, collagen type I, and collagen type III. RESULTS The macrophage count was statistically significantly lower in the HA-Ge group than in the saline group (p < .05) at Day 28. Results suggested that the HA-Ge injection did not induce inflammatory or rejection response. Myofibroblast counts and elastin were statistically insignificant across treatment groups at all time points. Increased elastin deposition was qualitatively observed in both HA groups from Day 3 to Day 28, and not in the saline group. Significantly more elastin was observed in the HA-bulk group than in the uninjured group at Day 28. Significantly more collagen type I was observed in the HA-bulk and HA-Ge groups than in the saline group (p < .05) at Day 28. The collagen type I concentration in the HA-Ge and saline groups was found to be comparable to that in the uninjured controls at Day 28. The concentration of collagen type III in all treatment groups was similar to that in uninjured controls at Day 28. CONCLUSION Local HA-Ge and HA-bulk injections for acute injured vocal folds were biocompatible and did not induce adverse response.

Fluid shear induced endothelial cell detachment from modified polystyrene substrata

Abstract In this study, human umbilical vein endothelial cells were seeded for 3 and 24 h on polystyrene (PS), tissue culture polystyrene (TCPS) and polystyrene modified by oxygen plasma treatment (PtPS) in order to investigate their detachment behaviour during exposure to fluid shear. All three materials have smooth surfaces at the submicron level. Equilibrium water contact angles were higher on PS (88°) than on TCPS (78°) and PtPS (79°). Furthermore, contact angle hysteresis, i.e. the difference between advancing and receding angles, was much larger on TCPS (39°) and PtPS (41°) than on PS (14°), indicating either a large surface heterogeneity or a possible reorientation of functional groups on TCPS and PtPS. X-ray photoelectron spectroscopy data revealed that both TCPS and PtPS had oxygen incorporated at their surfaces (18.6% and 9.9%, respectively), whereas only TCPS had a small amount of nitrogen (0.9%) at its surface. Cells seeded on these materials were exposed to various shear stresses in the range 88–352 dyn cm−2 in order to gain more insight into the influence of the specific physico-chemical surface properties of these polystyrene surfaces on cell retention, cell morphology and migration. The retention of cells having adhered during 24 h on TCPS and PtPS was not different from the retention of cells having adhered during 3 h, but retention on TCPS and PtPS was higher than on PS. At shear stresses of 88 and 176 dyn cm−2, however, differences in the morphology of cells adhered to TCPS during 3 and 24 h were observed, indicating that between 3 and 24 h changes in cell-substratum interactions occurred. Migration on TCPS and PtPS was accompanied by the presence of fibrillike structures left behind on the surface. Summarizing, this study shows that cell retention is higher on modified polystyrenes than on polystyrene, the only major difference between the polystyrenes being the higher contact angle hysteresis and oxygen content of the modified polystyrene surfaces.

Cellular responses to environmental stimuli: Influence of fluid shear and substratum hydrophobicity on adhesion of human fibroblasts

Morphological changes in human skin fibroblasts adhering to hydrophobic and hydrophilic substrata were studied in a parallel‐plate flow chamber using light‐ and scanning electron microscopy. Cells were seeded on the bottom plate of the flow chamber, which was made of the substratum being studied, and allowed to adhere for 3–6 h. Then, the seeded cells were exposed to an incrementally increased laminar flow. Light microscope observations were performed in situ in the flow chamber. Fixation for electron microscopy was also done in situ in order to avoid passing cells through a liquid‐air interface which would create a high shear stress relative to ones which remain submerged. Cells spread far less on the hydrophobic material than on the hydrophilic material and reached the point of detachment at a significantly lower shear stress (22 dynescm‐2 vs 324 dynescm‐2 on the hydrophilic substratum). Cells on the point of detachment had a similar morphology on both substrata, viz. rounded to a nearly spherical shape...

Influence of glutaraldehyde fixation of cells adherent to solid substrata on their detachment during exposure to shear stress.

In order to determine the response of fixed and nonfixed cells adherent to a solid substratum to shear stress, human fibroblasts were allowed to adhere and spread on either hydrophilic glass or hydrophobic Fluoroethylene-propylene (FEP-Teflon) and fixed with glutaraldehyde. Then, the cells were exposed to an incrementally loaded shear stress in a parallel plate flow chamber up to shear stresses of about 500 dynes/cm2, followed by exposure to a liquid-air interface passage. The cellular detachment was compared with the one of nonfixed cells. In case of fixed cells, 50% of the adhering cells detached from FEP-Teflon at a shear stress of 350 dynes/cm2, whereas 50% of the adhering, nonfixed cells detached already at a shear stress of 20 dynes/cm2. No fixed cells detached from glass for shear stresses up to at least 500 dynes/cm2. More than 50% of the nonfixed cells were detached from glass at a shear stress of 350 dynes/cm2. Furthermore, the shape and morphology of fixed cells did not change during the incrementally loaded flow, in contrast to the ones of nonfixed cells, which clearly rounded up prior to detachment.

Molecular signalling mechanisms of host–materials interactions

Biomaterials are being used in several different clinical applications worldwide. However, they often induce a nonspecific host immune response and are prone to infections due to microorganisms that adhere to their surface and adopt a biofilm phenotype. Modern biomaterials science provides a large array of biomaterial designs and surface modifications that modulate the host–material interactions to prevent an aggressive foreign-body response and at the same time avoid bacterial colonization. Furthermore, the use of biological motives in the new generations of biomaterials may elicit a specific immune response. Altogether, perfect biomaterials for the different clinical applications do not yet exist, and will always depend on the specific application. Therefore a better understanding of the molecular signalling mechanisms involved in the host–material interactions may provide important targets for further exploration in the field of biomaterials research. The following chapter provides an overview of signalling molecules involved in the host cell response to biomaterials and the latest advances in biomaterials designs using biological or biology-inspired molecules.