Willeke F. Daamen

Preparation and evaluation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds for tissue engineering.

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.

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.

Comparison of five procedures for the purification of insoluble elastin.

Elastin is an insoluble, highly cross-linked protein, providing elasticity to organs like lung. aorta, and ligaments. Despite its remarkable mechanical properties. elastin has found little use as a biomaterial. Purification of intact elastin from elastic fibres presents a major challenge, among others for the intimate interwoveness of elastin and microfibrils. Insoluble elastin preparations tend to calcify, which may be due to calcium-binding microfibrillar (e.g. fibrillin). In this study, elastin was purified from horse ligamentum nuchae using five different procedures. One procedure is based on treatment with 0.1 M NaOH, another on autoclaving and treatment with cyanogen bromide. Three other procedures are based on combinations of extraction steps and enzyme digestions. Purity of preparations was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, amino acid analysis, bright field immunofluorescence and transmission electron microscopy. The procedure involving extractions/enzymes combined with an early application of 2-mercaptoethanol and cyanogen bromide gives a highly pure elastin preparation. Electron microscopic analysis showed that this preparation is devoid of microfibrillar components. This procedure is therefore the method of choice for preparation of insoluble elastin as a biomaterial for tissue engineering.

The osteogenic effect of electrosprayed nanoscale collagen/calcium phosphate coatings on titanium.

For orthopedic and dental implants, the ultimate goal is to obtain a life-long secure anchoring of the implant in the native surrounding bone. To this end, nanoscale calcium phosphate (CaP) and collagen-CaP (col-CaP) composite coatings have been successfully deposited using the electrospray deposition (ESD) technique. In order to study to what extent the thickness of these coatings can be reduced without losing coating osteogenic properties, we have characterized the mechanical and biological coating properties using tape tests (ASTM D-3359) and in vitro cell culture experiments, respectively. Co-deposition of collagen significantly improved coating adhesive and cohesive strength, resulting in a remarkably high coating retention of up to 97% for coating thicknesses below 100 nm. In vitro cell culture experiments showed that electrosprayed CaP and col-CaP composite coatings enhanced osteoblast differentiation, leading to improved mineral deposition. This effect was most pronounced upon co-deposition of collagen with CaP, and these coatings displayed osteogenic effects even for a coating thickness of below 100 nm.

In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages

An increasing number of investigations supports that adult stem cells have the potential to differentiate into matured cell types beyond their origin, a property defined as plasticity. Previously, the plasticity of stem cells derived from dental pulp (DPSC) has been confirmed by culturing cells in lineage‐specific media in vitro. In the current study, the in vivo differentiation or maturation potential of DPSC was further analysed, by transplanting human DPSC/collagen scaffold constructs into subcutaneous tissue of immunocompromised mice. Cells received odontogenic, adipogenic or myogenic pre‐induction, whereas control samples received no stimulation. Also blank collagen scaffolds were implanted. The results indicated that seeded cells produced tissue within the implanted constructs after 3 weeks of implantation. According to morphological and phenotypical changes, the pre‐induced DPSC showed the ability to further differentiate along odontogenic, myogenic and adipogenic pathways in vivo. Moreover, DPSC without pre‐treatment were able to spontaneously differentiate along odontogenic and adipogenic directions in vivo. However, only limited mature morphological changes were detected in histology. In summary, stem cells derived from human dental pulp form a suitable source for tissue engineering and cell‐mediated therapy, although additional analyses should be considered. Copyright

Urethral reconstruction of critical defects in rabbits using molecularly defined tubular type I collagen biomatrices: key issues in growth factor addition.

Tubular type I collagen biomatrices with and without growth factors (GFs) were constructed and evaluated in a rabbit model for critical urethral defects. Porous tubular biomatrices with an inner diameter of 3  mm were prepared using highly purified collagen fibrils and were crosslinked with or without heparin. Heparinized biomatrices were supplemented with the heparin-binding GFs vascular endothelial GF, fibroblast GF-2, and heparin-binding epidermal GF. Biomatrices with and without GFs were used to replace a critical 1 cm urethral segment in rabbits (n = 32). All animals showed normal urination without urinary retention. General histology and immunohistology of graft areas (2, 4, 12, and 24 weeks after implantation) indicated that all biomatrices were replaced by urethra-like structures with normal appearing cytokeratin-positive urothelium surrounded by vascularized tissue. The GF-containing biomatrices showed an increase in extracellular matrix deposition, neovascularization, urothelium, glands, granulocytes, and fibroblasts, compared with biomatrices without GF. GFs substantially improved molecular features of healing but failed to be superior in functional outcome. Retrograde urethrography indicated a normal urethral caliber in case of biomatrices without GF, but a relative narrowing of the urethra at 2 weeks postsurgery and diverticula after 4 weeks in case of biomatrices with GF. In conclusion, tubular acellular type I collagen biomatrices were successful in repairing urethral lesions in artificial urethral defects, and inclusion of GF has a profound effect on regenerative processes.

A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials

Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.

Micro-computed tomographical imaging of soft biological materials using contrast techniques.

The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.

Tissue Engineered Tubular Construct for Urinary Diversion in a Preclinical Porcine Model

PURPOSE The ileal conduit has been considered the gold standard urinary diversion for patients with bladder cancer and pediatric patients. Complications are mainly related to the use of gastrointestinal tissue. Tissue engineering may be the technical platform on which to develop alternatives to gastrointestinal tissue. We developed a collagen-polymer conduit and evaluated its applicability for urinary diversion in pigs. MATERIALS AND METHODS Tubular constructs 12 cm long and 15 mm in diameter were prepared from bovine type I collagen and Vypro® II synthetic polymer mesh. Characterized tubes were sterilized, seeded with and without primary porcine bladder urothelial cells, and implanted as an incontinent urostomy using the right ureter in 10 female Landrace pigs. At 1 month the newly formed tissue structure was functionally and microscopically evaluated by loopogram and immunohistochemistry, respectively. RESULTS The survival rate was 80% with 1 related and 1 unrelated death. By 1 month the collagen was resorbed and a retroperitoneal tunnel had formed that withstood 40 cm H(2)O water pressure. In 5 cases the tunnel functioned as a urostomy. Histological analysis revealed a moderate immune response, neovascularization and urothelial cells in the construct lumen. The polymer mesh provoked fibroblast deposition and tissue contraction. No major differences were observed between cellular and acellular constructs. CONCLUSIONS After implanting the tubular constructs a retroperitoneal tunnel was formed that functioned as a urinary conduit in most cases. Improved large tubular scaffolds may generate alternatives to gastrointestinal tissue for urinary diversion.

Controlled fabrication of triple layered and molecularly defined collagen/elastin vascular grafts resembling the native blood vessel.

There is a consistent need for a suitable natural biomaterial to function as an arterial prosthesis in achieving arterial regeneration. Natural grafts are generally obtained by decellularization of native blood vessels, but batch to batch variations may occur and the nature/content of remaining contaminants is generally unknown. In this study we fabricated a molecularly defined natural arterial graft from scratch resembling the native three layered architecture from the fibrillar extracellular matrix components collagen and elastin. Using casting, moulding, freezing and lyophilization techniques, a triple layered construct was prepared consisting of an inner layer of elastin fibres, a middle (porous) film layer of collagen fibrils and an outer scaffold layer of collagen fibrils. The construct was carbodiimide cross-linked and heparinized. Characterization included biochemical/biophysical analyses, scanning electron microscopy, micro-computed tomography, (immuno)histology and haemocompatibility. Burst pressures were up to 400mm Hg and largely conferred by the intermediate porous collagen film layer. The highly purified type I collagen fibrils and elastin fibres used did not evoke platelet aggregation in vitro. Suturability of the graft in end to side anastomosis was successful and considered adequate for in vivo application.