Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.

Nonwoven fibrous matrices of collagen and elastin have been prepared by means of electrospinning. Variations in the morphology of the scaffolds have been evaluated as a function of the ratio of the two proteins. Scaffolds having an architecture resembling the media of natural blood vessels have been obtained. Crosslinking has been used as a valuable method to improve the stability of such matrices. The obtained nonwoven matrices can be used for tissue engineering applications.

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.

Tissue homeostasis, the balance between cell proliferation and apoptosis, is an important factor in tissue engineering. We describe a new method to analyze markers of both proliferation and apoptosis in a single assay to monitor growth behavior of cell cultures. Human vascular smooth muscle cells (VSMCs) were cultured either on gelatin-coated tissue culture polystyrene or in three-dimensional porous scaffolds composed of insoluble collagen and elastin. mRNA concentrations of cyclin E, as a marker of proliferation, and of tissue transglutaminase (tTG) as a marker of apoptosis, quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to porphobilinogen deaminase mRNA concentrations, were analyzed. tTG mRNA expression levels were increased when apoptosis was induced by tumor necrosis factor-alpha in combination with cycloheximide or by culturing the cells in serum-free culture medium. Cyclin E mRNA expression levels were less altered in these cell cultures. Results were compared with several reference tests to measure apoptosis including DNA fragmentation, annexin V staining, and light microscopy. This RT-PCR method could be used to characterize cell growth behavior of VSMCs in vitro. In addition, it was shown that this test is suitable to measure the balance between proliferation and apoptosis of VSMCs present in tissue-engineered constructs.

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (Mw=8×106) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (Td=79°C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14d of culture.

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-(alpha-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.

The balance between apoptosis and proliferation of vascular smooth muscle cells (SMCs) is responsible for mediating profound changes in vascular architecture in development and disease. New insights in the biology of SMCs can be important to our understanding of (patho) physiological mechanisms and for tissue-engineering (TE) applications. Here the development of a new method to characterise SMCs regarding proliferation versus apoptosis is described and the application of this method for TE purposes is showed. Proliferation and programmed cell death (apoptosis) of human umbilical vein SMCs were analysed by measuring cyclin E (1) and tissue transglutaminase (tTG) (2) mRNA expression levels with use of a semi-quantitative real-time RT-PCR method. Ratios of cyclin E and tTG were calculated to make a quantified comparison of proliferation versus apoptosis. In this way the viability of SMCs cultured on standard culture systems and on TE scaffolds for small diameter blood vessel constructs were analysed.

Extracellular matrix components are valuable building blocks for the preparation of biomaterials involved in tissue engineering, especially if their biological, chemical and physical characteristics can be controlled. In this study, isolated type I collagen fibrils, elastin fibres and chondroitin sulphate (CS) were used for the preparation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds. A total of 12 different scaffolds were prepared with four different ratios of collagen and elastin (1:9, 1:1, 9:1 and 1:0), with and without chemical crosslinking, and with and without CS. Collagen was essential to fabricate coherent, porous scaffolds. Electron microscopy showed that collagen and elastin physically interacted with each other and that elastin fibres were enveloped by collagen. By carbodiimide-crosslinking, amine groups were coupled to carboxylic groups and CS could be incorporated. More CS could be bound to collagen scaffolds (10%) than to collagen-elastin scaffolds (2.4-8.5% depending on the ratio). The attachment of CS increased the water-binding capacity to up to 65%. Scaffolds with a higher collagen content had a higher tensile strength whereas addition of elastin increased elasticity. Scaffolds were cytocompatible as was established using human myoblast and fibroblast culture systems. It is concluded that molecularly-defined composite scaffolds can be composed from individual, purified, extracellular matrix components. Data are important in the design and application of tailor-made biomaterials for tissue engineering.

The issue related to the development of scaffolds for tissue-engineered small-diameter blood vessels, is presented. Presently, tubes are being prepared by freeze drying a suspension of type I insoluble collagen from bovine Achilles tendon in a tubular mould. It is estimated that after once cultured on the scaffolds, smooth muscle cells (SMC) would grow and produce extracellular matrix components (ECM), and develop new blood vessels. Different crosslinking methods as well as varius crosslinked samples were studied from a physical and chemical point of view to ascertain the situation.

The use of melt spinning and fiber winding of trimethylenecarbonate (TMC)-based polymers for tissue engineering small diameter blood vessels was investigated in order to overcome the problems and poor performance of artificial blood vessels. For the study, porous tubular scaffolds were obtained by heating PTMC to a temperature of 220°C. It was found that it is possible to produce tubular TMC-based scaffolds by means of melt spinning. It was also found that combining the synthetic scaffold with collagen further enhances the structural integrity in time permitting to obtain a scaffold that resembles the mechanical properties of native blood vessels.

A technique developed to characterize and compare the cell growth of human vascular smooth muscle cells (SMC) cultured on tissue-engineered (TE) scaffolds composed of elastin and collagen was discussed. Proliferation and apoptosis of SMC were determined by measuring cyclin E and tissue trans glutaminase (tTG) mRNA expression levels respectively. It was found that with the method SMC cultured on porous films of elastin and collagen grow better than on gelatin-coated tissue culture polystyrene (g-TCPS). The results indicated the possibility to culture cells in a standardized way not only for obtaining an artificial media of TE blood vessel using SMC but also for all kinds of tissue-engineering purposes using other cell types.