André A. Poot

Biodegradable elastomeric scaffolds for soft tissue engineering

Elastomeric copolymers of 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone (CL) and copolymers of TMC and D,L-lactide (DLLA) have been evaluated as candidate materials for the preparation of biodegradable scaffolds for soft tissue engineering. TMC-DLLA copolymers are amorphous and degrade more rapidly in phosphate-buffered saline (PBS) of pH 7.4 at 37 degrees C than (semi-crystalline) TMC-CL copolymers. TMC-DLLA with 20 or 50 mol% TMC loose their tensile strength in less than 5 months and are totally resorbed in 11 months. In PBS, TMC-CL copolymers retain suitable mechanical properties for more than a year. Cell seeding studies show that rat cardiomyocytes and human Schwann cells attach and proliferate well on the TMC-based copolymers. TMC-DLLA copolymers with either 20 or 50 mol% of TMC are totally amorphous and very flexible, making them excellent polymers for the preparation of porous scaffolds for heart tissue engineering. Porous structures of TMC-DLLA copolymers were prepared by compression molding and particulate leaching techniques. TMC-CL (co)polymers were processed into porous two-ply tubes by means of salt leaching (inner layer) and fiber winding (outer layer) techniques. These grafts, seeded with Schwann cells, will be used as nerve guides for the bridging of large peripheral nerve defects.

Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds.

Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three‐dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze‐drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross‐linked by two different methods. A water‐soluble carbodi‐imide, alone or in combination with a diamine, was used for this purpose: zero‐ or non‐zero‐length cross‐links were obtained. The occurrence of cross‐linking was verified by monitoring the thermal behaviour and the free‐amino‐group contents of the scaffolds before and after cross‐linking. Smooth‐muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non‐cross‐linked scaffolds. As for the cross‐linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross‐linked using the two different methods. Cells cultured on the scaffolds were identified with an anti‐(α‐smooth‐muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.

Physical properties of high molecular weight 1,3-trimethylene carbonate and D,L-lactide copolymers.

High molecular weight statistical copolymers of 1,3-trimethylene carbonate (TMC) and D,L-lactide (DLLA) were synthesized and characterized with the aim of assessing their potential in the development of degradable and flexible materials for application in the biomedical field. Under the applied polymerization conditions (130 °C, 3 days using stannous octoate as a catalyst) monomer conversion was high or almost complete, and high molecular weight polymers (_Mn above 170 000) were obtained. Significant improvement of the mechanical performance of these materials was observed in comparison to results previously reported for TMC and DLLA based copolymers of lower molecular weight. For the entire range of compositions the polymers are amorphous with a glass transition temperature ranging between −17 °C for poly(TMC) and 53 °C for poly(DLLA). The polymers vary from rubbers to stiff materials as the content of TMC decreases. All polymers are hydrophobic with very low equilibrium water absorption (<1.5 wt %). Thermal analyses and tensile tests were performed on polymer samples after water uptake. Due to a plasticizing effect of the water, the thermal properties, and consequently the mechanical performance, of the copolymers with higher content of DLLA were the most affected. After water absorption, the polymer mechanical behavior can change from glassy to rubbery, as observed for the copolymer with 80 mol % of DLLA. The obtained results suggest that these copolymers are promising candidates as biomaterials in the preparation of degradable medical devices and systems.

A microfluidic wound-healing assay for quantifying endothelial cell migration

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-mum-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF(165)) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF(165) could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.

Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20 degrees -37 degrees C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells.

Controlled cavitation–cell interaction: trans-membrane transport and viability studies

Cavitation bubble dynamics close to a rigid surface gives rise to a rapid and transient fluid flow. A single bubble is created with a laser pulse at different stand-off distances from the rigid surface, where the stand-off distance gamma is defined by gamma = h/R(max), with h being the initial distance and R(max) being the maximum bubble radius. When the surface is covered with adherent cells, molecular delivery and cell detachment after single cavitation activity are observed at different locations. We find a maximum of cell detachment at a normalized stand-off distance of gamma approximately 0.65. In contrast, the maximum of the molecular uptake is found when gamma approaches 0. The single cavitation event has only little effect on the viability of cells in the non-detached area. We find apoptosis of cells only very close to the area of detachment and, additionally, the metabolism of the non-detached cells shows no pronounced difference compared to control cells according to an MTS assay. Thus, although the cavitation event is responsible for the detachment of cells, only few of the remaining cells undergo a permanent change.

In Vivo Recruitment of Hematopoietic Cells Using Stromal Cell–Derived Factor 1 Alpha–Loaded Heparinized Three-Dimensional Collagen Scaffolds

Implantable three-dimensional (3D) constructs to engineer tissue have great therapeutic potential in regenerative medicine and immunotherapy. However, autonomous recruitment of cells into the engineered scaffold in vivo is hampered by lack of attracting scaffolds. As a first step to engineering immune tissue, 3D collagen scaffolds were investigated for their ability to enhance in vivo recruitment and growth of various hematopoietic cells. Scaffolds containing immobilized heparin to trap the stem cell chemo-attractant stromal cell-derived factor 1 alpha (SDF1alpha) were implanted subcutaneously into C57Bl6 mice, and influx of cells was monitored using immunohistochemistry. Five weeks post-implantation, heparinized scaffolds were always populated by cells, but incorporating SDF1alpha considerably stimulated recruitment of cells. SDF1alpha could not exert this effect when the formation of a SDF1alpha gradient was abrogated. Scaffolds were mainly populated by CD11b+ and CD11c+ myeloid cells and fibroblasts. One week after implantation, scaffolds harbored only low numbers of cells. Apparently, not all CXCR4-expressing cells, like large numbers of granulocytes, migrate into the scaffold, but retransplantation of a 1-week-old scaffold from a CD45.2(+) into a CD45.1(+) mouse yielded a scaffold harboring mainly CD45.2(+) cells after 5 weeks. These data confirm that only a few progenitor cells are recruited early after implantation. These cells then proliferate and differentiate along different lineages and determine the outcome after 5 weeks.

Biodegradable elastomers for biomedical applications and regenerative medicine

Synthetic biodegradable polymers are of great value for the preparation of implants that are required to reside only temporarily in the body. The use of biodegradable polymers obviates the need for a second surgery to remove the implant, which is the case when a nondegradable implant is used. After implantation in the body, biomedical devices may be subjected to degradation and erosion. Understanding the mechanisms of these processes is essential for the development of biomedical devices or implants with a specific function, for example, scaffolds for tissue-engineering applications. For the engineering and regeneration of soft tissues (e.g., blood vessels, cardiac muscle and peripheral nerves), biodegradable polymers are needed that are flexible and elastic. The scaffolds prepared from these polymers should have tuneable degradation properties and should perform well under long-term cyclic deformation conditions. The required polymers, which are either physically or chemically crosslinked biodegradable elastomers, are reviewed in this article.

Hollow fibers of poly(lactide-co-glycolide) and poly(ε-caprolactone) blends for vascular tissue engineering applications.

At present the manufacture of small-diameter blood vessels is one of the main challenges in the field of vascular tissue engineering. Currently available vascular grafts rapidly fail due to development of intimal hyperplasia and thrombus formation. Poly(lactic-co-glycolic acid) (PLGA) hollow fiber (HF) membranes have previously been proposed for this application, but as we show in the present work, they have an inhibiting effect on cell proliferation and rather poor mechanical properties. To overcome this we prepared HF membranes via phase inversion using blends of PLGA with poly(ε-caprolactone) (PCL). The influence of polymer composition on the HF physicochemical properties (topography, water transport and mechanical properties) and cell attachment and proliferation were studied. Our results show that only the ratio PCL/PLGA of 85/15 (PCL/PLGA85/15) yielded a miscible blend after processing. A higher PLGA concentration in the blend led to immiscible PCL/PLGA phase-separated HFs with an inhomogeneous morphology and variation in the cell culture results. In fact, the PCL/PLGA85/15 blend, which had the most homogeneous morphology and suitable pore structure, showed better human adipose stem cell (hASC) attachment and proliferation compared with the homopolymers. This, combined with the good mechanical and transport properties, makes them potentially useful for the development of small-caliber vascular grafts.

Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.