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Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs., Statement of Significance: In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In skeletal muscle, the stem cell niche is important for controlling the quiescent, proliferation and differentiation states of satellite cells, which are key for skeletal muscle regeneration after wounding. It has been shown that type I collagen, often used as 3D-scaffolds for regenerative medicine purposes, impairs myoblast differentiation. This is most likely due to the absence of specific extracellular matrix proteins providing attachment sites for myoblasts and/or myotubes. In this study we investigated the differentiation capacity of primary murine myoblasts on type I collagen films either untreated or modified with elastin, laminin, type IV collagen, laminin/entactin complex, combinations thereof, and Matrigel as a positive control. Additionally, increased reactive oxygen species (ROS) and ROCK signaling might also be involved. To measure ROS levels with live-cell microscopy, fibronectin-coated glass coverslips were additionally coated with type I collagen and Matrigel onto which myoblasts were differentiated. On type I collagen-coated coverslips, myotube formation was impaired while ROS levels were increased. However, anti-oxidant treatment did not enhance myotube formation. ROCK inhibition, which generally improve cellular attachment to uncoated surfaces or type I collagen, enhanced myoblast attachment to type I collagen-coated coverslips and -films, but slightly enhanced myotube formation. Only modification of type I collagen films by Matrigel and a combination of laminin/entactin significantly improved myotube formation. Our results indicate that type I collagen scaffolds can be modified by satellite cell niche factors of which specifically laminin and entactin enhanced myotube formation. This offers a promising approach for regenerative medicine purposes to heal skeletal muscle wounds., Statement of Significance: In this manuscript we show for the first time that impaired myotube formation on type I collagen scaffolds can be completely restored by modification with laminin and entactin, two extracellular proteins from the satellite cell niche. This offers a promising approach for regenerative medicine approaches to heal skeletal muscle wounds., (Copyright © 2015. Published by Elsevier Ltd.)

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., (Copyright © 2010 John Wiley & Sons, Ltd.)

Wound contraction and scar formation after cleft palate repair lead to growth impairment of the maxilla and midface. Myofibroblasts play a key role in these processes. The application of an interferon-γ (IFN-γ)-loaded collagen scaffold after surgery might reduce the differentiation of myofibroblasts. In this study, the tissue response to IFN-γ-loaded collagen scaffolds was evaluated after implantation in the palate of rats. Scaffolds, with or without IFN-γ, were implanted submucoperiosteally in the palate of two groups of 25 five-week-old male Wistar rats. Groups of five rats were sacrificed at 1, 2, 4, 8, and 16 weeks post-implantation and processed for histological analyses. On haematoxylin and eosin-stained sections, the cell density and number of giant cells within the scaffolds were determined. Blood vessels, inflammatory cells, and myofibroblasts were detected by immunohistochemistry. The data for cell density, blood vessels, and giant cells were compared with a two-way analysis of variance. The scores for myofibroblasts and inflammation were compared by a rank sum test. A mild and rapidly subsiding inflammatory and foreign body response was found in both groups. Angiogenesis had already begun after 1 week, showed a peak after 4 weeks, and declined thereafter. IFN-γ induced a faster influx of host cells and a major reduction in myofibroblast numbers. The scaffolds might be suitable for future applications in oral surgery.

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.

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., (Copyright © 2010 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

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.

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.