Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention.

Extensive full-thickness skin loss, associated with deep burns or other traumata, represents a major clinical problem that is far from being solved. A promising approach to treat large skin defects is the use of tissue-engineered full-thickness skin analogues with nearly normal anatomy and function. In addition to excellent biological properties, such skin substitutes should exhibit optimal structural and mechanical features. This study aimed to test novel dermo-epidermal skin substitutes based on collagen type I hydrogels, physically strengthened by two types of polymeric net-like meshes. One mesh has already been used in clinical trials for treating inguinal hernia; the second one is new but consists of a FDA-approved polymer. Both meshes were integrated into collagen type I hydrogels and dermo-epidermal skin substitutes were generated. Skin substitutes were transplanted onto immuno-incompetent rats and analyzed after distinct time periods. The skin substitutes homogeneously developed into a well-stratified epidermis over the entire surface of the grafts. The epidermis deposited a continuous basement membrane and dermo-epidermal junction, displayed a well-defined basal cell layer, about 10 suprabasal strata and a stratum corneum. Additionally, the dermal component of the grafts was well vascularized.

Versatile wedge-based system for the construction of unidirectional collagen scaffolds by directional freezing: practical and theoretical considerations.

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.

Preparation of differently sized injectable collagen micro-scaffolds.

Collagen scaffolds have been widely used as biomaterials for tissue engineering. In general, application of scaffolds requires surgery. In this study, we describe a new and simple technique to prepare porous micro-scaffolds from type I collagen fibrils which can be injected, thus preventing surgery. The size of the micro-scaffolds could be easily controlled using sieves with varying cut-offs. EDC-NHS crosslinking was essential to stabilize the collagen micro-scaffolds. Micro-scaffolds were highly porous and could be injected through small diameter needles (18-21 gauge). Collagen micro-scaffolds may be used as injectables for the local delivery of effector molecules and/or cells, thus creating specific niches to enhance local tissue regeneration.

Organ-specific tubular and collagen-based composite scaffolds.

The body contains a number of organs characterized by a tubular shape. In this study, we explored several methodologies for the construction of collagenous tubular scaffolds and films with defined (ultra)structure, length, diameter, orientation, and molecular composition. Standardization of molding, casting, freezing, and lyophilizing techniques using inexpensive materials and methods resulted in controllable fabrication of a wide variety of tubular and tissue-specific tubular scaffolds and films. Analysis included immunohistochemical and (ultra)structural examination. Handling and suturability were found adequate for tissue engineering applications.

Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

OBJECTIVE: Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. METHODS: Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37°C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. RESULTS: Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 µm. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Histology confirmed the network formation, which was surrounded by thrombus. CONCLUSIONS: Collagen injectables were prepared and EtO sterilized without major loss of structural integrity and platelet activity. In vivo, the injectable collagen formed a dense network and triggered (partial) local hemostasis. Although optimization is needed, an injectable collagen may be used as a therapeutic agent for femoral PSA treatment.

An animal model for femoral artery pseudoaneurysms.

PURPOSE: To prepare a porcine model for femoral artery pseudoaneurysm via a one-step surgical procedure without the need for microsurgery. MATERIALS AND METHODS: This pseudoaneurysm model involves the preparation of an arteriovenous shunt between the femoral artery and femoral vein in which approximately 2 cm of the vein is segmented by proximal and distal closure with the use of ligatures. The femoral pseudoaneurysm models were evaluated by angiography, Doppler auscultation, and histologic examination. RESULTS: In seven of eight pigs, angiography and Doppler auscultation showed that the pseudoaneurysm models were open and that there was communication between the pseudoaneurysm model and the femoral artery. The mean length (+/-SD) of the pseudoaneurysm model was 1.9 cm +/- 0.3 (n= 7), with a neck region of 4 mm. Histologic analysis confirmed that pseudoaneurysm models were open and no thrombi were observed. CONCLUSIONS: The principal advantages of this model are the location of the pseudoaneurysm model, the short period of clamping, and the controllable size. The pig pseudoaneurysm model is straightforward and reproducible, and may serve as a useful tool in the evaluation of interventional strategies for treatment of pseudoaneurysms.

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.

Construction of collagen scaffolds that mimic the three-dimensional architecture of specific tissues.

Every tissue and organ has its own 3-dimensional (3D) extracellular matrix (ECM) organization. Cells in a 3D bioscaffold for tissue engineering typically align new ECM components according to the bioscaffold provided. Therefore, scaffolds with a specific 3D structural design resembling the actual ECM of a particular tissue may have great potential in tissue engineering. Here, we show that, using specific freezing regimes, 3D scaffolds that mimic the 3D architecture of specific tissues can be made from collagen. Three examples are given, namely, scaffolds resembling the cup-shaped parenchymal (alveolar) architecture of lung, scaffolds that mimic the parallel collagen organization of tendon, and scaffolds that mimic the 3D organization of skin. For the preparation of these tissue-specific scaffolds, we relied on simple techniques without the need for expensive or customized equipment. Freezing rate, type of suspension medium, and additives (e.g., ethanol) were found to be prime parameters in controlling scaffold morphology.

From molecules to matrix: construction and evaluation of molecularly defined bioscaffolds.

In this chapter, we describe the fundamental aspects of the preparation of molecularly-defined scaffolds for soft tissue engineering, including the tissue response to the scaffolds after implantation. In particular, scaffolds prepared from insoluble type I collagen fibres, soluble type II collagen fibres, insoluble elastin fibres, glycosaminoglycans (GAGs) and growth factors are discussed. The general strategy is to prepare tailor-made "smart" biomaterials which will create a specific microenvironment thus enabling cells to generate new tissues. As an initial step, all biomolecules used were purified to homogeneity. Next, porous scaffolds were prepared using freezing and lyophilisation, and these scaffolds were crosslinked using carbodiimides. Crosslinking resulted in mechanically stronger scaffolds and allowed the covalent incorporation of GAGs. Scaffold characteristics were controlled to prepare tailor-made scaffolds by varying e.g. collagen to elastin ratio, freezing rate, degree of crosslinking, and GAGs attachment. The tissue response to scaffolds was evaluated following subcutaneous implantations in rats. Crosslinked scaffolds maintained their integrity and supported the formation of new extracellular matrix. Collagen-GAG scaffolds loaded with basic fibroblast growth factor significantly enhanced neovascularisation and tissue remodelling. Animal studies of two potential applications of these scaffolds were discussed in more detail, i.e. for bladder and cartilage regeneration.